CN101629178A - Manually-combined Newcastle diseases virus F gene and recombining expression vector and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种人工改造的鸡新城疫病毒F基因及其重组表达载体和应用。本发明将NDV F基因ORF的密码子全部替换为鸡体内偏嗜的密码子，在上游引入EcoR V酶切位点和Kozak序列，下游引入Xbal I酶切位点和TGA终止密码子，并把终止密码子下游碱基A改为G，所得到的基因(SEQ ID NO：2)能够在真核细胞中高效表达，其表达效率比野生型F基因表达效率高，所表达的蛋白具有鸡新城疫病毒天然蛋白的免疫原性和生物学活性，并能刺激鸡体产生比野生型F基因DNA疫苗更加强烈的体液免疫应答和细胞免疫应答，使免疫鸡获得100％的抵抗高致病力新城疫病毒株的致死性攻击，可有效阻止病毒在体内的增殖。

The invention discloses an artificially transformed chicken Newcastle disease virus F gene and its recombinant expression vector and application. In the present invention, all the codons of NDV F gene ORF are replaced with codons favored in chickens, an EcoR V restriction site and a Kozak sequence are introduced upstream, an Xbal I restriction site and a TGA stop codon are introduced downstream, and the The downstream base A of the stop codon is changed to G, and the resulting gene (SEQ ID NO: 2) can be highly expressed in eukaryotic cells, and its expression efficiency is higher than that of the wild-type F gene, and the expressed protein has the characteristics of chicken Newcastle disease virus The immunogenicity and biological activity of the natural protein can stimulate the chicken to produce a stronger humoral immune response and cellular immune response than the wild-type F gene DNA vaccine, so that the immunized chicken can obtain 100% resistance to the highly pathogenic Newcastle disease virus The lethal attack of the strain can effectively prevent the proliferation of the virus in the body.

Description

Translated fromChinese

人工改造的鸡新城疫病毒F基因及其重组表达载体和应用Artificially modified chicken Newcastle disease virus F gene and its recombinant expression vector and application

技术领域technical field

本发明涉及一种cDNA，尤其涉及一种人工改造的鸡新城疫病毒E₄₈F₉株F基因以及含有该F基因的重组真核表达载体，本发明还涉及它们作为DNA疫苗在预防或治疗鸡新城疫疾病中的应用，属于鸡新城疫疾病的防制领域。The present invention relates to a cDNA, in particular to an artificially modified F gene of chicken Newcastle disease virus E₄₈ F₉ strain and a recombinant eukaryotic expression vector containing the F gene. The invention also relates to their use as DNA vaccines in preventing or treating chicken Newcastle disease The application of the invention belongs to the field of prevention and control of chicken Newcastle disease.

背景技术Background technique

新城疫病毒(Newcastle disease virus，NDV)属于副黏病毒科腮腺炎病毒属，是一种高致病性病毒，对禽类危害较大，是世界公认的两大重要禽病之一。本病的病死率极高，强毒力毒株常可以引起易感鸡群100％感染，并造成100％死亡，已经遍布世界各地。目前，我国对于鸡新城疫主要采用油佐剂灭活疫苗或弱毒活疫苗等传统疫苗进行免疫预防。灭活疫苗具有保护率高、免疫期长、研制周期短等优点，但其最大的不足是疫苗接种后诱导产生的抗体与自然感染鸡无法区分，难以用现行的血清学检测方法进行疫情监测，从而影响鸡新城疫的流行病学调查，不利于对疾病的发生和流行进行控制，而且灭活疫苗的成本高、接种反应大，存在因灭活不全而散毒的危险。常规弱毒活疫苗也存在散毒问题，某些疫苗株毒性较强，能造成免疫鸡群轻微感染以及低日龄鸡的发病，为疾病的流行和毒株变异埋下安全隐患。因此，有必要探寻新型疫苗以弥补上述不足，而DNA疫苗则为实现这一目标提供了可能，鸡新城疫DNA疫苗的开发和应用将为防制当前流行的新城疫提供有效的工具和手段。Newcastle disease virus (NDV) belongs to the genus Mumps virus in the Paramyxoviridae family. It is a highly pathogenic virus that is harmful to poultry and is recognized as one of the two important poultry diseases in the world. The case fatality rate of this disease is extremely high, and the highly virulent virus strain can often cause 100% infection of susceptible flocks, and cause 100% death, has spread all over the world. At present, traditional vaccines such as oil adjuvant inactivated vaccines or attenuated live vaccines are mainly used for immunization prevention against Newcastle disease in my country. Inactivated vaccines have the advantages of high protection rate, long immunization period, and short development cycle, but their biggest disadvantage is that the antibodies induced after vaccination are indistinguishable from those of naturally infected chickens, and it is difficult to monitor the epidemic situation with current serological detection methods. Thereby affecting the epidemiological investigation of Newcastle disease, it is not conducive to controlling the occurrence and prevalence of the disease, and the cost of the inactivated vaccine is high, the inoculation reaction is large, and there is a risk of loosening the virus due to incomplete inactivation. Conventional attenuated live vaccines also have the problem of loose toxins. Some vaccine strains are highly toxic, which can cause mild infection in immune flocks and disease in young chickens, laying a safety hazard for the epidemic of diseases and strain variation. Therefore, it is necessary to search for new vaccines to make up for the above-mentioned shortcomings, and DNA vaccines provide the possibility to achieve this goal. The development and application of chicken Newcastle disease DNA vaccines will provide effective tools and means for preventing and controlling the current epidemic of Newcastle disease.

对于DNA疫苗而言，抗原蛋白的表达水平对其免疫原性和保护效力起着十分关键的作用。有相当多的文献报道，免疫原基因密码子的优化可以显著地改善蛋白的表达效率。Leder等对HPV16L1、L2的密码子进行了优化，分别用哺乳动物细胞及植物细胞偏爱密码子取代原有基因的相应密码子，用合成基因转染哺乳动物细胞后，使用哺乳动物细胞偏爱密码子合成的基因蛋白表达水平较原野生型病毒基因增加了10⁴～10⁵倍。而用植物细胞偏爱密码合成的基因其蛋白表达水平也至少提高了100倍。Andre等人工合成人免疫缺陷病I型病毒(HIV)gp120基因，该基因中原来存在的大多数密码子被替换为人体偏好的密码子，体外试验表明改造后的基因表达效率比原始基因显著提高，在动物试验中合成基因所诱导的抗体滴度和CTL反应显著高于原始基因。随后，大量的研究证明用经过密码子优化的DNA疫苗接种模型动物，所诱导的免疫应答显著提高。密码子优化的人免疫缺陷病病毒的gp120、Gag、Gag-pol、Tat、Rev和Nef基因，用于构建DNA疫苗或重组病毒活载体疫苗后，其特异性细胞和体液免疫反应均获得了显著改善，目前几乎所有的HIV重组疫苗都采用了密码子优化免疫原基因。密码子优化免疫原基因在人的乳头瘤病毒、犬乳头瘤病毒、狂犬病病毒以及SARS冠状病毒DNA疫苗相继得到应用。密码子优化免疫原基因增强免疫反应的机制不仅仅涉及翻译过程密码子移动速度加快，真核生物偏嗜性密码子优化往往导致mRNA中CG含量的大幅度提高，因而彻底改变RNA的二级结构，一方面有可能增加mRNA的稳定性，另一方面还有可能增加mRNA转录后从细胞核向细胞浆的转运效率。此外，CG含量的增加还有可能增加CpG的出现频率，进一步发挥DNA疫苗本身免疫佐剂的作用。For DNA vaccines, the expression level of antigenic protein plays a key role in its immunogenicity and protective efficacy. There are quite a lot of literature reports that the optimization of immunogen gene codons can significantly improve the expression efficiency of proteins. Leder et al. optimized the codons of HPV16L1 and L2, and replaced the corresponding codons of the original genes with mammalian cell and plant cell preferred codons respectively. After transfecting mammalian cells with synthetic genes, mammalian cell preferred codons were used. The protein expression level of the synthesized gene is 10⁴ -10⁵ times higher than that of the original wild-type virus gene. And the protein expression level of genes synthesized with the plant cell's preferred code was at least 100 times higher. Andre et al. artificially synthesized the human immunodeficiency virus type 1 (HIV) gp120 gene, and most of the original codons in the gene were replaced with codons preferred by the human body. In vitro tests showed that the expression efficiency of the modified gene was significantly higher than that of the original gene , In animal experiments, the antibody titers and CTL responses induced by the synthetic gene were significantly higher than those of the original gene. Subsequently, a large number of studies have demonstrated that the immune response induced by inoculating model animals with codon-optimized DNA vaccines is significantly improved. Codon-optimized gp120, Gag, Gag-pol, Tat, Rev, and Nef genes of human immunodeficiency disease virus were used to construct DNA vaccines or recombinant virus live vector vaccines, and their specific cellular and humoral immune responses were significantly improved To improve, almost all HIV recombinant vaccines currently use codon-optimized immunogen genes. Codon-optimized immunogen genes have been successively applied in human papillomavirus, canine papillomavirus, rabies virus and SARS coronavirus DNA vaccines. The mechanism by which codon-optimized immunogenic genes enhance the immune response not only involves the acceleration of codon movement during translation, but eukaryote-biased codon optimization often leads to a substantial increase in the CG content in mRNA, thus completely changing the secondary structure of RNA , on the one hand, it may increase the stability of mRNA, on the other hand, it may also increase the translocation efficiency of mRNA from the nucleus to the cytoplasm after transcription. In addition, the increase of CG content may also increase the frequency of CpG, and further play the role of DNA vaccine itself as an immune adjuvant.

由于核酸疫苗可诱导机体产生全面的免疫应答，并且对不同亚型的病原体具有交叉防御作用，同时兼备安全、可靠、生产方便等优点，故被认为是继减毒活疫苗、灭活疫苗和基因工程亚单位疫苗之后的第三代疫苗。但是，DNA疫苗也存在其自身的局限性，目的基因的免疫原性较弱以及其蛋白表达水平不高严重制约了它的发展。Because nucleic acid vaccines can induce a comprehensive immune response in the body, and have cross-defense effects against different subtypes of pathogens, and have the advantages of safety, reliability, and convenient production, they are considered to be the successor to attenuated live vaccines, inactivated vaccines and gene vaccines. The third generation of vaccines after engineered subunit vaccines. However, DNA vaccines also have their own limitations. The weak immunogenicity of the target gene and the low level of protein expression seriously restrict its development.

