【作者】 王文东；

【导师】 冯书章；

【作者基本信息】 吉林大学 ， 预防兽医学， 2010， 博士

【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。更多还原

【Abstract】 Enteropathogenic Escherichia coli (EHEC) is an important food borne pathogen capable of causing bloody. EPEC infection was mainly transmitted by fecal–oral, with contaminated hands, foods or water. In developed countries, EPEC once caused frequent outbreaks of infant diarrhea. The EPEC infection outbreaks were often explosive, with up to 50% mortality. In developing countries, about 30~40 % of infants diarrhea caused by EPEC, especially in the 0-6 month-old infants. At present, for EPEC infection mainly treated with antibiotics. However, antibiotic treatment may cause some side-effects. The best method to prevent EPEC infection is to develop a vaccine.Bacterial ghost is a new type of bacterial vaccines developed in recent years. Bacterial ghosts usually produced by PhiX174 phage protein E at the bacterial surface to form a pore-like structure, and bacterial cell contents exhausted through the pore-like structure to form the non-cytoplasmic non-active bacteria.Bacteria ghosts were produced by non-denatured methods. The natural surface antigen and cell morphology of bacteria were well-preserved. Bacteria ghosts also have adjuvant activity on its own. Furthermore, bacteria ghosts could be used as vaccine vector. Bacteria ghosts production process is simple and safe.PhiX174 phage lysis gene E encodes a 91 amino acid membrane protein. Protein E can integrate internal and external membrane of Gram-negative bacteria, resulting in a hole-like channel formed on the surface of bacteria. The diameter of hole-like structure of bacteria ghosts is between the range of 40-200nm. Cytoplasmic of bacterial was exhausted through this pore-like structure to form the non-cytoplasmic non-active bacteria. But the membrane structure of bacteria was well-preserved.Staphylococcal aureus nuclease A (SNA), also known as Micrococcal nuclease enzyme, is a non-specific nuclease secreted by Staphylococcus aureus. SNA can degrade the host bacterial DNA. SNA has a strong ability to degrade single-stranded or double-stranded DNA or RNA. It can degrade bacteria DNA into small fragments less than 100bp.Plasmid pBV220 has a thermo-sensitive transcriptional control systemλpL/pR-cI857, which can inhibit protein expression at 28℃. The protein expression could be induced by transfer the temperature from 28 to 39~42℃.To develop a vaccine against Enteropathogenic E. coli, in this study, we intend to use the temperature-sensitive plasmid pBV220 to express PhiX174 lytic gene E and staphylococcal nuclease A to produce EPEC ghosts. And we further study the biological and immunological characteristics of the EPEC ghosts.A pair of primers was designed according to PhiX174 phage E gene sequence published in GenBank. The lysis gene E was amplified by PCR from PhiX174 phage DNA by use of primers LE-F and LE-R. PCR product was cloned into vector pMD18-T and then subcloned into pBV220 downstream of theλPL/PR-cI857 regulatory system between sites EcoRI and BamHI to form the temperature-sensitive bacteriolysis plasmid pBV220::E. The recombinant plasmid pBV220::E was transformed into host cell DH5α. The lysis curve and lysis efficiency of plasmid pBV220::E to host cell DH5αwere studied. Results showed recombinant plasmid pBV220::E could lysis DH5αafter the temperature shift from 28℃to 42℃. The lysis rate of culture was 99.76%.In order to improve the efficiency of generation of bacteria ghosts, Staphylococcal nuclease A (SNA) was amplified from Staphyloccocus aureus by PCR. Temperature regulation fragment CI-P was amplified from plasmid pBV220. These two fragments were ligated to form fusion fragment CI-P-SNA by PCR. The fusion fragment CI-P-SNA was subcloned into downstream sites BamHI and PstI of lysis gene E to generate a recombinant plasmid pBV220::E::CI-P-SNA. This recombinant plasmid was transformed into host cell DH5α. The lysis curve and lysis efficiency of plasmid pBV220::E::CI-P-SNA to host cell DH5αwere studied. Results showed recombinant plasmid pBV220::E::CI-P-SNA could lysis DH5αafter the temperature shift from 28℃to 42℃. The lysis rate of culture was 99.53%.Recombinant plasmid pBV220::E and pBV220::E::CI-P-SNA were transformed into EPEC reference strain E2348/69 (O127:H6). The resultant recombinant strains were named as F158 and F159, respectively.The lysis curve, lysis efficiency and bacterial ghosts morphology of strains F158 and F159 were studied. The results showed that strains F158 and F159 were grown at 28℃until mid log-phase, followed by incubation at 42℃to induce the expression of gene E and SNA to generate EPEC bacterial ghosts. The OD600 of culture began to decline 20 min after the temperature shift from 28℃to 42℃. The lysis rate of F158 and F159 culture were 99.92% and 99.95%, respectively. Electron microscopy observations indicated that EPEC ghosts have basic cell morphology of E. coli. The EPEC ghosts were shown to be intact cells with contents released to extracellular region. However, as bacterial cytoplasmic flow outside of bacteria, EPEC ghosts surface significantly shrink. There are few living bacteria surviving in EPEC ghosts. The living bacteria surviving in EPEC ghosts could be completely killed after treatment by freezing and thawing in 5% or 10% NaCl solution.This study has also established an animal model of mice infected with EPEC. We studied the safety and immune effects of EPEC ghosts using mice infection model. EPEC ghosts produced from F158 and F159 by temperature induction were used to infect mice. Negative control mice,inoculated with F158 or F159 EPEC ghosts grew normally. Mice inoculated with wild-type strain EPEC E2348/69 showed rapid weight loss and all mice were died within 4 days. The results showed that EPEC ghosts are safe to mice.Experiments of immunity and challenge showed that all mice of non-immunized control group died (20/20), 13 mice immunized with F158 EPEC ghosts were survive 13/20, 16 mice immunized with F159 EPEC ghosts were survive16/20. It is suggestion that EPEC ghosts have good immune protective effect.We can conclude from above study that recombinant plasmid pBV220::E and pBV220::E::CI-P-SNA could be used to generate EPEC ghosts, EPEC ghosts is safety and have good immune protection. This study showed that EPEC ghosts could be used as candidate vaccine against EPEC infection.更多还原