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

Abstract

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

CLC Number:

S828

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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Eukaryotic Expression and Differential Analysis of Japanese Encephalitis Virus NS1 and NS1-2A Proteins

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