中文摘要：

目的构建人颗粒溶素（GLS）活性肽（9ku-GLS）与增强型绿色荧光蛋白（EGFP）融合基因的真核表达载体，并检测其在小鼠黑色素瘤细胞B16中的表达。方法经PCR扩增9ku-GLS基因，插入质粒pBudCE4．1中，经酶切及测序鉴定正确后，亚克隆至质粒pEGFP-C1中，将融合基因真核表达质粒pEGFP-C1／S9K转染B16细胞，采用荧光显微镜及RT-PCR法检测融合蛋白的表达。结果融合基因真核表达质粒pEGFP—C1／S9K经双酶切鉴定证明构建正确，转染B16细胞24h后，荧光显微镜下可观察到绿色荧光，且经RT—PCR可扩增出267bp的目的基因片段。结论已成功构建9ku—GLS与EGFP融合基因的真核表达质粒，且在B16细胞中表达了融合蛋白。

英文摘要：

Objective To construct a eukaryotic expression vector for fusion gene of granulysin active peptide （9ku-GLS） and enhanced green fluorescent protein （EGFP） and observe its expression in murine melanoma B16 cells. Methods 9ku-GLS gene was amplified by PCR and insert into plasmid pBudCE4.1. The constructed recombinant plasmid pBudCE4.1/S9K was identified by restriction analysis and sequencing and subcloned to plasmid pEGFP-C1. B16 cells were transfected with the constructed recombinant plasmid pEGFP-C1/S9K and the expressed fusion protein was identified by fluorescent microscopy and RT-PCR. Results Restriction analysis proved that recombinant plasmid pEGFP-C1/S9K was constructed correctly. Green fluorescence was observed in the B16 cells 24 h after transfection with pEGFP-CI / S9K, and the target gene fragment at a length of 267 bp was amplified by RT-PCR. Conclusion The eukaryotic expression vector for fusion gene of 9ku-GLS and EGFP was successfully constructed and expressed in B16 cells.