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-1的研究  

Effective expression of human insulin like growth factor-1 in Bombyx mori larvae

作　　者：段宇[1] 汪承亚[1] 赵红[1] 陈家伟[1] 张志芳[2] John S Sussenbach 

机构地区：[1]南京医科大学附属第一医院,江苏南京210029 [2]中国农业科学研究院镇江蚕业研究所,江苏镇江212018 [3]荷兰Utrecht大学

出　　处：《中国生化药物杂志》2001年第6期274-277,共4页Chinese Journal of Biochemical Pharmaceutics

基　　金：国家自然科学基金 (39770 355)

摘　　要：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可溶性分泌型重组人胰岛素样生长因子 1(rhIGF 1)。方法将 15kD人IGF 1(hIGF 1)前体cDNA基因插入家蚕核型多角体病毒 (BmNPV)转移载体pBak 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BmNPV/IGF 1。以Bm NPV/IGF 1感染 5龄家蚕幼虫 ,分别在感染后 2 4、48、72、96、10 8、12 0h提取家蚕血淋巴液 ,以ELISA法测定不同时象家蚕血中IGF 1浓度 ,采用Western blot分析鉴定IGF 1免疫学活性 ,MTT法观察其对NIH3T3细胞增殖的影响。结果ELISA测定显示 ,BmNPV/IGF 1感染家蚕幼虫后 ,72h起蚕血中IGF 1浓度随感染时间延长而逐渐增高 (19.0 9～ 2 3.36 μg/ml) ,12 0hIGF 1含量达到最高值 (2 3 .36 μg/ml) ,Western blot分析发现表达产物在蚕体内被加工成 7.5kD成熟hIGF 1,并对NIH3T3细胞具有良好促增殖效应 ,其促细胞增殖能力明显优于来源于E .coli的IGF 1标准品。结论具有免疫学活性和生物学活性的rhIGFPurposeThe aim is to study the efficient expression and production of biologically active recombinant human insulin like growth factor 1 (rhIGF 1) in the silkworm system by the vector of Bombix mori Nuclear Polyhedrosis Virus (BmNPV).MethodsA full length IGF 1 cDNA with the start codon at position 25 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 1. rhIGF 1 concentration was detected by ELISA, and Western blot was used to find the size of IGF 1, while MTT pathway was involved to study the bioactivity of rhIGF 1 by stimulation the growth of NIH3T3 cell line .ResultsIt was found that from 72 h to 120 h post infection, the concentration of IGF 1 in silkworm hemolymph was increased gradually(19.09～23.74 μg/ml), and mature rhIGF 1 with a size of 7.5 kD was identified by Western blot analysis. It was also showed that rhIGF 1 had excellent bioactivity by stimulated NIH3T3 cell growth. Further verification was performed by specific inhibition of the biological effects using the IGF 1 receptor antibody, IGF 1Rα.ConclusionHuman IGF 1 was highly expressed by the BmNPV expression vector. This production showed satisfied both in bioactivity and immunol activity thus providing a very efficient way to produce soluble and biologically active mature rhIGF 1.

关 键 词：人胰岛素样生长因子-1 基因表达 重组家蚕核型多角体病毒 生物学活性 

分 类 号：R392－33[医药卫生—免疫学]