技术领域technical field
本发明所涉及的牛病毒性腹泻病毒(BVDV)E2蛋白表达及其亚单位疫苗制备方法，属兽用生物制品领域。The invention relates to bovine viral diarrhea virus (BVDV) E2 protein expression and a subunit vaccine preparation method thereof, belonging to the field of veterinary biological products.
背景技术Background technique
牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)可引起的牛的腹泻及繁殖失败。怀孕母牛感染BVDV后，可引起死产、流产以及胚胎畸形，或是分娩出持续感染犊牛。这种持续感染的犊牛虽不表现任何临床症状，但可终身带毒，持续排毒，成为重要传染源，在我国广泛存在，给我国养牛业造成重大的经济损失。Bovine viral diarrhea virus (BVDV) can cause diarrhea and reproductive failure in cattle. Pregnant cows infected with BVDV can cause stillbirth, miscarriage and embryo deformities, or give birth to persistently infected calves. Although this persistently infected calves do not show any clinical symptoms, they can carry the virus for life and continue to shed the virus, becoming an important source of infection and widely existing in my country, causing great economic losses to my country's cattle industry.
BVDV属于黄病毒科瘟病毒属，其基因组为单股正链RNA，全长约12.5Kb，仅含有一个能编码约4000个氨基酸多聚蛋白大的ORF，多聚蛋白经翻译和加工后，可形成11种成熟的蛋白，其中C、Erns、E1、E2是病毒的结构蛋白。E2是BVDV的囊膜蛋白，免疫原性最强，能同时诱导机体产生体液免疫和细胞免疫，并产生中和抗体，是制备检测抗原和疫苗的首选基因，对BVDV E2表达和免疫原性的研究已经进行了很多。Marzocca MP等将E2基因在黑腹果蝇里进行表达，重组的E2蛋白表现出良好的抗原性(Truncated E2ofbovine viral diarrhea virus(BVDV)expressed in Drosophila melanogaster cells:a candidateantigen for a BVDV ELISA.J Virol Methods,2007,144(1-2):49-56)；Ferrer F等用Rachiplusianuperos杆状病毒蛋白表达体系获得E2重组蛋白，经实验证明，该蛋白可诱导中和抗体的产生，可用于制备疫苗抗原(Induction ofvirus neutralizing antibodies by immunization withRachiplusia nuperos infected with a recombinant baculovirus expressing the E2glycoprotein ofbovine viral diarrhea virus.J Virol Methods，2007，146(1-2):424-427)。国内已采用了多种方式对其进行表达(如：徐兴然等,牛病毒性腹泻病病毒Changchun184株E2基因的克隆及在大肠杆中的高效表达)，但原核表达产物为包涵体，无正确结构，活性差。因此，需进一步研究E2蛋白的表达方法，以获取可溶的高活性蛋白。BVDV belongs to the genus Pestivirus of the family Flaviviridae. Its genome is a single-stranded positive-strand RNA with a total length of about 12.5Kb. It contains only one ORF that can encode a polyprotein of about 4000 amino acids. After the polyprotein is translated and processed, it can be Eleven mature proteins are formed, among which C, Erns, E1, and E2 are structural proteins of the virus. E2 is the envelope protein of BVDV. It has the strongest immunogenicity. It can induce the body to produce humoral immunity and cellular immunity at the same time, and produce neutralizing antibodies. It is the gene of choice for the preparation of antigens and vaccines. It has the greatest effect on the expression and immunogenicity of BVDV E2 Research has been done a lot. Marzocca MP et al. expressed the E2 gene in Drosophila melanogaster, and the recombinant E2 protein showed good antigenicity (Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: a candidate antigen for a BVDV ELISA.J Virol Methods , 2007,144(1-2):49-56); Ferrer F etc. obtained the E2 recombinant protein with the Rachiplusianuperos baculovirus protein expression system, and it was proved by experiments that the protein can induce the production of neutralizing antibodies and can be used to prepare vaccine antigens (Induction of virus neutralizing antibodies by immunization with Rachiplusia nuperos infected with a recombinant baculovirus expressing the E2glycoprotein of bovine viral diarrhea virus. J Virol Methods, 2007, 146(1-2):424-427). It has been expressed in various ways in China (such as: Xu Xingran et al., Cloning of the E2 gene of bovine viral diarrhea virus Changchun184 strain and its high-efficiency expression in Escherichia coli), but the prokaryotic expression product is an inclusion body without the correct structure , Poor activity. Therefore, it is necessary to further study the expression method of E2 protein in order to obtain soluble highly active protein.
