CN104711240A - Application of sigma A protein of avian reovirus and related biological materials of sigma A protein - Google Patents
Application of sigma A protein of avian reovirus and related biological materials of sigma A protein Download PDFInfo
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Abstract
The invention discloses an application of sigma A protein of an avian reovirus (ARV) and related biological materials of the sigma A protein. The invention discloses an application of the sigma A protein of the ARV or the related biological materials of the sigma A protein to the preparation of a product capable of increasing expression quantity of phosphorylated protein kinase B (P-Akt) in a cell. The invention, for the first time, proves that the sigma A protein and sigma NS protein of the ARV can activate a PI3K/AKt signal pathway and enable the expression quantity of the P-Akt in the cell to be increased, so that the activation of the PI3K/AKt signal pathway can be inhibited expectedly by inhibiting expression of the sigma A protein and/or the sigma NS protein of the ARV to inhibit ARV replication, and a new target point of a therapeutic drug for the ARV can be discovered. The application provides new direction and idea for ARV therapy.
Description
Technical field
The present invention relates to the application of a kind of Avianreovirus σ A albumen and relevant biological material thereof, belong to biological technical field.
Background technology
Many barss Signal Transduction Pathways is there is in eukaryotic cell; phosphatidyl-inositol 3-kinase (PI3K) signal path plays very important effect in numerous signal path; various kinds of cell function is regulated and controled by its downstream effect molecule protein kinase b (Akt); as cell proliferation and differentiation, inhibited apoptosis etc., virus infection can disturb this signal path usually.The dimer protein that PI3K is made up of catalytic subunit p110 and adjustment subunit p85, has the double activity of lipoid kinases and protein kinase.Research shows, (P is proline(Pro) by PXXP for viral protein or cell protein, X is arbitrary amino acid) or YXXXM/YXXM (Y is tyrosine, X is arbitrary amino acid, M is methionine(Met)) motif is combined with P85, thus activate PI3K/Akt signal path, Akt (P-Akt) expression amount of phosphorylation is raised, realizes the generation of inhibited apoptosis.
A large amount of research datas shows, when after Virus entry host cell, infected cell by the immune system recognition of host, and can be removed having infected viral cell by cell death inducing, thus limiting virus copies intracellular and propagate.But under the effect of selective pressure, much virus has been evolved out, and some can escape apoptotic mechanism, make virus can complete its replicative cycle before host cell death, these counter measure are normally mediated by viral protein.Much virus regulates subunit to be combined by some albumen of self with the P85 of PI3K, and then activates PI3K/Akt signal path, promotes the transcriptional expression of P-Akt.The rising of P-Akt expression amount, can the many signal proteins of phosphorylation, and regulate many downstream signal transduction paths relevant with cells survival, propagation, migration, differentiation and apoptosis by these albumen, as the activity by suppressing pro apoptotic protein, thus the apoptosis that apoptosis inhibit stimulus signal brings out, the also inhibited apoptosis by the expression of control apoptosis gene.
The research of Labrada and Shih etc. shows, Avianreovirus (Avian reovirus, ARV) cause cells infected occur apoptosis be occur in its infect middle and later periods.After Lin etc. use ARV vero cells infection, the expression amount of P-Akt is detected by Western blot, what confirm that ARV infects from the angle of virus have activated PI3K/Akt signal path in early days, thus inhibits the apoptosis of cells infected, completes its replicative cycle to be conducive to ARV.How ARV starts PI3K/Akt signal path, realize the apoptosis suppressing cells infected, not clear at present, find out the ARV material that can activate PI3K/Akt signal path, disclosing the molecule mechanism of causing a disease of ARV from the angle of cell signalling, new thinking can be provided for finding anti-ARV drug target.
Summary of the invention
Technical problem to be solved by this invention determines to activate the ARV albumen of PI3K/Akt signal path, and then can it can be used as the action target spot of anti-ARV medicine.