蛋白的表达水平与其引起的免疫反应直接相关，而编码氨基酸密码子的摇摆性导致了不同物种的细胞在蛋白翻译过程中具有密码子使用的偏嗜性，动物细胞在蛋白翻译过程中与病毒蛋白氨基酸密码子使用频率存在着明显差异。基因的修饰可以显著地改善蛋白的表达效率。为了探索解决这一问题的新途径，提高F基因的表达量从而增强其免疫原性。The expression level of the protein is directly related to the immune response it causes, and the swing of the coding amino acid codons leads to the preference of codon usage in cells of different species during protein translation, and animal cells interact with viral proteins during protein translation. There are significant differences in the frequency of amino acid codon usage. Gene modification can significantly improve protein expression efficiency. In order to explore a new way to solve this problem, increase the expression of F gene to enhance its immunogenicity.

研究表明，NDV F基因密码子的优化可显著提高F基因表达水平和新城疫病毒F₄₈E₉株DNA疫苗诱导的保护性抗体免疫反应水平，显著增强免疫保护效果。因此，通过基因修饰的新城疫病毒F₄₈E₉株DNA疫苗将有着良好的的应用前景。Studies have shown that the optimization of NDV F gene codon can significantly improve the expression level of F gene and the level of protective antibody immune response induced by DNA vaccine of Newcastle disease virus F₄₈ E₉ strain, and significantly enhance the immune protection effect. Therefore, the genetically modified Newcastle disease virus F₄₈ E₉ strain DNA vaccine will have a good application prospect.

原有的NDV F基因在真核细胞中的表达效率不高，需要对其进行分子改造和修饰，以提高其在真核细胞中的表达效率，以提高NDVF₄₈E₉株DNA疫苗在鸡体内的表达水平和免疫保护效果。The expression efficiency of the original NDV F gene in eukaryotic cells is not high, and it needs molecular transformation and modification to improve its expression efficiency in eukaryotic cells, so as to improve the NDV F₄₈ E₉ strain DNA vaccine in chickens. expression level and immune protection effect.

发明内容Contents of the invention

本发明首先所要解决的技术问题是克服现有技术的不足，将新城疫病毒F₄₈E₉株F基因进行分子改造和修饰，提供一种更适合在鸡体细胞中高效表达的新城疫病毒人工F基因。The first technical problem to be solved in the present invention is to overcome the deficiencies of the prior art, carry out molecular transformation and modification of the F gene of Newcastle disease virus F₄₈ E₉ strain, and provide a kind of artificial Newcastle disease virus that is more suitable for high-efficiency expression in chicken somatic cells. F gene.

本发明首先所要解决的技术问题是通过以下技术方案来实现的：The technical problem to be solved in the present invention is realized by the following technical solutions:

本发明在不改变原有新城疫病毒F基因(SEQ ID NO.1)所编码的氨基酸序列的前提下，按照鸡体内密码子的选择取向对鸡新城疫病毒F₄₈E₉株F基因密码子进行优化，规避了一些可能降低表达的序列，得到了一种新的适合在鸡体细胞中高效表达的鸡新城疫病毒人工F基因序列。On the premise of not changing the amino acid sequence encoded by the original Newcastle disease virus F gene (SEQ ID NO.1), the present invention optimizes the codon of the F gene of chicken Newcastle disease virus F₄₈ E₉ strain according to the selection orientation of the codon in the chicken , evaded some sequences that may reduce expression, and obtained a new artificial F gene sequence of chicken Newcastle disease virus that is suitable for high-efficiency expression in chicken somatic cells.

具体的，本发明将NDV F₄₈E₉株F基因ORF的密码子全部替换为鸡体内偏嗜的密码子，在上游引入EcoR V酶切位点和Kozak序列，下游引入XbalI酶切位点和TGA终止密码子，并把终止密码子下游碱基A改为G，得到一种适合在鸡体细胞中高效表达的鸡新城疫病毒人工F基因，其核苷酸序列为SEQ ID NO：2所示。Specifically, the present invention replaces all the codons of the F gene ORF of the NDV F₄₈ E₉ strain with codons favored in chickens, introduces an EcoR V restriction site and a Kozak sequence upstream, and introduces an XbalI restriction site and a Kozak sequence downstream. TGA stop codon, and change the downstream base of the stop codon from A to G to obtain a chicken Newcastle disease virus artificial F gene suitable for high-efficiency expression in chicken somatic cells, the nucleotide sequence of which is shown in SEQ ID NO:2.

优化后的F基因(SEQ ID NO：2)GC含量由原来优化前的44.28％变为65.46％，大大提高了F基因中的GC含量；同时，在起始密码子ATG上游引入了一段影响其起始效率的Kozak序列，并在3′端引入了鸡体偏嗜的终止密码子(TGA)，改变了可能影响其翻译终止效率终止密码子下游碱基。重新设计合成的基因也去除或改变了mRNA去稳定的序列，消除了稀有密码子，从而使蛋白的生产更有效、更经济。The GC content of the optimized F gene (SEQ ID NO: 2) changed from 44.28% before optimization to 65.46%, which greatly increased the GC content in the F gene; Kozak sequence for initiation efficiency, and a chicken-preferred stop codon (TGA) was introduced at the 3' end, and the downstream base of the stop codon that may affect its translation termination efficiency was changed. Redesigning synthetic genes also removes or alters mRNA destabilizing sequences, eliminating rare codons and thus making protein production more efficient and economical.

本发明所要解决第二个技术问题是提供一种含有SEQ ID NO.2所示核苷酸序列的重组真核表达载体。The second technical problem to be solved by the present invention is to provide a recombinant eukaryotic expression vector containing the nucleotide sequence shown in SEQ ID NO.2.

本发明所要解决第二个技术问题是通过以下技术方案来实现的：The second technical problem to be solved by the present invention is achieved through the following technical solutions:

一种重组真核表达载体，含有SEQ ID NO.2所示的核苷酸序列。A recombinant eukaryotic expression vector contains the nucleotide sequence shown in SEQ ID NO.2.

本发明的重组真核表达载体可通过本领域的常规方法构建而成，即：将SEQ ID NO.2所示的核苷酸序列插入到真核表达载体合适的限制性酶切位点之间，使SEQ ID NO：2所示的核苷酸序列可操作的与真核表达载体相连接并在真核表达载体相关元件的调控下获得正确表达。The recombinant eukaryotic expression vector of the present invention can be constructed by conventional methods in the art, that is, the nucleotide sequence shown in SEQ ID NO.2 is inserted between suitable restriction enzyme sites of the eukaryotic expression vector , so that the nucleotide sequence shown in SEQ ID NO: 2 is operably linked to the eukaryotic expression vector and is correctly expressed under the control of the related elements of the eukaryotic expression vector.

作为本发明的一个最优选的实施方案，优选为将SEQ ID NO：2所示的核苷酸序列插入到真核表达载体pVAX1多克隆位点区的EcoRV和XbaI限制性酶切位点之间，使该核苷酸序列位于CMV启动子的下游并受其调控，得到重组真核表达载体pVAX1-optiF。As a most preferred embodiment of the present invention, preferably the nucleotide sequence shown in SEQ ID NO:2 is inserted between EcoRV and the XbaI restriction enzyme site in the eukaryotic expression vector pVAX1 multiple cloning site region , make the nucleotide sequence located downstream of the CMV promoter and be regulated by it, and obtain the recombinant eukaryotic expression vector pVAX1-optiF.

本发明所构建的重组真核表达载体可通过常规的方法转染宿主细胞，所述的宿主细胞可为293或COS7细胞，再通过间接免疫荧光技术和Western-blot技术来鉴定其表达情况，表达出的目的蛋白具有新城疫病毒天然蛋白的生物学活性和免疫原性。The recombinant eukaryotic expression vector constructed in the present invention can be transfected into host cells by conventional methods, and the host cells can be 293 or COS7 cells, and then its expression can be identified by indirect immunofluorescence technology and Western-blot technology. The obtained target protein has the biological activity and immunogenicity of the natural protein of Newcastle disease virus.

本发明所要解决第三个技术问题是大量制备更适于转染鸡体细胞和刺激鸡体免疫系统的DNA疫苗。The third technical problem to be solved by the present invention is to prepare a large amount of DNA vaccines that are more suitable for transfecting chicken somatic cells and stimulating the immune system of chickens.

本发明所要解决第三个技术问题是通过以下技术方案来实现的：The third technical problem to be solved by the present invention is achieved through the following technical solutions:

提取含有SEQ ID NO：2所示的核苷酸序列的质粒DNA，纯化，即得DNA疫苗。The plasmid DNA containing the nucleotide sequence shown in SEQ ID NO: 2 is extracted and purified to obtain the DNA vaccine.

本发明优选的技术方案的整体描述：The overall description of the preferred technical solution of the present invention:

应用RT-PCR技术扩增自国内来航鸡分离的新城疫病毒F₄₈E₉株基因组。以NDV F₄₈E₉株的基因组cDNA为模版，经根据Genbank中发表的新城疫病毒F₄₈E₉株F基因(ID：AY508514)序列设计一对引物(上游：5′-ATA GAT ATC ATG GGC CCC AAA TCT TCT-3′；下游：5′-CTA TCT AGA GGC CAC TAC AAG AAT CTG A-3′)来扩增野生型F基因，将F基因克隆到pMD-18T载体中，然后通过酶切和测序鉴定正确后记为pMD-18T-F。RT-PCR technique was used to amplify the genome of Newcastle disease virus F₄₈ E₉ strain isolated from domestic Laihang chickens. Using the genomic cDNA of NDV F_{48E 9strain} as a template,_a pair of primers (upstream: 5′-ATA GAT ATC ATG GGC CCC AAA TCT TCT-3′; downstream: 5′-CTA TCT AGA GGC CAC TAC AAG AAT CTG A-3′) to amplify the wild-type F gene, clone the F gene into the pMD-18T vector, and then digest It is recorded as pMD-18T-F after being correctly identified by sequencing.

