人S-腺苷蛋氨酸脱羧酶α亚基的克隆、表达与纯化  

Cloning,expression and purification of human S-adenosylmethionine decarboxylase gene α subunit

作　　者：龚磊[1] 刘贤锡[1] 张冰[1] 张岩[1] 胡海燕[1] 赵志艺[2] 

机构地区：[1]山东大学医学院医学分子生物学实验中心,山东济南250012 [2]山东大学医学院临床医学系,山东济南250012

出　　处：《山东大学学报（医学版）》2006年第11期1184-1188,共5页Journal of Shandong University:Health Sciences

基　　金：山东省科技厅科技计划项目(032050111)

摘　　要：目的:构建人S-腺苷蛋氨酸脱羧酶(SAMDC)的α亚基的原核表达载体,诱导该质粒在大肠杆菌中表达并纯化表达的重组蛋白。方法:从大肠癌细胞中提取总RNA,RT-PCR方法扩增人SAMDCα亚基的cDNA片段801 bp,经TA克隆及亚克隆方法构建原核表达载体pTriEx-4-SAMDC-α。将重组表达质粒转化入E.coliJM109(DE3)中,经IPTG诱导表达,SDS-PAGE电泳和Western blot鉴定表达蛋白,并通过6×His.Tag,利用亲和层析法纯化表达的融合蛋白。结果:酶切鉴定和DNA测序显示,人SAMDCα亚基的cDNA片段成功插入表达载体pTriEx-4且方向正确,SDS-PAGE电泳显示表达出32kD的外源蛋白。Western blot检测结果显示,表达出的蛋白为6×His.Tag的融合蛋白,且Ni-NTA亲和层析法纯化了该重组蛋白。结论:成功构建、表达且纯化了重组SAMDC-α亚基,为制备抗SAMDC抗体、研究SAMDC基因与结直肠肿瘤的关系提供了必要的工具。Objective： To construct the prokaryotic expression plasmid of human S-adenosylmethionine decarboxylase （SAMDC） α subunit, and to induce and purify the recombined SAMDC-α protein. Methods： A 801 bp cDNA spanning the coding region of SAMDC α subunit was amplified by RT-PCR and subcloned into prokaryotic expression vector pTriEx-4. The resulted vector pTriEx-4-SAMDC-α was transformed into competence E. coli JM109（DE3）, then was induced by IFIG. The protein was verified by SDS-PAGE and Western blot with anti His· Tag monoclonal antibody. The fusion protein including 6 ×His· Tag was purified by Ni-NTA chromatographic column. Results： A 801 bp cDNA was successfully amplified by RT-PCR from colorectal cancer cells and confirmed by sequence. The prokaryotic expression plasmid pTriEx-4-SAMDC-α was successfully constructed, which was identified by digestion and sequence. An approximate 32kDa exogenous protein was observed on the SDS-PAGE and Western blot showed that the protein including 6 × His· Tag was purified successfully by Ni-NTA affinity chromatography. Conclusion： The prokaryotic expression plasmid pTriEx-4-SAMDC-α is constructed correctly and the fusion protein is expressed and purified successfully.

关 键 词：S-腺苷蛋氨酸脱羧酶 结直肠肿瘤 原核表达 

分 类 号：Q784[生物学—分子生物学]