猪传染性胸膜肺炎放线杆菌毒素apxH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

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HB03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Southern blot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。该突变株的构建为进一步研究突变株作为载体和疫苗奠定了坚实的基础。

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

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.