A group of antibiotic peptides, method for preparation and use thereof
A technology for antibacterial peptides and fungal infections, which is applied in antibacterial drugs, antifungal agents, chemical instruments and methods, etc. It can solve the problems of sequence modification and functional application, low bactericidal activity, and no hemolytic property of ParasinI
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Embodiment 1
[0030] Embodiment 1 solid-phase chemical synthesis preparation and separation and purification of antimicrobial peptides
[0031] GKV-1 to GKV-12 were prepared according to the above sequence, while GramicidinS, parasinI and magaininII were prepared as controls.
[0032] Sequence of GramicidinS:
[0033] Val Orn Leu Phe Pro Val Orn Leu Phe Pro
[0034] Sequence of ParasinI
[0035] Lys Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr Arg Ser Ser
[0036] Sequence of magaininII:
[0037] Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe Val Gly Glu
[0038] Ile Met Asn Ser
[0039] In this example, solid-phase chemical synthesis was adopted, and the instrument used was the Pioneer polypeptide synthesizer produced by Applied Biosystems, Inc. of the United States. The synthesized peptide was sheared by high-concentration TFA, then purified by reverse column, and the purified peptide was identified by mass spectrometry. The specific test...
Embodiment 2
[0049] Expression of embodiment 2 antimicrobial peptide GK V-4 gene in escherichia coli
[0050] First, design and synthesize the GKV-4 gene encoding the antibacterial peptide, clone it into the pGEX-4T1 vector (purchased from Amersham Pharmcia Biotech), then transform Escherichia coli JM109, induce the expression of GST-GKV-4 fusion protein by IPTG, and then After cleavage by thrombin, the antimicrobial peptide GK V-4 is obtained.
[0051] ATP, IPTG, T 4 polynucleotide kinase, T 4 DNA ligase, Klenow enzyme, and restriction endonuclease are all products of BIOLAB unless otherwise specified, the rubber tapping recovery kit is a product of Shanghai Sangon Company, and oligonucleotides are synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Thrombin cleavage kit was purchased from SIGMA.
[0052] Molecular cloning operations such as DNA isolation, purification, PCR amplification, enzymatic hydrolysis, plasmid transformation of host bacteria...
Embodiment 3
[0059] Expression of embodiment 3 antimicrobial peptide GK V-4 gene in yeast cell
[0060] ATP, IPTG, T 4 polynucleotide kinase, T 4 DNA ligase, Klenow enzyme, and restriction endonuclease are all products of BIOLAB unless otherwise specified, the rubber tapping recovery kit is a product of Shanghai Sangon Company, and oligonucleotides are synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Thrombin cleavage kit was purchased from SIGMA.
[0061] The GK V-4 gene encoding the antibacterial peptide was designed and synthesized, which was digested with BamHI and ligated with the DNA sequence encoding GST, and then ligated into the plasmid pBluescriptSKII (purchased from Stratagene, USA) to transform Escherichia coli DH5α (purchased from Wuhan University Bacillus Species Collection Center), the extracted plasmid was sequenced to prove that the sequence was correct, and the plasmid was digested with EcoRI and XhoI and connected to the pPIC9 v...
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