C3d-P28增强乙肝病毒基因免疫诱导的特异性细胞免疫应答

目的 :研究补体C3d中P2 8分子对HBV基因免疫诱导的细胞免疫应答的调节作用 ,为增强基因疫苗细胞免疫的效果寻求新方法。方法 :分别分离获取C3d P2 8和HBV preS2 /S编码基因 ,并克隆入真核表达载体 pVAON33中 ,构建相应重组质粒pVAON33 S2 /S (仅含HBV preS2 /S编码基因 )和pVAON33 S2 /S P2 8.4 (含HBV preS2 /S和 4拷贝C3d P2 8的编码基因 ) ,并以PCR、酶切和DNA序列测定进行鉴定。以肌肉注射法对BALB/c小鼠实施 3次基因免疫 ( 10 0 μg/10 0 μL·只 ) ,间隔 3wk ,并以空质粒免疫小鼠作为对照。免疫小鼠脾细胞体外经HBsAg刺激后 ,用3 H TdR掺入法和同位素释放法 ,分别检测特异性淋巴细胞增殖和CTL杀伤活性。结果 :pVAON33 S2 /S和 pVAON33 S2 /S P2 8.4免疫小鼠的脾细胞 ,均显示较强的特异性增殖活性和CTL杀伤活性 ,但后者显著强于前者 (P <0 .0 5 )。结论 :C3d P2 8可增强HBV preS2 /S基因免疫诱导的特异性细胞免疫应答

Enhancement of HBV gene-induced specific cell-mediated immunoresponse by C3d-P28

AIM:To investigate whether P28 derived from complement C3d can enhance the cell-mediated immunoresponse to HBV-preS2/S induced by direct injection of naked plasmid DNA containing four tandem repeats of C3d-P28 gene and HBV-preS2/S gene existed in fusion form. METHODS: Four copies of gene coding for C3d-P28, amplified by PCR and cut by restriction endonucleases digestion, were subcloned into a eukaryotic expression vector pVAON33 to construct pVAON33-P28.4. HBV-preS2/S gene was then introduced into the pVAON33 and pVAON33-P28.4 respectively to form pVAON33-S2/S and pVAON33-S2/S-P28.4. The recombinant plasmids were identified by PCR and restriction endonucleases digestion as well as DNA sequencing. BALB/c mice were immunized i.m. three times at 3 weeks' intervals with 100 microg of pVAON33-S2/S DNA, pVAON33-S2/S-P28.4 and mock DNA, respectively. Splenocytes from immunized mice were stimulated by HBsAg and then harvested to analyze the specific lymphocytic proliferative response and CTL cytotoxic activity by (3)H-TdR incorporation assay and isotopic release analysis, respectively. RESULTS: Specific lymphocytic proliferation and CTL cytotoxic activity against HBV-preS2/S were observed in mice immunized by both pVAON33-S2/S and pVAON33-S2/S-P28.4 in dose-dependent form. Specific lymphocytic proliferation and CTL response in mice immunized by pVAON33-S2/S-P28.4 were markedly stronger than those in mice immunized by pVAON33-S2/S. CONCLUSION: A C3d-P28 can enhance the cell-mediated immunoresponse induced by HBV-preS2/S gene immunization.