文献类型：期刊文献

中文题名：乙肝表面抗原突变体的表达及其初步应用

英文题名：Expression and preliminary applications of hepatitis B surface antigen mutants

作者：祝长城[1];罗春贞[2];王缦[2]

机构：[1]华东理工大学生物化学与分子生物学系,上海200237;[2]上海科华生物工程股份有限公司,上海200233

年份：2007

卷号：23

期号：12

起止页码：1136

中文期刊名：细胞与分子免疫学杂志

外文期刊名：Chinese Journal of Cellular and Molecular Immunology

收录：CSTPCD;;Scopus;北大核心:【北大核心2004】；CSCD:【CSCD2011_2012】；PubMed;

基金：上海市科学技术委员会科研计划项目(07DZ19502)

语种：中文

中文关键词：乙肝表面抗原突变体;毕赤酵母;免疫反应性

外文关键词：Hepatitis B surface antigen mutant;Pichia pastoris;immunoreactivity

摘要：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 HBsAgV2(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。
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 pastods 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 Westem 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.