CN109182380B - Preparation method and application of baculovirus-expressed swine fever E2 subunit vaccine - Google Patents

Abstract

Translated fromChinese

本发明涉及分子生物学及兽用生物制品技术领域，旨在提供一种杆状病毒表达的猪瘟E2亚单位疫苗，该疫苗是通过杆状病毒表达系统表达的含有信号肽的猪瘟SP‑E2蛋白，其中信号肽SP的序列如SEQ ID NO.1所示，表达E2蛋白的核酸序列如SEQ ID NO.2所示。本发明研制的SP‑E2亚单位疫苗免疫小鼠后能够优先于其QZ14‑E2、HZ08‑E2蛋白的产生猪瘟的特异性抗体，且中和抗体水平远高于QZ14‑E2、HZ08‑E2蛋白；免疫猪后可诱导产生特异性抗体。本发明的疫苗具有产量高，免疫原性强，安全性好，通过鉴别诊断能够区分感染及免疫动物，能够从根本上净化猪瘟病毒。

The invention relates to the technical field of molecular biology and veterinary biological products, and aims to provide a baculovirus-expressed swine fever E2 subunit vaccine, which is a swine fever SP-containing signal peptide expressed through a baculovirus expression system E2 protein, wherein the sequence of the signal peptide SP is shown in SEQ ID NO.1, and the nucleic acid sequence for expressing E2 protein is shown in SEQ ID NO.2. The SP-E2 subunit vaccine developed by the present invention can produce swine fever-specific antibodies in preference to QZ14-E2 and HZ08-E2 proteins after immunizing mice, and the level of neutralizing antibodies is much higher than that of QZ14-E2 and HZ08-E2 protein; specific antibodies can be induced after immunizing pigs. The vaccine of the invention has high yield, strong immunogenicity and good safety, can distinguish infected and immunized animals through differential diagnosis, and can fundamentally purify swine fever virus.

Description

Translated fromChinese

杆状病毒表达的猪瘟E2亚单位疫苗的制备方法及应用Preparation method and application of baculovirus-expressed swine fever E2 subunit vaccine

技术领域technical field

本发明属于分子生物学及兽用生物制品技术领域，具体涉及杆状病毒表达的猪瘟E2亚单位疫苗的制备方法及其应用。The invention belongs to the technical field of molecular biology and veterinary biological products, and particularly relates to a preparation method and application of a baculovirus-expressed swine fever E2 subunit vaccine.

背景技术Background technique

猪瘟(Classical swine fever，CSF)是由猪瘟病毒(Classical swine fevervirus，CSFV)引起猪的一种高度接触性、致死性的传染病，临床上以发病急、高热稽留、全身泛发性点状出血和脾梗死为主要特征，主要危害家猪和野猪，给世界养猪业造成了巨大的经济损失。猪瘟的流行趋势发生了很大变化，呈现典型猪瘟和非典型猪瘟共存，隐性感染和持续感染并现，且免疫失败的现象时有发生。这种新的流行形式给全世界养猪业提出了新的挑战。Swine fever (Classical swine fever, CSF) is a highly contagious and lethal infectious disease of pigs caused by classical swine fever virus (CSFV). Symptomatic hemorrhage and splenic infarction are the main features, which mainly endanger domestic pigs and wild boars, causing huge economic losses to the world pig industry. The epidemic trend of swine fever has changed a lot, showing the coexistence of typical swine fever and atypical swine fever, the coexistence of recessive infection and persistent infection, and the phenomenon of immune failure occurs from time to time. This new fashion poses new challenges to the pig industry around the world.

目前，疫苗接种仍然是防制猪瘟的重要措施，但是传统疫苗由于是弱毒疫苗，不能区分感染和免疫动物(Differentiating Infected from VaccinatedAnimals，DIVA)，因而不能满足防制猪瘟的需求。At present, vaccination is still an important measure to prevent swine fever, but traditional vaccines are attenuated vaccines and cannot differentiate between infected and immunized animals (Differentiating Infected from Vaccinated Animals, DIVA), so they cannot meet the needs of swine fever control.

发明内容SUMMARY OF THE INVENTION

本发明要解决的技术问题是，克服现有技术中的不足，提供了一种杆状病毒表达的猪瘟E2亚单位疫苗的制备方法及应用。The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a preparation method and application of a baculovirus-expressed swine fever E2 subunit vaccine.

为解决技术问题，本发明的解决方案是：For solving the technical problem, the solution of the present invention is:

提供一种杆状病毒表达的猪瘟E2亚单位疫苗，是通过杆状病毒表达系统表达的含有信号肽的猪瘟SP-E2蛋白，其中信号肽SP的序列如SEQ ID NO.1所示，表达E2蛋白的核酸序列如SEQ ID NO.2所示。Provided is a swine fever E2 subunit vaccine expressed by baculovirus, which is a swine fever SP-E2 protein containing a signal peptide expressed by a baculovirus expression system, wherein the sequence of the signal peptide SP is as shown in SEQ ID NO.1, The nucleic acid sequence for expressing E2 protein is shown in SEQ ID NO.2.

本发明所述的猪瘟E2亚单位疫苗的氨基酸序列如SEQ ID NO.3所示。The amino acid sequence of the swine fever E2 subunit vaccine of the present invention is shown in SEQ ID NO.3.

