The cloning, expression and bioactivity research of recombinant chicken IGF-Ⅰ

chinois. 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-PAGE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。

anglais. 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-SDS-PAGE result 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 blotting indicated that recombinant protein had the antigenicity of IGF-Ⅰ. The inclusion bodies were subsequently dissolved in 7 mol/L guanidine chloride and renatured with dilution in refolding buffer containing 0.5 mol/L arginine. In order to obtain pure protein, the renatured chicken IGF-Ⅰ was desalted by Hiprep 26/10 and purified by Hiprep Sephacryl S-200 chromatography. The biological activities of IGF-Ⅰproduct was assayed in NIH 3T3 cells and osteoblastic cells of embryonic chicken by using MTT method. The results showed that the expressed IGF-Ⅰ obviously stimulated NIH3T3 cells and osteoblastic cells to proliferate at the concentration ranging from 0.1 mg/ml、0.2 mgand#8226;ml-1、0.4 mg/ml、0.8 mg/ml, suggesting that the protein has its biological activities.