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  本文關(guān)鍵詞：HIV-1治療性重組DNA疫苗的構(gòu)建、純化及實驗免疫研究　出處：《吉林大學(xué)》2005年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 新型 HIV 治療 重組DNA 疫苗 構(gòu)建 純化 免疫

【摘要】：本論文利用分子生物學(xué)、免疫學(xué)、分子病毒學(xué)、生物化學(xué)、生物信息學(xué)等技術(shù)和手段進(jìn)行了新型治療性HIV-1 重組核酸疫苗的構(gòu)建、純化及實驗免疫學(xué)研究,內(nèi)容概述如下: 本實驗以選擇HIV-1 優(yōu)勢抗原表位為原則,提出以HIV-1 表位基因為基礎(chǔ),以HIV-1 巨分子顆粒p24 為支架載體分子,對抗原基因進(jìn)行人工分子設(shè)計,并輔以計算機模擬來研制治療性HIV 基因工程疫苗的新思路。該種HIV DNA 疫苗的設(shè)計在國內(nèi)外尚未見報道。將經(jīng)分子設(shè)計的疫苗抗原的核苷酸序列進(jìn)行人工合成,獲得新型HIV-1 治療性抗原基因(MEG);以PCR 法獲得HIV-1 衣殼蛋白基因p24,將其插入到MEG 基因中,獲得嵌合基因MEGp24。利用新型安全性真核表達(dá)載體質(zhì)粒pVAXI,構(gòu)建了新型HIV-1 治療性重組DNA 疫苗(pVAXI-MEG-p24),經(jīng)過哺乳動物細(xì)胞轉(zhuǎn)染結(jié)果表明,pVAXI-MEG-p24 成功地表達(dá)了MEGp24 抗原蛋白。 將已構(gòu)建好的治療性HIV-1 重組DNA 疫苗的質(zhì)粒(pVAXI-MEG-p24),經(jīng)發(fā)酵罐建立發(fā)酵工藝,通過陰離子交換層析方法(Q Sepharose XL)進(jìn)行純化,隨后將得到的質(zhì)粒DNA 通過排阻層析方法進(jìn)行進(jìn)一步的純化及脫鹽,濃縮。經(jīng)過質(zhì)量鑒定結(jié)果表明,該純化質(zhì)粒疫苗符合FDA 推薦的臨床用質(zhì)粒DNA 的質(zhì)量標(biāo)準(zhǔn),表明所建立的工藝是可行。 純化后的重組DNA 疫苗質(zhì)粒pVAX1-MEG-p24 進(jìn)行實驗免疫研究表明,純化后HIV-1 重組DNA 疫苗使小鼠和恒河猴,產(chǎn)生了特異性體液和細(xì)胞免疫應(yīng)答;治療性HIV-1 重組DNA 疫苗(pVAXI-MEG-p24)與HIV 全結(jié)構(gòu)蛋白基因疫苗(pVAXI-gag-gp120)比較研究表明,pVAXI-MEG-p24 能夠誘發(fā)比全結(jié)構(gòu)蛋白基因疫苗更強的表位特異性CTL 反應(yīng)和抗體反應(yīng)。 總之,本研究以新的研究思路,對HIV 治療性重組核酸疫苗的構(gòu)建進(jìn)行了
[Abstract]:In this paper, a novel therapeutic HIV-1 recombinant nucleic acid vaccine was constructed using molecular biology, immunology, molecular virology, biochemistry, bioinformatics and other techniques and methods. Purification and experimental immunological studies are summarized as follows: Based on the selection of HIV-1 epitopes, this study proposed that HIV-1 giant particle p24 should be used as scaffold carrier molecule based on HIV-1 epitope gene. Artificial molecular design of antigen gene. A new idea of developing therapeutic HIV gene engineering vaccine with computer simulation was also presented. The HIV DNA. The design of vaccine has not been reported at home and abroad. The nucleotide sequence of vaccine antigen designed by molecule was synthesized. A novel HIV-1 therapeutic antigen gene was obtained. HIV-1 capsid protein gene p24 was obtained by PCR and inserted into MEG gene. The chimeric gene MEGp24. using a novel safe eukaryotic expression vector plasmid pVAXI was obtained. A novel HIV-1 therapeutic recombinant DNA vaccine pVAXI-MEG-p24 was constructed and transfected into mammalian cells. PVAXI-MEG-p24 successfully expressed MEGp24 antigen protein. The plasmid pVAXI-MEG-p24 of therapeutic HIV-1 recombinant DNA vaccine was constructed and the fermentation process was established. The plasmid DNA was purified by anion exchange chromatography and then further purified and desalted by exclusion chromatography. The results of quality identification show that the purified plasmid vaccine meets the quality standard of clinical plasmid DNA recommended by FDA, and the established technology is feasible. The purified recombinant DNA vaccine plasmid pVAX1-MEG-p24 was used for experimental immunological study. The results showed that the purified HIV-1 recombinant DNA vaccine made mice and rhesus monkeys. Produced specific humoral and cellular immune responses; The comparative study of therapeutic HIV-1 recombinant DNA vaccine pVAXI-MEG-p24 and pVAXI-gag-gp120) showed that pVAXI-MEG-p24 and pVAXI-gag-gp120). PVAXI-MEG-p24 can induce a stronger epitope specific CTL reaction and antibody response than the whole structure protein gene vaccine. In conclusion, the construction of HIV therapeutic recombinant nucleic acid vaccine was carried out with new research ideas.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R392.1

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 付延軍;含塞姆利基森林病毒復(fù)制子的HIV多表位核酸疫苗的構(gòu)建與表達(dá)[D];延邊大學(xué);2007年

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本文編號：1384530