CN105693866A - Fusion antibacterial peptide and application thereof - Google Patents
Fusion antibacterial peptide and application thereof Download PDFInfo
- Publication number
- CN105693866A CN105693866A CN201610120163.2A CN201610120163A CN105693866A CN 105693866 A CN105693866 A CN 105693866A CN 201610120163 A CN201610120163 A CN 201610120163A CN 105693866 A CN105693866 A CN 105693866A
- Authority
- CN
- China
- Prior art keywords
- antibacterial peptide
- 3pocec
- pmd18
- double digestion
- inclusion body
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Insects & Arthropods (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of biological agents, and particularly relates to fusion antibacterial peptide and application thereof. The production method comprises the first step of building of a cloning vector, the second step of building of an expression vector, the third step of expression of 3Pocec inclusion body protein, the fourth step of purification of 3Pocec inclusion body protein, the fifth step of dissolution of the 3Pocec inclusion body protein, the sixth step of cutting of the 3Pocec inclusion body protein and the seventh step of purification of antibacterial peptide monomers. After the fusion antibacterial peptide is chemically cut, two kinds of antibacterial peptide monomers are obtained, the strong inhibiting effect on Gram-positive bacteria and Gram-negative bacteria is achieved, the cost is reduced to the great degree, and large-scale application of existing antibacterial peptide is achieved.
Description
Technical field
The invention belongs to biologic product technology field, particularly relate to a kind of fusion antibacterial peptide and application thereof。
Background technology
In recent years, due to antibiotic, the abuse of chemicals, residual, in feedstuff, the interpolation etc. of antibacterials causes that drug resistance occurs in antibacterial, even variation phenomenon。And antibiotic therapy is invalid to this, can only treat by corresponding monoclonal antibody。Therefore finding some and antibacterial has killing action, and don't cause the drug resistance of antibacterial, the biological therapeutic agent that environment is had no side effect is imperative。
Antibacterial peptide (antibacterialpeptides, ABPs), is that organism class of generation in the defense reaction process of opposing pathogenic microorganism has the micromolecule polypeptide of antimicrobial acivity。Antibacterial peptide be in animal body fluid immune system exist there is broad-spectrum sterilization, virus, some tumor, fungus are had a necessarily inhibiting class film activity polypeptide, are the important component part of body natural immune system。
The current method obtaining antibacterial peptide is mainly extraction and the gene engineering expression of natural antibacterial peptide。Owing to antibacterial peptide is Immune inducing in vivo synthesis, in organism, content is relatively low, causes that the difficulty of extracting directly is big in organism, the requirement of technology and cost is high, it is difficult to large-scale production;Antibacterial peptide is generally cationic polypeptide simultaneously, is easily easily degraded by proteases, and generally adds some protein tag, to improve expression and can simplify separation purge process when therefore expressing。But fusion protein typically requires protease or chemical cracking and after cutting protein tag, antibacterial peptide just can be played antibacterial action。These problems limit the large-scale application of antibacterial peptide to a certain extent。
Summary of the invention
The one that it is an object of the invention to overcome deficiency of the prior art and invent merges antibacterial peptide and application thereof。
The present invention is achieved in that a kind of fusion antibacterial peptide, it is characterised in that: include cecropin cm4 and pig derived antimicrobial peptide PR-39。
