CN107176979A - A kind of polypeptide and its application with antimicrobial antiphlogistic activity - Google Patents
A kind of polypeptide and its application with antimicrobial antiphlogistic activity Download PDFInfo
- Publication number
- CN107176979A CN107176979A CN201710445058.0A CN201710445058A CN107176979A CN 107176979 A CN107176979 A CN 107176979A CN 201710445058 A CN201710445058 A CN 201710445058A CN 107176979 A CN107176979 A CN 107176979A
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- Prior art keywords
- polypeptide
- lunasin
- application
- seq
- antimicrobial antiphlogistic
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention belongs to technical field of molecular biology, and in particular to a kind of polypeptide and its application with antimicrobial antiphlogistic activity.The nucleotide sequence of the polypeptide is as shown in SEQ ID NO.1, and amino acid sequence is as shown in SEQ ID NO.2.There is the polypeptide of antimicrobial antiphlogistic activity be improved based on polypeptide Lunasin amino acid structure for this, can in cell high efficient expression, it is easy to isolate and purify, it is active higher.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of to have the polypeptide of antimicrobial antiphlogistic activity and its answer
With.
Background technology
Antibacterial polypeptide is the small molecule beta-alexin 3 polypeptide of organism synthesis, with broad-spectrum bactericidal action, to gram-negative
Property bacterium and gram-positive bacteria have stronger killing action, to some fungies, mycoplasma, especially also have to drug tolerant bacteria
Good killing action, is the important member of animal inherent immunity system.General antibiotic is by blocking large biological molecule
Biosynthesis play a role, and antibacterial polypeptide can perforate on bacterial cell membrane and form ionic passage, cause thin
Bacterium membrane structure is destroyed, and is caused intracellular water-soluble substances largely to ooze out, is ultimately resulted in bacterial death.With conventional antibiotic
Widely use, many pathogens have produced drug resistance, therefore, active peptides just have a clear superiority in antibiosis.
In the prior art, scientific workers are from bacterium, fungi, higher plant, invertebrate, vertebrate
Find and separate to obtain the thousands of kinds of polypeptides with antibacterial activity into human body.Such as soybean biological active peptides Lunasin is
A natural polypeptides of identification is separated from soybean, molecular weight is about 5kDa, and 43 amino acid residues of total length transfect this gene
Mammalian cell, can block mitosis and cause chromosome breakage, ultimately result in Apoptosis, research shows, Lunasin
Function with inhibitor against colon carcinoma cells, breast cancer and prostate cancer, also with antiinflammatory action, has antioxidation to giant cell.
But naturally isolated active peptides Lunasin is present that content is low, the relatively low defect of activity, it is unfavorable for separation pure
Change, limit its application.
The content of the invention
A kind of polypeptide and its application with antimicrobial antiphlogistic activity that the present invention is provided, to existing polypeptide Lunasin structures
It is improved, the high efficient expression in cell, it is easy to isolate and purify, activity is higher.
First purpose of the present invention is to provide a kind of polypeptide with antimicrobial antiphlogistic activity, the nucleotides sequence of the polypeptide
Row are as shown in SEQ ID NO.1, and amino acid sequence is as shown in SEQ ID NO.2.
Second object of the present invention is to provide recombinant vector prepared by the polypeptide described in a kind of application claim 1, institute
It is being obtained in the nucleotide sequence insertion plasmid pET-32a (+) as described in SEQ ID NO.1 to state recombinant vector.
Third object of the present invention is to provide a kind of transformant of the recombinant vector comprising described in claim 2.
The polypeptide that fourth object of the present invention is to provide described in a kind of claim 1 is anti-in inflammation caused by suppression LPS
Application in answering.
The 5th purpose of the present invention is to provide a kind of the answering in anti-acne Propionibacterium of the polypeptide described in claim 1
With.
Compared with prior art, the present invention provide it is a kind of with antimicrobial antiphlogistic activity polypeptide and its application, with
Lower beneficial effect：
G-Lunasin is that existing polypeptide Lunasin amino acid structures are improved, and utilizes 32-37 GRDGRD's
Effect makes G-Lunasin enter in aim cell core, improves the joint efficiency with aim cell, compared with natural Lunasin, promotees
Enter high efficient expression, increase bioactivity.After GRDGRD add H amino acid, on the basis of polypeptide active is not influenceed, increase its with
The joint efficiency of affine Ni posts, is easy to isolate and purify.
