基于同源重组技术的表达CRE重组酶的减毒沙门菌的构建及功能初步研究  

Construction and preliminary study of recombinant regulated delayed lysis Salmonella expressing Cre based on homologous recombination

作　　者：高兴 刘晶 张赞 刘洋 许可 杨桂连 姜延龙 王春凤 GAO Xing;LIU Jing;ZHANG Zan;LIU Yang;XU Ke;YANG Gui-lian;JIANG Yan-long;WANG Chun-feng(Jilin Provincial Engineering Research Center of Animal Probiotics,Key Laboratory of Animal Production and Quality Security,Ministry of Education,College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]吉林农业大学动物科学技术学院吉林省动物微生态制剂工程研究中心动物生产及产品质量安全省部共建教育部重点实验室

出　　处：《中国兽医科学》2018年第10期1222-1227,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31602092,31672528);国家“十三五”重点研发计划项目(2017YFD0501000);吉林省教育厅科学技术研究项目(JJKH20170316KJ);吉林省科技发展计划项目(20160519011JH,20170204034NY);吉林省产业创新专项资金项目(2016C063)

摘　　要：为了构建表达Cre重组酶的减毒沙门菌,本试验首先将Cre重组酶表达框插入到自杀质粒pYA3599中,构建重组自杀质粒pYA3599-Cre,电转至感受态χ7232中,经酶切鉴定与测序后,电转至χ7213感受态中,得到含有自杀质粒pYA3599-Cre的重组菌χ7213。随后通过同源重组,经两步筛选,将Cre重组酶表达框整合至减毒沙门菌χ11802的基因组中,得到重组减毒沙门菌χ11802-Cre,通过Western-boltting技术检测重组酶Cre的表达,进而将报告质粒pYA4545-lox P电转至重组菌中验证Cre重组酶的功能。结果显示,成功构建了自杀质粒pYA3599-Cre,并基于同源重组技术成功将重组酶Cre表达框整合至χ11802基因组中,通过Western-blotting成功检测出重组酶Cre的表达,且报告质粒电转至重组菌中可被切除lox P位点之间序列。本试验成功构建出能表达重组酶Cre的减毒沙门菌,为后期以位点特异性重组酶Cre为基础的减毒沙门菌微环DNA疫苗的构建奠定了基础。To construct the regulated delayed lysis Salmonella to express Cre,in this study,the gene Cre was inserted into pYA3599 to construct the recombinant plasmid pYA3599-Cre and then pYA3599-Cre was transformed into χ7232.Eventually,we transformed pYA3599-Cre into χ7213 after restriction enzyme and sequencing analysis.Followed by homologous recombination with two step filter.The gene Cre was successfully integrated into χ11802 genome,and χ11802-Cre was yielded.The recombinant Salmonella was verified by Western-blotting and reporter plasmid pYA4545-lox P.As a result,we constructed the suicide plasmid pYA3599-Cre and inserted Cre into χ11802 genome successfully,Western-blotting detected the expression of Cre and the sequence between lox P of reporter plasmidpYA4545-lox P also be removed respectively.In conclusion,We constructed the attenuated Salmonella expressing Cre,indicating its possible application in minicircle DNA vaccine based on Cre.

关 键 词：CRE 自杀质粒 同源重组 减毒沙门菌 

分 类 号：S852.612[农业科学—基础兽医学]