卡介苗阿拉伯糖苷转移酶embC基因敲除株的构建和初步鉴定

Construction and identification of Mycobacterium BCG strain with the mutant embC gene

目的采用基因敲除技术构建了卡介苗embC基因缺失株。方法从卡介苗基因组中扩增出embC基因,定向插入自杀质粒p2NIL中,切除embC基因中约1000 bp片段使其失活,再定向插入标记片段,筛选鉴定阳性克隆,电穿孔转入卡介苗,筛选重组菌株。结果PCR和酶切鉴定证明构建成功用于基因打靶的置换型自杀质粒,并筛选成功获得重组卡介苗。结论获得了卡介苗embC基因敲除株,为进一步研究对卡介苗免疫活性的影响奠定了基础。

Objective To construct the mutant embC gene of Mycobacterium BCG strain using gene knockout. Method The complete embC gene was amplified from BCG genome and inserted into the suicide plasmid p2NIL, then an inner fragment of about 1000 bp was cut down form the mutant of embC gene and the mark caste was engaged to constract the gene-target vector. The recombinant vector was transformed into Mycobacterium BCG strain by electroporation method. Result PCR and restriction endonuclease map revealed the successful construction of the suicide vector and the mutant strain. Conclusion The recombinant BCG strain which lost embC gene function was constructed, laying a foundation for further research on rBCG immune function.