本文選題：幽門(mén)螺桿菌 + DNA疫苗�。� 參考：《第三軍醫(yī)大學(xué)》2005年博士論文

【摘要】： 目的: 幽門(mén)螺桿菌(Helicobacter pylori,Hp)在人群中呈現(xiàn)高感染率,嚴(yán)重危害人類(lèi)健康。用Hp核酸疫苗預(yù)防Hp感染的報(bào)道不多,且效果不甚理想。本研究擬構(gòu)建殼聚糖包裹的Hp粘膜核酸疫苗,采用與抗原共表達(dá)細(xì)胞因子的方式來(lái)增強(qiáng)和調(diào)控免疫反應(yīng),同時(shí)觀察疫苗誘導(dǎo)的免疫應(yīng)答與抗Hp定植之間的關(guān)系,進(jìn)一步揭示疫苗可能的保護(hù)性機(jī)制。 方法: 1.以Hp標(biāo)準(zhǔn)株NCTC11637基因組為模板,PCR擴(kuò)增hpaA及ureB基因,亞克隆至pMD18-T載體,酶切并測(cè)序鑒定后,克隆至真核表達(dá)載體pTCAE,構(gòu)建pT-H和pT-U,并分別轉(zhuǎn)化DH5α。抽提質(zhì)粒電穿孔法轉(zhuǎn)染CHO細(xì)胞,間接免疫熒光法及免疫印跡法檢測(cè)目的蛋白的表達(dá)。分別將pT-H、pT-U注射小鼠股四頭肌,檢測(cè)血清中特異性IgG水平。 2. RT-PCR法從小鼠脾臟中擴(kuò)增GM-CSF、IL4基因,亞克隆至pMD18-T載體,酶切并測(cè)序鑒定后,將細(xì)胞因子基因分別插入pT-U中,構(gòu)建真核表達(dá)載體pT-GU、pT-IU。抽提質(zhì)粒電穿孔法轉(zhuǎn)染CHO細(xì)胞,免疫印跡法檢測(cè)GM-CSF、IL4的表達(dá),并通過(guò)細(xì)胞因子依賴細(xì)胞測(cè)活。 3.發(fā)酵工程菌DH5α(pT-U),采用裂菌  Q Sepharose XL粗純化  Source 30 Q精細(xì)純化的方法純化質(zhì)粒。經(jīng)復(fù)合物凝聚法制備、比較不同濃度殼聚糖和質(zhì)粒形成納米粒子的形狀、大小、穩(wěn)定性,確定適合的濃度搭配。 4.發(fā)酵工程菌,純化pT、pT-GU、pT-IU,并制備殼聚糖包裹的納米粒子。將裸質(zhì)粒pT-U和殼聚糖包裹的pT、pT-U、pT-GU、pT-IU分別滴鼻或口服免疫BALB/c小鼠,測(cè)定小鼠血清特異性IgG、IgG1、IgG2a、IgA、糞便抽提物的sIgA,胃組織RT-PCR檢測(cè)GAPDH、IFN-γ、IL10、mBD1、mBD3的表達(dá)水平。將殼聚糖包裹的pT、pT-U、pT-GU、pT-IU分別滴鼻或口服免疫沙鼠,末次免疫后2周灌喂109CFU Hp動(dòng)物適應(yīng)株, 4周后檢測(cè)Hp定植情況。 結(jié)果:
[Abstract]:Objective: Helicobacter pylori Helicobacter pylori (Helicobacter pylori) has a high infection rate in the population and seriously endangers human health. There are few reports on the prevention of HP infection with HP nucleic acid vaccine, and the effect is not satisfactory. The aim of this study was to construct a chitosan-encapsulated nucleic acid vaccine of HP mucous membrane to enhance and regulate the immune response by co-expressing cytokines with antigens, and to observe the relationship between the immune response induced by the vaccine and the anti-HP colonization. To further reveal the possible protective mechanism of vaccines. Methods: 1. HpaA and ureB genes were amplified by PCR from the NCTC11637 genome of HP standard strain and subcloned into pMD18-T vector. After restriction endonuclease digestion and sequencing, they were cloned into eukaryotic expression vector pTCAE, pT-H and pT-U were constructed and transformed into DH5 偽, respectively. The expression of target protein was detected by indirect immunofluorescence and Western blotting. The specific IgG level in serum was detected by injecting pT-HU into quadriceps femoris of mice. 2. The GM-CSFU IL4 gene was amplified from mouse spleen by RT-PCR and subcloned into pMD18-T vector. After restriction enzyme digestion and sequencing, cytokine genes were inserted into pT-U to construct eukaryotic expression vector pT-GUU pT-IU. The plasmid was electroporated and transfected into CHO cells, and the expression of GM-CSFffnil 4 was detected by immunoblotting, and cytokine dependent cell viability was determined. 3. The plasmids were purified by the fine purification method of DH5 偽 -pT-UX by Q Sepharose XL. The recombinant plasmid was purified by the fine purification method of Source 30Q. The shape, size and stability of nanoparticles formed by different concentrations of chitosan and plasmids were compared by the method of complex agglomeration, and the suitable concentration was determined. 4. The purified pTT-GUU-pT-IUS and chitosan-coated nanoparticles were prepared by fermentation engineering bacteria. BALB/c mice were immunized with naked plasmid pT-U and chitosan-encapsulated pTT-UPT-GUPT-IU, respectively. The serum specific IgG1 IgG1 IgG2aI IgA, the fecal extract Siga, and the expression level of GAPDHIFN- 緯 IL10mBD1mBD3 in gastric tissue were detected by RT-PCR. Chitosan-coated pTT-U pT-GUPT-IU was administered to gerbils by nasal drip or oral administration. 109CFU HP adaptation strain was infused 2 weeks after the last immunization, and HP colonization was detected 4 weeks later. Results:
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R392

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本文編號(hào)：1878622