CN101265291A - Recombination pig origin antibiotic peptide PG4 and its biological synthesis method and application - Google Patents
Recombination pig origin antibiotic peptide PG4 and its biological synthesis method and application Download PDFInfo
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Abstract
The invention discloses a recombinant swine antibacterial peptide PG4, the amino acid sequence of which has amino acid sequence as shown in SEQ ID NO:1. The invention also discloses a method for synthesizing the recombinant swine antibacterial peptide PG4, which includes: (1) constructing antibacterial peptide PG4 prokaryotic expression vector, (2) transforming into Escherichia coli host cell, and constructing expression engineering bacteria; (3) fermenting and culturing the engineering bacteria, and performing inducible expression of inducer; and (4) purifying. The PG4 has the advantages of high expression efficiency, simple separation and purification, convenient operation, easy magnification, good stability, and suitability for large scale industrialized production. The PG4 is of important value for the research of novel high-efficiency antibacterial drug, and has wide application prospect in the fields of pharmacy industry and biological antibacterial material.
Description
Technical field
The present invention relates to the expression and the purifying of genetically engineered field recombinant protein, relate in particular to a kind of gene recombination pig derived antimicrobial peptide and biosynthetic means thereof.
Background technology
The Mammals antibacterial peptide is Mammals infecting of cause of disease material and the product of a series of non-specificity immune response that produces is the important component part of the congenital system of defense of host to external world.It has broad-spectrum antibacterial, and is thermally-stabilised, and strong basicity is soluble in water, and is positively charged, and molecular weight is low, and eukaryotic cell is not almost had toxic side effect, only kills the characteristics such as eukaryotic cell of prokaryotic cell prokaryocyte and generation pathology.Because it is by changing the eukaryotic cell that bacterium lipid membrane permeability is killed prokaryotic cell prokaryocyte and pathology is taken place, thereby bacterium is difficult for forming resistance, and traditional microbiotic is to play a role by the macromolecular biosynthesizing of blocking-up, causes more and more many bacteriums that it has been produced resistance.In addition, antibacterial peptide is a short chain polypeptides, and the amino acid of its composition for easily being absorbed by the human or animal has solved the residual problem of medicine.Therefore, the Mammals antibacterial peptide will replace the antimicrobial substance that traditional microbiotic becomes a new generation.
In mammalian body, find at present ten surplus the peptide of kind with anti-microbial activity all belong to Defension (alexin) and Cathelicidins two extended familys.The antibacterial peptide Protegrins of pig leucocyte source property (being called for short PG) comprises 5 kinds, and promptly PG 1～5, separates obtaining at first from pig leucocyte, also separates obtaining its mRNA in recent years in the Lymphoid tissue of piglet.PG is that a class is rich in Cys in the Cathelicidins family, and the small-molecular peptides of Arg only contains 16～18 amino acid, the about 2KD of relative molecular mass, and four halfcystines form two intramolecular disulfide bonds, to keep its antiparallel beta sheet stability of structure.PG has the killing microorganisms activity of wide spectrum, resists various Gram-negative bacterias, positive bacteria, fungi and togavirus, spreads disease germs and the biologic activity of unique anti-human HIV virus comprising diversity.External, PG can kill gram pathogenic bacteria such as intestinal bacteria, Pseudomonas aeruginosa, salmonella typhi, chlamydia trachomatis and mycobacterium tuberculosis etc. when concentration is 0.1～8 μ g/mL.The antimicrobial acivity of PG remains on physiological salt concentration, is not subjected to the inhibition of extracellular positively charged ion or serum composition.Steinberg etc. discover that PG also has fungicidal activity fast, can make 3 log units of CFU decline of bacterium in 10min, and conventional microbiotic gentamicin or norfloxicin reach identical effect then to be needed more than a few hours.Research finds also that in containing the MHB substratum of 1/2 concentration Pseudomonas aeruginosa and staphylococcus are divided 11 generations of supplementary biography and 18 generations continuously and do not produce resistance, and the MIC of norfloxicin then increases 10-190 doubly under the same terms.PG can also alleviate endotoxemia by in coming in conjunction with LPS and intracellular toxin.The antibacterial therapy medicine that everything makes PG become to have very big potentiality.