利用Genestar软件将NDV F₄₈E₉株F基因的ORF翻译为氨基酸序列，根据密码子使用频率表，将F基因的密码子全部替换为鸡体内偏嗜的密码子，以鸡偏嗜密码子将氨基酸序列反翻译为DNA序列；在上游引入EcoR V酶切位点和Kozak序列，下游引入Xbal I酶切位点和鸡体偏嗜的终止密码子TGA，紧接着终止密码子的下游碱基A改为G。利用Genestar软件核查设计的序列，进行氨基酸比对和酶切位点检查，合成最终序列(SEQ ID NO：2)并克隆到pMD-18T载体中，酶切和测序鉴定正确后记为pMD-18T-optiF。Using Genestar software, the ORF of the F gene of NDV F₄₈ E₉ strain was translated into an amino acid sequence. According to the codon usage frequency table, all the codons of the F gene were replaced with chicken preference codons, and chicken preference codons were used to The amino acid sequence is back-translated into a DNA sequence; the EcoR V restriction site and Kozak sequence are introduced upstream, the Xbal I restriction site and the stop codon TGA favored by chickens are introduced downstream, followed by the downstream base A of the stop codon Change to G. Use the Genestar software to check the designed sequence, perform amino acid alignment and restriction site inspection, synthesize the final sequence (SEQ ID NO: 2) and clone it into the pMD-18T vector, and mark it as pMD-18T- optiF.

将pMD-18T-F与pMD-18T-optiF分别经EcoR V和XbalI酶切处理后回收目的片段，并把回收的目的片段与经同样酶切处理的表达载体pVAX1连接，经酶切、鉴定后得到重组质粒分别命名为pVAX1-F和pVAX1-optiF，即得到新城疫病毒F基因DNA疫苗。pMD-18T-F and pMD-18T-optiF were digested with EcoR V and XbalI respectively, and the target fragments were recovered, and the recovered target fragments were connected with the expression vector pVAX1 that had been digested with the same enzymes. The obtained recombinant plasmids were respectively named pVAX1-F and pVAX1-optiF, and the Newcastle disease virus F gene DNA vaccine was obtained.

将pVAX1-F和pVAX1-optiF两种重组质粒分别在脂质体Lipofectamine^TM 2000介导下转染COS 7细胞，通过间接免疫荧光和Western-blot技术来检测重组质粒的体外表达情况；经间接免疫荧光试验、SDS变性聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western-blot检测证明重组质粒pVAX1-optiF在体外能正确表达，表达的重组蛋白具有鸡新城疫病毒天然蛋白的生物学活性和免疫原性。Two recombinant plasmids, pVAX1-F and pVAX1-optiF, were transfected intoCOS 7 cells under the mediation of liposome Lipofectamine^TM 2000, and the in vitro expression of the recombinant plasmids was detected by indirect immunofluorescence and Western-blot techniques; Fluorescence test, SDS-denatured polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot detection proved that the recombinant plasmid pVAX1-optiF can be correctly expressed in vitro, and the expressed recombinant protein has the biological activity and immunogenicity of the natural protein of chicken Newcastle disease virus .

在采用间接免疫荧光技术检测F基因的体外表达时发现，转染重组质粒pVAX1-optiF的细胞中荧光细胞数目明显多于pVAX1-F，这与Western-blot检测的结果是一致的。体外表达结果表明，密码子优化的F基因在鸡体细胞中表达蛋白的水平显著高于野生型F基因，由于将密码子改造为鸡体嗜好的密码子后，加快了翻译过程密码子移动速度，同时由于GC含量大幅度提高彻底改变了RNA的二级结构，这不仅增加了mRNA的稳定性，还可能增加mRNA转录后从细胞核向细胞浆的转运效率，最终使蛋白的表达量大大提高。When detecting the expression of F gene in vitro by indirect immunofluorescence technique, it was found that the number of fluorescent cells in cells transfected with recombinant plasmid pVAX1-optiF was significantly more than that of pVAX1-F, which was consistent with the results of Western-blot detection. The results of in vitro expression showed that the protein expression level of the codon-optimized F gene in chicken somatic cells was significantly higher than that of the wild-type F gene, because the codon movement speed in the translation process was accelerated after the codons were modified to the codons favored by the chicken body , At the same time, due to the substantial increase in GC content, the secondary structure of RNA is completely changed, which not only increases the stability of mRNA, but also may increase the transfer efficiency of mRNA from the nucleus to the cytoplasm after transcription, and finally greatly increase the expression of protein.

本发明的鸡新城疫病毒F₄₈E₉株F-DNA疫苗(pVAX1-optiF)能够在体外瞬时表达细胞COS 7高效表达，所表达的重组蛋白具有鸡新城疫病毒天然蛋白的生物学活性和免疫原性。将所得到的重组质粒(pVAX1-optiF)制成DNA疫苗，免疫鸡实验结果表明，本发明鸡新城疫病毒F48E9株优化的F-DNA疫苗能有效诱导机体产生特异性的体液和细胞免疫应答，在免疫剂量为200μg的条件下使免疫鸡获得100％的抵抗高致病力NDV致死性攻击的保护，并可有效阻止病毒在体内的增殖，试验结果说明，本发明的DNA疫苗能够有效防治鸡新城疫。The chicken Newcastle disease virus F₄₈ E₉ strain F-DNA vaccine (pVAX1-optiF) of the present invention can transiently expressCOS 7 in vitro and highly express it, and the expressed recombinant protein has the biological activity and immunogenicity of the chicken Newcastle disease virus natural protein. The obtained recombinant plasmid (pVAX1-optiF) is made into a DNA vaccine, and the results of the immunization chicken experiment show that the F-DNA vaccine optimized by the chicken Newcastle disease virus F48E9 strain of the present invention can effectively induce the body to produce specific humoral and cellular immune responses. Under the condition of a dose of 200 μg, the immunized chickens can obtain 100% protection against the lethal attack of highly pathogenic NDV, and can effectively prevent the proliferation of the virus in the body. The test results show that the DNA vaccine of the present invention can effectively prevent and treat Newcastle disease.

特异性抗体水平与新城疫免疫的保护率高度相关。将两种质粒免疫鸡后发现pVAX1-optiF免疫鸡的特异性抗体产生比较迅速，而且上升的幅度比较大，从一免后2周开始，诱导产生的抗体水平显著高于pVAX1-F免疫鸡，而且随后差距越来越大。由此可见，优化的F基因比野生型F基因表达出了更多的F蛋白，从而诱导产生出更高的特异性抗体。因此，在动物体内免疫原基因蛋白表达量的提高是改善DNA疫苗免疫效果的重要途径之一。The level of specific antibody is highly correlated with the protection rate of Newcastle disease immunity. After immunizing chickens with the two plasmids, it was found that the specific antibody production of pVAX1-optiF immunized chickens was relatively rapid, and the increase was relatively large. From 2 weeks after the first immunization, the level of induced antibodies was significantly higher than that of pVAX1-F immunized chickens. And then the gap widened. It can be seen that the optimized F gene expresses more F protein than the wild-type F gene, thereby inducing a higher specific antibody. Therefore, increasing the expression of immunogenic gene protein in animals is one of the important ways to improve the immune effect of DNA vaccines.

淋巴细胞转化率是考察机体细胞免疫状态的重要指标之一，本试验免疫鸡脾淋巴细胞特异性增殖结果显示，二免后2周，pVAX1-F和pVAX1-optiF免疫鸡后所测的OD₅₇₀值分别为0.3804±0.0344和0.5804±0.0789，PBS对照组和pVAX1对照组分别为0.2541±0.0535和0.2664^a±0.0410，两重组质粒免疫组淋巴细胞转化率都显著高于对照组，说明DAN免疫质粒确实在一定程度上激活了机体的细胞免疫系统，而pVAX1-optiF免疫鸡脾细胞对Con A表现出更明显的反应性，说明免疫原基因的密码子优化后诱发了较强的细胞免疫。CD4⁺是T淋巴细胞的一个亚群，是免疫应答的中心细胞。主要识别由MHC II类分子递呈的抗原，诱导Th I型细胞因子的分泌，辅助B细胞合成抗体；CD8⁺T细胞又称细胞毒性T淋巴细胞，其主要功能是特异性直接杀伤靶细胞，它主要识别由MHC I类分子递呈的抗原短肽，在T淋巴细胞免疫应答中发挥重要功能。因此，动物体内CD8⁺T细胞的含量是评价动物机体细胞免疫状况的一个重要指标。Lymphocyte transformation rate is one of the important indicators to investigate the state of cellular immunity of the body. The results of the specific proliferation of lymphocytes in the spleen of immunized chickens in this experiment showed that 2 weeks after the second immunization, the OD₅₇₀ measured after pVAX1-F and pVAX1-optiF immunized chickens The values were 0.3804±0.0344 and 0.5804±0.0789, respectively, and the PBS control group and pVAX1 control group were 0.2541±0.0535 and 0.2664^a ±0.0410 respectively. The lymphocyte transformation rates of the two recombinant plasmid immunization groups were significantly higher than those of the control group, indicating that the DAN immunization plasmid was indeed To a certain extent, the cellular immune system of the body was activated, while pVAX1-optiF immunized chicken splenocytes showed more obvious reactivity to Con A, indicating that the codon optimization of the immunogen gene induced stronger cellular immunity. CD4⁺ is a subset of T lymphocytes and is the central cell of the immune response. It mainly recognizes antigens presented by MHC class II molecules, induces the secretion of Th I type cytokines, and assists B cells to synthesize antibodies; CD8⁺ T cells are also called cytotoxic T lymphocytes, and their main function is to specifically and directly kill target cells. It mainly recognizes short antigenic peptides presented by MHC class I molecules and plays an important role in T lymphocyte immune response. Therefore, the content of CD8⁺ T cells in the animal body is an important indicator for evaluating the cellular immune status of the animal body.

