Study on the Cloning, Expression and Bioactivity of Recombinant Chicken IGF-Ⅰ

摘要

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 is 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 was expressed in the form of inclusion bodies in the E.coli cell with molecular weight of 7.6KD and was amount to 23 % of the whole protein in the E.coli cell, Western blotting indicated that recombinant protein had the antigenicity of IGF- Ⅰ. The inclusion bodies was subsequently dissolved in 7M guanidine chloride and renatured with dilution in refolding buffer containing 0.5M arginine. In order to obtain pure protein, the renatured chicken IGF- Ⅰ was desalting by Hiprep 26/10 and purified by Hiprep Sephacryl S-200 chromatography. The biological activities of IGF- Ⅰ product was assayed in NIH 3T3 cells and osteoblastic cells of embryonic chicken by using MTT method. The results show that the expressed IGF- Ⅰ can obviously stimulate NIH3T_3 cells and osteoblastic cells to proliferate at the concentration ranging from 0.1, 0.2, 0.4, 0.8 μg · ml~(-1), suggesting that the protein has its biological activities.