【摘要】： 目的:選擇暴露于細(xì)菌表面并與幽門螺桿菌(Helicobacter pylori, H.pylori)定植密切相關(guān)的黏附素Hp1188為目標(biāo),通過(guò)基因工程技術(shù)獲得純化的重組黏附素蛋白Hp1188(recombinant Hp1188 protein, rHp1188),在體外和小鼠模型中評(píng)價(jià)其免疫特性及對(duì)H. pylori感染的免疫保護(hù)效果,以確定rHp1188在H. pylori疫苗研制中的應(yīng)用價(jià)值。 方法: ①構(gòu)建重組質(zhì)粒pQE30-hp1188,DNA測(cè)序分析正確后,轉(zhuǎn)化大腸桿菌DH5α,IPTG誘導(dǎo)表達(dá)。Ni2+-NTA樹脂純化表達(dá)蛋白,SDS-PAGE分析其純度,Bradford法測(cè)定其濃度,Western blot分析其抗原特異性,雙向免疫擴(kuò)散檢測(cè)其效價(jià)。 ②以純化的rHp1188為抗原,建立檢測(cè)血清H.pylori Hp1188抗體的間接ELISA法,與科研診斷標(biāo)準(zhǔn)比較評(píng)價(jià)其應(yīng)用價(jià)值。 ③建立H. pylori感染小鼠模型,在該模型中評(píng)價(jià)rHp1188與黏膜佐劑霍亂毒素B亞單位(cholera toxin subunit B,CTB)結(jié)合預(yù)防H. pylori感染的效果:ELISA法檢測(cè)血清和小腸黏液中抗Hp1188抗體產(chǎn)生情況;胃組織H. pylori培養(yǎng)和組織切片Giemsa染色觀察細(xì)菌定植量的改變;HE染色檢查免疫后小鼠胃黏膜的病理學(xué)改變。 結(jié)果: ①成功構(gòu)建了重組質(zhì)粒pQE30-hp1188,序列分析顯示hp1188結(jié)構(gòu)基因由810bp組成,與基因文庫(kù)中的hp1188基因同源性達(dá)98%,開放讀碼框完整無(wú)中斷,編碼269個(gè)氨基酸。SDS-PAGE顯示表達(dá)產(chǎn)物相對(duì)分子量約30kD,可溶性表達(dá)占全菌蛋白的47%以上,純化后獲得純度達(dá)90%的rHp1188,濃度為1.0mg/ml。兔抗rHp1188血清雙向免疫擴(kuò)散檢測(cè)抗體效價(jià)為1:16;Western bolt結(jié)果顯示該蛋白可被兔抗H. pylori全菌抗體識(shí)別,在30kD附近出現(xiàn)反應(yīng)條帶。 ②對(duì)臨床血清標(biāo)本的ELISA檢測(cè)結(jié)果顯示,Hp1188抗體檢測(cè)的敏感性和特異性分別為87.5%和86.7%。 ③預(yù)防組小鼠在血清和腸黏液中檢測(cè)到高水平抗體(IgG、IgA);胃組織H. pylori培養(yǎng)陽(yáng)性率、H. pylori定植及炎癥程度均明顯低于陽(yáng)性對(duì)照組(P0.05)。預(yù)防組不僅可以減少H. pylori的定植,而且能減輕H. pylori造成的胃組織的局部炎癥反應(yīng)。 結(jié)論: 成功構(gòu)建了基因工程菌pQE30-hp1188-DH5α,并高效表達(dá)了rHp1188,表達(dá)蛋白具有良好的抗原性和免疫原性,在小鼠體內(nèi)能阻止H. pylori的定植,有望作為H.pylori疫苗的一個(gè)備選抗原。
[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 偽, 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 偽 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.
【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】：2009
【分類號(hào)】：R392.11

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