CN101696417B

CN101696417B - Method for producing recombined cationic antibacterial peptide by adopting co-expression anionic ligand

Info

Publication number: CN101696417B
Application number: CN 200910185208
Authority: CN
Inventors: 查向东; 周鹏; 王作利
Original assignee: 查向东
Current assignee: Anhui Xipu Biotechnology Co.,Ltd.
Priority date: 2009-11-02
Filing date: 2009-11-02
Publication date: 2012-12-26
Anticipated expiration: 2029-11-02
Also published as: CN101696417A

Abstract

The invention discloses a method for producing a recombined cationic antibacterial peptide by adopting a co-expression anionic ligand, which is characterized in that an independent anionic ligand with net negative charges is co-expressed at the same time of expressing the cationic antibacterial peptide, namely, a cationic antibacterial peptide gene and an encoded DNA sequence of the anionic ligand are cloned to a plasmid vector and expressed in a colibacillus host cell in an independent non-fusing way, and the toxicity of the cationic antibacterial peptide to the host cell is restricted by the anionic ligand so as to improve the expression yield; and in the subsequent separation and purification processes, the step of cutting a fusing head, which consumes manpower, physical resources and financial resources, is avoided, therefore, the separation and purification efficiency and the yield ratio are improved. The invention can remarkably shorten the production cycle to one third or a half of the original production cycle and reduce the cost above 50 percent. The invention provides a high-efficiency and low-cost way for the large-scale production of the cationic antibacterial peptide.

Description

The method of producing recombined cationic antibacterial peptide by adopting co-expression anionic ligand

Technical field

The present invention relates to the biological gene engineering field, specifically is a kind of method of producing recombined cationic antibacterial peptide by adopting co-expression anionic ligand.

Background technology

(English name cationic antimicrobial peptides AMPs), is one type of both sexes micromolecule polypeptide with clean positive charge to cationic antibacterial peptide, in animal, plant and mikrobe, extensively exists, and its sterilization spectrum width, immunogenicity are low; Cationic antibacterial peptide and bacterial cell membrane interaction form ionic channel on film, cause that cell content exosmoses and dead, are difficult for producing Resistant strain for the bactericidal mechanism of this uniqueness.And because zooblast film component and bacterial cell film component there are differences at aspects such as electric charges, cationic antibacterial peptide is less usually to the toxicity of zooblast.Outstanding day by day in current resistance problem, under the very urgent background of the demand of novel drugs, antibacterial peptide demonstrates tempting application prospect in fields such as medicine, agricultural, food, national defence.

Cationic antibacterial peptide content in vivo is atomic, if utilize biochemical method in organism, to extract natural antibacterial peptide, yield is low, and cost is high; If the chemical synthesis of using costs an arm and a leg at present, fragment is long all the more so.These issues limit to the research and the practical application of antibacterial peptide.It is an effective way that genetically engineered is produced cationic antibacterial peptide.But because the difference of cationic antibacterial peptide gene order, to problems such as the toxicity of host cell and separation and purification efficient, also need research and develop corresponding high efficiency recombinant expressed technology to different antibacterial peptide molecules, this is one of current domestic and international primary study content.

In the genetically engineered production technology, be that the prokaryotic expression system cost of representative is low with intestinal bacteria, be first-selected expression method always.Cationic antibacterial peptide brings a special difficult problem for the toxic effect of e. coli host cell for protokaryon is recombinant expressed, if directly express with nonfused mode, then expression product damage host cell influences conversely and expresses output.So, generally through with the anionic moiety amalgamation and expression suppressing in the expression process toxicity to host cell, but need excision to merge head behind the amalgamation and expression to recover active, could obtain activated recombined cationic antibacterial peptide.The method of head is merged in excision commonly used at present, mainly contains two kinds of cyanogen bromide chemical chop method and enzyme cutting methods.If with cyanogen bromide cutting, require target cationic antibacterial peptide itself not contain methionine(Met), and cyanogen bromide itself also is toxic substance, also possibly produce the problems such as chemically modified of antibacterial peptide; Enzyme cutting method then needs to stay unnecessary amino-acid residue (therefore the different pharmaceutical of recombinant production can have identical unnecessary amino-acid residue, and the repeated overlapping use can cause that immunoreation influences drug effect) and also exist non-specific cutting to disturb after expensive specific proteases, the cutting.In a word, chemical chop method and enzyme cutting method all exist bigger restriction and defective in production reality, genetically engineered is produced cationic antibacterial peptide formed bigger restriction.

