Construction Of Nisin-rbLF-N Fusion Gene And Expression In Escherichia Coli And Pichia Pastoris

Nisin is an antibacterial peptide produced by certain strain of L.Iactis. It is ribosomally synthesized as a precursor peptide that undergoes complex post-translational modifications. Nisin has a relatively wide antimicrobial spectrum and can inhibit the proliferation of most gram-positive (G+) bacteria, in which it is particularly effective in preventing the development of clostridial spores. It has been used as natural food preservative in a variety of food products worldwide.In view of that nisin has shortcomings of a narrow antibacterial spectrum and that rbLF-N has strong activity against a wide range of bacteria, Construct of fusion gene nisin- rbLF-N, were transformed into E.coli host BL21(DE3) and P. Pastoris GS115 respectively to the performance of expression researches. nisin and rbLF-N fusion gene nisin- rbLF-N was constructed to get protein with broadened antibacterial spectrum.Oligonucleotide primers were designed based on the DNA sequence of nisin and rbLF-N deposited in Genebank. Construction of Plasmid pMD19-T/nisin and genomic DNA was extracted from fresh bovine, Successfully obtained nisin-rbLF-N by PCR, and successfully constructed the recombinant plasmid pGEM-T easy/nisin- rbLF-N. The PCR products were sequenced to confirm the right gene sequence with 340 bp in length compared with the sequence reported by GeneBank.After cloning fusion gene nisin- rbLF-N into the expression vector pGEX-4T1, the gene was transformed into E. coli BL21 prokaryotic expression system and expressed at high level in prokaryotic cells, the expression of nisin- rbLF-N gene was detected by Tricine-SDS-PAGE protein electrophoresis and functional test. SDS-PAGE analysis of the IPTG induced expression of pGEX-4T1/ nisin- rbLF-N showed that the weight of target protein was correct, and it was approximately 38KDa. The result showed that after induction fusion protein was expressed at considerable amount , fusion protein was found mainly in inclusion bodies, after being solved in 8 mol/L urea and refolded showed antimicrobial activity against G+ bacteria and no activity against G- ones.Then the successful constructed eukaryotic recombinant expression plasmid pPIC9K/ nisin- rbLF-N, pPIC9K/ nisin- rbLF-N was linearized with SacI, and electroporated into P. pastoris GS115. 362 transformants were selected on the MD agar plates, and 40 highly expression transformant were screened by YPD plates containing G418. After expression of recombinant elastase in shaking flask by methanol induction, we found the size of recombinant elastase was approximately 10KDa by SDS-PAGE analysis. Removal of the fusion protein glycosylation, and then showed antimicrobial activity against G+ and G- bacteria .The experiment cloned a nisin- rbLF-N gene, constructed prokaryotic and eukaryotic expression plasmid and expressed the protein successfully. The results for the further construction of excellent antibacterial protein fusion to provide a certain amount of research material and theoretical basis.