本发明进一步提供了所述猪瘟E2亚单位疫苗在防治猪瘟病毒感染中的应用。The present invention further provides the application of the swine fever E2 subunit vaccine in preventing and treating swine fever virus infection.

本发明所述猪瘟SP-E2亚单位疫苗的制备方法，包括步骤如下：The preparation method of the swine fever SP-E2 subunit vaccine of the present invention comprises the following steps:

1)构建含有E2及其信号肽目的基因的穿梭质粒，将其转化DH10Bac感受态细胞，使其发生转座，通过蓝白斑筛选，获得重组杆粒；1) Construct a shuttle plasmid containing the target gene of E2 and its signal peptide, transform it into DH10Bac competent cells, make it transpose, and obtain recombinant bacmid through blue-white screening;

2)将重组杆粒转染昆虫细胞，获得杆状病毒，并将杆状病毒在昆虫细胞上进行传代；2) The recombinant bacmid is transfected into insect cells to obtain baculovirus, and the baculovirus is passaged on the insect cells;

3)将P3代病毒液感染大量的昆虫细胞，纯化并获取E2蛋白；3) Infect a large number of insect cells with the P3 generation virus liquid, purify and obtain the E2 protein;

4)利用SP-E2蛋白与seppic 206水佐剂充分乳化，制得猪瘟SP-E2亚单位疫苗。4) The SP-E2 protein was fully emulsified with seppic 206 water adjuvant to prepare the swine fever SP-E2 subunit vaccine.

进一步地，该方法的具体操作步骤如下所述：Further, the specific operation steps of the method are as follows:

(1)目的基因的扩增(1) Amplification of the target gene

根据Genbank设计引物SP-F、SP-R、E2-F和E2-R；Primers SP-F, SP-R, E2-F and E2-R were designed according to Genbank;

先以引物SP-F、SP-R扩增获得SP信号肽，以引物E2-F、E2-R扩增获得E2片段，再以SP信号肽、E2蛋白为模板，引物SP-F、E2-R扩增获得目的基因SP-E2；First, use primers SP-F and SP-R to amplify to obtain SP signal peptide, then use primers E2-F and E2-R to amplify to obtain E2 fragment, and then use SP signal peptide and E2 protein as templates, primers SP-F, E2- R amplification to obtain the target gene SP-E2;

引物SP-F、SP-R、E2-F和E2-R的序列如SEQ ID NO.4～7所示；The sequences of primers SP-F, SP-R, E2-F and E2-R are shown in SEQ ID NO.4-7;

(2)穿梭质粒的构建(2) Construction of shuttle plasmid

pFastBacHTB质粒以EcoRⅠ和XholⅠ限制性内切酶37℃酶切2h后回收，将酶切的载体与目的基因用同源重组酶37℃重组30min，转化Ecoli感受态细胞，挑斑鉴定，阳性菌送测序；The pFastBacHTB plasmid was digested with EcoRI and XholⅠ restriction enzymes at 37°C for 2 hours and recovered. The digested vector and the target gene were recombined with homologous recombinase at 37°C for 30 minutes, and transformed into Ecoli competent cells. sequencing;

(3)重组杆粒的获取(3) Acquisition of recombinant bacmid

将阳性质粒转化DH10Bac感受态细胞，48h后挑选白斑，用引物M13-F和M13-R进行PCR鉴定后抽提杆粒；The positive plasmid was transformed into DH10Bac competent cells, and leukoplakia was selected after 48 hours, and the bacmid was extracted after PCR identification with primers M13-F and M13-R;

引物M13-F和M13-R的序列如SEQ ID NO.8～9所示；The sequences of primers M13-F and M13-R are shown in SEQ ID NO.8-9;

(4)转染及杆状病毒的获取(4) Transfection and acquisition of baculovirus

用脂质体转染试剂

Ⅱ Reagent按照Invitrogen说明书进行转染SF9细胞，27℃培养96h，细胞出现病变，收集P1代病毒液，将P1代病毒液重新感染High Five细胞，27℃培养96h后收获P2代病毒液，同样的方法获取P3代病毒液；Transfection reagents with liposomes

Ⅱ Reagent was used to transfect SF9 cells according to Invitrogen's instructions, and cultured at 27°C for 96 hours. The cells became diseased. The P1 generation virus solution was collected, and the P1 generation virus solution was re-infected with High Five cells. After 96 hours of incubation at 27°C, the P2 generation virus solution was harvested. The same Methods to obtain P3 generation virus liquid;

(5)SP-E2的Western blot鉴定(5) Western blot identification of SP-E2

将P1代毒液加入长至70％-80％密度的High Five细胞的96孔板，27℃感染96h后进行Western blot鉴定，一抗为CSFV的阳性血清、阴性血清及单抗，二抗为HRP标记的兔抗猪二抗、FITC标记的羊抗鼠二抗；The P1 generation venom was added to a 96-well plate of High Five cells grown to a density of 70%-80%, and Western blot was performed after infection at 27°C for 96 hours. The primary antibody was CSFV positive serum, negative serum and monoclonal antibody, and the secondary antibody was HRP Labeled rabbit anti-pig secondary antibody, FITC-labeled goat anti-mouse secondary antibody;

