CN102260325A - Antibacterial peptide NX-16, and preparation method and application thereof - Google Patents
Antibacterial peptide NX-16, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an antibacterial peptide NX-16, and a preparation method and application thereof. The preparation method comprises the steps as follows: the amino acid sequence of the antibacterial peptide NX-16 is designed; the prokaryotic expression tandem gene of the antibacterial peptide NX-16 is designed according to the preference of Escherichia coli codon; a target gene is synthesized by a splicing by overlap extension PCR (polymerase chain reaction) method; an expression vector is built by an enzyme-linked method, the expression vector is transformed in Escherichia coli for expression, and the expression product is subjected to enzymolysis, thus obtaining the antibacterial peptide NX-16. In the preparation method provided by the invention, the gene of the antibacterial peptide is modified and is then subjected to efficient tandem expression in Escherichia coli (the expression product is a polymerized peptide of multiple molecules of the antibacterial peptide, does not have antibacterial activity and does not have killing or inhibiting effects on Escherichia coli), and then the antibacterial peptide molecule with antibacterial activity is obtained by an enzymolysis method; the obtained antibacterial peptide has broad application prospects in the pharmaceutical industry and animal husbandry; and the preparation method provided by the invention has the advantages of high expression efficiency, simple separation and purification, easy operation and good stability.
Description
Technical field
The present invention relates to a kind of antibacterial peptide, be specifically related to a kind of antibacterial peptide NX-16 and preparation method thereof and application.
Background technology
Antibacterial peptide is the micromolecule polypeptide of a kind of biologically active through inducing generation in the organism, contains 15-45 amino-acid residue usually.This class active polypeptide majority has characteristics such as strong basicity, thermostability and broad-spectrum antimicrobial.First antibacterial peptide is found from the sky silkworm chrysalis in 1980 by people such as Sweden scientist G. Boman in the world.Then, people find from bacterium, fungi, batrachians, insect, higher plant, Mammals and even the mankind in succession and separate the polypeptide that obtains to have anti-microbial activity.At first, it is found that this class active polypeptide has the broad-spectrum high efficacy fungicidal activity to bacterium.Along with carrying out in a deep going way of people's research work, find that some antibacterium peptide all has strong lethal effect to part fungi, protozoon, virus, tumour cell or cancer cells etc., some antibacterial peptide also has immunoloregulation function.
Antibiotic abuse has not only caused public health problems such as drug residue, and because of the appearance of Resistant strain, makes the control of bacteriosis become the human difficult problem of puzzlement once more.Simultaneously national relevant laws regulation, multiple microbiotic is disabled or limit the use of.Because the anti-microbial activity efficiently of the wide spectrum of antibacterial peptide and unique antibacterial mechanisms thereof and in the novel antibacterial drug research, manifested clear superiority.Particularly,, can design more effective antibacterial peptide in medicine industry and livestock industry in conjunction with the modern genetic engineering technology to deepening constantly that the antibacterial peptide gene expression and regulation mechanism is familiar with.Therefore, to can be used as that antibiotic favorable substitutes used clinically widely will be trend of the times to antibacterial peptide.
At present, many antibacterial peptides are being developed to medicine, but the natural output of antibacterial peptide is very low, and synthetic peptide price is quite expensive again, and these become a bottleneck of the antibiotic peptide medicament of exploitation.The gene engineering expression technology can efficiently express biological activity protein, and simple to operate, cost is low.Therefore, producing antibacterial peptide by genetic engineering technique has broad application prospects.By genetic engineering antibiotic peptides reasonably being transformed and being suddenlyd change, further increase its antibacterial and fungicidal activity, the relation of studying its mechanism of action and 26S Proteasome Structure and Function has important directive significance to the production practice of livestock industry and microbiotic alternative medicine.
At present, many investigators focus mostly on the antibacterial peptide gene expression method in eukaryotic expression system.Once had and report that the employing escherichia expression system goes out antibacterial peptide with the formal representation of fusion rotein, but the result not very good.Simultaneously, have the investigator to propose such problem, promptly the antibacterial peptide that goes out of escherichia coli expression has certain killing action to intestinal bacteria itself, can have influence on the expression of antibacterial peptide.
