人sCR1酵母分泌型表达载体构建及其高拷贝转化子的快速筛选

目的采用酵母分泌型载体表达人可溶性补体受体1型(sCR1),研究人sCR1高拷贝重组阳性转化子的快速筛选及鉴定。方法从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,然后将其克隆入毕赤酵母细胞分泌型表达载体pPIC9k中,构建含人sCR1的重组质粒(pPIC9k-sCR1),经测序鉴定正确,电转化入毕赤酵母细胞SMD1168中,将经G418抗性筛选出的重组sCR1酵母细胞株进行PCR鉴定,经甲醇诱导,表达产物经SDS-PAGE分析和Western blot鉴定,通过Ni2+-NTA agarose亲和层析纯化。结果获得毕赤酵母细胞分泌型表达载体pPIC9k-sCR1,经G418筛选及PCR鉴定得到高拷贝整合的重组酵母细胞株,经甲醇诱导含pPIC9k-sCR1的酵母SMD1168细胞表达出重组sCR1融合蛋白。此蛋白在SDS-PAGE上表现为Mr约31000的蛋白区带,在Western blot分析中可被sCR1的CD35单克隆抗体(mAb)识别。经Ni2+-NTA agarose亲和层析纯化后得到较纯的sCR1融合蛋白。结论人sCR1融合蛋白在酵母细胞表达系统中的高水平表达,并且有与人体天然蛋白相同的抗原性。

Construction of human sCR1 ferment cell secreting type carrier and rapid screen for its high copy carrier

Objective To express human soluble complement receptor type 1(sCR1)protein using ferment cell secreting type carrier and study the rapid screen and assessment for its high copy carrier.Method Total human RNA was extracted from peripheral blood.The full length cDNA of human sCR1 gene was obtained by RT-PCR and them,cloned into Pichia pastoris eukaryotic expression vector pPIC9k to construct the recombinant plasmid pPIC9k-sCR1 containing human sCR1.After identified by DNA sequencing,the recombinant plasmid pPIC9k-sCR1 was transformed into Pichia pastoris SMD1168.The ferment cell line of the recombinant sCR1 which was chosen by G418 resistance was identified by PCR.After methanol induction,the expressed protein products were verified by SDS-PAGE and Western blotting,purified by Ni2 +-NTA agarose affinity chromatography.Result The obtained Pichia pastoris secretion type yeast carrier pPIC9k-sCR1 was chosen by G418 and identified by PCR to get a highly copied and integral recombinant ferment cell line.The recombinant human sCR1 fusion protein was expressed by yeast cells containing pPIC9k-sCR1 induced by methanol.It was a protein band about Mr 31000 in gel,which could be identified by CD35 of anti-sCR1 protein monoclonal antibody with Western blotting technique.The highly purified sCR1 fusion protein was detected obtained by Ni2+-NTA agarose affinity chromatography.Conclusion The recombinant human sCR1 fusion protein can be highly expressed in the Pichia pastoris expression system,which resembles the human natural protein's antigenicity.