人微管蛋白失稳剂Oncoprotein18原核表达载体的构建及表达

Construction and expression of prokaryotic expression vector of human microtubule destabilizers Oncoprotein18

目的:原核表达人Oncoprotein18(Op18)蛋白,为 制备抗Op18的mAb作准备.方法:从人皮肤组织中用RT PCR扩增Op18cDNA的全长编码序列,克隆入表达载体pR SETA中,构建Op18的高表达工程菌,并以IPTG诱导表达目 的蛋白,通过亲和层析法纯化表达的Op18融合蛋白.结果: 酶切及测序鉴定证明,获得含有目的基因片段的重组质粒,表 达的Op18融合蛋白以可溶性的形式存在.结论:所获Op18 融合蛋白以可溶性的形式存在,为制备其mAb及进一步研究 Op18在创伤愈合及瘢痕形成中的作用打下了基础.

AIM: To construct and express prokaryotic expression vector of human microtubule destabilizers Oncoprotein18 (Op18) and to prepare monoclonal antibody (mAb) against Op18. METHODS: Full length sequence of Op18 gene was amplified from human skin by RT-PCR and was clones into the expression vector pRSET A. Op18 fusion protein was expressed in E.coli under IPTG induction and the expressed fusion protein was purified by glutathione agarose chromagraphy. RESULTS: The recombinant plasmid containing the target gene was constructed successfully. The fusion protein was expressed in E.coli in soluble form. CONCLUSION: The fusion protein is expressed in soluble form, which is a useful reagent for further preparation of monoclonal antibody and study of the function of Op18 in wound healing and scar excess.