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目的利用pJV53质粒编码的分枝杆菌重组工程系统将串取亲和纯化(TAP)标签敲入耻垢分枝杆菌基因组。方法从质粒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;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 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。
OBJECTIVE: The Mycobacterium smegmatis genome was knocked-in with the affinity purification (TAP) tag using the pJV53 plasmid-encoded Mycobacterium recombination engineered system. Methods The fragment of tap was amplified from plasmid pBS1479. The hyg fragment was amplified from plasmid pSMT3. The aasf gene and its 5 ’untranslated region (AASF5) and the 3’ end of aasf gene were amplified from Mycobacterium smegmatis genome. End non-coding region fragment AASF3, the above four fragments were spliced together by overlap PCR to form the final knock-in fragment A5THA3; the pJV53 plasmid was transferred into Mycobacterium smegmatis to express the recombinant protein to prepare recombinant protein with recombinant protein The competent cells were transformed into competent cells, and then recombined into M. smegmatis genome. PCR and DNA sequencing were used to identify the knock-in effect. Results The results of PCR and DNA sequencing confirmed that the TAP-tagged A5THA3 fragment was successfully knocked into Mycobacterium smegmatis genome. Conclusion TAP tag was successfully knocked into Mycobacterium smegmatis genome, which laid the foundation for the further study of gene function.