肝靶向肽修饰的人内皮抑制素rES-CSP融合蛋白可溶性表达及活性鉴定

Soluble expression and biological activity identification of CSP I-plus modified human endostatin rES-CSP

为了方便获得大量的有活性rES-CSP融合蛋白,通过优化诱导表达条件,实现融合蛋白rES-CSP的可溶性表达,并纯化后检测其生物学活性,使该融合蛋白作为药用蛋白大规模生产成为可能。首先在常规条件下诱导表达融合蛋白rES-CSP,通过SDS-PAGE电泳分析,选出最优菌株;继而优化诱导温度和诱导时间,Western Blot检测目的蛋白可溶性表达情况,Ni-NTA亲和层析柱进行纯化后RP-HPLC分析其纯度;CCK-8细胞增殖实验和流式细胞仪检测融合蛋白生物学活性。结果选出目的蛋白约占菌体总蛋白43.84%的单克隆菌株为研究对象,在诱导温度为25℃,诱导时间为20 h时,融合蛋白rES-CSP实现可溶性表达,并且可溶性表达量较高。经纯化后获得的rES-CSP融合蛋白纯度达到98%以上;制备的rES-CSP融合蛋白具有抑制肝癌细胞HepG2增殖作用,且与HepG2结合能力较强;为融合蛋白rES-CSP的应用研究奠定基础。

The soluble expression of the fusion protein rES-CSP was accomplished by condition optimization in order to obtain amount of active protein conveniently.Its biological activity was detected after purification.The fusion protein was made possible as a pharmaceutical.The rES-CSP was expressed under normal conditions,and the optimal strain was selected by SDS-PAGE.After optimization of induction time and temperature,the targeted protein was analyzed by Western Blot,purified by Ni-NTA,and the purity of the fusion protein was analyzed by RP-HPLC.The biological activity of fusion protein was detected by CCK-8 and flow cytometry.Results showed that the monoclonal strain with the target protein of about 43.84%of the total bacterial protein was selected as the research object.Soluble fusion protein rES-CSP was expressed at 25℃for 20 h,and the amount was higher.The purity of rES-CSP after purification was over 98%.Biological assay showed that rES-CSP inhibited the proliferation of hepatoma cell line HepG2 and had stronger binding ability to HepG2.