利用分枝杆菌的重组工程系统将TAP标签敲入耻垢分枝杆菌的方法研究

Knocking TAP tag in the genome of Mycobacterium smegmatis mc2155 by the recombineering system in Mycobacterium

目的利用pJV53质粒编码的分枝杆菌重组工程系统将TAP标签敲入耻垢分枝杆菌基因组。方法将pJV53质粒转入耻垢分枝杆菌,使其表达重组蛋白,制备带有重组蛋白的感受态细胞;从质粒pBS1479中扩增出tap片段,从质粒pSMT3中扩增hyg片段,从耻垢分枝杆菌基因组中分别扩增出aasf基因及其5′端非编码区片段AASF5和aasf基因下游3′端非编码区片段AASF3,利用重叠PCR将以上4个片段拼接在一起,形成最终的敲入片段A5THA3;将构建好的敲入片段转入感受态细胞,使其重组入耻垢分枝杆菌基因组中,PCR和DNA测序鉴定敲入效果。结果重叠PCR技术得到全长为3 200 bp的敲入片段,PCR与DNA测序结果证实带有TAP标签的A5THA3片段已成功敲入耻垢分枝杆菌基因组中。结论成功将TAP标签敲入耻垢分枝杆菌基因组,为下一步进行目的基因功能研究奠定了基础。

In order to knock the TAP tag in the genome of Mycobacterium smegmatis （M. smegmatis） mc^2 155 using the pJV53 plasmid encoding recombineering system, pJV53 plasmid was transformed into M. smegmatis mc^2 155 to make the M. smegmatis mc^2 155 express recombinant proteins and prepare the electrocompetent cells. The tap gene was amplified from pBS1479 plasmid and the hyg gene was amplified from pSMT3 plasmid. The aasf （anti-anti-sigma factor）gene with AASF5 fragment （5′-noneoding region of aasf gene） and AASF3 fragment （3′-noncoding region of aasf gene） were amplified from the genome of M. smegmatis mc^2 155. These four fragments were spliced together to form the knock-in fragment ASTHA3 by overlap PCR. The constructed fragment was transformed into the electroeompetent cells to make it recombinant into the genome of M. smegmatis mc^2 155. The recombinant effect was verified by PCR and DNA sequencing. The knock-in fragment of 3200bp was gotten by overlap PCR. It was confirmed by PCR and DNA sequencing that the A5THA3 fragment with TAP tag was knocked into the genome of M. smegmatis mc^2 155 correctly. The TAP tag was successfully knocked into the genome of M. smegmatis mc^2 155 and paved the way for further study of the functions of some target genes.