At present, many antibacterial peptides are being developed patent medicine, but the natural output of antibacterial peptide is very low, and prices are rather stiff for the chemosynthesis antibacterial peptide, and this becomes a bottleneck of exploitation patent medicine, have broad application prospects and produce antibacterial peptide by genetic engineering technique.
Summary of the invention
The purpose of this invention is to provide a kind of biosynthetic means of expressing pig origin antibiotic peptide PG 4 in the protokaryon system, the fusion rotein after the expression can obtain to have the reorganization PG4 polypeptide of antimicrobial acivity behind cyanogen bromide chemical chop, purifying.
Technical scheme of the present invention is: a kind of recombination pig origin antibiotic peptide PG 4, the aminoacid sequence of described PG4 are to have the aminoacid sequence shown in the SEQ ID NO:1.
The method of a kind of synthetic aforesaid recombination pig origin antibiotic peptide PG 4 of the present invention may further comprise the steps:
(1) the synthetic oligonucleotide is become double-helical target DNA monomer through anneal, use the DNA recombinant technology, make up antibacterial peptide PG4 prokaryotic expression carrier;
(2) antibacterial peptide PG4 prokaryotic expression carrier transformed into escherichia coli host cell, the construction expression engineering bacteria;
(3) fermentation culture engineering bacteria carries out the inductor abduction delivering;
(4) with step (3) gained expression product through affinitive layer purification, cyanogen bromide chemical chop, obtain recombination pig origin antibiotic peptide PG 4 after the repurity.
The described oligonucleotide of step (1) is:
PG4-1：
5’-ATTCAGGGATCCATGCGCGGTGGCCGTCTGTGCTATTGTCGCGGTTGGATCTGCTTCTGTGTGGGTCGTTAAAAGCTTAATTCG-3’；
PG4-2：
5’-CGAATTAAGCTTTTAACGACCCACACAGAAGCAGATCCAACCGCGACAATAGCACAGACGGCCACCGCGCATGGATCCCTGAAT-3’。
Step (1) specifically is that goal gene dna fragmentation and expression vector are carried out BamH I and HindIII double digestion, enzyme is cut product behind agarose gel electrophoresis, cut glue and reclaim purifying purpose fragment, connect through the T4 dna ligase, make up recombinant antibacterial peptide PG4 prokaryotic expression carrier, used prokaryotic expression carrier is pGEX-2TK-CMS.
Step (2) specifically be antibacterial peptide PG4 prokaryotic expression carrier that step (1) is made up through BamH I and HindIII double digestion, it is correct through the agarose gel electrophoresis checking that enzyme is cut product, is transformed into e. coli host cell, the construction expression engineering bacteria.
Step (3) specifically is to express engineering bacteria 1.5～4h (Best Times 2.5h) to OD with TB substratum, 25～37 ℃ of (37 ℃ of optimum tempss) fermentation culture
600Reach 0.4～1.0; Adding the IPTG inductor is 0.01～1mM (optimum concn is 0.1mM) to final concentration, inducing culture 2～5h.
Step (4) comprises that collecting step (3) induces the back engineering bacteria, and is centrifugal after ultrasonication, gets supernatant liquor; The separating medium of affinity chromatography is GSH-Sepharose Resin, and with the PBS damping fluid balance chromatography column of the pH7.4 of 10 times of column volumes, last sample, Wash buffer washes resin, and Elution buffer wash-out is collected elutriant, recombination pig origin antibiotic peptide PG 4.
The application of gene recombination pig origin antibiotic peptide PG 4 of the present invention in antibacterials and biological antibiotic material.
Usefulness of the present invention is:
(1) using gene engineering technique has efficiently expressed recombinant antibacterial peptide PG4 in prokaryotic host cell, and is inhibited to intestinal bacteria and streptococcus aureus through this recombination pig origin antibiotic peptide PG 4 of experimental verification.
(2) expression efficiency height of the present invention, separation and purification is simple, and is easy to operate, easily amplifies, and good stability is suitable for large-scale industrial production.Therefore, the present invention has important value to developing new and effective anti-microbial type medicine, has broad application prospects at pharmaceutical industry and biological antibiotic material field.
