人TRAF3IP3基因剪接异构体2的克隆表达和生物信息学分析  

Cloning,prokaryotic expression and bioinformatics analysis of human TRAF3IP3 gene isoform 2

作　　者：王沂[1] 杨文思[2] 赵俊暕[1] 石峻[1] 戚晓渊[1] 郑永强[1] 牛艳艳[1] 王洋[3] 

机构地区：[1]河北联合大学附属医院检验科,河北唐山063000 [2]中国科学院生物物理研究所,北京100101 [3]河北联合大学生命科学学院,河北唐山063000

出　　处：《生物技术》2015年第2期119-123,共5页Biotechnology

基　　金：河北省青年科学基金项目("蓝氏贾第鞭毛虫α-4贾第素细胞定位和功能的研究";No.C2012401039);唐山市科技局项目("人类TRAF3IP3基因启动子的鉴定及其转录调控的研究";No.13130295z)资助~~

摘　　要：[目的]克隆、原核表达并纯化人类TRAF3IP3基因剪接异构体2(TRAF3IP3iso2),对TRAF3IP3iso2蛋白进行生物信息学分析。[方法]从人骨髓单个核细胞c DNA中扩增TRAF3IP3iso2开放阅读框区,双酶切连入原核表达载体p ET-28a(+),重组质粒转化E.coli Rosetta(DE3),IPTG诱导表达,SDS-PAGE和Western blot鉴定表达效果,Ni-NTA亲和层析柱纯化目的蛋白;根据测序结果对TRAF3IP3iso2蛋白进行生物信息学分析。[结果]成功克隆了人类TRAF3IP3iso2编码区并构建了原核表达载体p ET-28a(+)-TRAF3IP3iso2,在大肠杆菌中诱导表达、纯化获得了相对分子量约22.7k Da的融合蛋白;生物信息学分析显示TRAF3IP3iso2蛋白二级结构以α螺旋为主,无TRAF3IP3iso1蛋白的跨膜区结构。[结论]证明了人类TRAF3IP3iso2的存在,为TRAF3IP3功能的研究提供了实验依据。[ Objective] To prokaryotically express, purify and bioinformatically analyze human TRAF31P3 gene isoform 2 （TRAFMP3iso2）. [ Methods] The full -length open reading frame of TRAF3IP3iso2 was obtained by PCR from cDNA of human bone marrow mononuclear cells. The PCR product was inserted into pET- 28a（ ＋ ） vector by double digestion. The recombinant vector was transformed into E. coli Rosetta（ DE3 ） and expressed by IPTG induction. The expression product was identified by SDS - PAGE and Western blot and purified by Ni- NTA affinity chromatography. The structural feature was analyzed by bioinformatics tools. [ Results] The TRAF31P3iso2 coding se- quence about 550bp was obtained and cloned into pET -28a（ ＋ ） successfully. The TRAF3IP3iso2 fusion protein about 22. 7kDa was ex- pressed by IPTG induction in pET -28a（ ＋ ） -TRAF3IP3iso2 transformed E. coli Rosetta（ DE3 ） and visualized by SDS -PAGE and Western blot. The purified fusion protein exhibited a single band in SDS - PAGE. Bioinformatics analysis showed that human TRAF3IP3iso2 protein had no transmembrane domain and its secondary structure was mainly a - helix. [ Conclusion ] Human TRAF3IP3iso2 fusion protein was successfully expressed in E. coli and analyzed by bioinformatics,which provided a foundation for further study of TRAF3IP3.

关 键 词：TRAF3IP3 剪接异构体 原核表达 生物信息学 

分 类 号：Q71[生物学—分子生物学]