CN105175524B - A kind of carp recombination Antagonistic protein and preparation method thereof - Google Patents
A kind of carp recombination Antagonistic protein and preparation method thereof Download PDFInfo
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- CN105175524B CN105175524B CN201510662032.2A CN201510662032A CN105175524B CN 105175524 B CN105175524 B CN 105175524B CN 201510662032 A CN201510662032 A CN 201510662032A CN 105175524 B CN105175524 B CN 105175524B
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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
- C07K14/435—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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention discloses a kind of carp recombination Antagonistic protein and preparation method thereof, the carp recombination Antagonistic protein has amino acid sequence shown in sequence table SEQ ID NO:1, and the base sequence as shown in sequence table SEQ ID NO:2 encodes.Gained Antagonistic protein of the invention can effectively inhibit the growth, breeding and survival of the microorganisms such as bacterium, fungi, widen the processing and utilization of the stock of fish.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to a kind of carp recombination Antagonistic protein and preparation method thereof.
Background technique
China waters is vast, and aquatic resources very abundant, the especially stock of fish are very rich, wherein having contained very rich
Rich genetic resources has high biological value.But for a long time the fishery cultivating in China and fish processing be in compared with
The developing stage of low level generates and has abandoned a large amount of aquatic products such as fish-egg, liver pancreas, spleen, hypophysis, serum and skin and adds
Work waste by-product, therefore the processing and utilization rate of the stock of fish is low, economic benefit is poor.
Applicant for many years has made intensive studies many-sided application of piscine organism resource, finds the part in carp
Protein product has significant bacteriostasis, the growth, breeding and survival of multiple microbial strains is able to suppress, if realized
Prepared by batch, by effective economic value for promoting fishery by-product, and providing for the scientific and reasonable utilization of fish by-product must
The Research foundation wanted is of great significance in various aspects such as environment, resource, science and economy.
Summary of the invention
Applicant is in the scientific research of many years, it was found that the intracorporal Antagonistic protein of carp, and suppression is realized by further investigation
The recombination and preparation of mycoprotein improve the processing and utilization rate of the stock of fish, avoid by-product from arbitrarily abandoning and to natural environment
Generated pressure improves the comprehensive benefit of fish processing.
Specifically, the present invention is achieved through the following technical solutions:
Firstly, the invention discloses a kind of carps to recombinate Antagonistic protein, there is amino shown in sequence table SEQ ID NO:1
Acid sequence, and the base sequence as shown in sequence table SEQ ID NO:2 encodes.
Secondly, specifically comprising the following steps: the invention discloses the recombination and preparation of above-mentioned Antagonistic protein
1) with upstream primers F 1:5 '-TAGGATCCACTGGGATTCCTGGAGGCCTTG-3 ', downstream primer R1:5 '-
GCCCTCGAGCATGCAGGTGTTTTCAG-3 ' passes through base sequence shown in PCR amplification sequence table SEQ ID NO:2；
2) PCR amplification genetic fragment is connected on pMD19-T carrier, is transferred to JM109 competent cell, extracts positive matter
Grain；
3) carrier is prepared with BamH I, Xho I double digestion positive plasmid and PET-30, connection gained prepares carrier and is transferred to
BL21 Escherichia coli and the recombinant protein that soluble form is prepared under inducer IPTG effect.
In the above-mentioned methods, the base sequence for encoding Antagonistic protein can both extract total serum IgE through RT- from Cyprinus Carpio
PCR processing obtains, and can also be obtained by artificial synthesized mode with base sequence shown in sequence table SEQ ID NO:2
Genetic fragment.
In the above-mentioned methods, skilled addressee readily understands that in order to improve fungistatic effect pair after obtaining recombinant protein
It is purified, and purifying process is using common method biologically, such as destination protein is collected by centrifugation, with Buffer A
(NaCl containing 0.4-0.5mol/L, 0.5-1mol/L urea, 5% glycerol, 50mmol/LTris-HCl, pH7-9) dialyses, is dense
Contracting, affinity chromatography are purified.
Finally, the invention also discloses above-mentioned albumen to inhibit the application in microbial bacteria.Microbial bacteria referred to herein
Refer to the microorganisms such as common bacterium, fungi, applicant confirms after study, and Antagonistic protein of the invention is to Pseudomonas
Bacterium, pseudomonas aeruginosa, Escherichia coli especially have an ideal inhibitory effect, thus application of the invention be preferably applied to it is above-mentioned
Three kinds of bacterium.
Technical solution of the present invention has successfully been obtained the Antagonistic protein that can efficiently inhibit microbial strains, improves fishing
The economic value and comprehensive utilization ratio of industry and fish processing.
Specific embodiment
In order to absolutely prove technical solution of the present invention, applicant provide several specific implementations.It is real provided by following
It applies that example is only schematical, specific implementation method composition of the invention is not particularly limited to.
Embodiment 1
Total serum IgE is extracted from Cyprinus Carpio, by obtaining single-stranded cDNA after RT-PCR, with 5 '-
TAGGATCCACTGGGATTCCTGGAGGCCTTG-3 ' is upstream primer, then with 5 '-
GCCCTCGAGCATGCAGGTGTTTTCAG-3 ' is downstream primer, obtains genetic fragment through PCR amplification.Wherein PCR reaction condition
It is set as 94 DEG C of initial denaturation 5min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of extension 1min, 35 circulations；72 DEG C of last extension 5min.
Amplification gained genetic fragment is connected on pMD19-T carrier, is transferred in JM109 competent cell, and is carried out blue white
It screens, picking positive colony, PCR detection is carried out after bacterium liquid activation.
