The Isolation, Identification, CDNA Cloning From Chinese Forest Frog, Rana Chensinensis And The Construction Of Expression Vector

Background & Objective Antimicrobial peptides named antimicrobial peptides or peptideantibiotics are typically 10-50 amino acid residues long. AMPs that are considered to be a mainelement of innate immunity, are a kind of small MW cationic polypeptide with broad-spectrumgrowth-inhibitory activity. Amphibians are constantly exposed to multiple harmful microbes, thecapacity to overcome infections is essential for their survival and, Therefore, they are endowedwith an excellent chemical defense system composed of pharmacological and antimicrobialpeptides. A considerable variety of antimicrobial peptides have been characterized fromamphibians. The skin gland of amphibian acts an important role in synthesizing antimicrobialpeptides. Antimicrobial peptides from mammals are less than Amphibian skin peptides, not onlyin category, but also in quantity. The foreign country has studied on defensive skin secretions ofamphibians for forty years over 500 species. In present, the frog-skin active peptides fromXenopus and Phyllomedusa are more than other species. Up to now there were a lot ofantimicrobial peptides family, include Magainin, Bombinin, Bombinin-like, Brevinin, Esculentin,Ranatuerin, Tempori, Ranalexin, Aurein, Caerin and Citropin. A variety of bio-active substancesfrom amphibian skins which play an important role in defense against invading factors haveattracted significant attention of the researchers all over the world, not only high antimicrobialactivities, wide activity spectrum, but also not easy to induce resistant pathogenic strains. Duringthe past decade, the emergence of multiple-drug resistant strains of many pathogenicmicro-organisms has stimulated the search for new classes of antimicrobial compounds that mayhave clinical applications. For the present study, a novel antimicrobial peptides gene (GenBanKAccession Number: DQ673105) was cloned from Chinese forest frog, Rana chensinensis by 3'RACE. We use bioinformatics method to analyses the nucleotide sequences and amino acidsequences, to predict the structure and the function ofprotein and Cloned the coden sequences of mature peptides into the expression vector ofPGEX-3X to construct Recombination plasmid to fusion expression. Set foundation to study on the level of molecular biology.Methods (1) The peptides of the secretion of Rana chensinensis were purified byreverse-phase high-pressure liquid chromatography and characterized by amino acid analysis andelectrospray ionization mass spectrometry. Antibacterial activity was examed by the radialdiffusion assay during the peptides purification procedure, sing the Gram-negative bacteriaEscherichia coli and Gram-negative bacterium Staphylococcus aureus as the test organism. Thepresence of predicted peptide structures in skin secretions was confirmed by automated Edmandegradation. (2) Using their amino acids sequence of N-terminatio as submitting sequence, theBLAST sever was applied to retrieve amphibiotic antimicrobial peptides in the protein databaseto design a degeneracy primer. (3) In the RNase-free condition, the total RNA of the skin ofChinese forest frog was prepared by Guanidine Thiocyanate. (4) the cDNA sequence ofantimicrobial peptides gene was cloned by the 3'RACE (Rapid Amplification Of cDNA Ends)and the total RNA of the skin of Chinese forest frog was used as cloning template. Thebioinformatics soil-ware was applied to analysis the cDNA sequence that was submitted toGenbanK by the tool of Bankit in the NCBI. such as open reading frame, homology andcorrelative biological function. (5) According to the full-length cDNA sequence to design andsynthesis the primer, we used PCR amplification to get the just sequence that is include no-signalpeptides with the site of restriction enzyme. (6) The target sequence was inserted into thepMD18-T simple cloning vector by T/A cloning. After identification by sequencing, the digestedDNA was cloned into prokaryotic expression vector PGEX-3X to construct Recombinationplasmid, expressed in E. coli BL 21 under induction of IPTG and identified by SDS-PAGE.Results (1) A peptide with antimicrobial activity was purified from the skin secretions ofRana Chensinensis by reverse-phase high-pressure liquid chromatography (RP-HPLC).(2) High-quality totle RNA was prepared by by Guanidine Thiocyanate. (3) A full-length cDNAsequence(GenBanK accession number: DQ673105)of the antimicrobial peptides of RanaChensinensis was cloned by 3'RACE.The cDNA consists of 297bp with 213bp open readingframe encoding 71 amino acids with 7810.9Da (pI=5.08) and 84bp 3' UTR.According to theanalysis of bioinformatics software, the conserved preproregion comprises a hydrophobic signalpeptide of 22 residues followed by a 16 residue acidic propiece which terminates by a typicalprohormone processing signal Lys-Arg, included one copy 33 residue acidic mature peptide. (4)The DNA sequence encoding mature peptide, was successfully cloned into the pMD 18-T simple cloning vector, and then inserted into PGEX-3X. The GST fusion protein with relative molecularweight of 30kDa, was expressed in the construction prokaryotic vector.Conclusion (1) the novel full-length cDNA of antimicrobial peptide CAMPs-3 has clonedfrom Chinese forest frog, Rana chensinensis.it hare about.90%-72%, 70%-53% identity with thecorresponding regions of ranatuerin and brevinin, respectively. (2) The GST fusion protein (30kDa)was expressed in in E. coli BL 21 under induction of IPTG.