猪源益生菌EP株突变株EP△asd的构建和初步应用  

The construction of probiotic Escherichia coli EP△asd deletion mutant and its primary application

作　　者：金铎[1,2] 原志伟 单玉平[4] 区炳明 羊扬[1,2] 朱军 朱国强[1,2] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [3]中华人民共和国荣成出入境检验检疫局,山东荣成264300 [4]江苏省连云港市畜牧兽医站,江苏连云港222001

出　　处：《中国预防兽医学报》2018年第1期5-8,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：江苏省重点研发计划(现代农业)BE2017342;国家自然科学基金(31672579;31270171;31072136;31502075;30771603);江苏省自然科学基金(BK20150442);江苏高校优势学科建设工程资助项目;课题号连云港市科技攻关项目(CN1505)

摘　　要：为研制更加安全的大肠杆菌疫苗株,并将其开发为新型活疫苗载体,本研究采用电转化将氯霉素抗性基因片段转化至益生菌EP株。采用同源重组技术使氯霉素抗性基因的PCR产物通过其两侧asd基因序列与益生菌EP基因组中的asd基因发生同源重组。通过氯霉素抗性平板筛选得到阳性克隆,对筛选得到的阳性克隆再导入编码Flp重组酶的质粒p CP20以去除抗性基因,从而构建成EP△asd突变株。该缺失突变株可以与表达链球菌asd基因的质粒形成功能性互补,转化有上述质粒的EP△asd突变株可长时间定居于仔猪肠道。本研究为进一步开发以益生菌EP△asd为染色体—质粒平衡致死系统宿主菌的用于预防仔猪肠道性传染病的细菌活载体疫苗研究奠定基础。In order to develop safer Escherichia coli vaccine strain and exploit EP as a novel live vaccine vector, chloramphenicol-resistance gene was amplified and electrotransformed into probiotic EP. Making the bilateral asd gene sequence of this resistance gene reacts with the asd gene sequence in EP genome by homologous recombination technique, and positive clones were selected from chloramphenicol-resistance agar plates. Then, plasmid pCP20 was transformed into these clones to delete the resistance gene and the deletion mutant strain EP△asd was contrusted. EP△asd could make complementation with the pYA3341 plasmids which could express asd gene of the streptococcus, and the EP△asd with these plasmids could colonize in the piglets’ intestine for a long time. The research layed the foundation for the live bacteria vaccine using the probiotic EP△asd as host bacteria vector through chromosome-plasmid balanced-lethal system to prevent the swine intestine infectious disease.

关 键 词：益生菌 RED重组系统 asd基因 平衡致死系统 

分 类 号：S852.61[农业科学—基础兽医学]