Summary of the invention
The technical problem to be solved in the present invention has provided a kind of antibacterial peptide NX-16 with anti-microbial activity, made up the recombinant expression vector that contains goal gene, and the antibacterial peptide gene that adopts the prokaryotic expression system express recombinant disclosed, obtain the preparation method of antibacterial peptide NX-16 again by enzymolysis process, this method expression efficiency height, separation and purification is simple, and is easy to operate, good stability.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
A kind of antibacterial peptide NX-16, it is the polypeptide with aminoacid sequence shown in the SEQ ID NO:1 in the sequence table, or by the aminoacid sequence shown in the SEQ ID NO:1 change mutually through modification, L-type amino acid and the D-type amino acid of cyclisation, N-terminal and/or C-terminal, at least a processing in the sequence terminal deletion and the polypeptide of the function equivalent that obtains.
A kind of polynucleotide, its nucleotide sequence:
(a) polynucleotide of the polypeptide shown in the coding SEQ ID NO:1; Or be
(b) with polynucleotide (a) complementary polynucleotide.
A kind of carrier that contains above-mentioned Nucleotide.
A kind of genetically engineered host cell that contains above-mentioned carrier.
The preparation method of described antibacterial peptide NX-16 comprises the steps:
(1) aminoacid sequence shown in the SEQ ID NO:1 in the sequence table is repeated series connection by 3, and according to the preferences of e. coli codon, design the tandem gene of this series connection aminoacid sequence, add nucleic acid restriction endonuclease recognition site and protectiveness base respectively at two ends again, form goal gene to be expressed;
(2) at the above-mentioned purpose gene, utilize four complementary primers of SOEingPCR primer design method design, contain overlapping base fragment in four primers, the synthetic goal gene of utilization overlap amplification splicing PCR method;
(3) be connected behind goal gene and prokaryotic expression carrier pET30a (+) double digestion, transformed into escherichia coli BL21, and identify;
(4) carry out abduction delivering with IPTG, thalline is analyzed expression product through ultrasonication with SDS-PAGE;
(5) expression product is used the kallikrein enzymolysis after dialysis, renaturation, obtains antibacterial peptide NX-16.
Described target gene sequences is shown in SEQ ID NO:2.
The sequence of described four complementary primers is shown in SEQ ID NO:3～6.
A kind of can with described antibacterial peptide NX-16 specificity bonded antibody.
A kind of pharmaceutical composition contains the described antibacterial peptide NX-16 of claim 1 and the medically acceptable carrier of safe and effective amount.
The application of described antibacterial peptide NX-16 in the preparation sterilant.
The present invention has actively useful effect:
Using gene engineering technique of the present invention at the prokaryotic host cell expression in escherichia coli recombinant antibacterial peptide gene, expression product behind enzymolysis, obtain the design antibacterial peptide, NX-16 has higher anti-microbial activity through this antibacterial peptide of verification experimental verification.Expression efficiency height of the present invention, separation and purification is simple, and is easy to operate, good stability.Therefore, the present invention has important value to developing new and effective anti-infectives, has broad application prospects in pharmaceutical industry and livestock industry.
The present invention is by behind the modified antimicrobial peptide gene, (expression product is the pdef polypeptide of a plurality of molecules of antibacterial peptide to carry out efficient tandem expression in intestinal bacteria, do not have bacteriostatic activity, intestinal bacteria itself are not had yet kill or restraining effect), obtain to have the antibacterial peptide molecule of anti-microbial activity then by enzymolysis process.
Description of drawings
Fig. 1 is the expression vector establishment synoptic diagram;
Fig. 2 is expression vector double digestion qualification result figure;
Fig. 3 is that the thalline PCR of recombinant vectors identifies;
Fig. 4 is the SDS-PAGE figure as a result of abduction delivering;
The anti-microbial activity analytical results figure of Fig. 5 purified product.
Embodiment
Embodiment 1: the amino acid of antibacterial peptide and gene design
(1) manually designs antibacterial peptide NX-16, its aminoacid sequence is ILPWHYPFFPWRRPFR, and PFR is kallikrein (Kallikrein) restriction enzyme site, the synthetic full sequence, have anti-microbial activity through the synthetic peptide of agarose plate diffusion process proof, see embodiment 6.