本试验在对免疫鸡外周血淋巴细胞亚群动态变化的检测中发现，用质粒pVAX1-optiF免疫鸡的CD4⁺和CD8⁺T淋巴细胞百分数在免疫后27d均显著高于pVAX1-F免疫鸡的，并且这两个免疫质粒试验组又显著高于pVAX1质粒免疫对照组和PBS空白对照组，说明构建的质粒在动物细胞中得到了表达，激活包括CD8⁺的CTL，CD4+的Th1细胞及产生IgG2a为主的B淋巴细胞。成熟T细胞表面可表达2种不同类型的T细胞抗原受体(TCR)，一种是TCRαβ，另一种是由γ链和δ链组成的异二聚体分子TCRγδ。外周血中的TCRγδT细胞仅占成熟T细胞的0.5％～10％，TCRγδ识别病原体表面抗原分子后，增殖分化为效应细胞，释放细胞毒性效应分子如穿孔素、颗粒酶，表达Fas/FasL以及分泌IFN-γ，最终清除感染细胞和病原微生物。全身性γδT细胞可以快速被募集并易于活化，它们可以在局部释放IL-2、IL-4、IL-5、IL-6、IL-10、IFN-7、GM-CSF、TNF-α等多种细胞因子，参与免疫调节，增强机体非特异性免疫防御功能。初免27d后，pVAX1-optiF免疫鸡外周血TCRγδT细胞百分率比pVAX1-F免疫鸡明显增高(9.1522％±1.2547％对6.3989％±1.1625％，P＜0.05)，并且这两个免疫质粒试验组又显著高于pVAX1质粒免疫对照组和PBS空白对照组。当机体在受到病毒感染时，γδT细胞活化并增殖，活化的γδT细胞识别并杀伤病毒感染的靶细胞，所以推测pVAX1-optiF免疫鸡机体内含有更多的TCRγδT细胞而具有更强的抗病毒能力。In this experiment, in the detection of dynamic changes of peripheral blood lymphocyte subsets of immunized chickens, it was found that the percentages of CD4⁺ and CD8⁺ T lymphocytes in chickens immunized with plasmid pVAX1-optiF were significantly higher than those in chickens immunized with pVAX1-F at 27 days after immunization , and these two immune plasmid test groups were significantly higher than the pVAX1 plasmid immune control group and PBS blank control group, indicating that the constructed plasmid was expressed in animal cells, activated CTL including CD8⁺ , CD4 + Th1 cells and produced IgG2a Predominantly B lymphocytes. Two different types of T cell antigen receptors (TCR) can be expressed on the surface of mature T cells, one is TCRαβ, and the other is TCRγδ, a heterodimeric molecule composed of γ chain and δ chain. TCRγδT cells in peripheral blood only account for 0.5% to 10% of mature T cells. After TCRγδ recognizes pathogen surface antigen molecules, it proliferates and differentiates into effector cells, releases cytotoxic effector molecules such as perforin and granzyme, expresses Fas/FasL and secretes IFN-γ, which ultimately clears infected cells and pathogenic microorganisms. Systemic γδT cells can be quickly recruited and easily activated, and they can locally release IL-2, IL-4, IL-5, IL-6, IL-10, IFN-7, GM-CSF, TNF-α, etc. A cytokine that participates in immune regulation and enhances the body's non-specific immune defense function. After 27 days of primary immunization, the percentage of TCRγδT cells in the peripheral blood of pVAX1-optiF-immunized chickens was significantly higher than that of pVAX1-F-immunized chickens (9.1522%±1.2547% vs. 6.3989%±1.1625%, P<0.05), and the two immune plasmid test groups also Significantly higher than pVAX1 plasmid immunization control group and PBS blank control group. When the body is infected by the virus, the γδT cells are activated and proliferate, and the activated γδT cells recognize and kill the virus-infected target cells, so it is speculated that pVAX1-optiF immunized chickens contain more TCRγδT cells and have stronger antiviral ability .

评价一个疫苗的好坏，最关键的还是看疫苗对免疫动物的保护效果，本发明二免后2周分别对所有试验鸡以10⁵EID₅₀NDV的F₄₈E₉强毒进行滴鼻攻毒后，最终PBS对照组和pVAX1对照组全部死亡，而pVAX1-F组12只存活2只，最终的保护率为16.7％；而pVAX1-optiF组的最终保护率为100％，达到完全保护。To evaluate the quality of a vaccine, the most important thing is to look at the protective effect of the vaccine on the immunized animals. Two weeks after the second immunization, all test chickens were challenged by nasal drops with the virulent F₄₈ E₉ of 10⁵ EID₅₀ NDV Finally, all of the PBS control group and pVAX1 control group died, while 2 of the 12 animals in the pVAX1-F group survived, with a final protection rate of 16.7%; while the final protection rate of the pVAX1-optiF group was 100%, reaching complete protection.

实验结果表明，采用F基因密码子的优化可显著提高F基因表达水平和新城疫病毒F₄₈E₉株DNA疫苗诱导的保护性抗体免疫反应水平，显著增强免疫保护效果。The experimental results show that the optimization of F gene codon can significantly improve the expression level of F gene and the level of protective antibody immune response induced by DNA vaccine of Newcastle disease virus F₄₈ E₉ strain, and significantly enhance the immune protection effect.

用优化的鸡新城疫病毒F₄₈E₉株F基因的新城疫DNA疫苗，其效果与传统疫苗一样。比起用SPF鸡胚或细胞培养物制备的传统NDV疫苗，DNA疫苗的制备简单、方便，整个制备过程的经济消耗也较低，表达产物经过粗提即可应用。因此，本发明新城疫DNA疫苗与现有的疫苗相比将具有更好的应用前景。The effect of the Newcastle disease DNA vaccine using the optimized Newcastle disease virus F₄₈ E₉ strain F gene is the same as that of the traditional vaccine. Compared with the traditional NDV vaccine prepared by SPF chicken embryo or cell culture, the preparation of DNA vaccine is simple and convenient, and the economic consumption of the whole preparation process is also low, and the expression product can be used after crude extraction. Therefore, compared with the existing vaccines, the Newcastle disease DNA vaccine of the present invention will have better application prospects.

DNA疫苗利用的仅仅是病毒的一段抗原基因，不是完整的病毒基因组，不具有感染性，是可以在动物细胞中表达抗原性蛋白的的安全疫苗，不存在散毒的危险。本发明DNA疫苗将为防制当前流行的鸡新城疫疾病提供有效的工具和手段，并可为防制畜禽其它疾病提供有效的参考和经验，投入应用后既能有效地防止新城疫流行造成的经济损失，同时也降低了养殖成本，具有重要的科学意义和应用价值，并能带来更好的经济效益和社会效益。DNA vaccines use only a segment of the antigenic gene of the virus, not the complete viral genome, and are not infectious. They are safe vaccines that can express antigenic proteins in animal cells, and there is no risk of spreading the virus. The DNA vaccine of the present invention will provide effective tools and means for the prevention and control of the current popular Newcastle disease disease, and can provide effective reference and experience for the prevention and control of other diseases of livestock and poultry. It also reduces the cost of breeding, has important scientific significance and application value, and can bring better economic and social benefits.

本发明DNA疫苗的用法与用量：用法：经由大腿肌肉注射。用量：雏鸡0.2mL/羽(浓度为1mg/mL)，成年鸡0.4mL/羽(浓度为0.5mg/mL)。Usage and dosage of the DNA vaccine of the present invention: usage: via thigh muscle injection. Dosage: 0.2mL/feather for chicks (concentration is 1mg/mL), 0.4mL/feather for adult chickens (concentration is 0.5mg/mL).

附图说明Description of drawings

图1鸡新城疫病毒F基因与载体构建图谱。Figure 1 Map of chicken Newcastle disease virus F gene and vector construction.

图2NDV F片段的PCR结果；1为DNA分子量标准DL2000(TaKaRa公司)；2为超纯水为模板PCR对照；3为NDV F基因1.7Kb。The PCR result of Fig. 2 NDV F fragment; 1 is DNA molecular weight standard DL2000 (TaKaRa company); 2 is that ultrapure water is template PCR control; 3 is NDV F gene 1.7Kb.

图3重组表达载体酶切鉴定结果；1为15000DNA分子标准；2和3分别为EcoR V和Xbal I消化pVAX1-F与pVAX1-optiF的产物，目的片段为1.7Kb。Figure 3 Recombinant expression vector enzyme digestion identification results; 1 is 15000 DNA molecular standard; 2 and 3 are the products of pVAX1-F and pVAX1-optiF digested by EcoR V and Xbal I respectively, the target fragment is 1.7Kb.

图4重组表达载体体外表达间接免疫荧光鉴定结果。Figure 4 The results of indirect immunofluorescence identification of recombinant expression vector expressed in vitro.

图5重组表达载体体外表达Western-blot鉴定结果；1：蛋白质分子质量标准；2和3：pVAX1-F和pVAX1-optiF在COS 7细胞瞬时表达产物；4：pVAX1空载体转染COS 7细胞对照；5：COS 7空细胞对照。Fig. 5 Western-blot identification results of recombinant expression vectors expressed in vitro; 1: Protein molecular mass standard; 2 and 3: Transient expression products of pVAX1-F and pVAX1-optiF inCOS 7 cells; 4: Control ofCOS 7 cells transfected with pVAX1 empty vector ; 5:COS 7 empty cell control.

图6DNA疫苗免疫鸡和对照鸡外周血中和抗体在免疫和攻毒前后的动态变化结果。Fig. 6 The results of dynamic changes of neutralizing antibodies in peripheral blood of DNA vaccine immunized chickens and control chickens before and after immunization and challenge.

图7DNA疫苗免疫鸡和对照鸡外周血CD4⁺T淋巴细胞含量在免疫和攻毒前后的变化结果。Fig. 7 Change results of CD4⁺ T lymphocyte content in peripheral blood of chickens immunized with DNA vaccine and control chickens before and after immunization and challenge.

图8DNA疫苗免疫鸡和对照鸡外周血CD8⁺T淋巴细胞含量在免疫和攻毒前后的变化结果。Fig. 8 Change results of CD8⁺ T lymphocyte content in peripheral blood of chickens immunized with DNA vaccine and control chickens before and after immunization and challenge.

