MCL-1原核表达载体构建及重组蛋白表达  

Preparation of Prokaryotic Expression Construct of Bufo japonicus formosus MCL-1 and Its Recombinant Protein Expression

作　　者：余美华[1] 张姝芳[2] 倪晓敏[1] 袁进强[2] 胡巧玲[1] 万文彬[1] 杨仙玉[1] 

机构地区：[1]浙江农林大学动物科技学院,浙江临安311300 [2]浙江农林大学林业与生物技术学院,浙江临安311300

出　　处：《安徽农业科学》2013年第18期7742-7744,7977,共4页Journal of Anhui Agricultural Sciences

基　　金：国家自然基金项目(31071181);2011年度浙江省科技创新活动计划项目(2011R412042)

摘　　要：[目的]构建日本蟾蜍(Bufo japonicus formosus)髓细胞白血病基因-1(myeloid cell leukemia-1,mcl-1)的原核表达载体并诱导其重组蛋白表达。[方法]通过PCR方法扩增日本蟾蜍皮肤mcl-1开放阅读框,将其插入到原核表达载体pET-28b中。经菌落PCR、DNA限制性内切酶消化筛选阳性克隆,并通过DNA测序确定原核表达载体pET-28b-mcl-1构建成功。将pET-28b-mcl-1转入大肠杆菌(E.coli)感受态细胞BL21(DE3)中,用IPTG诱导表达,并用SDS-PAGE对重组蛋白的表达进行检测。[结果]原核表达载体pET-28b-mcl-1构建成功;重组蛋白在大肠杆菌中成功表达。[结论]原核表达载体的构建及重组蛋白的表达,为后续MCL-1的纯化、抗血清的制备及生理功能检测奠定了基础。[Objective] Preparation of prokaryotic expression construct of Bufo japonicus formosus mcl-1 and the induction of its recombinant protein expression.[Method] The open reading frame（ORF） of B.japonicusformosus skin mcl-1 was amplified by polymerase chain reaction （PCR） and inserted into expression vector pET-28b,and the positive clones were screened by colony PCR and enzyme digestion,which were finally confirmed by DNA sequencing.pET-28b-mcl-1 was transformed into E.coli competent cell BL21 （DE3）,and the recombinant protein was induced by isopropylβ-D-1-thiogalactopyranoside （IPTG） and its expression was detected by dodecyl sulfate,sodium salt （SDS）-polyacrylamide gel electrophoresis （SDS-PAGE）.[Result] The prokaryotic expression construct of pET-28b-mcl-1 was obtained and the expression of the recombinant protein of MCL-1 was confirmed by SDS-PAGE.[Conclusion] This study will become the basis for purifying the recombinant protein and preparation of its antiserum,and the further studies on the biological functions of MCL-1.

关 键 词：MCL-1 原核表达载体 重组蛋白表达 

分 类 号：S188[农业科学—农业基础科学]