重组mOP-1原核细胞高效表达和纯化的研究

PROKARYOTIC EXPRESSION OF RECOMBINANT MOUSE OP-1 AND ITS PURIFICATION

①目的 探讨重组mOP 1 (rmOP 1 )的克隆化、原核高效表达载体的构建以及rmOP 1制备和纯化简单有效方法。②方法 于胎龄为 2周的CD 1小鼠胚胎组织中提取、纯化mRNA ,应用ligo(dT)引物反转录酶合成cDNA ,PCR筛选扩增OP 1全长开放读框 ,产物TA克隆入质粒载体pCR 3.1 Uni;亚克隆入高效原核表达载体pTrcHis2B ,经大肠杆菌JM 1 0 9、IPTG诱导表达rmOP 1蛋白 ;经Ni NTA树脂纯化 ,SDS PAGE分析。 ③结果限制性酶切分析、DNA测序确认mOP 1的全长开放读框 ,Ni NTA亲和层析纯化重组蛋白PAGE分析满意。④结论 胚胎期约 2周的CD 1小鼠有OP 1mRNA的高水平表达 ,RT PCR筛选OP 1全部开放读框 ,产物TA克隆化方法便捷 ,原核高效表达质粒 pTrcHis 2B能够方便、快速回收和纯化重组蛋白。

Objective To investigate the effective means of rmOP-1 cloning, construction of the prokaryotic expression vector, and its purification. Methods mRNAs were isolated and purified from a 2-week embryo of a CD-1 mouse. cDNAs were synthesized by Oligo(dT) primers with AMV. Full-length open reading frame of mOP-1 was screened by PCR. PCR products was inserted into pCR 3.1-Uni vector by TA cloning. Subcloned into pTrcHis 2B and induced by IPTG, recombinant OP-1 was expressed with prokaryotic expression system, E. coli, JM109. The recombinant protein was isolated and purified with Ni-NTA resin, and its osteogenesis was assayed in an ALP activity. Results Open reading frame was verified by both restrictive endonuleotidase analysis and DNA sequencing. rmOP-1 purified from Ni-NTA resin chromatography was analyzed by PAGE. Conclusion Over-expression of OP-1 mRNA exists in a 2-week embryo of a CD-1 mouse. The gene can be easily obtained by RT-PCR and TA cloning. The high efficiency of prokaryotic expression vector pTrcHis 2B would offer a fast and simple means of rmOP-1 production.