图9DNA疫苗免疫鸡和对照鸡外周血TCRγδT淋巴细胞含量在免疫和攻毒前后的变化结果。Fig. 9 Change results of TCRγδT lymphocyte content in peripheral blood of chickens immunized with DNA vaccine and control chickens before and after immunization and challenge.

图10DNA疫苗免疫鸡、对照鸡及正常未攻毒鸡在NDV F₄₈E₉株病毒攻击后死亡或7d后存活鸡胃、肠和法氏囊病理组织学变化。A1.胃的正常外观；A2.肠的正常外观；A3.法氏囊正常外观；B1.腺胃深层复管腺集合窦内有上皮细胞坏死的碎片；B2.肠腺间有大量嗜酸性粒细胞浸润；B3.法氏囊-部分滤泡淋巴细胞坏死减少出现小空泡，静脉淤血，间质水肿增宽；C1.浅层单管腺间有少量纤维素渗出；C2.肠-轻度淤血；C3.法氏囊无明显病理变化；D1.腺胃浅层单管腺及固有膜内有大量出血；D2.肠绒毛皮细胞坏死脱落，严重淤血出血；D3.法氏囊-淋巴滤泡体积缩小，淋巴细胞大量坏死缺失只剩网状纤维支架，小静脉淤血；E1.腺胃-深层复管腺上皮细胞坏死脱落至集合窦内及腺管间；E2.肠-大范围出血淤血及充血，上皮细胞坏死，大量淋巴细胞浸润；E3.法氏囊-淋巴细胞大量坏死减少，滤泡内呈空泡状，间质水肿增宽；H&E染色；20倍放大。Fig. 10 Histopathological changes of the stomach, intestine and bursa of the chickens immunized with DNA vaccine, control chickens and normal non-challenged chickens that died after NDV F₄₈ E₉ strain virus challenge or survived 7 days later. A1. The normal appearance of the stomach; A2. The normal appearance of the intestine; A3. The normal appearance of the bursa of Fabricius; B1. There are fragments of epithelial cell necrosis in the antrum of the glandular glands in the deep layer of the glandular gland; B2. There are a large number of eosinophils between the intestinal glands Cell infiltration; B3. Bursa of Fabricius-partial follicular lymphocyte necrosis decreased, small vacuoles appeared, venous congestion, and interstitial edema widened; C1. There was a small amount of cellulose exudation between superficial single-tube glands; C2. Intestine-mild Severe congestion; C3. No obvious pathological changes in the bursa of Fabricius; D1. Massive hemorrhage in the superficial single-tube gland and lamina propria of the glandular stomach; D2. Necrosis and shedding of intestinal villi cells, severe congestion and hemorrhage; D3. Bursa of Fabricius-lymphatic Follicles shrink in size, a large number of lymphocytes are necrotic and missing, only reticular fibrous scaffolds remain, and small veins are congested; E1. Glandular stomach-deep multiple duct glandular epithelial cells are necrotic and shed to the collecting sinus and between glandular ducts; E2. Intestine-extensive hemorrhage Congestion and congestion, epithelial cell necrosis, and a large number of lymphocyte infiltration; E3. Bursa of Fabricius - lymphocyte necrosis decreased, follicles were vacuolated, and interstitial edema was widened; H&E staining; 20 times magnification.

具体实施方式Detailed ways

下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

1材料和方法1 Materials and methods

1.1材料1.1 Materials

1.1.1质粒、细菌、病毒与细胞1.1.1 Plasmids, Bacteria, Viruses and Cells

质粒pVAX1购自invitrogen公司(Catalog Number：V26020)；NDV F₄₈E₉株病毒购自中国兽医药品监察所(菌种保存号：AV1611)；DH5α宿主菌、COS7细胞均由本实验室保存。Plasmid pVAX1 was purchased from Invitrogen (Catalog Number: V26020); NDV F₄₈ E₉ strain virus was purchased from China Veterinary Drug Administration (species preservation number: AV1611); DH5α host bacteria and COS7 cells were preserved by our laboratory.

1.1.2试剂1.1.2 Reagents

限制性内切酶EcoR V、Xba1 I和DNA分子质量标准(DL 2000和DL 15000)等均为宝生物工程(大连)有限公司产品；脂质体Lipofectamine2000转染试剂盒购自Invitrogen公司；RPMI1640和灭活过滤除菌的胎牛血清购自GIBCO公司。NDV阳性血清由本实验室保存；淋巴细胞分离液、MTT试剂盒、FITC标记兔抗鸡IgG、HRP标记羊抗鼠IgG购自Sigma公司；FITC标记的抗鸡CD4⁺、CD8⁺和TCRγδ表面标记的单克隆抗体购自Southernbiotech公司，蛋白质分子质量标准PageRuler^TM Protein Ladder为Fragment s公司产品；新城疫抗体检测试剂盒购自北京爱德士元亨生物科技有限公司。Restriction enzymes EcoR V, Xba1 I and DNA molecular quality standards (DL 2000 and DL 15000) are all products of Bao Bioengineering (Dalian) Co., Ltd.; liposome Lipofectamine2000 transfection kit was purchased from Invitrogen; RPMI1640 and Inactivated and filtered-sterilized fetal bovine serum was purchased from GIBCO. NDV-positive serum was preserved by our laboratory; lymphocyte separation fluid, MTT kit, FITC-labeled rabbit anti-chicken IgG, HRP-labeled goat anti-mouse IgG were purchased from Sigma; FITC-labeled anti-chicken CD4⁺ , CD8⁺ and TCRγδ surface markers The monoclonal antibody was purchased from Southernbiotech Company, the protein molecular mass standard PageRuler^TM Protein Ladder was a product of Fragment s Company; the Newcastle disease antibody detection kit was purchased from Beijing IDESI Yuanheng Biotechnology Co., Ltd.

1.1.3SPF鸡胚和SPF鸡1.1.3 SPF chicken embryo and SPF chicken

SPF鸡胚和SPF鸡由哈尔滨兽医研究所实验动物中心提供，2周龄SPF鸡实验期间饲养于负压隔离器中。SPF chicken embryos and SPF chickens were provided by the Experimental Animal Center of Harbin Veterinary Research Institute, and the 2-week-old SPF chickens were kept in a negative pressure isolator during the experiment.

1.2方法1.2 Method

1.2.1F基因的密码子优化和表达载体构建1.2.1F gene codon optimization and expression vector construction

将NDV F₄₈E₉株F基因ORF的密码子全部替换为鸡体内偏嗜的密码子，在上游引入EcoR V酶切位点和Kozak序列，下游引入Xba1I酶切位点和TGA终止密码子，并把终止密码子下游碱基A改为G。改造后的基因人工合成后并克隆到pMD-18T载体中，命名为pMD-18ToptiF。用EcoR V和Xba1 I对pMD-18ToptiF进行双酶切处理，将回收的optiF目的片段插入经过同样双酶切处理的pVAX1真核表达载体中，获得pVAX1-optiF(图1)。将NDV F₄₈E₉株F基因同样克隆到pMD-18T载体中(命名为pMD-18TF)，经过同样的酶切处理获得pVAX1-F真核表达载体。All the codons in the F gene ORF of the NDV F₄₈ E₉ strain were replaced with codons favored in chickens, the EcoR V restriction site and Kozak sequence were introduced upstream, and the Xba1I restriction site and TGA stop codon were introduced downstream. And change the base A downstream of the stop codon to G. The modified gene was artificially synthesized and cloned into the pMD-18T vector, named pMD-18ToptiF. pMD-18ToptiF was double digested with EcoR V and Xba1 I, and the recovered target fragment of optiF was inserted into the pVAX1 eukaryotic expression vector that had undergone the same double digestion to obtain pVAX1-optiF (Figure 1). The F gene of the NDV F₄₈ E₉ strain was also cloned into the pMD-18T vector (named pMD-18TF), and the pVAX1-F eukaryotic expression vector was obtained through the same restriction treatment.

1.2.2重组质粒的体外表达与鉴定1.2.2 In vitro expression and identification of recombinant plasmids

将pVAX1、pVAX1-F和pVAX1-optiF三种质粒分别在脂质体Lipofectamine^TM 2000介导下转染COS 7细胞，具体操作按说明书进行。The three plasmids pVAX1, pVAX1-F and pVAX1-optiF were respectively transfected intoCOS 7 cells under the mediation of liposome Lipofectamine^TM 2000, and the specific operation was carried out according to the instructions.

在6孔板中转染后72h的细胞，经PBS(pH7.4)洗2次，用中性甲醛固定15min后，PBS洗涤2次，加上适量1∶100稀释的NDV阳性血清，37℃作用60min，PBS洗涤3次，每次5min，然后加入适量1∶5000稀释的FITC标记的羊抗鸡二抗，37℃作用60min，PBS洗涤3次，加入适量碱性甘油，荧光显微镜下观察荧光。Cells 72 hours after transfection in a 6-well plate were washed twice with PBS (pH 7.4), fixed with neutral formaldehyde for 15 minutes, washed twice with PBS, added an appropriate amount of NDV-positive serum diluted 1:100, and kept at 37°C. For 60 minutes, wash with PBS for 3 times, each time for 5 minutes, then add an appropriate amount of FITC-labeled goat anti-chicken secondary antibody diluted at 1:5000, react for 60 minutes at 37°C, wash with PBS for 3 times, add an appropriate amount of alkaline glycerol, and observe the fluorescence under a fluorescence microscope .

同时将转染后72h后收集的转染细胞，经PBS洗1次，在细胞沉淀中加入适量的1×SDS-PAGE上样缓冲液混匀，煮沸8min，利用12％的SDS-PAGE胶电泳分离蛋白，采用半干转移转印硝酸纤维膜，5％的脱脂乳封闭60min，加入NDV阳性血清作为一抗作用60min，洗涤3次后，加入IRDye800标记的羊抗鸡IgG为二抗，避光作用60min，洗涤次3后，采用红外荧光扫描成像系统(Odyssey，LI-COR)进行扫描。At the same time, the transfected cells collected 72 hours after transfection were washed once with PBS, and an appropriate amount of 1×SDS-PAGE loading buffer was added to the cell pellet to mix well, boiled for 8 minutes, and electrophoresed on a 12% SDS-PAGE gel. Isolate protein, use semi-dry transfer transfer to nitrocellulose membrane, block with 5% skim milk for 60 minutes, add NDV positive serum as primary antibody for 60 minutes, after washing 3 times, add IRDye800-labeled goat anti-chicken IgG as secondary antibody, keep away from light After acting for 60 minutes and washing for 3 times, the infrared fluorescence scanning imaging system (Odyssey, LI-COR) was used for scanning.

