Research Article

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

Published: October 10, 2013
Genet. Mol. Res. 12 (4) : 4405-4412 DOI: 10.4238/2013.October.10.6

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.

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.