Molecular Cloning, Expression Vector Construction And Antibacterial Activity Characterization Of Caprine β-defensin-1

Defensins are a large family of antimicrobial peptides widely distributed in nature. They are small (the molecular weight is only 3-6 kD) arginine-rich cationic proteins. Defensins have high effective and broad spectrum antibacterial activity, can kill Gram-negative bacteria, Gram-negative bacteria, fungi, mycobacteria and several enveloped viruses, and they have no drug resistance. Extracting natural defensins from organisms is expensive and complex, so it is significant to produce defensins with high antibacterial activity by using genetic engineering technology.The tongue of Black-bone goat was used to obtain the total RNA, then 5’RACE and 3’RACE were used to get the full cDNA sequence of goatβ-defensin 1(GBD-1). Amino acid sequence of GBD-1 was analysised and the result indicated that GBD-1 have signal peptide cleavage site at 21st amino acid. Blast analysis of amino acid sequence of GBD-1 showed that Black-bone goat GBD-1 was homologous to that of Boer goat (98%), and the homology of GBD-1 and sheepβ-defensin 1 was 93%.Semi-quantitative RT-PCR was used to measure the expression leves of GBD-1 in different tissues. The results showed that there was no GBD-1 expression in gastro-intestinal system while the gene expressed highly in the respiratory system and the reproductive tract system. Quantitative Real-time RT-PCR revealed that GBD-1 gene was highly expressed in vagina, cervix and ovary.According to the cDNA sequence of goatβ-defensin 1 on GenBank and the Pichia pastoris codon preference,4 primers were designed to get GBD-1 gene which codes 38 amino acid by using Overlap PCR. The 1st primer had EcoR I and the 4th primer contained Not I restriction endoenzyme site. The amplified gene fragment was ligated to pPICZaA vector. The recombinant pPICZaA/GBD)-1 was identified by restriction enzyme digestion and sequencing, then the correct recombinant was lined with restriction enzyme Sac I. The lined recombinant was transfected into Pichia pastoris GS115 by electroporation. The recombinant was selected with antibiotic Zeocin substratum and identified by PCR. The positive recombinant strains were cultured and inducted by addition of 1% methanol every 24 h. The expression protein was displayed on the Tricine-SDS- PAGE electrophoresis and Western Blot and the molecular weight of the protein was approximately 4.3 kD. The activity of the samples were identified, the study of the GBD-1 in vitro had antibacterial activity against Escherichia coli and Staphylococcus aureus.