CN106084025A - The application in resisiting influenza virus of SLD5 albumen and encoding gene thereof - Google Patents
The application in resisiting influenza virus of SLD5 albumen and encoding gene thereof Download PDFInfo
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Abstract
The invention discloses a kind of SLD5 albumen and encoding gene application in resisiting influenza virus thereof.Application provided by the present invention is specially the protein or the application in the medicine preparing resisiting influenza virus or immunostimulant of its encoding gene being made up of the aminoacid sequence shown in SEQ ID NO:1.It is demonstrated experimentally that protein provided by the present invention can interact with Influenza matrix albumen M1, it is possible to suppression influenza virus duplication in host, reach to remove virus, the purpose for the treatment of disease, it is adaptable to the prevention of influenza virus and treatment, it is easy to large-scale promotion and application.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of SLD5 albumen and encoding gene thereof answering in resisiting influenza virus
With.
Background technology
The health of the mankind is caused high risks by influenza virus, and in 20th century pass by, the mankind occurred 4 big rule
The influenza pandemic of mould, be very popular including " spanish influenzas " of 1918, " Asia influenza " of nineteen fifty-seven be very popular, nineteen sixty-eight
" Mao flu " is very popular and " Russia's influenza " of 1977 is very popular.Being very popular so that human life's property of these influenzas
And economic development is subject to huge strike.
Influenza virus belongs to orthomyxoviridae family, is class minus-strand RNA virus segmented, tunicary, and infectiousness is strong
And easily morph, including first, second, the third three types, different hosts can be infected.Wherein influenza A virus can infect the mankind, fowl
Class and mammal, be the main pathogens causing seasonal epidemic sexuality to emit；B-mode, influenza virus C then main infection people
Class.Influenza A virus again can be according to its surface antigen haemagglutinin (Hemagglutinin, HA), neuraminidase
(Neuraminidase, NA) kind is divided into different subtype, by the end of having discovered that the HA of 17 kinds of hypotypes and 10 kinds so far
The NA of hypotype.Influenza A virus presents seasonal epidemic trend in China in recent years.
The surface of influenza virus particles comes from the lipid bilayer structure of host cell, and two kinds of major surfaces therein resist
Former HA and NA and a kind of ionophorous protein M2.Double-layer of lipoid is internal is the stromatin (Matrix from virus
Protein, M1).Virion core is 8 ribonucleoprotein complexs (Ribonucleoprotein Complex, RNP).
Play an important role on integrity maintaining virus shape.M1 albumen participates in the multiple link of virus replication, has regulation and control disease
The matter transportation effect transcribed between the nucleus of infected cell, cytoplasm of poison, and participate in the Budding process of virus,
It it is one of the main component of influenza virus.Well-conserved in view of in A type and influenza B, M1 albumen is considered as to set
Count the promising target of new antiviral drugs.Therefore, in research host, the albumen of interaction special with M1 contributes to new drug
Research and development.
Yeast-two hybrid technique can identify interaction albumen in group level.Tissue-specific by screening mouse lung
Yeast two-hybrid library, we identify the interaction albumen that SLD5 is M1.SLD5 comprises 223 aminoacid, in eukaryote
The SLD5 protein amino acid sequence concordance of high conservative, people and mice is 87.9%.SLD5 is that the composition of GINS complex becomes
Member, plays central role in GINS complex assembling process.GINS complex is made up of four albumen, respectively Psf1, Psf2,
Plf3 and SLD5.Synthesize beginning period, GINS complex and MCM complex and CDC45 protein binding at DNA, form CMG and replicate
Fork unwindase, starts DNA replication dna.Striking the cell line after low SLD5 and be stuck in the G0/G1 phase, cell growth is significantly inhibited.
Summary of the invention
The invention provides a kind of SLD5 albumen and encoding gene application in resisiting influenza virus thereof.
One aspect of the present invention, it is provided that protein or its encoding gene increase at the medicine or immunity preparing resisiting influenza virus
Application in strong agent；
Described protein is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in SEQ ID NO:1；
B aminoacid sequence shown in SEQ ID NO:1 through the replacement of one or several amino acid residue and/or is lacked by ()
Lose and/or add and have the derivative protein of anti-influenza virus activity.
