Preparation Of Antibacterial Peptide Abaecin And Effect Of Abaecin On Growth And Immunity In Broiler

The coding gene of Abaecin(ABA) was synthetized by Overlap-extension PCR method, expressed it in E.coli and B.subtilis shuttle expression system. In vitro antimicrobial trial,Studied the effect of reombinant expression products(rAbaecin) on growth performance,immune functions in broiler. Results are as bellow:1.The signal peptide sacB of Bacillus subtilis was cloned by Overlap-extension PCR, according genes sequence reported in Genebank(AL009126.3). The coding gene of Abaecin(ABA) was cloned, according genes sequence reported in Genebank(LOC406144). Full length Abaecin gene was cloned successfully by the two segments loned respectively and linked together. Results of agarose gel electrophoresis showed that molecular weight of Abaecin gene is approximately 189bp which was consistent to the molecular weight estimated according to its primary structure. Sequence results showed that its primary structure was consistent to the Abaecin gene sequence reported in Genebank. These results indicated that ABA gene was cloned correctly.2. The E. coli-B. subtilis shuttle vector pGplt was constructed. The ABA gene was inserted into the plasmid pGplt, resulting in the construction of pGplt-ABA. The plasmid pGplt-ABA that was extracted from the positive transformant was transformed into the expression vector B. subtilis 1A747 to secretory expression. Analysis of the purified Abaecin using Tricine-SDS-PAGE revealed that the molecular mass of Abaecin was about 3.9 kDa, which is consistent with the the theoretical molecular mass. These results indicted that antibacterial peptide abaecin was expressed successfully in B. subtilis 1A747.3. Antibacterial effect of the samples were tested by inhibition zone assay. The secretory product of rAbaecin was found that it inhibited growth of E. coli k88. The result demonstrate that recombination Abaecin has Antibacterial activity. Stability analysis results showed that temperatures as high as 60oC for 15 min did not influence the activity of rAbaecin. However, temperatures above 100oC (10min) decreased the antimicrobial activity of rAbaecin. Varying the pH from 4.0 to 9.0 did not appear to affect the activity of rAbaecin, but some loss of potency was observed at extremely low pH values. Under the challenge of several proteases (proteinase K, trypsin and pepsin), rAbaecin maintained functional activity.4. The toxicity of rAbaecin was tested with Caco-2 cell and epithelial cell of chicken intestine. These results demonstrate that rAbaecin had no effect to cell shape,cell proliferation and cell permeability. Therefore, expression product rAbaecin is safe.5. Antibacterial peptide rAbaecin was add to the feed of broilers, which could significantly boost body weight of broilers, and significantly decreased ration of feed/gain of broilers(36-42 days)(p<0.05); and significantly increased villus length and ratio of villus length and crypt-depth(p<0.05); had no harmful effects to Enzyme activity of entero-amylase chyme; and decreased Microbial population quantity of Escherichia coli, and increased Microbial population quantity of Bacillus acidi lactici.6. Antibacterial peptide rAbaecin was add to the feed of broilers, which had no harmful effect to the immune organ, and promoted lymphocyte transformation rate of peripheral blood, and increased contents of serum complement C3,C4,immunoglobulin IgG and lysozyme in broilers'serum. These results indicated that antibacterial peptide could boost immunity of animals, and the effect was better than antibiotics.