血管形成抑制因子HIAF-1在昆虫细胞中的高效表达和活性研究

High Expression of Human Angiogenesis Inhibitor HIAF-1 in Insect Cells and Biological Activity

背景与目的:大肠杆菌表达的人抑制血管生成因子-1(humaninhibitingangiogenesisfactor-1,HIAF-1)可抑制肿瘤血管的形成。本研究在昆虫细胞中表达HIAF-1,并研究其生物活性。方法:用DNA重组技术,构建了重组杆状病毒表达载体pAcuW51-HIAF-1,用Cellfectin将其与线性病毒DNA共转染昆虫细胞sf9,获得重组病毒。用SDS-PAGE电泳和Westernblot鉴定重组病毒感染的昆虫细胞中重组HIAF-1重组蛋白的表达。重组蛋白经亲和层析纯化后,在体外用MTT法检测其对内皮细胞的作用,用食管癌移植瘤研究其体内抑瘤活性。结果:在昆虫细胞中高效表达了分子量为26kDa的HIAF-1重组蛋白,其表达量可达昆虫可溶性蛋白的5%-10%。重组HIAF-1蛋白不仅在体外显著抑制内皮细胞的生长,IC50值为3.1μg/ml,而且显著抑制食管癌移植瘤的生长。结论:在昆虫细胞中高效表达了HIAF-1重组蛋白;HIAF-1重组蛋白的生物活性优于相应的原核重组蛋白。

Background &Objective:Human inhibiting angiogenesis factor-1(HIAF-1)expressed in E coli.shown the activity of inhibiting the angio genesis of tumors.This study was designed to investigate the expression of HIAF-1in insect cells and to elucidate its biologica l activity.Methods:Recombinant baculovirus expressio n vector pAcuW51-HIAF-1was constructed by recombinant DNA tech nique and cotransfected with linearized baculovirus DNA into sf9cells t o produce recombinant virus.The expression o f recombinant HIAF-1in insect cells infected by recombinant virus was detected by SDS-PAGE and Western blot.The recom binant protein was purified by affin ity chromatography.In vitro endoth elial proliferation inhibiting activity of recombinant HIAF-1was examined b y MTT method,its antitumor activity in vivo was studied in human esophageal cancer t ransplanted in nude mice.Results:HIAF-1was effectively expressed in insect cells as26KD fusing protein and its expression level was about 5-10%of insect cellular total soluble proteins.Recom binant HIAF-1protein could inhibit endoth elial cell proliferation in vitro wi th IC 50 value of 3.1μg /ml and inhibited remarkably growth of human esophageal cancer tr ansplanted in nude mice.Conclusions:The recombinant HIAF-1with better a ctivity was successfully expressed in insec t cells and establish a base for clini cal application.