CN104650218A - Fish prothymosin alpha and application thereof - Google Patents
Fish prothymosin alpha and application thereof Download PDFInfo
- Publication number
- CN104650218A CN104650218A CN201510096179.XA CN201510096179A CN104650218A CN 104650218 A CN104650218 A CN 104650218A CN 201510096179 A CN201510096179 A CN 201510096179A CN 104650218 A CN104650218 A CN 104650218A
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- China
- Prior art keywords
- prothymosin
- fish
- recombinant protein
- prothymosin alpha
- cynoglossus semilaevis
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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
- C07K14/575—Hormones
- C07K14/57581—Thymosin; Related peptides
-
- 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 the field of molecular biology and particularly relates to a fish (cynoglossus semilaevis gunther) prothymosin alpha and application thereof. The recombinant protein of fish prothymosin alpha is the recombinant protein of cynoglossus semilaevis gunther prothymosin alpha. The recombinant protein is formed as follows: by taking the cDNA of cynoglossus semilaevis gunther as a template, carrying out PCR (polymerase chain reaction) by using primers F1 and R1, amplifying the gene of prothymosin alpha, and inducing, expressing and purifying the amplification product. The protein of prothymosin alpha can be injected into the bodies of fishes to improve the antiviral ability of fishes significantly.
Description
Technical field
The present invention relates to biology field, specifically a kind of fish (Cynoglossus semilaevis) prothymosin α and application thereof.
Background technology
Prothymosin α (ProTthymosin alpha, ProTT α) is a kind of strongly-acid albumen, and molecular weight is about 12.5kDa.Lack aromatic series and sulfur-bearing amino acid in the amino acid composition of this albumen and there is no the native conformation fixed.Prothymosin α can be hydrolyzed by lysosome l-asparagine endopeptidase, thus produces one containing 28 amino acid whose active polypeptide-thymosin α1s.Prothymosin α mainly acts on propagation and the differentiation of cell.Increasing research shows, in Mammals, prothymosin α has the function of immunomodulatory and cytokine-like, such as, can stimulate the up-regulated expression of IL-2 acceptor, promote the maturation of dendritic cell, urge chemotactic and anti-infection activity etc.Mammals prothymosin α can the release of immune stimulatory regulatory factor I type Interferon, rabbit as the endogenous part of Tol l sample acceptor TLR4, and then effectively activate killer T cell and natural killer cell.But the function and application potential of fish prothymosin α it be unclear that.
Summary of the invention
The object of the invention is to provide a kind of fish (Cynoglossus semilaevis) prothymosin α and application thereof.
For achieving the above object, the technical solution used in the present invention is:
A kind of fish prothymosin α recombinant protein, fish prothymosin α recombinant protein is the prothymosin α recombinant protein of Cynoglossus semilaevis, it is for template with Cynoglossus semilaevis cDNA, pcr amplification prothymosin α gene is carried out with primers F 1 and R1, amplified production is recombinant protein through abduction delivering, purifying, and described F1 is 5 '-GATATCATGGCGGACAGCAAAG-3 '; R1 is 5 '-GATATCCTGGTCTGTCTTCTGCT-3 '.
An application for fish prothymosin α recombinant protein, the prothymosin α recombinant protein of described Cynoglossus semilaevis can be applicable to preparation and suppresses antiviral preparation.
Described virus is Megalocytivirus.
Tool of the present invention has the following advantages: the anti-virus ability that can significantly improve fish after prothymosin α injection fish of the present invention.
Accompanying drawing explanation
The prothymosin α protein electrophoresis figure of the purifying that Fig. 1 provides for the embodiment of the present invention.Swimming lane 1, molecular weight standard; Swimming lane 2, prothymosin α.
Embodiment
Below in conjunction with embodiment, the invention will be further described.Embodiment is intended to carry out citing to the present invention and describes, but not limits the invention in any form.
Experimental technique routinely involved in embodiments of the present invention is all adopted with the following method:
1. plasmid extraction, DNA (PCR) product purification, DNA fragmentation reclaim the corresponding reagent box all using " TIANGEN Biotech (Beijing) Co., Ltd. " from gel.
2. intestinal bacteria are with Hanahan method (Sambrook and Russell:MolecularCloning:A Laboratory Mannual.Cold Spring Harbor Laboratory Press2001).
3. all restriction enzymes and ligase enzyme are all purchased from " Niu Yinglun Bioisystech Co., Ltd ", Beijing.