(2) novel antimicrobial peptide sequence of design in (1) is repeated series connection by 3, according to the preferences of e. coli codon, the tandem gene of design series connection aminoacid sequence, add again at two ends respectively nucleic acid restriction endonuclease (
XhoI and
NcoI) recognition site and protectiveness base form goal gene to be expressed.
The series connection target gene sequences of design is: 5 '-G
GAATTCA
CCATGGATCCATTTCGTAT TCTGCCGTGGCATTACCCGTTCTTCCCGTGGAGAAGACCATTTCGTATTCTGCCGT GGCATTACCCGTTCTTCCCGTGGAGAAGACCATTTCGTATTCTGCCGTGGCATTAC CCGTTCTTCCCGTGGAGAAGACCATTTCGT
CTCGAGCGG-3 ', the total 178bp of total sequence, the front and back underscore is represented respectively
EcoRI,
NcoI and
XhoThe restriction enzyme site of I, wherein
NcoI is standby restriction enzyme site, represents atg start codon in the square frame.
Embodiment 2: the amplification of design of primers and goal gene
(1) utilize the method for SOEingPCR design primer that goal gene described in the embodiment 1 is designed to four complementary overlapping fragmentses, wherein contain 12 overlapping base fragments, overlapping fragments is respectively: G1 contains 57 bases, G2 and contains 56 bases, G3 and contain 55 bases, G4 and contain 54 bases.Wherein G1 and G2 contain 12 overlapping bases, and G2 and G3 contain 17 overlapping bases, and G3 and G4 contain 15 overlapping bases.Article four, the base sequence of overlapping fragments is respectively:
G1：G
GAATTC A
CCATGGATCCATTTCGTATTCTGCCGTGGCATTACCCGTTCTTCCCGTG
G2：GGAAGAACGGGTAATGCCACGGCAGAATACGAAATGGTCTTCTCCACGGGAAGAAC
G3：GCATTACCCGTTCTTCCCGTGGAGAAGACCATTTCGTATTCTGCCGTGGCATTAC
G4：CCG
CTCGAG ACGAAATGGTCTTCTCCACGGGAAGAACGGGATATGCCACGGCAG
(2) two superimposed sheets sections each other template each other primer carry out TD-PCR amplification.
Synthesize new gene fragment NG1 with overlapping fragments G1 and G2 reaction: reaction system (50 μ l) is 10 * PCR buffer, 5 μ l, dNTPs 1 μ l,
Pfu(2.5U/ μ l) 1 μ l, G1 1 μ l, G2 1 μ l, deionized water 42 μ l; Response procedures is 95 ℃ of pre-sex change 1min, 95 ℃ of 30sec then, and 70 ℃ of 30sec, 72 ℃ of 40sec carry out 5 circulations altogether, 95 ℃ of 30sec then, 54 ℃ of 30sec, 72 ℃ of 40sec carry out 25 circulations altogether, and 72 ℃ are extended 10min, 4 ℃ of insulations.
Synthesize new gene fragment NG2 with overlapping fragments G3 and G4 reaction: reaction system (50 μ l) is 10 * PCR buffer, 5 μ l, dNTPs 1 μ l,
Pfu(2.5U/ μ l) 1 μ l, G1 1 μ l, G2 1 μ l, deionized water 42 μ l; Response procedures is 95 ℃ of pre-sex change 1min, 95 ℃ of 30sec then, and 70 ℃ of 30sec, 72 ℃ of 40sec carry out 5 circulations altogether, 95 ℃ of 30sec then, 55 ℃ of 30sec, 72 ℃ of 40sec carry out 25 circulations altogether, and 72 ℃ are extended 10min, 4 ℃ of insulations.
