CN104017086A - Fusion antimicrobial peptide and preparation method thereof - Google Patents
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Abstract
The invention relates to a fusion antimicrobial peptide and a preparation method thereof. The fusion antimicrobial peptide is constructed by connecting porcine antimicrobial peptide (PAMP37) and bovine antimicrobial peptide (Indolicidin) in series. The DNA sequence of the fusion antimicrobial peptide is designed by utilizing the preference of an escherichia coli codon, and is constructed into an expression carrier, then the expression and purification of the fusion antimicrobial peptide are conducted, and the antimicrobial activity results show that the antimicrobial activity of the obtained fusion antimicrobial peptide is greatly improved. The fusion antimicrobial peptide provides an excellent candidate peptide for developing a novel antimicrobial immune preparation, and has important significance on effectively substituting or reducing the use of antibiotics as a feed additive, and improving the safety of livestock products.
Description
Technical field
The invention belongs to the technical field that in biological-pharmacy, genetically engineered is produced medicine, be specially a kind of fusion antibacterial peptide and preparation method thereof.
Background technology
Medium-term and long-term or the high dosage of diet adds microbiotic and causes microbiotic residual in livestock product, causes food safety hidden danger, and generation that also can inducible resistance bacterium, makes enteric flora disturbance in animal body, and animal autoimmune function declines, resistibility reduction etc.
Antibacterial peptide (Antibacterial Peptides), be again antimicrobial peptide (Antimicrobial Peptides) or host defense peptide (Host Defense Peptides), being the important composition composition of body congenital immunity, is the first barrier that many organisms are resisted external pathogenic bacterium invasion and attack.The extensive concern and the research that have caused scientific circles due to its unique and various biological function and mechanism of action, study on the one hand the Basic of Biology that antibacterial peptide plays a role in congenital immunity, on the one hand the molecular template using it as design novel antibacterial, anti-infectious preparation.Antibacterial peptide is the product of natural evolution, there have been 1 years together with microorganism, although through long-term common evolution, but antibacterial peptide is not lost its ability of killing or suppressing microorganism, or producing the ability that makes microorganism escape antibacterial peptide, antibacterial peptide is the important breakthrough of the new and effective Substitutes For Antibiotic of research and development thus.
Spiral antibacterial peptide is the antibacterial peptide the most widely that distributes, and probably accounts for 27% of all known secondary structure antibacterial peptides.When forming αhelix, common wetting ability residue and hydrophobic residue lay respectively at the both sides of spiral, therefore present the amphiphilic structure of Structure Dependence.Spiral antibacterial peptide does not generally have obvious constitutional features in water, but forms typical αhelix being rich under the cytolemma environment of lipid." cap " structure at C end and N end makes α spiral antibacterial peptide more stable, and is not subject to the impact of electrolyte concentration.This type of antibacterial peptide mainly, by destroying bacterial cell membrane performance germicidal action, causes the degraded step by step of film By Electrolysis matter or other semiochemicalses etc.
Antibacterial peptide market is widely used: in agricultural, can be used for fodder additives, and be applicable to the raising of various animals; Pharmaceutically, can be used as outstanding antibiotic substitute, be also considered to overcome in the future the medicine that cancer, acquired immune deficiency syndrome (AIDS) get a good chance of; Industrial, can be used as green sanitas; Human lives is upper, can be used as the substitute of healthcare products and antibiont.And, constantly perfect along with biological engineering method, the improvement of the extraction process of antibacterial peptide, further developing of transgenic technology, use engineering bacterium or yeast to carry out a large amount of productions of antibacterial peptide, the practical ranges of antibacterial peptide will further expand, and bring into play very important effect in daily life.
Yet, existing research shows, many natural antibacterial peptide activity are not high, or eukaryotic cell is existed to higher cytotoxicity, therefore before as the development of new antibiotic substitute, need further to improve its activity by molecular improvement, or reduce its toxicity.In addition, bacterium infects the appearance that has become major reason, especially a Resistant strain affecting livestock industry production, has increased especially the treatment difficulty of disease.Therefore, in the research of current antibacterial peptide, in the urgent need to transforming, have antibacterial peptide and design novel antimicrobial peptide molecule in order to create high vigor antibacterial peptide.
