Constructing prokaryotic vector of DOC-1 gene,expression and purification of its recombinant protein

Objective To clone DOC-1 gene, construct prokaryotic expression vector of DOC-1 gene, express and purify its recombinant protein. Methods The total RNA was extracted from the brain of human embryo. The segment of open read frame of DOC-1 was amplified by RT-PCR and was further cloned into the vector, pMD18-T, and the prokaryotic expression vector, pGEX-4T-1, by turns, to produce the new construct pGEX-4T-1-DOC-1. After restriction enzymes digestion analysis and sequenced, pGEX-4T-1-DOC-1 was transformed into E.coli BL21(DE3). GST-p12 recombinant protein was expressed under IPTG induction and purified by affinity column. Results The target sequences were specifically amplified through RT-PCR and demonstrated to be the same as that of DOC-1 gene in GenBank. After IPTG induction and affinity chromatography, a new protein band about Mr 38000 showed on SDS-PAGE and the high purity GST-p12 fusion protein was obtained. Conclusion The prokaryotic expression vector of DOC-1 gene has been successfully constructed. Further more, purified recombinant proteins are obtained through affinity chromatography,laying foundation for further study of p12DOC-1 protein.