研究发现，不同物种对氨基酸密码子的使用频率有很大差异，因此，本发明中将BVDVE2基因密码子优化为昆虫杆状病毒偏好密码子，以提高BVDV E2的表达量。Studies have found that the usage frequency of amino acid codons in different species is very different. Therefore, in the present invention, the BVDVE2 gene codons are optimized to insect baculovirus preferred codons to increase the expression of BVDV E2.
本发明的目的是建立BVDV E2蛋白及其亚单位疫苗制备方法。The purpose of the invention is to establish the preparation method of BVDV E2 protein and its subunit vaccine.
发明内容Contents of the invention
本发明的技术方案Technical scheme of the present invention
1.牛病毒性腹泻病毒E2蛋白亚单位疫苗制备方法，其特征在于该疫苗是由构建的已被命名为苜蓿银蚊夜蛾核型多角体病毒(简称杆状病毒)BacVDMoptiE2株作为生产毒株制备而成，该毒株已于2016年06月01日送交北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物保藏委员会普通微生物保藏中心，保藏号分别为：CGMCCNo.12545。1. the preparation method of bovine viral diarrhea virus E2 protein subunit vaccine is characterized in that this vaccine is to be named after the californica californica nucleopolyhedrosis virus (being called for short baculovirus) BacVDMoptiE2 strain as production strain Prepared, the virus strain was sent to the General Microorganism Collection Center of China Microbiology Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, on June 1, 2016, No. 1, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation numbers are: CGMCCNo. 12545.
2.权利要求1所述牛病毒性腹泻病毒E2蛋白亚单位疫苗制备方法，其特征在于其构建的杆状病毒BacVDMoptiE2株中携带MoptiE2-1(序列1)和MoptiE2-2(序列2)。2. The method for preparing the bovine viral diarrhea virus E2 protein subunit vaccine according to claim 1, characterized in that the constructed baculovirus BacVDMoptiE2 strain carries MoptiE2-1 (sequence 1) and MoptiE2-2 (sequence 2).
3.如权利要求1所述牛病毒性腹泻病毒(BVDV)E2蛋白亚单位疫苗制备方法，其特征在于用其构建的杆状病毒BacVDMoptiE2株进行表达获得可溶性E2蛋白，经灭活，加入适宜佐剂乳化后制备成亚单位疫苗。3. as claimed in claim 1, bovine viral diarrhea virus (BVDV) E2 protein subunit vaccine preparation method is characterized in that the baculovirus BacVDMoptiE2 strain of its construction is used to express and obtain soluble E2 protein, through inactivation, adding suitable adjuvant The subunit vaccine was prepared after emulsification.
本发明的主要技术原理Main technical principle of the present invention
参考BVDV E2基因序列，采用全基因合成的手段，合成优化的BVDV E2基因序列(optiE2)；再通过重叠延伸PCR技术分别将优化的BVDV E2基因序列(optiE2)与分泌信号肽Mels基因融合，获得转移载体；Referring to the BVDV E2 gene sequence, the optimized BVDV E2 gene sequence (optiE2) was synthesized by means of total gene synthesis; then the optimized BVDV E2 gene sequence (optiE2) was fused with the secretory signal peptide Mels gene by overlap extension PCR technology to obtain Transfer carrier;
将转移载体通过转座技术克隆至杆状病毒基因组中，构建重组杆状病毒，获得BVDVE2蛋白；Cloning the transfer vector into the baculovirus genome by transposition technology, constructing recombinant baculovirus, and obtaining BVDVE2 protein;
将获得的BVDV E2蛋白加上适宜佐剂，乳化后免疫动物，分析其做为亚单位疫苗的可行性；Add the obtained BVDV E2 protein with a suitable adjuvant, immunize animals after emulsification, and analyze its feasibility as a subunit vaccine;
本发明具体实施方式Specific embodiments of the invention
1.BVDV E2基因序列的获得1. Acquisition of BVDV E2 gene sequence
采用全基因合成的手段，合成优化的BVDV E2基因序列(optiE2)，克隆至pMD 18载体，命名为：质粒poptiE2。The optimized BVDV E2 gene sequence (optiE2) was synthesized by means of whole gene synthesis, cloned into the pMD 18 vector, and named: plasmid poptiE2.