The described aminoacid sequence merging antibacterial peptide is such as shown in SQEIDNO.1。
The nucleotide sequence of described fusion antibacterial peptide is such as shown in SQEIDNO.2。
A kind of expression vector containing the nucleotide sequence merging antibacterial peptide。
A kind of preparation method manufacturing above-mentioned fusion antibacterial peptide, it is characterised in that: include
The structure of step 1) cloning vehicle:
1.1 aminoacid sequences according to the cecropin cm4 announced and pig derived antimicrobial peptide PR-39, the gene order with E.coli standard codon for Reference Design antibacterial peptide;And the restriction enzyme site of Bgl II, BamH I isocaudarner and Xhol I, the gene order as shown in SEQIDNO.2 of chemosynthesis is introduced respectively at gene order two ends;The gene order synthesized with BamH I and Xhol I double digestion it is connected to the pMD18 carrier with BamH I and Xhol I double digestion and converts to E.coliDH5 α competent cell, obtaining pMD18-Pocec cloning vehicle。
1.2 by pMD18-Pocec cloning vehicle with BamH I and Xhol I double digestion, then by the pMD18-Pocec cloning vehicle after double digestion be attached with the synthetic gene sequence of Bgl II and Xhol I double digestion, product will be connected convert to E.coliDH5 α competent cell, obtain pMD18-2Pocec cloning vehicle。
1.3 by pMD18-2Poce cloning vehicle with BamH I and Xhol I double digestion, then by the pMD18-2Poce cloning vehicle after double digestion be attached with the synthetic gene sequence of Bgl II and Xhol I double digestion, namely the gene order as shown in SEQIDNO.3 is obtained, product will be connected convert to E.coliDH5 α competent cell, obtain pMD18-3Pocec cloning vehicle。
Step 2) structure of expression vector:
PMD18-3Pocec cloning vehicle and pET-32a carrier are used that Bgl II and Xhol I double digestion, then the pMD18-3Pocec cloning vehicle after double digestion, pET-32a carrier are connected, obtain pET-32a-3Pocec expression vector, pET-32a-3Pocec expression vector is converted to E.coliBL21(DE3) competent cell, the coating LB plate screening containing ampicillin, obtains E.coliBL21(DE3)/pET-32a-3Pocec。
The expression of step 3) 3Pocec inclusion body protein:
By E.coliBL21(DE3)/pET-32a-3Pocec is seeded in the LB culture medium containing ampicillin according to the ratio of 1:100 1:106, it is seeded to the LB culture medium containing ampicillin again according to 1:100 ratio after 37 DEG C of overnight incubation, when being cultured to 600nm place OD value 0.4-0.6 under 37 DEG C of conditions, add IPTG and make its final concentration of 0.2-0.3mmol/L with the expression of induced fusion antibacterial peptide inclusion body protein。
The purification of step 4) 3Pocec inclusion body protein:
Step 3) will add the E.coliBL21(DE3 of IPTG induction) centrifugal after/pET-32a-3Pocec microorganism collection, abandon supernatant, thalline is resuspended after washing 23 times, carries out ultrasonication;The condition of described ultrasonication is: power 250W, and work 5sec, interval 5sec, altogether 20min;Then by the centrifugal 20min in centrifuge temperature 4 DEG C, the 12000rpm when of the thalline after broken, discard supernatant, precipitation adds the PBS agitator treating 30min containing 1%TritonX-100, in 4 DEG C, 12000rpm is centrifuged 15min, abandoning supernatant, is repeated once;Again with PBS agitator treating once, in 4 DEG C, 12000rpm is centrifuged 15min, abandoning supernatant, obtains inclusion body protein precipitation。
The dissolving of step 5) 3Pocec inclusion body protein:
Being dissolved by the inclusion body protein precipitation 8M carbamide lysate obtained in step 4), and place 30min in room temperature, then 4 DEG C, 1500rpm is centrifuged 30min, leaves and takes supernatant。
Step 6) cutting of 3Pocec inclusion body protein:
