Response Of AvBD6 On Bacterial Infection And Stress And Exogenous AvBD6 On CLEAP-2

Beta-defensins are endogenous small molecule cationic antimicrobial peptides (CAMP) with broad-spectrum antimicrobial activity. The phagocytes of avian lack oxygen dependent bactericidal mechanisms, and it is more dependent on CAMP to remove bacteria, avian β-defensin 6 (AvBD6) as one of β-defensin family, it is a new promising alternatives to antibiotics.For the moment, the quantitative study of avian β-defensins is little, and quantitative study is the basis of understanding of pathophysiology and application of AvBD.Therefore, sandwich ELISA detection of AvBD6 was established, and the concentrations of chicken serum or tissue was detected in the different pathophysiological states; and the effect of exogenous AvBD6 on chicken liver expressed of antimicrobial peptide (LEAP-2) also was studied. The main findings are as follows.1、Establishment of sandwich ELISA method for detection of AvBD6The immunogen of coating antibody from mouse and detection antibody from rabbit, was the B-cell epitope of AvBD6 (PGPCRW-KLH) and N-terminal of AvBD6 (SPIHACRYQRGV-KLH)coupling with KLH. respectively. The optimum dilution of coating antibody (mouse anti-AvBD6). the detection antibody (rabbit anti-AvBD6) and horseradish peroxidase (HRP) labeled goat anti-rabbit IgG, was 2500,2000, and 10000 respectively. In the linear detection range from 20 to 320 ng/mL,the standard curve regression equation was Y=12024X-2583 (R2=0.9842), Where Y was the concentration of AvBD6, and X was the OD 450.2> Concentration of AvBD6 under different pathophysiology conditionsWith the extension of Salmonella infection. the concentration of AvBD6 in duodenum (from 34.885 to 122.645ng/mL). jejunum (from 45.015 to 183.395 ng/mL) and serum (from 7320.385 to 7855.56 ng/mL) were increased, and showed significant difference with control group at the same time (P<0.05). In Escherichia coli infection, the concentration of AvBD6 in serum (7641.49 ng/mL) was increased,and showed significant difference with control group at the same time (P<0.05). Under cold stress stimuli, with the time extension the concentrations of AvBD6 were increased (502.095-1525.755 ng/mL), and the concentration were significantly increased after 12h stimulation (3666.125ng/mL). Early in ACTH injection (25IU/kgBW), the concentration of AvBD6(1245.60 ng/mL) was slightly lower than the control group at the same time, after continuous injection of 5 days, the concentration of AvBD6 (4429.96 ng/mL) increased gradually, but the concentration had no significant difference with the control group at same time.3、Effect of exogenous AvBD6 on chicken liver expressed of antimicrobial peptideWhen the concentration of exogenous AvBD6 was increased from 20μg/kgBW to 40μg/kgBW, we detect the expression changes of cLEAP-2 in tissues by SABC, found that the expression of cLEAP-2 was significantly increased in liver and intestine, while the expression was only a little in the spleen, thymus and burs.In this study, a sandwich ELISA method was initially established to detect AvBD6, and the concentrations of AvBD6 was detected in the different pathophysiology states. The results show that the expression of AvBD6 could increase with the effect of Salmonella and Escherichia coli infections and cold stress, but the regulation of ACTH on the expression of AvBD6 was not obviously; With the exogenous AvBD6 increasing, the expression of cLEAP-2 in liver and intestine is promoted.