Description of drawings
Fig. 1 is an antibacterial peptide PG4 construction of prokaryotic expression vector synoptic diagram of the present invention;
Fig. 2 is the SDS-PAGE electrophorogram of fusion polypeptide expression amount before and after of the present invention the inducing;
Wherein, swimming lane 1 is the electrophoretic band of inductive BL21-PG4 engineering bacteria not; Swimming lane 2 is the electrophoretic bands of inducing thalline behind the 3h; Swimming lane 3 is the electrophoretic bands of inducing broken supernatant behind the 4h; Swimming lane 4 is to induce broken sedimentary electrophoretic band behind the 4h.
Fig. 3 is the SDS-PAGE electrophorogram of affinitive layer purification recombinant fusion polypeptide GST-PG4 of the present invention;
Wherein, swimming lane 1 is IPTG inductive BL21-PG4 engineering bacteria soluble polypeptide after ultrasonication; Swimming lane 2 is Wash buffer elutriants; Swimming lane 3 is the GST-PG4 fusion polypeptide that obtain through the GSH-Sepharose column separating purification; M is a protein molecular weight standard.
Fig. 4 is recombinant antibacterial peptide PG4 Chinese People's Anti-Japanese Military and Political College enterobacteria Escherichia coli (E.coli) effect of the present invention's preparation and the comparison of blank sample.
Wherein, 1 is the blank sample, and 2 is recombinant antibacterial peptide PG4.
Fig. 5 is anti-staphylococcus aureus Staphylococcusaureus (S.aureus) effect of recombinant antibacterial peptide PG4 of the present invention's preparation and the comparison of blank sample.
Wherein, 1 is the blank sample, and 2 is recombinant antibacterial peptide PG4.
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with embodiment, it should be understood that to the invention is not restricted to these embodiment.
Embodiment 1:
1, a kind of clone of recombination pig origin antibiotic peptide PG 4 gene
(1) design and synthetic two oligonucleotide: PG4-1, PG4-2 obtain one section duplex DNA segment through the annealing renaturation then, and its nucleotide sequence is respectively:
PG4-1：
5’-ATTCAGGGATCCATGCGCGGTGGCCGTCTGTGCTATTGTCGCGGTTGGATCTGCTTCTGTGTGGGTCGTTAAAAGCTTAATTCG-3’；
PG4-2：
5’-CGAATTAAGCTTTTAACGACCCACACAGAAGCAGATCCAACCGCGACAATAGCACAGACGGCCACCGCGCATGGATCCCTGAAT-3’。
Adopt the intestinal bacteria preference codon, two oligonucleotide fragments of chemosynthesis PG4-1 and PG4-2, PG4-1 and PG4-2 are SEQ ID NO4:Ile-Gln-Gly-Ser-Met-Arg-Gly-Gly-Arg-Leu-Cys-Tyr-Cys-Arg-Gly-Trp-Ile-Cys-Phe-Cys-Val-Gly-Arg-terminator-Lys-Leu-Asn-Ser through anneal gained duplex DNA corresponding to aminoacid sequence.
At two segmental two ends of oligonucleotide, BamH I and HindIII restriction endonuclease sites have been designed respectively.Introducing the methionine(Met) encoding gene in the structure gene upstream simultaneously is the cyanogen bromide recognition site, introduces the TAA terminator codon in the downstream.
(2) will anneal after product phosphorylation and pSIMPLE-19 EcoR V/BAP (precious biological available from Dalian) carrier is connected, called after pSIMPLE-PG4 is converted among the intestinal bacteria competence JM109 (available from Novagen), coating LB flat board incubated overnight thalline.From transforming the dull and stereotyped picking list bacterium colony of going up, with single bacterium colony shaking culture in 2ml LB substratum.With the alkaline lysis method of extracting plasmid, plasmid pSIMPLE-PG4 is inserted the transformant of correct foreign gene with the screening of BamH I/HindIII double digestion method.Prepare plasmid in a small amount with RapidPlasmid DNA Daily Mini-prep Kit, and confirm its sequence, get positive colony pSIMPLE-PG4 through dna sequencing.