By step 5) supernatant that obtains is directly added into hydroxylamine cleavage liquid, make final concentration of: oxammonium hydrochloride .-1.0mol/L, Ches-0.1mol/L, pH9.3(35 DEG C), mixed solution is placed in 35 DEG C of water, 8 9h in water-bath, obtains merging antibacterial peptide monomer solution, and the described aminoacid sequence merging antibacterial peptide is such as shown in SQEIDNO.1。
The purification of step 7) antibacterial peptide monomer:
Being the filter membrane of 1kd by the antibacterial peptide monomer solution interception that merges that cut in step 6), the Tris-HCl of the 20mmol/L of pH8.0 carries out ultrafiltration, removes hydroxylamine cleavage liquid and carbamide, obtains antibacterial peptide monomer。
The described application merging antibacterial peptide, is added in finished product or semi-finished product feedstuff as antibacterial and mildew removing agent。
Further, the described application merging antibacterial peptide, the described nucleotide sequence merging antibacterial peptide uses transgenic technology and eukaryotic expression technological sourcing in the nucleotide sequence of forage crop itself, makes the great expression of forage crop own go out to merge antibacterial peptide。
The invention have the advantages that
First; by gram positive bacteria will be had the cecropin cm4 of relatively high inhibition effect and gram negative bacteria has the nucleotide sequence of pig derived antimicrobial peptide PR-39 of relatively high inhibition effect be optimized combination; optimize and remove rare codon so that expressing; and protectiveness base is added at two ends; add isocaudarner (Bgl II simultaneously; BamH I) and the restriction enzyme site of Xhol I and Asn-Gly azanol chemical cleavage site, obtain designed gene order。
Then, the designed nucleotide sequence merging antibacterial peptide is obtained by chemical synthesis process。The described nucleotide sequence merging antibacterial peptide being connected on cloning vehicle, by the effect of isocaudarner, repeating to connect the nucleotide sequence merging antibacterial peptide 2 times thus obtaining the recombinant nucleotide sequence containing 3 sections of fusion antibacterial peptide nucleotide sequences。
Secondly, the nucleotide sequence of restructuring is connected on expression vector。
Finally, the expression vector transformed host cell of the nucleotide sequence merging antibacterial peptide will be contained, cultivate transformant, obtain from transformant culture containing the inclusion body protein merging antibacterial peptide。The inclusion body protein obtained is purified, namely obtains two kinds of antibacterial peptide monomers after chemical cleavage and renaturation。
Optimize through substantial amounts of experimentation, undertaken designed gene order 3 times repeating series connection, increase the molecular weight of expressed fusion protein, can express with the form of inclusion body protein on the one hand, thus avoiding the suppression that expression strain is produced by activated antibacterial peptide monomer after expression and the raising limiting expression;After on the other hand fusion protein being carried out chemical cleavage, the fusion antibacterial peptide monomer of obtainable repetition multiple so that the yield of end-product improves further。The suppression of gram positive bacteria and gram negative bacteria is had very strong inhibitory action again by monomer respectively that simultaneously obtain two kinds of antibacterial peptides after chemical cleavage, and this reduces cost to a great extent, solves the large-scale application problem of existing antibacterial peptide。
Accompanying drawing explanation
Fig. 1 is the PCR primer electrophoretogram of synthetic antibacterial peptide。
Fig. 2 is restructuring expression vector establishment process schematic。
Fig. 3 is the electrophoretogram of the double digestion qualification of expression vector。
