1株 asdA缺陷型减毒鼠伤寒沙门氏菌作为核酸疫苗载体的构建与鉴定

摘要

In this study ,a wild type Salmonella typhimurium (S .typhimurium) strain was isolated and identified in Hong Kong (S129) ,then the asdA gene was knocked out and replaced with kanamycin resistant gene in a Salmonella typhi-murium strain S129 using the λ RED-mediated recombination method .The constructed mutant asdAΔS129 was validated by culturing in the presence or absence of 2 ,6-diaminopimelic acid (DAP) growth in vitro and evaluating its virulence in BALB/c mice challenge assay .Therefore ,this study has demonstrated that an asdA mutant Salmonella typhimurium has been success-fully constructed .%目的：利用载体-宿主平衡致死系统，将1株香港分离的野生型沙门氏菌（S129）构建为减毒核酸疫苗载体。方法以细菌生化反应和血清学方法鉴定S129为鼠伤寒沙门氏菌；以λ-RED同源重组方法靶向敲除S129株的 asdA基因，并以卡那霉素抗性基因代替；通过菌落PCR鉴定后挑取阳性克隆 asdA缺陷型减毒鼠伤寒沙门氏菌（asdAΔS129），在含2，6-二氨基庚二酸（2，6-diaminopimelic acid ，DAP）或无DAP的LB培养基中培养，与野生株S129对比生长曲线以验证 asdAΔS129构建的成功；以S129和 asdAΔS129攻击BALB/c小鼠验证 asdAΔS129减毒情况；结果菌落PCR鉴定得到阳性克隆，asdAΔS129必需外源性添加DAP方能生长；asdAΔS129不能致死BALB/c小鼠，得到成功的减毒；结论成功将1株野生型沙门氏菌构建成 as d A缺陷型减毒核酸疫苗载体，并在体外和体内实验中验证，为下一步的疫苗呈递和肿瘤治疗实验提供了合适的载体。