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Cloning And Expression Of Antibacterial Peptide Cryptonin Gene In Pichia Pastoris

Posted on:2013-06-27Degree:MasterType:Thesis
Country:ChinaCandidate:Y Y ZhaoFull Text:PDF
GTID:2180330395463296Subject:Food Science
Abstract/Summary:PDF Full Text Request
Antibacterial Peptides are a class of short peptides, which are encoded by certain genes. They are very important to defend the pathogenic microorganisms. Antibacterial Peptides have the features of advantages in thermostability, water-solubility, and also have wide antibiogram, etc, and have stronge immunity in extreme environment, for example, low pH value, hight pH value and high ionic force. Antibacterial Peptides only act on rocaryotic cells and eucaryote cells which have pathologic change, and are not able to work on natural well-balanced eucaryote cells. Antibacterial Peptides would not cause the germ become the bacterial strain of the drug resistance. Nowadays, there are three ways to obtain Antibacterial Peptide. The way by using genetic engineering is an available method to receive Antibacterial Peptide.A new Antibacterial Peptide, Cryptonin, was isolated and characterized from the adult Korean blackish cicada, Cryptotympana dubia. It has the typical linear a-helical, consists of24amino acid residues and has a molecular weight of3,000Da. It can kill microbial cells effectually. In this experiment, based on the amino acid sequence of Cryptonin which is registered in GenBank (ID:GI:206557920), we selected it as the fragment. Selected trinucleotides, which is the Pichia pastoris preference, to design nucleotide sequence of the Cryptonin gene and its primers using in PCR. Then structured the new vector pPICZa A-Cryptonin. According to the result of its products of double digestion, the result of PCR with the target genes and gene sequencing, it could be approved that the vector of pPICZa A-Cryptonin was the target vector which structured by us.Chose Pichia pastoris GS115as the expression system of Cryptonin, linearlized the vector pPICZa A-Cryptonin by SacI, and transformed the vector into Pichia pastoris GS115by way of electrotransformation. Selected the high copy leve transformants by different gradient slope consistency of Zeocin. Identified transformants by PCR. Induced the GS115-pPICZa A-Cryptonin by using methanol, and made the bacteriostatic test by the expression supernatant. Purified the expression product by Sephadex G-50, and made the Tricine-SDS-PAGE experiment. At last, made a quantitative analysis of Antibacterial Peptide by means of Bradford method.Making a comprehensive survey of the study, we can find that the new vector pPICZa A-Cryptonin was structured and transformed it into Pichia pastoris GS115. The products had shown the function of bacteriostasis. The result of the Tricine-SDS-PAGE experiment showed that the expression products contained Cryptonin. The expression products could destroy and inhibit the growth of E.coli, Salmonella, S.aureus, their antibacterial circle diameter were2.3cm,2.2cm and1.3cm. After purified we got the purified Antibacterial Peptide, the expression level of Cryptonin was8.3mg/L.
Keywords/Search Tags:Cryptonin, Pichia pastoris, recombinant expression
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