CN1920020A - Gene engineering producing method of antibacterial peptide Pexiganan - Google Patents

Gene engineering producing method of antibacterial peptide Pexiganan Download PDF

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Publication number
CN1920020A
CN1920020A CNA2005100148334A CN200510014833A CN1920020A CN 1920020 A CN1920020 A CN 1920020A CN A2005100148334 A CNA2005100148334 A CN A2005100148334A CN 200510014833 A CN200510014833 A CN 200510014833A CN 1920020 A CN1920020 A CN 1920020A
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China
Prior art keywords
pexiganan
gene engineering
seq
antibacterial peptide
expression vector
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CNA2005100148334A
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Chinese (zh)
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杨旭
吴全忠
高峰
姜燕
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Tianjin Tasly Pharmaceutical Co Ltd
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Tianjin Tasly Pharmaceutical Co Ltd
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Priority to CNA2005100148334A priority Critical patent/CN1920020A/en
Publication of CN1920020A publication Critical patent/CN1920020A/en
Pending legal-status Critical Current

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Abstract

The invention relates the antibiosis peptide Pexiganan gene engineering expression method, comprising synthesized encoding Pexiganan DNA sequence, which is connected with the carrier to express and purify. The encoding Pexiganan DNA sequence adopts codons which is used high frequency, reducing the mismatch rate. The Tm value is moderate, and the Pexiganan expressed in procaryotic cell fusion expression system is 48-50% of total protein.

