重组乙型肝炎表面抗原插入突变体的真核表达及其抗原性的分析

Expression of recombinant inserting mutants of HBsAg in vitro and its antigenic analysis

目的研究与乙型肝炎病毒感染的临床进程相关的两种乙型肝炎表面抗原（ＨＢｓＡｇ）插入变异体的免疫学性状，以阐明在ＨＢｓＡｇ阴性的慢性乙型肝炎病毒感染中所起的作用。方法利用体外ＰＣＲ诱变方法，构建重组ＨＢｓＡｇ的插入突变体；借助ＥＢ病毒载体ｐＣＥＰ４和逆转录病毒载体ＰＸＴ１将重组插入变异Ｓ基因转染哺乳动物细胞；利用ＥＬＩＳＡ及Ｗｅｓｔｅｒｎｂｌｏｔ方法，探讨变异抗原与抗ＨＢｓ的结合力。结果（１）构建了ＨＢｓＡｇ的插入突变体（分别在ＨＢｓＡｇ的第１２２位与１２３位氨基酸间插入Ａｒｇ，Ａｌａ和第１２３位与１２４位氨基酸间插入Ａｒｇ，Ｇｌｙ，Ａｌａ）的真核表达载体；（２）将其转染哺乳动物细胞，最终建立了能表达野生型和变异型ＨＢｓＡｇ的ＨｅｐＧ２细胞系；（３）这些细胞系能在体外稳定地连续传代培养；（４）野生型ＨＢｓＡｇ可与抗ＨＢｓ结合，变异型ＨＢｓＡｇ不能与抗ＨＢｓ结合。结论研究表明这两种插入变异体影响了ＨＢｓＡｇ的空间结构，从而使其失去与乙型肝炎病毒表面抗体的结合力，因而可能逃避机体的免疫清除而持续存在于体内形成慢性感染。

Objective To study the immunological characteristics of two inserting variants of HBsAg as further to elucidate the role of inserting mutants in the development of HBsAg negative chronic hepatitis B. Methods The inserting mutants of HBsAg were constructed by PCR mediated mutagenesis in vitro. Epstein Barr virus vector pCEP4 and retrovirus vector PXT1 carrying the mutant genes of HBsAg were transfected into mamalian cells. The affinity of polyclonal and monoclonal antibodies against HBsAg for mutant antigens were studied by ELISA and western blot.Results Two insertions were introduced into s gene, and a six nucleotide insertion introduced two aminoacids (Arg and Ala) between codons 122 and 123 of the s polypeptide, whereas a nine nucleotide insertion introduced three aminoacids(Arg, Gly and Ala) between codons 123 and 124. These s genes were subcloned into two eukaryotic expression vectors (pCEP4 and PXT1), and the recombinant eukaryotic expression plasmids had been constructed, which could express s polypeptide in mammalian cells. Cell lines expressing wild type or mutant type HBsAg stably were obtained after transfection of these vectors into mammalian cells. The two inserting variants failed to react with polyclonal and monoclonal antibodies against HBsAg by ELISA and Western blot. Conclusion The insertions may result in a conformational change of the s protein, and affect its antigenicities, suggesting that the inserting mutations may be critical for immunoescape of HBV and in part responsible for chronicity of HBsAg negative patients.