牛源大肠杆菌O14菌蜕的制备及其免疫原性的研究

发布时间：2018-12-09 10:29

【摘要】：大肠杆菌(Escherichia coli),是一种重要的人畜共患病病原菌。该菌在环境中广泛存在,部分菌株具有较强的致病性,菌蜕(BGs)是革兰阴性菌被噬菌体PhiX174裂解基因E裂解后形成的完整细菌空壳,无细胞内容物。除可以作为疫苗外,还可作为佐剂、外源蛋白抗原、生物活性物质和DNA疫苗载体等多种功能。仅通过基因E裂解制备菌蜕,还可能具有病原岛或抗生素抗性基因水平转移等风险。因此,作为疫苗或疫苗载体的菌蜕应当更为安全。为了尽量消除裂解后细菌的活力并清除残留的遗传物质,本研究又将辅助致死基因SNA引入裂解表达系统中,并构建了新型温控表达质粒pBVE-CIP-SNA(pBVES),成功导入牛源大肠杆菌O14野生株,且进行反复多次溶菌动力学试验,质粒型大肠杆菌菌蜕裂解率高达99.65%,用透射电镜进行观察,与正常状态大肠相比菌蜕内容物外排彻底,形态及表面结构完整。为验证菌蜕免疫原性,研究设立菌蜕组、灭活组、灭活佐剂组为试验组,对照组为PBS组,通过测定不同免疫指标来比较四组的免疫原性。结果显示:随着免疫次数增加,试验组血清IgG含量明显高于对照组,小鼠淋巴细胞亚群试验结果表明菌蜕能刺激小鼠机体产生Th1型细胞免疫应答,而小鼠主要细胞因子检测结果表明菌蜕组的IFN-γ和IL-12分泌水平明显高于其他试验组和对照组,且试验组均高于对照组水平,但IL-4试验组的分泌水平与对照组差异不明显。攻毒保护结果显示,菌蜕组对小鼠机体保护率可达到70%。临床症状表明,在攻毒后,菌蜕组小鼠状态优于其他免疫组。综上所述,质粒型大肠杆菌菌蜕具有较好的免疫原性,研究结果为新型疫苗的研究提供依据,具有广阔的应用前景。
[Abstract]:E. coli (Escherichia coli), is an important zoonotic pathogen. Some strains have strong pathogenicity. Slough (BGs) is an intact bacterial shell formed by E cleavage of bacteriophage PhiX174 cleavage gene, and has no cell contents. It can also be used as adjuvant, foreign protein antigen, bioactive substance and DNA vaccine carrier. The preparation of mycelium slough by gene E cleavage may also have the risk of pathogenetic island or horizontal transfer of antibiotic resistant gene. Therefore, it should be safer to use as vaccine or vaccine carrier. In order to eliminate the viability of the bacteria and remove the residual genetic material, the co-lethal gene SNA was introduced into the lytic expression system, and a new thermo-controlled expression plasmid pBVE-CIP-SNA (pBVES), was constructed. The wild strain of Escherichia coli O14 from cattle was successfully introduced, and repeated bacteriolysis kinetics tests were carried out. The slough cleavage rate of plasmid type Escherichia coli was up to 99.65, which was observed by transmission electron microscope (TEM). Compared with the normal large intestine, the fungicidal shedding contents were completely discharged, and the morphology and surface structure were complete. In order to verify the immunogenicity of mycelium slough, the study established the mycelium slough group, the inactivated adjuvant group as the experimental group and the control group as the PBS group. The immunogenicity of the four groups was compared by measuring different immune indexes. The results showed that the level of serum IgG in the experimental group was significantly higher than that in the control group with the increase of immunization times. The results of lymphocyte subgroup test showed that the mycelium could stimulate the Th1 type cellular immune response in mice. The levels of IFN- 纬 and IL-12 in the mycelia group were significantly higher than those in the other test groups and control groups, and the levels in the test group were higher than those in the control group. However, the secretory level of IL-4 test group was not significantly different from that of control group. The results showed that the protective rate of mycelium slough group could reach 70%. The clinical symptoms showed that the state of the mice in the mycelia group was better than that in the other immune groups. To sum up, plasmid Escherichia coli slough has good immunogenicity, the research results provide the basis for the study of new vaccine, and have a broad application prospect.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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