人精脒/精胺N1乙酰基转移酶基因的克隆、表达和纯化  

Cloning,expression and purification of human spermidine/spermine N^1-acetyltransferase gene

作　　者：孙慧[1] 龚磊[1] 刘斌[1] 杨亚培[1] 徐春晓[1] 刘贤锡[1] 

机构地区：[1]山东大学医学院生物化学与分子生物学研究所,济南250012

出　　处：《山东大学学报（医学版）》2008年第2期128-131,共4页Journal of Shandong University:Health Sciences

基　　金：山东省自然科学重大项目(z2004c01);教育部博士点自助课题基金资助课题(20040422029)

摘　　要：目的构建人精脒/精胺N1乙酰基转移酶基因的原核表达载体,诱导该质粒在大肠杆菌中表达并纯化其表达的重组蛋白。方法从大肠癌组织中提取总RNA,采用RT-PCR法扩增人SSAT基因的cDNA片段,经TA克隆及亚克隆方法构建原核表达载体pTriEx-4-SSAT。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6His-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SSAT的cDNA片段成功插入表达载体pTriEx-4中,而且方向正确,SDS-PAGE电泳显示表达出20 kD的外源蛋白。Western blot检测结果显示,表达出的蛋白为6His-tag的融合蛋白,而且用Ni-NTA亲和层析法纯化了该重组蛋白。结论成功构建、表达和纯化了重组SSAT蛋白,为制备其抗体及研究该基因与结直肠肿瘤的关系提供了有力的工具。Objective To construct the prokaryotic expression plasmid of human spermidine/spennine N^1-aeetyltransferase （SSAT） and to induce and purify the recombined SSAT protein in E. coli JMI09 （DE3）. Methods The total RNA was extracted from colon cancer tissues and a 513 bp eDNA was amplified by RT-PCR with two primers, which span the coding region of SSAT. The synthesized cDNA was sub-cloned into the prokaryotic expression vector pTriEx-4. The constructed vector pTriEx-4- SSAT was transformed into competence E. coli JM109 （DE3） and then was induced by IPTG. The protein was verified by SDSPAGE and Western blot with the anti His-Tag monoclonal antibody. The fusion protein was purified by the Ni-NTA chromatographic column, Results The prokaryotic expression plasmid pTriEx-4-SSAT was successfully constructed, and identified by digestion and sequence. An approximate 20 kI） exogenous protein was observed on the SDS-PAGE. The protein was verified by Western blot with an anti His-Tag monoclonal antibody. The fusion protein including 6His-Tag was purified by the Ni-NTA chro- matographic column. Conclusion The SSAT prokaryote expression vector was successfully constructed and the purified protein can be applied for further research of the immunity of SSAT.

关 键 词：精脒/精胺N1乙酰基转移酶 原核表达 结直肠肿瘤 

分 类 号：Q784[生物学—分子生物学]