大肠杆菌O157重组弱毒疫苗的研究

发布时间：2018-02-09 18:25

本文关键词： 肠出血性大肠杆菌O157:H7 ler基因志贺毒素突变疫苗　出处：《吉林大学》2007年博士论文　论文类型：学位论文

【摘要】： 本研究首先建立肠出血性大肠杆菌O157:H7感染的动物模型,利用丝裂霉素对小鼠进行腹腔注射预处理,并通过灌胃接种和腹腔注射两种途径,建立了O157:H7感染的动物模型。然后对EHEC O157:H7 ler/stx基因缺失突变菌株的原噬菌体及其原始整合位点进行分析。通过对Stx A亚基毒性活性区和跨膜区的定点突变,消除或降低Stx的细胞毒性,并将Stx的无毒性或弱毒性突变体导入O157:H7 ler/stx基因缺失突变菌株,构建O157重组弱毒疫苗菌株。利用小鼠模型和vero细胞对O157重组弱毒疫苗菌株的vero细胞毒性、对小鼠的致病性、重组菌株的稳定性,以及被动免疫和主动免疫保护性进行了研究。结果表明,O157重组弱毒疫苗菌株具有良好的稳定性,对vero细胞的毒性作用降低了约104倍,丧失了对小鼠模型的致病性,能有效缩短小鼠感染O157:H7强毒株后的排毒时间(免疫组小鼠第6天后粪便内即检测不到强毒株,而未免疫对照组第13天仍可检测到)。用疫苗菌株对母鼠免疫,免疫母鼠所生的乳鼠通过吸吮母乳,能够获得良好的被动免疫保护作用(F25和F105的免疫保护率分别为75.2%和83.0%);通过腹腔注射的途径对小鼠进行免疫和攻毒,结果也表明O157重组弱毒疫苗菌株对小鼠具有良好的免疫保护作用(F25和F105的免疫保护作用分别为11/20和14/20)。
[Abstract]:In this study, the animal model of enterohemorrhagic Escherichia coli O157: H7 infection was established. The mice were pretreated by intraperitoneal injection of mitomycin, and were injected intraperitoneally and intraperitoneally. The animal model of O157: H7 infection was established. The phage and its original integration site of EHEC O157: H7 ler/stx gene deletion mutant strain were analyzed. The site-directed mutations of the toxic active region and transmembrane region of Stx A subunit were carried out. The cytotoxicity of Stx was eliminated or reduced, and the non-toxic or weakly toxic mutants of Stx were introduced into O157: H7 ler/stx gene deletion mutant. A recombinant attenuated vaccine strain O157 was constructed. The pathogenicity, stability, passive immunity and active immune protection of O157 recombinant attenuated vaccine strain were studied by using mouse model and vero cells to study the vero cytotoxicity of O157 recombinant attenuated vaccine strain. The results showed that the recombinant attenuated vaccine strain of YO157 had good stability, the toxicity to vero cells was reduced by about 104-fold, and the pathogenicity of the strain was lost to the mouse model. It can effectively shorten the detoxification time of mice infected with O157: H7 virulent strain (the mice in the immunized group could not detect the virulent strain in feces after 6 days, while the mice in the unimmunized control group could still be detected on the 13th day after inoculation with the vaccine strain. The immune protection rates of F25 and F105 were 75.2% and 83.0 by sucking breast milk, respectively, and the mice were immunized and poisoned by intraperitoneal injection. The results also showed that the recombinant attenuated strain O157 had a good protective effect on mice. The protective effects of F25 and F105 on mice were 11/20 and 14 / 20 respectively.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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