猪霍乱沙门氏菌C78-1株ΔcrpΔasd缺失株平衡致死载体系统的构建及生物学特性研究

Construction and characterization of ΔcrpΔasdmutant balanced-lethal host-vector system of Salmonella choleraesuis C78-1 strain

为开发减毒猪霍乱沙门氏菌的活疫苗载体,通过自杀性质粒介导的细菌同源重组技术,构建了猪霍乱沙门氏菌C78-1株ΔcrpΔasd双基因缺失株,并对其生物学特性进行探讨。首先构建含有缺失1 488bp asd基因的重组自杀性质粒pREΔasd,然后与已构建完成的ΔcrpC78-1缺失株进行接合转移,采用两步法筛选无抗性的C78-1的ΔcrpΔasd双缺失株。PCR鉴定结果表明,C78-1的ΔcrpΔasd双缺失株构建成功;进一步研究表明,该缺失株的生长需要外源的二氨基庚二酸(DAP),且能稳定遗传缺失的asd基因,与C78-1相比,其血清型未发生变化,但其生长速度明显减慢。ΔcrpΔasd C78-1双缺失株可接受asd+质粒作为宿主平衡致死系统高效稳定地表达外源基因,为开发以C78-1为载体的口服多价疫苗奠定了基础。

To develop a live vaccine vector of Salmonella choleraesuis,Δcrp Δasd double deletion mutant was constructed by the allelic exchange of the suicide plasmid and the biological characteristics of the mutant were also determined.Firstly,the recombination suicide vector pREΔasd with 1 488 bp-deleted asd gene was constructed and conjugated with ΔcrpC78-1 which had been successfully constructed,then the Δcrp Δasd double deletion mutant was selected by two-step method and the successfully result was identified with PCR.The further study indicated that foreign DAP must be supplied for the Δcrp Δasd mutant to grow,in addition,the asd gene was transmitted stably.Compared with C78-1 strain,the O and H antigens was identical to C78-1 strain,but the growth velocity was reduced significantly.The ΔcrpΔasd mutant can accept asd＋ plasmid to construct host-vector balance lethal system,and this system can be used to express foreign gene efficiently and to develop potential oral multivalent vaccines.