Preparation process of antibacterial peptide

An antibacterial peptide and engineering bacteria technology, applied in the directions of antibacterial drugs, introduction of foreign genetic material using a carrier, fermentation, etc., can solve the problems of high cost, complex peptide synthesis process, unsuitable for large-scale production, etc., to reduce the cumbersome and simplify. Follow-up purification work to achieve high-efficiency expression

Inactive Publication Date: 2005-01-05
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to disclose a method for preparing the antimicrobial peptide Adenoregulin by using genetic engineering technology, so as to overcome the shortcomings of the prior art that the peptide synthesis process is complex, the cost is high, and it is not suitable for large-scale production

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  • Preparation process of antibacterial peptide
  • Preparation process of antibacterial peptide
  • Preparation process of antibacterial peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: the preparation method of ADR polypeptide

[0046] In this protocol, Adenoregulin is expressed by the pET expression system of Novagen Company, the pET32a(+) plasmid is used as the expression vector, and the host cell is Escherichia coli.

[0047] 1 According to the sequence of SEQ ID NO: 1 (GENEBANK ACCESSION X70278 S58040), the antimicrobial peptide Adenoregulin gene was synthesized by chemical method and modified according to the preferred codons of Escherichia coli.

[0048] 2 Design and synthesize primers

[0049] Primer 1 (SEQ ID NO: 2) introduced a BamHI restriction site, and at the same time added a methionine encoding gene, ie, a cyanogen bromide recognition site, upstream of the structural gene. Primer 2 (SEQ ID NO: 3) introduces an XhoI restriction site, and at the same time introduces a methionine encoding gene, ie, a cyanogen bromide recognition site, downstream of the structural gene.

[0050] 3 PCR amplification of Adenore...

Embodiment 2

[0080] Embodiment 2: the preparation method of ADR polypeptide

[0081] In this protocol, the pET expression system of Novagen was used to express Adenoregulin, the pET32a(+) plasmid was used as the expression vector, the host cell was Escherichia coli, and multiple copies of the ADR gene were expressed in tandem.

[0082] 1. Design and synthesize primers

[0083] Design primers 3 and 4, introduce restriction endonuclease EcoRI, SalI recognition site and methionine codon in primer 3 (SEQ ID NO: 4), introduce methionine in primer 4 (SEQ ID NO: 5) Acid codons and XhoI, NotI recognition sites.

[0084] 2. Amplify ADR gene by PCR method

[0085] 1) Reaction composition:

[0086] 10xBuffer (Shanghai Sangong) 5μL

[0087] 4 dNTP mixes, each concentration 10mM 4μL

[0088] Primer 3 (5μM) 5μL

[0089] Primer 4 (5μM) 5μL

[0090] Template pET32a-adr (0.1 μM) 5 μL

[0091] Taq enzyme (5u / μL) 1μL

[0092] Add water to a final volume of 50 μL

[0093] 2) Put it i...

Embodiment 3

[0114] Embodiment 3: the preparation method of ADR polypeptide

[0115] In this example, the Pichia pastoris expression vector from Invitrogen Company, such as the pPIC9K plasmid, is used to express the ADR gene with yeast as the host cell.

[0116] 1. Construction of recombinant plasmids (see Figure 8 , Figure 9 )

[0117] 1) Digest pPIC9K plasmid with EcoRI and NotI, (5mL pPIC9K plasmid, 2mL 10' buffer (Roche Reagent Company), 0.5mL EcoRI, 0.5mL NotI, 12mL ultrapure water, 37°C, 2 hours).

[0118] 2) Digest pMD18-adr' with EcoRI and NotI, (5mL pMD18-adr'plasmid, 2mL10' buffer (Roche Reagent Company), 0.5mL EcoRI, 0.5mL NotI, 12mL ultrapure water, 37°C, 2 hours)

[0119] 3) Recover the large fragments of 1) and the small fragments of 2) by electrophoresis.

[0120] 4) Ligation of Fragment 1) and Fragment 2) (2.5mL ultrapure water, 1mL 10' ligation buffer (Dalian Bao Biological Co., Ltd.), 1mL Fragment 1), 5mL Fragment 2), 0.5mL ligase, 14°C, 20 hours) ....

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Abstract

The invention discloses a method of preparing antibiotic peptide Adenoregulin, chemically synthesizing an object gene, making amplification by PCR, constructing recombinant particles, converting them into host cells, obtaining bacteria making fusion expression on Adenoregulin polypeptide, fermenting and culturing the bacteria, at last making purification and separation to obtain the antibiotic peptide Adenoregulin.

Description

technical field [0001] The present invention relates to a preparation method of antibacterial peptide, more specifically relates to a kind of genetic engineering preparation method of antibacterial peptide Adenoregulin. Background technique [0002] Antimicrobial peptides are a type of membrane-active polypeptides with broad-spectrum bactericidal, antiviral, or tumor cell-inhibiting effects. Its unique mechanism of action is not easy to cause drug resistance in bacteria, and it is expected to be developed into a new generation of antibacterial, antiviral, and anticancer agents. drug. Adenoregulin is a broad-spectrum antibacterial peptide extracted from the skin secretion of the South American tree frog Phyllomedusa bicolor in 1992. It consists of 33 amino acids and has a similar precursor structure and antibacterial spectrum to Dermaseptin b. It is an antibacterial peptide Dermaseptin family. one of the. In addition, Adenoregulin has an anti-filamentous fungal effect that ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P31/04C12N15/63C12P21/02
Inventor 周宇荀曹魏魏东芝马昱澍王锦之
Owner EAST CHINA UNIV OF SCI & TECH
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