乙型脑炎病毒NS1和NS1-2A蛋白的真核表达及其差异性研究

doi:10.16431/j.cnki.1671-7236.2020.07.024

摘要/Abstract

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。

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

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.

Key words: Japanese encephalitis virus (JEV); NS1 gene; NS1-2A gene; cytokine; immune effect

中图分类号:

S828

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.

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 and Veterinary Medicine, 2020, 47(7): 2190-2199.

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