| 摘要: |
| 【目的】在昆虫细胞中表达猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV) S1-NTD蛋白，研究其免疫原性，为基于PEDV S蛋白的基因工程亚单位疫苗的研究奠定基础。【方法】PCR扩增PEDV S1-NTD基因，构建其重组表达质粒pFastBac Dual PEDV S1-NTD和重组穿梭质粒Ac-PEDV S1-NTD。将Ac-PEDV S1-NTD转染sf9昆虫细胞，利用昆虫杆状病毒表达系统制备重组杆状病毒rvAcPEDV S1-NTD。将P2代重组杆状病毒感染sf9细胞，Western blot鉴定重组蛋白的表达，空斑试验测定重组杆状病毒的滴度。在此基础上，分别以感染复数（multiplicity of infection，MOI）为0.1，1和3的P2代重组杆状病毒感染sf9细胞，并在感染后1，2，3，4和5 d收集细胞，通过Western blot和空斑试验分别检测目的蛋白的表达量和重组杆状病毒的增殖情况。将MOI为0.1的P3代重组杆状病毒皮下注射免疫BALB/c小鼠，每隔2周免疫1次，共免疫3次，以注射PBS、空载体Bacmid和PEDV商业疫苗为对照组，于首免后不同时间采集血液分离其血清，ELISA法检测血清中PEDV特异性抗体的水平；于首免后35 d剖取小鼠脾脏，分离其淋巴细胞；PEDV刺激后，MTT法检测小鼠脾淋巴细胞的增殖情况。【结果】PCR扩增获得了约738 bp的PEDV S1-NTD基因；成功构建了重组表达质粒pFastBac Dual PEDV S1-NTD、重组穿梭质粒Ac-PEDV S1-NTD和重组杆状病毒rvAcPEDV S1-NTD。利用杆状病毒表达系统在sf9细胞内成功表达了重组PEDV S1-NTD蛋白，且杆状病毒以MOI为1感染sf 9细胞后在第3天蛋白表达量达到最高。在首免后35 d，重组杆状病毒免疫组小鼠血清效价达到最高值，为1∶5 000，与商业疫苗组效价一致；淋巴细胞增殖试验表明，重组杆状病毒rvAcPEDV S1 NTD免疫组小鼠脾淋巴细胞对PEDV的刺激也发生了显著增殖（P<0.05），而PBS和空载体Bacmid对照组淋巴细胞未发生明显增殖（P>0.05）。【结论】成功构建了重组杆状病毒rvAcPEDV S1-NTD，在昆虫细胞中成功表达了PEDV S1-NTD蛋白，且具有较好的免疫原性。 |
| 关键词: 猪流行性腹泻病毒 S1蛋白 昆虫杆状病毒表达系统 免疫原性 基因工程疫苗 |
| DOI： |
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| 基金项目:安徽省自然科学基金项目（1708085MC83）；浙江省重点研发计划项目（2019C02043）；家畜疫病病原微生物学国家重点实验室开放基金项目（SKLVEB2016KFKT003）；安徽省质量工程项目“动物医学专业卓越兽医师教育培养计划”（2016zjjh022） |
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| Expression and immunogenicity of S1-NTD protein of porcine epidemic diarrhea virus in insect cells |
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SUN Pei,ZHANG Juan,SHU Jinqi,et al
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| Abstract: |
| 【Objective】The S1-NTD gene of porcine epidemic diarrhea virus (PEDV) was expressed in insect cells and its immunogenicity was analyzed to provide basis for subunit vaccine research.【Method】PEDV S1-NTD gene was amplified by PCR to construct recombinant expression plasmid (pFastBac Dual PEDV S1-NTD) and recombinant shuttle plasmid (Ac-PEDV S1-NTD).The recombinant shuttle plasmid Ac-PEDV S1-NTD was transfected into sf9 insect cells,and the expression system of insect baculovirus was used to construct rvAcPEDV S1-NTD.The expression of recombinant protein was confirmed by Western blot,and the titer of recombinant baculovirus was measured by plaque assay.The sf9 cells were then infected with P2 recombinant baculovirus with the ultiplicity of infection (MOI) of 0.1,1 and 3,respectively,and the cells were collected 1,2,3,4 and 5 days after infection.Western blot and plaque assay were performed to detect the expression of target protein and proliferation of recombinant baculovirus,respectively.BALB/c mice were subcutaneously injected with P3 recombinant baculovirus with MOI of 0.1,once every 2 weeks for a total of three times.Blood was collected at different times after the first dispensation,the serum was isolated,and the specific antibody level of PEDV in the serum of mice was detected by ELISA,and the spleen of mice was cut off 35 d after the first dispensation to isolate lymphocytes.After PEDV stimulation,the proliferation of mouse spleen lymphocytes was detected by MTT.【Result】The PEDVS1-NTD gene of about 738 bp was obtained by PCR amplification.The recombinant expression plasmid pFastBac Dual PEDV S1-NTD,the recombinant shuttle plasmid Ac-PEDV S1-NTD and the recombinant baculovirus rvAc-PEDV S1-NTD were successfully constructed.Recombinant PEDV S1-NTD protein was successfully expressed in sf9 cells by baculovirus expression system,and the recombinant protein reached the maximum when using baculovirus 1 MOI to infect sf9 cells and collecting the infected sf9 cells on the third day.At 35 days after the first immunization,the serum titer of mice in the recombinant baculovirus immunization group reached the highest value of 1∶5 000,which was same as the titer of the commercial vaccine group.Lymphocyte proliferation test showed that the splenic lymphocytes stimulated by recombinant baculovirus rvAcPEDV S1 NTD immunized mice also had significant proliferation to PEDV(P<0.05),while PBS and empty vector Bacmid control group had no significant proliferation (P>0.05).【Conclusion】The recombinant baculovirus rvAcPEDV S1 NTD was successfully constructed,and S1 NTD protein was successfully expressed in insect cells with good immunogenicity. |
| Key words: porcine epidemic diarrhea virus(PEDV) S1 protein baculovirus expression system immunogenicity genetically engineering vaccine |