Embodiment 1
The preparation of prothymosin α recombinant protein
1) structure of the plasmid pProT of prothymosin α recombinant protein is expressed:
Prothymosin α sequence of the present invention is announced (GenBank accession numberXM_008334972.1).Prothymosin α is made up of 103 amino acid, does not have fixing structural domain, and unique conservative feature is strongly-acid (iso-electric point 3.6).With Cynoglossus semilaevis cDNA for template, carry out pcr amplification prothymosin α gene with primers F 1 and R1.PCR condition is: 94 DEG C of 60s denaturation template DNAs, then 94 DEG C of 40s, 53 DEG C of 60s, 72 DEG C of 60s, again at 72 DEG C of extension 7-10min after 30 circulations.The PCR primer corresponding reagent box of sky root is purified.By expression vector pET259, (building process is see Hu YH, Zheng WW, Sun L.Identification and molecular analysisof a ferritin subunit from red drum (Sciaenops ocellatus) .FishShellfish Immunol 2010; 28:678 – 86) cut rear recovery 5.3kb fragment with restriction enzyme EcoRV enzyme, it is connected with T4DNA ligase enzyme with the PCR primer of above-mentioned purifying, connecting fluid is transformed into bacillus coli DH 5 alpha, 18-24 hour is being cultivated containing on the LB substratum of kantlex (100ug/ml), screening transformant extracts plasmid, called after pProT.By DNA sequencing analytical proof, pProT is the expression plasmid of the extrasin alpha sequence containing strongly-acid (iso-electric point 3.6) feature, wherein, in pProT plasmid, prothymosin α sequence is consistent with numberXM_008334972.1 sequence disclosed in GenBank access ion.
Described LB moiety is by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium-chlor, 97.5% distilled water.Described F1 is 5 '-GATATCATGGCGGACAGCAAAG-3 '; R1 is 5 '-GATATCCTGGTCTGTCTTCTGCT-3 '.
2) expression and purity of prothymosin α recombinant protein
By above-mentioned plasmid pProT ordinary method transformation of E. coli BL21 (DE3) (purchased from " Tian Gen biochemical technology company limited ", Beijing), LB solid medium containing kantlex (50ug/ml) cultivates 18-24 hour, picking transformant, by its called after BL21/pProT.
By BL21/pProT incubated overnight in the LB liquid nutrient medium containing kantlex (50ug/ml); Get 1ml spend the night after nutrient solution, add 100ml fresh containing kantlex (50ug/ml) LB liquid nutrient medium in, at 37 DEG C, rotating speed 200rpm wave and culture is to OD
600be 0.6, add the IPTG that final concentration is 1mM, 28 DEG C are continued with rotating speed 160rpm wave and culture 5h, and then with 5000g, 4 DEG C of centrifugal 10min, collect bacterium liquid, add 5ml lysate, slowly shake 1-2 hour in room temperature on shaking table, till bacteria suspension becomes clarification.By bacterium liquid with 10000g, 4 DEG C of centrifugal 30min, reclaim supernatant.Albumen affinity column His Trap HP Columns (being purchased from GEHealthcare company of the U.S.) in supernatant is reclaimed purifying.By the albumen of purifying through SDS-PAGE electrophoresis detection (under 8v/cm voltage electrophoresis 25-30min, subsequently electrophoresis 2-2.5h under 15v/cm voltage), measure its molecular size range (see Fig. 1).Find that its protein quality is identical with prothymosin α.
Described lysate is the 10mM NaH of final concentration
2pO
4, 10mM Tris and 8M urea, pH8.0.
Embodiment 2
The application of prothymosin α
Step 1) injection of prothymosin α
The prothymosin α albumen of above-described embodiment 1 purifying is diluted to 200ug/ml in PBS, is prothymosin α diluent.20 Cynoglossus semilaevis (heavily about 13.4g) are divided into 2 groups at random, often organize 10.By these 2 groups difference called after A and B.Every bar fish of A group is injected 100ul prothymosin α diluent respectively, every bar fish of B group (control group) is injected 100ul PBS respectively.
Described PBS moiety is by weight percentage: 0.8%NaCl, 0.02%KCl, 0.358%Na
2hPO
4.12H
2o, 0.024%NaH
2pO
4, surplus is water.
