本文選題：b型流感嗜血桿菌D蛋白　切入點：原核表達　出處：《暨南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：腦膜炎奈瑟雙球菌(Neisseria Meningitidis)是唯一能引起全球性細菌性腦膜炎大規(guī)模流行的細菌,疫苗接種是用于預(yù)防的主要手段,但是傳統(tǒng)的多糖疫苗產(chǎn)生抗體能力不足,對2歲以下的嬰幼兒更無法產(chǎn)生保護作用。實驗證明用細菌多糖和蛋白載體通過化學(xué)方法制備的結(jié)合疫苗,能有效克服多糖疫苗免疫時的各種缺點,誘導(dǎo)產(chǎn)生細胞免疫和加強免疫應(yīng)答,并且對所有年齡組的人群都能夠提供免疫保護作用�，F(xiàn)在國內(nèi)上市的結(jié)合疫苗大都使用破傷風類毒素作為載體,但其具有一定的局限性,可能會出現(xiàn)毒性回復(fù)或是產(chǎn)生載體抑制效應(yīng)等問題,所以尋找安全、有效的載體是結(jié)合疫苗領(lǐng)域的課題之一。流感嗜血桿菌D蛋白具有良好的免疫原性和安全性,并且作為疫苗載體己應(yīng)用于十價的肺炎結(jié)合苗中,在國外上市。目前,國內(nèi)尚沒有以D蛋白為載體疫苗的相關(guān)報道。 本文通過對b型流感嗜血桿菌D蛋白基因進行擴增,構(gòu)建重組質(zhì)粒pET30a/hpd并轉(zhuǎn)化進入大腸桿菌DH5a中,經(jīng)篩選保存陽性克隆菌,再轉(zhuǎn)化入大腸桿菌BL21(DE3),IPTG誘導(dǎo),并使重組D蛋白以包涵體形式表達,表達量約占菌體總蛋白的40%；經(jīng)一步過柱純化可得到純度達95%的重組D蛋白。 本文采用CDAP法活化A群和C群腦膜炎多糖(GAMP和GCMP),在三乙胺存在的條件下,制備A群和C群腦膜炎多糖和D蛋白結(jié)合物,并通過Sepharose 4FF柱層析,對結(jié)合物組分進行多糖和蛋白含量的測定。 本文將GAMP, GCMP, GAMP-D, GCMP-D, GAMP+D和GCMP+D混合物分別經(jīng)皮下免疫接種Balb/c小鼠,通過間接ELISA法測定小鼠體內(nèi)A群和C群腦膜炎球菌多糖特異的IgG, IgG1, IgG2a抗體水平,結(jié)果表明接種后結(jié)合物誘生的多糖特異性IgG, IgG1, IgG2a抗體水平顯著高于不結(jié)合組,初步證明重組D蛋白能夠起到載體的作用。 本研究采用分子克隆技術(shù)獲得了能夠產(chǎn)生重組D蛋白的菌株,并將純化的重組蛋白作為載體與A群和C群腦膜炎球菌多糖結(jié)合,動物實驗證明結(jié)合物具有良好的免疫原性,為進一步以重組D蛋白為載體制備其它結(jié)合疫苗奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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