b型流感嗜血杆菌D蛋白的克

发布时间：2018-03-20 12:04

本文选题：b型流感嗜血杆菌D蛋白　切入点：原核表达　出处：《暨南大学》2011年硕士论文　论文类型：学位论文

【摘要】：脑膜炎奈瑟双球菌(Neisseria Meningitidis)是唯一能引起全球性细菌性脑膜炎大规模流行的细菌,疫苗接种是用于预防的主要手段,但是传统的多糖疫苗产生抗体能力不足,对2岁以下的婴幼儿更无法产生保护作用。实验证明用细菌多糖和蛋白载体通过化学方法制备的结合疫苗,能有效克服多糖疫苗免疫时的各种缺点,诱导产生细胞免疫和加强免疫应答,并且对所有年龄组的人群都能够提供免疫保护作用。现在国内上市的结合疫苗大都使用破伤风类毒素作为载体,但其具有一定的局限性,可能会出现毒性回复或是产生载体抑制效应等问题,所以寻找安全、有效的载体是结合疫苗领域的课题之一。流感嗜血杆菌D蛋白具有良好的免疫原性和安全性,并且作为疫苗载体己应用于十价的肺炎结合苗中,在国外上市。目前,国内尚没有以D蛋白为载体疫苗的相关报道。本文通过对b型流感嗜血杆菌D蛋白基因进行扩增,构建重组质粒pET30a/hpd并转化进入大肠杆菌DH5a中,经筛选保存阳性克隆菌,再转化入大肠杆菌BL21(DE3),IPTG诱导,并使重组D蛋白以包涵体形式表达,表达量约占菌体总蛋白的40%；经一步过柱纯化可得到纯度达95%的重组D蛋白。本文采用CDAP法活化A群和C群脑膜炎多糖(GAMP和GCMP),在三乙胺存在的条件下,制备A群和C群脑膜炎多糖和D蛋白结合物,并通过Sepharose 4FF柱层析,对结合物组分进行多糖和蛋白含量的测定。本文将GAMP, GCMP, GAMP-D, GCMP-D, GAMP+D和GCMP+D混合物分别经皮下免疫接种Balb/c小鼠,通过间接ELISA法测定小鼠体内A群和C群脑膜炎球菌多糖特异的IgG, IgG1, IgG2a抗体水平,结果表明接种后结合物诱生的多糖特异性IgG, IgG1, IgG2a抗体水平显著高于不结合组,初步证明重组D蛋白能够起到载体的作用。本研究采用分子克隆技术获得了能够产生重组D蛋白的菌株,并将纯化的重组蛋白作为载体与A群和C群脑膜炎球菌多糖结合,动物实验证明结合物具有良好的免疫原性,为进一步以重组D蛋白为载体制备其它结合疫苗奠定了基础。
[Abstract]:Neisseria Meningitidisis (Neisseria Meningitidisis) is the only bacterium that can cause a global epidemic of bacterial meningitis. Vaccination is the main means of prevention, but the traditional polysaccharide vaccine has insufficient ability to produce antibodies. The experiment proved that the conjugated vaccine prepared by chemical method with bacterial polysaccharide and protein carrier can effectively overcome the shortcomings of polysaccharide vaccine immunization. To induce cellular immunity and enhance immune response, and to provide immune protection for all age groups. Nowadays, tetanus toxoid toxoid is widely used as a carrier of conjugated vaccines in China, but it has some limitations. There may be some problems such as toxicity recovery or vector inhibition. Therefore, finding a safe and effective vector is one of the topics in the field of vaccine. Haemophilus influenzae D protein has good immunogenicity and safety. And as a vaccine vector has been used in the decavalent pneumonia conjugate vaccine, listed in foreign countries. At present, there are no related reports of D protein vector vaccine in China. By amplifying the D protein gene of Haemophilus influenzae type b, the recombinant plasmid pET30a/hpd was constructed and transformed into Escherichia coli DH5a. The recombinant D protein was expressed in the form of inclusion body, which accounted for about 40% of the total bacterial protein, and the recombinant D protein with purity of 95% was obtained by one step column purification. In this paper, group A and group C meningitis polysaccharides were activated by CDAP method. In the presence of triethylamine, group A and group C meningitis polysaccharides and D-protein conjugates were prepared, and then purified by Sepharose 4FF column chromatography. The contents of polysaccharides and proteins were determined. The mixture of GAMPG, GCMP, GAMP-D, GCMP-D, GAMP D and GCMP D were inoculated subcutaneously into Balb/c mice. The specific IgG, IgG1, IgG2a antibody levels of group A and group C meningococcal polysaccharides in mice were determined by indirect ELISA method. The results showed that the levels of polysaccharides specific IgG, IgG1, IgG2a antibody induced by conjugate after inoculation were significantly higher than those of unconjugated group. It was preliminarily proved that recombinant D protein could act as a vector. In this study, the recombinant D protein was obtained by molecular cloning technique, and the purified recombinant protein was used as carrier to bind to group A and group C meningococcal polysaccharides. The animal experiments showed that the conjugate had good immunogenicity. It lays a foundation for further preparation of other conjugated vaccines using recombinant D protein as vector.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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