mMR-1基因的克隆和在毕赤酵母中分泌表达  

Cloning of mMR-1 Gene and Expression in Pichia pastoris Systems

作　　者：李天伯[1] 胡洋[2] 王以光[1] 夏焕章[2] 

机构地区：[1]中国医学科学院中国协和医科大学,北京100050 [2]沈阳药科大学,沈阳110015

出　　处：《生物工程学报》2005年第1期25-29,共5页Chinese Journal of Biotechnology

基　　金：北京市科技计划重大项目基金资助 (No .H0 2 0 2 2 0 0 2 0 3 10 )。~~

摘　　要：hMR 1为本实验室首次克隆发现的一个人类新基因 ,是一种和三种肌肉收缩蛋白及多种细胞信号蛋白具有相互作用的膜蛋白。经与鼠基因组数据库进行同源分析后设计合成引物 ,利用RT PCR技术从小鼠C5 7BL 6J脾脏T淋巴细胞总RNA中反转录得到鼠源MR 1基因 (mMR 1) ,提交GenBank ,收录号 (AY2 99972 )。序列分析证明其与人源MR 1基因 (hMR 1)同源性为 90 1%。构建表达载体pPIC9 mMR 1电转化PichiapastorisGS115 ,筛选得到整合分泌表达mMR 1蛋白的重组酵母菌株。表达目的蛋白分子量 2 5kD ,诱导 5d时产量达到 5 0mg L ,通过Westernblot验证了表达产物的正确性。本研究为进一步研究新基因MR 1的生物学功能奠定了基础。hMR 1 (Homo Myofibrillogenesis Regulator 1, AF417001) is a novel homo gene, which was firstly cloned in our la boratory. The former studies revealed that hMR 1is a transmembrane protein which shows protein interaction with sarcomeric proteins like myomesin I, myosin regulatory light chain,β enolase and some cell regulator proteins such as eukaryotic translation initiation factor3 subunit 5 (eIF3S5) and etc. In this work, we focused on cloning the homologous gene of hMR 1 from mouse C57BL/6J and exploring its expression using Pichia pastoris yeast system. Two pairs of primers were synthesized according to the hMR 1 gene homologous sequence on mouse genome chromosome 1 The mouse MR 1 gene (mMR 1) was cloned by PCR following the first round RT PCR from mouse C57BL/6J spleen total RNA. Sequence analysis verified that mMR 1 gene and amino acids sequence showed 90 4% and 90 1% identity with hMR 1, respectively. The prediction of hydrophobic transmembrane structure of mMR 1 suggested it is also a transmembrane protein. The mMR 1 Pichia pastoris expression vector pPIC9 mMR 1 was constructed by fusion of the flanking mMR 1 ORF in the pPIC9 plasmid. After linearization of pPIC9 mMR 1 with Sal Ⅰ, the 8 5kb DNA fragment was transformed into Pichia pastoris GS115 strain by electroporation. GS115/Mut +pPIC9 mMR 1 transformants were selected on minimal methanol medium. Integration of mMR 1 gene into the yeast genome in the recombinants was verified by PCR from the transformants total DNA. The mMR 1 protein was expressed by induction under the concentration of 0 5% methanol. The specific induced protein of 25 kD molecular mass in SDS PAGE was confirmed to be the mMR 1 protein by Western blot using hMR 1 polyclonal antibody. The expression level of this recombinant mMR 1 protein was about 50 mg/L. The successful expression of mMR 1 in the Pichia pastoris GS115 will facilitate the further functional analysis of the novel gene MR 1 in animal model.

关 键 词：MR-1 克隆与表达 毕赤酵母 

分 类 号：Q78[生物学—分子生物学]