Prokaryotic expression of human ubiquitous mitochondrial creatine kinase, preparation of its antiserum and clinical application / 第二军医大学学报

ABSTRACT

Objective:To clone the full-length cDNA of human ubiquitious mitochondrial creatine kinase(uMtCK) and express its protien in E. coli. The protein was used to immunize rabbit to prepare anti-uMTCR polyclonal antibody which was then used to detect the expression of uMtck in gastrointestinal carcinoma. Methods: A 1 062 bp segement was amplified from human HaLa cell by RT-PCR,which was then inserted into pMD18-T plasmid and proved to be the coding sequence of uMtCK by endonuclease digestion and sequencing. The coding sequence of uMtCK was inserted into pQE30 plasmid and recombinant plasmid pQE30-uMtCK was transformed into E. coli. The expressed protein was purified with a His. Bind column and was analyzed with SDS-PAGE and Westen blot and the activity of uMtCK was tested. An antiserum against uMtCK was perpetrated by immunizing rabbit with purified uMtCK. The expression of uMtCK in 59 patients with gastric and colonic cancer was detected by immunohistochemistry. Results:The coding sequence of uMtCK was successfully obtained by RT-PCR and the product was successfully inserted into pQE30 to construct a recombinant vector pQE30-uMtCK. The expression of uMtCK was effective and soluble, A good specific antiserum against uMtCK was proved by Western blot. The expression of uMtCK of gastric and colonic cancer was 76. 5%. Conclusion:The coding sequence of uMtCK is coined and expressed in E.coli. uMtCK is a prospective marker for the diagnosis of gastric and colonic cancer.