sCR1在原核中表达、纯化、复性及活性鉴定

目的采用原核表达载体pET28a在E.coli BL21(DE3)中获得高表达、高活性的重组人sCR1融合蛋白。方法从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,将其克隆入原核表达载体pET28a中,构建含人sCR1的重组质粒(pET28a-sCR1),经测序鉴定正确,转化入E.coli BL21(DE3)中,经IPTG诱导,表达产物经SDS-PAGE分析和Western blot鉴定,通过Ni2+-NTA agarose亲和层析纯化后进行复性及生物学活性鉴定。结果获得原核表达载体pET28a-sCR1,经PCR鉴定及测序鉴定,得到重组E.coli BL21(DE3)克隆菌株,经IPTG诱导含有pET28a-sCR1的E.coli BL21(DE3)克隆菌,表达出重组人sCR1融合蛋白。此蛋白在SDS-PAGE上表现为Mr大于29000的蛋白区带,在Western blot分析中可被sCR1的CD35单克隆单抗体(mAb)识别。经Ni2+-NTA agarose亲和层析纯化、复性后得到高纯度的sCR1融合蛋白表达及较高的生物学活性。结论人sCR1融合蛋白在E.coli BL21(DE3)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及其生物学活性。

Expression, purification, refolding and its biologic activity research of sCR1 in prokaryon

Objective To obtain highly expressed, highly actived recombinant human sCR1 fusion proteins in E. coli BL21 (DE3) using prokaryotic expression vector pET28a. Methods Total human RNA was extracted from peripheral blood. The full length cDNA of human sCR1 gene was obtained using RT-PCR and them, cloned into prokaryotic expression vector pET28a to construct the recombinant plasmid pET28a-sCR1 containing human sCR1. After identified by DNA sequencing, the recombinant plasmid pPIC9k-sCR1 was transformed into E. coli BL21(DE3). After IPTG induction, the expressed protein products were verified by SDS-PAGE and Western blotting, purified by Ni2+-NTA agarose affinity chromatography, refolding and its biologic activity was identified. Results The obtained prokaryotic expression vector pET28a -sCR1 was identified by PCR and DNA sequencing to get E. coli BL21(DE3) clone strain. The recombinant human sCR1 fusion protein was expressed by E. coli BL21(DE3) clone strain containing pET28a-sCR1 induced by IPTG. It was a protein band about Mr above 29 000 in gel, which could be identified by CD35 of anti-sCR1 protein monoclonal antibody with Western blotting technique. The highly purified sCR1 fusion protein and its biologic activity were detected obtained by Ni2+-NTA agarose affinity chromatography and refolding. Conclusion The recombinant human sCR1 fusion protein can be highly expressed in E. coli BL21(DE3) expression system, which resembles the human natural protein's antigenicity and biologic activity.