人类免疫缺陷病毒1型gp140包膜蛋白三聚体在哺乳动物细胞中的高效表达及其性质鉴定
发布时间：2018-11-15 21:53
【摘要】：本研究目的是通过优化1型人类免疫缺陷病毒(HIV-1)gp140编码基因的密码子和克隆设计,从而实现在293T哺乳动物细胞中高效表达,并对纯化获得的gp140蛋白进行抗原性质鉴定。在本研究中选择HIV-1B亚型NL4-3全基因序列为模板进行gp140克隆构建,通过密码子优化、信号肽替换、增加柔性linker、三聚体折叠序列等方法优化设计。通过HIV-1转录反式激活因子tat共转HEK293T细胞进行gp140蛋白表达,采用镍柱纯化。SDS-PAGE、Western blot、ELISA、负染电镜等结果显示目的蛋白纯度高于70%,每升培养基可获得0.5mg gp140蛋白,并且具有良好的抗原活性,电镜下呈现三聚体结构。通过弗氏佐剂与目的蛋白混合免疫Balb/c小鼠,检测小鼠免疫血清显示gp140蛋白能有效刺激机体产生免疫应答。本研究通过优化表达获得B亚型HIV-1NL4-3gp140蛋白,为HIV-1病毒包膜蛋白结构和重组疫苗研究奠定基础。
[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.
【作者单位】： 厦门大学公共卫生学院分子疫苗学和分子诊断学国家重点实验室;厦门大学生命科学学院国家传染病诊断试剂与疫苗工程技术研究中心;
【基金】：国家自然科学基金(项目号:81671645),题目:靶向CD4受体的人类免疫缺陷病毒中和抗体的作用机制和表位结构研究~~
【分类号】：R373
，
本文编号：2334502
[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.
【作者单位】： 厦门大学公共卫生学院分子疫苗学和分子诊断学国家重点实验室;厦门大学生命科学学院国家传染病诊断试剂与疫苗工程技术研究中心;
【基金】：国家自然科学基金(项目号:81671645),题目:靶向CD4受体的人类免疫缺陷病毒中和抗体的作用机制和表位结构研究~~
【分类号】：R373
，
本文编号：2334502
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