牛源空肠弯曲杆菌及其HSP43重组蛋白免疫原性的研究

发布时间：2018-07-04 17:09

本文选题：空肠弯曲杆菌 + HSP43重组蛋自　；参考：《黑龙江八一农垦大学》2010年硕士论文

【摘要】： 空肠弯曲杆菌(Campylobacter jejuni, C.jejuni)可引起牛冬痢,是一种重要的病原体。本试验以牛源空肠弯曲杆菌及其HSP43重组蛋白(rHSP43)为研究对象,制备免疫原,对鼠模型和奶牛进行免疫,研究其免疫原性。根据GenBank上已发表的C.jejuni的PorA基因设计—对特异性的引物,应用RCR方法扩增得到PorA蛋白基因,并克隆到pMD18-T载体上,筛选、鉴定阳性重组子pMD18-T-PorA,然后克隆到原核表达载体pQE-30a (+)上,经双酶切、PCR鉴定,获得重组表达质粒pQE-30a-PorA,经序列分析证实,构建的重组质粒pQE-30a-PorA中含有完整的PorA蛋白基因,将含有pQE-30a-PorA的重组菌BL21(DE3),经终浓度为1mmol/L的IPTG诱导,表达产物经SDS-PAGE分析,结果显示C.jejuni-DQ株PorA蛋白基因在原核表达系统中获得高效表达。Western blot检测结果表明表达的rHSP43具有良好的反应原性。利用表达并纯化后的rHSP43作为抗原,针对两种实验动物,分别建立了检测C.jejuni抗体的间接ELISA方法。检测小鼠血清抗体的间接ELISA方法的最佳反应条件为：抗原的最佳包被液为50mM pH9.6的碳酸盐缓冲液；抗原包被浓度为7.5μg/mL;封闭液为5%脱脂奶粉,封闭条件为37℃1h；血清稀释液为5%脱脂奶粉,血清稀释倍数为100倍；HRP-羊抗鼠IgG的最适[作浓度为1：5000。检测奶牛血清抗体的间接ELISA方法的最佳反应条件为：抗原的包被液为50mM pH9.6的碳酸盐缓冲液；抗原包被浓度为2.5μg/ml；最佳封闭液为5%脱脂奶粉,封闭条件为37℃lh；血清稀释液为5%脱脂奶粉,血清稀释倍数为200倍；HRP-羊抗牛IgG的最适工作浓度为1：5000。包被的重组抗原不与肠炎沙门氏菌、大肠埃希氏杆菌和金黄色葡萄球菌阳性血清发生交义反应,表明建立的ELISA方法具有良好的特异性。本研究采用常规方法分别制备白油、铝胶佐剂和不加佐剂的全菌体与rHSP43免疫原,分别以不同剂量经皮下、腹腔和灌胃途径免疫8周龄小鼠,应用间接ELISA方法检测两种免疫原免疫小鼠后抗体消长情况,并通过攻毒试验评价其保护效果。结果显示,在全菌体免疫原组中,5x1010CFU/mL组与5×109CFU/mL组产生的特异性抗体水平差异不显著,二者与5×107CFU/mL组相比,差异显著(p0.05)；腹腔与皮下注射产生的抗体水平差异不显著(p0.05),二者明显高于灌胃途径(P0.05)；白油佐剂组产生的抗体水平要高于铝胶佐剂组,二者明显高于未加佐剂组(P0.05)。rHSP43免疫原组中,免疫途径的效果比较和佐剂的效果比较结果与全菌体免疫原相似。在小鼠免疫后21 d以1×1010剂量的C.jejuni攻菌,以白油佐剂乳化的全菌体免疫原皮下注射能够提供完全保护,而rHSP43仅能提供75%的保护。将白油佐剂乳化的全菌体免疫原通过颈部皮下注射免疫奶牛,免疫后血清中产生了高水平的C.jejuni特异性IgG,与对照组差异极显著(p0.01)。小鼠和青年牛免疫试验表明,全菌体与rHSP43免疫原均能够诱导机体产生特异性抗体,小鼠攻毒试验证明两种免疫原均能有效预防C.jejuni攻击,但全菌体免疫原效果更好。
[Abstract]:Campylobacter jejuni (C.jejuni) is an important pathogen of bovine bacilli. In this experiment, the immunogenicity of Campylobacter jejuni and its HSP43 recombinant protein (rHSP43) was studied. Immunogen was prepared. The immunogenicity of rat model and cow was immunized and its immunogenicity was studied.
According to the PorA gene design of C.jejuni published on GenBank - specific primers, the PorA protein gene was amplified by RCR method and was cloned to pMD18-T vector. The positive recombinant pMD18-T-PorA was screened and identified and then cloned to the prokaryotic expression vector pQE-30a (+). The recombinant expression plasmid pQE-30a-Po was obtained by double enzyme cutting and PCR identification. The recombinant expression plasmid pQE-30a-Po was obtained. RA, sequence analysis confirmed that the recombinant plasmid pQE-30a-PorA contained a complete PorA protein gene, which contained the recombinant strain BL21 (DE3) containing pQE-30a-PorA, induced by IPTG of the final concentration of 1mmol/L, and the expression product was analyzed by SDS-PAGE. The results showed that the PorA protein gene of C.jejuni-DQ strain was highly expressed in the prokaryotic expression system. The results showed that the expressed rHSP43 had good reactivity.