2.E2基因与信号肽序列融合2. Fusion of E2 gene and signal peptide sequence
提取poptiE2，设计引物并加上合适酶切位点，通过重叠延伸PCR技术(重叠延伸PCR技术及其在基因工程上的应用[J].分子植物育种,2006,05:747-750)获得与信号肽Mels基因融合，克隆至pMD 18载体。构建的融合基因质粒分别命名为pMoptiE2-1、pMoptiE2-2。Extract poptiE2, design primers and add appropriate enzyme cutting sites, and obtain the same expression as The signal peptide was fused with the Mels gene and cloned into the pMD 18 vector. The constructed fusion gene plasmids were named pMoptiE2-1 and pMoptiE2-2, respectively.
融合基因质粒pMoptiE2-1中携带的序列，即：MoptiE2-1(序列1)：The sequence carried in the fusion gene plasmid pMoptiE2-1, namely: MoptiE2-1 (sequence 1):
上游添加BamHI酶切位点，下游添加终止密码子及HindIII位点A BamHI restriction site is added upstream, and a stop codon and HindIII site are added downstream
融合基因质粒pMoptiE2-2中携带的序列，即：MoptiE2-2(序列2)：The sequence carried in the fusion gene plasmid pMoptiE2-2, namely: MoptiE2-2 (sequence 2):
上游添加SmaI酶切位点，下游添加终止密码子及KpnI位点Add SmaI restriction site upstream, add stop codon and KpnI site downstream
3.亚克隆至杆状病毒转移载体3. Subcloning into Baculovirus Transfer Vectors
将pMoptiE2-1和载体pFastBacDual分别进行双酶切，纯化回收，酶切片断MoptiE2-1与pFastBacDual线性质粒连接，转化，鉴定，获得的新质粒命名为pFB-MoptiE2；再将pMoptiE2-2和载体pFB-MoptiE2分别进行双酶切，纯化回收，酶切片断MoptiE2-2与pFB-MoptiE2线性质粒连接，转化，鉴定，获得的新质粒命名为pFB-DMoptiE2(D表示双表达)。The pMoptiE2-1 and the vector pFastBacDual were subjected to double enzyme digestion, purified and recovered, the digested fragmented MoptiE2-1 was connected with the pFastBacDual linear plasmid, transformed and identified, and the obtained new plasmid was named pFB-MoptiE2; then pMoptiE2-2 and the vector pFB -MoptiE2 was subjected to double enzyme digestion, purification and recovery, restriction enzyme fragmentation of MoptiE2-2 and pFB-MoptiE2 linear plasmid connection, transformation, identification, and the new plasmid obtained was named pFB-DMoptiE2 (D indicates double expression).
4.转座获得重组杆粒DNA4. Transposition to obtain recombinant bacmid DNA
分别将pFB-MoptiE2、pFB-DMoptiE2转化DH10Bac构建出的杆粒DNA分别命名为：bMoptiE2、bDMoptiE2。The bacmid DNAs constructed by transforming pFB-MoptiE2 and pFB-DMoptiE2 into DH10Bac were respectively named as bMoptiE2 and bDMoptiE2.
