人颗粒溶素与EGFP基因融合表达载体的构建及其在鼠巨噬细胞RAW264.7中的表达

Construction of a fusion gene expression vector containing human granulysin and green fluorescent protein gene and its expression in murine macrophage RAW264.7

目的:克隆人天然颗粒溶素(granu lysin,GLS)的编码序列并构建与绿色荧光蛋白(EGFP)基因融合的真核表达载体,观察人GLS在鼠巨噬细胞株RAW 264.7中的表达.方法:提取培养并经同种异型抗原激活的人细胞毒性T淋巴细胞(CTL)细胞总RNA,经逆转录后以套式PCR方法扩增出GLS的编码序列,插入pEGFP-C1质粒,鉴定正确后转染RAW 264.7细胞,采用荧光显微镜及RT-PCR法检测融合蛋白及GLS的表达,采用免疫细胞化学法确证表达产物的免疫反应性.结果:成功扩增出了人GLS完整编码序列的cDNA,经PCR及酶切、测序鉴定证明得到了读码框正确的融合蛋白重组子并在靶细胞中得到了成功表达,表达产物具有良好的免疫反应性.结论:人天然GLS能够在鼠巨噬细胞株RAW 264.7中以融合蛋白的形式表达,其表达产物具有良好的免疫反应性.

AIM： To clone the human granulysin from activated cytotoxic T-lymphocytes （ CTL）, construct an eukaryotic expression vector containing the fusion gene of granulysin （GLS） and green fluorescent protein reporter and observe its expression in murine macrophage RAW264. 7 strain, METHODS： The coding sequence of the granulysin was amplified from the total RNA of human CTL activated by allogenic antigen after reverse transcription by Nested-PCR, inserted into pEGFP-C1 plasmid and then transfected RAW204, 7 cells. The expression of the fusion protein and GLS was detected by fluorescence microscope and RT-PCR. The immunoreactive of the product was confirmed by immunocytochemistry method, RESULTS： The whole coding sequence of GLS was successfully amplified as expected, The accurate open reading frame （ORF） of the fusion protein recon was confirmed by PCR, endonuclease digestion and sequencing. The fusion protein that was immunoreactive was successfully expressed in the targeted cells. CONCLUSION： Human granulysin can be expressed in murine macrophage RAW204. 7 strain in a fusion protein pattern and retain its immunoreactivity.