(6)SP-E2的IFA鉴定(6) IFA identification of SP-E2

将P1代毒液加入长至70％-80％密度的High Five细胞的96孔板，27℃感染48h后进行间接免疫荧光实验，一抗为CSFV的阳性血清、阴性血清及单抗，二抗为FITC标记的兔抗猪二抗、FITC标记的羊抗鼠二抗；The P1 generation venom was added to a 96-well plate of High Five cells grown to a density of 70%-80%, and the indirect immunofluorescence experiment was performed after 48 hours of infection at 27 °C. The primary antibody was CSFV positive serum, negative serum and monoclonal antibody, and the secondary antibody was FITC-labeled rabbit anti-pig secondary antibody, FITC-labeled goat anti-mouse secondary antibody;

(7)蛋白的表达及纯化(7) Expression and purification of protein

用50ml培养基在250ml摇瓶里培养High Five细胞，当细胞密度到达1.5×10⁶个/ml时，以MOI＝1感染细胞，27℃，115rpm/h培养96h，4000rpm离心10min，收集上清，用镍柱纯化蛋白，将纯化的蛋白进行定量及Western Blot鉴定。Culture High Five cells in 250ml shake flask with 50ml medium, when the cell density reaches 1.5×106 cells/ml, infect the cells with MOI=¹ , culture at 27°C, 115rpm/h for 96h, centrifuge at 4000rpm for 10min, collect the supernatant , the protein was purified with nickel column, and the purified protein was quantified and identified by Western Blot.

发明原理描述：Description of the principle of the invention:

猪瘟病毒为黄病毒科瘟病毒属成员，含有一个大的开放阅读框(Open readingframe，ORF)，该阅读框编码一个含3898个氨基酸的多聚蛋白，此多聚蛋白在宿主细胞和病毒自身蛋白酶作用下，加工形成4个结构蛋白(C、Erns、E1和E2)和8个非结构蛋白(Npro、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B)。其中囊膜糖蛋白E2是主要保护性抗原蛋白，可以诱导抗猪瘟的保护性免疫。CSFV is a member of the Pestivirus genus of the Flaviviridae family and contains a large open reading frame (ORF), which encodes a polyprotein containing 3898 amino acids. Under the action of protease, it is processed to form 4 structural proteins (C, Erns, E1 and E2) and 8 non-structural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). Among them, envelope glycoprotein E2 is the main protective antigenic protein, which can induce protective immunity against swine fever.

杆状病毒表达系统在近年来迅速发展，由于其操作简单、安全性高、可通过悬浮培养规模表达蛋白、有翻译后修饰作用、其蛋白的免疫原性及构想与天然蛋白相似等优点而得到广泛的应用。利用杆状病毒表达系统制备猪瘟E2亚单位疫苗免疫原性强，安全性好，通过鉴别诊断能够区分感染及免疫动物，能从根本上净化猪瘟病毒。The baculovirus expression system has developed rapidly in recent years, due to its simple operation, high safety, large-scale expression of proteins in suspension culture, post-translational modification, and the immunogenicity and conception of its proteins are similar to those of natural proteins. Wide range of applications. Using the baculovirus expression system to prepare the swine fever E2 subunit vaccine has strong immunogenicity and good safety. Through differential diagnosis, the infected and immunized animals can be distinguished, and the swine fever virus can be fundamentally purified.

由于杆状病毒表达系统表达不同的猪瘟E2蛋白，其表达产量、免疫效果等方面有较大差异，导致杆状病毒表达系统表达的猪瘟E2亚单位疫苗还未普及Because the baculovirus expression system expresses different swine fever E2 proteins, there are great differences in the expression yield and immune effect, so the swine fever E2 subunit vaccine expressed by the baculovirus expression system has not been popularized.

与现有技术相比，本发明的有益效果是：Compared with the prior art, the beneficial effects of the present invention are:

1、本发明研制的SP-E2亚单位疫苗免疫小鼠后能够优先于其QZ14-E2、HZ08-E2蛋白的产生猪瘟的特异性抗体，且中和抗体水平远高于QZ14-E2、HZ08-E2蛋白；免疫猪后可诱导产生特异性抗体。1. The SP-E2 subunit vaccine developed by the present invention can produce swine fever-specific antibodies in preference to QZ14-E2 and HZ08-E2 proteins after immunizing mice, and the level of neutralizing antibodies is much higher than that of QZ14-E2 and HZ08 -E2 protein; specific antibodies can be induced after immunization of pigs.

2、本发明的疫苗具有产量高，免疫原性强，安全性好，通过鉴别诊断能够区分感染及免疫动物，能够从根本上净化猪瘟病毒。2. The vaccine of the present invention has the advantages of high yield, strong immunogenicity and good safety, and can distinguish infected and immunized animals through differential diagnosis, and can fundamentally purify the swine fever virus.

附图说明Description of drawings

图1为SP-E2重组杆状病毒构建示意图。Figure 1 is a schematic diagram of the construction of SP-E2 recombinant baculovirus.

图2为目的基因PCR扩增及重组杆粒的PCR鉴定图(左图为目的基因的扩增图，右图为重组杆粒的PCR的鉴定图)。Figure 2 shows the PCR amplification of the target gene and the PCR identification of the recombinant bacmid (the left picture is the amplification picture of the target gene, and the right picture is the PCR identification picture of the recombinant bacmid).

