人PTP1B基因cDNA全长的克隆和原核系统表达

目的构建人蛋白酪氨酸磷酸酶1B(PTP1B)基因cD-NA全长的原核表达质粒(pET-28a(+)-hPTP1B)并在大肠杆菌中高效表达。方法取2型糖尿病人(BMI>28kg.m-2)的淋巴细胞提取总RNA,RT-PCR后的扩增产物回收构建克隆载体pMD-hPTP1B,测序后设计带酶切位点BamHⅠ和EcoRⅠ的引物,PCR后酶切连入pET-28a(+)中,转化DE3,IPTG诱导表达,SDS-PAGE后用Westernblot检测其特异表达,并进一步对rhPTP1B诱导表达时IPTG的浓度、时间和温度等条件进行了优化。结果测序证实所得的hPTP1BcDNA序列与其在GenBank中的序列一致,重组质粒pET-28a(+)-hPTP1B的双酶切结果与预期大小完全一致,IPTG诱导后高效表达的蛋白质是其不溶性的包涵体形式。IPTG诱导的最佳浓度是0.05mmol.L-1,时间为5h,温度为37℃。结论成功克隆了人PTP1B基因,构建了相应的原核表达载体并对原核体系诱导表达的条件进行了优化,使其能在DE3中高效表达,为筛选高特异的小分子抑制剂和制备相应的单克隆抗体打下坚实的基础。

Cloning and expression of human PTP1B cDNA in E.coli

Aim To construct the recombinant plasmid pET-28a(+)-hPTP1B with human protein tyrosine phosphatase 1B(PTP1B)full length cDNA sequence,and to express the rhPTP1B protein effectively in E.Coli.Methods Total RNA was isolated from the peripheral blood mononuclear cells of type2 diabetes.PTP1B were amplified by using two-step RT-PCR.The cloning plasmid pMD-hPTP1B was constructed and sequenced after retrieval of the amplification products,then PTP1B was subcloned with primers containing restriction endonucleases recognition sites of BamHⅠand EcoRⅠand ligated into pET-28a(+),transducted into DE3,induced with IPTG and expressed proteins were analyzed with SDS-PAGE and Western blot.Then induction conditions of high level expression were experimented.Results PTP1B was cloned and expressed in the form of insoluble inclusion body in E.Coli successfully,SDS-PAGE analysis showed the Mr was about 50 ku.Western blot result showed the highly expressed protein was real PTP1B.The best induction concentration of IPTG was 0.05 mmol·L-1,the best induction time was 5 h and the best induction temperature was 37℃.Conclusions The recombinant plasmid pET-28a(+)-hPTP1B was obtained,thus a basis was established for preparing recombinant human PTP1B monoclonal antibodies and screening highly specific small molecular inhibitors.