5.重组病毒的获得5. Acquisition of Recombinant Viruses
分别将重组杆粒bMoptiE2、bDMoptiE2用转染试剂Cellfectin转染sf9细胞，扩大培养，收集培养上清进行PCR鉴定。分别获得重组杆状病毒命名为杆状病毒BacVMoptiE2株和杆状病毒BacVDMoptiE2，该两毒株建议的分类命名均为苜蓿银蚊夜蛾核型多角体病毒，其中杆状病毒BacVDMoptiE2已于2016年06月01日送交北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物保藏委员会普通微生物保藏中心，保藏号分别为：CGMCC No.12545。The recombinant bacmids bMoptiE2 and bDMoptiE2 were respectively transfected into sf9 cells with the transfection reagent Cellfectin, expanded and cultured, and the culture supernatant was collected for PCR identification. The recombinant baculoviruses were named as baculovirus BacVMoptiE2 strain and baculovirus BacVDMoptiE2 respectively, and the proposed taxonomic names of the two strains are both Spodoptera californica nuclear polyhedrosis virus, of which baculovirus BacVDMoptiE2 was released on June 2016. It was sent to the General Microorganism Collection Center of China Microbiology Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, on March 01, and the preservation numbers are: CGMCC No.12545.
6.表达分析6. Expression Analysis
将鉴定好的病毒传代扩大培养，第二代记为P2Stock，第三代记为P3Stock。前三代均做为种毒，取第3代培养上清接种48孔板中的sf9细胞，同时设不接毒对照。60小时后弃上请，用预冷的固定液(丙酮:甲醇＝1:1)固定。弃固定液，加BVDV McAb(来自AHVLA)，37℃孵育50min，用PBS洗3遍，加FITC标记的羊抗鼠抗体，37℃孵育50min，用PBS洗3遍，荧光显微镜下观察。结果表明，接毒sf9细胞孔出现明显的亮绿色荧光(图1中图A，图B)，而未接毒sf9细胞孔未显荧光(图1中的图E)。说明重组杆状病毒能表达BVDV E2蛋白。The identified virus was subcultured and expanded, and the second generation was recorded as P2Stock, and the third generation was recorded as P3Stock. The first three generations were used as seed virus, and the culture supernatant of the third generation was used to inoculate sf9 cells in 48-well plates, and a non-infection control was set at the same time. Discard the supernatant after 60 hours, and fix with pre-cooled fixative (acetone:methanol=1:1). Discard the fixative, add BVDV McAb (from AHVLA), incubate at 37°C for 50 minutes, wash 3 times with PBS, add FITC-labeled goat anti-mouse antibody, incubate at 37°C for 50 minutes, wash 3 times with PBS, and observe under a fluorescent microscope. The results showed that the wells of sf9 cells inoculated with poison showed obvious bright green fluorescence (panels A and B in Figure 1), while the wells of sf9 cells not inoculated with poison showed no fluorescence (panel E in Figure 1). It shows that the recombinant baculovirus can express BVDV E2 protein.
7.乳化配苗7. Emulsified seedlings
以BacVDMoptiE2表达蛋白配苗为例。Take BacVDMoptiE2 expression protein vaccine as an example.
将鉴定正确的种毒用sf9细胞扩增至P6代，测定病毒滴度。用ExpressFive培养基(Invitrogen公司)悬浮培养High Five昆虫细胞(Invitrogen公司)(27℃，180r/min震荡培养)，待High Five昆虫细胞长至对数生长期时，更换新的ExpressFive培养基，调整细胞浓度为2～5×10-6/ml，将重组杆状病毒BacVDMoptiE2以1MOI剂量感染细胞，96小时后收获。2000r/min离心10min，取上清，进行SDS-PAGE电泳，计算目标产物含量。将BEI加入抗原中，终浓度为lmmol/L，置37℃灭活48h，即可将重组病毒灭活。The correctly identified sf9 cells were expanded to the P6 generation, and the virus titer was determined. Use ExpressFive medium (Invitrogen Company) to suspend culture High Five insect cells (Invitrogen Company) (27 ℃, 180r/min shaking culture), when High Five insect cells grow to logarithmic growth phase, replace new ExpressFive medium, adjust The cell concentration was 2-5×10-6 /ml, the cells were infected with the recombinant baculovirus BacVDMoptiE2 at a dose of 1 MOI, and harvested after 96 hours. Centrifuge at 2000r/min for 10min, take the supernatant, conduct SDS-PAGE electrophoresis, and calculate the content of the target product. The recombinant virus can be inactivated by adding BEI to the antigen at a final concentration of 1 mmol/L and inactivating it at 37°C for 48 hours.
