重组人Versican G1蛋白在昆虫细胞中的表达

Expression of human Versican G1 domain in insect cells

目的 :利用杆状病毒 昆虫细胞表达系统制备人蛋白聚糖VersicanG1区 (VG1)的重组蛋白。方法 :将编码人VG1蛋白 343个氨基酸的基因插入 pFastBac1载体 ,转化大肠杆菌DH10Bac ,提取重组Bacmids转染Sf9昆虫细胞 ,获取含重组人VG1蛋白的杆状病毒进一步感染昆虫细胞进行蛋白表达。通过Ni NTA层析柱对重组蛋白进行纯化。免疫印迹方法对昆虫细胞培养上清中的蛋白成分进行鉴定 ,采用透明质酸结合实验确定重组人VG1蛋白的生物活性。结果 :人VG1基因在昆虫细胞中获得高水平表达 ,重组人VG1蛋白具免疫原性和与透明质酸结合的生物活性。结论

Objective:To produce the recombinant human Versican G1 domain (VG1) in insect cells. Methods:The gene of VG1 which encodes 343 amino acids was inserted into pFastBac1 and the recombinant plasmid was transformed into DH10Bac competent cells for transposition. The recombinant baculovirus were transfected into Sf9 insect cells. The insect cells infected by recombinant baculovirus were determined by Western blot assays. The recombinant protein was purified with Ni-NTA agarose bead column, and its bioactivity was determined with hyaluronan binding assay. Results:The recombinant VG1 was successfully expressed in insect cells. VG1 reacted with the carboxyl-terminal His-tag sequence antibody and with the specific antibody to versican on Western blotting. VG1 bound directly to biotinylated hyaluronan and competitively with unlabeled hyaluronan, indicating that they are folded in a native conformation. Conclusion:Insect cells are suitable to express recombinant human VG1 in high efficiency.