Cloning and prokaryotic expression of beta-defensin 5 gene in dairy cattle

A pair of primers were designed and synthesized based on the published β-defensin 5 gene sequence of dairy cattle,and then β-defensin 5 gene was amplified using RT-PCR technique.Then,the PCR product was inserted into pGEM-T easy vector to construct the recombinant plasmid that was correct identified by agarose gel electrophoresis,PCR,enzymatic digestion and DNA sequencing.The mature peptide fragment of the target gene was amplified according to the recombinant plasmid template.After digested with EcoR I and Not I,the amplified product was ligated with PET28a expressive vector to form recombinant expressive plasmid.Sequence analysis showed that the inserted target gene was completely correct.Then,the recombinant plasmid was transformed into BL21(DE3) and induced with IPTG to express the fusion proteins with relative molecular mass of 6.4 kda.