The antibacterial peptide by the antibacterial peptide of pig and ox neutrophil leucocyte of the invention merges.The PAMP37 of pig is the antimicrobial polypeptide that contains 37 amino-acid residues,, containing halfcystine, its N end regions does not have strong basicity, can form αhelix, utilize this structure can on bacterial cell membrane, form perforation, therefore intestinal bacteria and streptococcus aureus are had to remarkable lethal effect.Indolicidin is the antimicrobial polypeptide that derives from ox neutrophil leucocyte, and intestinal bacteria and streptococcus aureus are all had to very strong fungicidal activity.Its aminoacid sequence is to comprise 13 amino acid whose polypeptide that contain 5 tryptophanes, and its C end is amidated.The demonstration of bacteriostatic activity experimental result, the more independent antibacterial peptide anti-microbial activity of antibacterial peptide of fusion significantly improves.
This research further obtains the fusion antibacterial peptide of high anti-microbial activity by molecular improvement, for the development of novel antibacterial immunological reagent provides good candidate's peptide, this is to research and development novel antibacterial immunological reagent, effectively substitute or reduce microbiotic as the use of fodder additives, to improve livestock product security significant.
Summary of the invention
The fusion antibacterial peptide that the object of this invention is to provide a kind of high anti-microbial activity.Described fusion antibacterial peptide comprises pig antibacterial peptide and ox antibacterial peptide.
Further, described fusion antibacterial peptide comprises pig antibacterial peptide PAMP37 and ox antibacterial peptide (Indolicidin).
Further, described fusion antibacterial peptide obtains pig antibacterial peptide (PAMP37) and ox antibacterial peptide (Indolicidin) series connection together.
A nearlyer step, described fusion antibacterial peptide aminoacid sequence is SEQ ID1 in sequence table.
The object of this invention is to provide a kind of recombinant DNA that merges antibacterial peptide, described recombinant DNA is codon optimization, be adapted at escherichia coli expression merges antibacterial peptide.
Further, the recombinant DNA sequence of described fusion antibacterial peptide is SEQ ID2 in sequence table.
The object of this invention is to provide a kind of carrier that can express above-mentioned fusion antibacterial peptide.Preferably use the above-mentioned fusion antibacterial peptide of pET30a vector expression.
The object of this invention is to provide a kind of clone that can express above-mentioned fusion antibacterial peptide.Preferably use e. coli bl21 to express above-mentioned fusion antibacterial peptide.
The object of this invention is to provide a kind of novel antibacterial immunological reagent, in described antibacterial immunity preparation, contain above-mentioned fusion antibacterial peptide.
The preparation method who the object of this invention is to provide a kind of fusion antibacterial peptide of high anti-microbial activity.
Further, the preparation method of described fusion antibacterial peptide comprises:
5) by the polypeptide series connection that comprises pig antibacterial peptide and ox antibacterial peptide, the amino acid order of the fusion antibacterial peptide obtaining;
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 merged to the expression of antibacterial peptide.
Further, described pig antibacterial peptide is PAMP37, and described ox antibacterial peptide is Indolicidin.
Further, the fusion antibacterial peptide of design can obtain by methods such as synthetic or multiplex PCRs.Preferably carry out sequence that multiple PCR primer adopts and be selected from one or several in SEQ ID4 in sequence table, SEQ ID5, SEQ ID6, SEQ ID7.
Further, recombinant DNA being cut to the modes such as link or homologous recombination by enzyme is connected on expression vector.
Further, the fusion antibacterial peptide of expressing is carried out to purifying.
The object of this invention is to provide and merge antibacterial peptide, can express and merge the carrier of antibacterial peptide and contain the application of antibacterial immunity preparation in preparing fodder additives, antibacterial medicine preparation, healthcare products, sanitas of merging antibacterial peptide.
Advantage of the present invention:
1) the invention carries out polypeptide series connection by the antibacterial peptide of pig antibacterial peptide PAMP37 and ox (Indolicidin), obtained the fusion antibacterial peptide with high anti-microbial activity, the development that described fusion antibacterial peptide is novel antibacterial immunological reagent provides good candidate's peptide, this is to research and development novel antibacterial immunological reagent, effectively substitute or reduce microbiotic as the use of fodder additives, to improve livestock product security significant;
2) the invention provides a kind of preparation method of fusion antibacterial peptide of high anti-microbial 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, further set forth the present invention, only for explaining the present invention, and can not be interpreted as limitation of the present invention.Those having ordinary skill in the art will appreciate that: in the situation that not departing from principle of the present invention and aim, can carry out multiple variation, modification, replacement and modification to these embodiment, scope of the present invention is limited by claim and equivalent thereof.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition or the condition examinations of advising according to manufacturer.