Fig. 4 is that 3Pocec inclusion body protein is to escherichia coli and staphylococcus aureus bacteriostatic test result。
Concrete facts mode
Embodiment 1:
The described preparation method merging antibacterial peptide, including
The structure of step 1) cloning vehicle:
As shown in Figure 1 and Figure 2,
Both are connected by 1.1 aminoacid sequences according to the cecropin cm4 announced and pig derived antimicrobial peptide PR-39, the gene order with E.coli standard codon for Reference Design antibacterial peptide;And the restriction enzyme site of Bgl II, BamH I isocaudarner and Xhol I, the gene order as shown in SEQIDNO.2 of chemosynthesis is introduced respectively at gene order two ends;The gene order synthesized with BamH I and Xhol I double digestion it is connected to the pMD18 carrier with BamH I and Xhol I double digestion and converts to E.coliDH5 α competent cell, obtaining pMD18-Pocec cloning vehicle。
1.2 by pMD18-Pocec cloning vehicle with BamH I and Xhol I double digestion, then by the pMD18-Pocec cloning vehicle after double digestion be attached with the synthetic gene sequence of Bgl II and Xhol I double digestion, product will be connected convert to E.coliDH5 α competent cell, obtain pMD18-2Pocec cloning vehicle。
1.3 by pMD18-2Poce cloning vehicle with BamH I and Xhol I double digestion, then by the pMD18-2Poce cloning vehicle after double digestion be attached with the synthetic gene sequence of Bgl II and Xhol I double digestion, namely the gene order as shown in SEQIDNO.3 is obtained, product will be connected convert to E.coliDH5 α competent cell, obtain pMD18-3Pocec cloning vehicle。
Step 2) structure of expression vector:
PMD18-3Pocec cloning vehicle and pET-32a carrier are used that Bgl II and Xhol I double digestion, then the pMD18-3Pocec cloning vehicle after double digestion, pET-32a carrier are connected, obtain pET-32a-3Pocec expression vector, pET-32a-3Pocec expression vector is converted to E.coliBL21(DE3) competent cell, the coating LB plate screening containing ampicillin, obtains E.coliBL21(DE3)/pET-32a-3Pocec。
The expression of step 3) 3Pocec inclusion body protein:
By E.coliBL21(DE3)/pET-32a-3Pocec is seeded in the LB culture medium containing ampicillin according to the ratio of 1:100, it is seeded to the LB culture medium containing ampicillin according to 1:100 ratio after overnight incubation, being cultured to 600nm place OD value is 0.6, adds IPTG(isopropylthiogalactoside) make its final concentration of 0.2mmol/L with the expression of induced fusion antibacterial peptide inclusion body protein;Under different condition, the SDS-PAGE electrophoresis result of induction expression protein is as described in Figure 3。
The purification of step 4) 3Pocec inclusion body protein:
Step 3) will add the E.coliBL21(DE3 of IPTG induction) centrifugal after/pET-32a-3Pocec microorganism collection, abandon supernatant, thalline is resuspended after washing 3 times, carries out ultrasonication;The condition of described ultrasonication is: power 250W, and work 5sec, interval 5sec, altogether 20min;Then by the thalline after broken in 4 DEG C of working environments of centrifuge, 12000rpm, centrifugal 20min, abandoning supernatant, add the PBS agitator treating 30min containing 1%TritonX-100 in precipitation, in 4 DEG C, 12000rpm is centrifuged 15min, abandoning supernatant, is repeated once;Again with PBS agitator treating once, in 4 DEG C, 12000rpm is centrifuged 15min, abandoning supernatant, obtains inclusion body protein precipitation。
The dissolving of step 5) 3Pocec inclusion body protein:
Being dissolved by the inclusion body protein precipitation 8M carbamide lysate obtained in step 4), and place 30min in room temperature, then 4 DEG C, 1500rpm is centrifuged 30min, leaves and takes supernatant。
Step 6) cutting of 3Pocec inclusion body protein:
