CN102675445A - Ubiquitin-like modified protein, preparation and application thereof - Google Patents
Ubiquitin-like modified protein, preparation and application thereof Download PDFInfo
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- CN102675445A CN102675445A CN2012101523708A CN201210152370A CN102675445A CN 102675445 A CN102675445 A CN 102675445A CN 2012101523708 A CN2012101523708 A CN 2012101523708A CN 201210152370 A CN201210152370 A CN 201210152370A CN 102675445 A CN102675445 A CN 102675445A
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Abstract
The invention relates to the field of molecular biology, in particular to cynoglossus semilaevis type ubiquitin-like modified protein and preparation and application thereof. The ubiquitin-like modified protein is shown as an amino acid sequence in the sequence table SEQ ID No.1. The preparation method comprises the following steps: with cynoglossus semilaevis cDNA (complementary deoxyribonucleic acid) as a template, performing PCR (polymerase chain reaction) amplification by using primers F1 and R1; connecting the PCR product to an expression vector to obtain plasmid pETG15; transforming Rosetta (DE3) with the plasmid to obtain RG15; inducing with isopropyl-beta-D-isopropylthiogalactoside, purifying recombinant protein with an affinity column to obtain the ubiquitin-like modified protein. The ubiquitin-like modified protein has remarkable anti-virus effect.
Description
Technical field
The present invention relates to biology field, a kind of specifically ubiquitin-like modified protein and preparation and application.
Background technology
(interferon-stimulated gene 15, ISG15) coding is the small molecular protein of the about 15kDa of a kind of molecular weight to ubiquitin-like modified protein ISG15, in animals such as people, mouse, ox, fish, finds at present by interferon-stimulated gene 15.ISG15 is very responsive to Interferon, rabbit, and Interferon, rabbit stimulates and causes that ISG15 expresses rise rapidly.The constitutional features of ISG15 is to contain two ubiquitin-like (ubiquitin-like) domains and a LRGG structure that is positioned at the C-end.Mammals ubiquitin-like modified protein has certain similarity with ubiquitin on mechanism of action, can modify a series of target proteins through the cascade enzyme reaction, thus the regulating cell function.In mouse, find multiple and ISG15 bonded target protein at present, comprised the signal conduction factor of immunoregulation path etc.In addition, ISG15 can also be secreted to born of the same parents except in cell, playing a role, and outside born of the same parents, brings into play various immunoregulation effects with the form of cytokine.Big in recent years quantity research shows, Mammals ubiquitin-like modified protein ISG15 has vital role in the host resists the process of various DNA and RNA viruses, and ISG15 genetically deficient can cause the host anti-virus ability drop.Compare with Mammals, the research of fish ubiquitin-like modified protein seldom, its effect and BA neither clear.
Summary of the invention
The object of the invention is to provide a kind of ubiquitin-like modified protein and preparation and application.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
A kind of ubiquitin-like modified protein, ubiquitin-like modified protein are shown in the aminoacid sequence among the sequence table SEQ ID No.1.
The preparation of ubiquitin-like modified protein,
1) structure of plasmid pETG15:
With Cynoglossus semilaevis cDNA is template, carries out pcr amplification with primers F 1 with R1, and the plasmid pET259 that cuts with the EcoRV enzyme behind the PCR product purification is connected with the T4DNA ligase enzyme, connects liquid and is transformed into intestinal bacteria, and the screening transformant extracts plasmid, is plasmid pETG15;
2) expression of ubiquitin-like modified protein and preparation
With above-mentioned steps 1) plasmid pETG15 transform Rosetta (DE3), cultivate containing on the LB substratum of kantlex, the screening transformant is RG15; After RG15 used the isopropyl-abduction delivering of final concentration as 1mM, adopt the affinity column purification of recombinant proteins, be the ubiquitin-like modified protein shown in the aminoacid sequence among the sequence table SEQ ID No.1.
F1 is 5 '-GATATCATGGAAATAACCATCACAA-3 ' in the said step 1); R1 is 5 '-GATATCGCCAATGACGGTGT-3 '.
The application of ubiquitin-like modified protein, the ubiquitin-like modified protein of the aminoacid sequence among the said sequence table SEQ ID No.1 can be used for preparing the virus immunity vaccine.The present invention has following advantage: ubiquitin-like modified protein preparation of the present invention is simple, has tangible antivirus action.
Description of drawings
(wherein, the ubiquitin-like modified protein that obtains of purifying is a swimming lane 2 to the ubiquitin-like modified protein electrophoretogram that the purifying that Fig. 1 provides for the embodiment of the invention obtains; Swimming lane 1: molecular weight marker.)。
Embodiment
Below in conjunction with embodiment the present invention is described further.Embodiment is intended to the description of giving an example to the present invention, but not limits the invention in any form.
Involved in embodiments of the present invention routine property experimental technique all adopts following method:
1. plasmid extraction, DNA (PCR) product purification, dna fragmentation reclaim the corresponding reagent box that all uses " TIANGEN Biotech (Beijing) Co., Ltd. " from gel.
