减毒沙门菌介导的apo-tPA融合基因在鸡体组织的分布与表达  

Distribution and expression of apo-tPA fusion gene in chick tissue delivered by attenuated Salmonella typhimurium

作　　者：王晓利[1] 李银聚[1] 程相朝[1] 张春杰[1] 吴庭才[1] 廖成水[1] 胡阿勇[1] 

机构地区：[1]河南科技大学动物科技学院,河南洛阳471003

出　　处：《中国兽医学报》2011年第4期485-488,共4页Chinese Journal of Veterinary Science

基　　金：河南省自然科学基金资助项目(031103100);洛阳市重大科技攻关项目(0801048A)

摘　　要：探讨以减毒沙门菌为载体,介导外源基因在动物体内的组织分布和表达的可行性。将编码人t-PA功能区的基因片段与鸡VLDLy组装前体apo-B100基因融合,定向克隆到荧光蛋白表达载体SV40启动子下游,构建pApo-tPA-GFP表达质粒。应用电转化法将pApo-tPA-GFP表达质粒转化减毒沙门菌,阳性减毒沙门菌经翅下静脉注入鸡体内,分别于注射后第1、2、3、4、5周随机剖检试验用鸡,无菌取内脏器官,经组织细菌培养、RT-PCR检测各脏器中基因分布,涂片荧光镜检基因表达情况。结果显示,在肝脏、脾脏和十二指肠均分离到沙门菌,PCR鉴定均为减毒沙门菌载体;RT-PCR检测在肝脏、脾脏、十二指肠中有目的基因存在;荧光检测在肝脏、脾脏中有荧光蛋白。结果表明,减毒沙门氏菌能够作为载体将目的基因有效的转入到动物体内,这一方法有望为动物转基因提供便捷、有效的途径。To evaluate the distribution and expression of apo-tPA fusion gene in vivo delivered by attenuated Salmonella typhimurium.A recombinant plasmid pApo-tPA-GFP combined human tissue-type plasminogen activator（t-PA） with apo-B100 and green fluorescent fusion protein（GFP） fusion gene was constructed.When the resultant constructing expression plasmid pApo-tPA-GFP was transformed into attenuated Salmonella typhimurium using electroporation method,and then was transfected henes by wing intravenous injection.Then henes were randomly anatomized on 1st,2nd,3rd,4th and 5th weeks after the injection,visceral organs such as muscle,liver,spleen,duodenum,oviduct,trachea and heart were collected with aseptic manipulation.The distribution of attenuated Salmonella typhimurium and fusion gene in organs were detected by bacterium cultures and PCR as well as RT-PCR;the expression of fusion gene was detected by smear fluorescence microscopy.The results showed that the attenuated Salmonella typhimurium were mainly distribute in liver,spleen and duodenum,the plasmid pApo-tPA-GFP may delivered in the organs and expressed t-PA and fluorescence protein.It was demonstrated that attenuated Salmonella typhimurium can be used as an effective vectors for exogenous gene transfer in to hen′s body,therefore the results provide useful perspectives for transgenic animals.

关 键 词：减毒沙门菌 T-PA 表达质粒 

分 类 号：S852.61[农业科学—基础兽医学]