牡蛎胰岛素相关多肽3克隆表达及纯化

Cloning expression and purification of insulin-related polypeptides 3 from Oyster

目的克隆构建含有胰岛素相关多肽3(IRP3)基因的BL21表达菌株,并对表达蛋白进行分离纯化、鉴定和复性。方法采用DNA重组技术克隆获得IRP3-pET30a阳性BL21菌株,双酶切后琼脂糖凝胶电泳显示IRP3-p ET30a质粒构建成功,利用镍金属亲和层析方法获得IRP3/HIS纯化蛋白,进一步利用SDS-PAGE和Western blot技术确认IRP3/HIS融合蛋白的纯化效果。结果成功克隆IRP3-pET30a质粒,筛选获得含有IRP3-pET30a质粒的BL21表达菌株。从包涵体裂解上清液中获得了纯化的IRP3/HIS融合蛋白并进行透析复性。结论IRP3基因克隆成功及IRP3/HIS融合蛋白表达纯化,为IRP3蛋白的抗体制备和作为体外生长因子添加剂的应用打下基础。

Objective To clone and construct a BL21 expressing strain containing insulin related polypeptide 3(IRP3)gene,and isolate,purifiy,identifiy and renaturat the expressed protein.Methods IRP3-pET30 a positive strain BL21 was cloned by DNA recombination technique,the plasmid IRP3-PET30 A was successfully constructed by agarose gel electrophoresis after double enzyme digestion,and IRP3/HIS purified protein was obtained by nickel affinity chromatography,SDS-PAGE and Western blot were further used to confirm the purification effect of IRP3/HIS fusion protein.Results Plasmid IRP3-PET30 A was successfully cloned and BL21 expressing strain containing plasmid IRP3-p ET30 a was screened,purified IRP3/HIS fusion protein was obtained from the supernatant of inclusion body lysis and renaturated by dialysis.Conclusion The successful cloning of IRP3 gene and the expression and purification of IRP3/HIS fusion protein lay a foundation for the preparation of IRP3 protein antibody and the application of IRP3 protein as in vitro growth factor additive.What we have done will surely be useful to the antibody preparation for the IRP3 protein and the application of growth factor additive in vitro.