重组人心肌肌红蛋白基因工程菌的构建  

Construction of gene engineering bacteria of recombinant human myocardial myoglobin

作　　者：张欣[1] 张春明[1] 王德芝[1] 

机构地区：[1]中国医学科学院放射医学研究所,天津300192

出　　处：《生物医学工程与临床》2008年第1期75-78,共4页Biomedical Engineering and Clinical Medicine

基　　金：天津市科技攻关培育项目(043102211)

摘　　要：目的构建稳定、高效表达重组人心肌肌红蛋白(rhMb)的基因工程菌株,为临床检测早期心肌损伤及预后提供诊断试剂,促进肌红蛋白诊断标准化的研究。方法采用RT-PCR技术从人心肌组织总RNA中克隆出全长的人心肌肌红蛋白基因,将其插入到PMD18-Tsimple克隆载体中,转化DH5α克隆菌株。经酶切和测序对目的基因进行分析,再插入到pET-21a(+)表达载体中,转化BL21(DE3)。对诱导表达的目的蛋白行SDS-PAGE和运用锐普心肌梗死仪检测重组蛋白的抗原性并准确定量。结果测序表明RT-PCR所得的肌红蛋白基因hMb序列与GenBank(NM005368.2)中报道的序列一致,目的蛋白表达量达菌体总蛋白的15%,重组蛋白抗原性良好。结论成功构建了稳定、高效表达重组人心肌肌红蛋白基因工程菌株。Objective To construct stable,high efficiency expressing gene engineering bacteria of recombinant human myocardial myoglobin, and provide diagnostic reagent for detection and prognosis of early myocardial damage. Methods The full-length human myocardial myoglobin （hMb） gene was amplified from the human cardiac total RNA by RT-PCR and inserted into clone vector pMD-18T simple for cloning in E.coli DH5α By sequencing and enzyme-incising analysis of target gene,then inserted into expression vector pET-21 a（＋） to form an expression plasmid and transformed it into E.coli BL21 （DE3）.The antigenicity and accurate amount of the recombinant protein was detected by SDS-PAGE and RAMP clinical Reader. Results The sequence of amplified hMb gene was consistent with that reported in GenBank （NM 005368.2）.The expressed product contained 15 % of total somatic protein. Conclusion The stable and high efficiency expressing gene engineering bacteria of recombinant human myoglobin was successfully constructed.

关 键 词：重组 心肌肌红蛋白 基因工程菌 

分 类 号：R346[医药卫生—基础医学]