Brief description of the drawings
Fig. 1 is the electrophoresis pattern of target gene fragment and plasmid fragments after gel is reclaimed；
Wherein, M swimming lanes are DNAmaker, and No. 1 swimming lane is purpose genetic fragment, and No. 2 swimming lanes are plasmid fragments；
Fig. 2 is that positive colony identifies collection of illustrative plates through Xba I and Pst I double digestions；
Wherein, M swimming lanes are DNAmaker, and No. 1 swimming lane is the empty plasmid after double digestion, and No. 2 swimming lanes are the weight after double digestion
Group plasmid；
Fig. 3 is SDS-PAGE electrophoresis detection results；
Wherein, M swimming lanes are DNAmaker, and No. 1 swimming lane is G-Lunasin Polypeptide expression products.
Embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings, but should not be construed as the limit of the present invention
System.Experimental method in following embodiments, is conventional method unless otherwise specified, material used, examination in following embodiments
Agent etc., unless otherwise specified, is commercially obtained.
A kind of polypeptide (referred to hereinafter as G-Lunasin) with antimicrobial antiphlogistic activity that the present invention is provided, the core of the polypeptide
Nucleotide sequence is as shown in SEQ ID NO.1, and amino acid sequence is as shown in SEQ ID NO.2, and the polypeptide is to active peptides
Lunasin amino acid structure is improved, and amino is added in the Lunasin amino acid sequences as shown in SEQ ID NO.3
Sour residue, increases original Lunasin activity and the expression efficiency in cell, reduces its difficulty isolated and purified.
Based on same inventive concept, present invention also offers a kind of G-Lunasin preparation method, including following step
Suddenly：
S1, builds recombinant vector
By one end addition Nde I of the nucleotide sequence as shown in SEQ ID NO.2 restriction enzyme site, other end addition
Xho I restriction enzyme site, obtains target gene, is inserted into pUC57 plasmids and preserves, obtains recombinant plasmid pUC57-G-
Lunasin。
With Nde I, Xho I double digestion recombinant plasmid pUC57-G-Lunasin, digestion system as shown in table 1, digestion condition
For 37 DEG C, 2h, gel reclaims the target gene fragment with G-Lunasin nucleotide sequences；
With Nde I, Xho I double digestion plasmid pET-32a (+), as shown in table 2, digestion condition is 37 DEG C to digestion system,
2h, gel reclaims the longer plasmid fragments of sequence；
Fig. 1 is the electrophoresis pattern of target gene fragment and plasmid fragments after gel is reclaimed, wherein, M swimming lanes are DNAmaker,
No. 1 swimming lane is purpose genetic fragment, and No. 2 swimming lanes are plasmid fragments, and band is clear, without miscellaneous band.
Target gene fragment and plasmid fragments are stayed overnight through the connection of T4 ligases, as shown in table 3, structure is obtained linked system
Recombinant vector pUC57-G-Lunasin-pET-32a.
Plasmid pET-32a (+) is widely used in the expression of fusion protein, polypeptide, and with histidine-tagged etc., is easy to
Downstream polypeptide protein is isolated and purified.
The recombinant plasmid pUC57-G-Lunasin of table 1 digestion system
The plasmid pET-32a (+) of table 2 digestion system
| Plasmid pET-32a (+) (30ng/ μ L) | 40μL |
| Nde I | 3μL |
| Xho I | 2μL |
| 10×buffer | 5μL |
| Total system | 50μL |
The linked system of table 3
| Reagent name | Usage amount |
| Target gene fragment (30ng) | 6μL |
| 10×T4DNA ligase Buffer | 2μL |
| Plasmid fragments (50ng) | 5μL |
| T4DNA ligase(5U/μL) | 1μL |
| ddH2O | 20μL |
S2, conversion prepares expression engineering bacteria
Recombinant vector pUC57-G-Lunasin-pET-32a is transformed into bacillus coli DH 5 alpha DE3 competent cells, choosing
Take positive transformant as expression engineering bacteria, method for transformation uses conventional transformation methods.The screening of positive transformant is also pressed
More solito is carried out.Picking positive colony is identified through Xba I and Pst I double digestions, as a result as shown in Fig. 2 result display weight
Group vector construction is correct.
S3, expression of polypeptides, purifying
S31, expression engineering bacteria is inoculated into LB (Amp resistances) culture medium, 37 DEG C, 220rpm concussion and cultivates are stayed overnight, and are obtained
To seed liquor, seed liquor is transferred in fresh LB (Amp resistances) culture medium by next day according to 2% volume ratio, continues to cultivate
To OD600=0.6, IPTG is then added, makes IPTG final concentration of 0.1mM, 30 DEG C, 220rpm concussion and cultivate 4h obtain polypeptide
Express bacterium solution；Expression of polypeptides bacterium solution 1mL, 5000rpm, 4 DEG C of centrifugation 5min are taken, thalline is collected；After IPTG induced expressions, expression production
Thing is through SDS-PAGE electrophoresis detections, and as a result as shown in figure 3, wherein, M swimming lanes are DNAmaker, No. 1 swimming lane is G-Lunasin tables
Up to product, visible clearly band near 6KDa.
