CN105175524A - Carp recombined antimicrobial protein and preparing method thereof - Google Patents
Carp recombined antimicrobial protein and preparing method thereof Download PDFInfo
- Publication number
- CN105175524A CN105175524A CN201510662032.2A CN201510662032A CN105175524A CN 105175524 A CN105175524 A CN 105175524A CN 201510662032 A CN201510662032 A CN 201510662032A CN 105175524 A CN105175524 A CN 105175524A
- Authority
- CN
- China
- Prior art keywords
- carp
- protein
- restructuring
- seq idno
- carrier
- 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.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses carp recombined antimicrobial protein and a preparing method thereof. The carp recombined antimicrobial protein has an amino acid sequence as shown in the SEQ ID NO:1 and basic group sequential codes as shown in the SEQ ID NO:2. By the adoption of the antimicrobial protein, growth, reproduction and survival of microorganisms such as bacteria and fungi can be effectively restrained, and the processing and utilization range of fish resources is widened.
Description
Technical field
The present invention relates to biology field, be specifically related to a kind of carp restructuring Antagonistic protein and preparation method thereof.
Background technology
China waters is vast, and aquatic resources are very abundant, and especially fish stock is very abundant, has wherein contained very abundant genetic resources, has had high biological value.But the fishery cultivating of China and fish processing are all in the developmental stage of lower level for a long time, produce and abandoned a large amount of processing of aquatic products waste by-product such as such as fish-egg, liver pancreas, spleen, hypophysis, serum and skin etc., therefore the processing and utilization rate of fish stock is low, and economic benefit is poor.
Applicant conducts in-depth research the many-side application of piscine organism resource for many years, find that the part protein product in carp has significant bacteriostatic action, the growth of multiple-microorganism bacterium, breeding and survival can be suppressed, if realized batch preparation, to effectively promote the economic value of fishery by product, and provide necessary Research foundation for the scientific and reasonable utilization of fish by product, significant in environment, resource, science and economic dispatch many-side.
Summary of the invention
Applicant is in scientific research for many years, find the Antagonistic protein in carp body, and restructuring and the preparation of Antagonistic protein is achieved through further investigation, improve the processing and utilization rate of fish stock, avoid by product arbitrarily to abandon and the pressure that produces physical environment, improve the comprehensive benefit of fish processing.
Specifically, the present invention is achieved through the following technical solutions:
First, the invention discloses a kind of carp restructuring Antagonistic protein, there is the aminoacid sequence shown in sequence table SEQ IDNO:1, and encoded by the base sequence shown in sequence table SEQ IDNO:2.
Secondly, the invention discloses the recombination and preparation of above-mentioned Antagonistic protein, specifically comprise the steps:
1) with upstream primers F 1:5 '-TA
gGATCCaCTGGGATTCCTGGAGGCCTTG-3 ', downstream primer R1:5 '-GCC
cTCGAGcATGCAGGTGTTTTCAG-3 ', by the base sequence shown in pcr amplification sequence table SEQ IDNO:2;
2) pcr amplification gene fragment, is connected on pMD19-T carrier, proceeds to JM109 competent cell, extracts positive plasmid;
3) prepare carrier with BamHI, XhoI double digestion positive plasmid and PET-30, connection gained is prepared carrier and is proceeded to BL21 intestinal bacteria and the recombinant protein preparing soluble form under inductor IPTG effect.
In the above-mentioned methods, the base sequence of coding Antagonistic protein both can extract total serum IgE and obtain through RT-PCR process from Cyprinus Carpio, also can obtain the gene fragment with the base sequence shown in sequence table SEQ IDNO:2 by the mode of synthetic.
In the above-mentioned methods, it will be readily appreciated by those skilled in the art that and in order to improve fungistatic effect, purifying is carried out to it after obtaining recombinant protein, purifying process adopts common method biologically, such as target protein is through collected by centrifugation, carry out purifying by BufferA (containing 0.4-0.5mol/LNaCl, 0.5-1mol/L urea, 5% glycerine, 50mmol/LTris-HCl, pH7-9) dialysis, concentrated, affinity chromatography.
Finally, the invention also discloses above-mentioned albumen and suppress the application in microbial bacteria.Microbial bacteria alleged herein makes a general reference the microorganisms such as common bacterium, fungi, applicant confirms through research, Antagonistic protein of the present invention especially has desirable inhibition to Pseudomonas fluorescens, Pseudomonas aeruginosa, intestinal bacteria, and therefore application of the present invention is preferably applicable to above-mentioned three kinds of bacterium.
Technical scheme of the present invention, have successfully been obtained the Antagonistic protein that efficiently can suppress microorganism strains, improves economic worth and the comprehensive utilization ratio of fishery and fish processing.
Embodiment
In order to absolutely prove technical scheme of the present invention, applicant provide some specific implementations.The following embodiment provided is only schematic, does not form specific embodiment of the invention method and is particularly limited to.
