利用重组杆状病毒在家蚕中表达人血管内皮细胞生长因子

家蚕作为“生物工厂”生产重组蛋白质具有很多优势 .构建携带编码人血管内皮细胞生长因子 (VEGF) 16 5个氨基酸的cDNA的重组杆状病毒 .将此重组病毒接种 5龄家蚕幼虫进行重组蛋白的表达生产 .时相表达分析表明 ,感染后大约 80h时表达水平达到最高 ,而且重组蛋白主要存在于血淋巴中 .从感染的幼虫收集血淋巴并用Nickle亲和层析纯化重组蛋白产物 .定量分析表明 ,每条家蚕幼虫的表达水平高达 4 2 6 μg左右 .通过细胞培养体外分析 ,发现与对照相比 ,加入纯化的重组VEGF(10ng ml和 10 0ng/ml)能够使人HUVEC细胞体外培养细胞数增加 1 8～ 3 3倍 ,说明家蚕幼虫表达的重组VEGF具有完全的生物活性 ,能够诱导内皮细胞在体外分裂增殖 .

Production of Human Vascular Endothelial Growth Factor in Silkworm by Using a Recombinant Baculovirus

Silkworm larva has a lot of advantages as a “biofactory” to produce recombinant proteins.A recombinant baculovirus,carrying cDNA encoding the 165 amino acid residues of human VEGF(vascular endothelial growth factor,VEGF) was constructed for the production of this protein using silkworms as an in vivo host.The fifth-instar silkworm larvae were inoculated with the recombinant virus.The time-course expression analysis indicated that the expression level was the highest at about 80-hour postinfection and the recombinant protein were found mainly in the larval haemolymph.The hemolymph was collected from the infected larvae and the recombinant protein was purified by using Nickel affinity chromatogrphy under native condition.The expression level was estimated to be as high as approximately 426 μg in esch larva.Compared with the conctrol,the number of HUVEC cells,treated with purified VEGF(10 ng/ml and 100 ng/ml),increased by 1.8～3.3 fold.The above evidences indicated that the recombinant protein was active in inducing endothelial cell proliferation in vitro.