CN104017087B - One boar derived antimicrobial peptide and preparation method thereof - Google Patents
One boar derived antimicrobial peptide and preparation method thereof Download PDFInfo
- Publication number
- CN104017087B CN104017087B CN201410284682.3A CN201410284682A CN104017087B CN 104017087 B CN104017087 B CN 104017087B CN 201410284682 A CN201410284682 A CN 201410284682A CN 104017087 B CN104017087 B CN 104017087B
- Authority
- CN
- China
- Prior art keywords
- antibacterial peptide
- fusion
- antibacterial
- preparation
- recombinant dna
- 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.)
- Expired - Fee Related
Links
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 89
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 108700042778 Antimicrobial Peptides Proteins 0.000 title abstract description 7
- 102000044503 Antimicrobial Peptides Human genes 0.000 title abstract description 7
- 230000004927 fusion Effects 0.000 claims abstract description 43
- 239000013604 expression vector Substances 0.000 claims abstract description 10
- 239000000654 additive Substances 0.000 claims abstract description 8
- 108020004705 Codon Proteins 0.000 claims abstract description 6
- 230000000844 anti-bacterial effect Effects 0.000 claims description 16
- 108020004511 Recombinant DNA Proteins 0.000 claims description 14
- 238000013461 design Methods 0.000 claims description 10
- 230000036039 immunity Effects 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 239000013598 vector Substances 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 abstract description 11
- 230000000845 anti-microbial effect Effects 0.000 abstract description 8
- 108010066857 PMAP-36 Proteins 0.000 abstract description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 6
- 108050004290 Cecropin Proteins 0.000 abstract description 5
- 241000588724 Escherichia coli Species 0.000 abstract description 5
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 4
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 230000000968 intestinal effect Effects 0.000 abstract description 4
- 244000144972 livestock Species 0.000 abstract description 4
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 13
- 238000000034 method Methods 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 210000000170 cell membrane Anatomy 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 102100029727 Enteropeptidase Human genes 0.000 description 2
- 108010013369 Enteropeptidase Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000862 Ion Channels Proteins 0.000 description 2
- 102000004310 Ion Channels Human genes 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 108010066849 PMAP-37 Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 101710145659 Antibacterial peptide PMAP-36 Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 241000446313 Lamella Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- 101100298055 Sus scrofa PMAP36 gene Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000012969 defense response to bacterium Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- ZPWVBRGFVGXRKZ-DMZHRTQBSA-N pmap-23 Chemical compound C([C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)C1=CC=CC=C1 ZPWVBRGFVGXRKZ-DMZHRTQBSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 108010027884 porcine myeloid antibacterial peptide 23 Proteins 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Landscapes
- Fodder In General (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to boar derived antimicrobial peptide and preparation method thereof.Is the present invention by pig antibacterial peptide cecropin? P1 and PMAP-36 series connection constructs a kind of fusion antibacterial peptide, and utilize the preferences of e. coli codon, devise the DNA sequence dna of described fusion antibacterial peptide, and be building up in expression vector, carry out the Expression and purification merging antibacterial peptide subsequently, anti-microbial activity result shows, and the fusion antibacterial peptide obtained all has good In Vitro Bacteriostatic to intestinal bacteria and streptococcus aureus.Antibacterial peptide provided by the invention can well improve the resistance against diseases of swinery, effectively can substitute or reduce the use of microbiotic as fodder additives, thus improves livestock product safety.
Description
Technical field
The invention belongs to genetically engineered in biological-pharmacy and produce the technical field of medicine, be specially boar derived antimicrobial peptide and preparation method thereof.
Background technology
Antibacterial peptide (antibacterialpeptides) is a kind of polypeptide with strong anti-microbial effect that organism produces, and is the important composition composition of biological congenital immunity.Up to now, discovery more than 600 kind of endogenous antibacterial active peptide in animal, plant and prokaryotic organism has been comprised at many biologies.Pig derived antimicrobial peptide is in pig body, be separated the antibacterial peptide obtained.
Because antibacterial peptide has broad-spectrum bactericidal action, relative molecular weight is less, thermally-stabilised, the advantages such as good water solubility, what is more important antibacterial peptide does not almost act on eukaryotic cell, only act on the eukaryotic cell of prokaryotic cell prokaryocyte and generation pathology, when current many pathogenic bacterias progressively produce resistance to original microbiotic, to the understanding of antibacterial peptide function spontaneous in pig body, and design a kind of method and just seem very important to regulate the function of natural antibacterial peptide in pig body with the research of substitute antibiotics, this plays positive promoter action by searching Substitutes For Antibiotic.
