【摘要】：本研究目的是通過優(yōu)化1型人類免疫缺陷病毒(HIV-1)gp140編碼基因的密碼子和克隆設(shè)計(jì),從而實(shí)現(xiàn)在293T哺乳動(dòng)物細(xì)胞中高效表達(dá),并對(duì)純化獲得的gp140蛋白進(jìn)行抗原性質(zhì)鑒定。在本研究中選擇HIV-1B亞型NL4-3全基因序列為模板進(jìn)行g(shù)p140克隆構(gòu)建,通過密碼子優(yōu)化、信號(hào)肽替換、增加柔性linker、三聚體折疊序列等方法優(yōu)化設(shè)計(jì)。通過HIV-1轉(zhuǎn)錄反式激活因子tat共轉(zhuǎn)HEK293T細(xì)胞進(jìn)行g(shù)p140蛋白表達(dá),采用鎳柱純化。SDS-PAGE、Western blot、ELISA、負(fù)染電鏡等結(jié)果顯示目的蛋白純度高于70%,每升培養(yǎng)基可獲得0.5mg gp140蛋白,并且具有良好的抗原活性,電鏡下呈現(xiàn)三聚體結(jié)構(gòu)。通過弗氏佐劑與目的蛋白混合免疫Balb/c小鼠,檢測(cè)小鼠免疫血清顯示gp140蛋白能有效刺激機(jī)體產(chǎn)生免疫應(yīng)答。本研究通過優(yōu)化表達(dá)獲得B亞型HIV-1NL4-3gp140蛋白,為HIV-1病毒包膜蛋白結(jié)構(gòu)和重組疫苗研究奠定基礎(chǔ)。
[Abstract]:The aim of this study was to optimize the codon and clone design of the gp140 encoding gene of human immunodeficiency virus (HIV-1) type 1, so as to express it efficiently in 293T mammalian cells and to identify the antigenic properties of the purified gp140 protein. In this study, the whole NL4-3 gene sequence of HIV-1B subtype was selected as the template to construct gp140 clone. Codon optimization, signal peptide replacement and flexible linker, trimer folding sequence were used to optimize the design. The gp140 protein was expressed by HIV-1 transactivator tat co-transfected HEK293T cells and purified by nickel column. The results of SDS-PAGE,Western blot,ELISA, negative staining electron microscopy showed that the purity of the target protein was higher than 70%. 0.5mg gp140 protein was obtained from each liter of culture medium and had good antigenic activity, which showed trimer structure under electron microscope. Balb/c mice were immunized with Freund's adjuvant and target protein. The results showed that gp140 protein could stimulate immune response effectively. In this study, subtype B HIV-1NL4-3gp140 protein was obtained by optimizing expression, which laid a foundation for the study of HIV-1 virus envelope protein structure and recombinant vaccine.
【作者單位】： 廈門大學(xué)公共衛(wèi)生學(xué)院分子疫苗學(xué)和分子診斷學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室;廈門大學(xué)生命科學(xué)學(xué)院國(guó)家傳染病診斷試劑與疫苗工程技術(shù)研究中心;
【基金】：國(guó)家自然科學(xué)基金(項(xiàng)目號(hào):81671645),題目:靶向CD4受體的人類免疫缺陷病毒中和抗體的作用機(jī)制和表位結(jié)構(gòu)研究~~
【分類號(hào)】：R373
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本文編號(hào)：2334502