人毛乳头细胞HSPC016基因在大肠杆菌中的表达和鉴定  

Expression and product identification of the HSPC 016 gene of human dermal papilla cell by Escherichia coli expression system

作　　者：邹锋[1] 郝飞[1] 宋志强[1] 易勇[2] 郝进[1] 钟华[1] 叶庆佾[1] 

机构地区：[1]第三军医大学西南医院皮肤科,重庆400038 [2]第三军医大学检验系临床微生物及免疫学教研室,重庆400038

出　　处：《免疫学杂志》2005年第3期212-215,共4页Immunological Journal

基　　金：国家自然科学基金资助项目(30200249)

摘　　要：目的构建人毛乳头细胞HSPC016基因的原核表达载体,诱导其表达并对目的蛋白基本生物学特性进行鉴定。方法采用PCR技术从pcDNA3.1(+)/HSPC016中扩增出195bp目的基因,克隆至pMD18-T载体,双酶切回收纯化目的片段后连接至同样酶切回收的表达载体pET-28a(+)质粒中。重组质粒经酶切及测序鉴定后转化工程菌E.coliBL21,IPTG诱导表达,SDS-PAGE检测,UVP图像扫描分析并对目的蛋白进行N-端氨基酸测序鉴定。结果采用PCR技术成功地从pcDNA3.1(+)/HSPC016中扩增出195bp目的基因,序列分析显示HSPC016基因ORF由195bp组成并与GenBank公布序列完全一致。原核表达载体pET-28a(+)/HSPC016转化工程菌E.coliBL21并经诱导后,SDS-PAGE电泳鉴定:目的蛋白Mr约为7200,与理论预测值相吻合;UVP扫描表明目的蛋白表达率约20%。N-端氨基酸测序结果与预期序列完全一致。结论HSPC016基因能通过原核载体pET-28a(+)及工程菌E.coliBL21进行高效、正确地表达,为研究HSPC016基因表达的生物学意义奠定了基础。Objective To express the HSPC016 gene, and study the basic biological properties/characteristics of the HSPC016 gene expression products. Methods The HSPC016 gene was amplified from the plasmid of pcDNA3.1 (+)/HSPC016 by polymerase chain reaction, then subcloned into the plasmid pMD-18T and digested by two restriction enzymes of Nco Ⅰ and Hin dⅢ. After purification, the HSPC016 gene was inserted into the prokaryotic expression vector pET-28a (+), which also digested by Nco Ⅰ and Hin dⅢ. Then, the pET-28a (+) /HSPC016 was transformed into E.coli BL21 and the recombinant protein was expressed after induction by isopropyl-1-thio-β-D-galactoside. The N-terminal amino acid residual of the protein was sequenced after SDS-PAGE analysis. Results The HSPC016 gene was cloned into the pET-28a (+) successfully. The results of SDS-PAGE showed that the relative molecular weight of the expressed product was 7 200, and the expression rate was about 15%. The result of the N-terminal amino acid residual sequencing was identical to that of the molecular design. Conclusion The results suggest that HSPC016 gene is constructed successfully and expressed well in this prokaryotic expression system.

关 键 词：毛乳头细胞 HSPC016基因 原核表达 

分 类 号：R346[医药卫生—基础医学]