Description

The genetic engineering process for preparing of antibacterial peptide Pexiganan
Technical field
The invention belongs to gene engineering technology field, relate in particular to the gene engineering expression method of antibacterial peptide.
Background technology
All kinds of diseases that antibiotic discovery makes people cause cause pathogeny imcrobe infection are no longer at a loss what to do, and the protection human health is made a great contribution.But, along with antibiotic widespread use, cause pathogenic strains constantly to produce resistance, some are the very effective controlled communicable disease of microbiotic in the past, and are revivable in recent years.And because chemical sproof continuous enhancing and spreading, it is powerless that existing microbiotic has seemed.Antibiotic in addition generally abuse and drug residue, problems such as some antibiotic anaphylaxis have been on the rise and have become increasingly conspicuous, and the antibiotic preparation that presses for new class is solved.
Face the urgency of bacterial drug resistance challenge greatly and current situation, investigators explore and the present diverse novel antibacterial medicine of mechanism of action with all strength.
Antibacterial peptide extensively is present in the multiple organism, be organism to external world pathogen infect and a series of immunoreactive product that produces, its molecular weight is little, stable performance has stronger broad-spectrum antimicrobial ability.This class antibacterial peptide acts on microbial film (cytolemma, adventitia), destroy its barrier and reach the effect of killing and wounding bacterial cell, has " traditional microbiotic " incomparable superiority simultaneously: can not induce the generation of drug resistance strain, promise to be antiseptic-germicide of new generation.Pexiganan is a micromolecule polypeptide of being made up of 22 amino-acid residues, its primary structure is: Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys-NN2, theoretical molecular is: 2.4Kda, derive from the analogue of xenopous laevis epithelium antibacterial peptide magainin family, correlative study shows, such antibacterial peptide has antimicrobial spectrum (the Yigong Ge of broad, Dorothy L.et al, In Vitro AntibacterialProperties of Pexiganan, an Analog of Magainin, Antimicrobial Agents and Chemotherapy, 1999,43:782-788.Bessalle, R., A.Kapitkovshy, et al.All D-magainin:chirality antimicrobial activity, andproteolytic resistance.FEBS Lett.274:151-155), to multiple gram-positive microorganism, Gram-negative bacteria, anerobe and fungi all have anti-microbial activity.Pexiganan has carried out the II clinical trial phase at present abroad, is mainly used in the treatment of diabetic infection.Because content is limited in the normal cell tissue, directly obtain very difficulty of Pexiganan.The Pexiganan of open report is the chemosynthesis product at present, the chemical synthesis complex process, and the cost height, its production process is used a large amount of organic solvents, contaminate environment.
Summary of the invention
Technical problem to be solved
The present invention is intended to solve the problem of utilizing engineered method efficient expression antimicrobial peptides Pexiganan.
Technical scheme
The invention provides a kind of genetically engineered and produce the method for Pexiganan.By analyzing the structure of Pexiganan, the codon of design intestinal bacteria preference, the dna sequence dna of composite coding Pexiganan, see sequence table SEQ ID NO.1 and SEQ IDNO.2, being connected to suitable expression vector expresses and purifying, this expression vector can be any carrier that is suitable for escherichia coli expression, molecular weight features of smaller according to antibacterial peptide, preferred fusion expression vector, as pBS, pTrx, pTrxFus etc., preferred especially GST fusion expression vector, the carrier of pGEX system and pET system (available from amersham pharmaciabiotech company) for example, because gst fusion protein is easy to the amalgamation and expression of small-molecular peptides, molecular weight has certain difference between purpose peptide and the fusion rotein in view of purifying needs, and the easier purifying of so little peptide is thorough.And for better being suitable for the cutting of purpose peptide.
SEQ ID NO.1 and SEQ ID NO.2 can adopt ordinary method synthetic with the nucleic acid automatic DNA synthesizer DNA.
The Pexiganan of gene engineering expression, 5 strain streptococcus aureus Staphylococcus aureus minimal bactericidal concentration (MIC) to different sources are 2~6ug/ml, to 5 Pseudomonas aeruginosa strain Pseudomonas aeruginosa (Pseudomonas aeruginosa) minimal bactericidal concentration (MIC), the 4~8ug/mL of different sources.Other all had good germicidal action as suis, Bacillus proteus, dysentery bacterium, Corynebacterium diphtheriae etc.
Beneficial effect
Genetically engineered provided by the invention is produced the method for Pexiganan, high abundance and the high codon of laws of use in intestinal bacteria have been adopted, repeat few between whole sequence of codon and the codon, reduced the mispairing probability in reproduction process, the Tm value is moderate, the Pexiganan that expresses in prokaryotic cell prokaryocyte amalgamation and expression system accounts for 48~50% of bacterial protein.
Description of drawings
The building process diagram of Pexiganan expression vector.
Embodiment
Below in conjunction with embodiment, it is bright to specify this law.
The used restriction endonuclease of the present invention, ligase enzyme, other molecular biology reagent and bacterial strain E.coli DH5 α are available from Promega company.The preparation of electrophoresis, competent cell, transform method that the equimolecular biological method adopts " molecular cloning experiment guide " and carry out that ((Sambrool J.) waits work to Sa nurse Bruce, and Huang Peitang etc. translate, molecular cloning experiment guide (the 3rd edition), Beijing, Science Press, 2002,8).Plasmid extracts, and adopts Shanghai to give birth to worker's test kit K192, carries out according to operation instruction.
