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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 changes of immunological characteristics after G145R mutation in hepatitis B virus surface antigen (HBsAg), the preS2 + S (intermediate protein) of HBsAg expressing G145R mutation was first secreted by Pichia pastoris yeast expression system and expressed in recombinant Product-immunized mice, enzyme-linked immunosorbent assay (ELISA) and Western blot experiments to study the antigenicity and immunogenicity and wild-type HBsAg similarities and differences. A cloned MC23 strain was screened from 150 positive clones. Western blot analysis showed that the main molecular weight of HBsAg in HBsAg was about 34kD and about 37kD, and the expression level was about 200μg / L. Using different HBsAg test reagents to detect their antigenicity found that G145R mutation HB-sAg, with most of the reagents are not well detected, the detection capacity of only wild-type HBsAg 50% or less, but with American Abbott’s reagent detection capacity up to 98% of wild virus. G145R mutant HBsAg protein immunized mice, the serum can be detected 1: 1600 specific surface antibody, the antibody and G145R mutant HBsAg “a” determinant peptide P2-145R cross-reaction can occur, The reaction titer is 1:80. However, the antibody did not react with the wild-type HBsAg protein and the wild-type “a” determinant peptide P1-wt. The above results indicated that the protein of HBsAg mutated by G145R was secreted in the Pichia pastoris yeast system and the expressed product still had good immunogenicity, but its antigenicity and immunogenicity were significantly changed compared with the wild-type HBsAg .