Embodiment 1
Total serum IgE is extracted, by obtaining strand cDNA after RT-PCR, with 5 '-TA from Cyprinus Carpio
gGATCCaCTGGGATTCCTGGAGGCCTTG-3 ' is upstream primer, then with 5 '-GCC
cTCGAGcATGCAGGTGTTTTCAG-3 ' is downstream primer, obtains gene fragment through pcr amplification.Wherein PCR reaction conditions is set as 94 DEG C of denaturation 5min; 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C extend 1min, 35 circulations; 72 DEG C finally extend 5min.
Amplification gained gene fragment is connected on pMD19-T carrier, proceeds in JM109 competent cell, and carries out indigo plant screening in vain, and picking positive colony, bacterium liquid activates laggard performing PCR and detects.
Extract recombinant plasmid, with BamHI, XhoI double digestion, through 1.2% agarose gel electrophoresis, cut glue and reclaim object fragment, prepare carrier with the PET-30 of BamHI, XhoI double digestion and be connected under T4 effect, proceed to BL21 intestinal bacteria.And in inductor 1mmol/LIPTG, under the inductive condition of 30-35 DEG C, 2-3h, prepare the recombinant protein of soluble form.
Prepare bacterial sediment after ultrasonication, the target protein that centrifugation supernatant obtains, with BufferA (containing 0.4-0.5mol/LNaCl, 0.5-1mol/L urea, 5% glycerine, 50mmol/LTris – HCl, pH7-9) carry out dialysing and after concentrating, affinitive layer purification target protein, SDS-PAGE presents the single band of about 14kD, and display the present invention successfully achieves the preparation of target protein.
By disc diffusion method qualification target protein to Pseudomonas fluorescens, Pseudomonas aeruginosa, colibacillary fungistatic effect, it is 0.21-0.42mg/ sheet that restructuring Antagonistic protein adds weight range.Experimental result shows, and Pseudomonas fluorescens antibacterial circle diameter scope is 8-16mm; Pseudomonas aeruginosa antibacterial circle diameter scope is 8-35mm; Intestinal bacteria antibacterial circle diameter scope is 8-25mm.
Embodiment 2
Synthetic has the base sequence shown in sequence table SEQ IDNO:2, with 5 '-TA
gGATCCaCTGGGATTCCTGGAGGCCTTG-3 ' is upstream primer, with 5 '-GCC
cTCGAGcATGCAGGTGTTTTCAG-3 ' is downstream primer, by pcr amplification gene fragment.PCR parameter setting is: 94 DEG C of denaturation 5min; 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C extend 1min, 33 circulations; 72 DEG C finally extend 5min, obtain gene fragment.
Gained amplified production is connected on pMD19-T carrier, proceeds in JM109 competent cell, picking positive colony, extracts recombinant plasmid.
Prepare carrier with BamHI, XhoI double digestion recombinant plasmid with PET-30 and be connected, proceeding to BL21 intestinal bacteria, and in the IPTG of inductor 0.5mmol/L, under the inductive condition of 25-30 DEG C, 2-3h, prepare the recombinant protein of soluble form.
Prepare bacterial sediment after ultrasonication, the target protein that centrifugation supernatant obtains, through BufferA (containing 0.4-0.5mol/LNaCl, 0.5-1mol/L urea, 5% glycerine, 50mmol/LTris – HCl, pH7-9) dialyse, affinitive layer purification target protein, target protein is detected as the single band of about 14kD through SDS-PAGE.
By disc diffusion method qualification target protein to Pseudomonas fluorescens, Pseudomonas aeruginosa, colibacillary fungistatic effect, it is 0.21-0.42mg/ sheet that restructuring Antagonistic protein adds weight range.Pseudomonas fluorescens antibacterial circle diameter scope is 8-16mm; Pseudomonas aeruginosa antibacterial circle diameter scope is 8-35mm; Intestinal bacteria antibacterial circle diameter scope is 8-25mm.
Claims (4)
1. a carp restructuring Antagonistic protein, is characterized in that the aminoacid sequence had shown in sequence table SEQ IDNO:1, and is encoded by the base sequence shown in sequence table SEQ IDNO:2.
2. the carp restructuring Antagonistic protein preparation method of claim 1, is characterized in that comprising the steps: 1) with upstream primers F 1:5 '-TA
gGATCCaCTGGGATTCCTGGAGGCCTTG-3 ', downstream primer R1:5 '-GCC
cTCGAGcATGCAGGTGTTTTCAG-3 ', by the base sequence shown in pcr amplification sequence table SEQ IDNO:2; 2) pcr amplification gene fragment, is connected on pMD19-T carrier, proceeds to JM109 competent cell, extracts positive plasmid; 3) prepare carrier with BamHI, XhoI double digestion positive plasmid and PET-30, connection gained is prepared carrier and is proceeded to BL21 intestinal bacteria and the recombinant protein preparing soluble form under inductor IPTG effect.
3. the carp restructuring Antagonistic protein of claim 1 is suppressing the application in microbial bacteria.
