乙型肝炎病毒核心抗原与表面抗原“a”决定簇融合疫苗的免疫效果

The immunization effects of HBV core antigen and surface antigen fusion protein in mice

目的评价乙型肝炎病毒核心蛋白作为类病毒颗粒载体融合表面抗原“a”决定簇的基因疫苗和蛋白疫苗的免疫效果，为寻求有效的慢性乙型肝炎治疗性疫苗奠定基础。方法体外分别扩增编码乙型肝炎病毒HBsAg 100～162aa部分、HBcAg1～78aa部分和HBcAg 83～144aa的基因序列，目的基因连接后定向克隆到真核表达载体pocDNA3的多克隆位点内。将嵌合基因亚克隆到原核表达载体，诱导表达并纯化获得融合蛋白。分别用真核表达质粒和纯化融合蛋白免疫BALB／c小鼠，加强免疫并定期采血，检测血清中特异性抗体并进行淋巴细胞增殖实验以评价体液和细胞免疫应答效果。实验结果进行重复测量资料的方差分析和单向方差分析。结果嵌合基因免疫后可以产生有效的抗-HBs“体液免疫”应答，免疫0、2、4、6、8、10、12、14周时的S/N值分别为1．05±0．20、2．69±0．38、4．48±0．50、6．61±0．49、6．95±0．19、7．43±0．31、8．11±0．14、7．43±0．31，F值分别为1202．021和200．059，P〈0．01。未引起明显的抗-HBc“体液免疫”应答却获得了明显的HBcAg特异性细胞免疫应答。融合蛋白免疫后也能获得有效的抗HBs“体液免疫”应答，但针对HBcAg的体液应答却较强；可以产生较强的HBcAg特异性增殖反应，而没有明显的HBsAg特异性增殖反应。嵌合基因免疫组较融合蛋白组的细胞免疫应答明显增强，体液免疫应答相对较弱。结论以该乙型肝炎病毒核心蛋白为载体的包含HBsAg“a”决定簇的融合基因疫苗相对于其融合蛋白具有良好的细胞和体液免疫效果。

Objective To evaluate the immunization effects of HBV core antigen and surface antigen fusion protein. Methods The DNA fragments encoding HBsAg 100-162 aa; HBcAg 1-78 aa and HBcAg 83-144 aa were PCR-amplified, and then cloned into pcDNA3 plasmid. The chimeric gene was subcloned into the prokaryotic vector, pRSET-B. The E.coli expressed recombinant protein purified. BALB/c mice were immunized with recombinant protein or eukaryotic expression plasmid, humoral response and cellular response were examined. Results The plasmid containing thte chimeric gene of HBsAg and HBcAg induced effective anti-HBs antibody response and strong HBcAg specific lymphocyte proliferative response, but could not induce anti-HBc antibody response. Fusion protein induced strong anti-HBs and anti-HBc antibody response, and it also casued significant HBcAg specific lymphocyte proliferation. Compared to the recombi- nant fusion protein, the plasmid containing thte chimeric gene of HBsAg and HBcAg can induce more effective cellullar response but weaker＇humoral response. Conclusion Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg is a more effective vaccine.