Another aspect of the present invention, it is provided that protein and encoding gene thereof are at following (a1) and/or (a2) and/or (a3)
In application:
(a1) preparation can suppress the medicine that influenza virus is replicated；
(a2) preparation can suppress the immunostimulant that influenza virus is replicated；
(a3) preparation can in conjunction with and suppress the medicine of influenza virus M1 albumen；
For the ease of the purification of albumen (SLD5), can be at the albumen being made up of the amino acid residue sequence of SEQ ID NO:1
The amino terminal of matter or carboxyl terminal connect upper label as shown in table 1.
Table 1: the sequence of label
Protein in above-mentioned (b) can synthetic, it is possible to first synthesize its encoding gene, then carries out biological expression and obtain.
The encoding gene of the protein in above-mentioned (b) can be by lacking one or several ammonia in the DNA sequence shown in SEQ ID NO:2
The codon of base acid residue, and/or carry out the missense mutation of one or several base pair.
In above-mentioned application, the encoding gene (SLD5 encoding gene) of described protein is arbitrary institute in following (1) to (3)
The DNA molecular stated:
(1) DNA molecular shown in SEQ ID NO:2；
(2) DNA molecule hybridize limited with (1) under strict conditions and the DNA molecular of code for said proteins；
(3) arbitrary described DNA molecular has more than 90% homology and code for said proteins with (1) or (2)
DNA molecular.
In above-mentioned application, described influenza virus can be A type or Type B influenza virus.
In one embodiment of the invention, described influenza virus is specially H1N1 type influenza virus A/PR8/1934 strain
The resisiting influenza virus function of described SLD5 albumen or its encoding gene is embodied in as follows: described SLD5 albumen energy
Enough combine influenza virus M1 albumen, thus suppress the duplication of influenza virus.
It is also another object of the present invention to provide the medicine of resisiting influenza virus containing the DNA molecular shown in SEQ ID NO:2
Thing or immunostimulant.
Another object of the present invention, it is provided that containing the DNA molecular shown in SEQ ID NO:2 and to be used for preparing anti-current susceptible
The medicine of poison or the recombinant vector of immunostimulant, expression cassette, transgenic cell line or recombinant bacterium.
It is still another aspect of the present invention to provide the medicine of resisiting influenza virus containing the protein shown in SEQ ID NO:1
Or immunostimulant.
The present invention, by research C57L/B6 mice host and Influenza matrix albumen M1 interaction mechanism, uses ferment
Female two-hybrid techniques has searched out in C57L/B6 mouse lung albumen and has interacted with influenza A virus PR8 matrix prote m1
Target protein SLD5, and by the interaction of experimental verification SLD5 albumen with Influenza matrix albumen M1, and it was found that
SLD5 albumen can suppress influenza virus levels of replication in influenza infection process (24-48h).The result of study of the present invention is
Develop antiviral drugs based on host cell gene target spot and provide new approach.
Accompanying drawing explanation
Fig. 1 is yeast two-hybrid result in the embodiment of the present invention 1；
Fig. 2 is SLD5 albumen and Influenza matrix albumen M1 co-immunoprecipitation result in the embodiment of the present invention 2；
Fig. 3 is overexpressing cell system (A549-SLD5) and compared with control cells system (A549-Vector) in the embodiment of the present invention 3
Immunoblot results；
Fig. 4 is overexpressing cell system (A549-SLD5) and compared with control cells system (A549-Vector) in the embodiment of the present invention 3
Influenza virus infection PR8 virus restrovirus titre comparison diagram.
Detailed description of the invention
Below in conjunction with drawings and Examples, the detailed description of the invention of the present invention is described in more details, in order to energy
The advantage being enough more fully understood that the solution of the present invention and its various aspects.But, specific embodiments described below and reality
Executing example is only descriptive purpose rather than limitation of the present invention.
