CAJ | 학술논문

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。
목적：이용재체-숙주평형치사계통，장1주향항분리적야생형사문씨균（S129）구건위감독핵산역묘재체。방법이세균생화반응화혈청학방법감정S129위서상한사문씨균；이λ-RED동원중조방법파향고제S129주적 asdA기인，병이잡나매소항성기인대체；통과균락PCR감정후도취양성극륭 asdA결함형감독서상한사문씨균（asdAΔS129），재함2，6-이안기경이산（2，6-diaminopimelic acid ，DAP）혹무DAP적LB배양기중배양，여야생주S129대비생장곡선이험증 asdAΔS129구건적성공；이S129화 asdAΔS129공격BALB/c소서험증 asdAΔS129감독정황；결과균락PCR감정득도양성극륭，asdAΔS129필수외원성첨가DAP방능생장；asdAΔS129불능치사BALB/c소서，득도성공적감독；결론성공장1주야생형사문씨균구건성 as d A결함형감독핵산역묘재체，병재체외화체내실험중험증，위하일보적역묘정체화종류치료실험제공료합괄적재체。
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 .