2, antibacterial peptide PG4 construction of prokaryotic expression vector
The pGEX-2TK carrier is a member in the pGEX of the GE company series prokaryotic expression carrier, and merging has gsh-s-transferring enzyme (GST) labelled protein, and the natural size of GST is 26KD, is a highly soluble albumen, can increase the solubility of foreign protein; In addition it can be in intestinal bacteria great expression, play a kind of effect that promotes that expression amount improves.Foreign protein and GST merge and form fusion rotein, act on the immobilized gsh in addition purifying under non-sex change condition by affinity chromatography, utilize the cutting action of the cyanogen bromide with identification specific site then, and the GST composition in the fusion rotein is removed.The GST molecular weight is bigger, for expressing the little target protein of molecular weight tool have great advantage-bigger relatively being not easy of fusion protein molecule amount be degraded.The pSIMPLE-PG4 plasmid double digestion that the present invention will clone from the JM109 cell makes up antibacterial peptide PG4 prokaryotic expression carrier (pGEX-PG4) (Fig. 1).
(1) with BamHI and HindIII plasmid pSIMPLE-PG4 and pGEX-2TK are carried out double digestion, enzyme is cut product carry out agarose gel electrophoresis, cut the agar block that contains target DNA fragment respectively, utilize dna gel to reclaim test kit (Gel Extraction Kit) (available from green skies company) purifying and reclaim dna fragmentation.
(2) insertion fragment and the carrier with above-mentioned purifying carries out ligation according to TaKaRa company ligation operation instructions.Connect product and change in the bacillus coli DH 5 alpha (available from Novagen), carry out clonal selection with penbritin.With clone strain incubated overnight in 2ml LB substratum, use the alkaline lysis method of extracting recombinant plasmid, carry out agarose gel electrophoresis through double digestion and identify correct.With this plasmid called after pGEX-PG4.
3, the expression of recombinant fusion polypeptide and purifying
(1) a small amount of of recombinant fusion polypeptide is expressed and is identified
The pGEX-PG4 plasmid of electrophoresis checking is transformed into competent escherichia coli cell BL21 (DE3) (available from Novagen), picking list colony inoculation is in 2ml LB liquid nutrient medium, 37 ℃ of shaking culture are spent the night, be inoculated in the 3ml TB liquid nutrient medium that contains penbritin with 1: 100 then and cultivate, work as OD
600When reaching 0.8～0.9 left and right sides, add inductor IPTG induced protein and express, the IPTG final concentration is 0.1mM, and inducing temperature is 28 ℃.Inducing culture 5 hours, every during this time interval 1h sampling once, through centrifugal collection thalline (8000g, 4 ℃ centrifugal 10 minutes), with the resuspended thalline of Tris-HCl (pH6.8) damping fluid, carry out the 12%SDS-PAGE gel electrophoresis, carry out coomassie brilliant blue staining behind the electrophoresis, analyze the relation of induction time and expression amount.
The gel images analytical system is observed behind the SDS-PAGE electrophoresis, and inducing the back to produce molecular weight is the specific band of 28KD, and with the prolongation of induction time, expression amount increases gradually, reaches maximum expression amount to 4h.
(2) fermentation culture of recombinant fusion polypeptide
In 1 liter of TB substratum that contains penbritin, 37 ℃ of about 2.5h of fermentation culture are to the OD of inoculum with the engineering bacteria of 1: 100 ratio inoculation express recombinant polypeptide
600Be about 0.8, add IPTG (final concentration 0.1mM), carry out the abduction delivering recombinant polypeptide, continue fermentation culture 4h at 28 ℃.8000g collected thalline in centrifugal 10 minutes.With 5 times of weight in wet base volumes thalline is resuspended in PBS (pH7.4) damping fluid, under condition of ice bath, carries out ultrasonic disruption thalline (non-stop run 2sec, interval 4sec, co-processing 30min).Under 4 ℃,, collect supernatant liquor and broken precipitation respectively with the centrifugal 20min of thalline solution 45000g after the fragmentation.Carry out the 12%SDS-PAGE gel electrophoresis, carry out coomassie brilliant blue staining behind the electrophoresis, detect the solvable character (Fig. 2) of polypeptide.
