Open AccessMolecular cloning and expression of the porcine S14R gene in Escherichia coli
Author(s) -
Yujie Guo,
G.Z. Liu,
C.M. Wang,
Y.Y. Wang,
H.J. Li,
Keyan Zhong,
Wang Lu,
Y.L. Wang,
Gangyi Yang
Publication year - 2013
Publication title -
genetics and molecular research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.356
H-Index - 48
ISSN - 1676-5680
DOI - 10.4238/2013.october.10.6
Subject(s) - microbiology and biotechnology , complementary dna , genbank , escherichia coli , molecular cloning , gene , biology , restriction enzyme , expression vector , recombinant dna , open reading frame , cloning (programming) , puc19 , plasmid , gene expression , genetics , peptide sequence , computer science , programming language
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.