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1 screens specificity antivirus albumen by yeast-two hybrid technique
(1) yeast-two hybrid technique is used, with Influenza matrix albumen M1 phase interaction in screening B6 mouse lung tissue
Albumen, find B6 mice host specificity antiviral protein.
(2) lung of three 6-8 week old female B6 mice (purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) is taken
Portion organizes liquid nitrogen grinding, uses Trizol method to extract total serum IgE.
(3) Clontech company Make Your Own " Mate & Plate is usedTM" Library System test kit will
Total serum IgE (1-2 μ g) reverse transcription is cDNA, specifically comprises the following steps that
1) following reagent is added in 0.25ml centrifuge tube, mix homogeneously, brief centrifugation；
The RNA obtained in reagent: 1-2 μ l step (2)；CDS III/6 primer of 1.0 μ l (5 '-
ATTCTAGAGGCCGAGGCGGCCGACATG-NNNNNN-3’)；1-2μl；Volume is supplied to 4.0 μ l with deionized water.
2) 72 DEG C of incubation 2min；It is placed on cooled on ice 2min again；Brief centrifugation.
3) centrifuge tube is placed at room temperature, after adding following reagent, raps mixing, brief centrifugation.
Reagent: 2.0 μ l 5X First-Strand Buffer；1.0μl DTT(20mM)；1.0μl dNTP Mix
(10mM)；1.0 μ l MMLV reverse transcriptases；Volume is supplied to 9.0 μ l with deionized water.
4) under the room temperature of 25 30 DEG C, incubation 10min；Again at 42 DEG C of incubation 10min.
5) 1.0 μ l BD SMART III oligonucleotide are added.
6) on air jet flow case or hot lid PCR instrument, 42 DEG C of incubations one hour.
7) 75 DEG C of placing response test tube, 10min, terminates first chain synthesis.
8) cool down under room temperature, adding 1.0 μ l (3 units) RNase H；At 37 DEG C of incubation 20min.
9) take 2 μ l liquid from first chain synthetic and join that one clean, pre-cooling, 0.5ml test tube, be placed on ice
Upper standby.
(4) the ds cDNA obtained by LD-PCR amplification first chain synthesis.
(5) by the ds cDNA obtained in (4) and pGADT7 plasmid (purchased from Clontech company) cotransformation Y187 yeast
(purchased from Clontech company) competent cell, sets up B6 mouse lung tissue yeast cDNA library.
1) convert Y187 yeast strain with ds cDNA and pGADT7-Rec (purchased from Clontech company) (to be purchased from
Clontech company) prepare yeast competent cell；The 15ml centrifuge tube following reagent of addition in aseptic pre-cooling: 20 μ l ds
cDNA；6μl pGADT7-Rec；20μl Herring Testes Carrier DNA.
Shift in 46 Herring DNA to centrifuge tubes of μ l and heat 5min at 100 DEG C.Immediately test tube is placed on ice
Bath cools down DNA.The most once heat and cooling step before adding DNA to 15ml reaction tube.Add 600 μ l Y187 yeast senses
By in state cell to Herring DNA, vortex concussion mixing gently, addition 2.5ml PEG/LiAc solution, vortex shakes gently
Mixing；30 DEG C of incubation 45min, mix a cell every 15min；Add 160 μ l DMSO, mixing, then holding test tubes to 42 DEG C
Water-bath, 20min.A cell is mixed every 10min；700 × g is centrifuged 5min, discards supernatant, with 3mlYPD Plus liquid culture
Base Eddy diffusion.30 DEG C of incubations, shake 90min.700 × g is centrifuged 5min, discards supernatant, with 30ml NaCl (0.9%) solution
Eddy diffusion.
2) on SD/ Leu flat board, transformant is selected
By 1) the Y187 yeast strain suspension that obtains paves plate, and each 150mm flat board is sprawled 150 μ l suspensions.
(note: detection transformation efficiency, spreads 100 μ l 1:10,1:100,1:1,000, and1:10, and 000 diluent is at 100-mm SD/ Leu
On flat board.)
