Research On The Fused Expression In Escherichia Coli Of The Antibacterial Peptide Dybowskin-2CDYa And HEGF

Antibacterial peptide(ABP),is expressed by immune defense systems of all kinds of organism to resist pathogen and malignant cells.ABP is a small molecule peptide generally containing 10-50 amino acids,with the features of broad-spectrum antibacterial activity,strong thermal stability.small molecular weight and immunogenicity and the bacteria difficult to produce resistant.Such peptides are attracted increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics.The living environment of the amphibians is complex and various,and their skin is moist and bareness,which created the condition for the invasion of the microorganism from the outside.In order to defense the invasion,they dissolve out a series of ABPs by the gland of skin.Research on the skin secretion of amphibians has lasted for more than forty years in aboard,and five hundred-kinds of ABPs have been reported.Different kinds of antibacterial bioactive molecule can be secreted by skin of different species.So far,a variety of animo acid sequence of ABPs were obtained by assay,and detailed research was done on their structure and function.The result indicated that the peptides catalogue of the amphibians'skin presented a variety of molecule and function beyond imagination.The Ranidae is the third big family of the Salienlia which distributes almost all over the world.So the class and the quantity of their skin peptides are all astonishing,which means the research on it has enormous value.In the previous work of our lab,we had obtained a new ABP from Rana dybowskii named Dybowskin-2CDYa(Included:EU827809),this ABP is composed of 18 amino acids,molecular weight of 2154.2,isoelectric point is 12.6 and have 10 positive charge.It has more positive charge:C terminal has not Amidation modified;it is basic peptide has a high pl;strong hydrophilia etc physicochemical character which is different with other ABP.According to the analysis of the characteristics of the peptide and structure prediction,Dybowskin-2CDYa is a series of ?-helix ABP which has strong affinity to the bacterial membrane,guess it may have strong antibacterial activity.Genetic engineering is the most effective way to get a mass of ABP.which mainly include Escherichia coli,Pichia pastoris,plant,insect etc expression system.Prokaryotic expression system is the first expression system of ABP.it is also the most widely used way.because of the strong toxicity to prokaryotic cells,expressed ABP in the shape of fusion protein in Escherichia coli expression system usually.Fusion expression can improve the expression level of ABP,neutralized its alkalinity,avoiding the degradation of enzyme,reduced the toxicity to the prokaryotic host by expressed with the inclusion body which has no activity,increased the stability of the production.At the same time,the expressed fusion protein has many specific labels,which could separated and purified it easily.Human Epdermal Growth factor(hEGF)is a member of the somatomedin family.it is the mitogen of sensitive cells like epidermis cell,epithelial cell and mesenchyme,hEGF is one effective factor of promote cell multiplication.hEGF recombinant protein has been widely used in skin burn,burn,corneal injury,gastric ulcer etc and effects obvious.It is also has a obviously effect in the formation of new blood vessels,promote the proliferation of epidermal cells and stimulation of nerve cell differentiation etc,so It can be used in the treatment of trauma,burns and accelerate wound healing and other diseases.In order to get the stably expressed active peptide which had antibacterial and Promoted wound healing activity,fusion expressed ABP Dybowskin-2CDYa and hEGF in Escherichia coli,study the antibacterial activity of fusion protein,then,lay foundation for the research of skin repair function.Firstly,we design the gene sequences of Rana dybowskii ABP and hEGF according to the codon preference of Escherichia coli,got the whole gene fragment by SOE-PCR.connected with the prokaryotic expression plasmid pET-30a(+),built reorganized expression plasmid.By sequencing analysis.plasmid sequences is identical with the design sequences,reading frame without error.Transformed the reorganized plasmid pET-30a-hEGF-Dybowskin-2CDYa to E.coli BL21(DE3),Con-structed prokaryotic expression system of fusion protein,tested the recombinant strain by vector primers.and induced the expression of fusion protein by IPTG.Analysed the expression by 12%SDS-PAGE,the result shown the aim stripe on 14.3 KD of protein standard,confirmed the expression of fusion protein by Western Blot.Dissolve the inclusion body of fusion protein which had no activity.isolated and purified by Nickel ions metal chelation affinity chromatography.removed salt and renatured the purified protein by dialysis,detected the antibacterial activity of fusion protein,the result shown that the fusion protein has inhibitory activity both to the Gram negative bacteria and Gram positive bacteria.The conclusion:Obtained the fusion protein gene sequence of Dybowskin-2CDYa and hEGF by SOE-PCR,and builted the recombinant expression plasmid named pET-30a-hEGF-Dybowskin-2CDYa of fusion protein.Obtained the stably expressed fusion protein using the prokaryotic bacterial strain which induced by IPTG.The fusion protein separated and purified by Nickel ions metal chelation affinity chromatography,SDS-PAGE proved the existence of purified protein.The renatured fusion protein has antibacterial activity both to the Gram negative bacteria and Gram positive bacteria.This paper laid theoretical foundation for the research of structure and function,also for the antibacterial mechanism of antibacterial peptide Dybowskin-2CDYa.