HIV-1 包膜蛋白V1V2区与GM-CSF双顺反子DNA疫苗诱导中和抗体研究
Construction of bicistronic DNA vaccine expressing HIV1-V1V2 and GM-CSF and its immune effects
摘要
gp120 是 HIV-1 感染细胞的关键分子，其中V1V2 结构域是诱导中和抗体的关键部位，诱导产生的抗体具有一定的中和能力，但该区免疫原性较低，很难诱导出识别该区域的广泛中和抗体。本研究通过构建HIV-1-V1V2与GM-CSF双顺反子DNA疫苗，利用mGM-CSF作为基因佐剂，提高HIV1-V1V2基因重组质粒的免疫效果，诱导高价的中和抗体。 本研究分别扩增V1V2基因与GM-CSF基因，连接进入含有IRES的真核表达载体中，构建双顺反子DNA疫苗pV1V2-IRES-mGM-CSF和pV1V2-IRES-EGFP。Lipofectamine2000介导转染293T细胞，反转录PCR检测V1V2和GM-CSF两个基因的转录情况。Western Blot检测细胞内V1V2基因的蛋白表达。通过肌肉注射免疫BALB／c 小鼠，肌肉注射后利用TERESA 核酸药物导入仪进行电穿孔加强。ELASA法测定清抗体效价，初步观察双顺反子核酸疫苗的免疫原性。 经酶切鉴定与序列测定证明共表达质粒中含有V1V2和GM-CSF两个基因且序列正确。转染细胞的反转录PCR可见V1V2和GM-CSF两个基因的转录。Western Blot印迹结果可见转染细胞的上清液在相对分子质量35KD位置有特异性条带。pV1V2-IRES-mGM-CSF组免疫小鼠的最高血清抗体效价为1：100，pV1V2-IRES-EGFP组免疫小鼠的最高血清抗体效价为1：2500。构建的实验组重组质粒pV1V2-IRES-mGM-CSF和对照组质粒pV1V2-IRES-EGFP具有生物学活性，能有效刺激小鼠特异性体液免疫应答；质粒pV1V2-IRES-mGM-CSF的效价低于对照组pV1V2-IRES-EGFP血清抗体效价。?
Abstract
gp120 is the key molecule and V1V2 is the key part which can also induce neutralizing antibodies. Due to the complicated structure, the immune efficacy of this part is low. In this paper, we tried to enhance the immune efficacy induced by V1V2 protein of HIV-1 by using granulocyte-macrophage-colony-stimulating-factor(GM-CSF) as a genetic adjuvant. V1V2 and mGM-CSF genes were amplified and cloned into bicistronic DNA vaccine containing internal ribosome entry site(IRES) to construct a plasmid co-expressions the two genes．After the co-expression plasmid pV1V2-IRES-mGM-CSF was transfected into 293T cells，V1V2 and mGM-CSF transcription were detected by RT-PCR. Western blotting was carried out to observe the expression of V1V2 protein．Finally，BALB/c mice were vaccinated with the recombinant DNA vaccine. Anti-V1V2 antibodies from immunized mice sera were detected by ELASA． Construction of the recombinant plasmid was proved successful by restriction enzymic degradation and sequencing．Transcription of both V1V2 and mGM-CSF was detected by RT-PCR. The end point titer of pV1V2-IRES-mGM-CSF was 1:100 and The end point titer of pV1V2-IRES-EGFP was 1:2500. The co-expression plasmids pV1V2-IRES-mGM-CSF and pV1V2-IRES-EGFP show good antigenicity of expressed V1V2, but V1V2 specific immune response can not be enhanced by GM-CSF expression．关键词
医学免疫学/DNA疫苗/HIV-1/佐剂Key words
Medical immunology/DNA vaccine/HIV-1/adjuvant引用本文复制引用
郑楠,陈雨寒,吴稚伟,楚鹰,李辰.HIV-1 包膜蛋白V1V2区与GM-CSF双顺反子DNA疫苗诱导中和抗体研究[EB/OL].(2017-05-09)[2026-04-05].http://www.paper.edu.cn/releasepaper/content/201705-582.学科分类
基础医学/生物科学研究方法、生物科学研究技术/分子生物学
评论