Structure design and the prediction of embodiment 1 antibacterial peptide
In order to strengthen the fungicidal activity of antibacterial peptide, antibacterial peptide (Indolicidin) series connection of pig antibacterial peptide (PAMP37) and ox is built together, and the aminoacid sequence of the fusion polypeptide obtaining is SEQ ID1:GLLSRLRDFLSDRGRRLGEKIERIGQKIKDLSEFFQSILPWKWPWWPWWRR.
Utilize the preferences of e. coli codon, designed the recombinant DNA sequence (SEQ ID2) of this sequence, through secondary structure prediction, obtaining this polypeptide N end is a α spiral, and C end is linear structure.
The structure of embodiment 2 peptide expression carriers
1) optimize DNA sequence dna and make it be adapted to colibacillary codon-bias, increase vector construction restriction enzyme site used, design fusion polypeptide DNA sequence dna (SEQ ID3) is as follows:
GACGACGACGACAAGGCCGGTCTGCTGTCTCGTCTGCGTGACTTCCTGTCTGACCGTGGTCGTCGTCTGGGTGAAAAAATCGAACGTATCGGTCAGAAAATCAAAGACCTGTCTGAATTCTTCCAGTCTATCCTGCCGTGGAAATGGCCGTGGTGGCCGTGGTGGCGTCGTTAA
The sequence of black underscore is restriction enzyme site (KpnI and XhoI).
DNA sequence dna is synthesized respectively to many primers, utilize the method for overlapping PCR, many primer amplifications are spliced into complete DNA sequence dna.Synthetic primer sequence is in Table 1:
The concrete sequence of table 1. synthetic primer
| Primer numbering | Sequence |
| P1-SEQ?ID4 | 5’-agggtaccgacgacgacgacaaggccggtctgctgtctcgtctgcgtgacttcctgtctg-3’ |
| P2-SEQ?ID5 | 5’-gaccgatacgttcgattttttcacccagacgacgaccacggtcagacaggaagtcac-3’ |
| P3-SEQ?ID6 | 5’-atcgaacgtatcggtcagaaaatcaaagacctgtctgaattcttccagtctatcctg-3’ |
| P4-SEQ?ID7 | 5’-gtctcgagttaacgacgccaccacggccaccacggccatttccacggcaggatagactgg-3’ |
2) overlapping pcr amplification target DNA sequence
The amplification that utilizes respectively P1 and P2 and P3 and P4 to carry out fusion rotein partial sequence (FP1 and FP2) is synthesized.
Reaction system (every pipe 50 microlitres of cumulative volume) is 2 * Pfu polymeric enzyme reaction liquid: 25 microlitres, primer 1:1 microlitre; Primer 2: 1 microlitre; Deionized water: 23 microlitres.Amplification condition is: 95 ℃ of denaturations 5 minutes; 95 ℃ of sex change 30 seconds, 65 ℃ of annealing 30 seconds, 72 ℃ are extended 30 seconds, totally 40 circulations.
Purifying reclaims amplified production separately, carries out the amplification of second step.
Reaction system (every pipe 50 microlitres of cumulative volume) is 2 * Pfu polymeric enzyme reaction liquid: 25 microlitres, and FP1 reclaims product: 1 microlitre; FP2 reclaims product: 1 microlitre; Primer P1:1 microlitre; Primer P4:1 microlitre; Deionized water: 21 microlitres.Amplification condition is: 95 ℃ of denaturations 5 minutes; 95 ℃ of sex change 30 seconds, 65 ℃ of annealing 30 seconds, 72 ℃ are extended 30 seconds, totally 40 circulations.Agargel electrophoresis is identified PCR product, and cuts glue and reclaim 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, reclaimed enzyme and cut after product, connect structure and obtain pET30a-P37-CIN expression vector, process as shown in Figure 1.By after the positive colony enlarged culturing that contains expression vector, carry out sequencing analysis, the sequence of sequencing result and design is in full accord, shows vector construction success.
