猪传染性胸膜肺炎放线杆菌毒素apxII基因无药物抗性标记突变株HBC^-/ GFP^+的构建及其生物学特性研究  

Research on Construction and Biological Characteristics of Actinobacillus pleuropneumoniae apxIIC Mutant Strain Lacking Drug Resistance Marker

作　　者：贝为成[1] 何启盖[1] 方六荣[1] 肖少波[1] 刘丽娜[1] 洪文洲[1] 刘正飞[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物医学院动物病毒室,武汉430070

出　　处：《生物工程学报》2004年第5期719-724,共6页Chinese Journal of Biotechnology

基　　金：国家自然科学基金 (No.3 0 2 0 0 0 11);湖北省科技攻关项目 (No .2 0 0 1AA2 0 1B0 2 )基金资助~~

摘　　要：通过使用枯草芽胞杆菌一个蔗糖敏感sacB基因发展了一种依靠蔗糖的负向筛选系统 ,这种方法允许没有标记突变的基因进入胸膜肺炎放线杆菌染色体。首先 ,构建了猪传染性胸膜肺炎放线杆菌毒素apxII基因GFP插入失活型的重组质粒pOSAKCG ,其中一个表达盒含有氨苄青霉素基因和以外膜蛋白omlA作为启动子表达sacB基因。重组质粒pOSAKCG通过电穿孔转化 ,它的突变apxIICA基因与野生型亲本菌株胸膜肺炎放线杆菌HB0 3染色体上野生型apxIICA基因发生同源交换 ,两步法筛选获得了apxII基因突变株HBC- GFP+ ,PCR和Southernblot对突变株进行初步鉴定 ,进一步对突变株的一些生物学特性 ,包括它的溶血活性、免疫原性、生长特性及其对小鼠的安全性进行了研究。结果表明 ,无药物抗性标记突变株的构建是成功的。Using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows introduction of unmarked mutation into APP chromosome.Recombinant plasmid pOSAKCG was constructed by inserting GFP gene. There are a cassette containing the Ampicilin resistance determinant(Amp r ) and the sacB gene expressed from the APP omlA promoter in the recombinant plasmid. When the plasmid pOSAKCG was introduced into APP by electroporation,the APP apxII-deficient mutant was prepared by homologous recombinant between the mutant apxIICA gene in the recombinant plasmid and the wild-type apxIICA gene in the chromosome of the parent strain HB03 The mutant strain HBC - /GFP + was obtained by two selections and identified primary by PCR and southern blot. Some biological activities such as the hemolytic activity,growth rate,immunitical activity,the genetic stabily and the safety were more investigated.The results indicated that construction of the mutant strain lacking of drug resistance is successful,which can provide a strong basis for further researching vector and genetic engineering vaccine.

关 键 词：猪 传染性胸膜肺炎放线杆菌 毒素 apxⅡ基因 负向选择 突变株 疫苗 

分 类 号：S852.61[农业科学—基础兽医学]