Among Fig. 2, swimming lane 1 is the electrophoretic band of inductive BL21-PG4 engineering bacteria not; Swimming lane 2 is the electrophoretic bands of inducing thalline behind the 3h; Swimming lane 3 is the electrophoretic bands of inducing broken supernatant behind the 4h; Swimming lane 4 is to induce broken sedimentary electrophoretic band behind the 4h.
Gel imaging is observed and is shown that polypeptide is a solubility expression behind the SDS-PAGE electrophoresis.
(3) purifying of recombinant fusion polypeptide
With sample GST chromatography column on the thalline supernatant liquor after the fragmentation, flow velocity is 0.5-1ml/min; After treating that the sample spontaneous current is crossed the post bed, the Wash buffer of 10 times of column volumes (20mM Tris-HCl, pH8.0,0.1M NaCl, 10%glycerol, 1%Triton X-100) washes resin to remove other soluble componentss of uncombined polypeptide and bacterium; With elution buffer (pH8.0 needs fresh preparation for 10mMglutathione, the 20mM Tris-HCl) wash-out of 3 times of column volumes, collect elutriant then.Dialysis is to remove the small molecules composition in the polypeptide solution in dialysis tubing.
As seen SDS-PAGE electrophoretic analysis, coomassie brilliant blue staining obtain the single band (Fig. 3) that molecular weight is 28KD after purified.
Among Fig. 3, swimming lane 1 is IPTG inductive BL21-PG4 engineering bacteria soluble polypeptide after ultrasonication; Swimming lane 2 is Wash buffer elutriants; Swimming lane 3 is the GST-PG4 fusion polypeptide that obtain through the GSH-Sepharose column separating purification; Swimming lane 4 is protein molecular weight standards.
(4) purifying of the cyanogen bromide chemical chop of recombinant fusion polypeptide and recombinant antibacterial peptide PG4
After the fusion polypeptide freeze-drying behind the dialysis desalting, heavily be dissolved in 70% formic acid solution, the polypeptide final concentration is 2mg/ml, adds isopyknic CNBr solution (CNBr is dissolved in 70% formic acid, and final concentration is 0.3g/ml) and mixes.Encase the centrifuge tube lucifuge that reaction mixture is housed with aluminium foil, room temperature reaction 24h in stink cupboard.Add isopyknic pure water and finish reaction in reaction mixture, the sample vacuum is drained.
Drain the back sample and heavily be dissolved in PBS (pH7.4) damping fluid, with above-mentioned working method, last sample GST chromatography column.Collect and flow out phase, obtain recombinant polypeptide PG4, elution buffer wash-out is collected elutriant then, obtains the GST labeling polypeptide, and recombinant polypeptide PG4 and GST labeling polypeptide are respectively through the dialysis desalting purifying.GST-PG4 fusion polypeptide and GST labeling polypeptide carry out the SDS-PAGE gel electrophoresis, and whether check cuts fully.
SDS-PAGE electrophoresis checking, coomassie brilliant blue staining as seen, the fusion polypeptide after the cutting is moved a segment distance forward, at the single specific band of the about 26KD of molecular weight place appearance, illustrates that the fusion polypeptide major part is cut open.
The test of recombinant antibacterial peptide PG4 germ resistance
1, the enterobacteria E.coli of recombinant antibacterial peptide PG4 Chinese People's Anti-Japanese Military and Political College test:
(1) cut-off directly is two of the filter papers of 1cm, and uv irradiating sterilization (irradiation time is 30 minutes) uses sterilized water and recombinant antibacterial peptide PG4 solution (0.1mg/mL) to soak into respectively.In Fig. 4, be followed successively by sample 1 and sample 2;
(2) above-mentioned two samples are put respectively to aseptic Tissue Culture Dish, respectively added 50 μ L intestinal bacteria nutrient solutions (10 subsequently
6CFU/mL), and in 37 ℃ of thermostat containers cultivated 2 hours;
(3) in each culture dish, add the 1mL phosphoric acid buffer, and with ultrasonic cleaning (64kHz, 2 minutes), respectively get 50 μ L gradient dilutions then, the different dilution bacterium damping fluids of 50 μ L are added in the culture dish, with spreading rod bacterium liquid is uniformly coated on the solid nutrient agar, and places 37 ℃ of thermostat containers to cultivate 24 hours;
(4) calculate the antibacterial ability of each sample according to the number of bacterium colony on the solid nutrient agar.