In order to solve above technical problem, the invention provides σ A albumen or the application of its relevant biological material in the product of the expression amount of the protein kinase B (P-Akt) of preparation raising Intracellular phosphorylation of Avianreovirus;
The relevant biological material of the σ A albumen of described Avianreovirus is following B1) to B9) in any one:
B1) to encode the nucleic acid molecule of σ A albumen of described Avianreovirus;
B2) containing B1) expression cassette of described nucleic acid molecule;
B3) containing B1) recombinant vectors of described nucleic acid molecule;
B4) containing B2) recombinant vectors of described expression cassette;
B5) containing B1) recombinant microorganism of described nucleic acid molecule;
B6) containing B2) recombinant microorganism of described expression cassette;
B7) containing B3) recombinant microorganism of described recombinant vectors;
B8) containing B4) recombinant microorganism of described recombinant vectors;
B9) reconstitution cell of Avianreovirus σ A albumen is expressed.
In order to solve above technical problem, the present invention also provides the σ A albumen of Avianreovirus or its relevant biomaterial preparing the application in activating cells in PI3K/Akt signal path product;
The relevant biological material of the σ A albumen of described Avianreovirus is following B1) to B9) in any one:
B1) to encode the nucleic acid molecule of σ A albumen of described Avianreovirus;
B2) containing B1) expression cassette of described nucleic acid molecule;
B3) containing B1) recombinant vectors of described nucleic acid molecule;
B4) containing B2) recombinant vectors of described expression cassette;
B5) containing B1) recombinant microorganism of described nucleic acid molecule;
B6) containing B2) recombinant microorganism of described expression cassette;
B7) containing B3) recombinant microorganism of described recombinant vectors;
B8) containing B4) recombinant microorganism of described recombinant vectors;
B9) reconstitution cell of Avianreovirus σ A albumen is expressed.
In above-mentioned arbitrary described biomaterial, B1) described in nucleic acid molecule be following 1) or 2) or 3) shown in nucleic acid molecule, i.e. σ A gene:
1) nucleotide sequence is cDNA molecule or the DNA molecular of SEQ ID No.2;
2) with 1) nucleotide sequence that limits has more than 75% or 75% identity, and the cDNA molecule of described σ A albumen of encoding or genomic DNA molecule;
3) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and the cDNA molecule of described σ A albumen of encoding or genomic DNA molecule;
Wherein, SEQ ID No.2 is made up of 1251 Nucleotide, the aminoacid sequence shown in nucleotide coding SEQ ID No.1 of SEQ ID No.2;
The aminoacid sequence of described σ A albumen is as shown in SEQ ID No.1;
Those of ordinary skill in the art can adopt known method easily, the method for such as orthogenesis and point mutation, suddenly change to the nucleotide sequence of coding σ A albumen of the present invention.Those are through manually modified, have and the nucleotide sequence 75% of coding σ A albumen of synthetic of the present invention or the Nucleotide of higher identity, as long as coding σ A albumen and there is the protein of identical function, be all be derived from nucleotide sequence of the present invention and be equal to sequence of the present invention;
Term used herein " identity " refers to the sequence similarity with native sequence nucleic acid.The nucleotide sequence that " identity " comprises the protein formed with the aminoacid sequence shown in the SEQ of coding ID No.1 of the present invention has 75% or higher, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher identity.Identity can with the naked eye or computer software evaluate.Use computer software, the identity between two or more sequence can represent with per-cent (%), and it can be used for evaluating the identity between correlated series.
In above-mentioned biomaterial, described stringent condition is in the solution of 2 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 5min at 68 DEG C, again in the solution of 0.5 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 15min at 68 DEG C;
More than above-mentioned 75% or 75% identity, can be the identity of more than 80%, 85%, 90% or 95%.
In above-mentioned biomaterial, the expression cassette (σ A expression casette) of the nucleic acid molecule of the σ A albumen containing coding Avianreovirus B2), refer to the DNA that can express σ A albumen in host cell, this DNA not only can comprise the promotor starting σ A genetic transcription, also can comprise the terminator stopping σ A genetic transcription.Further, described expression cassette also can comprise enhancer sequence.
In above-mentioned biomaterial, available existing expression vector establishment contains the recombinant vectors of described σ A expression casette.
In above-mentioned biomaterial, described carrier can be plasmid, glutinous grain, phage or virus vector.
In above-mentioned biomaterial, described microorganism can be prokaryotic micro-organisms or eukaryotic microorganisms; Described prokaryotic micro-organisms can be bacterium; Described bacterium can be Escherichia bacteria; Described Escherichia bacteria can be intestinal bacteria; Described eukaryotic microorganisms can be algae, fungi, protozoon; Described fungi can be unicellular fungi; Described unicellular fungi can be yeast.