S32, takes 1g thalline, add equivalent to thalline quality 5% Bingding Buffer solution As (20mM phosphate,
0.5M sodium chloride, 5mM imidazoles, pH7.4) and PMSF.PMSF is configured to 100mM storing liquid, PMSF work with absolute ethyl alcohol
Concentration is 1mM.Add Bingding Buffer solution As and PMSF and cell precipitation is resuspended, adding lysozyme, (working concentration is
0.3mg/mL), mix, 30min is placed on ice.400W sonicated cells on ice, ultrasonic 5s stops 5s, 40 times repeatedly.Add
10%TritonX-100, makes final concentration of 0.05%, 15min is placed in mixing on ice.12000rmp, 4 DEG C of centrifugation 20min, take
Supernatant, and with 0.22 μm of membrane filtration supernatant, obtain filtered fluid, save backup on ice.
S33, loads suitable chromatographic column, with the eluent equivalent to 10 times of volumes of filtered fluid by Ni Sepharose resins
Separation, through Bingding Buffer solution As (NaH containing 50mmol2PO4, 5mmol imidazoles, 500mmol NaCl, solvent is double steamings
Water, pH to 7.4), solution B (NaH containing 50mmol2PO4, 20mmol imidazoles, 500mmol NaCl, solvent is distilled water, and pH is extremely
7.4), solution C (contains 50mmolNaH2PO4, 60mmol imidazoles, 500mmol NaCl, solvent is distilled water, pH to 7.4), solution D
(NaH containing 50mmol2PO4, 100mmol imidazoles, 300mmol NaCl, solvent is distilled water, and 7.4) pH to eluting, and flow velocity is 12-
15mL/h collects the purity of G-Lunasin in eluent, identified eluent D up to 98.3%% respectively, and content reaches 0.05g/mL,
And the purity for the natural Lunasin that this method is extracted is 90.3%%, content is 0.03g/mL.
First, G-Lunasin toxicity detections
0,10 μM, 30 μM, 60 μM of G-Lunasin solution, stimulating expression of macrophage RAW264.7, dosing 24h are prepared respectively
MTT detects stimulating expression of macrophage activity afterwards, as a result respectively 100%, 104%, 110%, 98%, as a result show 0-60 μM of G-
Lunasin polypeptide solutions are non-toxic to cytomegalovirus.
2nd, influences of the G-Lunasin to NO concentration in cell culture medium
Detect that NO yield is 2.4 μM in NO cell culture substrates using sterilized water as negative control, after 24h.With 1 μ g/mL
LPS as positive controls, NO yield is 5.5 in detection NO cell culture substrates after stimulating expression of macrophage RAW264.7,24h
μM.Then added in the cell culture substrate that we stimulate to 4 LPS respectively 10 μM, 30 μM, 60 μM G-Lunasin solution,
After 100 μM of natural Lunasin solution, 24h detect cell culture substrate in NO difference concentration be 4.5 μM, 4.3 μM, 4.1 μM,
4.8μM.Illustrate that G-Lunasin solution can significantly reduce NO caused by LPS and raise phenomenon, with good biological activity,
Inflammatory reaction caused by LPS can effectively be suppressed, and G-Lunasin is more notable than natural Lunasin solution efficacies.
3rd, G-Lunasin antibacterial effects
The μ L intracutaneous injections of propionibacterium acnes bacteria suspension 50, which are given, in rat auricle center successfully builds rat Acne Model
Afterwards, Acne Model A groups do not give any treatment；The solution external curing of clindamycin 0.1% is given at B group skin damageds；At C group skin damageds
Give mass fraction 0.05%G-Lunasin solution external curings.Control group does not inject rat auricle center and gives acne propionic acid
Bacillus bacteria suspension.
Three groups of rat injection site skins start to thicken when 24h is injected, and gradually increase, and acne, mound occur in the 5th day
The acneform lesions such as rash, warts, color is red, and matter is hard；After B, C group are handled 2 weeks, abscess disappears substantially, and thickness is thinning, and color tends to just
Often.Each group Rat Right ear injection site skin histology is taken, HE dyeing finds that the left ear epiderm skin of rat of control group is relatively thin, can
See hair follicle, epidermis, corium have a common boundary clear；A group epidermal hyperkeratosis, hair follicle area expands, the infiltration of inflammatory cell diffusivity；B、C
Group follicular keratosis substantially mitigate, the visible a small amount of inflammatory cell of skin corium.Illustrate that G-Lunasin solution has good antibacterial effect
Really.B, C group rat do not observe toxicity.
