摘要
目的:构建绿色荧光蛋白(GFP)-去整合素(disintegrin)融合基因,表达并分析该重组蛋白的生物学活性。方法:利用HF-PCR扩增Ⅱ型蛇毒出血性金属蛋白酶agacutin基因的去整合素区,并与绿色荧光蛋白基因进行拼接;将GFP-去整合素融合基因克隆入表达载体pQE-30,IPTG诱导重组蛋白表达;流式细胞术(FCM)与荧光显微镜分析重组蛋白与细胞的结合能力。结果:GFP-去整合素融合蛋白的表达载体在大肠杆菌M15中得到有效表达,菌体呈绿色。表达的蛋白量占菌体总蛋白的38%左右,免疫印迹证实分子量约为39 kD;FCM分析显示该重组蛋白可与表达整合素的乳腺癌细胞MDA-MB-231和内皮细胞EA.hy926特异结合,在荧光显微镜下可见与重组蛋白结合的细胞发出绿色荧光。结论:GFP-去整合素融合蛋白可与表达整合素的细胞结合,并保留了各自的生物学活性,可用作肿瘤与血管生成等研究的新的分子标记物。
Objective：To construct green fluorencent protein（GFP）-disintegrin fusion gene for efficient expression and bioactivity analysis.Methods：Tbe agacutin gene fragment encoding its disintegrin region was amplified by HF-PCR and fused with GFP gene. The resulting fusion gene was then cloned into pQE-30 vector. The expression of fusion protein was induced by IPTG and identified by SDS-PAGE analysis and Western blot assay. The binding characterization of the recombinant protein was assayed with flow cytometry（FCM） and fluoressence microscope. Results：After induction of IPTG, recombinant protein was produced in E. coli M15 [ pREP4 ] with a major band at 39 kD, comprising up to 38% of the total cell protein, which was confirmed on SDS-PAGE and Western blotting. Recombiant GFP-disintegrin fusion protein could specifically bind to such integfin-expressing cells as breast cancer cell line MDA-MB-231 and endothelial cell line EA. hy926 in FCM analysis and these cells emit green fluorescence under the fluorescence microscope. Conclusion：Tbe recombinant GFP-disintegrin fusion protein maintains the activity of each of these two compounds, and can be used as a new molecular marker for study of tumor, angiogenesis, etc.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2005年第10期739-743,共5页
Chinese Journal of Immunology
基金
江苏省社会发展项目(BS2001070)
关键词
绿色荧光蛋白
去整合素
重组蛋白
血管生成
Disintegrin
Green fluorencent protein
Recombinant protein
Angiogenesis
