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Study on the Recombined Cationic Anti-Microbial Peptide

Author: WuWeiHua
Tutor: ChenJinChun
School: Beijing University of Chemical Technology
Course: Biochemical Engineering
Keywords: Cationic antimicrobial peptides Recombinant Optimization of fermentation condition of In vitro antibacterial activity
CLC: TQ464
Type: Master's thesis
Year: 2003
Downloads: 170
Quote: 0
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Abstract


This laboratory designed and synthesized by a length of 207bp of DNA sequence as a target gene fragment was cloned into the vector pBluescript -M13 , and measuring the vector nucleotide sequences, the physical map is drawn . Study , we chose Pichia pastoris as a gene expression system the secretion type plasmid pPIC9 of as a vector to construct genetically engineered bacteria . Endonuclease Bgl Ⅱ was linearized within the recombinant plasmid pPIC9-Atmp by restriction electroporation was transformed into the recipient strain Pichia pastoris GS115 . Filter obtained by the MD and MM plates his Muts phenotype positive clones JCC-2 23 Zhu . Small - scale cultivation of genetically engineered bacteria , JCC-2 induced expression of the target protein cationic antimicrobial peptides . Fermentation supernatant by Tricine-SDS-PAGE gel electrophoresis , from the molecular weight analysis presumed target protein exists in the form of aggregates . Protein aggregates after mercaptoethanol treatment , the Tricine-SDS-PAGE gel electrophoresis and found that the target protein in the presence of monomers , and the gel imaging system analysis , a monomer molecular weight of about 6000 , and the maximum expression reached 29.67mg / L . The the fermentation supernatant antibacterial activity in vitro experiments showed that : fermentation supernatant supernatant the pale yellow sarcina and E. coli poisoning force , 30μl, 96 -hour fermentation of a slightly yellow sarcina bacteriostatic diameter up to 2.6cm E. coli inhibition diameter of 2.36cm. The the fermentation supernatant antibacterial radius increase with the extension of the fermentation time . The inhibitory effect striped fermentation supernatant sarcina E. coli obvious . Optimization of fermentation conditions by engineering bacteria part get some shake flask fermentation optimal conditions are as follows : JCC - the best growth medium BMGY the growth medium BMGY best initial pH 6.0 , 70 hours maximum biological cell growth Cover 24.7g / l , LT ; WP = 4 GT ; , the amount of protein expression of growth in induction medium for 96 hours maximum, as 29.67mg / l . Screened by the present study , the the recombinant plasmid the pPIC9-Atmp linear after electrically transformed into the recipient strain Pichia pastoris GS115 , a positive clone strain and high - copy strain , to verify the correctness of the target protein , and measuring the the expression of the amount . Target protein in vitro antibacterial activity at the same time to verify and optimize the part of the shake-flask fermentation conditions , to lay the foundation for further study .

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