猪霍乱沙门氏菌C78-1株Δcrp缺失株的构建及其生物学特性初步研究

Construction and Characterization of a Salmonella Choleraesuis C78-1 Δcrp Deletion Mutant

通过自杀性质粒介导的等位交换技术构建猪霍乱沙门氏菌C78-1株的crp基因缺失株,并对其生物学特性进行初步研究。首先以猪霍乱沙门氏菌C78-1基因组为模板进行PCR,扩增出crp基因上下游片段(1 048和1 743 bp),并分别将其克隆入自杀性质粒pRE112上,构建含缺失320 bpcrp基因的重组自杀性质粒pREΔcrp。运用重组自杀性质粒介导的等位交换技术,两步法筛选C78-1的Δcrp缺失株。进一步的生物学特性研究表明,缺失株的血清型与亲本菌株C78-1一致,且能够稳定遗传缺失的crp基因,但其生化特性和生长速度与C78-1相比发生明显改变,小鼠致死性试验结果表明其毒力较C78-1降低约750倍。以上结果均证实,作者成功构建了猪霍乱沙门氏菌C78-1株的crp基因缺失突变株,缺失株遗传稳定,毒力显著降低,为进一步开发猪霍乱沙门氏菌的弱毒疫苗菌株奠定了基础。

In this study,a Δcrp mutant of Salmonella Choleraesuis C78-1 was constructed by the allelic exchange introduced by the transduction of the pRE112 suicide plasmid and characteristics of the mutant were also determined.Firstly,the upstream and downstream fragements of crp gene were amplified from Salmonella Choleraesuis C78-1 genome and then successively subcloned into suicide plasmid pRE112 to construct the recombinant suicide vector pREΔcrp with 320 bp-deleted crp fragment.Then,the C78-1 Δcrp mutant was constructed through the two-step method introduced by the transduction of recombinant suicide plasmid.The O and H antigens of the mutant was 6,7：C：1,5,identical to the parent C78-1 strain.The mutant was stabile with the recombinant Δcrp gene in vitro.However,fermentation patterns and growth rate of the mutant were differed from that of the parent C78-1 strain,obviously.The mouse lethal test showed that the virulence of the mutant was 750 times lower than C78-1.These results showed that the C78-1 Δcrp mutant was constructed successfully.It is likely that this Δcrp mutant could be adapted to develop attenuated Salmonella vaccine.