摘要
目的对分泌型毕赤酵母中表达乙肝表面抗原并进行亲和层析纯化。方法从已确诊的乙肝患者阳性血清中提取乙肝病毒DNA,PCR扩增乙肝病毒表面抗原编码基因S,将其插入含有α分泌信号肽序列和6个组氨酸纯化标签的毕赤酵母表达载体pPICZα,成功构建了重组表达载体pPICZα-HBVs。电转化酵母菌株GS115,得到重组乙肝表面抗原S的酵母表达菌株。结果诱导培养基BMMY中甲醇终浓度为1%,诱导表达48 h重组蛋白表达量及抗原性达到最高。非变性条件下亲和层析纯化后,薄层扫描分析得到纯度超过95%,表达量约为2 mg/L的重组蛋白。结论W estern-blot和ELISA分析表明,所得产物为乙肝表面抗原S蛋白,其纯度和产量足以免疫小鼠制备单克隆抗体。
Aim The expression and purification of HBsAg in secreted Pichia pastoris. Methods The HBV S gene was amplified by PCR from the patient and cloned into the Pichia pastoris expression vector pPICZα which has α signal sequence and 6 × His tag,through electroproration transformation of GS115 and zeocin selection, a strain of P. pastoris capable of expression S protein was constructed successfully. Results After induced in BMMY medium with 1% methnol for 48 h, the products were processed for Western-blot and ELISA, the results indicating that the recombinant protein is S protein which keeps the antigen activity and has got higher level expression. Conclusion The highly efficient expression and purification of S protein can be used to prepare monoclonal antibody and lay a foundation of developing diagnostic kit.
出处
《西北大学学报（自然科学版）》
CAS
CSCD
北大核心
2008年第4期605-608,共4页
Journal of Northwest University（Natural Science Edition）
基金
国家高技术研究发展计划基金资助项目(2005AA205220)
关键词
乙肝表面抗原
分泌表达
毕赤酵母
Hepatitis B surface protein （HBsAg）
Pichia pastoris
secretory expression
