本文選題：裂解基因E + 核酸酶A　； 參考：《中國預(yù)防獸醫(yī)學(xué)報》2017年03期

【摘要】：為制備高裂解效率的致禽腎臟、腸道病變大腸桿菌菌影,本研究通過編碼15個柔性氨基酸linker采用融合PCR法將噬菌體中Φ174的裂解蛋白E基因和金黃色葡萄球菌核酸酶A基因(SN)串聯(lián)(E-15L-SN),插入溫控表達(dá)質(zhì)粒pBV220,構(gòu)建重組溫控雙裂解表達(dá)質(zhì)粒(pBV-ES),采用PCR擴(kuò)增其溫控雙基因裂解表達(dá)盒(DLS-ES)插入E.coli-Pasteurella(大腸桿菌和巴氏桿菌)穿梭質(zhì)粒pBA1100,構(gòu)建重組溫控雙基因裂解穿梭質(zhì)粒pBA1100-DLS-ES。該質(zhì)粒可以通過溫控制備高裂解率的E.coli和Pasteurella兩種菌的菌影。本實(shí)驗將構(gòu)建的pBA1100-DLS-ES電轉(zhuǎn)化至致禽腎臟、腸道病變E.coli中,28℃集菌,42℃溫控誘導(dǎo)裂解蛋白E和核酸酶A表達(dá)。OD_(600nm)值及電鏡結(jié)果表明,雙基因裂解率高于單基因,同時收集菌影時間也比單基因裂解短,42℃誘導(dǎo)2 h含雙裂解基因的菌液處理菌體裂解率達(dá)到99.9999%,本實(shí)驗利用菌影形成機(jī)制將含青霉素抗性的致禽腎臟、腸道病變E.coli制備成菌影,為新型菌影疫苗的制備提供實(shí)驗依據(jù)。
[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，

本文編號：1836299