蛋白磷酸酶LY1基因原核表达载体的构建及蛋白的初步表达  

Construction of the prokaryotic expression vector and expression of protein phosphatase LY1 gene

作　　者：彭万仁[1] 陈兵[2] 倪芳[3] 鲁云霞[2] 郑红[3] 李菲菲[3] 汪思应[3] 

机构地区：[1]安徽医科大学第一附属医院肿瘤内科,安徽合肥230022 [2]安徽医科大学基础医学院生化教研室,安徽合肥230032 [3]安徽医科大学基础医学院病理生理教研室,安徽合肥230032

出　　处：《疾病控制杂志》2007年第5期456-458,共3页Chinese Journal of Disease Control and Prevention

基　　金：国家自然科学基金(30572206);安徽省自然科学基金(00044202;No01043709);安徽省教育厅自然科学基金(2005kj333zc);安徽省人才开发基金(2002Z035)

摘　　要：目的构建蛋白磷酸酶LY1基因的原核表达载体,并在E.coliBL21(DE3)中表达。方法PCR扩增LY1的CDs基因片段,并将其克隆入原核表达载体pET28a(+)中构建重组质粒pET28a(+)-LY1。经限制性内切酶Nhe I、XhoⅠ双酶切鉴定及序列测定后,转化E.coliBL21(DE3),经IPTG诱导表达组氨酸融合蛋白。SDS-PAGE、Western blot方法检测重组蛋白的表达。结果获得全长为1179bp的LY1基因片段,以构建的重组质粒pET28a(+)-LY1转化E.coliBL21(DE3)后,经IPTG诱导,表达出分子量约为42kD的重组蛋白。SDS-PAGE分析显示,表达的蛋白主要以不溶性包涵体的形式存在于E.coliBL21(DE3)的胞质中。Western blot方法检测出LY1融合蛋白的表达。结论成功构建了原核表达载体pET28a(+)-LY1,并表达出重组LY1蛋白,为进一步单克隆抗体的制备和生物信号转导的相关研究奠定了实验基础。　Objective To construct a prokaryotic expression vector for LY1 protein phosphatase gene,and to express the gene in E.coli BL21（DE3）.Methods LY1 gene fragment was amplified by PCR and cloned into the pET28a（＋）vector.The recombinant vector was identified by restriction endonuclease digestion analysis and DNA sequencing,and then transformed into E.coli BL21（DE3）by IPTG induction to express the target protein.The recombinant protein was detected by SDS-PAGE and Westernblot.Results A 1 179 bp of LY1 gene fragment was obtained.After recombinant vector pET28a（＋）-LY1 was transformed into E.coli BL21（DE3）and induced by IPTG induction,the recombinant protein with MW about 42 kD was obtained.SDS-PAGE analysis showed that the expressed product was mainly in inclusion bodies.Conclusions Recombinant expression vector pET28a（＋）-LY1 is constructed successfully.The expressed LY1 protein will be helpful to our further research.

关 键 词：基因 蛋白 基因表达 

分 类 号：R349.64[医药卫生—基础医学]