绿荧光蛋白基因在猪霍乱沙门氏菌C500株asd^-缺失株平衡致死载体系统中的表达  

GFP expression in asd^- mutant host-vector balanced lethal system of Salmonella choleraesuis C500 strain

作　　者：徐引弟[1] 郭爱珍[1] 刘维红[1] 贾爱卿[1] 陈焕春[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室湖北省预防兽医学重点实验室,湖北武汉430070

出　　处：《中国兽医学报》2007年第4期493-496,502,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30571386);湖北省科技攻关计划资助项目(2006AA205A02);中国教育部留学回国人员科研基金资助项目

摘　　要：猪霍乱沙门氏菌C500株是用化学方法致弱用于预防仔猪副伤寒病的弱毒疫苗株,毒力弱且免疫原性良好。为将猪霍乱沙门氏菌开发为适于粘膜免疫的疫苗活载体并保持原有免疫原性,构建了以C500为亲本菌,用负向选择的重组自杀性质粒介导接合转移的方法构建了无抗性标记的asd-缺失株。首先构建asd(天冬氨酸β-半乳糖脱氢酶)基因缺失1408bp的带有蔗糖敏感基因sacB的重组自杀性质粒,与C500接合转移,两步法筛选无抗性的asd-缺失株,通过PCR鉴定。该缺失株在无外源DAP(二氨基庚二酸)条件下溶菌死亡,它的生长必需外源DAP。PCR扩增绿荧光蛋白突变体4基因,克隆到带有asd基因的原核表达载体pYA3493中,电转化C500asd-缺失株,转化子在LB中生长,并激发强的绿荧光。上述结果表明,该asd-缺失株的构建是成功的,并且可以用来作为宿主载体平衡致死系统来高效表达外源基因,为开发C500为载体的口服多价疫苗奠定了基础。Salmonella choleraesuis C500 strain was an attenuated vaccine strain by chemical methad and used to prevent the piglet paratyphoid. It has feeble virulence and good immunogenicity. In order to exploit C500 as a vector for oral immunization and keep its good immunogenicity,unmarked asd^- deleted mutant was constructed by transfer conjugation with recombination suicide plasmid. First,the recombination suicide vector with sacB gene and 1 408-bp-deleted asd （β-aspartic semialdehyde dehydrogenase） gene was developed and conjugated with C500. The unmarked asd deleted strain was selected by two-step method and identified by PCR. The growth of asd deleted mutant needs DAP （diaminopimelic acid） in the medium or it would die. The GFP （Green fluorescence protein mutant 4） gene was amplified by PCR and cloned into prokaryotic expression plasmid pYA3493 with asd gene and electroporated into asd-C500. The transfer of asd^- C500 with pYAGFP grew in LB and aroused great green fluorescence. In above all,the construction of asd deleted mutant was successful and can be used as a host-vector balanced lethal system to express foreign genes efficiently. This also established a foundation to exploit C500 as an oral multivalent vaccine.

关 键 词：猪霍乱沙门氏茵C500株 asd^-缺失株 绿荧光蛋白表达 

分 类 号：S852.61[农业科学—基础兽医学]