利用杆状病毒载体高效表达人胰岛素样生长因子-1及其纯化的研究

Effective Expression and Purification of Human Insulin Like Growth Factor-1 in Bombyx Mori Nuclear Polyhedrosis Virus System

目的 :利用杆状病毒载体在家蚕幼虫中研制有生物学活性的重组人胰岛素样生长因子 1(rhIGF 1)。方法 :将 15kDhIGF 1前体cDNA基因插入家蚕核型多角体病毒 (BmNPV)转移载体pBakPAK8中 ,构建重组病毒BmNPV/IGF 1。用BmNPV/IGF 1感染家蚕幼虫 ,提取家蚕血淋巴液 ,采用亲和层析法纯化rhIGF 1,ELISA法测定rhIGF 1含量 ,用SDS PAGE和Western blot分析鉴定rhIGF 1纯度及免疫学活性 ,MTT法观察其对MCF 7细胞增殖的影响。结果 :ELISA测定显示 ,BmNPV/IGF 1感染家蚕幼虫 12 0hrhIGF 1含量达最高值 ( 2 3 36 μg/ml)。rhIGF 1纯度为 4 0 % ,Western blot分析发现主要纯化产物为 7 5kD成熟hIGF 1。细胞活性刺激实验显示 ,纯化后rhIGF 1对MCF 7细胞促增殖能力明显优于来源于大肠杆菌的rhIGF 1产品。结论 :实现了具有免疫学活性及生物学活性rhIGF 1在杆状病毒表达系统的高效表达 ,并使rhIGF

AIM:To study the efficient expression and production of biologically active recombinant human insulin like growth factor 1 (rhIGF 1) in silkworm system using the vector of bombyx mori nuclear polyhedrosis virus (BmNPV). METHOD: A full length IGF 1 cDNA was inserted into BmNPV genome under the control of polyhedrin promoter. Silkworm larvae (JY1) were used as hosts for infection with recombinant BmNPV/IGF 1 rhIGF 1 concentration was tested by ELISA. Immunoaffinity chromatography was used to purify rhIGF 1 expressed in hemolymph. The molecular size was identified by Western blot,and MTT pathway was involved in the study of its bioactivity assay via stimulating the growth of MCF 7 cell line which is a specific useful target cell for testing biological function of IGF 1 RESULT:It was found that the concentration of rhIGF 1 in silkworm hemolymph was gradually increased (19 09～23 74 μg/ml) from 72 h to 120 h post infection,its purity is about 40%. The molecular weight of purified rhIGF 1 was confirmed as 7 5 kD by Western blot analysis and its bioactivity was demonstrated by stimulation of MCF 7 cell growth. CONCLUSION:Human IGF 1 was highly expressed in BmNPV system. It provided a very efficient way to produce soluble and biologically active mature rhIGF 1