Summary of the invention

Produce the above-mentioned defective of cationic antibacterial peptide technology existence for overcoming current genetically engineered, the present invention provides a kind of new method, can shorten the production cycle, simplifies production sequence, reduces production costs, and improves and expresses output, and save this step of cutting fusion head.It is characterized in that when expressing cationic antibacterial peptide anion ligand of coexpression; Cationic antibacterial peptide and anion ligand be independent separately the expression in same host cell, and the relation of anion ligand and target cationic antibacterial peptide is non-fusion, is trans.Technology after use improving, anion ligand can the neutralizing cation antibacterial peptide to the toxicity of host cell, thereby improved expression output; And owing to there is not covalent linkage to connect between anion ligand and the cationic antibacterial peptide; In the separation and purification after expression; No longer need any cutting process, directly promptly obtain the higher antibacterial peptide of purity, improved purification efficiency and yield with the Zeo-karb purifying.

Technical scheme of the present invention is:

The method of producing recombined cationic antibacterial peptide by adopting co-expression anionic ligand; It is characterized in that: produce in the process of cationic antibacterial peptide in genetically engineered; Express an independently anion ligand simultaneously, described anion ligand is the aminoacid sequence of one section band net negative charge; Just the dna sequence dna with cationic antibacterial peptide gene and coding anion ligand is cloned into plasmid vector, in e. coli host cell, induces coexpression; The anion ligand that expression obtains can suppress the toxicity of cationic antibacterial peptide to host cell; Be non-fusion between anion ligand and the cationic antibacterial peptide, no covalent linkage connects.

Described method is characterized in that: cationic antibacterial peptide is meant the polypeptide with anti-microbial activity that has clean positive charge.

Described method; It is characterized in that: described clone and conversion, can use a co-expression plasmid carrier, cationic antibacterial peptide gene and coding anion ligand dna sequence dna are inserted into two MCSs of this carrier respectively; The transformed into escherichia coli competent cell is induced coexpression; Also can use two different expression vectors; They carry different antibiotic resistant genes respectively; Cationic antibacterial peptide gene and coding anion ligand dna sequence dna are cloned into this two carriers respectively; The cotransformation competent escherichia coli cell with containing two kinds of antibiotic screening of medium and culturing cell simultaneously, is induced coexpression.

Described method is characterized in that: described intestinal bacteria and plasmid vector can be substituted by yeast and corresponding plasmid vector thereof.

The principles of science of technical scheme institute foundation:

The positive charge of cationic antibacterial peptide is essential for its BA, and they help antibacterial peptide molecule and target combining of bacterial cell membrane or nucleic acid molecule for example.Anion ligand of coexpression; Positive charge that can the neutralizing cation antibacterial peptide, or weaken the tendency that the cationic antibacterial peptide basic aminoacids carries proton, and pH value in the statocyst; Thereby suppress the toxicity of the cationic antibacterial peptide of abduction delivering, improve and express output host cell.And, can directly use the Zeo-karb separation and purification behind the smudge cells because cationic antibacterial peptide and anion ligand are independent the expression, save this step of cutting.

Technique effect of the present invention:

1. improved quality product: the coexpression method has been avoided in the cyanogen bromide cutting process, in the solution urea or cyanogen bromide to the modification of alkaline amino acid residue for example formylation modify, and alkaline amino acid residue is very important for keeping anti-microbial activity.The coexpression method has also been avoided the unnecessary amino-acid residue that enzyme cutting method produced.

2. shortened the production cycle: under the situation of amalgamation and expression, use chemical chop and relevant treatment to need 1-2 days time; Enzyme cutting method also needs 0.5-1 days time, and this does not also comprise cutting forward and backward dialysis treatment about 2-3 days time.