The abduction delivering of embodiment 3 antibacterial peptides
1) transform according to a conventional method above-mentioned expression vector and enter e. coli bl21, the bacterium after transforming is coated to LB flat board, be placed in 37 ℃ of incubator overnight incubation.Can be observed obvious positive colony grows on flat board.
2) single bacterium colony of the picking positive LB liquid culture to OD600 be 0.4-0.6, then add the IPTG of 0.5mM, in 37 ℃ of 250rpm spend the night induction target protein expression, do a contrast that does not add IPTG simultaneously.
3) collect thalline, utilize 10000g within centrifugal 15 minutes, to discard supernatant liquor.Ultrasonication thalline in 1MTris-HCl solution, 20000g, centrifugal 30 minutes, supernatant liquor is carried out to SDS-PAGE analysis, polyacrylamide gel is carried out to visible significantly target protein soluble-expression band after coomassie brilliant blue staining.
The purifying of embodiment 4 antibacterial peptides
1) expression and purification of fusion rotein
5 liters of culturing engineering bacterium, the centrifugal collection thalline of 10000g.Adopt the broken thalline of ultrasonic method method, thalline after centrifugal breaking, collects supernatant liquor.Directly load in His-Tag affinity column, use 50mM/L imidazoles wash-out foreign protein to 1-2 times of column volume, then the fusion rotein of expressing with 100mM/L imidazoles wash-out, collect and concentrated fusion rotein.
2) enzymolysis of fusion rotein and the purifying of antibacterial peptide
The fusion rotein of above-mentioned acquisition is carried out to the endonuclease reaction of enteropeptidase, actual conditions is 23 ℃ hatches 12 hours, and every milligram of fusion rotein adds the enteropeptidase of 2-3 unit to carry out endonuclease reaction.Utilize molecular weight cut-off for dialysis tubing more than 10Kd carries out albumen dialysis, the antibacterial peptide in the dialyzate obtaining is concentrated again, obtain the antimicrobial peptide protein of the design after purifying.
The activity of embodiment 5 antibacterial peptides detects
Adopt the minimal inhibitory concentration (MIC) of plate dilution assay method antibacterial peptide, the OD value of measuring bacterial concentration shows, designed fusion antibacterial peptide concentrated solution all has In Vitro Bacteriostatic to intestinal bacteria and streptococcus aureus.Specifically see the following form.
The bacteriostatic activity of table 2. antibacterial peptide
| Antibacterial peptide | Intestinal bacteria MIC (μ g/L) | Streptococcus aureus MIC (μ g/L) |
| Merge antibacterial peptide | 2-8 | 0.5-2 |
| PAMP37 | 8-32 | 0.5-2 |
| indolicidin | 4-16 | 1-4 |
Result shows, the pig antibacterial peptide (PAMP37) that the anti-microbial activity of the fusion antibacterial peptide of the present invention's design is more independent and the antibacterial peptide (Indolicidin) of ox all increase significantly.For the development of novel antibacterial immunological reagent provides good candidate's peptide, this is to research and development novel antibacterial immunological reagent, effectively substitute or reduce microbiotic as the use of fodder additives, to improve livestock product security significant.
Claims (10)
1. merge an antibacterial peptide, comprise pig antibacterial peptide and ox antibacterial peptide.
2. fusion antibacterial peptide according to claim 1, is characterized in that, described pig antibacterial peptide is PAMP37, and described ox antibacterial peptide is Indolicidin.
3. according to the fusion antibacterial peptide described in claim 1 or 2 any one, it is characterized in that, described fusion antibacterial peptide is obtained together with the series connection of ox antibacterial peptide by pig antibacterial peptide.
4. according to the fusion antibacterial peptide described in claim 1-3 any one, it is characterized in that, described fusion antibacterial peptide aminoacid sequence is SEQ ID1 in sequence table.
5. a recombinant DNA that merges antibacterial peptide, is characterized in that, obtains the fusion antibacterial peptide described in claim 1-4 any one after described recombinant dna expression.
6. recombinant DNA according to claim 5, is characterized in that, described recombinant DNA sequence is SEQ ID2 in sequence table.
7. a carrier, is characterized in that, described carrier contains recombinant DNA described in claim 5-6 any one or expresses the fusion antibacterial peptide described in claim 1-4 any one.
8. an antibacterial immunity preparation, contains the fusion antibacterial peptide described in claim 1-4 any one in described antibacterial immunity preparation.