1.2.3SPF鸡的免疫1.2.3 Immunization of SPF chickens

将2周龄SPF鸡随机分为4组，每组18只，前3组分别免疫pVAX1、pVAX1-F和pVAX1-optiF，采用股四头肌多点注射，每只鸡200μg，第4组注射PBS作为空对照；一免后两周以相同的剂量加强免疫。一免后12d和二免后12d每组各杀3只鸡取脾做淋巴细胞增殖试验。加强免疫两周后以10⁵EID₅₀的F₄₈E₉株NDV强毒进行滴鼻攻毒(每组12只)，每只鸡的攻毒剂量为100μL，攻毒后连续观察14d计算发病率、死亡率。试验期间对鸡的体液免疫和细胞免疫状况进行监测。The 2-week-old SPF chickens were randomly divided into 4 groups, with 18 chickens in each group. The first 3 groups were immunized with pVAX1, pVAX1-F and pVAX1-optiF respectively. The quadriceps muscle was injected at multiple points, 200 μg per chicken, and the fourth group was injected with PBS was used as the empty control; two weeks after the first immunization, the same dose was used to boost the immunization. 12 days after the first immunization and 12 days after the second immunization, 3 chickens in each group were killed to take spleens for lymphocyte proliferation test. Two weeks after the booster immunization, the F₄₈ E₉ strain NDV with 10⁵ EID₅₀ was used for nasal drop challenge (12 birds in each group), and the challenge dose for each chicken was 100 μL, and the incidence rate was calculated by continuous observation for 14 days after the challenge ,mortality rate. The humoral immunity and cellular immunity of the chickens were monitored during the experiment.

1.2.4免疫鸡血清特异性IgG抗体测定(ELISA)1.2.4 Determination of specific IgG antibody in immunized chicken serum (ELISA)

试验期间每周采集免疫鸡血清，用间接ELISA测定血清中抗F蛋白的抗体效价，具体操作按新城疫抗体检测试剂盒说明书进行，待测血清均作1∶100稀释。During the test period, the serum of immune chickens was collected every week, and the antibody titer of anti-F protein in the serum was determined by indirect ELISA. The specific operation was carried out according to the instructions of the Newcastle disease antibody detection kit.

1.2.5免疫鸡外周血T淋巴细胞亚类的检测1.2.5 Detection of T lymphocyte subtypes in peripheral blood of immunized chickens

自第一次免疫后每隔9d和攻毒后9d采集外周抗凝血，用淋巴细胞分离液分离出淋巴细胞，以荧光素标记的抗鸡CD4⁺、CD8⁺和TCRγδ表面标记的单克隆抗体与淋巴细胞作用后，利用流式细胞技术(BD，FACSCalibur)进行淋巴细胞亚群和TCRγδT细胞数量的检测。Collect peripheral anticoagulant blood every 9 days after the first immunization and 9 days after challenge, separate lymphocytes with lymphocyte separation medium, and use fluorescein-labeled monoclonal antibodies against chicken CD4⁺ , CD8⁺ and TCRγδ surface markers After interacting with lymphocytes, flow cytometry (BD, FACSCalibur) was used to detect the number of lymphocyte subsets and TCRγδT cells.

1.2.6免疫鸡淋巴细胞增殖试验1.2.6 Lymphocyte proliferation test of immunized chicken

每次免疫后12d每组剖杀3只鸡，取脾细胞，参照淋巴细胞增殖试验MTT试剂盒操作说明书进行淋巴细胞增殖试验。其主要过程是：无菌取试验鸡脾脏，用铜网(200目)研磨挤压制成单细胞悬液，用台盼蓝法进行细胞活力测定，计数；以RPMI1640培养液(含200IU/青霉素、200μg/mL链霉素、2mmol/L L-谷氨酰胺)稀释至2×107个/mL。取制备的细胞悬液，以每孔80μl加入96孔培养板，每孔再加入20μl含75μg/mL ConA的RPMI 1640培养液，每个样品做4个重复孔，试验细胞在CO₂箱中37℃培养60h后加MTT，继续培养3h后，小心吸净每孔内的液体，保留底部的沉淀，然后每孔加入MTTSOLVENT溶液100μl，室温震荡15min。待结晶充分溶解后，测定每孔OD₅₇₀，结果以4孔平均值和标准差表示。12 days after each immunization, 3 chickens in each group were slaughtered, splenocytes were collected, and the lymphocyte proliferation test was carried out according to the operation manual of the lymphocyte proliferation test MTT kit. Its main process is: take test chicken spleen aseptically, grind and extrude with copper mesh (200 mesh) to make single cell suspension, carry out cell viability measurement with trypan blue method, count; , 200μg/mL streptomycin, 2mmol/L L-glutamine) diluted to 2×107 cells/mL. Take the prepared cell suspension, add 80 μl per well to a 96-well culture plate, add 20 μl RPMI 1640 culture solution containing 75 μg/mL ConA to each well, make 4 replicate wells for each sample, and test cells in a_CO2 box for 37 After culturing at ℃ for 60 hours, add MTT, continue culturing for 3 hours, carefully aspirate the liquid in each well, keep the sediment at the bottom, then add 100 μl of MTTSOLVENT solution to each well, and shake at room temperature for 15 minutes. After the crystals were fully dissolved, the OD₅₇₀ of each well was measured, and the results were expressed as the average value and standard deviation of 4 wells.

1.2.7数据处理1.2.7 Data processing

所有统计分析均采用SPSS17.0统计软件，统计学上的差异显著性是通过均数t检验来确定的，P＜0.05就被认为是差异显著。All statistical analyzes were performed using SPSS 17.0 statistical software, and the statistical significance of the differences was determined by the mean t test, and P<0.05 was considered significant difference.

2实验结果2 Experimental results

2.1载体的构建及目的基因的体外表达2.1 Construction of vector and expression of target gene in vitro

2.1.1人工合成密码子优化的F基因2.1.1 Artificially synthesized codon-optimized F gene

优化后的基因中GC含量由原来优化前的44.28％变为65.46％，优化前后两个基因的核苷酸同源性为71.3％。The GC content in the optimized gene changed from 44.28% before optimization to 65.46%, and the nucleotide homology of the two genes before and after optimization was 71.3%.

2.1.2载体的构建和鉴定2.1.2 Construction and identification of vectors

酶切和测序鉴定结果表明，获得的真核表达质粒pVAX1-F和pVAX1-optiF与预期的结果一致，经EcoR V和Xba1 I双酶切后都得到了1700bp左右的条带(图2、图3)。The results of enzyme digestion and sequencing identification showed that the obtained eukaryotic expression plasmids pVAX1-F and pVAX1-optiF were consistent with the expected results, and a band of about 1700bp was obtained after EcoR V and Xba1 I double digestion (Fig. 2, Fig. 3).

2.1.3pVAX1-F和pVAX1-optiF的体外表达2.1.3 In vitro expression of pVAX1-F and pVAX1-optiF

间接免疫荧光结果显示，在重组质粒pVAX1-F和pVAX1-optiF转染的细胞中均可观察到特异性荧光，转染pVAX1质粒的COS 7细胞和未转染的COS 7细胞均没有检测到荧光信号(图4)。pVAX1-F和pVAX1-optiF的体外表达结果表明，optiF转染的细胞视野中阳性细胞数量多于F₄₈E₉株的F基因转染的细胞。The results of indirect immunofluorescence showed that specific fluorescence could be observed in cells transfected with recombinant plasmids pVAX1-F and pVAX1-optiF, and no fluorescence was detected inCOS 7 cells transfected with pVAX1 plasmid or innon-transfected COS 7 cells signal (Figure 4). The in vitro expression results of pVAX1-F and pVAX1-optiF showed that the number of positive cells in the visual field of optiF-transfected cells was more than that of F gene-transfected cells of F₄₈ E₉ strain.

从Western-blot结果看到，2种重组质粒都检测到了分子量约为58ku的表达蛋白(图5)，optiF的基因表达量要高于F₄₈E₉株的F基因；而未转染的COS-7细胞和转染pVAX1的COS 7细胞均没有检测到目的蛋白的表达。From the results of Western-blot, it can be seen that the expression protein with a molecular weight of about 58ku was detected in both recombinant plasmids (Fig. 5), and the gene expression of optiF was higher than that of the F gene of the F₄₈ E₉ strain; while the untransfected COS -7 cells andCOS 7 cells transfected with pVAX1 did not detect the expression of the target protein.

2.2动物试验2.2 Animal testing

2.2.1免疫及强毒攻击前后的IgG抗体变化2.2.1 IgG antibody changes before and after immunization and virulent challenge

图6的结果显示，pVAX1-F和pVAX1-optiF免疫后，免疫鸡血清IgG抗体逐渐升高，特别是pVAX1-optiF组的抗体滴度从免疫初时，特异性抗体滴度上升的幅度就较快，一免后两周开始，pVAX1-optiF组的特异性抗体水平就显著高于对照组和pVAX1-F组的(P＜0.05)。而且在二免后两周IgG抗体滴度迅速提高，pVAX1-F和pVAX1-optiF二免后2周免疫鸡血清IgG抗体ELISA检测结果OD₆₅₀平均值分别为0.2838和0.7624，极显著高于两对照组的0.0805和0.0924(P＜0.01)，而且pVAX1-optiF组免疫鸡的特异性抗体水平也极显著高于pVAX1-F组免疫鸡的(P＜0.01)。攻毒后pVAX1-optiF组和pVAX1-F组免疫鸡的抗体水平比攻毒前有较大提高，其攻毒后1周免疫鸡血清IgG抗体ELISA检测结果OD₆₅₀平均值分别为0.9473和1.0619。The results in Figure 6 show that after immunization with pVAX1-F and pVAX1-optiF, the serum IgG antibody of the immunized chickens gradually increased, especially the antibody titer of the pVAX1-optiF group increased rapidly from the initial stage of immunization. , two weeks after the first immunization, the specific antibody level of the pVAX1-optiF group was significantly higher than that of the control group and the pVAX1-F group (P<0.05). Moreover, the IgG antibody titer increased rapidly two weeks after the second immunization, and the OD₆₅₀ values of the serum IgG antibody ELISA test results of pVAX1-F and pVAX1-optiF 2 weeks after the second immunization were 0.2838 and 0.7624, which were significantly higher than those of the two controls group 0.0805 and 0.0924 (P<0.01), and the specific antibody level of pVAX1-optiF group immunized chickens was significantly higher than that of pVAX1-F group immunized chickens (P<0.01). After challenge, the antibody levels of immunized chickens in pVAX1-optiF group and pVAX1-F group were greatly improved compared with those before challenge. The average OD₆₅₀ of serum IgG antibody ELISA test results of immunizedchickens 1 week after challenge were 0.9473 and 1.0619, respectively.

