Studies On Prokaryotic Expression And Biological Activity Of Duck IFN-α

According to the sequence of PB V220-DulFN-αORF which was cloned and constructed by our lab, primers were designed by permer5.0, then, the gene of duck IFN-αmature fragment was obtained by PCR and cloned into PMD18-T vector. After sequencing, it was subcloned into pET32a⁺ to construct recombinant expression plasmid. Then it was transformed into Escherichia coli BL21(DE3) and expressed after induced by IPTG The molecular weight of recombinant was 37kDa, and the expression product were non-soluble inclusion body which was 30% of total protein in BL21(DE3). The recombinant was purified by metal immobilization affinity chromatography(MIAC). The inclusion body was refolded by the methods of dilution and MIAC, then the activity of the recombinant was detected by anti VSV in DEE In the dilution refolding, the mature protein of duck IFN-αwas refolded preliminarily and its activity against VSV was up to 800U. And the activity of the mature protein which was refolded by MIAC was up to 12800U.The activity against DPV was detected in the DEF which was protected by 30U duck IFN-αmature protein by a quantitative PCR assay. The result shows that: in the 15 hours after the DEF infected by DPV, there is a little difference(p＞0.05) in the copies of virus between the positive group and the negative group; After 23 hours, the copies of virus of the group which was protected by duck IFN-αmature protein is lower than the negative one. The difference is up to the most between 47-55 hours after infected, and there is conspicuous difference(P＞0.05). Compared with the copies of DPV of the positive group, the group which was protected by duck IFN-αmature protein can reduce 10^8.83 copies, and the relatively suppressing rate is 80 %. These results imply that duck IFN-αmature protein can suppress the duplication of DPV in log phase, and can be applied to the clinical.The gene of DuIFN-αORF was cut from the recombinant plasmid of PBV220-DuIFN-αORF. in the some methods, the recombinant bacterium of BL21 (DE3) /pET32a⁺-DuIFN-αORF was constructed. After induced by IPTG, the inclusion body ofpET32a⁺-DuIFN-αORF was expressed, its molecular weight was 42kDa. Then, it was purificated-refolded by Ni-MIAC and the activity against VSV in DEF was up to 450U/mg.Compared with the activity against VSV of DuIFN-αORF and the activity against VSV of DuIFN-α,which refolded by MIAC ,the result shows:the signal peptide of DuIFN-αORF resisted its refoding.