Recombinant plasmid is extracted, with BamH I, XhoI double digestion, through 1.2% agarose gel electrophoresis, gel extraction purpose piece
Section, with the PET-30 of BamH I, XhoI double digestion prepare carrier T4 effect under connect, be transferred to BL21 Escherichia coli.And in luring
It leads agent 1mmol/LIPTG, 30-35 DEG C, under the inductive condition of 2-3h, prepares the recombinant protein of soluble form.
Bacterial sediment is prepared after ultrasonication, the destination protein that centrifuge separation supernatant obtains (contains 0.4- with Buffer A
0.5mol/L NaCl, 0.5-1mol/L urea, 5% glycerol, 50mmol/LTris-HCl, pH7-9) dialysed and be concentrated after,
Affinitive layer purification destination protein, is presented the single band of about 14kD on SDS-PAGE, and the display present invention is successfully realized mesh
Albumen preparation.
Suppression of the destination protein to Pseudomonas fluorescens, pseudomonas aeruginosa, Escherichia coli is identified by disc diffusion method
Bacterium effect, recombination Antagonistic protein dosage range are 0.21-0.42mg/ piece.Experimental result shows that Pseudomonas fluorescens inhibition zone is straight
Diameter range is 8-16mm；Pseudomonas aeruginosa antibacterial circle diameter range is 8-35mm；Escherichia coli inhibition zone diameter range is 8-
25mm。
Embodiment 2
It is artificial synthesized that there is base sequence shown in sequence table SEQ ID NO:2, with 5 '-
TAGGATCCACTGGGATTCCTGGAGGCCTTG-3 ' is upstream primer, with 5 '-GCCCTCGAGCATGCAGGTGTTTTCAG-
3 ' be downstream primer, passes through PCR amplification genetic fragment.PCR parameter setting are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of 30s, 53 DEG C
30s, 72 DEG C of extension 1min, 33 circulations；72 DEG C of last extension 5min, obtain genetic fragment.
Gained amplified production is connected on pMD19-T carrier, is transferred in JM109 competent cell, and picking positive colony mentions
Take recombinant plasmid.
Carrier is prepared with BamH I, XhoI double digestion recombinant plasmid and PET-30 and is connected, and BL21 Escherichia coli are transferred to, and
In the IPTG of inducer 0.5mmol/L, 25-30 DEG C, under the inductive condition of 2-3h, the recombinant protein of soluble form is prepared.
Bacterial sediment is prepared after ultrasonication, the destination protein that centrifuge separation supernatant obtains (contains 0.4- through Buffer A
0.5mol/L NaCl, 0.5-1mol/L urea, 5% glycerol, 50mmol/LTris-HCl, pH7-9) it dialyses, affinity chromatography
Destination protein is purified, destination protein is detected as the single band of about 14kD through SDS-PAGE.
Suppression of the destination protein to Pseudomonas fluorescens, pseudomonas aeruginosa, Escherichia coli is identified by disc diffusion method
Bacterium effect, recombination Antagonistic protein dosage range are 0.21-0.42mg/ piece.Pseudomonas fluorescens antibacterial circle diameter range is 8-
16mm；Pseudomonas aeruginosa antibacterial circle diameter range is 8-35mm；Escherichia coli inhibition zone diameter range is 8-25mm.
Claims (4)
1. a kind of carp recombinates Antagonistic protein, which is characterized in that the amino acid sequence of the carp recombination Antagonistic protein is SEQ
ID NO.1, the nucleotides sequence of the carp recombination Antagonistic protein are classified as SEQ ID NO.2.
2. the carp of claim 1 recombinates Antagonistic protein preparation method, it is characterised in that include the following steps:
1) with upstream primers F 1:5 '-TAGGATCCACTGGGATTCCTGGAGGCCTTG-3 ', downstream primer R1:5 '-
GCCCTCGAGCATGCAGGTGTTTTCAG-3 ' passes through base sequence shown in PCR amplification sequence table SEQ ID NO:2；
2) PCR amplification genetic fragment is connected on pMD19-T carrier, is transferred to JM109 competent cell, extracts positive plasmid；
3) carrier is prepared with BamH I, Xho I double digestion positive plasmid and PET-30, connection gained prepares carrier, and to be transferred to BL21 big
Enterobacteria and the recombinant protein that soluble form is prepared under inducer IPTG effect.
3. the carp recombination Antagonistic protein of claim 1 is inhibiting the application in microbial bacteria.
4. application according to claim 3, which is characterized in that the microbial bacteria includes Pseudomonas fluorescens, P. aeruginosa
Bacterium, Escherichia coli.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510662032.2A CN105175524B (en) | 2015-10-14 | 2015-10-14 | A kind of carp recombination Antagonistic protein and preparation method thereof |
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| CN201510662032.2A CN105175524B (en) | 2015-10-14 | 2015-10-14 | A kind of carp recombination Antagonistic protein and preparation method thereof |
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| CN105175524A CN105175524A (en) | 2015-12-23 |
| CN105175524B true CN105175524B (en) | 2019-02-26 |
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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 |
|---|
| Expression of Soluble Form Carp (Cyprinus carpio) Ovarian Cystatin in Escherichia coli and Its Purification.;Shinn-Shuenn Tzeng et al.;《J. Agric. Food Chem》;20011231;第49卷;4224-4230 |
| 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;李树红 等;《食品科学》;20150630;第36卷(第23期);6-11 |
| 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;陈海 等;《食品科学》;20141231;第35卷(第21期);133-137 |
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