图3为SP-E2蛋白在昆虫细胞中的表达结果(左图为SDS-PAGE结果，右图为Westernblot图)。Figure 3 shows the expression results of SP-E2 protein in insect cells (the left picture is the SDS-PAGE result, the right picture is the Western blot picture).

图4为SP-E2蛋白在昆虫细胞中的IFA鉴定结果(Sf9细胞感染Bac-SP-E2 48h后，用猪瘟标准阳性血清、标准阴性血清，单抗IFA鉴定SP-E2在昆虫细胞的表达情况)。Figure 4 shows the results of IFA identification of SP-E2 protein in insect cells (after Sf9 cells were infected with Bac-SP-E2 for 48h, the standard positive serum and standard negative serum of swine fever were used to identify the expression of SP-E2 in insect cells by monoclonal antibody IFA Happening).

图5为SP-E2、E2、CSP-CE2、SP-CE2、CSP-E2蛋白纯化鉴定结果(左图为蛋白10倍浓缩图，右图为蛋白2倍浓缩图)。Figure 5 shows the results of protein purification and identification of SP-E2, E2, CSP-CE2, SP-CE2, and CSP-E2 (the left picture shows the 10-fold concentration of protein, and the right picture shows the 2-fold concentration of protein).

图6为小鼠免疫SP-E2亚单位疫苗0、7、14、21、28天的血清的中和效价、ELISA及IFA结果。(图中A为血清的ELISA结果，B为血清的中和效价，C为免疫7天的血清的IFA结果，D为免疫14天的血清的IFA结果)Figure 6 shows the neutralization titer, ELISA and IFA results of sera of mice immunized with SP-E2 subunit vaccine at 0, 7, 14, 21, and 28 days. (A is the ELISA result of the serum, B is the neutralization titer of the serum, C is the IFA result of the immunized serum for 7 days, D is the IFA result of the immunized serum for 14 days)

图7为猪免疫SP-E2亚单位疫苗0、7、14、21、28天的血清阻断ELISA结果。Figure 7 shows the serum blocking ELISA results of swine immunization with SP-E2 subunit vaccine at 0, 7, 14, 21, and 28 days.

具体实施方式Detailed ways

以下实施例用于说明本发明，而不意图限制本发明的范围。The following examples serve to illustrate the present invention and are not intended to limit the scope of the present invention.

本发明蛋白的表达过程中所用引物的序列如下表所示：The sequences of primers used in the expression process of the protein of the present invention are shown in the following table:

实施例1猪瘟SP-E2蛋白的表达Example 1 Expression of swine fever SP-E2 protein

1)目的基因的扩增1) Amplification of the target gene

根据Genbank设计引物SP-F、SP-R、E2-F、E2-R，先以SP-F、SP-R扩增获得SP信号肽，以E2-F、E2-R扩增获得E2片段，再以SP、E2为模板，SP-F、E2-R引物扩增获得目的基因SP-E2(如图2所示)。Primers SP-F, SP-R, E2-F, and E2-R were designed according to Genbank. SP-F and SP-R were used to amplify the SP signal peptide, and E2-F and E2-R were used to amplify the E2 fragment. Then SP and E2 were used as templates, and SP-F and E2-R primers were amplified to obtain the target gene SP-E2 (as shown in Figure 2).

2)穿梭质粒的构建2) Construction of shuttle plasmid

pFastBacHTB质粒以EcoRⅠ和XholⅠ限制性内切酶37℃酶切2h后回收，将酶切的载体与目的基因用同源重组酶37℃重组30min，转化Ecoli感受态细胞，挑斑鉴定，阳性菌送测序。The pFastBacHTB plasmid was digested with EcoRI and XholⅠ restriction enzymes at 37°C for 2 hours and recovered. The digested vector and the target gene were recombined with homologous recombinase at 37°C for 30 minutes, and transformed into Ecoli competent cells. Sequencing.

3)重组杆粒的获取3) Acquisition of recombinant bacmid

将阳性质粒转化DH10Bac感受态细胞，48h后挑选白斑，用M13引物进行PCR鉴定(如图2所示)后抽提杆粒。The positive plasmid was transformed into DH10Bac competent cells, and leukoplakia was selected after 48 hours, and the bacmid was extracted after PCR identification with M13 primer (as shown in Figure 2).

4)转染及杆状病毒的获取4) Transfection and acquisition of baculovirus

用脂质体转染试剂

Ⅱ Reagent按照Invitrogen说明书进行转染SF9细胞，27℃培养96h，细胞出现病变，收集P1代病毒液，将P1代病毒液重新感染High Five细胞，27℃培养96h后收获P2代病毒液，同样的方法获取P3代病毒液。Transfection reagents with liposomes

Ⅱ Reagent transfected SF9 cells according to Invitrogen’s instructions, and cultured at 27°C for 96 hours. The cells became diseased. The P1 generation virus solution was collected, and the P1 generation virus solution was re-infected with High Five cells. After 96 hours of incubation at 27°C, the P2 generation virus solution was harvested. The same Methods The P3 generation virus liquid was obtained.

