本文關(guān)鍵詞： ETEC F41 滴鼻免疫 口服免疫 干酪乳桿菌 保護(hù)率　出處：《黑龍江八一農(nóng)墾大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】：產(chǎn)腸毒素性大腸桿菌(Enterotoxigenic Escherichia coli,ETEC)是引起人和幼畜(初生仔豬、犢牛、羔羊、斷奶仔豬)腹瀉的重要病原之一。我國(guó)乃至世界因ETEC引起的新生幼畜腹瀉發(fā)病率和死亡率都很高,給畜牧業(yè)帶來了極大危害,造成了巨大的經(jīng)濟(jì)損失。 本試驗(yàn)選擇干酪乳酸菌作為受體菌株表達(dá)外源基因F41,即可將乳酸菌的益生功能和外源基因的特異性免疫相結(jié)合,從而研制F41基因工程乳酸菌。為開發(fā)研制預(yù)防F41的基因工程乳酸菌口服疫苗奠定基礎(chǔ)。 為了有效的預(yù)防ETEC的感染,我們采用了一種新型的表面展示系統(tǒng),利用多聚谷氨酸跨膜蛋白pgsA基因作為錨定系統(tǒng),使外源抗原穩(wěn)定高效的表達(dá)在乳桿菌細(xì)胞表面。將構(gòu)建重組表達(dá)載體pLA-F41,電轉(zhuǎn)化至L. casei中,在MRS培養(yǎng)基中培養(yǎng)后, SDS PAGE、Western blotting檢測(cè),有約73kDa蛋白得到了表達(dá),表達(dá)蛋白的大小與理論值相符且可被抗血清所識(shí)別,間接免疫熒光試驗(yàn)及流式細(xì)胞術(shù)結(jié)果表明, pgsA能夠?qū)⑷诤系鞍壮晒Φ卣故驹诰w表面。將重組菌接種于人工模擬的胃腸液中,通過平板計(jì)數(shù)觀察其存活能力。結(jié)果表明,重組干酪乳桿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,為實(shí)驗(yàn)動(dòng)物模型的建立奠定了基礎(chǔ)。 將獲得的陽性重組菌株pLA-F41/L.casei口服或滴鼻免疫BALB/c SPF級(jí)小鼠,初免后不同時(shí)間采集血液樣品,測(cè)定小鼠產(chǎn)生抗F41的特異性IgG及其亞型,收集小鼠肺部、腸道、陰道沖洗液及糞便樣品測(cè)定小鼠產(chǎn)生抗F41的特異性sIgA,同時(shí)初免后4周和10周,運(yùn)用ELISPOT進(jìn)行細(xì)胞因子分析,并對(duì)主動(dòng)免疫和被動(dòng)免疫小鼠進(jìn)行攻毒保護(hù)性試驗(yàn)。結(jié)果表明重組干酪乳桿菌pLA-F41/L.casei免疫小鼠能夠產(chǎn)生明顯的抗體,對(duì)IgG亞型分析表明主要是IgG1,其次是IgG2a和IgG2b。ELISPOT結(jié)果、黏膜免疫產(chǎn)生明顯的sIgA抗體和系統(tǒng)免疫主要產(chǎn)生的IgG1亞型表明免疫類型偏向Th2型免疫反應(yīng)。口服主動(dòng)免疫組保護(hù)率在90%以上,滴鼻主動(dòng)免疫組保護(hù)率在85%以上,對(duì)照組均全部死亡。口服被動(dòng)免疫組保護(hù)率達(dá)90%,滴鼻被動(dòng)免疫組保護(hù)率達(dá)80%,對(duì)照組保護(hù)率均為5%。為進(jìn)一步研究ETEC F41基因工程乳酸菌口服疫苗奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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