hp1188基因的克隆表达及免疫特性研究

发布时间：2018-12-15 02:35

【摘要】： 目的:选择暴露于细菌表面并与幽门螺杆菌(Helicobacter pylori, H.pylori)定植密切相关的黏附素Hp1188为目标,通过基因工程技术获得纯化的重组黏附素蛋白Hp1188(recombinant Hp1188 protein, rHp1188),在体外和小鼠模型中评价其免疫特性及对H. pylori感染的免疫保护效果,以确定rHp1188在H. pylori疫苗研制中的应用价值。方法: ①构建重组质粒pQE30-hp1188,DNA测序分析正确后,转化大肠杆菌DH5α,IPTG诱导表达。Ni2+-NTA树脂纯化表达蛋白,SDS-PAGE分析其纯度,Bradford法测定其浓度,Western blot分析其抗原特异性,双向免疫扩散检测其效价。 ②以纯化的rHp1188为抗原,建立检测血清H.pylori Hp1188抗体的间接ELISA法,与科研诊断标准比较评价其应用价值。 ③建立H. pylori感染小鼠模型,在该模型中评价rHp1188与黏膜佐剂霍乱毒素B亚单位(cholera toxin subunit B,CTB)结合预防H. pylori感染的效果:ELISA法检测血清和小肠黏液中抗Hp1188抗体产生情况;胃组织H. pylori培养和组织切片Giemsa染色观察细菌定植量的改变;HE染色检查免疫后小鼠胃黏膜的病理学改变。结果: ①成功构建了重组质粒pQE30-hp1188,序列分析显示hp1188结构基因由810bp组成,与基因文库中的hp1188基因同源性达98%,开放读码框完整无中断,编码269个氨基酸。SDS-PAGE显示表达产物相对分子量约30kD,可溶性表达占全菌蛋白的47%以上,纯化后获得纯度达90%的rHp1188,浓度为1.0mg/ml。兔抗rHp1188血清双向免疫扩散检测抗体效价为1:16;Western bolt结果显示该蛋白可被兔抗H. pylori全菌抗体识别,在30kD附近出现反应条带。 ②对临床血清标本的ELISA检测结果显示,Hp1188抗体检测的敏感性和特异性分别为87.5%和86.7%。 ③预防组小鼠在血清和肠黏液中检测到高水平抗体(IgG、IgA);胃组织H. pylori培养阳性率、H. pylori定植及炎症程度均明显低于阳性对照组(P0.05)。预防组不仅可以减少H. pylori的定植,而且能减轻H. pylori造成的胃组织的局部炎症反应。结论: 成功构建了基因工程菌pQE30-hp1188-DH5α,并高效表达了rHp1188,表达蛋白具有良好的抗原性和免疫原性,在小鼠体内能阻止H. pylori的定植,有望作为H.pylori疫苗的一个备选抗原。
[Abstract]:Objective: to obtain recombinant adhesion protein Hp1188 (recombinant Hp1188 protein, rHp1188) by genetic engineering, which is closely related to the colonization of Helicobacter pylori (Helicobacter pylori, H.pylori) and exposed to bacterial surface. In vitro and in mice model, the immunological properties and the protective effect of rHp1188 on H. pylori infection were evaluated to determine the application value of rHp1188 in the development of H. pylori vaccine. Methods: 1 the recombinant plasmid pQE30-hp1188,DNA was constructed and analyzed correctly, then transformed into Escherichia coli DH5 伪, IPTG induced expression. Ni2 NTA resin was used to purify the expressed protein, SDS-PAGE was used to analyze its purity, and its concentration was determined by Bradford method. The antigen specificity was analyzed by Western blot and the titer was detected by two-way immunodiffusion. (2) using purified rHp1188 as antigen, an indirect ELISA method for detection of serum H.pylori Hp1188 antibody was established and compared with the diagnostic criteria of scientific research to evaluate its application value. (3) to establish a mouse model of H. pylori infection and evaluate the effect of rHp1188 combined with mucosal adjuvant cholerae B subunit (cholera toxin subunit) on the prevention of H. pylori infection: ELISA assay was used to detect the production of anti-Hp1188 antibodies in serum and intestinal mucus; The changes of bacterial colonization quantity were observed by Giemsa staining in gastric tissue and Giemsa staining in gastric tissue, and the pathological changes of gastric mucosa were detected by HE staining. Results: 1Recombinant plasmid pQE30-hp1188, sequence analysis showed that the hp1188 structure gene was composed of 810bp, which had 98 homology with the hp1188 gene in the gene library, and the open reading frame was intact without interruption. SDS-PAGE showed that the relative molecular weight of the expressed product was about 30kDand the soluble expression accounted for more than 47% of the total bacterial protein. The purified rHp1188, with purity of 90% was obtained and the concentration of rHp1188, was 1.0 mg / ml. The bidirectional immunodiffusion assay of rabbit anti-rHp1188 serum showed that the antibody titer was 1: 16. Western bolt showed that the protein could be recognized by rabbit anti-H. pylori antibody, and there were reaction bands near 30kD. 2 the sensitivity and specificity of Hp1188 antibody detection were 87.5% and 86.7%, respectively. 3High level antibody (IgG,IgA) was detected in serum and intestinal mucus of the mice in the prevention group, and the positive rate of, H. pylori colonization and inflammation in gastric tissue was significantly lower than that in the positive control group (P0.05). The prophylaxis group not only reduced the colonization of H. pylori, but also alleviated the local inflammation of gastric tissue caused by H. pylori. Conclusion: the genetically engineered strain pQE30-hp1188-DH5 伪 was successfully constructed, and the highly expressed rHp1188, protein had good antigenicity and immunogenicity. It could prevent the colonization of H. pylori in mice. It is expected to be an alternative antigen for H.pylori vaccine.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.11

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