8.BVDV亚单位疫苗的制备及动物免疫8. Preparation of BVDV subunit vaccine and animal immunization
以BacVDMoptiE2为例，将BacVDMoptiE2表达上清浓缩10倍后按1:1(v/v)比例加入ISA206佐剂，乳化后免疫大耳白兔，皮下多点注射，每只1ml，28天后加免一次。加免14天后采血，分离血清，56℃灭活30min。用分离的血清做中和试验。方法：稀释BVDVOregonC24至300TCID50/ml，将血清用MEM按2×、4×、8×、16×、32×、64×、128×、256×稀释，37℃作用60min，然后接种于准备好的48孔MDBK细胞板中，37℃吸附90min。然后吸干，每孔加入含2％FBS的MEM培养液0.5ml，37℃CO2培养箱培养72h。吸干，用PBS漂洗3遍，吸干，用BVDV荧光抗体液染色。有典型绿色荧光信号的孔表示未中和。Taking BacVDMoptiE2 as an example, the BacVDMoptiE2 expression supernatant was concentrated 10 times, then added ISA206 adjuvant at a ratio of 1:1 (v/v), emulsified and immunized with big-eared white rabbits, injected subcutaneously at multiple points, 1ml each, and added immunization 28 days later. once. Blood was collected 14 days after addition and immunization, serum was separated, and inactivated at 56°C for 30 minutes. Neutralization test was done with isolated serum. Method: Dilute BVDVOregonC24 to 300TCID50 /ml, dilute the serum with MEM at 2×, 4×, 8×, 16×, 32×, 64×, 128×, 256×, act at 37°C for 60 minutes, and then inoculate in the prepared Into the 48-well MDBK cell plate, adsorb at 37°C for 90min. Then suck dry, add 0.5 ml of MEM culture solution containing 2% FBS to each well, and culture in a CO2 incubator at 37°C for 72 hours. Blot dry, rinse 3 times with PBS, blot dry, and stain with BVDV fluorescent antibody solution. Wells with a typical green fluorescent signal indicate no neutralization.
结果表明，血清中和效价可达1:128，说明BVDV亚单位疫苗免疫兔子后能诱导产生中和抗体，可以做为候选疫苗使用。The results showed that the serum neutralizing titer could reach 1:128, which indicated that the BVDV subunit vaccine could induce neutralizing antibodies after immunizing rabbits, and could be used as a candidate vaccine.
本发明涉及的微生物资源信息Microorganism resource information involved in the present invention
本发明构建获得的重组杆状病毒BacVDMoptiE2株，父本为来自DH10Bac菌(Invitrogen公司)中的杆状病毒基因组，通过将外源基因BVDV E2基因与分泌信号肽Mels的融合基因全序列克隆入pFastBacDual质粒2个读码框后，与DH10Bac菌中的杆状病毒基因组重组后获得的新杆状病毒BacVDMoptiE2株，该毒株建议的分类命名均为苜蓿银蚊夜蛾核型多角体病毒，并于2016年06月01日送交北京市朝阳区北辰西路1号院3号中国科学院微生物研究所中国微生物保藏委员会普通微生物保藏中心，保藏号分别为：CGMCC No.12545。The recombinant baculovirus BacVDMoptiE2 strain obtained by the present invention, the male parent of which is the baculovirus genome from DH10Bac bacteria (Invitrogen Company), is cloned into pFastBacDual by cloning the full sequence of the fusion gene of the exogenous gene BVDV E2 gene and the secretion signal peptide Mels After two reading frames of the plasmid, the new baculovirus BacVDMoptiE2 strain obtained after recombination with the baculovirus genome in the DH10Bac bacteria, the proposed taxonomic names of the strains are all S. californica nuclear polyhedrosis virus, and were released on On June 1, 2016, it was sent to the General Microbiology Collection Center of China Microbiology Collection Committee, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing. The preservation numbers are: CGMCC No.12545.
