Molecular Cloning And Eukaryon Expression Preliminary Study Of Lingual Antimicrobial Peptide Of Chinese Holstein Cow

Defensins are a class of cysteine-rich cationic endogenous antimicrobial peptides widely distributed in animals and plants. The defensins play an important role in the innate immune system of host defense mechanisms. The research found a number ofβ-defense genes make gland expression in the mammary when the cows infected with mastitis, it shows that theβ-defensins genes may play a role in the galactophore's local defense at the time of mastitis. The study had carried out of the cloning analysis ofβ-defensin-gene LAP, constructed the eukaryotic expression vector of LAP gene, and successfully made the expression of the eukaryon confluence expression.This experiment selected the Holstein dairy cows sufferring from mastitis as the experimental material, extracted the total RNA from mammary epithelial cells, designed the primers of 5 'end with EcoRⅠand BamHⅠrestriction site according to the cowβ-defensin-LAP sequence of the cDNA which registered under the GenBank, used the RT-PCR technology to amplify the sequence of cDNA of the LAP gene, cloned it into pMD19-T vector for sequencing, renamed the correct clone plasmid identified by sequencing "pMD19-T-LAP". After bioinformatic analysis, the results showed that the study had successfully amplified the coding region sequence ofβ-defensin-LAP gene from bovine, the size of the sequence was 195bp, it had the homology of 98% with the cowβ-defensin-cDNA sequence which had been reported. Derived that the sequence encoded 64 amino acids, and contained the characteristic molecular structure ofβ-defensin, it was said that this sequence had six conserved cysteine residues(C) in the specific locations. With the reportedβ-defensin-gene comparison of amino acid sequences of LAP(Accession Number: EF630358.1), there were three base pair mutation occurred, they were the A-G mutation of Section 196, the T-C mutation of Section 199 and 201 of nucleotide sequence. Caused the mutation of Arg→Lys of amino acid sequence's No.63. The nucleotide homology was 98%, the deduced amino acid sequence homology was 95%, the sequence covered the full-length of coding region. This difference of nucleotide sequence might be caused by the difference between different species of cattle.With EcoRI and BamHI double digested of recombinant plasmid clones pMD19-T-LAP, then inserted the gene coding region into EcoRⅠand BamHⅠsites of the pEGFP-C1 vector. Constructed the recombinant eukaryotic expression plasmid pEGFP-C1-LAP of the cowβ-defensin-LAP. After plasmid PCR, double digestion and sequencing, the results showed that the recombinant expression plasmid pEGFP-C1-LAP had been built successfully. The recombinant plasmid was transformed into COS7 cells by using Lipofectamine reagent, detected the enhanced green fluorescent by using fluorescence microscopy. Separately extracted the total RNA of the cells after transfected from the recombinant plasmid group and empty vector control group. It had showed that the recombinant plasmid group LAP gene mRNA in COS7 cells had been transcribed by RT-PCR analysis.The results of this test had laid a foundation for further study ofβ-defensin-cow LAP gene's expression characteristics, gene function and antibacterial activity of the expression product., and provided the based information of the treatment of recombinant polypeptide and the prevention of cow mastitis at the same time.