Expression Of Recombinant Chicken β-defensin-2 In E.coli And Partial Biological Activity Research

Gallinacins, a group of small cationic antimicrobial peptid, have antimicrobial activities against G ram-positive,G ram-negative bacterium, fungus and virus. In order to investigate the partial biological activity of the gallinacins, This study has been successfully cloned in the chickenβ-defensin-2(Gal-2) expression plasmid on the basis of the successful expression and purification of recombinant Gal-2, after a in vitro antibacterial activity, lymphocyte proliferation activity and the immune organ index detection, access to a large number of Gal-2 and to lay the foundation for the production. The results are as follows:Have been cloned into the pMAL-c2X-Gal-2 in E.coli, expression conditions were optimized, the optimal induction conditions were as follows: at 15℃, host bacteria A600 for 1.0, IPTG concentration 0.8mmol/L, induced by 8h expression. IPTG-induced protein were purified by Amylose resin, BCA measured the concentration of purified samples, and then were cut by Factor Xa and purified by Amylose resin, tested by SDS-PAGE. The results showed that the purification sample was MBP-Gal-2 fusion protein, SDS-PAGE electrophoresis and 50KD grayscale scan revealed the location in a clear strip of inducible expression, the recombinant protein accounted for 36.24 of total bacterial proteins, the fusion protein in soluble form, the concentration can be achieved 0.468mg/mL. However, after cutting a few purified after SDS-PAGE only be put on MBP-Gal-2 and MBP, no Gal-2.To the number of medium-term growth for the detection of E. coli bacteria, the spread of the use of thin-layer agarose plate were used to be detecting the antibacterial activity of recombinant MBP-Gal-2, and also under the conditions of 20℃~100℃and pH4~pH10. The results showed that: Recombinant MBP-Gal-2 fusion protein has antibacterial activity against E. coli and in addition to 20℃~100℃and pH4~pH10.Chicken peripheral blood lymphocytes in vitro using the MTT method do not have the concentration of MBP-Gal-2 on lymphocyte proliferation function. The results showed that: in the dose of MBP-Gal-2(100μg/mL) to promote the PHA-P or LPS stimulated the proliferation of chicken peripheral blood lymphocytes, and was positively correlated with the dose. However, high doses of MBP-Gal-2(200μg/mL) inhibited the proliferation of peripheral blood lymphocytes.In vivo animal experiments to study the MBP-Gal-2 on lymphocyte proliferation function of conversion. The results showed that: Subject to section 10d experimental group compared with the control group, significant differences in other groups no significant difference.In vivo animal experiment, also examined the MBP-Gal-2-related immune system Chick index. The results showed that: defensins chicken on strengthening the immune function of the immune system have a certain role.The experimental results show that from the above: the Gal-2 both in antibacterial activity and immune system responses, are stronger with higher biological activity, the test for the reorganization of the chicken-defensin-depth study of biological functions and applications a theoretical reference.