In above-mentioned biomaterial, described recombinant vectors is for replacing with the DNA shown in SEQ ID No.2 by the DNA between the Xho I of pcAGEN and Not I site, keep all the other sequences of pcAGEN constant, the recombinant expression vector of the σ A albumen of the described Avianreovirus of the expression obtained.
In order to solve above technical problem, the present invention also provides the application in the cell model of reconstitution cell material of Avianreovirus σ A protein-active in the cell of preparation screening suppression infection Avianreovirus of expressing Avianreovirus σ A albumen;
The reconstitution cell of described expression Avianreovirus σ A albumen can be the recombinant mammalian cells of expressing Avianreovirus σ A albumen herein, as expressed the restructuring Vero cell of Avianreovirus σ A albumen.
In order to solve above technical problem, the present invention also provides and suppresses the material of Avianreovirus σ A protein-active preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes.
In order to solve above technical problem, the present invention also provides the application of material in the preparation suppression growth of Avianreovirus and/or the product of propagation suppressing Avianreovirus σ A protein-active.
In order to solve above technical problem, the present invention also provides and suppresses the material of Avianreovirus σ A protein expression preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes.
In order to solve above technical problem, the present invention also provides the application of material in the preparation suppression growth of Avianreovirus and/or the product of propagation suppressing Avianreovirus σ A protein expression.
In order to solve above technical problem, the present invention also provides and suppresses the material of Avianreovirus σ A genetic expression preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes.
In order to solve above technical problem, the present invention also provides the application of material in the preparation suppression growth of Avianreovirus and/or the product of propagation suppressing Avianreovirus σ A genetic expression.
In order to solve above technical problem, the material that it is action target spot that the present invention also provides with Avianreovirus σ A albumen is preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes;
Or,
In order to solve above technical problem, the application of material in the preparation suppression growth of Avianreovirus and/or the product of propagation that it is action target spot that the present invention also provides with Avianreovirus σ A albumen;
Or,
In order to solve above technical problem, the material that it is action target spot that the present invention also provides with Avianreovirus σ A gene is preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes;
Or,
In order to solve above technical problem, the application of material in the preparation suppression growth of Avianreovirus and/or the product of propagation that it is action target spot that the present invention also provides with Avianreovirus σ A gene.
In above-mentioned arbitrary described application, the aminoacid sequence of described σ A albumen is as shown in SEQ ID No.1, and described σ A gene is following 1) or 2) or 3) shown in nucleic acid molecule:
1) nucleotide sequence is cDNA molecule or the DNA molecular of SEQ ID No.2;
2) with 1) nucleotide sequence that limits has more than 75% or 75% identity, and the cDNA molecule of described σ A albumen of encoding or genomic DNA molecule;
3) under strict conditions with 1) or 2) nucleotide sequence hybridization that limits, and the cDNA molecule of described σ A albumen of encoding or genomic DNA molecule;
Those of ordinary skill in the art can adopt known method easily, the method for such as orthogenesis and point mutation, suddenly change to the nucleotide sequence of coding σ A albumen of the present invention.Those are through manually modified, have and the nucleotide sequence 75% of coding σ A albumen of synthetic of the present invention or the Nucleotide of higher identity, as long as coding σ A albumen and there is the protein of identical function, be all be derived from nucleotide sequence of the present invention and be equal to sequence of the present invention;
Term used herein " identity " refers to the sequence similarity with native sequence nucleic acid.The nucleotide sequence that " identity " comprises the protein formed with the aminoacid sequence shown in the SEQ of coding ID No.1 of the present invention has 75% or higher, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher identity.Identity can with the naked eye or computer software evaluate.Use computer software, the identity between two or more sequence can represent with per-cent (%), and it can be used for evaluating the identity between correlated series;
Described stringent condition is in the solution of 2 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 5min at 68 DEG C, again in the solution of 0.5 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 15min at 68 DEG C;
More than described 75% or 75% identity, can be the identity of more than 80%, 85%, 90% or 95%.
In above-mentioned arbitrary described application, described Avianreovirus σ A protein-active is specially the activity of PI3K/Akt signal path in its expression amount improving the protein kinase B (P-Akt) of Intracellular phosphorylation and/or activating cells.