Synthesize target gene fragment with NG1 and NG2 reaction: reaction system (50 μ l) is 10 * PCR buffer, 5 μ l, dNTPs 1 μ l,
Pfu(2.5U/ μ l) 1 μ l, G1 2 μ l, G2 2 μ l, deionized water 40 μ l; Response procedures is 95 ℃ of pre-sex change 1min, 95 ℃ of 40sec then, and 70 ℃ of 30sec, 72 ℃ of 50sec carry out 5 circulations altogether, 95 ℃ of 40sec then, 55 ℃ of 30sec, 72 ℃ of 60sec carry out 25 circulations altogether, and 72 ℃ are extended 10min, 4 ℃ of insulations.
(3) the PCR product is identified with 2% agargel electrophoresis, and cuts glue and reclaim.
Embodiment 3: the structure of expression vector and evaluation
The building mode of expression vector is referring to Fig. 1.
(1) extracts plasmid pET30a (+) with conventional plasmid extraction kit, carry out double digestion then.Enzyme is cut system (50 μ l): plasmid 30 μ l, and 10 * Buffer, 10 μ l,
Xho I 3 μ l,
Nco I 1 μ l, deionized water 6 μ l analyze with 1% agarose gel electrophoresis after 37 ℃ of enzymes are cut 2h.
(2) double digestion of goal gene.Enzyme is cut system (50 μ l): gained goal gene 30 μ l among the embodiment 2, and 10 * Buffer, 10 μ l,
Xho I 3 μ l,
NcoI 1 μ l, deionized water 6 μ l.After cutting 45min, 37 ℃ of enzymes analyze with 1% agarose gel electrophoresis.
(3) goal gene and carrier is connected.Reaction system (10 μ l): reclaim dna segment 4ml, enzyme is cut the carrier 1ml that reclaim the back, T4 dna ligase (350U/ μ l) 0.5ml, and ligase enzyme damping fluid 1ml, deionized water 3.5 ml, 16 ℃ connect 2h in thermostat water bath.
(4) according to ordinary method above-mentioned connection product is transformed into e. coli bl21, the bacterium after transforming is coated the LB flat board, cultivate 12～16h in 37 ℃.The mono-clonal that picking is suspicious, LB liquid culture 12～16h extracts plasmid with conventional plasmid extraction kit, carries out double digestion then and identifies that (the results are shown in Figure 2) and thalline PCR identify (the results are shown in Figure 3).Double digestion system (10 ml): recombinant plasmid 10 μ l, 10 * Buffer, 4 μ l,
Xho I 1 μ l,
Nco I 1 μ l, deionized water 4 μ l, 37 ℃ of enzymes that spend the night are cut, 1% agarose gel electrophoresis analysis.Thalline PCR: reaction system (50ml) is 10 * buffer, 5 μ l, dNTPs 1 μ l,
Pfu(2.5U/ μ l) 1 μ l, bacterium liquid 1 μ l, primer 11 μ l, primer 21 μ l, deionized water 41 μ l; Response procedures is 95 ℃ of pre-sex change 1min, 95 ℃ of 40sec then, and 67 ℃ of 30sec, 72 ℃ of 50sec, totally 35 circulations, 72 ℃ are extended 10min, 4 ℃ of insulations then; Primer is that its sequence is primer 1:5-TCCGAATTCACCATGGCT-3, primer 2: 5-GTGGTGGTGGTGCTCGAG-3 according to the design of the gene order of recombinant vectors.
(5) double digestion and PCR are identified send biotech firm to carry out sequencing analysis bacterium liquid after being male clone enlarged culturing.After sequential analysis,, carry out next step test operation with the sequence of design when in full accord.
Embodiment 4: abduction delivering and SDS-PAGE thereof analyze
(1) getting among the embodiment 3 through being accredited as the male bacterial strain is 0.5 in the LB liquid culture to OD600, the IPTG that adds different concns then spends the night in 37 ℃ of 120rpm and to induce, and does a contrast that does not add IPTG simultaneously, 4000rpm is centrifugal, and 15min discards supernatant liquor, the results thalline.
(2) according to a conventional method bacterium liquid is carried out SDS-PAGE and analyze, the results are shown in Figure 4.
Embodiment 5: dialysis renaturation and enzymolysis
(1) the gained thalline promotes solubilization of inclusion bodies with urea among the embodiment 4 after ultrasonication, and solution is dialysed concentrated with dialysis tubing, and dialyzate adds in the triethanolamine solution.
