C3d对基因免疫诱导的HBV特异性体液免疫应答的调节作用

观察补体C3d对HBV基因免疫诱导的特异性体液免疫应答的调节作用 ,为增强HBV基因疫苗免疫效果寻求新途径。将HBV preS2 /S编码基因分别插入真核表达载体TR4 2 1和含有 3拷贝C3d编码基因的TR4 2 1 C3d3质粒 ,构建重组质粒TR4 2 1 preS2 /S和TR4 2 1 preS2 /S C3d3。采用肌肉注射法对BALB/c小鼠实施基因免疫 (10 0 μg/ 10 0 μl/只小鼠 ) ,以空质粒为对照 ,定期采集血清。ELISA法检测免疫小鼠血清特异性抗 HBs IgG及其亚型 ,并采用NaSCN竞争ELISA法检测其亲合力。结果表明 ,TR4 2 1 preS2 /S C3d3重组质粒免疫组诱导的特异性抗 HBs IgG水平明显高于TR4 2 1 preS2 /S重组质粒免疫组 (P <0 0 5 ) ;而且TR4 2 1 preS2 /S C3d3重组质粒免疫组诱导的抗 HBs IgG抗体的亲合力 (ED50 :1 375 )显著高于TR4 2 1 preS2 /S重组质粒组 (ED50 :0 875 ) ,但C3d并不改变基因免疫诱导的特异性抗HBs IgG各亚型水平的格局 ,仍以IgG2b和IgG2a为主。提示C3d可增强基因免疫诱导的HBV特异性体液免疫应答 ,并促进特异性抗体亲和力的成熟 ,这为提高HBV基因疫苗的免疫效果提供了新的途径。

Regulation of the Humoral Immune Response against HBV-preS2/S Following Gene Immunization by C3d Molecule

To investigate whether C3d can enhance the immune response to HBV-preS2/S that was induced by direct injection of naked plasmid containing three copies of C3d and HBV-preS2/S in fusion form, HBV-preS2/S-coding sequence were introduced into the eukaryotic expression vectors TR421 and TR421-C3d3 respectively and identified by PCR and DNA sequencing analysis. Female BALB/c mice were primed by intramuscular gene immunization with different recombinant plasmids on day 0 and day 28, and levels of specific IgG and subclass in sera collected at the indicated dafes from each group were determined by ELISA. The avidity of specific IgG were determined by a specific NaSCN-displacement ELISA. It was found that HBsAg specific antibody response was elicited in groups primed with plasmids TR421-preS2/S-C3d3 and TR421-preS2/S. However, the response against HBsAg in the group primed with TR421-preS2/S-C3d3 was significant higher than that in TR421-preS2/S group (P<0.05). Analyses of subclass and avidity maturation of the raised antibody indicated that immunizations with HBV-preS2/S-3C3d-expressing DNA accelerated the avidity maturation of antibody to HBsAg, but didn't change the pattern of subclass of anti-HBs-IgG, mainly IgG2b. These results indicated three copies C3d could enhance humoral immune response against HBV induced by gene immunization and accelerate the avidity maturation of specific antibodies.