利用groupⅡ intron构建产气荚膜梭菌突变体  

Construction of plc gene of Clostridium perfringens mutant by groupⅡ intron

作　　者：马臣杰[1] 宋孚洋[1] 高姗[2] 康琳[2] 王玉炯[1] 王景林[2] 

机构地区：[1]宁夏大学生命科学学院,银川750021 [2]军事医学科学院微生物流行病研究所,北京100071

出　　处：《军事医学》2012年第4期285-288,共4页Military Medical Sciences

基　　金：病原微生物生物安全国家重点实验室开放课题资助项目(PBS2009B-05)

摘　　要：目的通过插入失活α毒素基因获得产气荚膜梭菌(Clostridium perfringens)突变体。方法利用MobilegroupⅡintron的自我剪切作用,对本室保藏的1株野生型C.perfringens的α基因plc插入失活。通过菌落PCR检测,以及观察突变株在哥伦比亚血琼脂培养基和脑心浸液培养基(4%卵黄)平板上的形态变化,进行阳性筛选。蛋白质免疫印迹分析α毒素蛋白活性。结果 PCR检测到α毒素突变体的plc基因中,存在Mobile groupⅡintron片段,并且观察到该菌在哥伦比亚鲜血琼脂平板培养基上的α毒素溶血环消失,在脑心浸液培养基(4%卵黄)平板上无白晕。蛋白质免疫印迹分析表明,plc突变体不表达α毒素蛋白。结论成功构建C.perfringensα毒素突变体,为后期探索C.perfringens口服疫苗打下了重要基础。Objective To construct the gene of alpha toxin of Clostridium perfringens.Methods plc genes were knocked out without any further modifications by group Ⅱ intron.Results PCR results showed that the intron RNA had been inserted successfully in the plc genes of the transformants.The colonies did not have any white halo around them on BHI agar containing egg yolk,and their phenotype was a different form of wild type C.perfringens on Columbia blood agar.Western blotting demonstrated that the plc mutant did not produce alpha toxin protein anymore.Endospore formation and transformability were as stable as those of the wild type.Conclusion The alpha toxin mutant is stable,and can be used as a delivery vehicle to develop mucosal vaccines.

关 键 词：产气荚膜梭菌 Α毒素 细菌毒素类 口服疫苗 

分 类 号：R378.8[医药卫生—病原生物学] Q344[医药卫生—基础医学]