摘要
目的:在大肠杆菌中克隆表达人线粒体肌酸激酶广泛型亚(uMtCK)的蛋白,免疫家兔制备抗uMtCK的多克隆抗体,检测胃肠肿瘤组织中uMtCK的表达。方法:采用RT-PCR从培养的肿瘤细胞(HeLa细胞)中成功扩增出1062bp的uMtCK基因片段,构建重组质粒pQE30-uMtCK,转化大肠杆菌表达酶融合蛋白,对表达产物进行SDS-PAGE、Western印迹鉴定及酶活性测定。纯化的uMtCK蛋白免疫家兔制备了抗uMtCK多抗。免疫组化法检测59例胃及直肠肿瘤组织切片中uMtCK的表达。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;在大肠杆菌M15中实现了高效、可溶性的融合表达。免疫家兔制备的抗uMtCK多抗经Western免疫印迹分析适合作uMtCK的进一步分析。免疫组化检测临床病理标本结果显示,59例胃及直结肠肿瘤标本uMtCK的阳性率为76.5％。结论:成功克隆了人uMtCK的编码基因,并将其在大肠杆菌中成功表达;制备了抗uMtCK多抗,免疫组化检测临床病理标本的结果提示uMtCK有助于胃肠肿瘤的诊断。
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.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2004年第12期1320-1324,共5页
Academic Journal of Second Military Medical University
基金
上海市科委基金(034119939)
关键词
线粒体肌酸激酶
基因表达
胃肠肿瘤
ubiquitous mitochondrial creatine kinase
gene expression gastrointestinal neoplasms
