asdA缺陷型减毒鼠伤寒沙门氏菌载体协助外源蛋白在Caco-2细胞和鸡小肠细胞中的表达  被引量：1

作　　者：于斌[1] 罗语思[2,3] 张宝中[3] 彭晓[1] 袁硕峰[3] 牛憨笨[1] 屈军乐[1] 张科[3,4] 

机构地区：[1]深圳大学光电工程学院光电子器件与系统(教育部/广东省)重点实验室,广东深圳518060 [2]贵州医科大学附属医院急诊内科,贵州贵阳550004 [3]香港大学李嘉诚医学院微生物学系,中国香港999077 [4]贵州医科大学人体寄生虫学教研室,贵州贵阳550025

出　　处：《中国病原生物学杂志》2015年第12期1091-1096,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31200639)

摘　　要：目的测试1株asdA缺陷型鼠伤寒沙门氏菌作为潜在疫苗载体在体内及体外的外源蛋白传递能力。方法以含真核表达质粒pIRES-eGFP的asdAΔS129感染人结肠癌细胞Caco-2,在添加DAP的情况下检测其对绿色荧光蛋白表达的调节;以该株细菌感染15d龄幼鸡,检测鸡小肠绿色荧光蛋白的表达。结果成功构建asdAΔS129-pIRESeGFP,在感染后的Caco-2细胞中检测到绿色荧光蛋白表达,且与DAP用量呈量效关系;在感染后第3d和第4d的幼鸡小肠均检测到绿色荧光蛋白表达,且表达自十二指肠向结肠发展。结论 asdA缺陷型减毒株能传递外源蛋白基因,并证实有外源蛋白表达,为特异性疫苗呈递和肿瘤治疗研究提供了潜在候选载体。Objective The aim of this study was to evaluate the ability of an asdA mutant strain of Salmonella typhimurium（asdAΔS129）to act as a potential vaccine vector to deliver a heterologous antigen in vitro and in vivo. Methods pIRES-eGFP,a eukaryotic promoter-driven plasmid,was transformed into asdAΔS129to generate asdAΔS129-pIRESeGFP.After co-incubation of asdAΔS129-pIRES-eGFP and Caco-2cells in the presence or absence of 2,6-diaminopimelic acid（DAP）,the expression of eGFP was detected with Western blotting.asdAΔS129-pIRES-eGFP was also inoculated into 15day-old chicks via oral lavage,and the expression of eGFP in intestinal mucosa samples was evaluated with Western blotting on day 3and day 4post-infection.The stability of pIRES-eGFP was measured in fecal samples from chicks that were treated with antibiotics. Results The plasmid asdAΔS129-pIRES-eGFP was successfully constructed.Western blotting indicated that Caco-2cells infected with asdAΔS129-pIRES-eGFP expressed eGFP in the presence of DAP in a dose-dependent fashion,but little or no expression of eGFP was noted in the absence of DAP.The expression of eGFP was also presented in the intestinal mucosa samples of young chicks at the day 3and day 4post-infection.Expression of eGFP spread from the duodenum to the colon.Stable pIRES-eGFP was detected in fecal samples from chicks and the ratio of asdAΔS129-pIRES-eGFP to asdAΔS129was（86.84±5.57）% on day 1post-infection,（66.29±7.89）% on day 2,（26.84±3.09）% on day 3,and（4.37±1.22）% on day 4. Conclusion This study identified an attenuated and mutated Salmonella typhimuriumisolate,asdAΔS129,that possesses exceptional ability to deliver a heterologous antigen in both mammalian cells and in chickens.This isolate could be used as a potential vaccine or vector for delivery of anti-cancer drugs in the future.

关 键 词：asdA缺陷型鼠伤寒沙门氏菌 疫苗载体 真核表达质粒 

分 类 号：R378.22[医药卫生—病原生物学]