人IgG Fc标签融合表达载体的构建及H5 HA1融合蛋白的表达  

Construction of Fusion Expression Vector for Human IgG Fc Tag and Expression of H5 HA1 Fusion Protein

作　　者：赵荣茂[1] 郭丽[1] 吴超[1] 王健伟[1] 洪涛[1] 

机构地区：[1]北京协和医学院中国医学科学院病原生物学研究所病毒基因工程国家重点实验室,北京100730

出　　处：《中国生物制品学杂志》2011年第10期1130-1133,共4页Chinese Journal of Biologicals

基　　金：国家科技重大专项子课题(2008ZX10004-002)

摘　　要：目的构建人IgG Fc标签融合表达载体,表达并纯化H5 HA1融合蛋白。方法以pCAGGS载体为基础,依次插入IL-2信号肽及人IgG Fc片段,构建人IgG Fc标签融合表达载体。将H5 HA1克隆至含人IgG Fc标签的融合表达载体pCAGGS-F-IL-2/Fc中,瞬时转染293T细胞,进行融合蛋白的表达。表达的蛋白经Protein G亲和层析一步法纯化。结果人IgG Fc标签融合表达载体经酶切及测序证明构建正确;在293T细胞中分泌表达的H5 HA1与人IgG Fc的融合蛋白HA1-Fc相对分子质量约75 000,转染后72和96 h表达量最高;纯化的融合蛋白纯度达90%以上,含量为1.63μg/ml。结论成功构建了人IgG Fc标签融合表达载体,表达并纯化了HA1-Fc融合蛋白,为流感病毒侵入及免疫机制的研究奠定了基础。Objective To construct a fusion expression vector for human IgG Fc tag and express and purify H5 HA1 fusion protein.Methods IL-2 signal peptide and human IgG Fc fragment were inserted into vector pCAGGS in turn to construct a fusion expression vector for human IgG Fc tag.H5 HA1 gene was cloned into fusion expression vector pCAGGS-F-IL-2 / Fc for human IgG Fc tag,and the constructed recombinant plasmid was transiently transfected to 293T cells for expression of fusion protein.The expressed product was purified by one-step Protein G affinity chromatography.Results Restriction analysis and sequencing proved that the fusion expression vector for human IgG Fc tag was constructed correctly,and HA1-Fc fusion protein,with a relative molecular mass of about 75 000,was successfully expressed in 293T cells in secretory form in 293T cells.The expression level of fusion protein in the cells 72 and 96 h after transfection reached peak values.The purified fusion protein reached a purity of more than 90%,and the recombinant protein content in cell culture was 1.63 μg / ml.Conclusion The fusion expression vector for human IgG Fc tag was successfully constructed,and HA1-Fc fusion protein was expressed and purified,which laid a foundation of study on invasion and immune mechanism of influenza virus.

关 键 词：免疫球蛋白Fc片段 血凝素糖蛋白类 流感病毒 融合蛋白 

分 类 号：Q78[生物学—分子生物学]