Molecular cloning and expression of the porcine S14R gene in Escherichia coli

Abstract

We amplified S14R protein gene cDNA of porcine, cloned it into a prokaryotic expression plasmid, and expressed it in Escherichia coli. A pair of primers was designed based on the cDNA sequence of the porcine S14R gene in GenBank. The target gene fragment from porcine liver tissue was amplified by RT-PCR. Confirmed by auto-sequencing, the target gene fragment was subcloned into an expression vector of pET28a. The pET28a-S14R construct was subsequently transformed into E. coli BL21 (DE3). This construct was verified by restriction endonuclease digestion and sequencing. Using isopropyl β-D-1-thiogalactopyranoside induction, a new recombinant protein with the expected relative molecular mass of 24 kDa appeared. The result was identified by SDS-PAGE electrophoresis. Porcine S14R includes 549bp (GenBank No. JN793537), with an open reading frame of 549 bp coding 182 amino acids.