【摘要】：一個(gè)成功的HIV-1 疫苗應(yīng)該既能產(chǎn)生有效的中和抗體又能產(chǎn)生持久的細(xì)胞免疫,同時(shí)又要具有良好的安全性。傳統(tǒng)的HIV-1 疫苗總是存在著這樣或那樣的欠缺,而VLP 疫苗以其安全性高、抗原性強(qiáng)等優(yōu)點(diǎn)為HIV-1 疫苗的研究提供了一個(gè)新的思路。 本論文利用哺乳動(dòng)物細(xì)胞系統(tǒng)構(gòu)建HIV-1 病毒樣顆粒(VLP)疫苗。該疫苗表達(dá)HIV-1 的主要結(jié)構(gòu)蛋白gagpol 和env,并研究其免疫原性,為其成為新一代艾滋病的治療性疫苗提供重要依據(jù)。 主要采用共轉(zhuǎn)染及G418 抗性篩選的方法篩選單克隆細(xì)胞株,通過(guò)Western-blot 檢測(cè)蛋白表達(dá)情況;經(jīng)蔗糖墊密度梯度離心檢測(cè)上清中的VLP 的分泌情況;SDS-PAGE 電泳及LOWRY 法初步確定分泌濃度;透射電鏡觀察VLP 的形態(tài);免疫印跡及ELISA、CTL 及ELISPOT、細(xì)胞增殖等實(shí)驗(yàn)檢測(cè)其動(dòng)物體液及細(xì)胞免疫情況。 結(jié)果成功篩選出了高效、穩(wěn)定表達(dá)HIV-1 結(jié)構(gòu)蛋白gagpol 和env 的哺乳動(dòng)物細(xì)胞株,該細(xì)胞株表達(dá)的結(jié)構(gòu)蛋白可自我組裝成病毒樣顆粒(VLP)并分泌至培養(yǎng)上清中,透射電鏡觀察可見(jiàn)典型病毒樣顆粒,該VLP(不加任何佐劑)在動(dòng)物實(shí)驗(yàn)中表現(xiàn)了很好的免疫原性。在實(shí)驗(yàn)進(jìn)程中分三級(jí)凍存了細(xì)胞種子庫(kù),初步確定了疫苗的質(zhì)量控制標(biāo)準(zhǔn),為今后的大規(guī)模疫苗生產(chǎn)打下了堅(jiān)實(shí)的基礎(chǔ)。 上述結(jié)果表明HIV-1 結(jié)構(gòu)蛋白gagpol 和env 能夠在哺乳動(dòng)物細(xì)胞中高效、穩(wěn)定表達(dá),并能自我組裝成VLP 分泌至上清中,動(dòng)物實(shí)驗(yàn)證明了該HIV-1VLP 具有良好的免疫原性,可望成為新一代HIV-1 疫苗。
[Abstract]:A successful HIV-1 vaccine should produce both an effective neutralizing antibody and a sustained cellular immunity with good safety. The traditional HIV-1 vaccine is always lacking in one way or another, but the VLP vaccine provides a new idea for the research of HIV-1 vaccine because of its high safety and strong antigenicity. In this paper, HIV-1 virus-like granulocyte (VLP) vaccine was constructed by mammalian cell system. This vaccine expresses the main structural proteins of HIV-1, gagpol and env. and studies its immunogenicity, which provides an important basis for the new generation of AIDS therapeutic vaccine. The monoclonal cell lines were screened by co-transfection and G418 resistance screening, the protein expression was detected by Western-blot, the secretion of VLP in the supernatant was detected by density gradient centrifugation with sucrose pad, and the secretory concentration was determined by SDS-PAGE and LOWRY method. The morphology of VLP was observed by transmission electron microscope, and the humoral and cellular immunity was detected by immunoblotting, ELISAL, Elispot, cell proliferation and so on. Results mammalian cell lines with high efficiency and stable expression of HIV-1 structural proteins gagpol and env were successfully screened. The expressed structural proteins could self-assemble into virus-like particles (VLP) and secreted into the culture supernatant. The typical virus-like particles were observed by transmission electron microscope (TEM). The VLP (without any adjuvant) showed good immunogenicity in animal experiments. In the course of experiment, the cell seed bank was frozen in three stages, and the quality control standard of vaccine was preliminarily determined, which laid a solid foundation for the large-scale vaccine production in the future. These results suggest that HIV-1 structural proteins gagpol and env can be expressed efficiently and stably in mammalian cells and can self-assemble into VLP secreting supernatant. Animal experiments have proved that the HIV-1VLP has good immunogenicity and is expected to be a new HIV-1 vaccine.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 吳芬芳;新城疫病毒樣顆粒免疫效力的初步研究[D];新疆農(nóng)業(yè)大學(xué);2007年

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本文編號(hào)：2175549