丙型肝炎病毒NS3基因疫苗质粒的构建、表达及其体液免疫应答

Construction and Expression of HCV-NS3 Plasmid Vaccine and Humoral Immune Response Induced

目的构建基于Ii分子的丙型肝炎病毒(HCV)内源性靶向基因疫苗质粒,并检测其在真核细胞中的表达及免疫小鼠诱导的体液免疫应答。方法通过3轮PCR,以HCV-NS3的Th1表位(1248～1261aa)取代Ii链CLIP片段编码基因,构建基于Ii分子的HCV内源性靶向基因疫苗质粒,转染COS-7细胞,RT-PCR法检测其在mRNA水平的表达;用内源性靶向基因疫苗pHCV-NS3-Th1和非靶向基因疫苗pHCV-NS3经股四头肌肌肉免疫BALB/c小鼠,以生理盐水、pCI-neo和pCI-neo-Ii作为对照,检测小鼠的体液免疫应答水平。结果转染细胞中,内源性靶向基因疫苗质粒在mRNA水平获得表达;对BALB/c小鼠的免疫结果显示,只有pHCV-NS3组小鼠可以检测到针对HCV-NS3的特异性抗体,抗体滴度可达1:1024。结论已成功构建了基于Ii分子的HCV内源性靶向基因疫苗质粒,其能够在真核细胞中表达,但不能刺激小鼠产生HCV-NS3特异性抗体。

Objective To construct a plasmid vaccine based on invariant chain CLIP substitution,express in eukaryotic cells and induce humoral immune response in mice.Methods HCV-NS3 Th1 plasmid vaccine was constructed by substitution of the gene encoding CLIP fragment of invariant chain（I）iwith the Th1 epitope（1248～1261aa）of HCV-NS3 through three cycles of PCR,then transfected to COS-7 cells and determined for expression by RT-PCR.BALB/c mice were immunized i.m.with the constructed vaccine pHCV-NS3-Th1 and non-targeting gene vaccine pHCV-NS3 respectively,using physiological saline,pCI-neo and pCI-neo-Ii as control,and determined for humoral immune response levels.Results The constructed plasmid vaccine pHCV-NS3-Th1 was expressed in transfected cells at mRNA level.However,the specific antibody against HCV-NS3,at a titer of 1：1 024,was only detected in the mice immunized with pHCV-NS3.Conclusion The plasmid vaccine based on invariant chain CLIP substitution was successfully constructed and expressed in eukaryotic cells,but induced no specific antibody against HCV-NS3 in mice.