C3d-P28增强乙型肝炎病毒特异性基因免疫效果的研究

目的　观察补体C3d P2 8对基因免疫的调节作用及其不同拷贝数对调节作用的影响 ,为增强基因免疫效果寻求新方法。方法　PCR法获得补体C3d P2 8编码基因并以头尾串连方式将1～ 4拷贝C3d P2 8编码基因克隆至pVAON33,构建pVAON33 P2 8.1～ 4 ]重组质粒 ,然后将HBV preS2 S编码基因分别插入pVAON33和pVAON33 P2 8.1～ 4 ]质粒获得pVAON33 S2 S和pVAON33 S2 S P2 8.1～ 4 ]重组质粒。肌肉注射各重组质粒DNA(每只 10 0 μg 10 0 μl)初次免疫小鼠 ,并以pVAON33为对照 ;12周后皮下注射HBsAg蛋白加强免疫各组小鼠 ,ELISA法检测免疫小鼠血清特异性抗 HBs IgG。结果　pVAON33 S2 S重组质粒免疫小鼠可诱导产生特异性抗 HBs IgG ,含不同拷贝C3d P2 8编码基因的重组质粒可诱导更高的特异性抗体 ,其中pVAON33 S2 S P2 8.4重组质粒诱导的抗体水平最高 (P <0 .0 1)。蛋白加强免疫后 ,含C3d P2 8编码基因重组质粒免疫组抗 HBs IgG迅速上升 ,并明显高于pVAON33 S2 S重组质粒免疫组 (P <0 .0 5 ) ,pVAON33 S2 S P2 8.4重组质粒诱导的抗体仍维持最高水平。结论　不同拷贝的C3d P2 8能不同程度地增强HBV preS2 S基因免疫诱导的特异性体液免疫及其蛋白加强后的回忆反应 ,其中 4拷贝C3d P2 8的增强作用较为显著。

P28 derived from C3d enhances the immune responses against HBV-preS2/S induced by gene immunization

Objective To investigate the possibility of P28 derived from C3d to enhance the immune response to HBV-preS2/S that is induced by direct injection of naked plasmids containing different repeats of P28 and HBV-preS2/S in fusion form. Methods Copies of C3d-P28 coding gene, amplified by PCR and modified by restriction endonucleases digestion, were subcloned into a eukaryotic expression vector pVAON33 to construct pVAON33-P28.1-4], respectively. HBV-preS2/S coding sequence was then introduced into pVAON33, pVAON33-P28.1-4] and then identified by both PCR and DNA sequencing. BALB/c mice were primed by im gene immunization with different recombinant plasmids on day 0 and were boosted by sc inoculation with HBsAg protein 12 weeks post-priming. The levels of specific IgG in sera collected at the indicated times from each group were determined by ELISA. Results HBsAg specific antibody response was elicited in groups primed with plasmids pVAON33-S2/S-P28.1-4] and pVAON33-S2/S. However, the response against HBsAg in the groups primed with pVAON33-S2/S-P28.1-4] was significant higher than that in pVAON33-S2/S group, the highest level of the specific antibody response was observed in the group primed with pVAON33-S2/S-P28.4 (P<0.01). The booster immunization with HBsAg protein accelerated and increased specific antibody in all groups except pVAON33 control, a stronger response was still found in the group vaccinated with pVAON33-S2/S-P28.1-4] than that in pVAON33-S2/S group (P<0.05). Conclusion Different repeats of C3d-P28 enhanced both primary and secondary response induced by gene immunization, in which 4 copies of C3d-P28 sequence showed the strongest effect of enhancement.