Cecropins is first Antibacterial Peptide From Animals be found, 1989, and the people such as Lee divide high to cecropinP1 from chitterlings, and describe cecropins extensively may exist in animal.This polypeptide contains 31 amino-acid residues, and not containing halfcystine, its N end regions has strong basicity, can form parents' spirane structure of almost Perfect, and is linear structure at C end, and the C end of most polypeptide is amidated, and amidation has vital role to its anti-microbial activity.Cecropins has very strong lethality to gram positive organism, negative bacterium portion.Lot of experiments research proves, the anti-microbial activity of cecropins depends on its amphipathic a-spirane structure, this structure makes it on prokaryotic cell prokaryocyte plasma membrane, to form huge timeliness and the dependent ionic channel of electromotive force, intraor extracellular osmotic pressure is caused to change, entocyte especially potassium ion oozes out in a large number, and bacterium is therefore dead.CecropinP1 sterilization process is as follows: first the positive charge on the amphipathic alpha-helix of antibacterial peptide molecule and the negative charge on bacterium plasma membrane phospholipid molecule are attracting and close by electrostatic, the flexibility of the continuous structure between then holding by means of N end and C in molecule, the hydrophobic side in antibacterial peptide molecule is inserted in plasma membrane; Then its amphipathic alpha-helix is also inserted in people's plasma membrane, destroy the original ordered structure of double-layer of lipoid, because the amphipathic antibacterial peptide molecule that makes of alpha-helix is flocked together mutually by displacement intermolecular in film, thus ionic channel is formed on film, bacterium is finally lethal because not keeping normal osmotic pressure.
PMAPs belongs to pig marrow antibiotic peptide, is divided into PMAP-23, PMAP-36 and PMAP-37 according to its textural difference, respectively containing 23,36 and 37 amino acid.Its structure prediction and stratographic analysis are shown that PMAP-36 and PMAP-37 has amphiphilicα-helix.The PMAP36 of pig is the antimicrobial polypeptide containing 36 amino-acid residues, not containing halfcystine, its N end regions has strong basicity, can form αhelix, utilize this structure can form perforation on bacterial cell membrane, therefore have remarkable lethal effect to intestinal bacteria and streptococcus aureus.
But, existing research shows, many natural antibacterial peptide activity are not high, or there is higher cytotoxicity to eukaryotic cell, therefore needed to improve its activity further by molecular improvement before as the development of new antibiotic substitute, or reduce its toxicity.In addition, bacteriological infection has become the major reason affecting Animal husbandry production, and the especially appearance of Resistant strain is more the increase in the treatment difficulty of disease.Therefore, in the research of current antibacterial peptide in the urgent need to transforming existing antibacterial peptide and design novel antimicrobial peptide molecule in order to create high vigor antibacterial peptide.
Along with the development of Protocols in Molecular Biology, by genetic engineering technique, to the research of antibacterial peptide in pig body, by pig antibacterial peptide gene cloning and expression.The invention antibacterial peptide cecropinP1 and PMAP-36 of pig is merged, antibacterial peptide after fusion improves anti-microbial activity, improves the codon thus obtain merging antibacterial peptide simultaneously and is suitable for mass-produced novel, the environmental protection of industry, highly active fusion antibacterial peptide.
Utilize pig antibacterial peptide provided by the invention as class fodder additives, the resistance against diseases of swinery can well be improved, effectively can substitute or reduce the use of microbiotic as fodder additives, thus improve livestock product safety.The antibacterial peptide feedstuff additive of pig provided by the invention will have very wide application prospect in husbandry sector.
Summary of the invention
The object of this invention is to provide a kind of fusion antibacterial peptide with high antibacterial activity.Described fusion antibacterial peptide comprises two or more pig antibacterial peptide.
Further, described fusion antibacterial peptide comprises pig antibacterial peptide cecropinP1 and PMAP-36.
Further, described fusion antibacterial peptide obtains together with pig antibacterial peptide cecropinP1 with PMAP-36 series connection.
A nearlyer step, described fusion antibacterial peptide aminoacid sequence is SEQ ID 1.
