中文摘要：

目的：构建含有人类免疫缺陷病毒I型（human immunodeficiency virus type Ⅰ,HIV-1）负性调节因子（nega-tive regulation factor,Nef）和增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）基因的重组融合蛋白分泌性表达载体,并检测该融合蛋白在真核细胞中的表达及其在培养上清中分泌情况,为进一步研究Nef功能奠定基础。方法：利用PCR扩增出HIV-1 Nef和EGFP基因,插入到分泌性表达载体pSecTag2B中。构建的pSecTag2B-Nef-EGFP融合蛋白表达质粒,转染293T细胞,荧光显微镜下观测细胞中Nef-EGFP融合蛋白的表达。收集细胞及上清液,蛋白质印迹和ELISA法分别检测Nef-EGFP融合蛋白的表达及其分泌。结果：限制性内切酶酶切鉴定和核酸序列测定证实,成功构建了含有Nef-EGFP基因的分泌性表达载体,该质粒转染293T细胞后,蛋白质印迹法能够检测到Nef-EGFP融合蛋白的表达条带,ELISA法检测上清液中目的蛋白分泌量为1.7 ng/ml。结论：成功构建含有Nef-EGFP的融合蛋白表达质粒,融合蛋白Nef-EGFP在293T细胞中获得表达,并能分泌到胞外。

英文摘要：

Objective： To construct the secretory expression vector containing human immunodeficiency virus type Ⅰ（HIV-1） negative regulation factor（Nef） and enhanced green fluorescent protein（EGFP） gene,and appraise the expression of Nef-EGFP fusion protein in the eukaryotic cell and the secretion into the supernatant.Methods： The Nef and EGFP fragment was amplified by PCR,then cloned into the secretory expression vector pSecTag2B.The recombinant vector pSecTag2B-Nef-EGFP was transfected into the 293T cells,observed the expression of GFP,then harvested the culture media and cells,the fusion protein Nef-EGFP was confirmed by Western blot and ELISA.Results： The recombinant secretory expression vector carried the Nef and EGFP gene was successfully constructed.Western blot analysis revealed that the Nef-EGFP fusion protein can be correctly transcripted and translated in 293T cells,ELISA analysis discovered it can be secreted into the supernatant,the level was 1.7 ng/ml.Conclusion： The recombinant secretory expression vector pSecTag2B-Nef-EGFP was constructed successfully,Nef-EGFP fusion protein could be expressed in 293T cells,and secreted to the supernatant.