By step 5) supernatant that obtains is directly added into hydroxylamine cleavage liquid, make final concentration of: oxammonium hydrochloride .-1.0mol/L, Ches-0.1mol/L, pH9.3,35 DEG C, mixed solution is placed in 35 DEG C of water, 9h in water-bath, obtaining merging antibacterial peptide monomer solution, the described aminoacid sequence merging antibacterial peptide is such as shown in SQEIDNO.1。
Being the filter membrane of 1kd by the antibacterial peptide monomer solution interception that merges that cut in step 6), the Tris-HCl of the 20mmol/L of pH8.0 carries out ultrafiltration, removes hydroxylamine cleavage liquid and carbamide, obtains antibacterial peptide monomer。
Embodiment 2: bacteriostatic activity detection and application
The detection of 1.1 bacteriostatic activities
Adopt agarose plate diffusion method detection antibacterial activity。By escherichia coli and staphylococcus aureus amplification cultivation in test tube respectively, it is respectively coated after bacterium solution is suitably diluted on MHA flat board, on flat board, rustless steel Oxford cup is put into after drying, cup is separately added into the antibacterial peptide 20 μ L obtained, 28 DEG C of incubated overnight also observe inhibition zone size, as shown in Figure 4, escherichia coli and staphylococcus aureus are had In Vitro Bacteriostatic after showing the cleaved purification of antibacterial peptide fusion protein of the design by result。
The application prospect of 1.2 fusion protein 3Pocec
In view of the fusion antibacterial peptide fusion protein expression of the design is big, and after chemical cleavage, gram positive bacteria and gram negative bacteria are respectively provided with stronger inhibitory action, it is contemplated that it can be used as the feed additive of a kind of high-quality to use。Except can being added in finished product or semi-finished product feedstuff as antibacterial and mildew removing agent, transgenic and eukaryotic expression technology can be used long term by importing in forage crop by preferred antibacterial peptide gene sequence, forage crop itself is made to get final product great expression, thus reducing operating procedure to obtain highly efficient production efficiency。
Sequence table
<110>Henan Ou Pu bio tech ltd
<120>antibacterial peptide and application thereof are merged
<160>3
<170>PatentInVersion3.3
<210>1
<211>89
<212>PRT
<213>artificial sequence
<400>1
GluAspIleThrValValGlyLysGerGerLysLysGerLysLys
151015
IleValArgThrGerValThrGerValIleLysIleValArgIle
202530
IleIleIleIleValArgIleIleProGerThrValValValVal
354045
ArgValArgArgThrCysArgValIleValIleIleArgGerGer
505560
ArgArgValCysArgArgValTyrHisGlnGlaPheHisGlnGly
657075
GerGlaGlaAspGerArgThrGlaAspProAsnGerGerGer
808589
<210>2
<211>269
<212>DNA
<213>artificial sequence
<400>2
GAAGATCTAACGGTCGTTGGAAAATCTTCAAAAAAATCGAAAAAGTTGGTCAGAACATCC60
GTGACGGTATCGTTAAAGCTGGTCCGGCTGTTGCTGTTGTTGGTCAGGCTGCTACCATCA120
ACGGTCGTCGTCGTCCGCGTCCGCCGTACCTGCCGCGTCCTCGTCCTCCTCCGTTCTTCC180
CGCCGCGTCTGCCGCCGCGTGCTTTCCACCAAGGTTCCCAAAGGTTCCCAAAGGTTCCCA240
CGAACGGCGGATCCTAATAGCTCGAGCGG269
<210>3
<211>761
<212>DNA
<213>artificial sequence
<400>3
GAAGATCTAACGGTCGTTGGAAAATCTTCAAAAAAATCGAAAAAGTTGGTCAGAACATCC60
GTGACGGTATCGTTAAAGCTGGTCCGGCTGTTGCTGTTGTTGGTCAGGCTGCTACCATCA120
ACGGTCGTCGTCGTCCGCGTCCGCCGTACCTGCCGCGTCCTCGTCCTCCTCCGTTCTTCC180
CGCCGCGTCTGCCGCCGCGTGCTTTCCACCAAGGTTCCCAAAGGTTCCCAAAGGTTCCCA240
CGAACGGCGGATCTAACGGTCGTTGGAAAATCTTCAAAAAAATCGAAAAAGTTGGTCAGA300
ACATCCGTGACGGTATCGTTAAAGCTGGTCCGGCTGTTGCTGTTGTTGGTCAGGCTGCTA360
CCATCAACGGTCGTCGTCGTCCGCGTCCGCCGTACCTGCCGCGTCCTCGTCCTCCTCCGT420
TCTTCCCGCCGCGTCTGCCGCCGCGTATACCACCAGGCTTTCCACCAAGGTTCCCACCAC480
GATTCCCGAACGGCGGATCTAACGGTCGTTGGAAAATCTTCAAAAAAATCGAAAAAGTTG540
GTCAGAACATCCGTGACGGTATCGTTAAAGCTGGTCCGGCTGTTGCTGTTGTTGGTCAGG600
CTGCTACCATCAACGGTCGTCGTCGTCCGCGTCCGCCGTACCTGCCGCGTCCTCGTCCTC660
CTCCGTTCTTCCCGCCGCGTCTGCCGCCGCGTATACCACCAGGCTTTCCACCAAGGTTCC720
CACCACGATTCCCGAACGGCGGATCCTAATAGCTCGAGCGG761
Claims (8)
1. one kind merges antibacterial peptide, it is characterised in that: include cecropin cm4 and pig derived antimicrobial peptide PR-39。