Wherein, recombinant antibacterial peptide PG4 can effectively kill 95.7% intestinal bacteria E.coli.
2, the anti-staphylococcus aureus S.aureus test of recombinant antibacterial peptide PG4:
(1) cut-off directly is two of the filter papers of 1cm, and uv irradiating sterilization (irradiation time is 30 minutes) uses sterilized water and recombinant antibacterial peptide PG4 solution (0.1mg/mL) to soak into respectively.In Fig. 5, be followed successively by sample 1 and sample 2;
(2) above-mentioned two samples are put respectively to aseptic Tissue Culture Dish, respectively added 50 μ L staphylococcus aureus nutrient solutions (10 subsequently
6CFU/mL), and in 37 ℃ of thermostat containers cultivated 2 hours;
(3) in each culture dish, add the 1mL phosphoric acid buffer, and with ultrasonic cleaning (64kHz, 2 minutes), respectively get 50 μ L gradient dilutions then, the different dilution bacterium damping fluids of 50 μ L are added in the culture dish, with spreading rod bacterium liquid is uniformly coated on the solid nutrient agar, and places 37 ℃ of thermostat containers to cultivate 24 hours;
(4) calculate the antibacterial ability of each sample according to the number of bacterium colony on the solid nutrient agar.
Wherein, recombinant antibacterial peptide PG4 can effectively kill 90.9% staphylococcus aureus S.aureus.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ?ID?NO?1：
15?Arg?Gly?Gly?Arg?Leu?Cys?Tyr?Cys?Arg?Gly?Trp?Ile?Cys?Phe?Cys
18?Val?Gly?Arg
SEQID?NO?2：
60?attcagggat?ccatgcgcgg?tggccgtctg?tgctattgtc?gcggttggat?ctgcttctgt
84?gtgggtcgtt?aaaagcttaa?ttcg
SEQ?ID?NO?3：
60?cgaattaagc?ttttaacgac?ccacacagaa?gcagatccaa?ccgcgacaat?agcacagacg
84?gccaccgcgc?atggatccct?gaat
SEQ?ID?NO?4：
15?Ile?Gln?Gly?Ser?Met?Arg?Gly?Gly?Arg?Leu?Cys?Tyr?Cys?Arg?Gly
27?Trp?Ile?Cys?Phe?Cys?Val?Gly?Arg?Lys?Leu?Asn?Ser
Claims (8)
1, a kind of recombination pig origin antibiotic peptide PG 4 is characterized in that: the aminoacid sequence of described PG4 is to have the aminoacid sequence shown in the SEQ ID NO:1.
2, a kind of method of synthetic recombination pig origin antibiotic peptide PG 4 as claimed in claim 1 is characterized in that may further comprise the steps successively:
(1) the synthetic oligonucleotide is become double-helical target DNA monomer through anneal, use the DNA recombinant technology, make up antibacterial peptide PG4 prokaryotic expression carrier;
(2) antibacterial peptide PG4 prokaryotic expression carrier transformed into escherichia coli host cell, the construction expression engineering bacteria;
(3) fermentation culture engineering bacteria carries out the inductor abduction delivering;
(4) with step (3) gained expression product through affinitive layer purification, cyanogen bromide chemical chop, obtain recombination pig origin antibiotic peptide PG 4 after the repurity.
3, the synthetic method of recombination pig origin antibiotic peptide PG 4 according to claim 2 is characterized in that: the described oligonucleotide of step (1) is:
PG4-1：
5’-ATTCAGGGATCCATGCGCGGTGGCCGTCTGTGCTATTGTCGCGGTTGGATCTGCTTCTGTGTGGGTCGTTAAAAGCTTAATTCG-3’；
PG4-2：
5’-CGAATTAAGCTTTTAACGACCCACACAGAAGCAGATCCAACCGCGACAATAGCACAGACGGCCACCGCGCATGGATCCCTGAAT-3’。
4, according to the synthetic method of claim 2 or 3 described recombination pig origin antibiotic peptide PG 4s, it is characterized in that: step (1) specifically is that goal gene dna fragmentation and expression vector are carried out BamH I and HindIII double digestion, enzyme is cut product behind agarose gel electrophoresis, cut glue and reclaim purifying purpose fragment, connect through the T4 dna ligase, make up recombinant antibacterial peptide PG4 prokaryotic expression carrier, used prokaryotic expression carrier is pGEX-2TK-CMS.
