摘要
目的 克隆人胰岛素样生长因子Ⅰ型 (hIGF 1) ,构建真核表达载体并进行表达及活性检测 ,为IGF 1基因治疗糖尿病奠定基础。 方法 提取胎儿肝脏总RNA ,RT PCR法扩增IGF 1cDNA片段 ,重组于pUCM T载体 ,测序正确后构建表达载体 ,转染猴肾成纤维细胞系COS 7,用原位杂交和免疫组织化学检测表达 ,收集培养上清以胰岛素刺激释放实验进行活性测定。 结果 扩增得到 710bp带有Kozak序列的IGF 1cDNA片段 ;成功构建了真核表达载体pCI neo hIGF 1;IGF 1在COS 7细胞得到了表达 ,并具有刺激胰岛素分泌的活性。 结论 新构建的载体pCI neo hIGF 1能在COS 7细胞中表达、分泌 ,且所分泌的IGF 1具有生物活性。
Objective In order to investigate the roles of hIGF\|1 in treatment of diabetes mellitus and diabetic syndromes,the gene of human insulin like growth factor type Ⅰ(IGF\|1) was cloned and constructed into eukaryotic expression vector,then the expression and activity were determined. Methods Total cellular RNA of human fetal liver was abstracted and the RT\|PCR amplification of the cDNA fragment was performed.The fragment was cloned into pUCM\|T vector and sequenced.The eukaryiotic expression vector was recombined and transfected into fibroblast cell line,COS\|7.The expression of hIGF\|1 was examined by in situ hybridization and immunohistochemistry.The effect of hIGF\|1 on cultured islet cells was observed by glucose\|stimulated insulin release assay. Results The cDNA fragment of 710bp with additional Kozak sequence was amplified by RT\|PCR.Eukaryiotic expression vector pCI\|neo/hIGF\|1 was constructed and IGF\|1 gene expressed in COS\|7.The biological activity of hIGF\|1 was proved by increasing inslin secretion from islet cells.Conclusion\ The newly constructed vector,pCI\|neo/hIGF\|1 could be transfected into COS7 cells and its expressed product showed to have the biology activity of hIGF\|1.\;[
出处
《解剖学报》
CAS
CSCD
北大核心
2003年第3期306-311,共6页
Acta Anatomica Sinica