5)SP-E2的Western blot鉴定5) Western blot identification of SP-E2

将P1代毒液加入长至70％-80％密度的High Five细胞的96孔板，27℃感染96h后进行Western blot鉴定，一抗为CSFV的阳性血清、阴性血清及单抗，二抗为HRP标记的兔抗猪二抗、FITC标记的羊抗鼠二抗，结果如图3所示。The P1 generation venom was added to a 96-well plate of High Five cells grown to a density of 70%-80%, and Western blot was performed after infection at 27°C for 96 hours. The primary antibody was CSFV positive serum, negative serum and monoclonal antibody, and the secondary antibody was HRP Labeled rabbit anti-pig secondary antibody and FITC-labeled goat anti-mouse secondary antibody, the results are shown in Figure 3.

6)SP-E2的IFA鉴定6) IFA identification of SP-E2

将P1代毒液加入长至70％-80％密度的High Five细胞的96孔板，27℃感染48hThe P1 generation venom was added to a 96-well plate of High Five cells grown to a density of 70%-80%, and infected at 27°C for 48h

后进行间接免疫荧光实验，一抗为CSFV的阳性血清、阴性血清及单抗，二抗为FITC标记的兔抗猪二抗、FITC标记的羊抗鼠二抗，结果如图4所示。Then, indirect immunofluorescence experiment was performed. The primary antibody was CSFV positive serum, negative serum and monoclonal antibody, and the secondary antibody was FITC-labeled rabbit anti-pig secondary antibody and FITC-labeled goat anti-mouse secondary antibody. The results are shown in Figure 4.

7)蛋白的表达及纯化7) Expression and purification of protein

用50ml培养基在250ml摇瓶里培养High Five细胞，当细胞密度到达1.5×10⁶个/ml时，以MOI＝1感染细胞，27℃，115rpm/h培养96h，4000rpm离心10min,收集上清，用镍柱纯化蛋白，将纯化的蛋白进行定量及Western Blot鉴定。Culture High Five cells in 250ml shake flask with 50ml medium, when the cell density reaches 1.5×106 cells/ml, infect the cells with MOI=¹ , culture at 27℃, 115rpm/h for 96h, centrifuge at 4000rpm for 10min, collect the supernatant , the protein was purified with nickel column, and the purified protein was quantified and identified by Western Blot.

如图5所示，SP信号肽可使E2蛋白呈分泌表达，且能够提高CE2、E2的蛋白表达量。As shown in Figure 5, the SP signal peptide can secretely express the E2 protein, and can increase the protein expression levels of CE2 and E2.

实施例2本发明的猪瘟SP-E2蛋白亚单位疫苗的制备及动物免疫实验。Example 2 Preparation and animal immunization experiment of the swine fever SP-E2 protein subunit vaccine of the present invention.

将实施例1表达的SP-E2蛋白与Seppic 206水佐剂以1：1的比例350rpm混匀10min,密封4℃避光保存。The SP-E2 protein expressed in Example 1 was mixed with Seppic 206 water adjuvant at a ratio of 1:1 at 350 rpm for 10 min, and sealed at 4°C and stored in the dark.

试验例1Test Example 1

16只6周龄的雌性BALB/c小鼠随机分为四组，每组4只，第一组皮下注射SP-E2蛋白50μg,第二组皮下注射HZ08-E2蛋白50μg，第三组皮下注射QZ14-E2蛋白50μg，第四组皮下注射PBS，首免14天时进行二次免疫，分别于首免后的0d、7d、14d、21的、28d采血，分别用间接ELISA、间接免疫荧光，血清的中和实验，如图6所示，实验表明SP-E2亚单位疫苗免疫小鼠后能够优先于其QZ14-E2、HZ08-E2蛋白的产生猪瘟的特异性抗体，且中和抗体水平远高于QZ14-E2、HZ08-E2蛋白如图6所示。Sixteen 6-week-old female BALB/c mice were randomly divided into four groups, with 4 mice in each group. The first group was subcutaneously injected with SP-E2 protein 50 μg, the second group was subcutaneously injected with HZ08-E2 protein 50 μg, and the third group was subcutaneously injected QZ14-E2 protein 50μg, the fourth group was subcutaneously injected with PBS, and the second immunization was performed on the 14th day after the first immunization. The neutralization experiment shown in Figure 6 shows that the SP-E2 subunit vaccine can produce swine fever-specific antibodies in preference to QZ14-E2 and HZ08-E2 proteins after immunizing mice with the SP-E2 subunit vaccine, and the level of neutralizing antibodies is far The protein higher than that of QZ14-E2 and HZ08-E2 is shown in Figure 6.

试验例2Test Example 2

12头仔猪，随机分为4组，免疫组每组3头，免疫剂量分别为5μg、15μg、30μg，对照组1头，免疫DMEM培养基，分别于免疫0d、7d、14d、21、28d采血,进行阻断ELISA试剂盒检测，如图7所示，免疫5μg的猪在免疫21d时抗体转阳性，而免疫15μg、30μg的猪在免疫14d时抗体部分转阳性，21d时抗体全部转阳性，由此可以看出猪瘟SP-E2蛋白亚单位疫苗的免疫效果良好。12 piglets were randomly divided into 4 groups, 3 piglets in the immunization group, the immunization doses were 5 μg, 15 μg, and 30 μg respectively, and 1 piglet in the control group was immunized with DMEM medium, and blood was collected on 0d, 7d, 14d, 21, and 28d of immunization. , Perform blocking ELISA kit detection, as shown in Figure 7, the immunized pigs with 5 μg turned positive for antibodies at 21 d, while the pigs immunized with 15 μg and 30 μg turned positive for antibodies at 14 d, and all antibodies turned positive at 21 d. It can be seen that the immune effect of the swine fever SP-E2 protein subunit vaccine is good.