5, the synthetic method of recombination pig origin antibiotic peptide PG 4 according to claim 2, it is characterized in that: step (2) specifically is that antibacterial peptide PG4 prokaryotic expression carrier that step (1) is made up is through BamHI and HindIII double digestion, it is correct through the agarose gel electrophoresis checking that enzyme is cut product, be transformed into e. coli host cell, the construction expression engineering bacteria.
6, the synthetic method of recombination pig origin antibiotic peptide PG 4 according to claim 2 is characterized in that: step (3) specifically is to express engineering bacteria 1.5～4h (Best Times 2.5h) to OD with TB substratum, 25～37 ℃ of (37 ℃ of optimum tempss) fermentation culture
600Reach 0.4～1.0; Adding the IPTG inductor is 0.01～1mM (optimum concn is 0.1mM) to final concentration, inducing culture 2～5h.
7, the synthetic method of recombination pig origin antibiotic peptide PG 4 according to claim 2 is characterized in that: step (4) comprises that collecting step (3) induces the back engineering bacteria, and is centrifugal after ultrasonication, gets supernatant liquor; The separating medium of affinity chromatography is GSH-Sepharose Resin, and with the PBS damping fluid balance chromatography column of the pH7.4 of 10 times of column volumes, last sample, Wash buffer washes resin, and Elution buffer wash-out is collected elutriant, recombination pig origin antibiotic peptide PG 4.
8, the application of a kind of gene recombination pig origin antibiotic peptide PG 4 as claimed in claim 1 or 2 in antibacterials and biological antibiotic material.
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Cited By (6)
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| CN102703457A (en) * | 2012-05-18 | 2012-10-03 | 中国农业科学院兰州兽医研究所 | Method for preparing and expressing antibacterial peptide gene |
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| CN107090473A (en) * | 2017-05-19 | 2017-08-25 | 华南农业大学 | Antibacterial peptide PR39 and PG1 coexpression vector and turn PR39 and PG1 DNA murine preparation methods |
| CN114276413A (en) * | 2022-01-26 | 2022-04-05 | 中国石油大学(华东) | Preparation method of artificial antibacterial peptide G3 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102703457A (en) * | 2012-05-18 | 2012-10-03 | 中国农业科学院兰州兽医研究所 | Method for preparing and expressing antibacterial peptide gene |
| CN104341497A (en) * | 2014-11-05 | 2015-02-11 | 青岛农业大学 | Novel pig-source antibacterial peptide mutant, preparation method and application thereof |
| CN104341497B (en) * | 2014-11-05 | 2017-03-01 | 青岛农业大学 | A kind of Novel pig derived antimicrobial peptide mutant and its preparation method and application |
| CN104448006A (en) * | 2014-12-13 | 2015-03-25 | 青岛宏昊生物科技有限公司 | Hybrid antibacterial peptide CE-PR and application thereof |
| CN105777889A (en) * | 2016-03-25 | 2016-07-20 | 东北农业大学 | Heterozygosis alpha spiral swine antibacterial peptide, and preparation method and application thereof |
| CN105777889B (en) * | 2016-03-25 | 2019-02-01 | 东北农业大学 | A kind of heterozygosis α screw type pig derived antimicrobial peptide and its preparation method and application |
| CN107090473A (en) * | 2017-05-19 | 2017-08-25 | 华南农业大学 | Antibacterial peptide PR39 and PG1 coexpression vector and turn PR39 and PG1 DNA murine preparation methods |
| CN114276413A (en) * | 2022-01-26 | 2022-04-05 | 中国石油大学(华东) | Preparation method of artificial antibacterial peptide G3 |
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