C3d增强基因免疫诱导的小鼠抗乙型肝炎病毒特异性免疫应答  

C3d enhances the immune response against HBV-preS2/S following gene immunization in mice

作　　者：关庆东[1] 王立新[1] 郭强[1] 张进平[1] 徐薇[1] 王缨[1] 熊思东[1] 

机构地区：[1]复旦大学上海医学院免疫学系教育部分子医学重点实验室上海基因免疫与疫苗研究中心,200032

出　　处：《中华医学杂志》2005年第2期101-105,共5页National Medical Journal of China

基　　金：国家高技术研究发展计划(863计划)资助项目(2004AA215242);国家杰出青年科学基金资助项目(39925031)

摘　　要：目的探讨C3d对乙型肝炎病毒(HBV)基因免疫诱导的特异性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。方法将HBVpreS2/S编码基因分别插入真核表达载体TR421和含有三拷贝C3d编码基因的TR421C3d3质粒,构建重组质粒TR421preS2/S和TR421preS2/SC3d3。采用肌肉注射法对BALB/c小鼠实施基因免疫,以空质粒TR421为对照,定期采集血清。ELISA法检测免疫小鼠血清特异性抗HBsIgG,3HTdR掺入法检测其特异性淋巴细胞增殖活性。结果TR421preS2/SC3d3重组质粒免疫组诱导的特异性抗HBsIgG水平明显高于TR421preS2/S重组质粒免疫组(P<005),而且TR421preS2/SC3d3重组质粒基因免疫诱导的特异性淋巴细胞增殖活性也显著高于TR421preS2/S重组质粒组(P<005)。结论C3d可增强基因免疫诱导的HBV特异性体液和细胞免疫应答,为提高HBV基因疫苗的免疫效果提供了新的途径。Objective To investigate whether C3d can enhance the immune response to HBV-preS2/S induced by direct injecting naked plasmid containing three copies of C3d and HBV-preS2/S in fusion form.Methods HBV-preS2/S coding sequence was introduced into the eukaryotic expression vectors TR421 and TR421-C3d3 containing 3 copies of C3d respectively. PCR was used to identify the direction of insertion, DNA sequencing analysis was used on the positive clones. The recombinant plasmids TR421-preS2/S and TR421-preS2/S-C3d3 were transfected into the human breast cancer cells of the line 4T1 as a transient expression system. Semi-quantitative RT-PCR was used to detect the expression of HBV-preS2/S protein. TR421-preS2/S and TR421-preS2/S-C3d3 were injected into the anterior tibial muscles of female BALB/c mice, 6 in each group, 3 times at an interval of 3 weeks. Six mice were injected with blank plasmid TR421 as controls. The total blood was collected from the mice. ELISA was used to detect the level of specific anti-HBs antibody. The splenocytes of the immunized mice were collected and stimulated with HbsAg protein and then harvested to analyze the specific lympho-proliferative response by 3H-thymidine incorporation assay. CR2 positive Raji cells were added with plasmid transfected and mouse anti-HBs serum. Flow cytometry was used to detect the integration of preS2/S-C3d3 fusion protein. Results The 4T1 cells transfected with TR421-preS2/S and TR421-preS2/S-C3d3 effectively expressed HBV-preS2/S, with the expression level of TR421-preS2/S-C3d3 lower by 15.25% than that of TR421-preS2/S. Twelve weeks after immunization, the specific anti-HBs-IgG level (A_~490nm ) was 0.81±0.09 in the TR421-preS2/S primed mice, significantly higher than that in the blank plasmid injected mice (0.49±0.02, P<0.05); and the specific anti-HBs-IgG level (A_~490nm ) was 1.24±0.29 in the TR421-preS2/S-C3d3 immunized mice, significantly higher than that of the TR421-preS2/S primed mice (P<0.001). The lympho-proliferative response of the TR421-preS2/S pri

关 键 词：基因 疫苗 补体 乙型肝炎表面抗原 乙型肝炎病毒 基因免疫 

分 类 号：R392[医药卫生—免疫学]