猪传染性胃肠炎病毒S-N融合双基因疫苗的构建及其免疫原性分析

Construction and immunogenicity analysis of the S-N fusion gene vaccine against porcine transmissible gastroenteritis virus

为了构建TGEV S-N融合双基因疫苗并分析其免疫原性,从S、N基因克隆质粒中以PCR扩增了S基因(2.1kb,含A、B、C、D抗原位点)和N基因(1.2kb),将S基因插入pVAX1载体构建了pVAX-S质粒,再将N基因插入pVAX-S中S基因末端,构建了融合表达S、N双基因的重组质粒pVAX-S-N,将pVAX-S-N转染COS7细胞以免疫荧光试验进行S、N双基因的表达鉴定。用纯化的pVAX-S-N和作为对照的pVAX-S、pVAX1、PBS免疫BALB/c小鼠,共免疫3次,分别测定免疫后第0、14、28、42天的小鼠血清IgG抗体,测定免疫后第42天小鼠外周血T淋巴细胞亚群(CD3+、CD4+、CD8+)的数量。结果,融合质粒pVAX-S-N可在COS7细胞特异性表达S、N两个蛋白,pVAX-S-N免疫小鼠后第14天即可诱导产生抗TGEV的特异性IgG,但pVAX-S-N诱导的抗体水平一直低于pVAX-S诱导的抗体水平,在免疫后第42天差异极显著(P<0.01);pVAX-S-N可激发小鼠产生细胞免疫应答,但pVAX-S-N组的CD3+、CD4+、CD8+数量均低于pVAX-S免疫组。研究结果表明,融合双基因疫苗pVAX-S-N具有免疫原性,但免疫效果却不如单基因疫苗pVAX-S的理想。

To construct a fusion gene vaccine coexpressing S gene and N gene of porcine transmissible gastroenteritis virus（TGEV） and analyze its immunogenicity,the S gene（2.1 kb,encoding A,B,C and D antigen sites of S protein） and N gene（1.2 kb） were amplified respectively by PCR from the cloning plasmids with S gene or N gene.The S gene was inserted into the eukaryotic expression vector pVAX1 to construct pVAX-S,and the N gene was subsequently linked into the pVAX-S to form the recombinant plasmid pVAX-S-N.The pVAX-S-N was transfected into COS7 cells and the expressions of the two target genes were detected by indirect immunofluorescence assay.The purified plasmid pVAX-S-N,control plasmids（pVAX-S and pVAX1） and PBS were respectively used to intramuscularly immunized BALB/c mice in triplicate,and the specific anti-TGEV IgG were detected by indirect ELISA on day 0, 14,28 and 42 post the 1st-immunization.The percentage of CD3＋,CD4＋ and CD8＋ T-lymphocytes from the peripheral blood of the immunized mice were detected on day 42 post the 1st-immunization. In result,the plasmid pVAX-S-N was successfully constructed. The pVAX-S-N could indue specific anti-TGEV IgG on day 14 post the 1st-immunization,but the IgG level of pVAX-S-N group was lower than the pVAX-S group throughout the immunization period,and the antibody level was significant different（P0.01） on day 42 post the 1st-immunization.The pVAX-S-N could effectively elicit cellular immune responses in mice,but the level was lower than that of the pVAX-S group. Therefore,the S-N fusion gene vaccine pVAX-S-N had immunogenicity,but the immune efficacy was relatively lower compared with the pVAX-S.