摘要
目的表达野生及变异的乙型肝炎病毒(HBV)表面抗原,为评价其免疫诊断试剂的敏感性提供依据。方法利用含有野生型和3种突变型乙型肝炎病毒表面抗原(HBsAg)序列为模板PCR进行扩增,将目的片段胶回收后,进行酶切与酵母表达载体pPICZA和pPICZα连接。电转化酵母菌株GS115,通过使用抗生素Zeocine加压筛选,获得高拷贝菌株。挑选多个单克隆菌株在BMMY中诱导表达,选择表达量高的菌株用于后续研究。结果构建野生型HBsAg以及突变型T126N、D144A、G145R三种重组质粒。通过对157株菌株筛选,获得1株野生型和3株突变型表达量高的菌株。表达产物经雅培Architect i2000的检测试剂、确证试剂以及WB(Western blotting)检测,结果均为阳性。结论成功表达了野生及变异的HBsAg,为评价国产HBsAg诊断试剂对变异株的检测能力提供了依据。
Objective To express surface antigen （ HBsAg） proteins from the wild-type hepatitis B virus and HBV variants for evaluating the sensitivity of HBsAg diagnostic kit.Methods Amplified PCR products that were by using of genomes from wild-type and three variant forms of HBV as templates were employed to construct the plasmid of pPICZA and pPICZα.The plasmids were transferred into the yeast strain GS115.The high copy strains were acquired in the presence of Zeocine.The strains for high-level expression were selected for a further study.Results One wild and three variant plas-mids of HBsAg were constructed.The strains for high-level expression were selected by screening 157 strains.The ex-pressed HBsAg were positive revealed by the ARCHITECT i2000 and Western blotting detection.Conclusion The wild and variant type HBsAg proteins were successfully expressed.These expressed HBsAg proteins provide a reference basis in evaluation of HBsAg diagnostic kit.
出处
《微生物学免疫学进展》
2015年第6期6-9,共4页
Progress In Microbiology and Immunology
关键词
乙型肝炎病毒
变异体
毕赤酵母
表达
Hepatitis B virus（HBV）
Variant
Pichia pastoris
Expression
