前列腺素脱氢酶在大肠杆菌中的表达纯化及鉴定  

Expression Purification and Identification of 15-Hydroxyproglandin Dehydrogenase Gene in Escherichia coli

作　　者：李美宁[1] 解军[1] 常冰梅[1] 张悦红[1] 殷云勤[2] 程牛亮[1] 

机构地区：[1]山西医科大学生物化学与分子生物学教研室,太原030001 [2]山西医科大学第一医院消化检验中心,太原030001

出　　处：《中国生物工程杂志》2008年第5期41-45,共5页China Biotechnology

基　　金：山西省科技攻关资助项目(2006031087-02)

摘　　要：15-羟基前列腺素脱氢酶(PGDH)属于抑癌基因,在多种肿瘤中表达缺失,在肿瘤的发生发展中起着重要作用。提取人正常大肠黏膜组织总RNA,利用RT-PCR方法扩增得到PGDH基因的编码序列,克隆入原核表达载体pBV220,测序鉴定正确后转化E.coliDH5α,经温控诱导表达,表达产物进行SDS-PAGE和Western blot,证实为相对分子质量约为29000的PGDH-His6蛋白,表达产物以包涵体形式存在,3h诱导表达量最高,约占菌体总蛋白的30%。经Ni2+配体亲和层析纯化得到纯度大于95%的目的蛋白。重组PGDH简单复性后具有一定的生物活性,约为3.7×104U/mg,为下一步研究其在肿瘤中的作用奠定了基础。15-hydroxyproglandin dehydrogenase （PGDH）is a tumor suppressor gene. PGDH transcript and protein are both highly expressed by normal tissue but are nearly undetectable in several tumors. Total RNA was isolated from human normal colonic epithelia. Then the PGDH cDNA gene was amplified by reverse transcript polymerase chain reaction （RT-PCR ）. The amplified fragment was orientationly linked into the prokaryotic expression vector pBV220 by T4 DNA ligase and then sequenced. The result indicated that the cloned gene was a corrected PGDH. Then the recombined expression plasmid was transduced into DH5ct by TSS, and the expression of PGDH protein was induced by temperature for 3 hours at 42℃, and the fusion protein of PGDH -His6 was analyzed by SDS-PAGE and Western blot after lysis of bacteria. The SDS-PAGE result showed that cloned recombinant protein was expressed in the inclusion body with molecular weight of approximated 29kDa. The PGDH fusion protein containing about 30% of total somatic protein, was stably expressed in E. coli. After uhrasonication and purification by immobilized metal chelation affinity chromatography, homogenous protein was obtained and reached 95 % purity as proved by SDS-PAGE. Western blot identified its specificity. The expressed PGDH protein had highly bioactivity,and its enzyme activity is about 3.7 × 10^4U/mg.

关 键 词：大肠癌 15-羟基前列腺素脱氢酶 克隆 原核表达 

分 类 号：Q786[生物学—分子生物学]