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Purification, Characterization, Molecular Cloning and Diversity Analysis of Anti-Infective Peptides from Rana Pleuraden Skin

Author: WangXu
Tutor: LaiZuo
School: Nanjing Agricultural College
Course: Zoology
Keywords: Amphibians Rana Antimicrobial peptides Anti-infective Diversity
CLC: S865.49
Type: Master's thesis
Year: 2008
Downloads: 131
Quote: 4
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Abstract

Antimicrobial peptides (Antimicrobial peptides, AMPs) are a class of small molecule peptides (molecular weight less than 10 kDa), was first discovered and named, and later because it is not easy to induce microorganisms develop resistance caused worldwide with broad-spectrum antimicrobial activity widespread concern. With the deepening of the study, it was found: the innate immune peptides are one of the end-effector, the defense and the elimination of foreign pathogens (bacteria, fungi, viruses, protozoa, etc.) of the invasion and destruction of the body to maintain the body's normal physiological function has played an important role, is an the activity important factor in the inflammatory response, immune activation and wound healing process. Plants, animals (insects, amphibians, humans), and even the microorganisms found the presence of antimicrobial peptides (sample). Anti-infective peptide (Anti-infective peptides) proposal of the concept, the functional correction of antimicrobial peptides in the name. Amphibian skin secretion first defensive barrier against microbial infection, Zasloff et al 1987 Magainin has been isolated from the skin of Xenopus laevis, more and more extensive and in-depth research of scholars from various countries of the amphibian skin secretion up. The frog (Rana pleuraden), is endemic in China, mainly distributed in China's Yunnan, Guizhou, Sichuan, medicinal and economic value has not yet been discovered, there is a broad space for the development and utilization. In this paper, we use the means of biochemistry and molecular biology of the frog skin anti-infective peptide. The liquid component of the frog skin secretion were isolated and purified by a two step method (Sephadex G-50 and RP-HPLC), we obtain two kinds of a polypeptide having antimicrobial activity, named pleurain-A1 and pieurain-A2.Edman degradation method Determination The whole sequence, respectively, is SIITMTKEAKLPQLWKQIACRLYNTC and SIITMTKEAKLPQSWKQIACRLYNTC. use matrix assisted laser dissociation ionization technique (MALDI-TOF-MS) Determination of the molecular weight of 3048.5 Da and 3024.8 Da of. The CLONTECH company Creator ^{TM SMART ^{TM cDNA Library Construction Kit build of the frog skin cDNA library, based on protein sequence alignment, similar to the antibacterial peptide signal peptide N end primers were designed sequence, we from the Yunnan frog skin anti-infection polypeptide was subcloned library, screened 135 anti-infection by the antimicrobial peptide signal peptide encoded polypeptide cDNA nucleic acid sequence (GenBank accession accession no.: EF613243-EF613259, EF621707-EF621709 EU138029-EU138143), encoding 34 polypeptides, belonging to 12 antimicrobial peptides family pleurain-As (4), pleurain-Bs (4), pleurain-Cs (2), pleurain-Ds (unit) the pleurain-E (1), pleurain-F (1) pleurain-Gs (nine), pleurain-Js (2) pleurain-K (1), Lividin-RP (1) pleurain- Ms (2) pleurain-N (1 unit). Study found the molecular diversity of these anti-infective peptide precursor sequence of 12 family of antimicrobial peptides SPD area (Signal and PropieceDomain, SPD) is very conservative, the Mega and DAMBE molecular genetic analysis shows that they may have originated in the same ancestral gene. Synonymous mutation rate and a missense mutation rate detected, they may be subjected to different selective pressures. Randomly selected 10 family of anti-infective polypeptide the synthetic study its biological functional diversity found: by the pleurain-A1 pleurain-D4, pleurain-G7, pleurain-M1 having a broad spectrum of anti-microbial activity, pleurain-J1 exhibit a narrow antimicrobial spectrum (anti-Staphylococcus aureus), while other peptides of the family does not directly have the ability to kill microorganisms. Anti-infection of the same family the polypeptide pleurain-G1 and pleurain-G7 exhibit significantly different anti-bacterial activity, antibacterial peptide, pleurain-G1 in the activity in the present experimental procedure is not detected. In addition, we also found that the anti-infective peptides generally have 10 family promote histamine release, the promotion of mast cell degranulation, aggregation of red blood cells, remove one or several of the biological activity of free radicals and other immune-related, indicates that antimicrobial peptides (sample ) is present may be provided with the body's immune response is closely related. With the deepening of antimicrobial peptide research, with the further understanding and awareness of the body's innate immune. In this study, alternative medicine - drugs of antimicrobial peptides (anti-infective) is looking for a new type of traditional antibiotics provide more design template.}}

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