MCL-1原核表达载体构建及重组蛋白表达

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。

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

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.