The object of this invention is to provide a kind of recombinant DNA merging antibacterial peptide, described recombinant DNA be through codon optimized, be adapted at escherichia coli expression merge antibacterial peptide.
Further, the recombinant DNA sequence of described fusion antibacterial peptide is SEQ ID 2.
The object of this invention is to provide a kind of carrier can expressing above-mentioned fusion antibacterial peptide.The above-mentioned fusion antibacterial peptide of preferred use pET30a vector expression.
The object of this invention is to provide a kind of clone can expressing above-mentioned fusion antibacterial peptide.Preferred use e. coli bl21 expresses above-mentioned fusion antibacterial peptide.
The object of this invention is to provide a kind of novel antibacterial immunological reagent, containing above-mentioned fusion antibacterial peptide in described antibacterial immunity preparation.
The object of this invention is to provide a kind of preparation method of fusion antibacterial peptide of high antibacterial activity.
Further, the preparation method of described fusion antibacterial peptide comprises:
5) two or more pig antibacterial peptide is connected, the amino acid sequence of the fusion antibacterial peptide obtained;
6) according to the preferences of codon, the recombinant DNA sequence of antibacterial peptide is merged in design;
7) recombinant DNA sequence is building up on expression vector;
8) expression vector transfered cell is carried out the expression of merging antibacterial peptide.
Further, described pig antibacterial peptide is antibacterial peptide cecropinP1 and antibacterial peptide PMAP-36.
Further, the fusion antibacterial peptide of design obtains by methods such as synthesis or multiplex PCRs, directly synthesis.Preferably carry out that sequence that multiple PCR primer adopts is selected from SEQ ID 4, SEQID5, SEQID6, SEQID7 one or several.
Further, recombinant DNA is cut the modes such as link or homologous recombination by enzyme to be connected on expression vector.
Further, purifying is carried out to the fusion antibacterial peptide of expressing.
The object of this invention is to provide fusion antibacterial peptide, the carrier of fusion antibacterial peptide can be expressed and preparing the application in fodder additives, antibacterial medicine preparation, healthcare products, sanitas containing the antibacterial immunity preparation merging antibacterial peptide.
Advantage of the present invention:
1) the invention pig antibacterial peptide cecropinP1 and PMAP-36 is carried out polypeptide series connection, obtain the fusion antibacterial peptide with high antibacterial activity, described fusion antibacterial peptide can well improve the resistance against diseases of swinery, effectively can substitute or reduce the use of microbiotic as fodder additives, thus improving livestock product safety.；
2) the invention provides a kind of preparation method of fusion antibacterial peptide of high antibacterial activity, adopt this preparation method can efficiently obtain fusion antibacterial peptide.
Accompanying drawing explanation
Fig. 1 is the building process of recombinant expression vector
Embodiment
Below in conjunction with specific embodiment, setting forth the present invention further, only for explaining the present invention, and can not limitation of the present invention be interpreted as.Those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition examinations of advising according to manufacturer.
The structure design of embodiment 1 antibacterial peptide and prediction
In order to strengthen the fungicidal activity of antibacterial peptide, the C terminal sequence series connection of pig antibacterial peptide (PAMP36) and CercropinP1 built together, its aminoacid sequence building the fusion polypeptide obtained is SEQID1:VGRFRRLRKKTRKRLKKIGKVLKWIPPIVGSIPLGCGPEGIAIAIQGGP R.
Utilize the preferences of e. coli codon, devise the recombinant DNA sequence (SEQID2) of this sequence, obtaining this polypeptide N end through secondary structure prediction is a α spiral, and C end is the structure of β lamella.
The structure of embodiment 2 peptide expression carrier
1) optimizing DNA sequence dna makes it be adapted to colibacillary codon-bias, increases the restriction enzyme site that vector construction is used, and design fusion polypeptide DNA sequence dna (SEQID3) is as follows:
GGTACCGTTGGTCGTTTCCGTCGTCTGCGTAAAAAAACCCGTAAACGTCTGAAAAAAATCGGTAAAGTTCTGAAATGGATCCCGCCGATCGTTGGTTCTATCCCGCTGGGTTGCGGTCCGGAAGGTATCGCTATCGCTATCCAGGGTGGTCCGCGTTAA
CTCGAG
The sequence of black underscore is restriction enzyme site (KpnI and XhoI).