2. fusion antibacterial peptide according to claim 1: it is characterized in that: the described aminoacid sequence merging antibacterial peptide is such as shown in SQEIDNO.1。
3. fusion antibacterial peptide according to claim 1, it is characterised in that: the nucleotide sequence of described fusion antibacterial peptide is such as shown in SQEIDNO.2。
4. the expression vector of the nucleotide sequence containing fusion antibacterial peptide according to claim 3。
5. the preparation method of a fusion antibacterial peptide according to claim 1, it is characterised in that: include
The structure of step 1) cloning vehicle:
1.1 aminoacid sequences according to the cecropin cm4 announced and pig derived antimicrobial peptide PR-39, the gene order with E.coli standard codon for Reference Design antibacterial peptide;And the restriction enzyme site of Bgl II, BamH I isocaudarner and Xhol I, the gene order as shown in SEQIDNO.2 of chemosynthesis is introduced respectively at gene order two ends;The gene order synthesized with BamH I and Xhol I double digestion it is connected to the pMD18 carrier with BamH I and Xhol I double digestion and converts to E.coliDH5 α competent cell, obtaining pMD18-Pocec cloning vehicle;
1.2 by pMD18-Pocec cloning vehicle with BamH I and Xhol I double digestion, then by the pMD18-Pocec cloning vehicle after double digestion be attached with the synthetic gene sequence of Bgl II and Xhol I double digestion, product will be connected convert to E.coliDH5 α competent cell, obtain pMD18-2Pocec cloning vehicle;
1.3 by pMD18-2Poce cloning vehicle with BamH I and Xhol I double digestion, then by the pMD18-2Poce cloning vehicle after double digestion be attached with the synthetic gene sequence of Bgl II and Xhol I double digestion, namely the gene order as shown in SEQIDNO.3 is obtained, product will be connected convert to E.coliDH5 α competent cell, obtain pMD18-3Pocec cloning vehicle;
Step 2) structure of expression vector:
PMD18-3Pocec cloning vehicle and pET-32a carrier are used that Bgl II and Xhol I double digestion, then the pMD18-3Pocec cloning vehicle after double digestion, pET-32a carrier are connected, obtain pET-32a-3Pocec expression vector, pET-32a-3Pocec expression vector is converted to E.coliBL21(DE3) competent cell, the coating LB plate screening containing ampicillin, obtains E.coliBL21(DE3)/pET-32a-3Pocec;
The expression of step 3) 3Pocec inclusion body protein:
By E.coliBL21(DE3)/pET-32a-3Pocec is seeded in the LB culture medium containing ampicillin according to the ratio of 1:100 1:106, it is seeded to the LB culture medium containing ampicillin again according to 1:100 ratio after 37 DEG C of overnight incubation, when being cultured to 600nm place OD value 0.4-0.6 under 37 DEG C of conditions, add IPTG and make its final concentration of 0.2-0.3mmol/L with the expression of induced fusion antibacterial peptide inclusion body protein;
The purification of step 4) 3Pocec inclusion body protein:
Step 3) will add the E.coliBL21(DE3 of IPTG induction) centrifugal after/pET-32a-3Pocec microorganism collection, abandon supernatant, thalline is resuspended after washing 23 times, carries out ultrasonication;The condition of described ultrasonication is: power 250W, and work 5sec, interval 5sec, altogether 20min;Then by the centrifugal 20min in centrifuge temperature 4 DEG C, the 12000rpm when of the thalline after broken, discard supernatant, precipitation adds the PBS agitator treating 30min containing 1%TritonX-100, in 4 DEG C, 12000rpm is centrifuged 15min, abandoning supernatant, is repeated once;Again with PBS agitator treating once, in 4 DEG C, 12000rpm is centrifuged 15min, abandoning supernatant, obtains inclusion body protein precipitation;
The dissolving of step 5) 3Pocec inclusion body protein:
Being dissolved by the inclusion body protein precipitation 8M carbamide lysate obtained in step 4), and place 30min in room temperature, then 4 DEG C, 1500rpm is centrifuged 30min, leaves and takes supernatant;
