颗粒溶素活性肽与增强型绿色荧光蛋白融合基因真核表达质粒的构建及其在小鼠黑色素瘤细胞中的表达

Construction of Eukaryotic Expression Vector for Fusion Gene of Granulysin Active Peptide 9ku-GLS and Enhanced Green Fluorescent Protein and Its Expression in Murine Melanoma B16 Cells

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。

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.