人sCR1转化克隆菌的快速筛选和鉴定

Rapid screening and identification of human sCR1 from engineered yeast cells

目的用G418快速筛选及鉴定重组人可溶性补体受体1型(sCR1)高拷贝转化克隆菌。方法采用的sCR1重组质粒,经电转化将其克隆入酵母SMD1168细胞株中,利用G418的抗性,快速筛选转化克隆菌,并对克隆菌株提取基因组DNA进行聚合酶链反应(PCR)鉴定;该克隆菌经甲醇诱导培养后,对其表达产物进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot鉴定分析。结果从G418快速筛选的毕赤酵母克隆菌株中提取的基因组DNA进行PCR鉴定,获得了高拷贝整合的重组毕赤酵母克隆菌株,经诱导培养后,对其表达产物进行SDS-PAGE及Western blot鉴定分析,该表达蛋白在SDS-PAGE上表现为相对分子质量大于30 000的蛋白区带,在Western blot分析中可被sCR1的CD35单克隆抗体(mAb)识别,成功获得高拷贝整合的重组毕赤酵母细胞株,可表达出重组人sCR1融合蛋白。结论经高浓度G418药物快速筛选的高拷贝毕赤酵母SMD1168细胞株,用于表达人sCR1融合蛋白。

Objective To obtain highly copied and integrally recombinant Pichiapastoris secretion type yeast carrier pPIC9K- sCR1 by G418 screening. Methods The recombinant plasmid encoding human sCR1 was transfected into SMD1168 ceils. After identification by DNA sequencing, the cell line carrying the recombinant sCR1 was chosen by G418 resistance and identified by polymerase chain reaction （PCR）. After methanol induction, the expressed protein was identified by sodium dodecy sulfate polyacrylamide gel electrophoiesis（SDS-PAGE） and Western blotting. Results The obtained Pichiapastor＇s secretion type yeast carrier pPIC9K-sCR1 was chosen by G418 and identified by PCR to get a highly copied and integral recombinant cell line. The sCR1 gene was highly expressed as inclusion body and the unique band was found at the position corresponding to around 30 000 Dahons by SDS-PAGE. Western blot analysis showed the protein could combine CD35 antibody. Conclusion The highly copied Pichia pastor＇s secretion type yeast carrier pPIC9K-sCR1 chosen by G418 could express the recombinant protein of interest.