【摘要】 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asdA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。更多还原

【Abstract】 In this study,a wild type Salmonella typhimurium(S.typhimurium)strain was isolated and identified in Hong Kong(S129),then the asdAgene was knocked out and replaced with kanamycin resistant gene in a Salmonella typhimuriumstrain S129 using theλRED-mediated recombination method.The constructed mutant asdAΔS129was 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 asdAmutant Salmonella typhimuriumhas been successfully constructed.更多还原