附图说明Description of drawings
图1其中图A-D显示接毒sf9细胞孔出现明显的亮绿色荧光，图A.为BacVME2，图B.为BacVDME2，图C.为BacVMoptiE2,图D.为BacVDMoptiE2；E.为未接毒sf9细胞的对照未显荧光Figure 1. Figures A-D show obvious bright green fluorescence in the wells of sf9 cells exposed to poison. Figure A. is BacVME2, Figure B. is BacVDME2, Figure C. is BacVMoptiE2, and Figure D. is BacVDMoptiE2; E. is uninfected sf9 cells The control did not show fluorescence
图2本发明的技术路线Fig. 2 technical route of the present invention
本发明的优点Advantages of the invention
本发明涉及牛病毒性腹泻病毒E2蛋白亚单位疫苗制备方法。其优点如下：1.建立了高效获取BVDV E2蛋白的表达方法；2.用BVDV的E2蛋白表达上清免疫兔子，证明有良好的免疫反应，可用于制备BVDV亚单位疫苗。The invention relates to a preparation method of a bovine viral diarrhea virus E2 protein subunit vaccine. Its advantages are as follows: 1. An expression method for efficiently obtaining BVDV E2 protein is established; 2. The expression supernatant of BVDV E2 protein is used to immunize rabbits, which proves to have a good immune response and can be used to prepare BVDV subunit vaccines.
实施例Example
实施例1Example 1
——重组杆状病毒的构建——Construction of recombinant baculovirus
按照本发明同样的原理，已同时构建出含天然BVDV E2基因的重组杆状病毒。According to the same principle of the present invention, a recombinant baculovirus containing natural BVDV E2 gene has been constructed at the same time.
1.BVDV E2基因序列的获得1. Acquisition of BVDV E2 gene sequence
BVDV BA株(CVCC AV69)感染材料中提取病毒RNA，通过常规RT-PCR技术，获得BVDV E2基因，克隆至pMD 18载体，命名为：质粒pE2。Viral RNA was extracted from the infection material of BVDV BA strain (CVCC AV69), and the BVDV E2 gene was obtained by conventional RT-PCR technology, and cloned into pMD 18 vector, named: plasmid pE2.
2.E2基因与信号肽序列融合2. Fusion of E2 gene and signal peptide sequence
提取pE2，设计引物并加上合适酶切位点，通过重叠延伸PCR技术获得与信号肽Mels基因融合，克隆至pMD 18载体。构建的融合基因质粒分别命名为pME2-1、pME2-2。Extract pE2, design primers and add appropriate restriction sites, obtain fusion with signal peptide Mels gene by overlap extension PCR technology, and clone into pMD 18 vector. The constructed fusion gene plasmids were named pME2-1 and pME2-2, respectively.
融合基因质粒pME2-1中携带的序列，即：ME2-1(序列3)：The sequence carried in the fusion gene plasmid pME2-1, namely: ME2-1 (SEQ ID NO: 3):
上游添加BamHI酶切位点，下游添加终止密码子及HindIII位点A BamHI restriction site is added upstream, and a stop codon and HindIII site are added downstream
融合基因质粒pME2-2中携带的序列，即：ME2-2(序列4)：The sequence carried in the fusion gene plasmid pME2-2, namely: ME2-2 (SEQ ID NO: 4):
上游添加SmaI酶切位点，下游添加终止密码子及KpnI位点Add SmaI restriction site upstream, add stop codon and KpnI site downstream
3.亚克隆至杆状病毒转移载体3. Subcloning into Baculovirus Transfer Vectors
将pME2-1和载体pFastBacDual分别进行双酶切，纯化回收，酶切片断ME2-1与pFastBacDual线性质粒连接，转化，鉴定，获得的新质粒命名为pFB-ME2；再将pME2-2和载体pFB-ME2分别进行双酶切，纯化回收，酶切片断ME2-2与pFB-ME2线性质粒连接，转化，鉴定，获得的新质粒命名为pFB-DME2(D表示双表达)；The pME2-1 and the carrier pFastBacDual were subjected to double enzyme digestion, purified and recovered, the digested ME2-1 was connected with the pFastBacDual linear plasmid, transformed and identified, and the obtained new plasmid was named pFB-ME2; then pME2-2 and the vector pFB -ME2 was subjected to double enzyme digestion, purification and recovery, enzyme digestion fragmentation of ME2-2 and pFB-ME2 linear plasmid connection, transformation, identification, and the obtained new plasmid was named pFB-DME2 (D indicates double expression);
4.转座获得重组杆粒DNA4. Transposition to obtain recombinant bacmid DNA
分别将pFB-ME2、pFB-DME2转化DH10Bac构建出的杆粒DNA分别命名为：bME2、bDME2。The bacmid DNA constructed by transforming pFB-ME2 and pFB-DME2 into DH10Bac was respectively named as bME2 and bDME2.