(2) add the kallikrein 200 μ l of 100mg/mL in mixed solution, in 37 ℃ of water-bath 1.0,2.0 and 3.0h respectively down, the solution after cutting with enzyme is done the detection of anti-microbial activity.
Embodiment 6: the agarose plate diffusion process detects the enzymolysis product activity
(1) gets respectively and tried Gram negative intestinal bacteria O111 of microorganism and Gram-positive streptococcus aureus NCTC4163 in right amount, be suspended in lower floor's agarose substratum (the 10 mL trypticase soybean broths about 50 ℃, the ultrapure agar of 10 g, 1 L distilled water, pH 7.4) on.
(2) with the punch tool punching, about 2 mm in aperture add 5 μ L specimen (solution after embodiment 5 gained enzymes are cut) in every hole; Positive control adopts general microbiotic: PXB is at gram-negative bacteria, and nisin is at gram positive organism; Negative control adopts 0.01% acetate.
(3) flat board is inverted 3 h in 37 ℃ of incubators, test fluid is diffused in the agarose.
(4) add one deck nutrient agar again.
(5) flat board places 37 ℃ of cultivations, spends the night.
(6) diameter of register hole periphery inhibition zone the results are shown in Table 1 and Fig. 5.This shows that enzymolysis product has anti-microbial activity, and the enzymolysis product activity of enzymolysis 2h is best.
The bacteriostatic activity of table 1 enzymolysis product is unit as a result: mm
| ? | 1 | 2 | 3 | 4 | 5 | 6 | 2F1 | Positive control | Negative control |
| |
4 | 3 | 9.5 | 11.8 | 9.3 | 0 | 10 | 19 | 0 |
| Streptococcus aureus | 0 | 0 | 2 | 8.2 | 5 | 0 | 7.2 | 12 | 0 |
The positive contrast of mark "+" among Fig. 5, "-" negative contrast.
Left side figure is to colibacillary bacteriostatic test, the 1st hole (culture dish lid mark " 1 " is located) is for expressing the centrifugal supernatant of back bacterium liquid, (culture dish lid mark " 2 " is located in the 2nd hole, be the purifying expression product of not enzymolysis down together), the 3rd hole is the product of expression product enzymolysis 1h, the 4th hole is the product of expression product enzymolysis 2h, the 5th hole is the product of expression product enzymolysis 3h, the 2F1 hole is that (patent publication No.: CN101607992), the 6th hole is the contrast of antibacterial peptide diluent to isolating antibacterial peptide from bovine blood.
Right figure is the bacteriostatic test to streptococcus aureus, (culture dish lid mark " 1 " is located in the 1st hole, down together) for expressing the centrifugal supernatant of back bacterium liquid, the 2nd hole is the purifying expression product of not enzymolysis, the 3rd hole is the product of expression product enzymolysis 1h, and the 4th hole is the product of expression product enzymolysis 2h, and the 5th hole is the product of expression product enzymolysis 3h, the 2F1 hole is that (patent publication No.: CN101607992A), the 6th hole is the contrast of antibacterial peptide diluent to isolating antibacterial peptide from bovine blood.