2. intestinal bacteria are with Hanahan method (Sambrook and Russell:Molecular Cloning:A Laboratory Mannual.Cold Spring Harbor Laboratory Press2001); Yeast conversion is pressed the method (Catalog no.V175-20) of Invitrogen company; Polyacrylamide gel electrophoresis (SDS-PAGE) is according to Sambrook and Russell:Molecular Cloning:A Laboratory Mannual.Cold Spring Harbor Laboratory Press2001.
3. all restriction enzymes and ligase enzyme are all available from " Niu Yinglun Bioisystech Co., Ltd ", Beijing.
Ubiquitin-like modified protein of the present invention is the aminoacid sequence among the sequence table SEQ ID No.1.Sequence table SEQ ID No.1 is:
MEITITMLNGNSCTLMVQPQENVGSLRQRIHQKLNVTPERQRLVFDNGQRKDLSDNSQNIAFYGLRSGSKVSLLVIEPVTIQVFLKNVKNVVSAYDITPDETVANFKRRVQHREGVAESQQRLVFQGQEMTQGKLSDYNVQALSTIELLLRLRGGRGHTVIGE
(a) sequence signature:
● length: 162
● type: aminoacid sequence
● chain: strand
● topological framework: linearity
(b) molecule type: protein
(c) suppose: not
(d) antisense: not
(e) initial source: Cynoglossus semilaevis
Constructional feature: this albumen expection contains two ubiquitin-like domains (being made up of with 81-151 amino acid/11-76 respectively) and a LRGG sequence (amino acid position 152-155).
The preparation method of ubiquitin-like modified protein:
1) structure of plasmid pETG15:
With Cynoglossus semilaevis cDNA is template, carries out pcr amplification with primers F 1 with R1.The PCR condition is: 94 ℃ of preparatory sex change template DNAs of 60s, 94 ℃ of 40s then, 49 ℃ of 60s, 72 ℃ of 60s change 94 ℃ of 40s into after 5 circulations, 58 ℃ of 60s, 72 ℃ of 60s, after 30 circulations again at 72 ℃ of extension 7-10min.The PCR product is with the corresponding reagent box purifying of day root.(the pET259 building process is referring to Zheng, W.J., Hu with expression vector pET259; Y.H., Sun, L.; 2010.Cloning and analysis of a ferritin subunit from turbot (Scophthalmus maximus) .Fish.Shellfish.Immunol.28 829-836) cuts the back with restriction enzyme EcoRV enzyme and reclaims the 5.4kb fragment, and it is connected with the T4DNA ligase enzyme with the PCR product of above-mentioned purifying; Connect liquid and be transformed into bacillus coli DH 5 alpha; On the LB substratum that contains kantlex (30ug/ml), cultivated 18-24 hour, the screening transformant extracts plasmid, is pETG15.
Said LB moity is by weight percentage: 1.0% peptone, 0.5% yeast powder, 1.0% sodium-chlor, 97.5% zero(ppm) water.Said F1 is 5 '-GATATCATGGAAATAACCATCACAA-3 '; R1 is 5 '-GATATCGCCAATGACGGTGT-3 '.
2) expression of ubiquitin-like modified protein and preparation
The plasmid pETG15 of step 1) is transformed Rosetta (DE3) (purchasing in " Beijing hundred Tyke Bioisystech Co., Ltd "), on the LB substratum that contains kantlex (30ug/ml), cultivate, the screening transformant is RG15.RG15 is cultured to OD600 in 37 ℃ in containing the LB substratum of kantlex be 0.6; Adding isopropyl-makes its final concentration reach 1mM; 18 ℃ were continued wave and culture 10-12 hour; (chromatography condition: (Wash buffer) washes post twice with the 4-5ml lavation buffer solution then to use the nickel-nitrilotriacetic acid affinity column purification of recombinant proteins of GE Healthcare (U.S.) company; Use 0.5-1ml elution buffer (Elution buffer) to wash post subsequently, collect all elutriants).Recombinant protein in the elutriant is carried out mass spectroscopy, the ubiquitin-like modified protein shown in the aminoacid sequence among its sequence table SEQ ID No.1.Recombinant protein is analyzed the molecular weight consistent (referring to Fig. 1) of sequence in finding its molecular weight and showing SEQ ID No.1 with polyacrylamide gel electrophoresis (SDS-PAGE).
Above-mentioned Wash buffer composition is: 50mM NaH
2PO
4, 300mM NaCl, 20mM imidazole, pH 8.0; Above-mentioned Elution buffer composition is: 50mM NaH
2PO
4, 300mM NaCl, 250mMimidazole, pH 8.0.