2.2.2免疫鸡脾淋巴细胞增殖反应2.2.2 Proliferative response of immunized chicken spleen lymphocytes

淋巴细胞增殖试验结果显示，和对照组pVAX1及PBS相比，PVAX1-optiF和PVAX1-F接种组对ConA均表现出较明显的反应性，其中PVAX1-optiF免疫鸡脾细胞的增殖反应要比pVAX1-F免疫鸡强，特别是在二免后两周，PVAX1-optiF免疫鸡的脾细胞增殖反应显著强于pVAX1-F免疫组的(P＜0.05)(见表1)The results of the lymphocyte proliferation test showed that compared with the control group pVAX1 and PBS, the PVAX1-optiF and PVAX1-F inoculation groups showed obvious reactivity to ConA. -F immunized chickens were strong, especially two weeks after the second immunization, the splenocyte proliferation response of PVAX1-optiF immunized chickens was significantly stronger than that of pVAX1-F immunized group (P<0.05) (see Table 1)

表1鸡免疫后脾淋巴细胞的增殖反应Table 1 The proliferative response of spleen lymphocytes after chicken immunization

注：数据经t检验分析，均以x±s表示，表中同一列标有不同字母的数值之间差异显著(P＜0.05)Note: The data were analyzed by t test, all expressed as x±s, and the values marked with different letters in the same column in the table are significantly different (P<0.05)

2.2.3免疫鸡外周血CD4⁺、CD8⁺和TCRγδT细胞数量的动态变化2.2.3 Dynamic changes of CD4⁺ , CD8⁺ and TCRγδT cell numbers in peripheral blood of immunized chickens

免疫鸡外周血T淋巴细胞亚类的检测结果显示：初免后9天，所有接种鸡外周血CD4⁺T淋巴细胞含量(见图7)、CD8⁺T淋巴细胞含量(见图8)以及TCRγδT细胞含量(见图9)没有明显差异；初免后18天，pVAX1-F和pVAX1-optiF接种组的CD4⁺、CD8⁺及TCRγδT淋巴细胞含量都高于对照组，但四组之间都没有显著差异；初免后27d(即二免后13d)，pVAX1-optiF接种鸡外周血CD4⁺、CD8⁺和TCRγδT细胞含量都显著高于pVAX1-F接种组(P＜0.01)，并且两重组质粒接种鸡的这3个指标都显著高于pVAX1对照组和PBS空白对照组(P＜0.01)。攻毒后9d，pVAX1-optiF和pVAX1-F试验鸡外周血CD4⁺、CD8⁺、TCRγδT淋巴细胞含量与攻毒前相比有明显提高，差异显著(P＜0.01)。The detection results of T lymphocyte subsets in the peripheral blood of immunized chickens showed: 9 days after the initial immunization, the CD4⁺ T lymphocyte content (see Figure 7), the CD8⁺ T lymphocyte content (see Figure 8) and the TCRγδT The content of cells (see Figure 9) had no significant difference; 18 days after primary immunization, the content of CD4⁺ , CD8⁺ and TCRγδT lymphocytes in the pVAX1-F and pVAX1-optiF vaccination groups were higher than those in the control group, but there was no difference among the four groups Significant difference; 27 days after the primary immunization (i.e. 13 days after the second immunization), the contents of CD4⁺ , CD8⁺ and TCRγδ T cells in the peripheral blood of chickens inoculated with pVAX1-optiF were significantly higher than those in the pVAX1-F inoculated group (P<0.01), and the two recombinant plasmids The three indicators of the vaccinated chickens were significantly higher than those of the pVAX1 control group and the PBS blank control group (P<0.01). 9 days after challenge, the contents of CD4⁺ , CD8⁺ , TCRγδT lymphocytes in the peripheral blood of pVAX1-optiF and pVAX1-F test chickens were significantly increased compared with those before challenge (P<0.01).

2.2.4试验鸡对NDV F₄₈E₉株攻击的保护作用2.2.4 Protective effect of test chickens challenged by NDV F₄₈ E₉ strain

二免后2周分别对所有试验鸡以10⁵EID₅₀的F₄₈E₉株NDV强毒进行滴鼻攻毒。结果显示，所有的试验组在攻毒后前3d都没有出现发病鸡，攻毒4d后，除pVAX1-optiF组外，所有组都出现了症状明显的发病鸡。最终PBS对照组和pVAX1对照组全部发病，并且在攻毒后6d内全死亡，pVAX1-F组12只存活2只，最终的保护率为16.7％；而pVAX1-optiF组的最终保护率为100％(见表2)。Two weeks after the second immunization, all test chickens were challenged by nasal drops with 10⁵ EID₅₀ of the virulent F₄₈ E₉ strain NDV. The results showed that no diseased chickens appeared in all the experimental groups in the first 3 days after the challenge, and 4 days after the challenge, all groups except the pVAX1-optiF group had obvious symptoms of diseased chickens. In the end, all the PBS control group and the pVAX1 control group became ill, and all died within 6 days after the challenge. In the pVAX1-F group, 12 survived and 2 survived, and the final protection rate was 16.7%. The final protection rate of the pVAX1-optiF group was 100%. % (see Table 2).

表2pVAX1-F和pVAX1-optiF免疫鸡对NDV F₄₈E₉株强毒攻击的保护作用Table 2 Protective effect of pVAX1-F and pVAX1-optiF immunized chickens against NDV F₄₈ E₉ strain virulent challenge

序列表sequence listing

<110>中国农业科学院哈尔滨兽医研究所<110> Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences

<120>人工改造的鸡新城疫病毒F基因及其重组表达载体和应用<120>Artificially modified chicken Newcastle disease virus F gene and its recombinant expression vector and application

<130>KLPI08078<130>KLPI08078

<160>2<160>2

<170>PatentIn version 3.1<170>PatentIn version 3.1

<210>1<210>1

<211>1662<211>1662

<212>DNA<212>DNA

<213>Newcastle disease virus<213>Newcastle disease virus

<400>1<400>1

atgggcccca aatcttctac caatgtccca gcacctctga tgctgaccgt caggattgcg   60atgggcccca aatcttctac caatgtccca gcacctctga tgctgaccgt caggattgcg 60

ctggcactga gctgtgtccg tctgacaaat tctctcgatg gaaggcctct tgcagctgca  120ctggcactga gctgtgtccg tctgacaaat tctctcgatg gaaggcctct tgcagctgca 120

gggattgtag taacgggaga caaagcagtc aacatataca cctcatctca gacagggtca  180gggattgtag taacggggaga caaagcagtc aacatataca cctcatctca gacagggtca 180

ataatagtca agttactccc aaatatgcct aaggataaag aggcgtgtgc aaaagccccg  240ataatagtca agttactccc aaatatgcct aaggataaag aggcgtgtgc aaaagccccg 240

ttggaggcat acaacaggac actgactact ttgctcaccc cccttggtga ttctatccgc  300ttggaggcat acaacaggac actgactact ttgctcaccc cccttggtga ttctatccgc 300

aggatacaag agtctgcgac tacgtccgga ggaaggaggc agagacgctt tataggtgcc  360aggatacaag agtctgcgac tacgtccgga ggaaggaggc agagacgctt tataggtgcc 360

attatcggca gtgtagctct tggggttgcc acagatgccc agataacagc agcctcagct  420attatcggca gtgtagctct tggggttgcc agatgccc agataacagc agcctcagct 420

ctgatacaag ccaaccagaa tgctgccaac atcctccggc ttaaagagag cattgctgca  480ctgatacaag ccaaccagaa tgctgccaac atcctccggc ttaaagagag cattgctgca 480

actaatgaag ctgtacatga agtcactgac ggattatcgc aactagcagt ggcagttggg  540actaatgaag ctgtacatga agtcactgac ggattatcgc aactagcagt ggcagttggg 540

aagatgcagc agtttgttaa tgaccagttt aataacacag ctcaggaatt ggactgtata  600aagatgcagc agtttgttaa tgaccagttt aataacacag ctcaggaatt ggactgtata 600

aaaattacac agcaggttgg tgtagaactc aacctgtacc taactgaatt gactacagta  660aaaattacac agcaggttgg tgtagaactc aacctgtacc taactgaatt gactacagta 660

ttcgggccac aaatcacttc ccctgcctta actcagctga ctatccaggc gctttacaat  720ttcgggccac aaatcacttc ccctgcctta actcagctga ctatccaggc gctttacaat 720

ctagctggtg gtaatatgga ttatttgttg actaagttag gtgttgggaa caaccaactc  780ctagctggtg gtaatatgga ttattgttg actaagttag gtgttgggaa caaccaactc 780

agctcattaa tcggtagcgg cttgatcacc ggtaacccta ttctgtacga ttcacagact  840agctcattaa tcggtagcgg cttgatcacc ggtaacccta ttctgtacga ttcacagact 840

caactcttag gtatacaggt aactttaccc tcagtcggta acctaaataa tatgcgtgct  900caactcttag gtatacaggt aactttaccc tcagtcggta acctaaataa tatgcgtgct 900