9. merge a preparation method for antibacterial peptide, described preparation method comprises:
1) by the polypeptide series connection that comprises pig antibacterial peptide and ox antibacterial peptide, the amino acid order of the fusion antibacterial peptide obtaining;
2), according to the preferences of codon, the recombinant DNA sequence of antibacterial peptide is merged in design;
3) recombinant DNA sequence is building up on expression vector;
4) expression vector transfered cell is merged to the expression of antibacterial peptide.
10. the fusion antibacterial peptide described in claim 1-4 any one, carrier claimed in claim 7 and the application of antibacterial immunity preparation claimed in claim 8 in preparing fodder additives, antibacterial medicine preparation, healthcare products, sanitas.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106432513A (en) * | 2016-11-29 | 2017-02-22 | 东北农业大学 | Efficient hybrid antibacterial peptide LI and preparation method and application thereof |
| CN114196631A (en) * | 2020-09-02 | 2022-03-18 | 江苏申琅生物科技有限公司 | Antibacterial functional polypeptide and preparation method and application thereof |
| CN115947815A (en) * | 2022-10-10 | 2023-04-11 | 天津科技大学 | Recombinant antibacterial peptide PMAP-37 and preparation method and application thereof |
| CN115960197A (en) * | 2022-11-14 | 2023-04-14 | 北京大北农科技集团股份有限公司 | Pig-derived antibacterial peptide PAMP-37-BT3 and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1896107A (en) * | 2006-06-13 | 2007-01-17 | 南京农业大学 | Cecropin A-magainin hybrid gene engineering bactericidal peptide |
| CN101063101A (en) * | 2006-12-28 | 2007-10-31 | 中国科学院亚热带农业生态研究所 | Cattle lactoferrin engineering bacterium and preparation method for antibiotic peptide cattle lactoferrin |
| 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 |
| CN101717737A (en) * | 2009-12-11 | 2010-06-02 | 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 |
| 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 |
| CN103435701A (en) * | 2013-07-24 | 2013-12-11 | 山西农业大学 | Pig antibacterial peptide cystatin11 fusion protein, and encoding gene and application thereof |
-
2014
- 2014-06-24 CN CN201410284681.9A patent/CN104017086B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1896107A (en) * | 2006-06-13 | 2007-01-17 | 南京农业大学 | Cecropin A-magainin hybrid gene engineering bactericidal peptide |
| CN101063101A (en) * | 2006-12-28 | 2007-10-31 | 中国科学院亚热带农业生态研究所 | Cattle lactoferrin engineering bacterium and preparation method for antibiotic peptide cattle lactoferrin |
| 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 |
| CN101717737A (en) * | 2009-12-11 | 2010-06-02 | 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 |
| 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 |
| CN103435701A (en) * | 2013-07-24 | 2013-12-11 | 山西农业大学 | Pig antibacterial peptide cystatin11 fusion protein, and encoding gene and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| SELSTED ME等: "indolicidin=antimicrobial peptide [cattle, neutrophils[Bos taurus]", 《GENBANK:AAB21494.1》 * |
| TOSSI A等: "RecName: Full=Antibacterial peptide PMAP-37;antibacterial peptide 37; Flags: Precursor", 《UNIPROTKB/SWISS-PROT》 * |
| 黎观红等: "抗菌肽的抗菌作用及其机制", 《动物营养学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106432513A (en) * | 2016-11-29 | 2017-02-22 | 东北农业大学 | Efficient hybrid antibacterial peptide LI and preparation method and application thereof |
| CN114196631A (en) * | 2020-09-02 | 2022-03-18 | 江苏申琅生物科技有限公司 | Antibacterial functional polypeptide and preparation method and application thereof |
| CN115947815A (en) * | 2022-10-10 | 2023-04-11 | 天津科技大学 | Recombinant antibacterial peptide PMAP-37 and preparation method and application thereof |
| CN115960197A (en) * | 2022-11-14 | 2023-04-14 | 北京大北农科技集团股份有限公司 | Pig-derived antibacterial peptide PAMP-37-BT3 and application thereof |
| CN115960197B (en) * | 2022-11-14 | 2023-06-30 | 北京大北农科技集团股份有限公司 | Pig-derived antibacterial peptide PAMP-37-BT3 and application thereof |
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