以上所述仅是本发明的实施方式。The above are merely embodiments of the present invention.

应当指出，对于本技术邻域的技术人员，在不脱离本发明技术原理的前提下，还可以做出若干改进，这些改进也视为本发明的保护范围。It should be pointed out that for those skilled in the art, without departing from the technical principle of the present invention, several improvements can be made, and these improvements are also regarded as the protection scope of the present invention.

序列表sequence listing

<110> 浙江大学<110> Zhejiang University

<120> 杆状病毒表达的猪瘟E2亚单位疫苗的制备方法及应用<120> Preparation method and application of baculovirus-expressed swine fever E2 subunit vaccine

<160> 9<160> 9

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 72<211> 72

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 1<400> 1

atgataaaag tattaagagg gcaggtcgtg caaggtataa tatggctgct gctggtgacc 60atgataaaag tattaagagg gcaggtcgtg caaggtataa tatggctgct gctggtgacc 60

ggggcacaag gg 72ggggcacaag gg 72

<210> 2<210> 2

<211> 993<211> 993

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 2<400> 2

cggctgtcct gcaaggaaga ctacaggtat gcgatatcat caaccaatga gatagggccg 60cggctgtcct gcaaggaaga ctacaggtat gcgatatcat caaccaatga gatagggccg 60

ctaggggctg aaggcctcac caccacctgg agagagtata gccatggttt gcagctggat 120ctaggggctg aaggcctcac caccacctgg agagagtata gccatggttt gcagctggat 120

gacgggactg tcagggccat ctgcactgcg gggtccttca aagttatagc acttaatgtg 180gacgggactg tcagggccat ctgcactgcg gggtccttca aagttatagc acttaatgtg 180

gtcagtagga ggtacctggc atcattacac aagagggctt tgcccacctc agtaacattt 240gtcagtagga ggtacctggc atcattacac aagagggctt tgcccacctc agtaacattt 240

gaactcctat ttgatgggac tagcccagca attgaggaga tgggagatga ctttggattt 300gaactcctat ttgatgggac tagcccagca attgaggaga tgggagatga ctttggattt 300

gggctgtgcc cttttgacac aaccccagtg gtcaaaggga agtacaatac cactctatta 360gggctgtgcc cttttgacac aaccccagtg gtcaaaggga agtacaatac cactctatta 360

aacggcagtg ctttctacct agtctgccca ataggatgga cgggtgtcat agagtgcacg 420aacggcagtg ctttctacct agtctgccca ataggatgga cgggtgtcat agagtgcacg 420

gcagtgagcc ccacaacctt aagaacagaa gtggtgaaga ccttcaagag agagaagcct 480gcagtgagcc ccacaacctt aagaacagaa gtggtgaaga ccttcaagag agagaagcct 480

ttcccacaca gagtggattg cgtgaccact atagtagaaa aagaagacct gttctactgc 540ttcccacaca gagtggattg cgtgaccact atagtagaaa aagaagacct gttctactgc 540

aagtgggggg gtaattggac atgtgtgaaa ggcaacccgg tgacctacat gggggggcaa 600aagtgggggg gtaattggac atgtgtgaaa ggcaacccgg tgacctacat gggggggcaa 600

gtaaaacaat gcaggtggtg cggttttgac ttcaaggagc ccgatgggct cccacactac 660gtaaaacaat gcaggtggtg cggttttgac ttcaaggagc ccgatgggct cccacactac 660

cccataggca agtgcatcct agcaaatgag acgggttaca gggtagtgga ttccacagac 720cccataggca agtgcatcct agcaaatgag acgggttaca gggtagtgga ttccacagac 720

tgcaatagag atggcgtcgt tatcagcact gaaggagaac acgagtgctt gattggtaac 780tgcaatagag atggcgtcgt tatcagcact gaaggagaac acgagtgctt gattggtaac 780

accaccgtca aggtgcacgc gttggatgga agactgggcc ctatgccgtg cagacccaaa 840accaccgtca aggtgcacgc gttggatgga agactgggcc ctatgccgtg cagacccaaa 840

gaaatcgtct ctagcgcggg acctgtaagg aaaacttcct gtactttcaa ttacacaaag 900gaaatcgtct ctagcgcggg acctgtaagg aaaacttcct gtactttcaa ttacacaaag 900

acactaagaa acaagtacta tgagcccagg gacagctatt ttcagcaata tatgcttaag 960acactaagaa acaagtacta tgagcccagg gacagctatt ttcagcaata tatgcttaag 960

ggcgagtacc aatactggtt tgacctggac gtg 993ggcgagtacc aatactggtt tgacctggac gtg 993

<210> 3<210> 3

<211> 355<211> 355

<212> PRT<212> PRT

<213> 编码猪瘟E2-SP的氨基酸序列(Artificial Sequence)<213> Amino acid sequence encoding swine fever E2-SP (Artificial Sequence)

<400> 3<400> 3

Met Ile Lys Val Leu Arg Gly Gln Val Val Gln Gly Ile Ile Trp LeuMet Ile Lys Val Leu Arg Gly Gln Val Val Gln Gly Ile Ile Trp Leu

