| 乙型脑炎病毒NS1和NS1-2A蛋白的真核表达及其差异性研究 |
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| 引用本文： | 刘巧玲,黄涛,汤德元,曾智勇,石柯,林泠,任杰.乙型脑炎病毒NS1和NS1-2A蛋白的真核表达及其差异性研究[J].中国畜牧兽医,2020,47(7):2190-2199. |
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| 作者姓名： | 刘巧玲 黄涛 汤德元 曾智勇 石柯 林泠 任杰 |
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| 作者单位： | 贵州大学动物科学学院, 贵阳 550025 |
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| 基金项目： | 贵州省2016年科学技术基金（黔科合支撑[2016]1048号）；青年教师国家自然科学基金培育项目建议资助项目（黔科合平台人才[2017]5788-70）；贵州大学引进人才科研项目（贵大人基合字[2015]33） |
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| 摘 要： | 为深入研究乙型脑炎病毒（JEV）NS1和NS1-2A蛋白的表达和免疫效果差异，本试验构建并扩增C-端含Flag标签的NS1和NS1-2A基因，利用T4 DNA连接酶分别连接到质粒pcDNA3.1（+）上，构建重组质粒pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag。将这两种真核表达质粒分别转染BHK-21细胞，利用RT-PCR、IFA和Western blotting检测NS1和NS1-2A蛋白在体外的表达情况，用pcDNA3.1-NS1-Flag、pcDNA3.1-NS1-2A-Flag和pcDNA3.1（+）免疫BALB/c小鼠，检测这两种蛋白在体内的表达差异。结果显示，试验成功构建了NS1和NS1-2A基因的真核表达载体pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag，IFA和Western blotting鉴定NS1和NS1-2A蛋白成功表达，重组质粒pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag免疫小鼠后可诱发机体产生特异性体液免疫，pcDNA3.1-NS1-2A-Flag联合免疫组小鼠血清抗体效价和INF-γ细胞因子分泌水平比pcDNA3.1-NS1-Flag免疫组高，且与pcDNA3.1（+）空载体免疫组差异极显著（P<0.01）；pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag免疫组小鼠体内的INF-γ分泌量会增多，免疫第4周达到最高后逐渐降低。pcDNA3.1-NS1-Flag和pcDNA3.1-NS1-2A-Flag免疫小鼠能够刺激JEV特异性抗体的分泌和增强机体的细胞免疫功能，且NS1-2A联合基因的免疫效果优于NS1单一基因，为进一步研究JEV的非结构蛋白功能、研发NS1和NS1-2A基因疫苗奠定基础。
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| 关 键 词： | 乙型脑炎病毒炎(JEV) NS1基因 NS1-2A基因 细胞因子 免疫效果 |
| 收稿时间： | 2019-12-20 |
Eukaryotic Expression and Differential Analysis of Japanese Encephalitis Virus NS1 and NS1-2A Proteins |
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| LIU Qiaoling,HUANG Tao,TANG Deyuan,ZENG Zhiyong,SHI Ke,LIN Ling,REN Jie.Eukaryotic Expression and Differential Analysis of Japanese Encephalitis Virus NS1 and NS1-2A Proteins[J].China Animal Husbandry & Veterinary Medicine,2020,47(7):2190-2199. |
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| Authors: | LIU Qiaoling HUANG Tao TANG Deyuan ZENG Zhiyong SHI Ke LIN Ling REN Jie |
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| Institution: | College of Animal Science, Guizhou University, Guiyang 550025, China |
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| Abstract: | In order to further study the expression and immune effect differences of Japanese encephalitis virus (JEV) NS1 and NS1-2A proteins,eukaryotic expression plasmids of pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag were constructed by connecting T4 DNA ligase to pcDNA3.1(+),which contained NS1 and NS1-2A genes with Flag tag at C-terminal.Two eukaryotic expression plasmids were transfected into BHK-21 cells,respectively.RT-PCR,IFA and Western blotting were used to detect the expression of NS1 and NS1-2A proteins in vitro.BALB/c mice were immunized with pcDNA3.1-NS1-Flag,pcDNA3.1-NS1-2A-Flag and pcDNA3.1(+) to detect the expression difference of the two proteins in vivo.The results showed that the experiments successfully constructed the eukaryotic expression vectors pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag of NS1 and NS1-2A genes.The expression of NS1 and NS1-2A proteins were successfully identified by IFA and Western blotting.Recombinant plasmids pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag could induce specific humoral immunity after immunizing mice.The serum antibody titer and INF-γ cytokine secretion level of pcDNA3.1-NS1-2A-Flag combined immunization group were higher than that of pcDNA3.1-NS1-Flag immunization group.Moreover,the pcDNA3.1-NS1-2A-Flag group were extremely significantly different from pcDNA3.1(+) empty vector immune group (P<0.01).The final IFN-γ of mice immunized with pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag immunized group increased,and they showed a trend of first increase and then decrease.Mice immunized with pcDNA3.1-NS1-Flag and pcDNA3.1-NS1-2A-Flag could stimulate JEV-specific antibodies secretion and enhance the body's cellular immune function,and the immune effect of NS1-2A combined gene was better than NS1 single gene.The results laid a foundation for further research on the function of non-structural proteins of JEV and the vaccines of NS1 and NS1-2A genes. |
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| Keywords: | Japanese encephalitis virus (JEV) NS1 gene NS1-2A gene cytokine immune effect |
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