Studies On The Cloning, Sequencing, Expression And Biologic Activity Of Gene Encoding Antibacterial Peptide PMAP-37

To study the gene expression and biological function of the antibacterial peptides(ABPs), a pair of RT-PCR primers were designed and synthesized based on the published nucleotide sequence of porcine myeloid antimicrobial peptide37(PMAP-37) gene;Total RNA, isolated from fresh small intestine of porcine,was used as template to generate complementary DNA by reverse transcription.The PMAP-37DNA fragment was amplified by polymerase chain reaction.Sequencing results demonstrated that PMAP-37gene contained516bp with a504bp open reading fragment(ORF),and the sequence homology was99.6%.The cloned gene fragment was inserted into expression vector pBV220by BamHI/EcoRI enzymatic digestion.The recombinant plasmid was transformed into DH5α strain by CaCl2. SDS-PAGE results showed that recombinant protein was notgot or expressed. The antimicrobial activity(ABA) of natural ABPs extracted by acetic acid, ethanol, methanol and ion chromatography were tested, results showed that theOvis aries leucocyte ABP was the best; Biologic activity research indicated that theOvis aries leucocyte ABP could resist high temperature and protease digestion to a certain extent, and its ABA, which was better than streptomycin and penicillinum and worse than florfenicol, was step down when the pH value was step up. Good animal experiments could be obtained using the therapeutic efficacy of Ovis aries leucocyte ABP against fowl disease caused by E.coli.