1 5 10 151 5 10 15

Leu Leu Val Thr Gly Ala Gln Gly Arg Leu Ser Cys Lys Glu Asp TyrLeu Leu Val Thr Gly Ala Gln Gly Arg Leu Ser Cys Lys Glu Asp Tyr

20 25 30 20 25 30

Arg Tyr Ala Ile Ser Ser Thr Asn Glu Ile Gly Pro Leu Gly Ala GluArg Tyr Ala Ile Ser Ser Thr Asn Glu Ile Gly Pro Leu Gly Ala Glu

35 40 45 35 40 45

Gly Leu Thr Thr Thr Trp Arg Glu Tyr Ser His Gly Leu Gln Leu AspGly Leu Thr Thr Thr Trp Arg Glu Tyr Ser His Gly Leu Gln Leu Asp

50 55 60 50 55 60

Asp Gly Thr Val Arg Ala Ile Cys Thr Ala Gly Ser Phe Lys Val IleAsp Gly Thr Val Arg Ala Ile Cys Thr Ala Gly Ser Phe Lys Val Ile

65 70 75 8065 70 75 80

Ala Leu Asn Val Val Ser Arg Arg Tyr Leu Ala Ser Leu His Lys ArgAla Leu Asn Val Val Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Arg

85 90 95 85 90 95

Ala Leu Pro Thr Ser Val Thr Phe Glu Leu Leu Phe Asp Gly Thr SerAla Leu Pro Thr Ser Val Thr Phe Glu Leu Leu Phe Asp Gly Thr Ser

100 105 110 100 105 110

Pro Ala Ile Glu Glu Met Gly Asp Asp Phe Gly Phe Gly Leu Cys ProPro Ala Ile Glu Glu Met Gly Asp Asp Phe Gly Phe Gly Leu Cys Pro

115 120 125 115 120 125

Phe Asp Thr Thr Pro Val Val Lys Gly Lys Tyr Asn Thr Thr Leu LeuPhe Asp Thr Thr Pro Val Val Lys Gly Lys Tyr Asn Thr Thr Leu Leu

130 135 140 130 135 140

Asn Gly Ser Ala Phe Tyr Leu Val Cys Pro Ile Gly Trp Thr Gly ValAsn Gly Ser Ala Phe Tyr Leu Val Cys Pro Ile Gly Trp Thr Gly Val

145 150 155 160145 150 155 160

Ile Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val ValIle Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val

165 170 175 165 170 175

Lys Thr Phe Lys Arg Glu Lys Pro Phe Pro His Arg Val Asp Cys ValLys Thr Phe Lys Arg Glu Lys Pro Phe Pro His Arg Val Asp Cys Val

180 185 190 180 185 190

Thr Thr Ile Val Glu Lys Glu Asp Leu Phe Tyr Cys Lys Trp Gly GlyThr Thr Ile Val Glu Lys Glu Asp Leu Phe Tyr Cys Lys Trp Gly Gly

195 200 205 195 200 205

Asn Trp Thr Cys Val Lys Gly Asn Pro Val Thr Tyr Met Gly Gly GlnAsn Trp Thr Cys Val Lys Gly Asn Pro Val Thr Tyr Met Gly Gly Gln

210 215 220 210 215 220

Val Lys Gln Cys Arg Trp Cys Gly Phe Asp Phe Lys Glu Pro Asp GlyVal Lys Gln Cys Arg Trp Cys Gly Phe Asp Phe Lys Glu Pro Asp Gly

225 230 235 240225 230 235 240

Leu Pro His Tyr Pro Ile Gly Lys Cys Ile Leu Ala Asn Glu Thr GlyLeu Pro His Tyr Pro Ile Gly Lys Cys Ile Leu Ala Asn Glu Thr Gly

245 250 255 245 250 255

Tyr Arg Val Val Asp Ser Thr Asp Cys Asn Arg Asp Gly Val Val IleTyr Arg Val Val Asp Ser Thr Asp Cys Asn Arg Asp Gly Val Val Ile

260 265 270 260 265 270

Ser Thr Glu Gly Glu His Glu Cys Leu Ile Gly Asn Thr Thr Val LysSer Thr Glu Gly Glu His Glu Cys Leu Ile Gly Asn Thr Thr Val Lys

275 280 285 275 280 285

Val His Ala Leu Asp Gly Arg Leu Gly Pro Met Pro Cys Arg Pro LysVal His Ala Leu Asp Gly Arg Leu Gly Pro Met Pro Cys Arg Pro Lys

290 295 300 290 295 300

Glu Ile Val Ser Ser Ala Gly Pro Val Arg Lys Thr Ser Cys Thr PheGlu Ile Val Ser Ser Ala Gly Pro Val Arg Lys Thr Ser Cys Thr Phe

305 310 315 320305 310 315 320

Asn Tyr Thr Lys Thr Leu Arg Asn Lys Tyr Tyr Glu Pro Arg Asp SerAsn Tyr Thr Lys Thr Leu Arg Asn Lys Tyr Tyr Glu Pro Arg Asp Ser

325 330 335 325 330 335

Tyr Phe Gln Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe AspTyr Phe Gln Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp

340 345 350 340 345 350

Leu Asp ValLeu Asp Val

355 355

<210> 4<210> 4

<211> 37<211> 37

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 4<400> 4

aagcgcgcgg aattcatgat aaaagtatta agagggc 37aagcgcgcgg aattcatgat aaaagtatta agagggc 37

