单核细胞增多症李斯特菌InlC基因缺失株的构建与鉴定

Construction and Identification of InlC-Deleted Strains of Listeria monocytogenes

为探讨单核细胞增生性李斯特菌(Lm)InlC基因在Lm致病性中的作用,本研究对其进行缺失。以单核细胞增多症李斯特菌4b血清型临床分离株LM-90SB2DNA为模板,扩增出用于缺失InlC基因的上、下游同源臂。运用SOE-PCR技术,将上、下游同源臂融合扩增得到△InlC基因缺失片段。将△InlC基因缺失片段与自杀性质粒p KSV-7连接,构建p KSV7-△InlC质粒。电转p KSV7-△InlC于LM-90SB2感受态细胞中,经过10μg/m L氯霉素和41℃下连续传代15代,获得单交换重组株,然后在41℃氯霉素中传至第108代,获得双交换重组株,菌液用旁侧引物进行PCR鉴定,然后30℃无氯霉素条件下连续传代培养30代丢失自杀性质粒并检测其遗传稳定性。结果显示:获得LM90SB2-△InlC缺失突变株,旁侧引物扩增只有1条1480 bp大小的片段,同时,对缺失片段进行测序验证,结果表明成功对InlC基因进行了缺失,并且缺失株遗传稳定。LM90SB2-△InlC的构建为进一步研究InlC基因在LM毒力中的作用奠定了基础。

To determi ne the effect of InlC gene on Listeria monocytogenes（Lm） pathogenisis.The upstream and downstream homology arms of InlC gene were amplified from 4b LM-90SB2.The two homology arms was connected together to get the△InlC fragment through SOE-PCR.The △InlC fragment was inserted into the suicide plasmid p KSV7 to construct the recombinant suicide vector p KSV7-△InlC.The recombinant vector was transformed by electroporation into the competent cells of the Lm-90SB2. The positive transformants were subcultured for 15 generations in Chloramphenicol（10 μg/m L） at 41 ℃,to get the single-exchange strain.Then the strain was continued to be passaged to the 108 th generation in the absence of Chloramphenicol at 41 ℃ to get the double-exchange strain,and the detection primers were used to identify for the mutant.In order to lost plasmid and identify genetic stability of LM90SB2- △InlC,subcultured was stilled for 30 generations without chloramphenicol at 30 ℃. LM90SB2-△InlC was constructed.The detection primers were used to detect for the mutant which can get only one target fragment of 1480 bp by PCR,meanwhile,the sequence technology successfully verified the mutant.At the same time, the mutant had genetic stability.The successful mutant construction provided the necessary experimental materials for the function analysis of InlC.