The present invention proves, compares with empty carrier pcAGEN group with negative cells control group, and the expression amount of the P-Akt of the Vero cell of transfection σ A-pcAGEN or σ NS-pcAGEN obviously raises.And P-Akt detected when adding PI3K specific inhibitor LY294002 before transfection σ A-pcAGEN or σ NS-pcAGEN in Vero cell culture fluid, to compare with empty carrier pcAGEN group with negative cells control group and do not raise, illustrate that the phosphorylation of Akt depends on the activation of PI3K, the present invention confirms the σ A albumen of ARV first, σ NS albumen can activate PI3K/Akt signal path, and the expression amount of cell P-Akt is raised.
Activation in view of PI3K/Akt signal path can suppress the apoptosis of host cell, to be conducive to copying of virus, in actual applications, can using the σ A albumen of ARV and/or the σ NS albumen action target spot as anti-ARV medicine, infected the expression of ARV σ A albumen and/or σ NS albumen in the cell of ARV by suppression, suppress the activation of PI3K/Akt signal path, promote the apoptosis of the cell infecting ARV, and then suppressing copying of ARV, the present invention is that the treatment of ARV provides new direction and thinking.Also can the cell model of the material of ARV σ A albumen and/or σ NS albumen in the cell of infection ARV be suppressed to prevent and/or treat the product of the disease that Avianreovirus causes with screening as screening in the expression σ A albumen of ARV and/or the reconstitution cell of σ NS albumen.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis result of σ A gene fragment 1, σ NS gene fragment 1, μ A gene fragment 1, μ 1 B gene fragment 1 and μ NS gene fragment 1.
Fig. 2 is the indirect immunofluorescene assay result of each group of cell after transfection 6h.
Fig. 3 is the Western blot detected result of the target protein of each group of cell after transfection 6h.
Fig. 4 is the expression of results of the Flow cytometry P-Akt of each group of cell after transfection 6h.Percentage ratio in figure is the percentage of P-Akt positive cell.
Fig. 5 is the expression of results of the Western blot detection P-Akt after transfection 6h.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Experiment in following embodiment is 3 repetitions if no special instructions.
Avianreovirus S1133 standard virulent strain (ARV-S1133) (Avian reovirus) is China Veterinery Drug Inspection Office's product, and catalog number is AV2311.
Carrier for expression of eukaryon pcAGEN is at document " Weng Y; Lu W; Harmon A; Xiang X; Deng Q; SongM, et al.The cellular endosomal sorting complex required for transport pathwayis not involved in avian metapneumovirus budding in a virus-like-particleexpression system.J Gen Virol.2011; 92:1205-13 " in be disclosed, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
Vero cell is China typical culture collection center product.
PMD18-T carrier is precious biotechnology (Dalian) company limited product.
Lipofectamine
tM2000 is Invitrogen Products.
The goat anti-rabbit igg that goat-anti chicken IgY, Alexa fluor 488 that Alexa fluor 488 marks marks is abcam Products, and catalog number is respectively ab150173, ab150081.
Phospho-Akt monoclonal antibody is CST Products, and catalog number is 2965.
Cytofix fixes/and rupture of membranes test kit is BD Products.
PI3K specific inhibitor LY294002 is CST Products.
Embodiment 1, Avianreovirus σ A albumen and σ NS protein activation PI3K/Akt signal path
One, the Design and synthesis of primer
According to the gene order of σ A, the σ NS of Avianreovirus (ARV), μ A, μ B and μ NS, design and synthesize 5 pairs of primers of table 1.
Table 1 primer sequence
In table 1, italic is Xho I or Not I recognition site, and the sequence shown in underscore is Kozak sequence.
Two, the structure of σ A-pcAGEN, σ NS-pcAGEN, μ A-pcAGEN, μ B-pcAGEN and μ NS-pcAGEN carrier
(1) total serum IgE of Avianreovirus S1133 standard virulent strain (ARV-S1133) is extracted, and reverse transcription is cDNA, take cDNA as template, with σ A-F and σ A-R for primer carries out pcr amplification, obtain the PCR primer containing the σ A gene shown in SEQ ID No.2, this PCR primer is designated as σ A gene fragment 1; Or carry out pcr amplification with σ NS-F and σ NS-R for primer, obtain the PCR primer containing σ NS gene, this PCR primer is designated as σ NS gene fragment 1; Or carry out pcr amplification with μ A-F and μ A-R for primer, obtain the PCR primer containing μ A gene, this PCR primer is designated as μ A gene fragment 1; Or carry out pcr amplification with μ B-F and μ B-R for primer, obtain the PCR primer containing μ 1 B gene, this PCR primer is designated as μ 1 B gene fragment 1; Or carry out pcr amplification with μ NS-F and μ NS-R for primer, obtain the PCR primer containing μ NS gene, this PCR primer is designated as μ NS gene fragment 1.