, but those skilled in the art once know basic creation although preferred embodiments of the present invention have been described
Property concept, then can make other change and modification to these embodiments.So, appended claims are intended to be construed to include excellent
Select embodiment and fall into having altered and changing for the scope of the invention.
Obviously, those skilled in the art can carry out the essence of various changes and modification without departing from the present invention to the present invention
God and scope.So, if these modifications and variations of the present invention belong to the scope of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to comprising including these changes and modification.
Sequence table
<110>Henan Engineering College
<120>A kind of polypeptide and its application with antimicrobial antiphlogistic activity
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 141
<212> DNA
<213>Artificial sequence
<400> 1
tccaaatggc agcaccagca agacagctgc cgcaagcagc tccagggggt gaacctcacg 60
ccttgcgaga agcacatcat ggagaagatc caaggccgcg gcggccgcgg ccacgatgac 120
gatgatgatg atgacgacga c 141
<210> 2
<211> 47
<212> PRT
<213>Artificial sequence
<400> 2
Ser Lys Trp Gln His Gln Gln Asp Ser Cys Arg Lys Gln Leu Gln Gly
1 5 10 15
Val Asn Leu Thr Pro Cys Glu Lys His Ile Met Glu Lys Ile Gln Gly
20 25 30
Arg Asp Gly Arg Asp His Asp Asp Asp Asp Asp Asp Asp Asp Asp
35 40 45
<210> 3
<211> 43
<212> PRT
<213>Soybean
<400> 3
Ser Lys Trp Gln His Gln Gln Asp Ser Cys Arg Lys Gln Leu Gln Gly
1 5 10 15
Val Asn Leu Thr Pro Cys Glu Lys His Ile Met Glu Lys Ile Gln Gly
20 25 30
Arg Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp
35 40
Claims (5)
1. a kind of polypeptide with antimicrobial antiphlogistic activity, it is characterised in that the nucleotide sequence of the polypeptide such as SEQ ID NO.1
Shown, amino acid sequence is as shown in SEQ ID NO.2.
2. recombinant vector prepared by the polypeptide described in a kind of application claim 1, it is characterised in that the recombinant vector is by such as
Obtained in nucleotide sequence insertion plasmid pET-32a (+) described in SEQ ID NO.1.
3. a kind of transformant of the recombinant vector comprising described in claim 2.
4. application of the polypeptide in inflammatory reaction caused by LPS is suppressed described in a kind of claim 1.
5. application of the polypeptide in anti-acne Propionibacterium described in a kind of claim 1.
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| CN201710445058.0A CN107176979B (en) | 2017-06-13 | 2017-06-13 | Polypeptide with antibacterial and anti-inflammatory activity and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710445058.0A CN107176979B (en) | 2017-06-13 | 2017-06-13 | Polypeptide with antibacterial and anti-inflammatory activity and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN107176979A true CN107176979A (en) | 2017-09-19 |
| CN107176979B CN107176979B (en) | 2020-09-22 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102793931A (en) * | 2012-08-09 | 2012-11-28 | 上海交通大学 | CT contrast media for specific targeting tumor neovascularization and preparation method thereof |
| CN103936838A (en) * | 2014-04-10 | 2014-07-23 | 武汉启瑞科技发展有限公司 | Micro-molecule polypeptide TAT-p53DM and application thereof to preparing medicine for treating or preventing ischemic stroke |
| CN105418735A (en) * | 2015-12-05 | 2016-03-23 | 李斯文 | Angiogenesis inhibitor polypeptide HS-1 and application thereof |
-
2017
- 2017-06-13 CN CN201710445058.0A patent/CN107176979B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102793931A (en) * | 2012-08-09 | 2012-11-28 | 上海交通大学 | CT contrast media for specific targeting tumor neovascularization and preparation method thereof |
| CN103936838A (en) * | 2014-04-10 | 2014-07-23 | 武汉启瑞科技发展有限公司 | Micro-molecule polypeptide TAT-p53DM and application thereof to preparing medicine for treating or preventing ischemic stroke |
| CN105418735A (en) * | 2015-12-05 | 2016-03-23 | 李斯文 | Angiogenesis inhibitor polypeptide HS-1 and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| J. LIU等: ""Lunasin as a promising health-beneficial peptide"", 《EUROPEAN REVIEW FOR MEDICAL AND PHARMACOLOGICAL SCIENCES》 * |
| LAILA SHIRBEIGI ET AL.,: ""A Review of Acne Etiology and Treatment in Iranian Traditional"", 《J SKIN STEM CELL》 * |
| 陈琛: ""抗肿瘤肽露那辛研究进展"", 《中国生化药物杂志》 * |
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| CN107176979B (en) | 2020-09-22 |
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