共表达ETEC K99、K88重组干酪乳杆菌的构建及其免疫研究

发布时间：2018-03-28 08:45

本文选题：产肠毒素性大肠杆菌　切入点：K88菌毛蛋白　出处：《黑龙江八一农垦大学》2011年硕士论文

【摘要】：产肠毒素性大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起新生仔猪和断奶仔猪黄痢、白痢、水肿等腹泻疾病的主要病原菌之一,给我国乃至全世界养猪业带来了巨大的经济损失。ETEC的主要致病因子有黏附素(又称定居因子抗原)和肠毒素,黏附素与小肠上皮细胞上的特异性受体相结合而定植于黏膜是引起仔猪腹泻的前提条件。引起仔猪腹泻的菌毛抗原主要有K88、K99、987P、F41及F17等类型,其中以K88、K99黏附素抗原最为常见。本研究将PCR扩增的K99和K88-LTB基因片段串联克隆到可在大肠杆菌和干酪乳杆菌之间穿梭的表面表达载体pLA上,构建得重组质粒pLA-K99-K88-LTB,经鉴定正确后,电转入干酪乳杆菌,获得了表达融合蛋白pLA-K99-K88-LTB的重组干酪乳杆菌表达系统。经过SDS-PAGE、Western blot检测,约有90 kDa的融合蛋白得到表达,蛋白大小与理论值相符合。间接免疫荧光技术和流式细胞术的检测结果表明,融合蛋白成功地利用多聚谷氨酸跨膜蛋白pgsA基因展示在干酪乳杆菌菌体表面。将重组干酪乳杆菌pLA-K99-K88-LTB/L.casei口服接种SPF级C57BL/6小鼠,免疫后不同时间检测肠道、肺部和阴道冲洗液及粪便中抗K88、K99特异性sIgA抗体和血清中抗K88、K99特异性IgG水平;并测定免疫小鼠脾淋巴细胞增殖情况;然后对免疫后的小鼠进行攻毒保护性试验。间接ELISA结果显示重组干酪乳杆菌pLA-K99-K88-LTB/L.casei免疫小鼠后能在血清中检测到明显的抗K88、K99特异性IgG抗体,粪便及洗液中检测到明显的抗K88、K99特异性sIgA抗体;淋巴细胞增殖试验测定结果显示,免疫组可产生明显的细胞免疫应答。攻毒后小鼠免疫组的保护率均高于80%,表明该重组菌对小鼠具有保护作用。此外,ELISA结果显示,重组菌pLA-K99/L.casei、pLA-K88/L.casei免疫小鼠后体液免疫和细胞免疫水平均比重组干酪乳杆菌pLA-K99-K88-LTB/L.casei高。结果表明该重组干酪乳杆菌pLA-K99-K88-LTB/L.casei可诱导小鼠产生黏膜免疫应答和系统免疫应答,能有效地预防ETEC K88、K99的感染,为ETEC重组乳酸菌多价口服疫苗的研制提供了重要的物质基础。
[Abstract]:Enterotoxigenic Escherichia coli is one of the main pathogens causing diarrhea such as yellow dysentery, white dysentery and edema in newborn piglets and weaned piglets. The main pathogenic factors of ETEC are adhesion factor (also called settlement factor antigen) and enterotoxin. Binding of adhesion to specific receptors on small intestinal epithelial cells and colonization of mucous membrane is a prerequisite for diarrhea in piglets. The main types of bacterial hair antigens causing diarrhea in piglets are K88N K99987Pf41 and F17, among which K88K99 adhesion antigen is the most common. In this study, the K99 and K88-LTB gene fragments amplified by PCR were cloned into the surface expression vector pLA, which could shuttle between E. coli and Lactobacillus casei. The recombinant plasmid pLA-K99-K88-LTB was constructed. The recombinant plasmid pLA-K99-K88-LTB was successfully identified and electrotransferred to Lactobacillus casei. A recombinant Lactobacillus casei expression system expressing fusion protein pLA-K99-K88-LTB was obtained, and about 90 kDa fusion protein was detected by SDS-PAGEG Western blot. The results of indirect immunofluorescence and flow cytometry showed that the fusion protein was successfully displayed on the surface of Lactobacillus casei by polyglutamic acid transmembrane protein pgsA gene. The recombinant Lactobacillus casei (pLA-K99-K88-LTB/L.casei) was inoculated orally into SPF C57BL/6 mice. The levels of anti-K88K99 specific sIgA antibody in intestinal tract, lung, vaginal flushing fluid and feces were detected at different time after immunization, and the levels of anti-K88K99 specific IgG in serum were also detected. The results of indirect ELISA showed that the recombinant Lactobacillus casei pLA-K99-K88-LTB/L.casei immunized mice could detect anti-K88K99 specific IgG antibody in serum. The specific sIgA antibody against K88K99 was detected in feces and lotions, and the lymphocyte proliferation test showed that, The protective rate of the immunized group was higher than that of the control group, which indicated that the recombinant strain had protective effect on the mice. In addition, the results of Elisa showed that the protective rate of the recombinant strain was higher than that of the control group. The humoral and cellular immunity levels of the recombinant strain pLA-K99 / L.caseiia-pLA-K88 / L.casei were higher than that of the recombinant Lactobacillus casei pLA-K99-K88-LTB/L.casei. The results showed that the recombinant Lactobacillus casei pLA-K99-K88-LTB/L.casei could induce mucosal immune response and systemic immune response in mice. It can effectively prevent the infection of ETEC K88 K99 and provide an important material basis for the development of multivalent oral vaccine against ETEC recombinant lactic acid bacteria.
【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】：2011
【分类号】：R378

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