重组ETEC F41干酪乳杆菌诱导免疫应答及保护性研究

发布时间：2018-02-10 04:48

本文关键词： ETEC F41 滴鼻免疫口服免疫干酪乳杆菌保护率　出处：《黑龙江八一农垦大学》2009年硕士论文　论文类型：学位论文

【摘要】：产肠毒素性大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起人和幼畜(初生仔猪、犊牛、羔羊、断奶仔猪)腹泻的重要病原之一。我国乃至世界因ETEC引起的新生幼畜腹泻发病率和死亡率都很高,给畜牧业带来了极大危害,造成了巨大的经济损失。本试验选择干酪乳酸菌作为受体菌株表达外源基因F41,即可将乳酸菌的益生功能和外源基因的特异性免疫相结合,从而研制F41基因工程乳酸菌。为开发研制预防F41的基因工程乳酸菌口服疫苗奠定基础。为了有效的预防ETEC的感染,我们采用了一种新型的表面展示系统,利用多聚谷氨酸跨膜蛋白pgsA基因作为锚定系统,使外源抗原稳定高效的表达在乳杆菌细胞表面。将构建重组表达载体pLA-F41,电转化至L. casei中,在MRS培养基中培养后, SDS PAGE、Western blotting检测,有约73kDa蛋白得到了表达,表达蛋白的大小与理论值相符且可被抗血清所识别,间接免疫荧光试验及流式细胞术结果表明, pgsA能够将融合蛋白成功地展示在菌体表面。将重组菌接种于人工模拟的胃肠液中,通过平板计数观察其存活能力。结果表明,重组干酪乳杆菌在人工消化液中均具有良好的存活性能,符合益生菌的基本特征,为实验动物模型的建立奠定了基础。将获得的阳性重组菌株pLA-F41/L.casei口服或滴鼻免疫BALB/c SPF级小鼠,初免后不同时间采集血液样品,测定小鼠产生抗F41的特异性IgG及其亚型,收集小鼠肺部、肠道、阴道冲洗液及粪便样品测定小鼠产生抗F41的特异性sIgA,同时初免后4周和10周,运用ELISPOT进行细胞因子分析,并对主动免疫和被动免疫小鼠进行攻毒保护性试验。结果表明重组干酪乳杆菌pLA-F41/L.casei免疫小鼠能够产生明显的抗体,对IgG亚型分析表明主要是IgG1,其次是IgG2a和IgG2b。ELISPOT结果、黏膜免疫产生明显的sIgA抗体和系统免疫主要产生的IgG1亚型表明免疫类型偏向Th2型免疫反应。口服主动免疫组保护率在90%以上,滴鼻主动免疫组保护率在85%以上,对照组均全部死亡。口服被动免疫组保护率达90%,滴鼻被动免疫组保护率达80%,对照组保护率均为5%。为进一步研究ETEC F41基因工程乳酸菌口服疫苗奠定基础。
[Abstract]:Enterotoxigenic Escherichia coli is one of the most important pathogens causing diarrhea in human and young animals (newborn piglets, calves, lambs, weaning piglets). The morbidity and mortality of diarrhea caused by ETEC in China and the world are very high. It has brought great harm to animal husbandry and caused huge economic losses. In this experiment, we selected the caseous lactic acid bacteria as the receptor strain to express the exogenous gene F41, which could combine the probiotic function of lactic acid bacteria with the specific immunity of exogenous genes. Thus, the genetic engineering lactic acid bacteria F41 was developed, which laid a foundation for the development of oral vaccine against F41 genetically engineered lactic acid bacteria. In order to effectively prevent ETEC infection, we have adopted a new surface display system, using polyglutamic acid transmembrane protein pgsA gene as the anchoring system. The recombinant expression vector pLA-F41 was transformed into L. casei. After cultured in MRS medium, the expression of 73 kDa protein was detected by Western blotting. The size of the expressed protein is consistent with the theoretical value and can be recognized by the antiserum. The results of indirect immunofluorescence assay and flow cytometry showed that pgsA could successfully display the fusion protein on the cell surface. The recombinant bacteria were inoculated into simulated gastrointestinal fluid and their viability was observed by plate counting. The recombinant Lactobacillus casei has good viability in artificial digestible fluid, which accords with the basic characteristics of probiotics and lays a foundation for the establishment of experimental animal model. BALB/c SPF mice were immunized by oral or nasal immunization with the positive recombinant strain pLA-F41/L.casei. The blood samples were collected at different times after the first immunization, and the specific IgG and its subtypes against F41 were determined, and the lungs and intestines of the mice were collected. Vaginal flushing fluid and fecal samples were used to detect the specific Siga produced by mice against F41, and the cytokines were analyzed by ELISPOT at 4 and 10 weeks after the first immunization. The results showed that the recombinant Lactobacillus casei pLA-F41/L.casei immunized mice could produce obvious antibodies, and the IgG subtypes were mainly IgG1, followed by IgG2a and IgG2b.ELISPOT. The obvious sIgA antibody produced by mucosal immunity and the IgG1 subtype produced by systemic immunity showed that the immune type was inclined to Th2 type. The protective rate of oral active immunization group was more than 90%, and that of nasal active immunization group was more than 85%. The protective rate of oral passive immunization group was 90%, that of nasal passive immunization group was 80%, and that of control group was 5%, which laid a foundation for further study of ETEC F41 gene engineering lactic acid bacteria oral vaccine.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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