旋毛虫与人骨肉瘤细胞MG-63相关抗原基因TCTP的原核表达与鉴定

Prokaryotic expression and identification of TCTP,a Trichinella spiralis antigen and gene in MG-63 human osteosarcoma cells

目的对旋毛虫(Trichinella spiralis)与人骨肉瘤细胞MG-63相关抗原基因TCTP进行原核表达,对表达产物进行鉴定及反应原性分析。方法利用抗MG-63细胞全蛋白抗血清筛选旋毛虫肌幼虫cDNA表达文库得到旋毛虫TCTP基因。PCR扩增TCTP基因,连接至pET-32a(+)载体,构建重组质粒pET-32a(+)-TCTP,转化入感受态细胞BL21(DE3)后用IPTG诱导表达,利用His标签镍离子蛋白纯化柱对表达产物进行纯化,采用SDS-PAGE和Western blot鉴定重组蛋白的表达及反应原性。结果经酶切鉴定和测序鉴定,重组质粒pET-32a(+)-TCTP构建正确。SDS-PAGE检测重组蛋白TCTP在原核表达系统中主要以可溶性形式表达,融合蛋白的相对分子质量约为43×10^(3)。Wetern blot检测重组蛋白TCTP可被抗MG-63细胞多抗血清识别。结论成功构建了重组质粒pET-32a(+)-TCTP,表达的重组蛋白TCTP具有反应原性,为该蛋白生物学功能的研究奠定了基础。

Objective To create a system for prokaryotic expression of TCTP,a Trichinella spiralis antigen and a gene in MG-63 human osteosarcoma cells,and to identify the expressed product and analyze its reactogenicity.Methods A cDNA expression library of T.spiralis muscle larvae was screened with anti-MG-63 cell antiserum to obtain the gene TCTP.The TCTP gene was amplified using PCR and ligated to a pET-32 a(+)vector to construct the recombinant plasmid pET-32 a(+)-TCTP.The recombinant plasmid pET-32 a(+)-TCTP was transformed into competent BL21(DE3)cells,and its expression was induced with 1 mol/L IPTG.The expressed product was purified on a His-tag nickel ion protein affinity column.12%SDS-PAGE and Western blotting were used to identify the protein and analyze its reactogenicity.Results 1%agarose gel electrophoresis and sequencing indicated that the T.spiralis TCTP gene was successfully amplified.Restriction enzyme identification and sequencing verified that the recombinant plasmid pET-32 a(+)-TCTP was successfully constructed.SDS-PAGE indicated that the recombinant protein TCTP was mainly expressed in a soluble form.The molecular weight of the fusion protein TCTP was about 43×10^(3).Western blotting indicated that the purified recombinant protein TCTP produces a single band and is recognized by an anti-MG-63 cell polyclonal antibody.Conclusion The recombinant plasmid pET-32 a(+)-TCTP was successfully constructed,and the recombinant protein TCTP was expressed and purified.The recombinant protein has reactogenicity,laying the foundation for the study of the biological function of this protein.