致病性大肠杆菌菌壳的研究

发布时间：2018-05-15 01:29

  本文选题：致病性大肠杆菌 + 裂解基因E　； 参考：《吉林大学》2010年博士论文

【摘要】： 致病性大肠杆菌(EPEC)是一类重要的食物源性病原菌。对EPEC感染主要用抗生素进行治疗,但抗生素治疗会带来一些副作用。目前还没有疫苗来预防EPEC的感染。菌壳是一种无胞质内容物的空壳状的无活性的细菌结构,完好地保留了细菌菌体和各种抗原结构,是一种具有良好应用前景的灭活疫苗。 为了制备致病性大肠杆菌菌壳,首先从PhiX174噬菌体扩增裂解E基因,克隆到温敏质粒pBV220,并转化到大肠杆菌DH5α,构建了重组质粒pBV220::E,该质粒对DH5α的裂解效率为99.76%。为实现E基因和葡萄球菌核酸酶A基因的双温控调节表达,扩增温控调节片段和葡萄球菌核酸酶A基因融合片段CI-P-SNA,并克隆到质粒pBV220::E,重组质粒pBV220::E::CI-P-SNA对菌株DH5α的裂解效率为99.53%。然后分别将重组质粒pBV220::E和pBV220::E::CI-P-SNA电转化到EPEC E2348/69 (O127:H6),转化后的重组菌株分别命名为F158和F159。 对F158和F159细菌制备菌壳的生物学和免疫学特性进行研究,结果表明重组质粒对F158和F159均有很高的裂解率,裂解率分别为99.92%和99.95%。EPEC菌壳经5%或10%的NaCl溶液冻融处理后,可将其中残存的活菌完全灭活。经42℃升温诱导裂解制备的EPEC菌壳保持有细菌的基本形态,具有完整的细菌外膜结构,由于细胞内容物外流,细菌形态变圆,表面发生明显皱缩。利用小鼠感染模型进行的实验显示F158菌壳和F159菌壳对小鼠具有良好的安全性,F158菌壳免疫保护率为13/20,F159菌壳免疫保护率为16/20。 本研究结果表明EPEC菌壳具有良好的安全性和免疫保护效果,可作为预防EPEC感染的候选疫苗,为最终研究出致病性大肠杆菌疫苗奠定了基础。
[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 伪 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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