acctacttgg agaccttgtc tgtaagcaca accaagggat ttgcctcagc acttgtccca  960acctacttgg agaccttgtc tgtaagcaca accaagggat ttgcctcagc acttgtccca 960

aaagtggtga cacaggtcgg gtctgtgata gaggaacttg acacctcata ctgtgtagag 1020aaagtggtga cacaggtcgg gtctgtgata gaggaacttg acacctcata ctgtgtagag 1020

accgatttgg atttatattg tacaagaata gtgacattcc ctatgtctcc tggtatttat 1080accgatttgg atttatattg tacaagaata gtgacattcc ctatgtctcc tggtatttat 1080

tcctgtctga gcggtaatac atcagcttgc atgtattcaa agactgaagg tgcacttact 1140tcctgtctga gcggtaatac atcagcttgc atgtattcaa agactgaagg tgcacttact 1140

acgccatata tgactatcaa gggctcagtt attgccaatt gcaagatgac aacatgcaga 1200acgccatata tgactatcaa gggctcagtt attgccaatt gcaagatgac aacatgcaga 1200

tgtgcagacc ctccgggtat catatcgcaa aattatggag aagctgtgtc tctaatagat 1260tgtgcagacc ctccgggtat catatcgcaa aattatggag aagctgtgtc tctaatagat 1260

aggcactcat gcaatgtctt atccttagac gggataactt tgaggctcag tggagaattt    1320aggcactcat gcaatgtctt atccttagac gggataactt tgaggctcag tggagaattt 1320

gacgtaactt atcaaaagaa tatctcaata ttagattctc aggtaatagt gacaggcaat    1380gacgtaactt atcaaaagaa tatctcaata ttagattctc aggtaatagt gacaggcaat 1380

ctcgatatct caactgaact tgggaatgtc aacaactcga taagtaatgc tttggataag    1440ctcgatatct caactgaact tgggaatgtc aacaactcga taagtaatgc tttggataag 1440

ttagaggaaa gcaatagcaa acttgacaaa gtcaatgtca agctgaccgg cacgtctgct    1500ttagaggaaa gcaatagcaa acttgacaaa gtcaatgtca agctgaccgg cacgtctgct 1500

ctcattacct atatagtttt aactatcata tctcttgttt gtggtatact tagcctggtt    1560ctcattacct atatagtttt aactatcata tctcttgttt gtggtatact tagcctggtt 1560

ctagcatgct atctgatgta taagcaaaag gcgcaacaaa agaccttatt atggcttggg    1620ctagcatgct atctgatgta taagcaaaag gcgcaacaaa agaccttatt atggcttggg 1620

aataataccc taaatcagat gagggccact acaagaatct ga                       1662aataataccc taaatcagat gagggccact acaagaatct ga 1662

<210>2<210>2

<211>1671<211>1671

<212>DNA<212>DNA

<213>artifical sequence<213>artificial sequence

<400>2<400>2

atagatatcg ccaccatggg gcccaagagc agcaccaacg tgcccgcccc cctgatgctg     60atagatatcg ccaccatggg gcccaagagc agcaccaacg tgcccgcccc cctgatgctg 60

accgtgcgca tcgccctggc cctgagctgc gtgcgcctga ccaacagcct ggacggccgc    120accgtgcgca tcgccctggc cctgagctgc gtgcgcctga ccaacagcct ggacggccgc 120

cccctggccg ccgccggcat cgtggtgacc ggcgacaagg ccgtgaacat ctacaccagc    180cccctggccg ccgccggcat cgtggtgacc ggcgacaagg ccgtgaacat ctacaccagc 180

agccagaccg gcagcatcat cgtgaagctg ctgcccaaca tgcccaagga caaggaggcc    240agccagaccg gcagcatcat cgtgaagctg ctgcccaaca tgcccaagga caaggaggcc 240

tgcgccaagg cccccctgga ggcctacaac cgcaccctga ccaccctgct gacccccctg    300tgcgccaagg cccccctgga ggcctacaac cgcaccctga ccaccctgct gaccccctg 300

ggcgacagca tccgccgcat ccaggagagc gccaccacca gcggcggccg ccgccagcgc    360ggcgacagca tccgccgcat ccaggagagc gccaccacca gcggcggccg ccgccagcgc 360

cgcttcatcg gcgccatcat cggcagcgtg gccctgggcg tggccaccgc cgcccagatc    420cgcttcatcg gcgccatcat cggcagcgtg gccctgggcg tggccaccgc cgcccagatc 420

accgccgcca gcgccctgat ccaggcccac cagaacgccg ccaacatcct gcgcctgaag    480accgccgcca gcgccctgat ccaggcccac cagaacgccg ccaacatcct gcgcctgaag 480

gagagcatcg ccgccaccaa cgaggccgtg cacgaggtga ccgacggcct gagccagctg    540gagagcatcg ccgccaccaa cgaggccgtg cacgaggtga ccgacggcct gagccagctg 540

gccgtggccg tgggcaagat gcagcagttc gtgaacgacc agttcaacaa caccgcccag    600gccgtggccg tgggcaagat gcagcagttc gtgaacgacc agttcaacaa caccgcccag 600

gagctggact gcatcaagat cacccagcag gtgggcgtga agctgaacct gtacctgacc    660gagctggact gcatcaagat cacccagcag gtgggcgtga agctgaacct gtacctgacc 660

gagctgacca ccgtgttccg cccccagatc accagccccg ccctgaccca gctgaccatc    720gagctgacca ccgtgttccg cccccagatc accagccccg ccctgaccca gctgaccatc 720

caggccctgt acaacctggc cggcggcaac atggactacc tgctgaccaa gctgggcgtg    780caggccctgt acaacctggc cggcggcaac atggactacc tgctgaccaa gctgggcgtg 780

ggcaacaacc agctgagcag cctgatcggc agcggcctga tcaccggcaa ccccatcctg    840ggcaacaacc agctgagcag cctgatcggc agcggcctga tcaccggcaa ccccatcctg 840

tacgacagcc agacccagct gctgggcatc caggtgaccc tgcccagcgt gggcaacctg    900tacgacagcc agacccagct gctgggcatc caggtgaccc tgcccagcgt gggcaacctg 900

aacaacatgc gcgccaccta cctggagacc ctgagcgtga gcaccaccaa gggcttcgcc    960aacaacatgc gcgccaccta cctggagacc ctgagcgtga gcaccaccaa gggcttcgcc 960

agcgccctga tccccaaggt ggtgacccag gtgggcagcg tgatcgagga gctggacacc   1020agcgccctga tccccaaggt ggtgacccag gtgggcagcg tgatcgagga gctggacacc 1020

agctactgcg tggagaccga cctggacctg tactgcaccc gcatcgtgac cttccccatg   1080agctactgcg tggagaccga cctggacctg tactgcaccc gcatcgtgac cttccccatg 1080

agccccggca tctacagctg cctgagcggc aacaccagcg cctgcatgta cagcaagacc   1140agccccggca tctacagctg cctgagcggc aacaccagcg cctgcatgta cagcaagacc 1140

gagggcgccc tgaccacccc ctacatgacc atcaagggca gcgtgatcgc caactgcaag   1200gagggcgccc tgaccacccc ctacatgacc atcaagggca gcgtgatcgc caactgcaag 1200

atgaccacct gccgctgcgc cgaccccccc ggcatcatca gccagaacta cggcgaggcc   1260atgaccacct gccgctgcgc cgaccccccc ggcatcatca gccagaacta cggcgaggcc 1260

gtgagcctga tcgaccgcca cagctgcaac gtgctgagcc tggacggcat caccctgcgc   1320gtgagcctga tcgaccgcca cagctgcaac gtgctgagcc tggacggcat caccctgcgc 1320

ctgagcggcg agttcgacgt gacctaccag aagaacatca gcatcctgga cagccaggtg   1380ctgagcggcg agttcgacgt gacctaccag aagaacatca gcatcctgga cagccaggtg 1380

atcgtgaccg gcaacctgga catcagcacc gagctgggca acgtgaacaa cagcatcagc   1440atcgtgaccg gcaacctgga catcagcacc gagctgggca acgtgaacaa cagcatcagc 1440

aacgccctgg acaagctgga ggagagcaac agcaagctgg acaaggtgaa cgtgaagctg   1500aacgccctgg acaagctgga ggagagcaac agcaagctgg acaaggtgaa cgtgaagctg 1500

accggcacca gcgccctgat cacctacatc gtgctgacca tcatcagcct ggtgtgcggc    1560accggcacca gcgccctgat cacctacatc gtgctgacca tcatcagcct ggtgtgcggc 1560

atcctgagcc tggtgctggc ctgctacctg atgtacaagc agaaggccca gcagaagacc    1620atcctgagcc tggtgctggc ctgctacctg atgtacaagc agaaggccca gcagaagacc 1620

ctgctgtggc tgggcaacaa caccctgaac cagatgcgta agagatctct a             1671ctgctgtggc tgggcaacaa caccctgaac cagatgcgta agagatctct a 1671

Claims (8)

1, manually-combined Newcastle diseases virus F gene is characterized in that: it is the nucleotide sequence shown in the SEQ IDNO:2.

2, the recombinant expression vector that contains the described manually-combined Newcastle diseases virus F gene of claim 1.

3, according to the described recombinant expression vector of claim 2, it is characterized in that: described recombinant expression vector is a recombinant eukaryon expression vector.

4, according to the described recombinant expression vector of claim 3, it is characterized in that: described recombinant eukaryon expression vector is pVAX1-optiF; Wherein, the nucleotides sequence of described optiF is classified as shown in the SEQ ID NO:2.

5, the host cell that contains any one described recombinant expression vector of claim 2-4.

6, the purposes of the described manually-combined Newcastle diseases virus F gene of claim 1 in the dna vaccination of preparation prevention or treatment newcastle disease disease.

7, the purposes of any one described recombinant expression vector of claim 2-4 in the dna vaccination of preparation prevention or treatment newcastle disease disease.

8, the vaccine composition of a kind of treatment or prevention newcastle disease disease, it is characterized in that: any one described recombinant expression vector of claim 2-4 and pharmaceutically acceptable adjuvant, carrier or excipient by significant quantity are formed.

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