Sequence table
SEQUENCE?LISTING
＜110〉Henan Science and Technology College
＜120〉antibacterial peptide NX-16 and preparation method thereof and application
<130> /
<160> 6
<170> PatentIn?version?3.2
<210> 1
<211> 16
<212> PRT
＜213〉synthetic
<400> 1
Ile?Leu?Pro?Trp?His?Tyr?Pro?Phe?Phe?Pro?Trp?Arg?Arg?Pro?Phe?Arg
1 5 10 15
<210> 2
<211> 178
<212> DNA
＜213〉artificial design
<400> 2
ggaattcacc?atggatccat?ttcgtattct?gccgtggcat?tacccgttct?tcccgtggag 60
aagaccattt?cgtattctgc?cgtggcatta?cccgttcttc?ccgtggagaa?gaccatttcg 120
tattctgccg?tggcattacc?cgttcttccc?gtggagaaga?ccatttcgtc?tcgagcgg 178
<210> 3
<211> 57
<212> DNA
＜213〉artificial design
<400> 3
ggaattcacc?atggatccat?ttcgtattct?gccgtggcat?tacccgttct?tcccgtg 57
<210> 4
<211> 56
<212> DNA
＜213〉artificial design
<400> 4
ggaagaacgg?gtaatgccac?ggcagaatac?gaaatggtct?tctccacggg?aagaac 56
<210> 5
<211> 55
<212> DNA
＜213〉artificial design
<400> 5
gcattacccg?ttcttcccgt?ggagaagacc?atttcgtatt?ctgccgtggc?attac 55
<210> 6
<211> 54
<212> DNA
＜213〉artificial design
<400> 6
ccgctcgaga?cgaaatggtc?ttctccacgg?gaagaacggg?atatgccacg?gcag 54
Claims (10)
1. antibacterial peptide NX-16, it is characterized in that, it is the polypeptide with aminoacid sequence shown in the SEQ ID NO:1 in the sequence table, or by the aminoacid sequence shown in the SEQ ID NO:1 change mutually through modification, L-type amino acid and the D-type amino acid of cyclisation, N-terminal and/or C-terminal, at least a processing in the sequence terminal deletion and the polypeptide of the function equivalent that obtains.
2. polynucleotide is characterized in that, its nucleotide sequence:
(a) polynucleotide of the polypeptide shown in the coding SEQ ID NO:1; Or be
(b) with polynucleotide (a) complementary polynucleotide.
3. a carrier is characterized in that, contains the described polynucleotide of claim 2.
4. a genetically engineered host cell is characterized in that, contains the described carrier of claim 3.
5. the preparation method of the described antibacterial peptide NX-16 of claim 1 comprises the steps:
(1) aminoacid sequence shown in the SEQ ID NO:1 in the sequence table is repeated series connection by 3, and according to the preferences of e. coli codon, design the tandem gene of this series connection aminoacid sequence, add nucleic acid restriction endonuclease recognition site and protectiveness base respectively at two ends again, form goal gene to be expressed;
(2) at the above-mentioned purpose gene, utilize four complementary primers of SOEingPCR primer design method design, contain overlapping base fragment in four primers, the synthetic goal gene of utilization overlap amplification splicing PCR method;
(3) be connected behind goal gene and prokaryotic expression carrier pET30a (+) double digestion, transformed into escherichia coli BL21, and identify;
(4) carry out abduction delivering with IPTG, thalline is analyzed expression product through ultrasonication with SDS-PAGE;
(5) expression product is used the kallikrein enzymolysis after dialysis, renaturation, obtains antibacterial peptide NX-16.
6. according to the preparation method of the described antibacterial peptide NX-16 of claim 5, it is characterized in that described target gene sequences is shown in SEQ ID NO:2.
7. according to the preparation method of the described antibacterial peptide NX-16 of claim 5, it is characterized in that the sequence of described four complementary primers is shown in SEQ ID NO:3～6.
8. energy and the described antibacterial peptide NX-16 of claim 1 specificity bonded antibody.
9. a pharmaceutical composition is characterized in that, it contains the described antibacterial peptide NX-16 of claim 1 and the medically acceptable carrier of safe and effective amount.
10. the application of the described antibacterial peptide NX-16 of claim 1 in the preparation sterilant.
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| CN105175508A (en) * | 2015-10-19 | 2015-12-23 | 河南科技学院 | Antimicrobial peptide HJH-1 and application thereof |
| CN108314718A (en) * | 2018-04-19 | 2018-07-24 | 贵州医科大学 | A kind of house fly antibiotic peptide MAF-1A peptides aggressiveness, its encoding gene and its expression and application |
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| CN108314718A (en) * | 2018-04-19 | 2018-07-24 | 贵州医科大学 | A kind of house fly antibiotic peptide MAF-1A peptides aggressiveness, its encoding gene and its expression and application |
| CN108314718B (en) * | 2018-04-19 | 2021-12-17 | 贵州医科大学 | Housefly antibacterial peptide MAF-1A peptide polymer, encoding gene thereof, expression and application thereof |
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| Publication number | Publication date |
|---|---|
| CN102260325B (en) | 2013-05-29 |
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