利用杆状病毒载体在家蚕幼虫中高效表达人胰岛素样生长因子-2(hIGF-2)

Effective Expression of Human Insulin Like Growth Factor-2 in Bombyx Mori Larvae

目的 :研制有生物活性的可溶性分泌型重组人胰岛素样生长因子 -2 (rh IGF-2 )。方法 :将 14 k Dh IGF -2前体 c DNA基因插入家蚕核型多角体病毒 (Bm NPV )转移载体 p Bak PAK8中 ,经与野生型病毒 DNA共转染家蚕细胞后 ,通过体内同源重组的方式获得重组病毒 Bm NPV/ IGF-2。以 Bm NPV/ IGF-2感染 5龄家蚕幼虫 ,分别在感染后 2 4,48,72 ,96,10 8,12 0 h提取家蚕血淋巴液 ,以 ELISA法测定不同时象家蚕血中 IGF-2浓度 ,采用 Western-blot分析鉴定 IGF -2免疫学活性 ,MTT法观察其对 NIH3 T3细胞增殖的影响。结果 :ELISA测定显示 ,Bm NPV/ IGF-2感染家蚕幼虫 96h后 IGF-2表达率最高 ,每毫升血淋巴中约含 IGF-2 14 μg,Western-blot分析发现表达产物在蚕体内被加工成 7.0 k D成熟 h IGF-2 ,并对 NIH3 T3细胞具有良好促增殖效应 ,其促细胞生长能力明显优于来源于 E.coli的 IGF-2标准品。结论 :本研究实现具有免疫学活性及生物学活性的rh

AIM The purpose is to study the efficient expression and production of biologically active recombinant human insulin like growth factor 2 (rhIGF 2) in the silkworm system by the vector of Bombyx mori Nuclear Polyhedrosis Virus (BmNPV). METHODS A full length IGF 2 cDNA with the start codon was inserted into the BmNPV genome under the control of the polyhedrin promoter. Silkworm larvae (JY1) were used as hosts for the infection with recombinant BmNPV /IGF 2. rhIGF 2 concentration were detected by ELISA, and Western blot was used to find the size of IGF 2, while MTT pathway was involved to study the bioactivity of rhIGF 2 by stimulation the growth of NIH3T3 cell line. RESULTS It was found that after 48 h after infection, the concentration of IGF 2 in silkworm hemolymph could be detected(1 μg/ml),while at 72 h after infection, the level of rhIGF 2 was tested for 14.00 μg/ml). Mature rhIGF 2 with a size of 7.0 kD was identified by Western blot analysis. It was also showed that rhIGF 2 has excellent bioactivity by stimulated NIH3T3 cell growth. CONCLUSION Human IGF 2 was highly expressed by the BmNPV expression vector, and this production showed satisfied results both in bioactivity and immunol activity thus providing a very efficient way to produce soluble and biologically active mature rhIGF 2.