Step 6) cutting of 3Pocec inclusion body protein:
By step 5) supernatant that obtains is directly added into hydroxylamine cleavage liquid, make final concentration of: oxammonium hydrochloride .-1.0mol/L, Ches-0.1mol/L, pH9.3(35 DEG C), mixed solution is placed in 35 DEG C of water, cultivating 8 9h in water-bath, obtain merging antibacterial peptide monomer solution, the described aminoacid sequence merging antibacterial peptide is such as shown in SQEIDNO.1。
6. the preparation method of fusion antibacterial peptide according to claim 5, it is characterised in that: also include the purification of antibacterial peptide monomer:
Being the filter membrane of 1kd by the antibacterial peptide monomer solution interception that merges that cut in step 6), the Tris-HCl of the 20mmol/L of pH8.0 carries out ultrafiltration, removes hydroxylamine cleavage liquid and carbamide, obtains antibacterial peptide monomer。
7. the application merging antibacterial peptide according to any one in claim 1,2, it is characterised in that: it is added in finished product or semi-finished product feedstuff as antibacterial and mildew removing agent。
8. the application of fusion antibacterial peptide according to claim 3, it is characterized in that: the described nucleotide sequence merging antibacterial peptide uses transgenic technology and eukaryotic expression technological sourcing in the nucleotide sequence of forage crop itself, makes the great expression of forage crop own go out to merge antibacterial peptide。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610120163.2A CN105693866A (en) | 2016-03-03 | 2016-03-03 | Fusion antibacterial peptide and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610120163.2A CN105693866A (en) | 2016-03-03 | 2016-03-03 | Fusion antibacterial peptide and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN105693866A true CN105693866A (en) | 2016-06-22 |
Family
ID=56223851
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610120163.2A Pending CN105693866A (en) | 2016-03-03 | 2016-03-03 | Fusion antibacterial peptide and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105693866A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
CN107266585A (en) * | 2017-07-13 | 2017-10-20 | 陕西科技大学 | A kind of MLH fusions antibacterial peptide and its preparation method and application |
CN110951807A (en) * | 2019-12-31 | 2020-04-03 | 成都大学 | Fusion expression and purification method of cecropin antibacterial peptide |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101319216A (en) * | 2008-07-10 | 2008-12-10 | 南京师范大学 | Preparation for recombined cultivated silkworm antimicrobial peptide CM4 and purification process |
CN102229934A (en) * | 2011-06-14 | 2011-11-02 | 华南农业大学 | Efficient production method and application of antimicrobial peptide PR-39 of pig small intestine in pichia pastoris |
CN102532325A (en) * | 2011-02-25 | 2012-07-04 | 山东省科学院中日友好生物技术研究中心 | Method for preparing cecropins AD and frog Buforin II fusion antimicrobial peptide by using hydroxylamine cutting method, and uses thereof |
CN104017085A (en) * | 2014-06-24 | 2014-09-03 | 任建廷 | PAMP37-PR39 fused antibacterial peptide and preparation method thereof |
CN104987367A (en) * | 2015-08-16 | 2015-10-21 | 刘诚 | Preparation method of artificial antibacterial peptide PR39-R1T and application thereof |
-
2016
- 2016-03-03 CN CN201610120163.2A patent/CN105693866A/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101319216A (en) * | 2008-07-10 | 2008-12-10 | 南京师范大学 | Preparation for recombined cultivated silkworm antimicrobial peptide CM4 and purification process |
CN102532325A (en) * | 2011-02-25 | 2012-07-04 | 山东省科学院中日友好生物技术研究中心 | Method for preparing cecropins AD and frog Buforin II fusion antimicrobial peptide by using hydroxylamine cutting method, and uses thereof |