Step 2) viral suspension preparation
By Megalocytivirus RBIV-C1, (concrete preparation method is shown in Zhang M, Xiao Z, Hu Y, SunL.Characterization of a megalocytivirus from cultured rock bream, Oplegnathus fasciatus (Temminck & Schlege), in China.Aquac Res.2012; 43:556 – 64) in PBS, be diluted to 10
6copies/ml, is viral suspension.
Step 3) attack poison infection
In above-mentioned steps 1) after injection the 5th hour, every bar fish of A and B group is injected 100ul above-mentioned steps 2) viral suspension.6 days after infection, get fish spleen and renal tissue.Utilize DNA extraction kit (be purchased from TIANGEN Biotech (Beijing) Co., Ltd. ") to extract DNA from tissue, detect viral level (concrete grammar is shown in above-mentioned reference) in tissue by absolute quantitation PCR method.Result shows, the viral number of A group fish spleen and kidney (is respectively 2x10
4and 8x10
3) significantly (P<0.01) (be respectively 1x10 lower than the viral number of B group fish spleen and kidney
5and 2x10
4).
These results show, prothymosin α significantly can strengthen fish and support antiviral infecting.
Claims (3)
1. a fish prothymosin α recombinant protein, it is characterized in that: fish prothymosin α recombinant protein is the prothymosin α recombinant protein of Cynoglossus semilaevis, it is for template with Cynoglossus semilaevis cDNA, pcr amplification prothymosin α gene is carried out with primers F 1 and R1, amplified production is recombinant protein through abduction delivering, purifying, and described F1 is 5 '-GATATCATGGCGGACAGCAAAG-3 '; R1 is 5 '-GATATCCTGGTCTGTCTTCTGCT-3 '.
2. an application for fish prothymosin α recombinant protein according to claim 1, is characterized in that: the prothymosin α recombinant protein of described Cynoglossus semilaevis can be applicable to preparation and suppresses antiviral preparation.
3. the application of fish prothymosin α recombinant protein according to claim 2, is characterized in that: described virus is Megalocytivirus.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107082804A (en) * | 2017-05-15 | 2017-08-22 | 海南大学 | A kind of egg-shaped pompano β thymosin extrasins and its application |
| CN108157676A (en) * | 2017-12-20 | 2018-06-15 | 厦门大学 | Application of the recombined extrasin alpha 1 alpha originals in functional feed |
| CN111909947A (en) * | 2020-08-04 | 2020-11-10 | 辽宁省海洋水产科学研究院 | Preparation and application of apostichopus japonicus beta-thymosin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7754687B2 (en) * | 2005-02-24 | 2010-07-13 | Mount Sinai School Of Medicine | Methods of inhibiting viral infection |
| WO2013131032A1 (en) * | 2012-03-02 | 2013-09-06 | Icahn School Of Medicine At Mount Sinai | Variants of prothymosin alpha and methods of using same |
| CN103497243A (en) * | 2013-10-14 | 2014-01-08 | 中国科学院海洋研究所 | High-mobility fish group protein and application thereof |
-
2015
- 2015-03-04 CN CN201510096179.XA patent/CN104650218A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7754687B2 (en) * | 2005-02-24 | 2010-07-13 | Mount Sinai School Of Medicine | Methods of inhibiting viral infection |
| WO2013131032A1 (en) * | 2012-03-02 | 2013-09-06 | Icahn School Of Medicine At Mount Sinai | Variants of prothymosin alpha and methods of using same |
| CN103497243A (en) * | 2013-10-14 | 2014-01-08 | 中国科学院海洋研究所 | High-mobility fish group protein and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| GENBANK: "XM_008334972.1", 《GENBANK》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107082804A (en) * | 2017-05-15 | 2017-08-22 | 海南大学 | A kind of egg-shaped pompano β thymosin extrasins and its application |
| CN107082804B (en) * | 2017-05-15 | 2020-12-08 | 海南大学 | Trachinotus ovatus beta-thymosin and application thereof |
| CN108157676A (en) * | 2017-12-20 | 2018-06-15 | 厦门大学 | Application of the recombined extrasin alpha 1 alpha originals in functional feed |
| CN111909947A (en) * | 2020-08-04 | 2020-11-10 | 辽宁省海洋水产科学研究院 | Preparation and application of apostichopus japonicus beta-thymosin |
| CN111909947B (en) * | 2020-08-04 | 2022-06-07 | 辽宁省海洋水产科学研究院 | Preparation and application of apostichopus japonicus beta-thymosin |
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