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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2007年第12期1136-1139,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
上海市科学技术委员会科研计划项目(07DZ19502)
关键词
乙肝表面抗原突变体
毕赤酵母
免疫反应性
Hepatitis B surface antigen mutant
Pichia pastoris
immunoreactivity