人脑蛋白酪氨酸磷酸酶SHP-1基因克隆与原核表达  

Cloning and Prokaryotic Expression of Protein Tyrosine Phosphatase SHP-1 from Human Brain

作　　者：李晓婷[1,2] 谢欣梅[1] 赵清辉[1] 庞晓斌[1] 杜冠华[2] 

机构地区：[1]河南大学药学院,河南开封475004 [2]中国医学科学院北京协和医学院药物研究所国家药物筛选中心,北京100050

出　　处：《河南大学学报（自然科学版）》2013年第4期435-438,共4页Journal of Henan University:Natural Science

基　　金：国家自然科学基金资助(No.81273652)

摘　　要：从人脑组织中提取mRNA,通过RT-PCR扩增出目标cDNA,构建蛋白酪氨酸磷酸酶SHP-1-pEASY-E1重组质粒.将重组质粒转化进E.coli TOP10感受态细胞中,筛选阳性克隆进行验证,将测序正确的质粒转化到E.coli Transetta感受态细胞中表达SHP-1.进一步对SHP-1表达的诱导温度和IPTG浓度等条件进行优化.结果显示,所克隆的SHP-1基因片断经PCR鉴定和测序分析,与目标基因完全相符.通过蛋白表达条件的优化,发现20℃,0.4mmol/L IPTG对表达菌株进行诱导时,SHP-1可溶性蛋白表达量最大.With human brain mRNA as template,SHP-1 gene fragments were obtained by RT-PCR.The recombinant prokaryotic expression vector SHP-1-pEASY-E1 was constructed and transformed into E.coli TOP10.Selecting positive clonings and sequencing were conducted and recombinant plasmids were transformed into E.coli Transetta to express the SHP-1 protein.Optimizing the expressive condition of SHP-1 protein,the results showed that PCR screen and sequencing confirmed that the cDNA sequence of SHP-1 had been correctly inserted into the plasmid pEASY-E1.The optimal induction concentration of IPTG was 0.4 mmol/L,the best temperature for induction was 20 ℃.

关 键 词：SHP-1 基因克隆 蛋白表达 

分 类 号：Q986[生物学—人类学]