3. improved production efficiency: chemical chop and enzyme are cut and all are difficult to reach the cutting fully to substrate, and enzyme hits and also has non-specific cutting damage antibacterial peptide, and loss is also arranged in the dialysis procedure, and these factors have all reduced yield.The coexpression method is simplified working process, and raises the efficiency.Compare with not using the direct expression method of anion ligand, output improves more than 50%; With fusion expression method relatively, reduced the loss of operation and downstream purification, and when considering amalgamation and expression the molecular weight of antibacterial peptide to account for whole fusion rotein ratio less, net yield be higher than fusion expression method 2-3 doubly more than.

4. reduce cost: present high-quality cyanogen bromide price is not cheap, and considers optional equipment and the safeguard protection problem that requires in the middle of the cutting process, adds not little pressure to production cost; And with methods such as expensive specific proteases such as enteropeptidase, can only use in the laboratory, the extensive this kind of enzyme blanking method cost that uses is too high in the production.

5. enlarged production range: for the antibacterial peptide that has methionine(Met), also protokaryon is recombinant expressed easily.

Embodiment

The method of producing recombined cationic antibacterial peptide by adopting co-expression anionic ligand:

1, gene amplification: the cationic antibacterial peptide gene that uses the round pcr amplification to express; Simultaneously according to the sequence characteristic of this antibacterial peptide, with the anionic peptides section of the band net negative charge of a correspondence as part, the dna sequence dna of this anion ligand of amplification coding.

2, clone and conversion: select a colibacillary co-expression plasmid carrier; The recombinant DNA technology that is connected with ligase enzyme through restriction enzyme cutting; The cationic antibacterial peptide gene is inserted into a MCS of this expression vector; The anion ligand dna sequence dna is inserted into another MCS of this plasmid vector, then with the recombinant plasmid transformed competent escherichia coli cell that builds; Also can use two different expression plasmid carriers; They carry different antibiotic resistant genes respectively; Antibacterial peptide gene and anion ligand gene are cloned into this two carriers respectively; The cotransformation competent escherichia coli cell is with containing two kinds of antibiotic screening of medium and culturing cell simultaneously.

3, abduction delivering: the shaking culture genetic engineering bacterium adds inductor then and induced 2-20 hour to logarithmic phase.Culture temperature is in 15-42 degree centigrade of scope.Temperature is low more, and induction time prolongs.The gene of cationic antibacterial peptide is regulated and control by separately promotor and operator with coding anion ligand DNA, have ribosome bind site separately, is induced the back independently to transcribe and translate.

4, separation and purification: broken Bacillus coli cells.Broken liquid is used cationic exchange resin adsorption, and wash-out obtains cationic antibacterial peptide.

Embodiment:

Produce cationic antibacterial peptide NKLF-2, its aminoacid sequence is: VCDKMKILRGVCKKIMRSFLRR

According to the aminoacid sequence of NKLF-2, synthetic cationic antibacterial peptide gene behind pcr amplification, is cloned into the MCS of carrier pACYCDuet-1; Simultaneously the dna sequence dna of a coding anion ligand is cloned into another MCS of this carrier, the aminoacid sequence of band net negative charge anion ligand is MGDDDDKVPMHELEIFEF.Behind the transformed into escherichia coli cell, in the liquid nutrient medium that is containing paraxin under 37 degrees centigrade, shake bacterium, through isopropyl-(IPTG) abduction delivering.Compare with not using the direct expression method of anion ligand, express output and improve more than 100%; Compare with fusion expression method, merge this step of head owing to save cutting, reduce cost 50%, in 3 days shortening cycles, actual final yield is raising greatly still.

Claims

1. the method for producing recombined cationic antibacterial peptide by adopting co-expression anionic ligand; It is characterized in that: produce in the process of cationic antibacterial peptide that aminoacid sequence is VCDKMKILRGVCKKIMRSFLRR in genetically engineered; Express an independently anion ligand simultaneously, described anion ligand is the aminoacid sequence of one section band net negative charge: MGDDDDKVPMHELEIFEF; Just the dna sequence dna with cationic antibacterial peptide gene and coding anion ligand is cloned into the pACYCDuet-1 plasmid vector, in e. coli host cell, induces coexpression; The anion ligand that expression obtains can suppress the toxicity of cationic antibacterial peptide to host cell; Be non-fusion between anion ligand and the cationic antibacterial peptide, no covalent linkage connects.