摘要
目的通过重叠延伸PCR法,定点突变微生物产谷氨酰胺转氨酶(Microbial transglutaminase,MTG)基因。方法根据GenBank中登录的Streptomyces sp.H197基因中MTG序列及重叠延伸PCR定点突变技术的原理,利用DNA-MAN5.0软件设计引物,以提取的Streptomyces sp.H197基因组DNA为模板,PCR扩增MTG基因,以扩增的MTG基因片段为模板,采用重叠延伸PCR技术对一个位点进行定点突变,胶回收PCR产物,与克隆载体pMD19-T连接后,转化感受态E.coli DH5α,提取质粒,经PCR及单、双酶切鉴定,并测序。结果重叠延伸PCR产物可见含有酶切位点和突变位点的特异性片段;重组质粒pMD19-T-MTG经PCR及单、双酶切鉴定,证明构建正确;测序结果表明,与野生型序列比对,仅有一个碱基发生改变,即第773位腺嘌呤突变为胞嘧啶,突变位点与预期一致,实现了目标位点的定点突变。结论重叠延伸PCR法对MTG基因序列预定位点突变成功,证明阳性克隆子含突变位点,为下阶段突变型功能表达奠定基础。
Objective To investigate the site-directed mutagenesis of microbial transglutaminase（MTG）by overlap extenision PCR.Methods Primers were designed by using DNAMAN5.0 software based on the MTG sequence of Streptomyces sp.H197 in GenBank and the principle of overlap extension PCR-based site-directed mutagenesis,with which MTG gene was amplified by PCR and used as a template for site-directed mutagenesis by overlap extension PCR. PCR product was recovered and inserted into vector pMD-19T,and the constructed recombinant plasmid pMD-19T-MTG was transformed to E.coli DH5α for identification by PCR,restriction analysis and sequencing.Results A specific gene fragment containing restriction and mutation sites was observed on electrophoretic profile of overlap extension PCR product.PCR and restriction analysis proved that recombinant plasmid pMD19-T-MTG was constructed correctly.Sequencing result showed mutation at only one site（A→G at site 773）as compared with the sequence of wild type MTG,which was consistent with the expected result.Conclusion The site-directed mutagenesis of MTG gene was successfully achieved by overlap extension PCR,and the positive clones contained mutation site,which laid a foundation of study on function of mutant.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第5期670-674,共5页
Chinese Journal of Biologicals
基金
湖北省自然科学基金资助项目(2010CDZ046)
关键词
谷氨酰胺转氨酶
重叠延伸聚合酶链反应
点突变
Microbial transglutaminase（MTG）
Overlap extension polymerase chain reaction
Site-directed mutagenesis
