人线粒体肌酸激酶广泛型亚型在大肠埃希菌中的表达及抗体制备

目的　获得人线粒体肌酸激酶广泛型亚型 (uMtCK)的全长基因 ,在大肠埃希菌中表达 ,研制抗uMtCK多克隆抗体。方法　采用逆转录PCR从培养的肿瘤细胞 (Hela细胞 )中成功扩增出 10 6 2bp的uMtCK的基因片段 ,插入到质粒pMD18 T中 ,经酶切和测序证实为uMtCK的基因编码序列 ,再插入到质粒pQE30 ,转化大肠埃希菌 ,诱导重组质粒pQE30 uMtCK表达酶融合蛋白。经亲和层析纯化 ,并进行SDS PAGE、westernblot鉴定及酶活性测定。纯化的uMtCK蛋白免疫家兔制备抗uMtCK多抗。结果　RT PCR扩增出的片段经酶切鉴定和测序证实为uMtCK的基因 ;在大肠埃希菌M15中实现了高效、可溶性的融合表达 ,表达量约 17%。重组uMtCK具有CK样催化活性 ,表明原核表达的uMtCK具有类似于天然蛋白质的生物学活性。经免疫印迹分析 ,制备的抗uMtCK多抗适合作uMtCK的进一步分析之用。结论　人uMtCK在大肠埃希菌中实现了高效表达 ,制备了针对uMtCK的抗体。

Expression of human ubiquitous mitochondrial creatine kinase in Escherichia coli and preparation of antiserum

Objective To clone the whole gene of human ubiquitous mitochondrial creatine kinase(uMtCK),express uMtCK in E.coli and prepare antiserum against uMtCK.Methods The sequence of 1062bp amplified by RT-PCR from human Hale cell was inserted into pMD18-T plasmid and proved to be the coding sequence of uMtCK by enzyme cutting and sequencing.The coding sequence of uMtCK was inserted into pQE30 plasmid and recombinant plasmid pQE30-uMtCK was transformed into E.coli.The expressing protein was purified with a His.Bind column and was analyzed by SDS-PAGE and Western blot.The activity of uMtCK was tested.The antiserum againstuMtCK was prepered by immunizing rabbit with purified uMtCK.Result The coding sequence of uMtCK was obtained successfully by RT-PCR from the cDNA of Hela cell and then successfully inserted into pQE30 to construct a recombinant expressive vector pQE30-uMtCK.The expression of uMtCK was effective and soluble, and held up to 17% of the total protein of E.coli M15.The biological activity of fusion protein covered some characteristics of native protein of uMtCK. The specificity of the antiserum to uMtCK was proved by analysis of Western blot.Conclusion The recombinant protein of uMtCK was expressed and purified.The antibody against uMtCK was prepared.