G145R突变后乙型肝炎病毒表面抗原在酵母系统的表达及其免疫学特性分析

Secreted Expression of G145R Mutant HBsAg PreS2+S Gene in Pichia pastoris Expression System and the Study of Its Antigenicity and Immunogenicity

为了研究乙型肝炎(乙肝)病毒表面抗原(HBsAg)发生G145R突变后的免疫学特性改变情况,首先利用Pichiapastoris酵母表达系统分泌表达G145R突变后的HBsAg的preS2+S(中蛋白),用重组表达产物免疫小鼠,酶联免疫吸附试验(ELISA)和Westernblot实验等研究其抗原性和免疫原性与野毒型HBsAg的异同。从150个阳性表达克隆中筛选出一株表达量最高的克隆株MC23,Westernblot检测显示,表达的HBsAg中蛋白单体主带分子量在34kD、37kD左右,表达量约为200μg/L。用不同的HBsAg检测试剂检测其抗原性发现,G145R突变后的HB-sAg,用绝大多数试剂都不能很好地检出,检出能力只有野毒型HBsAg的50%或更低,但用美国雅培公司的试剂检出能力可达野毒的98%。G145R突变后的HBsAg中蛋白免疫小鼠后,血清中可检测到1∶1600的特异性表面抗体,该抗体与G145R突变后的HBsAg“a”决定簇合成肽P2-145R也能发生交叉反应,反应滴度为1∶80。但该抗体和野毒型HBsAg蛋白以及野毒“a”决定簇合成肽P1-wt均不反应。上述结果表明,G145R突变后的HBsAg中蛋白在Pichiapastoris酵母系统得到了分泌表达,表达产物仍具有较好的免疫原性,但和野毒HBsAg相比,其抗原性和免疫原性发生了明显改变。

In order to study the antigenicity and immunogenicity of G145R mutant HBsAg, G145R mutant hepatitis B surface antigen (HBsAg) preS2+S gene was secretively expressed in Pichia pastoris system. The expressed middle protein of G145R mutant HBsAg was used to immunize mice. The antigenicity and immunogenicity (preS2+S) of G145R mutant HBsAg were studied with ELISA and Western blot tests. The highest-level expression clone MC23 was selected from 150 positive clones. Specific HBsAg middle protein band could be detected with Western blot test. The molecular weight of the monomer was about 34kD and 37kD. The expression level was about 200g/L culture medium when estimated with the AUZYME MONOCLONAL KIT. The antigenicity was tested with 7 commercial HBsAg detection kits and mice polyclonal antisera immunized with HBsAg standard. The results indicated that the detection level of the G145R mutant HBsAg could only be about 50% or less of the wild-type HBsAg in most of the HBsAg detection kits when they were in the same concentration. Only with the AUZYME MONOCLONAL KIT, we could equally detect the G145R mutant HBsAg and that of wild-type. After immunized with G145R mutant HBsAg protein, specific anti-HBs antibody could be detected in the mouse sera, the average titer was about 1:1 600. This anti-sera were also reactive with synthetic peptide P2-145R, which is specific to the 'a' determinant of G145R mutant HBsAg. But the anti-HBs did not react with wild-type HBsAg, or with synthetic peptide P1-wt which is specific to 'a' determinant of wild-type HBsAg. Further studies with Western blot supported the result too. All the results above suggested that the preS2+S gene of G145R mutant HBsAg could be expressed in the Pichia pastoris system successfully. The G145R mutant HBsAg still had a good immunogenicity. But the antigenicity and immunogenicity when compared with wild-type HBsAg had changed significantly after one amino acid mutation from Glycine to Arginine at amino acid site 145.