Prokaryoflc Expression And Purification Of Chiken Liver Expressed Antimicrobial Peptide-2 And Its Molecular Cloning

Liver expressed antimicrobial peptide-2 is one of natural antibiotics in nature, with the broad-spectrum antibacterial activity, antifungal, antitumor and antiviral function. But it has no-toxic to animal cells. So it has important research value. In recent years about the poultry LEAP-2 related exploration has just begun. We prepared recombinant chicken LEAP-2 using prokaryotic expression system for better study the function of LEAP-2. We used network information repository and biology software to predict primary structure and function of this protein, based on the NCBI information about the LEAP-2 mRNA and protein sequences of chicken. According to GenBank in the LEAP-2 cDNA sequences, using Premier 5.0 software to design a pairs of specific primer, total RNA was extracted from chincken Liver tissue by RT-PCR cloning colony LEAP-2 cDNA fragment. After PCR amplification, LEAP-2 gene is ligated with pGEX-6P-1. The recombinant pGEX-6P-1-LEAP-2 which was identical with GeneBank was transformed into E.coli BL21. The expression of chicken LEAP-2 protein was analzed by SDS-PAGE after IPTG induction. We Used Western blot to identify the Recombinant fusion protein. Then after the isolation and purification of the fusion protein we cleaved the fusion protein. At last we do in-vitro determination of antibacterial activity of the GST-LEAP-2 and LEAP-2. Results:The host cells with the fusion expression vector pGEX-6p-LEAP-2 successfully expressed the chicken LEAP-2 fusion protein. Fusion expression product possessed some activities against staphylococcus aureus and escherichia coli; the fusion protein after cleaving the fusion partner shows stronger antibacterial activity. Conclusion:Bioinformatics analysis provides us the related theoretical basis to explore the other functions of LEAP-2 in chicken and subsequent experiment Successfully built the chicken LEAP-2 fusion expression system, the molecular mass of LEAP-2 is about 8KD, the in-vitro determination of antibacterial activity showed that the LEAP-2 possess some antibacterial ctivity; successfulliy establishing a sets of stable expression method provides a basis for subsequent purification and similar expression of antimicrobial peptides in other animails.