人氨肽酶N基因克隆和原核表达  

Molecular cloning and prokaryotic expression of human aminopeptidase N

作　　者：涂向东[1] 谢飞[1] 吴玉水[1] 兰风华[1] 朱忠勇[1] 

机构地区：[1]南京军区福州总医院解放军医学检验中心实验科,福建福州350025

出　　处：《医学研究生学报》2004年第8期691-693,共3页Journal of Medical Postgraduates

摘　　要：目的 :构建人氨肽酶N(APN) 谷胱甘肽巯基转移酶 (GST)融合蛋白 (APN GST) ,并进行原核表达。　方法 :根据人APNmRNA序列设计合成特异性引物 ,从人肝组织中提取总RNA并反转录成cDNA第一链 ,RT PCR扩增人APN基因 ,并插入融合蛋白原核表达载体 pGEX 4T ,转化宿主菌BL2 1(DE3)细胞 ,异丙基锍基半乳糖(IPTG)诱导表达APN蛋白。包涵体经尿素变性、重折叠和亲和层析纯化。　结果 :重组质粒测序和酶切结果显示 :APN基因已正确插入 pGEX 4T ,重组蛋白及纯化产物SDS PAGE在 135 0 0 0处有一条明显的蛋白表达条带。　结论 :人APN基因已被成功地克隆、表达和纯化。Objective:To obtain recombinant human aminopeptidase N protein. Methods:Human APN cDNA was synthesized by RT-PCR using total RNA from human liver tissues and a couple of primers were designed according to the know sequence of human APN mRNA and inserted into the prokaryotic expression plasmid pGEX-4T vector and introduced into a strain of E.coli BL21(DE3), The protein expressed under the induction of IPTG. The inclusion bodies expressed in E.coli were washed,denatured?refolded and purified to a high purity. Results :Sequence and restriction analysis revealed APN gene was cloned in frame into pGEX-4T,SDS-PAGE profile showed a clear protein band with a relative molecular weight of 135 000. Conclusion:Human APN gene was successfully cloned ? expressed and purification .

关 键 词：氨肽酶N 基因克隆 原核表达 纯化 

分 类 号：Q785[生物学—分子生物学]