重组人胰岛素样生长因子-1纯化及其鉴定的研究  

Identification and Purification of rhIGF-1 Expressed in Hemolymph from Bombyx Mori Larvae

作　　者：段宇[1] 赵红[1] 汪承亚[1] 陈家伟[1] 

机构地区：[1]南京医科大学第一附属医院内分泌科,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2004年第6期557-560,共4页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助项目(BK39770355)

摘　　要：目的:利用亲和层析法将家蚕幼虫中高效表达的重组人胰岛素样生长因子(recombinanthumaninsulin鄄likegrowthfactor鄄1,rhIGF鄄1)纯化并鉴定,以得到具有免疫学活性和生物学活性的rhIGF鄄1生物制品。方法:蚕血淋巴中rhIGF鄄1初步提纯去除杂质,采用亲和层析法纯化rhIGF鄄1。以ELISA法测定纯化产物中rhIGF鄄1含量,用SDS鄄PAGE和Western鄄blot分析鉴定rhIGF鄄1纯度及免疫学活性,4-甲基偶氮唑蓝(tetrazoliumsalt,MTT)法观察其对MCF鄄7细胞增殖的影响。结果:纯化后rhIGF鄄1纯度约为40%,Westernblot分析发现,主要纯化产物相对分子质量为7.5ku的成熟hIGF鄄1,所含非目的产物与hIGF鄄1无免疫交叉反应。细胞活性刺激实验显示,rhIGF鄄1纯品对MCF鄄7细胞有较好的刺激活性,促增殖能力优于来源于大肠杆菌的rhIGF鄄1产品。结论:蚕血淋巴中的rhIGF鄄1经亲和层析后初步得到纯化,并显示良好的免疫学活性和生物学活性。Objective: To purify and identify rhIGF-1 expressed in bombyx mori larvae. Methods: Immunoaffinity chromatography was used to purify rhIGF-1 expressed in hemolymph. The concentration of purified rhIGF-1 was tested by ELISA, the molecular size was identified by Western blot analysis while MTT colorimetric assay was used for determing the effect of rhIGF-1 on the growth of MCF-7 cell line which was a specific useful target cell for testing biological function of IGF-1. Results: The purity of rhIGF-1 was about 40%. Its molecular weight was confirmed as 7.5 ku by Western blot analysis and its bioactivity of stimulant effect on MCF-7 cell growth was demonstrated. Conclusion: The recombinant human insulin like growth factor-1 expressed in hemolymph from Bombyx Mori larvae can be successfully purified by immunoaffinity chromatography. Its biological and immunological activities are both verified.

关 键 词：重组人胰岛素样生长因子—1 家蚕幼虫 亲和层析 纯化产物 活性 

分 类 号：R587[医药卫生—内分泌] Q74[医药卫生—内科学]