乙肝表面抗原突变体的表达及其初步应用

目的：构建和表达乙肝表面抗原（HBsAg）突变体用于HBsAg抗原性的深入研究。方法：利用定点突变技术构建HBsAg突变体，然后转化毕赤酵母GS115，菌落PCR、高浓度Zeocin抗性筛选鉴定转化子，表达产物经SDS—PAGE分析和Western blot分析，利用AxSYM HBsAg V2（Abbott）酶免试剂盒检测重组表面抗原的活性。结果：通过序列分析确定突变体构建成功，SDS—PAGE显示突变体能在毕赤酵母中有效表达，在Western blot试验中HBsAg突变体被特异性多克隆抗体识别，相对分子质量（Mr）约为38000，AxSYM试剂盒检测结果表明HBsAg突变体具有一定生物活性。结论：利用毕赤酵母表达系统高效表达出具有一定免疫反应性的HBsAg突变体。对于目前市售试剂盒的质量控制和临床应用有较高的实用价值。

Expression and preliminary applications of hepatitis B surface antigen mutants

AIM:To clone and express HBsAg mutant in the Pichia pastoris.METHODS:The cloned wild type pGAP-S was used as the DNA template to generate mutant type pGAP-MS with a single or double nucleotide changes incorporated in complementary oligonucleotide primers.The product was linearized with BspH I and transformed into Pichia pastoris strain GS115,and stable multicopy integrants were screened in medium containing different concentrations of Zeocin.RESULTS:The pGAP-MS expression vector was successfully constructed and stable numbers integrated strains with high copy number were obtained.The expression of HBsAg mutant protein was identified by SDS-PAGE and Western blot with specific polyclonal antibody.The molecular weight of recombinant HBsAg mutant was 38 kDa.AxSYM HBsAg V2(Abbott)assays demonstrated all 10 HBsAg mutants were reactive.CONCLUSION:The recombinant HBsAg mutant with immunoreactivity was successfully expressed in Pichia pastoris,and it was of practical value on the quality control and clinical applications of commercial assays.