Research On Two Types Of Expression Of Antibacterial Peptide Metchnikowin In Escherichia Coli

Antibacterial peptide is a kind of small molecular peptide that can inhibit bacteria.Up to now, more than 2600 kinds of antibacterial peptide was separated and identified.Antibacterial peptides not only has good antibacterial effect, but also does not produce resistance, is a good alternative to antibiotics, and gradually become a hot research.Antibacterial peptide Metchnikowin(Metch) which are rich in proline, composed of 26 amino acid residues, can inhibit the growth of gram-positive bacteria and fungi.The main purpose and main work of this paper were researching the secreted expression and intracellular soluble expression of Metch in BL21(DE3). Using the Metch gene cloned by our laboratory and Yeb( e Yeb) secreted protein gene in e. coli,we constructed the expression vector p ET28b-sp-e Yeb-RS-Metch( P-1) which can express in BL21(DE3) suitably.After the inducing of IPTG,antibacterial peptide Metch was successful secreted to the outside of the cell by the guidance of secreted protein e Yeb.The best secreted expression conditions of fusion protein e Yeb-Metch were 30?, 200 rpm, IPTG concentration 0.3 m M and expressiom time 24 h.As secreted protein,Yeb protein played an important role in secretory expression quantity of antibacterial peptide.In order to study the effect of different Yeb protein expression of secretion, using another Yeb(s Yeb)protein which comes from salmonella and Metch to build a new vector p ET28b-sp-s Yeb-RS-Metch(P-2).Through the comparision between s Yeb-Metch and e Yeb-Metch in the expression quantity,we found that the quantity of s Yeb-Metch was higher than the e Yeb-Metch.Yeb-Metch protien which was cut by TEV cleavage enzyme, had antibacterial activity for Staphylococcus aureus and Micrococcus.We also researched the fusion expression of Metch in BL21(DE3).Using ubiquitin(Ub) as a carrier protein,we constructed expression vector p ET28b-His-Ub-RS-Metch(P-4).The best secreted expression conditions of fusion protein Ub-Metch were 20?, 150 rpm, IPTG concentration 0.1 m M and expressiom time 24 h.The fusion protein His-Ub- RS- Metch was effectively purified by nickel column and antibacterial peptide Metch was released from the purified fusion protein which was cut by TEV cleavage enzyme specifically.Bacteriostatic circle method of inspection revealled that fusion antibacterial peptide Metch had antibacterial activity for Staphylococcus aureus.In this experiment,the quantity of secreted expression could be increased by taking the place of Yeb protein;comparing with the secreted expression, the quantity was further improved by intracellular expression when we used ubiquitin as a carrier protein and the purification system of fusion protein was established preliminary in Escherichia coli.