重组肺癌抑癌基因1蛋白的表达、纯化及其多克隆抗体的制备

目的表达、纯化重组人肺癌抑癌基因1(Tumorsuppressor in lung cancer1,TSLC1)蛋白,并制备其多克隆抗体。方法采用RT-PCR法扩增TSLC1基因全长编码区序列,克隆入原核表达质粒pQE30,转化大肠杆菌M15,IPTG诱导表达,表达的重组蛋白经Ni2+-NTA亲和层析纯化后,免疫家兔,ProteinA亲和层析纯化抗血清,并经Westernblot分析其反应原性。结果重组表达质粒pQE30-TSLC1经双酶切及测序鉴定证明构建正确。重组TSLC1蛋白的表达量约占菌体总蛋白的14%,主要以包涵体形式存在。纯化的重组蛋白纯度为93.4%,可与小鼠抗His-Tag单克隆抗体发生特异性反应。以其制备的多克隆抗体具有良好的抗原识别特异性。结论已成功制备TSLC1多克隆抗体,为深入研究TSLC1分子的生物学活性奠定了基础。

Expression and Purification of Recombinant Tumor Suppressor in Lung Cancer 1 Protein and Preparation of Its Polyclonal Antibody

Objective To express and purify tumor suppressor in lung cancer 1(TSLC1)protein and prepare its polyclonal antibody.Methods The full-length sequence of encoding region of TSLC1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pQE30,and the constructed recombinant plasmid pQE30-TSLC1 was transformed to E.coli M15 for expression under induction of IPTG.The expressed protein was purified by Ni2+-NTA affinity chromatography and used for the immunization of rabbits.The antisera were purified by Protein A affinity chromatography and analyzed for reactogenicity by Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmid pQE30-TSLC1 was constructed correctly.Recombinant TSLC1 protein mainly existed in a form of inclusion body and contained about 14% of total somatic protein.Purified TSLC1 protein reached a purity of 93.4% and showed specific reaction with mouse anti-His-Tag monoclonal antibody.The polyclonal antibody prepared with purifed TSLC1 protein showed high specificity in antigen recognition.Conclusions The polyclonal antibody against TSLC1 was successfully prepared,which laid a foundation of further study on the biological activity of TSLC1 molecules.