【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價其免疫特性及對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)用價值。 方法: ①構(gòu)建重組質(zhì)粒pQE30-hp1188,DNA測序分析正確后,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法測定其濃度,Western blot分析其抗原特異性,雙向免疫擴散檢測其效價。 ②以純化的rHp1188為抗原,建立檢測血清H.pylori Hp1188抗體的間接ELISA法,與科研診斷標(biāo)準(zhǔn)比較評價其應(yīng)用價值。 ③建立H. pylori感染小鼠模型,在該模型中評價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,與基因文庫中的hp1188基因同源性達(dá)98%,開放讀碼框完整無中斷,編碼269個氨基酸。SDS-PAGE顯示表達(dá)產(chǎn)物相對分子量約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血清雙向免疫擴散檢測抗體效價為1:16;Western bolt結(jié)果顯示該蛋白可被兔抗H. pylori全菌抗體識別,在30kD附近出現(xiàn)反應(yīng)條帶。 ②對臨床血清標(biāo)本的ELISA檢測結(jié)果顯示,Hp1188抗體檢測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到高水平抗體(IgG、IgA);胃組織H. pylori培養(yǎng)陽性率、H. pylori定植及炎癥程度均明顯低于陽性對照組(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)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392.11

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 韓飛;楊致邦;;幽門螺桿菌hp1188基因的原核表達(dá)[J];重慶醫(yī)科大學(xué)學(xué)報;2010年06期

2 韓飛;楊致邦;;幽門螺桿菌重組1188蛋白的表達(dá)[J];重慶醫(yī)學(xué);2011年03期

3 曾慶鎰;癌基因(Oncogenes)及其表達(dá)產(chǎn)物[J];腫瘤;1985年01期

4 張平，鮑依稀，康格非;癌基因及其表達(dá)產(chǎn)物──新一代腫瘤標(biāo)志物[J];重慶醫(yī)科大學(xué)學(xué)報;1994年02期

5 丁建中;;乙型肝炎病毒基因及表達(dá)產(chǎn)物研究進(jìn)展[J];湖北省衛(wèi)生職工醫(yī)學(xué)院學(xué)報;1992年02期

6 方政,余新炳,劉彥文,羅樹紅;惡性瘧原蟲FCC1/HN株CSP抗原基因表達(dá)產(chǎn)物在小鼠體內(nèi)的免疫應(yīng)答研究[J];中國寄生蟲學(xué)與寄生蟲病雜志;1999年06期

7 蔡亮;楊秋林;伍和平;劉傳愛;王可耕;張愉快;;弓形蟲RH株膜蛋白P30的原核表達(dá)與鑒定[J];中國血吸蟲病防治雜志;2006年05期

8 彭佳;王國華;陳坤;宋曉國;張賀秋;周見遠(yuǎn);何競;;核心鏈霉親和素基因的克隆及其在大腸埃希氏菌中的表達(dá)[J];現(xiàn)代檢驗醫(yī)學(xué)雜志;2008年04期

9 周志成;孫莉;吳英松;廖小青;郝文波;;間日瘧原蟲乳酸脫氫酶GST融合表達(dá)載體的構(gòu)建及表達(dá)[J];分子診斷與治療雜志;2010年02期

10 張丹;朱中元;王海波;;結(jié)核分枝桿菌CFP-10蛋白的克隆及表達(dá)[J];中國熱帶醫(yī)學(xué);2011年10期

相關(guān)會議論文 前10條

1 余子全;孫明;喻子牛;;蘇云金芽胞桿菌中殺線蟲晶體蛋白基因cry6A的負(fù)表達(dá)調(diào)控[A];第二屆中國青年學(xué)者微生物遺傳學(xué)學(xué)術(shù)研討會論文集[C];2006年

2 陳玉飛;祁克宗;彭開松;涂健;;雞β-防御素-8 cDNA的克隆及在畢赤酵母中的表達(dá)[A];中國畜牧獸醫(yī)學(xué)會獸醫(yī)病理學(xué)分會第十六次學(xué)術(shù)研討會、中國病理生理學(xué)會動物病理生理專業(yè)委員會第十五次學(xué)術(shù)研討會論文集[C];2009年

3 張錦秀;晁生玉;史利軍;候紹華;李剛;;豬圓環(huán)病毒Ⅱ型修飾Cap蛋白基因的原核表達(dá)及純化[A];中國畜牧獸醫(yī)學(xué)會家畜傳染病學(xué)分會第七屆全國會員代表大會暨第十三次學(xué)術(shù)研討會論文集（上冊）[C];2009年

4 廖富，

本文編號：2379803