包涵体蛋白LAP-F的制备、提取、纯化、验证  

Preparation,extraction,purification and verification of inclusion body protein LAP-F

作　　者：刘增瑞 宋旭东 初彦辉 LIU Zeng-rui(Medical research center of Mudanjiang Medical University,Mudanjiang 157011,China)

机构地区：[1]牡丹江医学院医药研究中心,黑龙江牡丹江157011

出　　处：《牡丹江医学院学报》2020年第2期33-37,共5页Journal of Mudanjiang Medical University

基　　金：国家自然科学基金资助项目(81573068;81200305)。

摘　　要：目的探究包涵体蛋白LAP-F制备、提取、纯化以及验证它对TGF-β诱导的HK-2细胞在形态学上的影响。方法通过高浓度尿素溶解包涵体,离心后将上清通过透析使蛋白复性,经镍琼脂糖亲和层析纯化重组蛋白,通过SDS-PAGE凝胶定量蛋白含量;将重组蛋白LAP-F作用于TGF-β诱导的HK-2细胞,并培养48h后通过显微镜观察细胞形态。结果诱导了LAP-F在包涵体的表达,并筛选出了高表达菌株。结论原核表达载体构建成功,成功筛选并纯化了目的蛋白;重组蛋白LAP-F可抑制TGF-β对HK-2细胞的纤维化,为后续研究LAP-F对TGF-β活化过程的干扰作用及对器官纤维化的影响奠定基础。Objective To explore the preparation,extraction,purification and verification of the inclusion body protein LAP-F,and investigate its effect on morphological changes of TGF-βinduced HK-2 cells.Methods The inclusion body was dissolved by high concentration urea,the protein was renatured by dialysis after centrifugation,the recombinant protein was purified by nickel agarose affinity chromatography,and the protein content was quantified by SDS-PAGE gel.The TGF-βinduced HK-2 cells were treated with LAP-F for 48 hours,and the cell morphology was observed by microscope.Results The expression of LAP-F in inclusion bodies was induced,and high-expression strains were selected.Conclusion The prokaryotic expression vector was successfully constructed,and the target protein was successfully screened and purified.The recombinant protein LAP-F could inhibit the fibrosis of TGF-βon HK-2 cells,which would lay the foundation for the follow-up studies on the interference effects of LAP-F on the activation process of TGF-βand organ fibrosis.

关 键 词：包涵体 复性 重折叠 纤维化 

分 类 号：R34[医药卫生—基础医学]