30 DEG C of Incubate plates are until (3-4 days) occurs in clone downwardly over.
Calculate transformation efficiency.Expected result: >=1 × 106transformants/3μg pGADT7-Rec。
3) transformant is collected
4 DEG C cool down flat board 3-4 hour, and each flat board adds 5ml freezing culture medium；Use aseptic Glass rod stirring gently
Move and make cell enter liquid；Collect all of liquid in flask, mix homogeneously.Numeration meter is used to calculate the density of cell.Often
1ml equal portions are saved at 80 DEG C.
(6) M1cDNA of H1N1 influenza A virus PR8 strain is connected to pGBKT7 carrier (purchased from Clontech company)
(the T4DNA ligase test kit of Promega company is used, according to examination as bait plasmid between EcoRI and SalI restriction enzyme site
The description concrete operations of agent box), utilize MATCHMAKER Two-Hybrid System screening and influenza virus M1 albumen phase
The mice host protein of interaction, specifically comprises the following steps that
1) host bacteria and bait bacterial strain are merged:
A) bait plasmid pGBKT7-M1 is converted Y2H GOLD bacterial strain (purchased from Clontech company), spread SD/-Trp flat board
Cultivate 2-3 days for 30 DEG C.
B) choose a diameter 2-3mm to be cloned in 50 milliliters of SD/ Trp fluid mediums, 250 270rpm, 30 DEG C of cultivations
It is 0.8 (16 20 hours) to OD value.Centrifugal thalline of collecting, resuspended with 4-5 milliliter SD/-Trp fluid medium.
C) take the B6 mouse lung tissue yeast cDNA library obtained in 1ml step (5) and place defrosting in 30 DEG C of water-baths.
CDNA Yeast libraries after thawing mixes with Y2H GOLD bacterium solution obtained in the previous step in the triangular flask adding 2L.
D) 45ml 2X YPDA/Kan (50 μ g/ml) is added.It is shaken gently for.
E) 45rpm rotating speed, 30 DEG C of incubations 20 24 hours.
F), after within 20 hours, merging, check a fusion culture under the microscope, if zygote exists, carry out next step
Operation.If zygote does not exists, continue to cultivate 4 hours.
G) integrative mixture is transferred to bottom the centrifuge tube of an aseptic 100ml.1,000 × g is centrifuged 10min.With
0.5X YPDA/Kan (50 μ g/ml) rinses and merges twice of flask (every time rinsing with 50ml).Collect flushing liquor and again hang with it
Floating cell.
H) 1,000 × g is centrifuged 10min.Add 10ml 0.5XYPDA/Kan (50 μ g/ml) culture medium.
2) the yeast diploid of selection expression interaction protein:
Measure fusion efficiencies, spread the 1:10 of 100 μ l, 000,1:1,000,1:100, and the fusion culture of 1:10 dilution in
Three culture medium (100mm flat board): SD/ Leu, SD/ Trp and SD/ Leu/ Trp.Sprawl remaining fusion culture in TDO
Or QDO flat board (200 μ l cells/150mm flat board).On TDO or QDO flat board, be possible with for the yeast monoclonal of growth
The host protein of influenza virus M1 protein-interacting, continues order-checking and identifies.
3) by 2) in the yeast strain clone expressing interaction protein that filters out carry out extraction of plasmid DNA, and convert
E.colidh5αcell, checks order the most again.Sequencing result enters in http://blast.ncbi.nlm.nih.gov website
Row sequence alignment, found that the gene order submitted to is SLD5.
As it is shown in figure 1, the yeast of M1 and SLD5 cotransformation can lack in culture medium add Aba and X-a-Gal four
Grow and become blue, showing that both can interact.And individually convert the yeast of M1 and SLD5 and empty plasmid add Aba and
The four of X-a-Gal lack and can not grow in culture medium.
