表达丙型肝炎病毒非结构蛋白3的重组腺病毒免疫小鼠的研究

Immunization effect of a recombinant adenovirus expressing the non-structural protein 3 of hepatitis C virus on mice

目的构建表达HCV非结构蛋白3(NS3)的重组腺病毒RAd/NS3,探讨其免疫小鼠后诱导机体产生特异性免疫应答的能力。方法PCR扩增编码NS3蛋白(329～935位氨基酸)的基冈片段,定向克隆至重组腺病毒AdEasy-1系统的穿梭质粒pAdTrack-CMV上,与腺病毒基因组质粒pAdEasy-1共转化BJ5183菌,利用细菌内同源重组获得重组腺病毒质粒,转染293细胞,以包装、扩增重组腺病毒。将重组腺病毒以1×10~8 pfu剂量经皮下注射免疫BALB/c小鼠,并以100μg重组质粒pRC/ NS3经肌内注射途径免疫小鼠为对照。ELISA法检测免疫小鼠血清抗体水平,^(51)Cr释放法检测免疫小鼠细胞毒性T淋巴细胞体外杀伤功能。结果重组腺病毒经皮下免疫小鼠后可刺激机体产生较强的特异性体液免疫应答和细胞免疫应答,且免疫效果强于重组质粒pRC/NS3组,初次免疫后2周,前者20只小鼠抗-HCV全部阳转,而后者10只小鼠仅3只阳转.未观察到明显的不良反应。结论表达HCV NS3抗原的重组腺病毒载体疫苗能够诱导机体产生有效的体液和细胞免疫应答。

Objective To construct a recombinant adenovirus expressing the non-structural protein（NS） 3 of hepatitis C virus （HCV） and to evaluate its immunization effect on BALB/c mice. Methods The HCV NS3 gene encoding 329-935 amino acid was amplified from plasmid pRC/NS3 by polymerase chain reaction （PCR） and cloned into the transfer vector pAdTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transfected into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombinant machinery presented in bacteria, the recombinant adenoviral backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant adenovirus was propagated in 293 cells with high titers. BALB/c mice were injected subcutaneously with 1 × 10^8 pfu recombinant adenovirus and intramuscularly with 100 μg pRC/NS3 recombinant plasmids. The antibody immune responses were measured by enzyme-linked immunosorbentassay （ELISA） and a standard 4-hour ^51 Cr release assay was utilized to determine cytotoxicity T lymphocyte （CTL） activity after immunization. Results The humoral and cellular immune responses to HCV- NS3 antigen were higher in mice immunized with recombinant adenovirus infection than those in mice immunized with recombinant pRC/NS3 plasmids. Antibody positive rate was 100% （20/20） in recombinant adenovirus RAd/NS3 group and was 30% （3/10） in pRC/NS3 group after 2 weeks immunization. No obvious side effect was observed. Conclusion The recombinant adenovirus expressing HCV NS3 protein can stimulate specific humoral and cellular immune response in mice.