猪霍乱沙门菌缺失株ΔcyaC78-1和ΔcrpΔcyaC78-1的构建及其生物学特性比较

Construction of Salmonella choleraesuis mutant ΔcyaC78-1 and ΔcrpΔcyaC78-1 and comparison of their biological characteristics

为开发更加安全有效且遗传背景清晰的减毒猪霍乱沙门菌活疫苗,运用自杀性质粒介导的等位交换技术缺失cya基因,分别构建猪霍乱沙门菌缺失株ΔcyaC78-1和ΔcrpΔcyaC78-1,并将两者的生物学特性与ΔcrpC78-1和疫苗株C500进行比较。以猪霍乱沙门菌C78-1为模板,分别扩增cya基因的上臂和下臂,然后通过中间转移载体pBluescriptⅡSK(+)克隆至自杀性质粒pRE112中,构建重组自杀性质粒pREΔcya,再与C78-1和ΔcrpC78-1分别进行接合转移,通过两步法筛选ΔcyaC78-1和ΔcrpΔcyaC78-1。PCR及测序结果表明,ΔcyaC78-1和ΔcrpΔcyaC78-1构建成功。生物学特性检测结果表明,所构建的ΔcyaC78-1和ΔcrpΔcyaC78-1的血清型和生化特性与ΔcrpC78-1保持一致,并能够稳定遗传Δcya基因;两者的生长速度明显慢于疫苗株C500,且ΔcrpΔcyaC78-1的生长速度最慢。小鼠毒力试验表明,ΔcyaC78-1的毒力高于ΔcrpC78-1约1.7倍,较疫苗菌株C500下降至58.2%,但ΔcrpΔcyaC78-1的毒力较ΔcrpC78-1下降至0.194%,较C500下降至0.065%。构建的猪霍乱沙门菌缺失株ΔcyaC78-1、ΔcrpΔcyaC78-1遗传稳定,背景清晰,毒力降低,尤其是ΔcrpΔcyaC78-1双缺失株的毒力较疫苗株C500降低显著,为筛选更加高效的减毒猪霍乱沙门菌疫苗和进一步开发适用于猪的口服活疫苗载体奠定了良好基础。

To develop safer and more effective live attenuated Salmonella choleraesuis vaccines,ΔcyaC78-1andΔcrpΔcyaC78-1were constructed by the method of suicide plasmid-mediated allelic exchange and their characteristics were also compared withΔcrpC78-1and vaccine strain C500.Firstly,the upstream and downstream fragments of cyagene were amplified fromS.choleraesuis C78-1genome and the two fragments were cloned into suicide plasmid pRE112by the general transfer vector pBluescriptⅡSK(+).Then,the recombinant suicide plasmid pREΔcya was constructed.Next ΔcyaC78-1 andΔcrpΔcyaC78-1were selected by the two-step method after being conjugated with C78-1andΔcrpC78-1,respectively.PCR and sequencing results showed thatΔcyaC78-1andΔcrpΔcyaC78-1were successfully constructed.Biological characteristics showed that the serotypes and biochemical characteristics ofΔcyaC78-1andΔcrpΔcyaC78-1were consistent withΔcrpC78-1and theΔcyagene was inherited stably.Both of their growth speeds were obviously slower than the vaccine strain C500,and the growth speed ofΔcrpΔcyaC78-1was the slowest among the four strains.The mice virulence test showed that the virulence ofΔcyaC78-1was 1.7times higher thanΔcrpC78-1and decreased to 58.2%compared with vaccine strain C500.Compared withΔcrpC78-1and C500,the virulence ofΔcrpΔcyaC78-1decreased to 0.194% and0.065%,respectively.The constructed strainsΔcyaC78-1andΔcrpΔcyaC78-1shared less virulence,high genetic stability and clear genetic background,especially the double mutantΔcrpΔcyaC78-1with significantly reduced virulence compared with the vaccine strain C500,which laid foundations for selection of more effective live attenuated S.Choleraesuis vaccines and further development as a live oral vaccine vector suitable for pigs.