本文選題：致病性大腸桿菌 + 裂解基因E�。� 參考：《吉林大學》2010年博士論文

【摘要】： 致病性大腸桿菌(EPEC)是一類重要的食物源性病原菌。對EPEC感染主要用抗生素進行治療,但抗生素治療會帶來一些副作用。目前還沒有疫苗來預防EPEC的感染。菌殼是一種無胞質內容物的空殼狀的無活性的細菌結構,完好地保留了細菌菌體和各種抗原結構,是一種具有良好應用前景的滅活疫苗。 為了制備致病性大腸桿菌菌殼,首先從PhiX174噬菌體擴增裂解E基因,克隆到溫敏質粒pBV220,并轉化到大腸桿菌DH5α,構建了重組質粒pBV220::E,該質粒對DH5α的裂解效率為99.76%。為實現(xiàn)E基因和葡萄球菌核酸酶A基因的雙溫控調節(jié)表達,擴增溫控調節(jié)片段和葡萄球菌核酸酶A基因融合片段CI-P-SNA,并克隆到質粒pBV220::E,重組質粒pBV220::E::CI-P-SNA對菌株DH5α的裂解效率為99.53%。然后分別將重組質粒pBV220::E和pBV220::E::CI-P-SNA電轉化到EPEC E2348/69 (O127:H6),轉化后的重組菌株分別命名為F158和F159。 對F158和F159細菌制備菌殼的生物學和免疫學特性進行研究,結果表明重組質粒對F158和F159均有很高的裂解率,裂解率分別為99.92%和99.95%。EPEC菌殼經5%或10%的NaCl溶液凍融處理后,可將其中殘存的活菌完全滅活。經42℃升溫誘導裂解制備的EPEC菌殼保持有細菌的基本形態(tài),具有完整的細菌外膜結構,由于細胞內容物外流,細菌形態(tài)變圓,表面發(fā)生明顯皺縮。利用小鼠感染模型進行的實驗顯示F158菌殼和F159菌殼對小鼠具有良好的安全性,F158菌殼免疫保護率為13/20,F159菌殼免疫保護率為16/20。 本研究結果表明EPEC菌殼具有良好的安全性和免疫保護效果,可作為預防EPEC感染的候選疫苗,為最終研究出致病性大腸桿菌疫苗奠定了基礎。
[Abstract]:Pathogenic Escherichia coli (EPEC) is an important foodborne pathogen. EPEC infection is mainly treated with antibiotics, but antibiotic therapy can bring some side effects. There is no vaccine to prevent EPEC infection. The bacterial shell is a kind of empty shell structure with no cytoplasmic contents. It is a kind of inactivated vaccine with good prospects for application. It retains the bacterial bacteria and various antigenic structures perfectly. In order to prepare pathogenic Escherichia coli shell, the E. coli gene was amplified from PhiX174 phage and cloned into the thermo-sensitive plasmid pBV220. the recombinant plasmid pBV220: 1: Ewas constructed. The cleavage efficiency of the plasmid was 99.76%. In order to regulate the expression of E gene and staphylococcal nuclease A gene, the fusion fragment CI-P-SNAand the fusion fragment of staphylococcal nuclease A gene were amplified. The recombinant plasmid pBV220: 1: Ewas cloned into plasmid pBV220: 1. The lytic efficiency of recombinant plasmid pBV220::E::CI-P-SNA on DH5 偽 was 99.53. Then the recombinant plasmids pBV220::E and pBV220::E::CI-P-SNA were transformed into EPEC E2348 / 69 O127: H6 respectively. The transformed recombinant strains were named F158 and F159respectively. The biological and immunological characteristics of the bacterial shell prepared by F158 and F159 were studied. The results showed that the recombinant plasmid had a high cleavage rate of F158 and F159, the cleavage rate was 99.92% and the 99.95%.EPEC shell was freeze-thawed with 5% or 10% NaCl solution, respectively. The remaining live bacteria can be completely inactivated. The EPEC shell prepared by induced pyrolysis at 42 鈩，

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