To construct the mutant of Salmonella typhimurium SL1344 (SL1344 △erp△cya), E. coli donor strain X7213{pRE △ctp) was conjugated with the recipient of SL1344 △cya. The SL1344 △crp△cya mutant was generated through the two-step method introduced by the transduction of recombiaant suicide plasmid which was determined by PCR and sequence analysis. The phenotype analysis of the SL1344△crp△cya mutant showed that the serotype of SL1344 △crp△cya mutant was ideaticat to the parent SL1344 △cya and tie SL1344 wild strain. The mutant was stable with the recombinant △crp gene in vitro. The carbohydrate fermentation or utilization assays of the mutant were identical with the parent SL1344 △cya, but obviously different from the SL1344 wild strain. However, the growth velocity of the mutant was much slow. The chicken lethal test showed that the virulence of the SL1344△crp△cya motent strain was 106 times lower than the SL1344 strain. In conclusion, the SL1344 △crp △cya mutant was constructed successfully, and it suggests the △crp△cya mutant was potential to be developed as attenuated Salmonella vaccine.%为研制更加安全并保持良好免疫原性的弱毒株鼠伤寒沙门氏菌(S.typhimurium)及疫苗活载体,本研究通过自杀性质粒介导的细菌同源重组技术,将已缺失cya基因的SL1344株进一步缺失crp基因,构建S.typhimurium SL1344△crp△cya双基因缺失突变株,并对其生物学特性进行初步研究.双基因缺失株的构建是以含有重组自杀性质粒pRE△crp的大肠杆菌X7213为供体菌,SL1344△cya为受体菌进行接合转移,两步法筛选crp/cya基因双缺失突变株,并通过PCR和测序鉴定.对双缺失菌株的生物学特性鉴定表明,SL1344△crp△cya 血清型与亲本株SL1344△cya及野生株SL1344保持一致,并且能够稳定遗传缺失后的crp基因；其生化特性与亲本株基本相近,但与野毒株相比发生明显变化,生长速度也更为缓慢；对雏鸡致病性测定试验显示,双缺失株毒力比SL1344至少降低了约106倍.实验结果表明,S.typhimurium SL1344△crp△cya缺失株具有良好的遗传稳定性,毒力明显降低,为深入研究以S.typhimurium为载体的口服疫苗奠定了基础.
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