5.重组病毒的获得5. Acquisition of Recombinant Viruses
分别将重组杆粒bME2、bDME2用转染试剂Cellfectin转染sf9细胞，扩大培养，收集培养上清进行PCR鉴定。分别获得重组杆状病毒命名为杆状病毒BacVME2株和BacVDME2株，该两毒株建议的分类命名均为苜蓿银蚊夜蛾核型多角体病毒。The recombinant bacmid bME2 and bDME2 were respectively transfected into sf9 cells with the transfection reagent Cellfectin, expanded and cultured, and the culture supernatant was collected for PCR identification. The recombinant baculoviruses were named as baculovirus BacVME2 strain and BacVDME2 strain respectively, and the proposed taxonomic names of the two strains were both S. californica nuclear polyhedrosis virus.
实施例2Example 2
——重组病毒表达分析——Recombinant virus expression analysis
将鉴定好的病毒传代扩大培养，第二代记为P2Stock，第三代记为P3Stock。前三代均做为种毒，取第3代培养上清接种48孔板中的sf9细胞，同时设不接毒对照。60小时后弃上请，用预冷的固定液(丙酮:甲醇＝1:1)固定。弃固定液，加BVDV McAb(来自AHVLA)，37℃孵育50min，用PBS洗3遍，加FITC标记的羊抗鼠抗体，37℃孵育50min，用PBS洗3遍，荧光显微镜下观察。结果表明，接毒sf9细胞孔出现明显的亮绿色荧光(图1中图C和图D)，而未接毒sf9细胞孔未显荧光(图1中的图E)。说明重组杆状病毒能表达BVDV E2蛋白。The identified virus was subcultured and expanded, and the second generation was recorded as P2Stock, and the third generation was recorded as P3Stock. The first three generations were used as seed virus, and the culture supernatant of the third generation was used to inoculate sf9 cells in 48-well plates, and a non-infection control was set at the same time. Discard the supernatant after 60 hours, and fix with pre-cooled fixative (acetone:methanol=1:1). Discard the fixative, add BVDV McAb (from AHVLA), incubate at 37°C for 50 minutes, wash 3 times with PBS, add FITC-labeled goat anti-mouse antibody, incubate at 37°C for 50 minutes, wash 3 times with PBS, and observe under a fluorescent microscope. The results showed that the wells of sf9 cells inoculated with poison showed obvious bright green fluorescence (Panel C and D in Figure 1), while the wells of sf9 cells not inoculated with poison showed no fluorescence (Panel E in Figure 1). It shows that the recombinant baculovirus can express BVDV E2 protein.
结果表明，血清中和效价可达1:128，说明BVDV亚单位疫苗免疫兔子后能诱导产生中和抗体，可以做为候选疫苗使用。The results showed that the serum neutralizing titer could reach 1:128, which indicated that the BVDV subunit vaccine could induce neutralizing antibodies after immunizing rabbits, and could be used as a candidate vaccine.
| Application Number | Priority Date | Filing Date | Title |
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| CN201610517156.6ACN105924506A (en) | 2016-07-04 | 2016-07-04 | Preparation method of bovine viral diarrhea virus E2 protein subunit vaccine |
| Application Number | Priority Date | Filing Date | Title |
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| CN201610517156.6ACN105924506A (en) | 2016-07-04 | 2016-07-04 | Preparation method of bovine viral diarrhea virus E2 protein subunit vaccine |
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| CN105924506Atrue CN105924506A (en) | 2016-09-07 |
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| CN201610517156.6APendingCN105924506A (en) | 2016-07-04 | 2016-07-04 | Preparation method of bovine viral diarrhea virus E2 protein subunit vaccine |
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