Embodiment 2 co-immunoprecipitation checking interaction
By influenza A virus PR8 strain matrix prote m1 and SLD5 albumen (nucleotide sequence is shown in SEQ ID NO:1) total length
CDNA be building up to respectively in eukaryon expression plasmid pcDNA-FLAG, pCI-GFP (Ogawa, K., Tanaka, Y., Uruno, T.,
Duan,X.F.,Harada,Y.,Sanematsu,F.,Yamamura,K.,Terasawa,M.,Nishikimi,A.,Cote,
J.F.,et al.(2014).DOCK5functions as a key signaling adaptor that links Fc
epsilon RI signals to microtubule dynamics during mast cell
Degranulation.Journal of Experimental Medicine 211,1401-1413. plasmid pcDNA-FLAG,
The pCI-GFP public can obtain for 20 years from the applying date of the application from Institute of Microorganism, Academia Sinica), concrete steps bag
Include:
(1) design influenza A virus PR8 strain matrix prote m1 total length primer, two ends, left and right interpolation restriction enzyme site EcoRI,
XhoI restriction enzyme site (M1-F:5 '-GCCGAATTCatgagtcttctaaccga-3 '；M1-R:5’-
CAGCTCGAGcttgaaccgttgcat-3 '), it is connected in pCI-FLAG plasmid, it is thus achieved that pCI-M1-FLAG.
(2) design SLD5 full length protein primer, two ends, left and right add respectively EcoRI, XhoI restriction enzyme site (primer sequence:
SLD5-F:5’-cgGAATTCatgacggaggttctg-3’；SLD5-R:5 '-tg aCTCGAGttatattagctgaac-3 '),
It is connected in pCI-GFP plasmid, it is thus achieved that pCI-GFP-SLD5.
Two plasmids of the cDNA sequence containing M1 and SLD5 albumen respectively built are transfected jointly 293T cell:
After 48h, cell lysis extracts albumen, usesM2Affinity Gel purified
Immunoglobulin (Sigma-Aldrich (Shanghai) trade Co., Ltd) carries out co-immunoprecipitation (IP), by IP product with
Total protein uses SLD5 antibody to carry out whether western blot, checking SLD5 have interaction with influenza virus M1 albumen；
Result: as in figure 2 it is shown, when cell is cleaved under the conditions of non denatured, the many albumen existed in intact cell
Interaction between matter-protein has been retained, if precipitating FLAG with the antibody mediated immunity of protein FLAG (M1)
(M1), then protein S LD5 being combined in vivo with SLD5 also can precipitate (see swimming lane 4).Otherwise, negative control group does not has
Having transfection M1, therefore SLD5 precipitated cannot get off (see swimming lane 3).
The structure of embodiment 3 SLD5 stable expression cell line and infection experiment
Lentiviral pCDH-CMV (purchased from System Biosciences, Inc company) is used to build SLD5 steady
Determine express cell system, specifically comprise the following steps that
(1) design XbaI, EcoRI restriction enzyme site at SLD5 two ends, insert in pCDH-CMV carrier, it is thus achieved that carrier pCDH-
SLD5；
(2) use PEI transfection reagent pCDH-CMV Yu pCDH-SLD5 is transfected respectively to A549 cell (Liu, D.,
Sheng,C.J.,Gao,S.J.,Yao,C.,Li,J.D.,Jiang,W.,Chen,H.M.,Wu,J.X.,Pan,C.C.,Chen,
S.,et al.(2015).SOCS3Drives Proteasomal Degradation of TBK1and Negatively
Regulates Antiviral Innate Immunity.Molecular and Cellular Biology 35,2400-
2413. public can obtain for 20 years from the applying date of the application from Institute of Microorganism, Academia Sinica), concrete operations are such as
Under:
A) prepare 40%-80% A549 cell (6-well, 5 × 105/ well), negative control (pCDH-CMV) and mesh
The each hole of gene (pCDH-SLD5)；
B) in 400ul serum-free DMEM culture fluid, 2 μ g plasmids, mix homogeneously are added；
C) in mixed liquor b), add the PEI transfection reagent (1mg/ml) of 4 μ l, mix homogeneously；Room temperature stands 10 minutes；
D) in mixed liquor c), add the 2%FBS/DMEM fluid medium of 1.1ml, mix homogeneously；
E) blot cell culture fluid, mixed liquor is all added；
F) cultivate 3 hours at 37 DEG C, then change normal incubation medium (10%FBS/DMEM) into；Detected after 24-48 hour
Gene expression.
