Abstract: The aim of this study was to clone IGF-1 mature peptide gene from Tibetan pig and study the IGF-1 prokaryotic expression.The total RNA was extracted by using TRIzol from the Tibetan pig liver and used as template to amplify IGF-1 gene by RT-PCR, the amplified fragment was cloned into pMD19-T vector to construct pMD19T- IGF-1 plasmid, which was identified by sequencing. The pMD19-T-IGF-1plasmid was used as a template to amplify IGF-1 mature peptide fragment which was cloned into vector pET32α to obtain recombinant plasmid pET-32α- IGF-1 and transformed into E. coli BL21(DE3).The pET-32α-IGF-1fusion protein was induced to express with different IPTG concentration and induction time. Then the expression culture was analyzed for it's solubility and was prepared to purify pET-32α- IGF-1 fusion protein with Ni-NTA Sefinose^TM Resin.Finally,the expressing culture and purified protein was identified with SDS-PACE analysis and Western Blot. The Tibetan pig IGF-1 mature peptide gene was 315 bp,restriction enzyme mapping and sequencing showed that expression vector was constructed successfully.The pET-32α- IGF-1 fusion protein induced in E. coli BL21(DE3) and could be expressed by IPTG induction with 0.5 mmol/L IPTG induction 10 hours for well expression; The fusion protein expressed in an insoluble form of inclusion bodies and a high-purity fused protein was obtained with Ni-NTA agarose purification. The expressing culture and purified protein were proved to be the pET-32α- IGF-1 fusion protein with SDS-PACE and Western Blot analysis. IGF-1 mature peptide gene was cloned and expressed, the fusion protein has the antigen antibody reactivity.

Key words: Tibetan pig, IGF-1, Prokaryotic expression, Protein purification, Western Blot

CLC Number: 

• Q78

• S828