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

发布时间：2018-05-03 01:12

  本文选题：裂解基因E + 核酸酶A　； 参考：《中国预防兽医学报》2017年03期

【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。
[Abstract]:In order to prepare Escherichia coli with high cleavage efficiency of poultry kidney and intestinal lesions, In this study, the cleavage protein E gene of 桅 174 and the nuclease A gene of Staphylococcus aureus in phage were connected with E-15L-SNN by encoding 15 flexible amino acid linker and inserted into the thermo-controlled expression plasmid pBV220. the recombinant thermo-controlled double cleavage was constructed. The expressed plasmid pBV-ESN was amplified by PCR and inserted into E. coli-Pasteurella shuttle plasmid pBA1100. The recombinant shuttle plasmid pBA1100-DLS-ESwas constructed. The plasmid can be used to prepare E.coli and Pasteurella bacteria with high lytic rate by temperature control. In this experiment, the constructed pBA1100-DLS-ES was transformed into poultry kidney. The expression of nuclease A and protein E and nuclease A in intestinal E.coli were induced by temperature control at 42 鈩，

本文编号：1836299