Expression And Purification Of Antimicrobial Peptide Dermaseptin S4 Gene In Baculovirus Expression System

Objective:We express and purify the antimicrobial peptide Dermaseptin S4 (DS4) in Bac-to-Bac baculovirus expression system.It layed the foundation in order to study the structure and function of antimicrobial peptides Dermaseptin S4Methods:1.According to amino acid sequences of antimicrobial peptides Dermaseptin S4 and eukaryotic preferred codons genes sequence,we inducted the genes of Dermaseptin S4,and we connected 6 Dermaseptin S4 with 5 connecting peptides.2.the fragment was cloned into the pFASTBac1 vector of BAC-TO-BAC recombinant baculovirus expression system to construct a recombinant expression vector.3.The plasmid pFASTBac-Dermaseptin S4 (DS4) was then transformed into DH10BACTM competent cell. High molecular weight DNA, The recombinant bacmid was prepared from the ovemight cultures of selected Eschefichia coli DH10BACTM colonies. We extracted recomplasmid and identified it with universal primer RV as primers for PCR cation.4.By transfecting spodoptem fragiperda 9 (SD)cells with the recombinant bacmid, the recombinant baculovirus from the supernatant was obtained, which could further infect SD cells and express the target protein.Identify the expressed protein by SDS-PAGE and Western blot.5.We purified the protein of Dermaseptin S4 with ProBondTM purification system on non-denaturing conditions.Results:PCR amp lification and DNA sequencing analys is confirmed that the gene was correctly inserte d into the vector. Relative molecular masses of 27000 were expressed by SDS-PAGE and accounted for 20.1%。Conclus:The correct tandem of Dermaseptin S4 gene is constructed and expressed in insect cells by baculovirus expression system successfully.We Purified the protein furtherly and we made the basis to study the structure and function of the protein again.