幽门螺杆菌粘膜核酸疫苗的制备及免疫学效应研究

发布时间：2018-05-12 12:28

本文选题：幽门螺杆菌 + DNA疫苗　；参考：《第三军医大学》2005年博士论文

【摘要】： 目的: 幽门螺杆菌(Helicobacter pylori,Hp)在人群中呈现高感染率,严重危害人类健康。用Hp核酸疫苗预防Hp感染的报道不多,且效果不甚理想。本研究拟构建壳聚糖包裹的Hp粘膜核酸疫苗,采用与抗原共表达细胞因子的方式来增强和调控免疫反应,同时观察疫苗诱导的免疫应答与抗Hp定植之间的关系,进一步揭示疫苗可能的保护性机制。方法: 1.以Hp标准株NCTC11637基因组为模板,PCR扩增hpaA及ureB基因,亚克隆至pMD18-T载体,酶切并测序鉴定后,克隆至真核表达载体pTCAE,构建pT-H和pT-U,并分别转化DH5α。抽提质粒电穿孔法转染CHO细胞,间接免疫荧光法及免疫印迹法检测目的蛋白的表达。分别将pT-H、pT-U注射小鼠股四头肌,检测血清中特异性IgG水平。 2. RT-PCR法从小鼠脾脏中扩增GM-CSF、IL4基因,亚克隆至pMD18-T载体,酶切并测序鉴定后,将细胞因子基因分别插入pT-U中,构建真核表达载体pT-GU、pT-IU。抽提质粒电穿孔法转染CHO细胞,免疫印迹法检测GM-CSF、IL4的表达,并通过细胞因子依赖细胞测活。 3.发酵工程菌DH5α(pT-U),采用裂菌 Q Sepharose XL粗纯化 Source 30 Q精细纯化的方法纯化质粒。经复合物凝聚法制备、比较不同浓度壳聚糖和质粒形成纳米粒子的形状、大小、稳定性,确定适合的浓度搭配。 4.发酵工程菌,纯化pT、pT-GU、pT-IU,并制备壳聚糖包裹的纳米粒子。将裸质粒pT-U和壳聚糖包裹的pT、pT-U、pT-GU、pT-IU分别滴鼻或口服免疫BALB/c小鼠,测定小鼠血清特异性IgG、IgG1、IgG2a、IgA、粪便抽提物的sIgA,胃组织RT-PCR检测GAPDH、IFN-γ、IL10、mBD1、mBD3的表达水平。将壳聚糖包裹的pT、pT-U、pT-GU、pT-IU分别滴鼻或口服免疫沙鼠,末次免疫后2周灌喂109CFU Hp动物适应株, 4周后检测Hp定植情况。结果:
[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 伪, 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 伪 -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- 纬 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:
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

【参考文献】