4. application according to claim 3, is characterized in that described microbial bacteria comprises Pseudomonas fluorescens, Pseudomonas aeruginosa, intestinal bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510662032.2A CN105175524B (en) | 2015-10-14 | 2015-10-14 | A kind of carp recombination Antagonistic protein and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510662032.2A CN105175524B (en) | 2015-10-14 | 2015-10-14 | A kind of carp recombination Antagonistic protein and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105175524A true CN105175524A (en) | 2015-12-23 |
| CN105175524B CN105175524B (en) | 2019-02-26 |
Family
ID=54898029
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510662032.2A Expired - Fee Related CN105175524B (en) | 2015-10-14 | 2015-10-14 | A kind of carp recombination Antagonistic protein and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105175524B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899457A (en) * | 2010-05-21 | 2010-12-01 | 四川农业大学 | Recombinant carp GLS gene, recombinant carp GLS protein, and preparation methods, detection methods and application thereof |
| CN103060347A (en) * | 2013-02-06 | 2013-04-24 | 四川农业大学 | Recombinant carp PKC delta gene and recombinant carp PKC delta protein as well as preparation method, detection method and application thereof |
| CN103074348A (en) * | 2013-02-06 | 2013-05-01 | 四川农业大学 | Recombinant carp Nrf2 (NF-E2-related factor 2) gene, protein, preparation and detection methods and application of recombinant carp Nrf2 gene |
-
2015
- 2015-10-14 CN CN201510662032.2A patent/CN105175524B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899457A (en) * | 2010-05-21 | 2010-12-01 | 四川农业大学 | Recombinant carp GLS gene, recombinant carp GLS protein, and preparation methods, detection methods and application thereof |
| CN103060347A (en) * | 2013-02-06 | 2013-04-24 | 四川农业大学 | Recombinant carp PKC delta gene and recombinant carp PKC delta protein as well as preparation method, detection method and application thereof |
| CN103074348A (en) * | 2013-02-06 | 2013-05-01 | 四川农业大学 | Recombinant carp Nrf2 (NF-E2-related factor 2) gene, protein, preparation and detection methods and application of recombinant carp Nrf2 gene |
Non-Patent Citations (3)
| Title |
|---|
| SHINN-SHUENN TZENG ET AL.: "Expression of Soluble Form Carp (Cyprinus carpio) Ovarian Cystatin in Escherichia coli and Its Purification.", 《J. AGRIC. FOOD CHEM》 * |
| 李树红 等: "鲢鱼卵中两种高分子半胱氨酸蛋白酶抑制因子的纯化与鉴定", 《食品科学》 * |
| 陈海 等: "鲢鱼重组Cystatin的原核表达、鉴定及对铜绿假单胞菌的抑制作用", 《食品科学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105175524B (en) | 2019-02-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105420175B (en) | The method of withered grass engineering bacteria Bacillus subtilis and its structure and enzyme preparation of being lost hair or feathers using its production | |
| CN115216462B (en) | Complex enzyme preparation for preparing hermetia illucens oil and application thereof | |
| CN105420158A (en) | Special fermentation microbial inoculum for harmless treatment of solid organic waste | |
| CN104328078A (en) | Brevibacillus borstelensis for producing neutral protease | |
| CN101254969A (en) | Method for preparing microbial flocculant by using byproduct bacterial of fermentation industry | |
| Zin et al. | Purification and characterization of a carboxymethyl cellulase from Artemia salina | |
| WO2022257391A1 (en) | Strain capable of degrading collagen and use thereof | |
| CN105918609A (en) | Preparation method of polypeptide protein powder from salted duck egg white | |
| CN107265612A (en) | Aerobic biochemical water purification agent | |
| CN102242092A (en) | Isolation and identification for bombyx mori intestinal tract probiotics, and cloning and expression of enzyme gene produced by bombyx mori probiotics | |
| CN103966191B (en) | The tryptic preparation method of a kind of recombinant bovine source property | |
| CN105175524A (en) | Carp recombined antimicrobial protein and preparing method thereof | |
| CN101838636B (en) | High-specific-activity xylanase XYN11F63 and genes and application thereof | |
| CN104017086A (en) | Fusion antimicrobial peptide and preparation method thereof | |
| CN103641900A (en) | Sea urchin antibacterial peptide and preparation method thereof | |
| CN102643849A (en) | Method for embedded membrane expression and aquaporins (AqpZ) purification in escherichia coli | |
| CN117660232A (en) | Algae and fungus combined preparation for cultivating Babylonia quadricarina and application thereof | |
| CN102020709A (en) | Mytilus coruscus antibacterial peptide and preparation method and application thereof | |
| CN101724613B (en) | Alkali proof intermediate temperature xylanase XYNAM6, gene thereof and application thereof | |
| JP7011132B2 (en) | New chitosanase CHI1, its coding gene and its use | |
| CN101368175B (en) | Novel phytase, encoding gene, cell and feedstuff additive including the enzyme | |
| CN101948837A (en) | Method for producing human insulin growth factor-1 in recombinant Escherichia coli | |
| CN110663813B (en) | Fish meal microbial fermentation and enzymolysis method | |
| CN101892208B (en) | High-temperature acidic xylanase XYN10J88, gene and application thereof | |
| CN102154246B (en) | Acid glucanase CEL7G5 and gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190226 Termination date: 20201014 |