Cloning And Prokaryotic Expression Of Oryctolagus Cuniculus CD147 In E.coli

CD147 is a member of immuno-globulin superfamily (IgSF), CD147 is a cell surface adhesion molecule which is discovered recently. As a transmembrane protein, CD147 had abroad expression pattern on epithelial cells and endothelial cells and involved in various physiological and pathological processes. The CD147 is closely associated with development of tumor cells ,sperm development, amd nervous system function, especially in the gut diseases , CD147 promote the epithelial cells and stromal cells to produce large amounts of matrix-degrading enzyme(matrix metalloproteinases), these matrix-degrading enzyme play an important role in the gut inflammation and cancer.To investigate if CD147 play the same roles in the Oryctolagus cuniculus, we cloned Oryctolagus cuniculus CD147 and obtained cognate fusion protein. The study was divided into two series.Ⅰ. Cloning,Sequence Analysis and structure prediction of Oryctolagus cuniculus CD147. A pair of cloning primers were designed according to the cloning principle of homologous sequence. Total RNA extracted from mucosal tissue of Oryctolagus cuniculus duodenum was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into E.coli JM109 competent cells. The positive clone was identified and the sequence was sequenced,analyzed and structure prediction. The Oryctolagus cuniculus CD147 gene was successfully cloned in present study, the sequence has been submitted to GenBank(Accession EU650668). The open reading frame of Oryctolagus cuniculus CD147 consists of 810bp and encodes 270 amino acids .The signal peptide of deduced protein sequence is 1~16 amino acid . The predicted peptide shared 62.5%, 65.9%, 59.5% and 66.3% homology with that of Oryctolagus cuniculus,Bos Taurus,Homo sapiens,Rattus norvegicus,Sus scrofa. Tertiary structure predicts that CD147 contains two extracellular Ig-like domains.Ⅱ. Prokaryotic Expression of Oryctolagus cuniculus CD147 in E.coli a pair of expression primers were designed according to the cloned sequence. A fragment of Oryctolagus cuniculus CD147 cDNA containing BamHI/HindⅢwas amplified from recombinant vector by PCR. The fragment digested by BamHI/HindⅢwas subcloned into pET32a﹙+﹚ vector to construct a recombinant prokaryotic expression vector, pET32a﹙+﹚～CD147, the recombinant vector was transformed into E.coli BL2l competent cells. The fusion protein induced by IPTG was analyzed by SDS-PAGE. The prokaryotic expression vector pET32a﹙ +﹚～CD147 was successfully constructed. The fusion protein expressed in E.coli BL21 was about 49.45kDa and mainly existed as inclusion bodies. It provides the experiment basis for further researching its biological function.