鸡类胰岛素生长因子Ⅰ的克隆、表达及其表达产物的生物学活性研究.pdfVIP

中国农业科学 2005,38(10):2129-2133 Scientia Agricultura Sinica 鸡类胰岛素生长因子Ⅰ的克隆、表达及其表达产物的 生物学活性研究 张建峰，侯加法，张 皎，姚 静 (南京农业大学动物医学院，南京 210095) 摘要：应用 RT-PCR 方法对雏鸡肝脏总 RNA 中鸡类胰岛素生长因子Ⅰ基因 cDNA 进行扩增，然后将特异性片段 连接到 pMD18-T 载体，测序结果表明，与已知序列同源性为 100%。然后将特异性片段连在 pRLC 载体上进行表达， 经 Tricine-SDS分析，原核表达产物为 7.6 kD 的重组蛋白，占菌体总蛋白的23%，Western 印迹表明重组蛋 白具有 IGF-Ⅰ抗原活性。表达产物以包涵体形式存在，经 7 mol·L-1 -1 盐酸胍的变性液溶解及 0.5 mol·L 精氨酸复性 液处理，表达产物随后进行脱盐、凝胶层析纯化、MTT 法分析其对 NIH3T3 和鸡胚成骨细胞的增殖效应，结果表明 重组鸡 IGF-Ⅰ具有较高的生物学活性。 关键词：鸡；类胰岛素生长因子Ⅰ基因；原核表达；MTT 法 The Cloning, Expression and Bioactivity Research of Recombinant Chicken IGF- Ⅰ ZHANG Jian-feng, HOU Jia-fa, ZHANG Jiao, YAO Jing （College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095 ） Abstract: With the reverse transcription polymerase chain reaction (RT-PCR), the DNA sequence encoding the chicken’s insulin-like growth factor Ⅰ (IGF- Ⅰ) was amplified, which was then cloned into vector pMD18-T and sequenced. The sequencing result showed that there was 100% homology among the documented sequences and sequence reported here, which was successfully inserted into the expressing plasmid pRLC and was highly expressed in E coli . The Tricine-SDSresult showed that the cloned recombinant protein expressed in the form of inclusion bodies in the E.coli cell with molecular weight of 7.6 kD and amounted to 23 % of the whole protein in the E.coli cell, and Western blot