Embodiment 3
The antitoxin sick effect of ubiquitin-like modified protein
Cynoglossus semilaevis head-kidney lymphocyte (is purchased the HyClone in Thermo Scientific, Beijing, the dull and stereotyped cultivation in 96-hole China) in containing the L-15 substratum.The extraction of head-kidney lymphocyte, cultural method are referring to reference Hu Y; Chen L; Sun L.CXCL8 of Scophthalmus maximus:expression, biological activity, and immunoregulatory effect.Dev Comp Immunol.2011; 35:1030-7.The ubiquitin-like modified protein is diluted to 500ng/ml in L-15.Add 20ul ubiquitin-like modified protein diluent or L-15 (contrast) in the above-mentioned lymphocyte, 25 ℃ of insulation 2h.Lymphocyte after the insulation is added 10
4Individual cell enlargement virus, 25 ℃ of insulation 4h.Cell is washed 3 times with PBS.(this method is specifically referring to Zhang M with the absolute quantitation PCR method; Xiao ZH; Hu YH; Sun L.Characterization of a megalocytivirus from cultured rock bream, Oplegnathus fasciatus (Temminck & Schlege), in China.Aquac Res.2012.doi:10.1111/j.1365-2109.2011.02861.x) the interior viral number that infects of calculating born of the same parents.This experiment repetition 3 times.The result shows, the viral mean number of cell that adds the ubiquitin-like modified protein is than the viral mean number low 2.5 times (having significant difference, P＜0.05) of control cells, explain that the ubiquitin-like modified protein can protect viral the infecting of cell resistance.
Said PBS moity is by weight percentage: 0.8%NaCl, 0.02%KCl, 0.358%Na
2HPO
4.12H
2O, 0.024%NaH
2PO
4, surplus is a water.
Claims (4)
1. ubiquitin-like modified protein, it is characterized in that: the ubiquitin-like modified protein is shown in the aminoacid sequence among the sequence table SEQ ID No.1.
2. the preparation of the said ubiquitin-like modified protein of claim 1 is characterized in that:
1) structure of plasmid pETG15:
With Cynoglossus semilaevis cDNA is template, carries out pcr amplification with primers F 1 with R1, and the plasmid pET259 that cuts with the EcoRV enzyme behind the PCR product purification is connected with the T4DNA ligase enzyme, connects liquid and is transformed into intestinal bacteria, and the screening transformant extracts plasmid, is plasmid pETG15;
2) expression of ubiquitin-like modified protein and preparation
With above-mentioned steps 1) plasmid pETG15 transform Rosetta (DE3), cultivate containing on the LB substratum of kantlex, the screening transformant is RG15; After RG15 used the isopropyl-abduction delivering of final concentration as 1mM, adopt the affinity column purification of recombinant proteins, be the ubiquitin-like modified protein shown in the aminoacid sequence among the sequence table SEQ ID No.1.
3. by the preparation of the described ubiquitin-like modified protein of claim 2, it is characterized in that: F1 is 5 '-GATATCATGGAAATAACCATCACAA-3 ' in the said step 1); R1 is 5 '-GATATCGCCAATGACGGTGT-3 '.
4. the application of the described ubiquitin-like modified protein of claim 1 is characterized in that: the ubiquitin-like modified protein of the aminoacid sequence among the said sequence table SEQ ID No.1 can be used for preparing the virus immunity vaccine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091498A (en) * | 2013-01-08 | 2013-05-08 | 中国科学院遗传与发育生物学研究所 | Plant in-vitro ubiquitin protein degradation system and application thereof |
| CN104749143A (en) * | 2013-12-31 | 2015-07-01 | 深圳先进技术研究院 | Detection method for sumoylated modification of proteins and application thereof |
| CN109358145A (en) * | 2018-07-24 | 2019-02-19 | 甘肃农业大学 | Osmotic stress ubiquitination protein screeing methods |
-
2012
- 2012-05-16 CN CN2012101523708A patent/CN102675445A/en active Pending
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| 《EMBL-BANK》 20110310 Sha Z.等 GH231767.1 第1-3页 1-4 , * |
| OVERGARD,A.C. 等: "AER92772.1", 《GENBANK》 * |
| OVERGARD,A.C. 等: "AER92773.1", 《GENBANK》 * |
| SHA Z.等: "GH231767.1", 《EMBL-BANK》 * |
| 万新星 等: "类泛素修饰蛋白质ISG15及其修饰酶系的功能", 《中国生物化学与分子生物学报》 * |
| 刘畅 等: "类泛素蛋白ISG15及其在先天免疫中的应用", 《生物化学与生物物理近战》 * |
| 等: "Crystal Structure of Human ISG15 Protein in Complex with Influenza B Virus NS1B", 《J. BIOL. CHEM.》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091498A (en) * | 2013-01-08 | 2013-05-08 | 中国科学院遗传与发育生物学研究所 | Plant in-vitro ubiquitin protein degradation system and application thereof |
| CN104749143A (en) * | 2013-12-31 | 2015-07-01 | 深圳先进技术研究院 | Detection method for sumoylated modification of proteins and application thereof |
| CN109358145A (en) * | 2018-07-24 | 2019-02-19 | 甘肃农业大学 | Osmotic stress ubiquitination protein screeing methods |
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Application publication date: 20120919 |