DNA sequence dna is synthesized many primers respectively, utilizes the method for over-lap PCR, many primer amplifications are spliced into complete DNA sequence dna.The primer sequence of synthesis is in table 1:
The concrete sequence of table 1. synthetic primer
| Primer numbers | Sequence |
| S1-SEQ ID 4 | 5’-agggtaccgttggtcgtttccgtcgtctgcgtaaaaaaacccgtaaacgtctgaa-3’ |
| A1-SEQ ID 5 | 5’-aacgatcggcgggatccatttcagaactttaccgatttttttcagacgtttacggg-3’ |
| F1-SEQ ID 6 | 5’-ggatcccgccgatcgttggttctatcccgctgggttgcggtccggaaggtatcgct-3’ |
| R1-SEQ ID 7 | 5’-tactcgagttaacgcggaccaccctggatagcgatagcgataccttccggac-3’ |
2) over-lap PCR amplification target DNA sequence
S1 and A1 and F1 and R1 is utilized to carry out the amplification synthesis of fusion rotein partial sequence (P1 and P2) respectively.
Purifying reclaims respective amplified production, carries out the amplification of second step.Reaction system (cumulative volume is pipe 50 microlitre often) is 2XPfu polymeric enzyme reaction liquid: 25 microlitres, and P1 reclaims product: 1 microlitre; P2 reclaims product: 1 microlitre; Deionized water: 21 microlitres.Amplification condition is: 95 DEG C of denaturations, 5 minutes; 95 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, totally 5 circulations.Then primer S1:1 microlitre is added; Primer R1:1 microlitre, 95 DEG C of sex change 30 seconds, 62 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, totally 35 circulations.Agargel electrophoresis qualification PCR primer, and cut glue recovery DNA fragmentation.
3) structure of peptide expression carrier
PET30a carrier and PCR end product (cutting the DNA fragmentation that glue reclaims in step 2) are cut with KpnI and XhoI enzyme respectively, after reclaiming digestion products, connect structure and obtain pET30a-P36-CP1 expression vector, process as shown in Figure 1.After the positive colony enlarged culturing containing expression vector, carry out sequencing analysis, the sequence of sequencing result and design is completely the same, shows vector construction success.
The abduction delivering of embodiment 3 antibacterial peptide
1) transform above-mentioned expression vector according to a conventional method and enter e. coli bl21, the bacterium after transforming is coated LB flat board, is placed in 37 DEG C of incubator overnight incubation.Can be observed obvious positive colony to be grown on flat board.
2) single bacterium colony of the picking positive is 0.4 in LB liquid culture to OD600, then adds the IPTG of 0.5-0.8mM, in the expression 16-18 hour that 37 DEG C of 250rpm induce target proteins, does the contrast that does not add IPTG simultaneously.
3) collect thalline, utilize 10000g to discard supernatant liquor in centrifugal 15 minutes.Ultrasonication thalline in 1MTris-HCl solution, 20000g, centrifugal 30 minutes, carries out SDS-PAGE analysis to supernatant liquor, visible significantly target protein soluble-expression band after carrying out coomassie brilliant blue staining to polyacrylamide gel.
The purifying of embodiment 4 antibacterial peptide
1) expression and purification of fusion rotein
Culturing engineering bacterium 5 liters, 10000g collected by centrifugation thalline.Adopt the broken thalline of ultrasonic method method, thalline after centrifugal breaking, collects supernatant liquor.Directly load in His-Tag affinity column, with 50mM/L imidazoles wash-out foreign protein to 1-2 times of column volume, then with the fusion rotein that 100mM/L imidazoles wash-out is expressed, collect and concentrate fusion rotein.
2) enzymolysis of fusion rotein and the purifying of antibacterial peptide
The fusion rotein of above-mentioned acquisition is carried out the endonuclease reaction of enteropeptidase, actual conditions is 23 DEG C and hatches 12 hours, and the enteropeptidase that every milligram of fusion rotein adds 2-3 unit carries out endonuclease reaction.The dialysis tubing utilizing molecular weight cut-off to be more than 10Kd carries out albumen dialysis, is concentrated by the antibacterial peptide in the dialyzate obtained again, and obtains the antimicrobial peptide protein of the design after purifying.
