Preliminary Purification Of Antibacterial Peptide Shiva-1 And Construction Of Prokaryotic Fusion Expression Vector Of ABPS1 And Interferon A-2b

The antibacterial peptide (ABPS1) is a small peptide which has a wide range of biological activities such as antimicrobial activities, antivirus and antitumor activities. And interferon is a key member of the glycosylated cytokines that share the capacity to inhibit viral replication and to exert effects on immune function in the host immunity. In this study, in order to obtain the fusion protein of IFNα -2b and ABPS1, the co-expression vector of IFNα -2b and ABPS1 genes was constructed and expressed in E.coli.On the base of high expression of ABPS1-GFP fusion protein in E.coli, the purification of expression product was performed. The fusion protein was highly expressed in the form of inclusion body. The bodies of engineering E. coli were harvested by centrifugation and disturbed by sonication, then inclusion body was extracted and washed with buffer consisting of urea and TritonX-100 for several times. The inclusion body was dissolved in the denature buffer using urea as denaturant, and then purified under urea denature condition using nickel chelate affinity chromatography method. A higher purity of the denatured inclusion body protein was attained. After renaturation through gradient dialysis method, a ideal renatured protein was obtained.However, there exists CNBr which is a kind of toxin and is difficult to buy. Furthermore, it is forbidden in production of gene engineering product.So we constructed Prokaryotic Fusion Expression Vector of ABPS1 and Interferon a-2b.The antibacterial peptide Shiva-1 gene and Interferon a-2b gene were subcloned by PCR respectively and inserted into pET-28b(+) expression plasmid in turn by restriction endonuclease.Then the multimer was constructed by a pair of isoschizomers, BamHI and Bgl II, which could produce the same cohesive end (GATC) when cut its target DNA sequence, their digested fragments could be ligated by T4 ligase and could not be excised by Bgl II or BamHI again. The recombinant plasmids containing IFN-a-2b and two copies of ABPS1 gene was transformed into Escherichia coli BL21 (DE3) plysS and then induced by IPTG to express fusion protein. The multimers of IFN α -2b and ABPS1 has been successfully constructed by restriction endonuclease and direct DNA sequencing. This benefits preparation of IFNα -2b and ABPS1 in a large scale and provides an experimental data for the production of other small bioactive peptides.