本文關(guān)鍵詞： HIV-1疫苗 DNA疫苗 病毒樣顆粒疫苗 CTL:細(xì)胞毒性T細(xì)胞 ELISPOT:酶聯(lián)免疫斑點法 中和抗體　出處：《吉林大學(xué)》2007年博士論文　論文類型：學(xué)位論文

【摘要】： 自1981年發(fā)現(xiàn)第一例病例以來,艾滋病(AIDS)一直以驚人的速度在全球蔓延,成為世界上危害人類健康最嚴(yán)重的病毒性疾病,迫切需要研制安全、有效、價廉的艾滋病疫苗預(yù)防艾滋病病毒(HIV)的傳播,或降低HIV病毒載量,延緩HIV感染者發(fā)病。艾滋病疫苗必須通過科學(xué)設(shè)計的臨床試驗,考察其安全性、免疫原性及保護性。 隨著人類對艾滋病認(rèn)識的不斷提高,越來越多的證據(jù)顯示陽性CD8介導(dǎo)的CTL在控制艾滋病毒感染中起舉足輕重的作用,有效地誘導(dǎo)針對HIV-Gag等較保守區(qū)域的CTL是設(shè)計有效的艾滋病疫苗重要手段。另一方面,誘導(dǎo)產(chǎn)生有效的中和抗體是一個疫苗成功與否的重要指標(biāo),也是十幾年來艾滋病疫苗研究的主攻方向之一,這是因為中和抗體的產(chǎn)生在阻止病毒感染過程中起著關(guān)鍵的作用。 本研究首次利用兩種質(zhì)粒共轉(zhuǎn)染的方法,成功構(gòu)建并篩選出了高效、持續(xù)表達(dá)HIV-1結(jié)構(gòu)蛋白Gagpol和Env的哺乳動物細(xì)胞株,監(jiān)測了細(xì)胞培養(yǎng)基中病毒樣顆粒(virus like particles,VLPs)的分泌情況,分離純化后確立了成品的質(zhì)量控制標(biāo)準(zhǔn),檢測了VLPs誘導(dǎo)小鼠機體產(chǎn)生細(xì)胞免疫的能力和中和抗體的水平,同時完成了HIV-1病毒樣顆粒疫苗初步的安全性評價。結(jié)果表明:①獲得的高效表達(dá)HIV-1結(jié)構(gòu)蛋白Gagpol和Env的重組293細(xì)胞系能夠持續(xù)分泌兩種目的蛋白;②分泌在培養(yǎng)上清中的目的產(chǎn)物經(jīng)分離純化后,透射電鏡觀察其能夠組裝成病毒樣顆粒(VLP);③該病毒樣顆粒能夠誘導(dǎo)機體產(chǎn)生良好的體液免疫和細(xì)胞免疫水平;④安全性評價表明,HIV-1病毒樣顆粒疫苗無明顯的毒副作用。
[Abstract]:Since the first case was discovered in 1981, HIV / AIDS AIDShas been spreading at an alarming rate all over the world, and has become the most serious viral disease in the world, which urgently needs to be developed safely. Effective and inexpensive AIDS vaccine to prevent the spread of HIV / AIDS, or reduce the load of HIV virus, to delay the onset of HIV infection. AIDS vaccine must be scientifically designed clinical trials. Its safety, immunogenicity and protection were investigated. With the increasing awareness of AIDS, more and more evidence shows that CTL mediated by positive CD8 plays an important role in the control of HIV infection. Effective induction of CTL targeting more conservative regions such as HIV-Gag is an important means of designing an effective AIDS vaccine. Induction and production of effective neutralizing antibodies is an important indicator of the success of a vaccine, but also one of the main directions of AIDS vaccine research in the past ten years. This is because the production of neutralizing antibodies plays a key role in preventing virus infection. In this study, we successfully constructed and screened mammalian cell lines with high efficiency and persistent expression of HIV-1 structural proteins Gagpol and Env by two plasmid cotransfection methods. The secretion of virus-like granulovirus like particlesus VLPsin cell culture medium was monitored and the quality control standard of the finished product was established after isolation and purification. The ability of VLPs to induce cellular immunity and the level of neutralizing antibody in mice were measured. At the same time, the preliminary safety evaluation of HIV-1 virus-like granular vaccine was completed. The results showed that:. 1Recombinant 293 cell line which highly expressed HIV-1 structural protein Gagpol and Env could continuously secrete two kinds of target proteins; (2) the target product secreted in the culture supernatant was isolated and purified, and it was observed by transmission electron microscope that the product could be assembled into virus-like particles. (3) the virus-like particles can induce good humoral and cellular immunity; 4 the safety evaluation showed that the HIV-1 vaccine had no obvious side effects.
【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】：2007
【分類號】：R392

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