鼠伤寒沙门菌SL1344株cAMP受体蛋白基因缺失株的构建及其生物学特性

Construction and characterization of the cAMP receptor protein gene deletion mutant of Salmonella typhimurium SL1344 strain

通过自杀性质粒介导的细菌同源重组技术,构建鼠伤寒沙门菌SL1344株的crp基因缺失疫苗候选菌株,并对其生物学特性进行初步研究。首先构建含缺失321bp crp基因的重组自杀性质粒pREΔcrp,然后利用重组自杀性质粒介导的等位基因交换技术,两步法筛选SL1344的Δcrp缺失株,用PCR鉴定结果表明该缺失株构建成功。生物学特性研究发现,缺失株ΔcrpSL1344保留了亲本菌株SL1344的血清型1,4,5,12:i:1,2,且能够稳定遗传缺失321bp的crp基因;但是,与亲本株SL1344相比,其生化特性发生明显改变,生长速度明显减慢。缺失株ΔcrpSL1344口服感染1日龄雏鸡的LD50为7.40×109 CFU,毒力较亲本株SL1344降低32 456倍。以上研究结果表明,SL1344株crp基因缺失株构建成功,且遗传稳定,毒力明显降低,为进一步将其开发为鼠伤寒沙门菌弱毒疫苗候选菌株奠定基础。

The cAMP receptor protein gene（crp） deletion mutant of Salmonella typhimurium SL1344 strain was constructed by the allelic exchange introduced by the transduction of suicide plasmid.In addition,the biological characteristics of the mutant were determined.Firstly,the upstream and downstream fragments of crp gene were amplified from SL1344 strain genome.The two fragments were successively cloned into the suicide pRE112 vector to construct the recombinant suicide vector pREΔcrp harboring the 321 bp-deleted crp fragment.The recombination suicide vector was conjugated with SL1344 and the unmarked crp deleted strain without resistance was selected by two-step method and crp deletion on the genome was determined by PCR.The serotype of the mutant was 1,4,5,12：i：1,2,identical to the parent SL1344.The mutant was stable with the recombinant Δcrp gene in vitro.However,the carbohydrate fermentation or utilization assays of the mutant were differed from the parent SL1344 strain,obviously.The growth velocity of the mutant was more slowly compared with SL1344.The chicken lethal test showed that the virulence of the SL1344 Δcrp mutant strain with LD50 of 7.40×109 CFU was 32 456 times lower than the parent SL1344 strain with LD50 of 2.28×105 CFU.These results showed that the SL1344 Δcrp mutant was constructed successfully.It is likely that this Δcrp mutant could be adapted to develop attenuated Salmonella vaccine.