The Activity determination of embodiment 5 antibacterial peptide
Agarose plate diffusion process is adopted to detect anti-microbial activity, by intestinal bacteria and streptococcus aureus amplification cultivation in test tube respectively; Dilution bacterial concentration is to 10
5bacterium/ml, then be applied to respectively on MHA flat board, dry 3-4 minute; Then on flat board, put into stainless steel Oxford cup respectively, antibacterial peptide concentrated solution 10 microlitre is added 28 DEG C of overnight incubation in the cup of Oxford.Observe the size of inhibition zone.Result shows that designed antibacterial peptide concentrated solution all has obvious In Vitro Bacteriostatic to intestinal bacteria and streptococcus aureus.
The anti-microbial activity of the fusion antibacterial peptide of the present invention's design is improved.Development for novel antibacterial immunological reagent provides excellent candidate peptide, and this is to research and development novel antibacterial immunological reagent, effectively substitute or reduce microbiotic as fodder additives use, to improve livestock product safety significant.
Claims (8)
1. merge an antibacterial peptide, it is characterized in that, described fusion antibacterial peptide aminoacid sequence is SEQ ID NO.1.
2. merge a recombinant DNA for antibacterial peptide, it is characterized in that, after described recombinant dna expression, obtain fusion antibacterial peptide according to claim 1.
3. recombinant DNA according to claim 2, is characterized in that, described recombinant DNA sequence is SEQ ID NO.2.
4. a carrier, is characterized in that, described carrier contains the recombinant DNA described in claim 2-3 any one.
5. a carrier, is characterized in that, described vector expression fusion antibacterial peptide according to claim 1.
6. an antibacterial immunity preparation, containing fusion antibacterial peptide according to claim 1 in described antibacterial immunity preparation.
7. merge a preparation method for antibacterial peptide, described preparation method comprises:
1) according to the preferences of Host Strains codon, the recombinant DNA sequence of design coding fusion antibacterial peptide according to claim 1;
2) recombinant DNA sequence is building up on expression vector;
3) expression vector transfered cell is carried out the expression of merging antibacterial peptide.
8. the application in fodder additives, antibacterial medicine preparation, healthcare products, sanitas prepared by fusion antibacterial peptide according to claim 1 or antibacterial immunity preparation according to claim 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410284682.3A CN104017087B (en) | 2014-06-24 | 2014-06-24 | One boar derived antimicrobial peptide and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410284682.3A CN104017087B (en) | 2014-06-24 | 2014-06-24 | One boar derived antimicrobial peptide and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104017087A CN104017087A (en) | 2014-09-03 |
| CN104017087B true CN104017087B (en) | 2016-03-09 |
Family
ID=51434121
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410284682.3A Expired - Fee Related CN104017087B (en) | 2014-06-24 | 2014-06-24 | One boar derived antimicrobial peptide and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104017087B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104448006B (en) * | 2014-12-13 | 2015-11-18 | 青岛宏昊生物科技有限公司 | A kind of heterozygous antibacterial peptide CE-PR and application thereof |
| CN105153281B (en) * | 2015-09-18 | 2018-04-03 | 广州傲农生物科技有限公司 | Antibacterial peptide HP10 and its preparation method and application |
| 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 |
| CN108570103B (en) * | 2018-04-03 | 2019-07-30 | 东北农业大学 | One kind is rich in tryptophan antibacterial peptide WK12 and its preparation method and application |
| CN108314722B (en) * | 2018-04-27 | 2020-08-11 | 九江牧威利元科技中心（普通合伙） | Antibacterial peptide and application thereof |
| CN110283253B (en) * | 2019-07-12 | 2020-04-10 | 东北农业大学 | Pig-derived hybrid antibacterial peptide MDP-2 and preparation method and application thereof |
| CN113024653B (en) * | 2021-03-30 | 2022-06-17 | 山东中科康源生物科技有限公司 | Pig-derived antibacterial peptide PMAP-23 variant and application thereof in preparation of feed |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509007A (en) * | 2008-12-10 | 2009-08-19 | 东北农业大学 | Synthesis of LfcinB15-Mag12 encoding gene and expression method in colon bacillus |
| CN102220255A (en) * | 2010-04-14 | 2011-10-19 | 深圳市圣西马生物技术有限公司 | Recombinant antimicrobial peptide (AMP) as well as genetic engineering preparation method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717737B (en) * | 2009-12-11 | 2012-05-30 | 中国科学院亚热带农业生态研究所 | Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof |
