Expression And Identification Of The Antibacterial Peptide Dybowskin-2DAMa From Rana Amurensis

Antibacterial peptide (ABP), also called antimicrobial peptide (AMP) or peptide antibiotics, is expressed by immune defense systems of all kinds of organism to resist pathogen and malignant cells. Antibacterial peptide is a small molecule peptide generally containing amino acids from 10-50, 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 amphibian’s skin is moist, nudity, which can apply a chance for the intrusion of microorganism. Their skin glands secrete a series of antibacterial peptide to defense pathogen. Ranidae is the third family of anura, and its distribution throughout all the continents, and their skin contains many kinds of antibacterial peptide, which means the research of skin antibacterial peptide of Ranidae has great signification.In the previous work of our lab, we had obtained a series of cDNA sequence of antibacterial peptide from Rana amurensis. dybowskin-2CAMa, a new type antibacterial peptide , which is different from other reported Ranidae antibacterial peptide, still have distinct characteristic, such as more positive charge, high isoelectric point and strong hydrophilic, eccept the specificity of sequence. Acording to its structure prediction and amimo acid distribution analysis, dybowskin-2CAMa is anα-helix cationic antibacterial peptide, with the high affinity of bacteria membrane. To further understand the biological characteristic and function of dybowskin-2CAMa , the primers were designed based on the sequence of Dybowskin-2CAMa gene and its gene was coned using SOE-PCR. The PCR product which encoded Dybowskin-2CAMa peptide was ligated into pPICZα-A and the recombinant plasmid pPICZαA-Dybowskin-2CAMa was constructed. The recombinant expression plasmid was linearized with SacI and then transformed into the competent P. pastoris X33 cells by electroporation. Positive recombinant strains were selected by the method of Zeocin-resistant and PCR. Positive transformation expression was induced by methanol, then the total genome of the induced yeast cells was extracted, amplified byα-factor primer and 3 ’AOX primer, obtained the 300bp product, which is as large as the target gene product dybowskin - 2CAMa. The data showed the target gene had already inserted in the chromosomes of the host. The total RNA of the yeast which was induced by methanol was extracted, then was detected by RT-PCR method, the results showed that the antimicrobial peptides dybowskin-2CAMa genes has already transcription.The product of expression was concentrated, and then purified by the method of YMC*GEL ODS-A and SephadexTM G-25 Fine.The molecular weight of the purified Dybowskin-2CAMa analyzed by MALDE-TOF-MS is.the 2456.8, which was the same with the molecular weight of dybowskin-2CAMa. Hemolytic activity analysis and antibacterial activity analysis showed recombination dybowskin-2CAMa inhibited the proliferation of Gram-negative bacteria and Gram-positive bacterial, according to the result,we can infered that the antibacterial activity on the Gram-positive bacteria was stronger than the Gram-negative bacteria. The minimum antibacterial concentrations of the recombination dybowskin-2CAMa to the drug-resistant strains of E.coli EBSL is 4μg/mL and had no hemolytic activity at a concentration of 500μg/mLConclusion: The Dybowskin-2CAMa gene can be expressed correctly by using Pichia pastoris expression system. Antibacterial activity analysis showed recombination Dybowskin-2CAMa inhibited the proliferation of Gram-negative bacteria and Gram-positive bacterial, and according to the result we can infer that the antibacterial activity on the Gram-positive bacteria stronger than the Gram-negative bacteria. Hemolytic activity analysis showed recombination Dybowskin-2CAMa had no hemolytic activity at a concentration of 500μg/mL. The study provided the stable source of material for further study on the mechanism of antibacterial and supression of tumor cell, also accumulated research experience for altering amino acid sequenece of the natural antimicrobial peptides.