重组可溶性补体1型受体在酵母SMD1168中表达纯化及鉴定

目的在酵母细胞SMD1168中表达人可溶性补体1型受体(sCR1),并对其重组蛋白进行纯化以探讨较为接近人体天然蛋白的表达方式,从而为临床诊断及治疗提供方便。方法从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,将其克隆入真核表达载体pPIC9K,构建含人sCR1的重组质粒(pPIC9K-sCR1);经测序鉴定正确后,将重组质粒转化入毕赤酵母菌细胞SMD1168中,经甲醇诱导,表达产物经SDS-PAGE分析及Western blot鉴定,并通过Ni2+-NTA agarose亲和层析纯化。结果经甲醇诱导的含pPIC9K-sCR1的酵母细胞表达出重组人sCR1的融合蛋白,48-72hsCR1融合蛋白表达量最高。此蛋白在凝胶上表现为大于31kD的蛋白区带,在Western blot实验中可被sCR1的CD35单克隆抗体识别。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活性。

Expression,purification and identification of recombinant soluble complement receptor type 1 in Pichia pastoris SMD1168

Objective Through expressing human sCR1 in Pichia pastoris SMD1168 and purifing the recombinant protein product,so as to provide convenience for clinical diagnosis and therapy with the expression similar to human natural protein. Methods Human total RNA was extracted from peripheral blood.The full length cDNA of human sCR1 gene was obtained using RT-PCR,cloned into eukaryotic expression vector pPIC9K to construct the recombinant plasmid pPIC9K-sCR1 containing human sCR1, identified by DNA sequencing,then the recombinant plasmid pPIC9K-sCR1 was transformed into Pichia pastoris SMD1168. After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blotting analysis,and purified by Ni2+-NTA agarose affinity chromatography.Results The recombinant human sCR1 fusion protein was expressed by yeast cells containing pPIC9K-sCR1 induced by methanol.The expression level was the highest in 48h-72h.It was a protein band >31KDa in gel which could be identified by CD35 of anti-sCR1 protein monoclonal antibod with Western blotting technique.Highly purified sCR1 fusion protein was obtained by Ni2+-NTA agarose affinity chromatography. Conclusion The recombinant human sCR1 fusion protein can be highly expressed in the Pichia pastoris expression system, resembling the human natural protein's antigenicity.