上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

重组人亲环蛋白CypB的制备及活性鉴定

章琪，吴佳伟，周爱武   

  1. 上海交通大学医学院细胞分化与凋亡教育部重点实验室，上海 200025
  • 出版日期:2016-12-28 发布日期:2016-12-29
  • 通讯作者: 周爱武，电子信箱：aiwu.zhou@googlemail.com。
  • 作者简介:章琪(1988—)，男，硕士生；电子信箱：zhangqi88612@aliyun.com。

Preparation and activity identification of human cyclophilin B

ZHANG Qi, WU Jia-wei, ZHOU Ai-wu   

  1. Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
  • Online:2016-12-28 Published:2016-12-29

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摘要：

目的 ·构建人源亲环蛋白B（hCypB）的表达载体，通过原核表达体系制备有活性的hCypB。方法 ·通过双酶切法将编码hCypB基因构建到pGEX-6p-1表达载体中,采用亲和层析法一步纯化hCypB,最后通过GST-pulldown验证过表达的hCypB的活性。结果 ·经DNA测序验证，成功构建了hCypB的表达质粒。通过GST柱纯化可以从1 L过夜诱导的大肠埃希菌菌液中获得26 mg，纯度达到90%的GST-hCypB融合蛋白，且GST-pulldown试验证明该重组蛋白可以与脯氨酸-3-羟化酶1（P3H1）及软骨相关蛋白（CRTAP）形成三元复合物。结论 ·利用该实验方法可以在短期内获得大量高纯度、有活性的人源亲环蛋白CypB。

关键词: 亲环蛋白B, P3H1/CRTAP/CypB复合物, 蛋白重组, 原核表达体系

Abstract:

Objective · To construct an expression vector for human cyclophilin B (hCypB) and prepare active CypB via the prokaryotic expression system. Methods · The DNA sequence enconding hCypB was amplified by PCR and inserted into expression vector pGEX-6P-1 by double enzyme digestion. hCypB was further purified with affinity chromatography and the activity of over-expressed hCypB was verified with GST-pulldown. Results · The recombinant CypB expression plasmid was successfully constructed and verified with DNA sequencing. GST-CypB fusion protein was purified to a purity of 90% by affinity chromatography with a yield of 26 mg of fusion protein from a liter of overnight E. coli culture. GST-pulldown further confirmed that this GST-hCypB fusion protein could form the ternary complex with prolyl 3-hydroxylase 1 (P3H1) and cartilage-associated protein (CRTAP). Conclusion · Large amount of active and highly purified hCypB can be obtained via this experiment method within short time period.

Key words: cyclophilin B, P3H1/CRTAP/CypB complex, protein recombination, prokaryotic expression system