<210> 5<210> 5

<211> 22<211> 22

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 5<400> 5

cccttgtgcc ccggtcacca gc 22cccttgtgcc ccggtcacca gc 22

<210> 6<210> 6

<211> 37<211> 37

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 6<400> 6

accggggcac aagggcggct gtcctgtaag gaagact 37accggggcac aagggcggct gtcctgtaag gaagact 37

<210> 7<210> 7

<211> 38<211> 38

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 7<400> 7

ctcgacaagc ttctacacgt ccaggtcaaa ccagtatt 38ctcgacaagc ttctacacgt ccaggtcaaa ccagtatt 38

<210> 8<210> 8

<211> 18<211> 18

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 8<400> 8

tgtaaaacga cggccagt 18tgtaaaacga cggccagt 18

<210> 9<210> 9

<211> 18<211> 18

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 9<400> 9

caggaaacag ctatgacc 18caggaaacag ctatgacc 18

Claims (4)

1. A swine fever E2 subunit vaccine expressed by baculovirus is characterized in that the vaccine is swine fever SP-E2 protein containing signal peptide expressed by a baculovirus expression system, wherein the nucleotide sequence for coding the signal peptide SP is shown as SEQ ID NO.1, and the nucleotide sequence for coding the E2 protein is shown as SEQ ID NO. 2.

2. The classical swine fever E2 subunit vaccine of claim 1, wherein the amino acid sequence of the classical swine fever E2 subunit vaccine is shown in SEQ ID No. 3.

3. The method for preparing the baculovirus-expressed classical swine fever E2 subunit vaccine of claim 1, comprising the steps of:

1) constructing shuttle plasmids containing E2 and signal peptide target genes thereof, transforming the shuttle plasmids into DH10Bac competent cells to enable the cells to generate transposition, and obtaining recombinant bacmids through blue-white spot screening;

2) transfecting the recombinant bacmid into an insect cell to obtain a baculovirus, and carrying out passage on the baculovirus on the insect cell;

3) infecting a large number of insect cells with the P3 generation virus liquid, purifying and obtaining E2 protein;

4) the SP-E2 protein and seppic 206 water adjuvant are fully emulsified to prepare the swine fever SP-E2 subunit vaccine.

4. The preparation method according to claim 3, wherein the specific operation steps of the preparation method are as follows:

(1) amplification of target Gene

Primers SP-F, SP-R, E2-F and E2-R were designed according to Genbank;

firstly, obtaining SP signal peptide by amplification of a primer SP-F, SP-R, obtaining an E2 fragment by amplification of a primer E2-F, E2-R, and obtaining a target gene SP-E2 by amplification of a primer SP-F, E2-R by taking the SP signal peptide and E2 protein as templates;

the sequences of the primers SP-F, SP-R, E2-F and E2-R are shown as SEQ ID NO. 4-7;

(2) construction of shuttle plasmid

The pFastBacHTB plasmid is digested for 2h by EcoRI and XholI restriction endonuclease at 37 ℃, then recovered, the digested vector and the target gene are recombined for 30min by homologous recombinase at 37 ℃, Ecoli competent cells are transformed, the spots are picked and identified, and positive bacteria are sequenced;

(3) obtaining recombinant bacmids

Transforming the positive plasmid into DH10Bac competent cells, selecting white spots after 48h, performing PCR identification by using primers M13-F and M13-R, and extracting bacmids;

the sequences of the primers M13-F and M13-R are shown as SEQ ID NO. 8-9;

(4) transfection and baculovirus acquisition

Transfecting SF9 cells by using a liposome transfection Reagent Cellffectin II Reagent according to Invitrogen specifications, culturing the cells at 27 ℃ for 96 hours until the cells are diseased, collecting P1 generation virus liquid, re-infecting P1 generation virus liquid into High Five cells, culturing the cells at 27 ℃ for 96 hours, then harvesting P2 generation virus liquid, and obtaining P3 generation virus liquid by the same method;

(5) western blot identification of SP-E2

Adding P1-generation venom into a 96-well plate of High Five cells with the density of 70-80%, infecting for 96h at 27 ℃, and then carrying out Western blot identification, wherein primary antibodies are positive serum, negative serum and monoclonal antibodies of CSFV, and secondary antibodies are rabbit anti-pig secondary antibodies marked by HRP and goat anti-mouse secondary antibodies marked by FITC;

(6) IFA identification of SP-E2

Adding the P1-substituted venom into a 96-well plate of High Five cells with the density of 70-80%, infecting for 48h at 27 ℃, and then performing indirect immunofluorescence experiment, wherein primary antibody is positive serum and negative serum of CSFV and monoclonal antibody, and secondary antibody is FITC-labeled rabbit anti-pig secondary antibody and FITC-labeled goat anti-mouse secondary antibody;

(7) expression and purification of proteins

Culturing High Five cells in 250ml shake flask with 50ml culture medium until the cell density reaches 1.5X 10⁶At each ml, cells were infected at MOI =1, cultured at 27 ℃ at 115rpm/h for 96h, centrifuged at 4000rpm for 10min, the supernatant was collected, the protein was purified by a nickel column, and the purified protein was quantified and identified by Western Blot.

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