Cloning And Expression Of Chicken Gal-11 And Gal-12 Gene And Biological Activity Detection

β-defensin Gallinacin is the most important member of a large family of antimicrobial peptides in chicken.β-defensin Gallinacin displays a strong antimicrobial activity against bacteria and fungi.Furthermore,it also plays indispensable function in triggering adaptive immune responses.So it could act as a substitute for antibiotic.In this paper,systems for high-level expression of theβ-defensin fusions in Escherichia toll were constructed,and expression regulation and the technology of protein purification were investigated.There are four parts in this paper:①Cloning of chicken Gal-ll gene:A pair of primers were designed and synthesized based on the publised gene sequence of Gal-11(AY621326).The target DNA,which contains 315bps,were amplified by RT-PCR on the template of tatal RNA isolated from kidney.The sequencing result displaied the target DNA was homologous with AY621326 by 99%.②Cloning of chicken Gal-12 mature peptides gene:A pair of primers were designed and synthesized based on the publised gene sequence of Gal-12(AY621327). The target DNA were amplified by PCR on the template of genome DNA isolated from blood.The sequencing result was completely consistent with AY621327,AY534898 and DQ858309.③Expression of chicken Gal-11 and Gal-12 gene:Gal-11 and Gal-12 gene was expressed in the form of fusion protein useing pET32a as expression vectors and BL21 (DE3)pLySs as expression host.Expression products with molecular weight of approximately 27.175KD and 22.896KD were examined by SDS-PAGE.Top yield of recombinant Gal-11 and Gal-12 could be achieved with as less as 0.2 mmol/L IPTG induction after 2 hours.④Biological activity detection of the purified protein:Recombinant fusion protein was extracted roughly from dissolved BL21 and purified with Ni-NTA resin affinity column.The biological activity was measured by the minimum inhibitory concentrations (MICs)the radial diffusion plate assay method.The result demonstrated that the purified protein has no antimicrobial activity against Escherichia.coli O78,Candida.albicans ATCC10231 and Staphylococcus.aureus 1056MRSA.Further study will be continued based on the results of this research.