The Design, Cloning And Expression In E.coli Of A Novel Fusion Protein Cecropin B-Human Lysozyme Gene

Anti-bacterial peptide (abbrevitated form-ABP) is a kind of small size molecular peptides that are encoded by a special gene, still is important molecular protective screen of that the host defend pathogenic bacteria invaders. These peptides have broad spectrum of antibacterial effect and inhibit DNA replication of some virus. They can kill some cancer cells selectively. Insect cecropin has a specific chemical structure and has a new target point to bacterial cell membrane, which is different from other general antibiotic. It may destroy the cell membrane directly, and there is no toxic effect to antimal and human body. While human lysozyme also have the antibacterial and antivirus activity. Because human lysozyme is a protein from human cell, so it is compatible with human body. Using in clinic, human lysozyme is safer than other lysozyme and has no irratation and minus effects. The aim of this research is that Cecropin B and human lysozyme gene were fused and subcloned into the expression vector through gene recombinant, and then maybe expressed the fusion protein with more effective antibacterial ability.In order to establish the cloning vector of fusion gene cecropin B - human lysozyme, Cecropin B gene was amplified through using gene splicing by overlap extension (gene SOEing) method and human lysozyme gene was gained from plasmid pUC118-hLyso by PCR. Then Cecropin B gene was genetically fused to the 5' end of hLyso gene and the termination codon was designed at the 3' end of hLyso gene. Finally, cecropin B gene and hLyso gene were subcloned into cloning plasmid pBS-T. The recombinant vector was verified with sequence analysis. The results showed that fusion gene cecropin B - human lysozyme was cloned correctly into pBS. It concludes that the vector was constructed successfully. Then the recombinant plasmid was turn into the E.coli to make the fusion gene cecropin B - human lysozyme largely amplified.After amplified by PCR, the fusion gene cecropin B - human lysozyme wasgained from electorphoresis. The expression vector pET32a and the fusion gene cecropin B - human lysozyme were cutted with the same restrict enzymes and were ligated together with T4 Ligase. The restriction analysis results showed that the fusion gene cecropin B - human lysozyme was ligated correctly into pET32a.The recombinant expression plasmid pET32a~CB-hLyso was transformed into E.coli BL21(DE3) and BL21(DE3)pLysS, then expressed the fusion protein, and was tested the antibacterial activity of the new fusion protein Cecropin B- humanlysozyme.