Embodiment 1
The gene engineering expression of Pexiganan
The structure of expression vector and the acquisition of transformant
According to a conventional method respectively with nucleic acid SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQID NO.5 synthesize on the plasmid pUC18 carrier, SEQ IDNO.1-pUC18 enzyme is cut with SmaI (ccc/ggg)+EcoRI (g/aattc) according to specification sheets, low melting-point agarose method (" molecular cloning ") reclaims small pieces, with PhsAI (gacNN/NNgtc)+EcoRI (g/aattc) carrier pET42a (+) being carried out enzyme cuts, the low melting-point agarose method reclaims big segment, connect the recovery product according to specification sheets with the T4DNA ligase enzyme, can obtain to contain the expression vector of dna sequence dna of Pexiganan of encoding, expression vector is transformed into the competent cell of freshly prepd bacillus coli DH 5 alpha, screening is also identified transformant, ordinary method is extracted plasmid, and is errorless through the sequencing sequence.
Abduction delivering detects and identifies
The LB flat board that transformant contrast pET42a (+) BL21 (DE3) plys S rules respectively and contains kantlex 30ug/ml, the single colony inoculation of 37 ℃ of cultivations contains the LB meat soup 20ml of kantlex 30ug/ml; And blank BL21 (DE3) plys S colony inoculation does not contain antibiotic LB meat soup 20ml.When the OD value reaches 0.6, add IPTG respectively to final concentration 1mM, 37 ℃ of abduction deliverings 2~3 hours.5, centrifugal 5 minutes of 000g keeps supernatant, usefulness 20mM TrisHCl (pH8.0) washing, and 5, centrifugal 5 minutes of 000g, last cleer and peaceful precipitation is stored in-80 ℃ and is used for electrophoresis detection.At 28~29KDa. molecular weight band is arranged, be fusion rotein (the about 28.4KDa of molecular weight), fusion protein expression is respectively 48%, 50%, 35%, 45% and 40%.
Purifying
GSTrap FF post is connected use with HiTrap Benzamidine FF (high sub) (GE Healthcare-formerly AmershamBiosciences-Products) post, use AKTA Prime (amersham pharmacia biotech company instrument) online treatment, sample cutting and Factor Xa remove a step and finish.Press GSTrap FF post and HiTrap Benzamidine FF (high sub) specification sheets, be used the Prime online treatment, concentrate and obtain pexiganan, according to the SDS-PAGE method (Shi Jihong etc. of little peptide molecule, use the discussion of SDS-PAGE demonstration micromolecule polypeptide technology, the biotechnology progress, 2001, Vol21, No.1,38~41), SDS-PAGE electrophoresis silver dyes its molecular weight of detection and is about 2.4KDa, and sequencing is consistent with bibliographical information.
Embodiment 2
Antibacterial effect detects
Method: measure (United States Patent (USP) 6,337, on January 8th, 314,2002) by inhibition zone and bacteriocidal concentration.
Obtain the Pexiganan of gene engineering expression according to the method for embodiment 1, to different sources (reference culture streptococcus aureus S.aureus, CMCC (B) 26003 separates staphylococcus aureus strains with the hospital clinical case) 5 strain streptococcus aureus Staphylococcus aureus minimal bactericidal concentration (MIC) be 2~6ug/ml (abroad the experiment and chemosynthesis product MIC bacteriocidal concentration be 16~32ug/ml), (abroad experiment and chemosynthesis product MIC bacteriocidal concentration are 8~32ug/mL) to 5 strain Pseudomonasaeruginosa Pseudomonas aeruginosa (Pseudomonas aeruginosa) minimal bactericidal concentration (MIC), the 4~8ug/mL of different sources (reference culture Pseudomonas aeruginosa Pseudomonas aeruginosaMignla, CMCC (B) 10104 separates pseudomonas aeruginosa strains with the hospital clinical case).
The nucleotides sequence tabulation
SEQ?ID?NO.1
gggatcggca?aattcctgaa?gaaagcgaag?aaattcggca?aggcgttcgt?gaaaatcctg 60
aagaaa 66
SEQ?ID?NO.2
gggatcggca?agttcctgaa?gaaggcgaag?aagttcggca?aggcgttcgt?gaagatcctg 60
aagaag 66
SEQ?ID?NO.3
gggataggta?agtttcttaa?aaaggccaaa?aagtttggaa?aagcatttgt?aaagatactt 60
aaaaag 66
SEQ?ID?NO.4
gggatcggta?agttcctgaa?gaaggctaag?aaattcggta?aggctttcgt?gaagatcctg 60
aagaag 66
SEQ?ID?NO.5
gggatcggta?aattcctgaa?aaaagctaaa?aaattcggta?aggctttcgt?gaaaatcctg 60
aaaaaa 66

Claims (3)

1. the genetic engineering process for preparing of a Pexiganan, the dna sequence dna that comprises composite coding Pexiganan, be connected to suitable expression vector and express and purifying, the dna sequence dna of the Pexiganan that wherein encodes is sequence table SEQ ID NO.1 or SEQ ID NO.2.
2. according to the method for the production Pexiganan of claim 1 or 2, wherein used expression vector is a fusion expression vector.
3. according to the method for the production Pexiganan of claim 4, wherein used expression vector is the GST carrier.
CNA2005100148334A 2005-08-24 2005-08-24 Gene engineering producing method of antibacterial peptide Pexiganan Pending CN1920020A (en)

Priority Applications (1)

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Application Number Priority Date Filing Date Title
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CN1920020A true CN1920020A (en) 2007-02-28

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011069280A1 (en) * 2009-12-11 2011-06-16 Wu Xiaoyan Antimicrobial pexiganan analogue and preparation process thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011069280A1 (en) * 2009-12-11 2011-06-16 Wu Xiaoyan Antimicrobial pexiganan analogue and preparation process thereof
CN102656184A (en) * 2009-12-11 2012-09-05 吴晓琰 Antimicrobial pexiganan analogue and preparation process thereof

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