【摘要】：腸產(chǎn)毒性大腸桿菌(Enterotoxigenic Escherichia coli,,ETEC)是引起幼畜腹瀉的主要病原之一。據(jù)統(tǒng)計(jì)，國內(nèi)大約有35%的仔豬腹瀉是因感染ETEC引起的；美國等國家新生仔豬腹瀉中，由ETEC引起的占45%以上。ETEC所導(dǎo)致的腹瀉發(fā)病率和死亡率都很高，給畜牧產(chǎn)業(yè)的發(fā)展造成了極其嚴(yán)重的經(jīng)濟(jì)損失。目前，世界各國都在致力于開發(fā)有效的疫苗來防治ETEC所引起的腹瀉。 ETEC致病因子主要包括腸毒素(enterotoxins)和黏附素(定居因子,fimbriae)。當(dāng)病原菌感染宿主后，ETEC通過菌毛黏附到小腸黏膜上皮細(xì)胞刷狀緣受體上，使ETEC在腸道內(nèi)定居繁殖，釋放腸毒素，與小腸細(xì)胞膜上的特異受體結(jié)合，引發(fā)細(xì)胞內(nèi)水、電解質(zhì)失衡，造成細(xì)胞吸收障礙，導(dǎo)致腹瀉的發(fā)生。豬源ETEC的黏附素類型主要有K88、K99、987P、F17，F(xiàn)18、F41、F42;其中，引起仔豬腹瀉的菌毛主要以K88為主。腸毒素包括不耐熱腸毒素（Heat-labileenterotoxin，LT）和耐熱腸毒素（Heat-stable enterotoxin，ST）兩種。 LTb是大腸桿菌不耐熱腸毒素的亞單位，因其具有無毒性且有很好黏膜免疫佐劑效果而被廣泛研究應(yīng)用。鼻黏膜組織分布著鼻相關(guān)淋巴組織和豐富的血管，通過鼻腔免疫能夠產(chǎn)生有效的免疫反應(yīng)，且免疫途徑方便。在本試驗(yàn)研究中，將K88和LTb通過鼻腔免疫BALB/c小鼠，期望能夠刺激機(jī)體產(chǎn)生特異性的黏膜和系統(tǒng)免疫反應(yīng)。為開發(fā)一種針對ETEC的安全有效、免疫接種方便的新型基因工程疫苗提供新的研究思路。 目的：將產(chǎn)腸毒素大腸桿菌K88菌毛蛋白與大腸桿菌不耐熱腸毒素LTb分別重組到原核表達(dá)載體中，得到純化后的重組蛋白后經(jīng)鼻腔接種免疫小鼠，觀察免疫效果。 方法：用PCR技術(shù)擴(kuò)增出不含信號(hào)肽的K88和LTb基因，，重組到表達(dá)載體pQE30上，轉(zhuǎn)化到大腸桿菌M15中進(jìn)行表達(dá)，將表達(dá)產(chǎn)物K88、LTb進(jìn)行純化、復(fù)性，分別用K88、K88與LTb滴鼻免疫BALB/c小鼠，ELISA分析各組特異性抗體水平。結(jié)果：重組蛋白K88、LTb在大腸桿菌M15中獲得高效表達(dá)，經(jīng)親和層析后蛋白純度大于95!。滴鼻免疫BALB/c小鼠試驗(yàn)表明， K88與LTb聯(lián)合滴鼻免疫組血清特異性IgG及鼻腔、小腸黏膜沖洗液中特異性IgA水平均明顯增高，與K88單獨(dú)免疫組和對照組相比較差異顯著(P＜0.01)。結(jié)論：K88與LTb滴鼻免疫BALB/c小鼠后，不僅可以增強(qiáng)特異性血清抗體反應(yīng)，而且誘導(dǎo)了黏膜部位產(chǎn)生免疫應(yīng)答。通過本試驗(yàn)研究可以為將來開發(fā)新型的ETEC基因工程疫苗奠定基礎(chǔ)。
[Abstract]:Enterotoxigenic Escherichia coli (Enterotoxigenic Escherichia coli,,ETEC) is one of the main pathogens causing diarrhea in young animals. According to statistics, about 35% of piglets' diarrhea in China is caused by infection with ETEC, and the incidence and mortality of diarrhea caused by ETEC in more than 45% of newborn piglets' diarrhea in the United States and other countries are very high. To the development of animal husbandry industry caused extremely serious economic losses. At present, countries all over the world are devoting themselves to developing effective vaccines to prevent and cure diarrhea caused by ETEC. The main pathogenic factors of ETEC include enterotoxin (enterotoxins) and colonizing factor fimbriae. When the pathogen infects the host, ETEC adheres to the brush edge receptor of the intestinal mucosal epithelial cells through the fimbriae, which makes ETEC settle and reproduce in the intestine, release enterotoxin, bind to the specific receptors on the small intestinal cell membrane, and cause intracellular water and electrolyte imbalance. Causes the cell absorption barrier, causes the diarrhea occurrence. The main types of adhesives in porcine ETEC were K88987 Pf17F17F18F41F42, among which K88 was the main cause of piglet diarrhea. Enterotoxins include heat-labile enterotoxins (Heat-labileenterotoxin,LT) and heat-resistant enterotoxins (Heat-stable enterotoxin,ST). LTb is a subunit of Escherichia coli heat-labile enterotoxins and has been widely used for its nontoxic and good mucosal immune adjuvant effect. Nasal mucosal tissue distributes rhino-associated lymphoid tissue and abundant blood vessels, which can produce an effective immune response through nasal immunization, and the immune pathway is convenient. In this study, BALB/c mice were immunized with K88 and LTb through nasal cavity, which was expected to stimulate specific mucosal and systemic immune responses. It provides a new research idea for developing a new genetic engineering vaccine which is safe and effective for ETEC and easy to inoculate. Aim: to recombine enterotoxigenic Escherichia coli K88 pili protein and Escherichia coli heat-labile enterotoxin (LTb) into prokaryotic expression vector, and then immunize mice by nasal inoculation. Methods: the K88 and LTb genes without signal peptide were amplified by PCR technique, and were recombined into the expression vector pQE30, and then transformed into E. coli M15 for expression. The expression product K88Tb was purified and renatured. BALB/c mice were immunized with K88 K88 and LTb respectively by Elisa. Results: the recombinant protein K88 LTb was highly expressed in Escherichia coli M15. The purity of the recombinant protein was more than 95! F by affinity chromatography. BALB/c mice immunized by nasal drip showed that the levels of serum specific IgG, nasal cavity, and specific IgA in small intestinal mucosa flushing fluid in K88 / LTb combined nasal immunization group were significantly higher than those in K88 alone and control group (P < 0. 01). Conclusion inoculation of BALB/c mice with LTb and K88 not only enhances the specific serum antibody response, but also induces an immune response at the mucosal site. This study can lay a foundation for the development of new ETEC genetic engineering vaccine in the future.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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