本文關(guān)鍵詞： 惡性瘧疾 DNA疫苗 基因槍 活體電穿孔 脂質(zhì)體肌肉注射 免疫原性　出處：《中國(guó)協(xié)和醫(yī)科大學(xué)》2007年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 瘧疾是目前世界上對(duì)人類(lèi)危害最嚴(yán)重的蚊媒寄生原蟲(chóng)病。全世界每年有5億人感染瘧原蟲(chóng)，約三百萬(wàn)人死于惡性瘧疾，其中80％是五歲以下的兒童和孕婦。惡性瘧原蟲(chóng)及蚊媒抗藥性的不斷增加和蔓延，使得瘧疾的防治成為世界性難題之一。瘧原蟲(chóng)生活周期復(fù)雜，抗原具有階段特異性、蟲(chóng)株特異性和高度變異性等特點(diǎn)，長(zhǎng)期以來(lái)形成研制抗瘧原蟲(chóng)疫苗的技術(shù)瓶頸。因此，針對(duì)惡性瘧原蟲(chóng)不同生活時(shí)期的免疫反應(yīng)特點(diǎn)，選取誘導(dǎo)產(chǎn)生多種免疫反應(yīng)類(lèi)型的不同抗原構(gòu)建多表位疫苗已經(jīng)成為研究抗瘧疫苗的熱點(diǎn)。 本課題組在前期工作中借鑒了分子育種技術(shù)(即DNA改組)的隨機(jī)重組原理，建立了表位改組技術(shù)并構(gòu)建了多表位基因庫(kù)，用庫(kù)免疫血清篩選出高抗原性的陽(yáng)性克隆，經(jīng)實(shí)驗(yàn)證實(shí)其中VR312(后改名為M．RCAg-1)基因克隆及其編碼蛋白不僅能在小鼠模型中誘導(dǎo)交叉免疫保護(hù)，而且能在家兔模型中誘導(dǎo)抑制抗惡性瘧原蟲(chóng)生長(zhǎng)的抗體。 由于蛋白質(zhì)疫苗成本昂貴，不適宜在非洲等落后地區(qū)推廣使用；單一DNA疫苗的免疫原性較低，難以達(dá)到預(yù)期的免疫反應(yīng)強(qiáng)度和令人滿意的免疫保護(hù)效果。因此本研究試圖采用不同的免疫策略以觀察對(duì)DNA疫苗免疫原性的影響，并分析其免疫反應(yīng)機(jī)制，重點(diǎn)觀察了基因槍免疫中不同粒徑金顆粒對(duì)免疫佐劑效應(yīng)的影響。初步證實(shí)將惡性瘧疾多表位DNA疫苗M．RCAg-1D通過(guò)基因槍皮內(nèi)免疫小鼠，能夠獲得高滴度特異性抗體水平，誘生明顯的CD4~+T細(xì)胞反應(yīng)，而且在用P．yoelii攻擊時(shí)能明顯保護(hù)小鼠延長(zhǎng)其存活時(shí)間。提示基因槍皮內(nèi)免疫能夠顯著提高基因疫苗的免疫原性，這可能與其提高外源基因的轉(zhuǎn)染及表達(dá)有關(guān)。與此同時(shí)，應(yīng)用活體電穿孔免疫小鼠，也能夠獲得較高滴度特異性抗體水平和誘生明顯的CD4~+T細(xì)胞反應(yīng)，但是不能對(duì)P．yoelii產(chǎn)生交叉保護(hù)。而脂質(zhì)體混合基因疫苗M．RCAg-1DDNA后肌肉注射所誘導(dǎo)產(chǎn)生的免疫原性盡管顯著高于裸DNA肌肉注射和裸DNA皮下注射，但是誘導(dǎo)產(chǎn)生的抗體不能對(duì)P．yoelii產(chǎn)生交叉免疫保護(hù)。基因槍免疫時(shí)誘導(dǎo)產(chǎn)生的抗體水平受到金粉粒徑的影響，其中1.0μm金顆粒作為質(zhì)粒DNA載體進(jìn)行基因免疫時(shí)，在小鼠體內(nèi)誘導(dǎo)產(chǎn)生的特異性抗體水平最高。以上工作對(duì)DNA疫苗的應(yīng)用和改進(jìn)均提供了有價(jià)值的實(shí)驗(yàn)依據(jù)。
[Abstract]:Malaria is the most serious mosquito - borne protozoan disease in the world . More than 500 million people are infected with Plasmodium falciparum every year , about 3 million people die from malignant malaria , and 80 percent of them are children and pregnant women under the age of five . Based on the principle of random recombination of molecular breeding technology ( i.e . , DNA shuffling ) in the previous work , this group established the epitope shuffling technique and constructed a multi - epitope gene pool , screened the positive clones with high antigenicity by using the library immune serum , and confirmed the VR312 ( renamed as M ) . RCAg - 1 ( RCAg - 1 ) gene clone and its encoded protein not only induce cross - immune protection in mouse model , but also induce anti - P . falciparum growth antibody in rabbit model . Because the cost of protein vaccine is expensive , it is not suitable to be popularized in such areas as Africa and so on . The immunogenicity of single DNA vaccine is low , it is difficult to achieve the expected immune response intensity and the satisfactory immune protection effect . Therefore , the present study attempts to adopt different immune strategies to observe the effect of different particle size gold particles on the immune adjuvant effect of DNA vaccine . RCAg - 1D was able to obtain high titer specific antibody level and induce obvious CD4 ~ + T cell reaction . It was suggested that the immunity of gene vaccine could significantly improve the immunogenicity of the gene vaccine , which could enhance the immunogenicity of the gene vaccine . At the same time , it was possible to obtain high titer specific antibody level and induce obvious CD4 ~ + T cell response when challenged with P.yoelii , but it was not possible to cross - protect P.yoelii . The antibody level induced by intramuscular injection after RCAg - 1DDNA was significantly higher than that of naked DNA intramuscular injection and naked DNA injection , but the antibody produced by induction could not cross - immune protection against P.yoelii . The antibody level induced by induction of gene gun was affected by the particle size of gold powder .

【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 吳巍;藺亞暉;馬瑞森;席玨敏;王恒;;電穿孔法提高惡性瘧疾DNA疫苗免疫原性[J];中國(guó)生物醫(yī)學(xué)工程學(xué)報(bào);2011年01期

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本文編號(hào)：1461913