(3) after green fluorescence occurring, use puromycin (5 μ g/ml) screening positive cell, about 2 weeks.Then use
Selected by flow cytometry apoptosis GFP high expressing cell, is A549-SLD5 (process LAN) and A549-CMV (comparison) cell line.
Protein blot experiment is utilized to verify SLD5 protein level, as it is shown on figure 3, in overexpressing cell system, SLD5 albumen
Level is significantly raised.
Respectively SLD5 express cell system A549-SLD5 and compared with control cells system A549-CMV is infected PR8 virus (MOI=
0.01), after, within 24 hours and 48 hours, virus titer is measured by plaque analytic process (Plaque assay), from figure after infection
Can be seen that after infecting 24 and 48 hours, the cell line virus titer of SLD5 process LAN have dropped about 50% compared to matched group.Knot
Really: as shown in Figure 4, display SLD5 can suppress influenza virus levels of replication.
It is last that it is noted that obviously above-described embodiment is only for clearly demonstrating example of the present invention, and also
The non-restriction to embodiment.For those of ordinary skill in the field, can also do on the basis of the above description
Go out change or the variation of other multi-form.Here without also cannot all of embodiment be given exhaustive.And thus drawn
What Shen went out obviously changes or changes among still in protection scope of the present invention.
Claims (6)
1. protein or the application in the medicine preparing resisiting influenza virus or immunostimulant of its encoding gene, its feature exists
In:
Described protein is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in SEQ ID NO:1；
B aminoacid sequence shown in SEQ ID NO:1 is passed through replacement and/or the disappearance of one or several amino acid residue by ()
And/or add and have the derivative protein of anti-influenza virus activity.
2. protein and encoding gene application in following (a1) and/or (a2) and/or (a3) thereof:
(a1) preparation can suppress the medicine that influenza virus is replicated；
(a2) preparation can suppress the immunostimulant that influenza virus is replicated；
(a3) preparation can in conjunction with and suppress the medicine of influenza virus M1 albumen.
Application the most according to claim 1 and 2, it is characterised in that: the encoding gene of described protein be following (1) extremely
(3) arbitrary described DNA molecular in:
(1) DNA molecular shown in SEQ ID NO:2；
(2) under strict conditions with the DNA molecule hybridize described in (1) and the DNA molecular of code for said proteins；
(3) DNA that arbitrary described DNA molecular has more than 90% homology and code for said proteins with (1) or (2) divides
Son.
4. contain medicine or the immunostimulant of the resisiting influenza virus of the DNA molecular shown in SEQ ID NO:2.
5. contain the DNA molecular shown in SEQ ID NO:2 and for preparing the medicine of resisiting influenza virus or the weight of immunostimulant
Group carrier, expression cassette, transgenic cell line or recombinant bacterium.
6. contain medicine or the immunostimulant of the resisiting influenza virus of the protein shown in SEQ ID NO:1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111714617A (en) * | 2019-03-21 | 2020-09-29 | 中国科学院微生物研究所 | UBL7 protein and application of encoding gene thereof in resisting virus infection |
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| CN104013954A (en) * | 2014-06-11 | 2014-09-03 | 中国科学院微生物研究所 | Application of ISG20 protein and encoding gene of ISG20 protein in anti-influenza virus |
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| CN104013954A (en) * | 2014-06-11 | 2014-09-03 | 中国科学院微生物研究所 | Application of ISG20 protein and encoding gene of ISG20 protein in anti-influenza virus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111714617A (en) * | 2019-03-21 | 2020-09-29 | 中国科学院微生物研究所 | UBL7 protein and application of encoding gene thereof in resisting virus infection |
| CN111714617B (en) * | 2019-03-21 | 2023-02-17 | 中国科学院微生物研究所 | UBL7 protein and application of coding gene thereof in resisting virus infection |
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