Using the expressed and purified rHSP43 as an antigen, the indirect ELISA method for detecting C.jejuni antibody was established for two experimental animals. The best reaction condition for the indirect ELISA method for detecting the serum antibody of mice was that the best envelope of the antigen was 50mM pH9.6, the concentration of the antigen was 7.5 mu g/mL; For 5% skim milk powder, the closed condition was 37 1H, the serum dilution was 5% skimmed milk powder and the serum dilution multiplier was 100 times. The optimum reaction condition for the indirect ELISA method of HRP- Sheep anti mouse IgG was as follows: the antigen inclusion fluid was the carbonate buffer solution of 50mM pH9.6; the antigen inclusion concentration was the concentration. The best closed liquid was 5% skimmed milk powder, the closed condition was 37 LH, and the serum diluent was 5% skimmed milk powder, and the serum dilution multiple was 200 times; the optimum working concentration of HRP- Sheep anti bovine IgG for 1:5000. package was not with Salmonella enteritis, Escherichia coli and Staphylococcus aureus positive serum. The results showed that the established ELISA method had good specificity.
In this study, white oil, aluminum gum adjuvant and non adjuvant all bacteria and rHSP43 immunogen were prepared by routine methods. 8 weeks old mice were immunized with different doses of subcutaneous, peritoneal and intraperitoneal methods respectively. Indirect ELISA method was used to detect the antibody growth of two immunogenic mice, and the protective effect was evaluated by attack test. The results showed that there was no significant difference in the level of specific antibody produced by the group 5x1010CFU/mL and the 5 x 109CFU/mL group in the whole bacterial immunogen group. The difference was significant (P0.05) compared with the 5 x 107CFU/mL group, and the difference in the level of antibody produced by the intraperitoneal and subcutaneous injection was not significant (P0.05), and the two were significantly higher than the intragastric pathway (P0.05); the white oil adjuvant group was produced. The level of antibody was higher than that of the aluminum gel adjuvant group. The two was significantly higher than that in the.RHSP43 immunogen group without the adjuvant group (P0.05). The results of the immunization pathway and the effect of the adjuvant were similar to those of the whole bacteria immunogen. The 21 d after the immunization of the mice was 1 * 1010 doses of C.jejuni, and the whole bacterial immunogen emulsified with the white oil adjuvant was injected subcutaneously. Complete protection can be provided, and rHSP43 can provide only 75% protection. The whole bacterial immunogen of the emulsified oil adjuvant is immunized by the subcutaneous injection of the cow. The high level of C.jejuni specific IgG is produced in the serum after immunization. The difference is very significant (P0.01) with the control group (P0.01). The mice and the green year bovine immune test showed that the whole fungus and the rHSP43 immunogen Both of them could induce specific antibody production in the body. The attack test showed that all two immunogens could effectively prevent C.jejuni attacks, but the whole cell immunogen was better.
【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】：2010
【分类号】：R392

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