关键词: 鸡, 类胰岛素生长因子Ⅰ基因, 原核表达, MTT法

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-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-1 guanidine chloride and renatured with dilution in refolding buffer containing 0.5 mol·L-1 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-1、0.2 mg·ml-1、0.4 mg·ml-1、0.8 mg·ml-1, suggesting that the protein has its biological activities.

Key words: Chicken, Insulin-like growth factor Ⅰ gene, Prokaryotic expression, MTT