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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
OBJECTIVE: To study the regulatory effect of P2 8 molecule in complement C3d on cellular immune response induced by HBV gene immunity, and to find a new way to enhance the effect of gene vaccine on cellular immunity. Methods: The C3d P2 8 and HBV preS2 / S genes were separately isolated and cloned into the eukaryotic expression vector pVAON33 to construct the corresponding recombinant plasmid pVAON33 S2 / S (HBV preS2 / S encoding gene only) and pVAON33 S2 / S P2 8.4 (containing HBV preS2 / S and 4 copies of C3d P2 8 encoding genes) and identified by PCR, restriction enzyme digestion and DNA sequencing. BALB / c mice were immunized three times with intramuscular immunization (10 0 μg / 100 μL · day) at an interval of 3 weeks, and mice were immunized with empty plasmid as a control. After stimulated with HBsAg in vitro, the splenocytes of immunized mice were tested for specific lymphocyte proliferation and CTL cytotoxicity by 3 H TdR incorporation and isotope release assay. Results: The splenocytes of mice immunized with pVAON33 S2 / S and pVAON33 S2 / S P2 8.4 both showed strong specific proliferative activity and CTL cytotoxic activity, but the latter were significantly stronger than the former (P <0.05). Conclusion: C3d P2 8 can enhance the specific cellular immune response induced by HBV preS2 / S gene