论文部分内容阅读
目的:构建肠道病毒71型(EV71)多表位-mGITRL真核表达质粒并对其免疫原性进行研究。方法:串联已证实的VP1表位并合成(VP1’);PCR扩增获得mGITRL,克隆至真核共表达载体pIRES中。对重组质粒进行测序鉴定后,以脂质体介导法转染至COS7细胞,通过Western blot方法检测其在COS7细胞中的表达。将构建好的重组质粒免疫小鼠,应用ELISA法检测小鼠血清中抗VP1的抗体滴度。结果:PCR扩增出目的基因,测序证实真核共表达载体pIRES-VP1’-mGITRL构建成功,Western blot结果显示目的基因在COS7细胞和肌细胞中过表达。小鼠血清中检测出高滴度的抗VP1抗体。结论:成功扩增出目的基因并构建VP1表位的重组真核共表达载体pIRES-VP1’-mGITRL,在COS7细胞和肌细胞中过表达,并获得高滴度的抗VP1抗体。
Objective: To construct enterovirus 71 (EV71) polytope-mGITRL eukaryotic expression plasmid and study its immunogenicity. Methods: VP1 epitope was confirmed and synthesized (VP1 ’); mGITRL was amplified by PCR and cloned into eukaryotic co-expression vector pIRES. The recombinant plasmid was identified by sequencing and transfected into COS7 cells by liposome-mediated method. The expression of COS7 cells was detected by Western blot. The constructed recombinant plasmids were used to immunize mice, and the antibody titer of anti-VP1 in serum of mice was detected by ELISA. Results: The target gene was amplified by PCR and confirmed by sequencing. The eukaryotic co-expression vector pIRES-VP1’-mGITRL was constructed successfully. Western blot results showed that the target gene was overexpressed in COS7 cells and muscle cells. High titer of anti-VP1 antibody was detected in mouse serum. CONCLUSION: The recombinant eukaryotic co-expression vector pIRES-VP1’-mGITRL, which successfully amplifies the target gene and constructs the VP1 epitope, is overexpressed in COS7 cells and muscle cells and high titer anti-VP1 antibody is obtained.