致禽肾脏、肠道病变大肠杆菌菌影的制备及其裂解效率的测定  

Production and lysis efficiency of ghosts of the Escherichia coli induced nephritis and enteritis in chicken

作　　者：赵星[1,2] 程伊洛 卢琴[2] 邵华斌[2] 杨玉莹[1] 罗青平[2] 

机构地区：[1]长江大学动物科学学院,湖北荆州434025 [2]湖北省农业科学院畜牧兽医研究所,湖北武汉430064

出　　处：《中国预防兽医学报》2017年第3期186-190,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：公益性农业行业科研专项:家禽主要细菌病防控技术研究与示范(201303044);现代农业产业技术体系建设专项资金资助(CARS-42-G11);规模化养鸡场主要疫病防控关键技术研究(2015ABA039)

摘　　要：为制备高裂解效率的致禽肾脏、肠道病变大肠杆菌菌影,本研究通过编码15个柔性氨基酸linker采用融合PCR法将噬菌体中Φ174的裂解蛋白E基因和金黄色葡萄球菌核酸酶A基因(SN)串联(E-15L-SN),插入温控表达质粒pBV220,构建重组温控双裂解表达质粒(pBV-ES),采用PCR扩增其温控双基因裂解表达盒(DLS-ES)插入E.coli-Pasteurella(大肠杆菌和巴氏杆菌)穿梭质粒pBA1100,构建重组温控双基因裂解穿梭质粒pBA1100-DLS-ES。该质粒可以通过温控制备高裂解率的E.coli和Pasteurella两种菌的菌影。本实验将构建的pBA1100-DLS-ES电转化至致禽肾脏、肠道病变E.coli中,28℃集菌,42℃温控诱导裂解蛋白E和核酸酶A表达。OD_(600nm)值及电镜结果表明,双基因裂解率高于单基因,同时收集菌影时间也比单基因裂解短,42℃诱导2 h含双裂解基因的菌液处理菌体裂解率达到99.9999%,本实验利用菌影形成机制将含青霉素抗性的致禽肾脏、肠道病变E.coli制备成菌影,为新型菌影疫苗的制备提供实验依据。The purpose of this study is to prepare high efficiency ghost of the E.coli induced nephritis and enteritis in chicken. Fusion PCR method was adopted to realize lysis protein E and nuclease A gene SN series of genes （E-15L-SN） through 15 flexible amino acids, then inserted into the temperature controlled expression vector pBV220 to construct pyrolysis temperature controlled double gene expression system pBV-ES, then the temperature controlled double gene cracking box （DLS-ES） was inserted into the shuttle vector pBA1100 of E.coli-Pasteurella to build temperature controlled double gene cracking shuttle vector pBA1100-DLS-ES, which was able to produce high efficiency ghost of Pasteurella multocida and Escherichia coli by the temperature control system. The pBA1100-DLS-ES was transfromed into the E.coli, and incubation temperature was shifted from 28℃ to 42℃ to induce cracking protein expression to prepare ghost of E.coli. The results of OD600nm and electron microscope examiniation showed that the rate of double gene lysis was higher than that of single gene, and the time of collecting bacteria was shorter than that of single gene, and the cell lysis rate was up to 99.9999% after 2 hrs of inducing. This study use cell lysis mechanism to prepare ghost of the E.coli, which provide the basis for the new bacterial ghost vaccine preparation.

关 键 词：裂解基因E 核酸酶A 致禽肾脏 肠道病变大肠杆菌菌影 穿梭质粒 

分 类 号：S852.61[农业科学—基础兽医学]