产肠毒素大肠杆菌K88菌毛蛋白与LTb的原核表达及其鼻腔免疫的研究

发布时间：2018-08-30 12:02

【摘要】：肠产毒性大肠杆菌(Enterotoxigenic Escherichia coli,,ETEC)是引起幼畜腹泻的主要病原之一。据统计，国内大约有35%的仔猪腹泻是因感染ETEC引起的；美国等国家新生仔猪腹泻中，由ETEC引起的占45%以上。ETEC所导致的腹泻发病率和死亡率都很高，给畜牧产业的发展造成了极其严重的经济损失。目前，世界各国都在致力于开发有效的疫苗来防治ETEC所引起的腹泻。 ETEC致病因子主要包括肠毒素(enterotoxins)和黏附素(定居因子,fimbriae)。当病原菌感染宿主后，ETEC通过菌毛黏附到小肠黏膜上皮细胞刷状缘受体上，使ETEC在肠道内定居繁殖，释放肠毒素，与小肠细胞膜上的特异受体结合，引发细胞内水、电解质失衡，造成细胞吸收障碍，导致腹泻的发生。猪源ETEC的黏附素类型主要有K88、K99、987P、F17，F18、F41、F42;其中，引起仔猪腹泻的菌毛主要以K88为主。肠毒素包括不耐热肠毒素（Heat-labileenterotoxin，LT）和耐热肠毒素（Heat-stable enterotoxin，ST）两种。 LTb是大肠杆菌不耐热肠毒素的亚单位，因其具有无毒性且有很好黏膜免疫佐剂效果而被广泛研究应用。鼻黏膜组织分布着鼻相关淋巴组织和丰富的血管，通过鼻腔免疫能够产生有效的免疫反应，且免疫途径方便。在本试验研究中，将K88和LTb通过鼻腔免疫BALB/c小鼠，期望能够刺激机体产生特异性的黏膜和系统免疫反应。为开发一种针对ETEC的安全有效、免疫接种方便的新型基因工程疫苗提供新的研究思路。目的：将产肠毒素大肠杆菌K88菌毛蛋白与大肠杆菌不耐热肠毒素LTb分别重组到原核表达载体中，得到纯化后的重组蛋白后经鼻腔接种免疫小鼠，观察免疫效果。方法：用PCR技术扩增出不含信号肽的K88和LTb基因，，重组到表达载体pQE30上，转化到大肠杆菌M15中进行表达，将表达产物K88、LTb进行纯化、复性，分别用K88、K88与LTb滴鼻免疫BALB/c小鼠，ELISA分析各组特异性抗体水平。结果：重组蛋白K88、LTb在大肠杆菌M15中获得高效表达，经亲和层析后蛋白纯度大于95!。滴鼻免疫BALB/c小鼠试验表明， K88与LTb联合滴鼻免疫组血清特异性IgG及鼻腔、小肠黏膜冲洗液中特异性IgA水平均明显增高，与K88单独免疫组和对照组相比较差异显著(P＜0.01)。结论：K88与LTb滴鼻免疫BALB/c小鼠后，不仅可以增强特异性血清抗体反应，而且诱导了黏膜部位产生免疫应答。通过本试验研究可以为将来开发新型的ETEC基因工程疫苗奠定基础。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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