CN102229934A (en) * | 2011-06-14 | 2011-11-02 | 华南农业大学 | Efficient production method and application of antimicrobial peptide PR-39 of pig small intestine in pichia pastoris |
CN104017085A (en) * | 2014-06-24 | 2014-09-03 | 任建廷 | PAMP37-PR39 fused antibacterial peptide and preparation method thereof |
CN104987367A (en) * | 2015-08-16 | 2015-10-21 | 刘诚 | Preparation method of artificial antibacterial peptide PR39-R1T and application thereof |
Non-Patent Citations (3)
Title |
---|
AGERBERTH,B.等: "PR-39=proline-arginine rich antibacterial peptide [swine, intestine, Peptide, 39 aa", 《GENBANK DATABASE》 * |
张双全等: "中国家蚕抗菌肽ABP-CM4在E.coli中的融合表达及活性分析", 《分子细胞生物学报》 * |
杨卫华: "PR-39,一种多功能的抗菌肽", 《生命的化学》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
CN107266585A (en) * | 2017-07-13 | 2017-10-20 | 陕西科技大学 | A kind of MLH fusions antibacterial peptide and its preparation method and application |
CN107266585B (en) * | 2017-07-13 | 2019-08-06 | 陕西科技大学 | A kind of MLH fusion antibacterial peptide and its preparation method and application |
CN110951807A (en) * | 2019-12-31 | 2020-04-03 | 成都大学 | Fusion expression and purification method of cecropin antibacterial peptide |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111607003B (en) | SARS-CoV-2N/S1 (RBD) recombinant protein and its preparation method and application | |
US7579005B2 (en) | Process for recombinant expression and purification of antimicrobial peptides using periplasmic targeting signals as precipitable hydrophobic tags | |
CN112143747B (en) | Phage lyase, gene thereof, gene recombination expression vector and application | |
CN110951715B (en) | Anti-staphylococcus broad-spectrum phage encoding lyase as well as preparation method and application thereof | |
CN105695440B (en) | Streptococcus suis phage lyase with enhanced antibacterial activity and preparation method thereof | |
EP2776560A2 (en) | Endonuclease for genome editing | |
CN102220303B (en) | Xylanase XynAHJ3 with protease resistance and gene thereof | |
CN113549610B (en) | Antibacterial peptide P104 with broad-spectrum lytic activity, lyase LysP53 and application thereof | |
CN105693866A (en) | Fusion antibacterial peptide and application thereof | |
CN110592057B (en) | Chimeric lyase ILTphg and polynucleotides encoding same | |
CN101392249A (en) | Antibiotic peptide gene, preparation method thereof and construction of expression plasmid of the same in pichia vector | |
CN104830825A (en) | Endolysin sourced from salmonella bacteriophage and application thereof | |
CN104017084A (en) | Heterozygous antibacterial and antiviral polypeptide as well as preparation method and application thereof | |
CN103088050B (en) | Mature human beta-defensin-2 (HBD-2) and preparation method thereof | |
CN104530215A (en) | Antibacterial peptide VIP (vasoactive intestinal peptide)-like peptide and preparation method thereof | |
Sun et al. | Generation of newly discovered resistance gene mcr-1 knockout in Escherichia coli using the CRISPR/Cas9 system | |
CN107266585B (en) | A kind of MLH fusion antibacterial peptide and its preparation method and application | |
CN107267537B (en) | Preparation method of hybrid antibacterial peptide M L H | |
CN104531712B (en) | The preparation and application of Bemisia tabaci peptidoglycan recognition protein with bactericidal activity | |
CN109021088B (en) | Penaeus monodon antibacterial peptide ALFpm10 and preparation method thereof | |
CN104987367A (en) | Preparation method of artificial antibacterial peptide PR39-R1T and application thereof | |
CN109706136B (en) | Lyase used as PCR preservative and preparation method and application thereof | |
CN107459569A (en) | A kind of cecropin B antibacterial peptide mutant | |
CN103484467B (en) | Diamondback moth cecropin 3, preparation method and application thereof | |
CN101899469B (en) | Shuttle plasmid and method for efficiently expressing cecropin and lysozyme genes |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160622 |
|
RJ01 | Rejection of invention patent application after publication |