| CN103435701B (en) * | 2013-07-24 | 2014-09-17 | 山西农业大学 | Pig antibacterial peptide cystatin11 fusion protein, and encoding gene and application thereof |
-
2014
- 2014-06-24 CN CN201410284682.3A patent/CN104017087B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509007A (en) * | 2008-12-10 | 2009-08-19 | 东北农业大学 | Synthesis of LfcinB15-Mag12 encoding gene and expression method in colon bacillus |
| CN102220255A (en) * | 2010-04-14 | 2011-10-19 | 深圳市圣西马生物技术有限公司 | Recombinant antimicrobial peptide (AMP) as well as genetic engineering preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104017087A (en) | 2014-09-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104017087B (en) | One boar derived antimicrobial peptide and preparation method thereof | |
| JP6594293B2 (en) | Purification of triple helix proteins | |
| Ramos et al. | Recombinant expression and purification of the antimicrobial peptide magainin‐2 | |
| CN105219779B (en) | Red claw crayfish coagulogen and preparation method and application | |
| CN104017086B (en) | A kind of fusion antibacterial peptide and preparation method thereof | |
| Yu et al. | Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner | |
| Yue et al. | Separation and purification of a keratinase as pesticide against root-knot nematodes | |
| CN104017085A (en) | PAMP37-PR39 fused antibacterial peptide and preparation method thereof | |
| CN106632664B (en) | Apolipoprotein II/I and preparation method, biological function and application thereof | |
| CN103621795A (en) | Application of hybrid antibacterial peptide as the feed supplement | |
| CN103833839B (en) | C-type agglutinin and its preparation method and application | |
| CN101948839B (en) | Antimicrobial peptide gene from pinctada fucata and application | |
| CN101319216B (en) | Preparation for recombined cultivated silkworm antimicrobial peptide CM4 and purification process | |
| CN103641901A (en) | Antibacterial peptide | |
| CN107475222B (en) | Genetically engineered heat-resistant human lysozyme | |
| WO2007033529A1 (en) | A gene encoding antibacterial protein of fenneropenaeus chinensis and its recombination expression method and uses | |
| CN106478812B (en) | Serine protease inhibitor-3, and function, preparation method and application thereof | |
| CN101265291A (en) | Recombination pig origin antibiotic peptide PG4 and its biological synthesis method and application | |
| Yang et al. | Efficient extracellular expression of phospholipase D in Escherichia coli with an optimized signal peptide | |
| CN102094024A (en) | Plutella xylostella cecropin gene, encoded protein, corresponding expression system and application | |
| CN102532325A (en) | Method for preparing cecropins AD and frog Buforin II fusion antimicrobial peptide by using hydroxylamine cutting method, and uses thereof | |
| CN102276729A (en) | Antibacterial peptide bovine lactoferricin-thanatin (LF-TH) and Escherichia coli recombination preparation method thereof | |
| CN101914546B (en) | Amphioxus saccharide pattern-recognition molecule Bjfcn1 gene and application thereof | |
| CN116143903B (en) | Peptidoglycan recognition protein-3, preparation method and application thereof | |
| Tak et al. | Gene expression profiling of coelomic cells and discovery of immune-related genes in the earthworm, Eisenia andrei, using expressed sequence tags |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: KEMEI BORUI TECHNOLOGY (BEIJING) CO., LTD. Free format text: FORMER OWNER: REN JIANTING Effective date: 20150320 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20150320 Address after: 100085 No. 1, building 5, building ten, No. 8, 805 street, Beijing, Haidian District Applicant after: Kemei Borui Technology (Beijing) Co.,Ltd. Address before: 100085 splendid international ten, 5-805 street, Beijing, Haidian District Applicant before: Ren Jianting |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information |
Inventor after: Yang Xiaolan Inventor before: Ren Jianting |
|
| CB03 | Change of inventor or designer information | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20180521 Address after: 100096 Changping District 11, 3, unit 11, No. 30 building, No. 30, Yu Zhi Dong Road, Beijing. Patentee after: Beijing Botai Rui Kang Technology Co., Ltd. Address before: 100085, 5 floor 8, 1 Street, ten Street, Haidian District, Beijing. Patentee before: Kemei Borui Technology (Beijing) Co.,Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160309 Termination date: 20190624 |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| RR01 | Reinstatement of patent right |
Former decision: termination of patent right due to unpaid annual fee Former decision publication date: 20200623 |
|
| RR01 | Reinstatement of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160309 Termination date: 20210624 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |