| Defesins are small molecular antibacterial peptides with positive charge that widely distributed in animals and plants. Defesins are able to inhibit and kill a broad spectrum of bacteria,fungus,virus and tumor cells, thus play important roles in innate immune system against pathogenic microbes invading.Blunt snout bream (Megalobrama amblycephala) is an herbivorous freshwater fish species native to China and has been recognized as a main aquaculture species in the Chinese freshwater polyculture system with high economic value.. In present study, defesin core cDNA sequence of megalobrama amblycephala has been cloned, the distribution in tissues before and after infection with Aeromonas hydrophila were analysed, respectively. pGEX-KG-β-defensin vector and the pPICZaA-β-defensin vector were successfully constructed, transformed into the procaryotic and eukaryotic expression system, the recombinant protein has been acquired, respectively. Overall, the main results are as follows:1. Cloning and expression analysis of β-defesin in megalobrama amblycephala Sequence analysis shows that the length of megalobrama amblycephala defesin is334bp, defesin open reading frame is204bp, The cDNA sequence is highly similar to Zebrafish with the similarity of90.2%and common carp with the similarity of86.3%after blast on NCBI. Amino acid sequences revealed that defensin possessed a typical arrangement of six conserved cysterine residues, the defesin was highly expressed in liver, spleen and kidney. The β-defein expression level was not significantly changed in liver and intestine after challenging with Aeromonas hydrophila(p>0.05). The level of β-defein expression up-regulated in kidney was much stronger after bacterial infection12h and24h while the up-regulation in spleen appeared after72h (p<0.05), respectively.2. The pGEX-KG-β-defensin vector has been successfully transformed into the prokaryotic cell BL21and expressed by the chemical inducer IPTG, the fushion protein don’t shows bacteriostatic activity after separation and purification. Acquire the polyclonal antibody of the fusion protein by the immunity to rabbit and measure the titer of the antibody. The result shows that the titer of the antibody is more than1:3200, and the antibody can specifically bind to the target gene. 3. The pPICZaA-β-defensin vector has been successfully transformed into the pichia pastoris and expressed by the chemical inducer methyl alcohol, enrich the expression product by the ammonium sulfate precipitation, then identify the bacteriostatic activity by the Oxford-cup tests, use the expression product of non-carrier pPICZaA as the negative control, the results show that the fushion protein has an antimicrobial spectrum to staphylococcus aureus, escherichia coli, streptococcus agalactiae, aeromonas sobria. And the effect to aeromonas hydrophila is evident and rests are not so obvious. |
