Cloning Of Defensin Gene And It's Expression In Escherichia Coli

According to the sequence of rabbit defensin NP-1 and porcine defensin pBD-1 gene published in GenBank,We selected respectively prokaryon or eukaryotic expression vector to construct recombinant expression plasmid hoping for getting expression protein in E.coli or COS-7 cells, All these laid down a foundation in research of these two antibacterial peptides.In experiment I, We selected the codon usage bias of E.coli, removed influence factors of expression in E.coli ,and modified intrinsic sequence of NP-1 gene with the help of computer,but amino acid sequence encoded by modified NP-1 gene was unchanged yet.The sequence of new NP-1 gene was divided into two pieces which complemented each other with 13bp.Th.ese two pieces each of which contained 59bp were synthesized chemically,and stretched into a complete chain by PCR.Two primers with Xba Ⅰ and HindⅢ restriction enzymes sites were designed for NP-1 gene being correctly linked to expression vector pMAL-p2X.At first,NP-l gene with enzymes sites was cloned into pGEM-T easy Vector,and then connected with pMAL-p2X vector by the same enzyme digestion.At last ,recombinant pMAL-p2X expression plasmid was transformed into E.coli TB1 which was induced by 0.1mM IPTG.The result of a some 4.0KD specific protein band appearing in 12% SDS-PAGE electrophoresis demonstrated that rabbit NP-1 gene expressed successfully in E.coli TB1.In experiment Ⅱ ,according to sequence of porcine defensin pBD-1 gene, We designed a couple of primers with restriction enzyme site Xba Ⅰ ,and HindⅢ by computer.After total RNA being isolated from porcine tongue tissue cells,The first cDNA chain could be gotten from it by RT-PCR,and a length of 213bp target gene fragment could be gained by amplifing further the first cDNA chain with a couple of special primers too.The target gene fragment was cloned into pGEM-T easy Vector with T4DNA enzyme,and then inserted into eukaryotic expression vector pcDNA3.1 (+) which was digested by Xba Ⅰ and HindⅢ enzymes.It was affirmed that porcine defensin pBD-1 gene had been successfully ligated to expression plasmid by restriction enzyme analysis and DNA sequencing.The result of DNA sequencing demonstrated that a base of porcine defensin pBD-1 gene was different as compared to the sequence published in GenBank,that was A→G mutation happening at position 104.this changes caused amino acid substitution in the mature peptide sequence at position 35 where aLys was changed into a Arg.This difference may be result of pig breed or error in PCR.The successful construction of pBD-1 gene expression vector laid down the foundation in research of antibacterial activities,and antibacterial mechanism.