摘要
人可溶性 gp190 (sgp190 )是白血病抑制因子 (LIF)的可溶性受体 ,可能在LIF行使众多生物学功能中扮演着重要的角色。为获得这一蛋白以研究它在人体内的生物学活性 ,在酵母Pichiapastoris中实现了重组sgp190分泌表达。利用基因重组技术 ,将编码人可溶性 gp190 (LIF受体α亚基 gp190胞外区 )cDNA克隆到毕赤酵母Pichiapastoris分泌表达载体 pPIC9K ,构建了重组表达质粒 pPIC9K sgp190。原生质体法转染PichiapastorisGS115菌株 ,经过G4 18筛选 ,得到了高效分泌表达sgp190蛋白的毕赤酵母菌株GS115 / pPIC9K sgp190 ,sgp190蛋白占摇瓶培养表达上清中总蛋白质的 2 6 %。经初步纯化 ,对表达产物的性质鉴定表明 ,其分子量约 12 5kD ,具有免疫活性。
The cDNA of N-terminal extracellular domain of human leukemia inhibitory factor receptor α-subunit(gp190) ,which encoded soluble gp190(sgp190),was cloned into the methylotrophic yeast Pichia pastoris secreted expression vector pPIC9K.The linearized plasmid sgp190-pPIC9K was then integrated with GS115 genome by spheroplasts transformation.MD medium and PCR method were used for screening positive transformants.The transformants(His^+Mut^+) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418 and induced by 0.5% methanol in shaking flask to express sgp190.The sgp190 expressed level could reach up to 26% of total proteins in culture supernatant.SDS-PAGE and Western blotting analysis showed that the molecular weight of sgp190 is about 125 kD and could be specifically recognized by polyclonal antibodies against human sgp190,which indicated good antigenicity of this secreted expressed protein.Biological activity assay showed that purified sgp190 could inhibit the effect of LIF on hCG biosynthesis of cytotrophoblast in vitro.
基金
重庆市科委资助项目 (编号 :2 0 0 1 5 4 46)
重庆市教委资助项目 (编号 :2 0 0 1 12 )~~