The agarose gel electrophoresis result of σ A gene fragment 1, σ NS gene fragment 1, μ A gene fragment 1, μ 1 B gene fragment 1 and μ NS gene fragment 1 as shown in Figure 1.
In Fig. 1, M is DNA molecular amount standard DL2500bp Ladder; 1-5 swimming lane is respectively σ A gene fragment 1, σ NS gene fragment 1, μ A gene fragment 1, μ 1 B gene fragment 1 and μ NS gene fragment 1.
(2) respectively σ A gene fragment 1, σ NS gene fragment 1, μ A gene fragment 1, μ 1 B gene fragment 1 and μ NS gene fragment 1 are inserted pMD18-T, obtain σ A-pMD18-T, σ NS-pMD18-T, μ A-pMD18-T, μ B-pMD18-T and μ NS-pMD18-T recombinant plasmid, send order-checking by σ A-pMD18-T, σ NS-pMD18-T, μ A-pMD18-T, μ B-pMD18-T and μ NS-pMD18-T recombinant plasmid, result is correct.Sequencing result shows that σ A-pMD18-T, σ NS-pMD18-T, μ A-pMD18-T, μ B-pMD18-T and μ NS-pMD18-T recombinant plasmid for correctly to insert σ A gene fragment 1, σ NS gene fragment 1, μ A gene fragment 1, μ 1 B gene fragment 1 and μ NS gene fragment 1 respectively in pMD18-T.
(3) Xho I and Not I double digestion σ A-pMD18-T, σ NS-pMD18-T, μ A-pMD18-T, μ B-pMD18-T and μ NS-pMD18-T respectively, obtains σ A gene fragment 2, σ NS gene fragment 2, μ A gene fragment 2, μ 1 B gene fragment 2 and μ NS gene fragment 2 respectively; Xho I and Not I double digestion pcAGEN, obtains carrier large fragment; σ A gene fragment 2, σ NS gene fragment 2, μ A gene fragment 2, μ 1 B gene fragment 2 are connected with carrier large fragment respectively with μ NS gene fragment 2, obtain recombinant plasmid σ A-pcAGEN, σ NS-pcAGEN, μ A-pcAGEN, μ B-pcAGEN and μ NS-pcAGEN respectively, send order-checking by σ A-pcAGEN, σ NS-pcAGEN, μ A-pcAGEN, μ B-pcAGEN and μ NS-pcAGEN, result is correct.Sequencing result shows that σ A-pcAGEN, σ NS-pcAGEN, μ A-pcAGEN, μ B-pcAGEN and μ NS-pcAGEN are respectively σ A, σ NS, μ A, μ B and μ NS gene fragment 2 and correctly substituted for DNA between the Xho I of pcAGEN and Not I site respectively, all the other sequences of pcAGEN remain unchanged, the recombinant vectors of expressing σ A, σ NS, μ A, μ B and μ NS respectively obtained.
Recombinant plasmid σ A-pcAGEN contains the σ A gene shown in SEQ ID No.2, and recombinant plasmid σ A-pcAGEN expresses the σ A albumen shown in SEQ ID No.1.
Three, cell transfecting
With reference to Lipofectamine
tMthe specification sheets of 2000 test kits, by the Vero cell monolayer that σ A-pcAGEN, σ NS-pcAGEN, μ A-pcAGEN, μ B-pcAGEN, μ NS-pcAGEN and pcAGEN respectively transfection 6 orifice plate grow up to, correspondence obtains σ A-pcAGEN, σ NS-pcAGEN, the Vero groups of cells of μ A-pcAGEN, μ B-pcAGEN and μ NS-pcAGEN transfection and empty carrier pcAGEN group, set up negative cells control group (above-mentioned plasmid is replaced with isopyknic ultrapure water, and all the other steps are constant) simultaneously.
Four, indirect immunofluorescence and Western blot detect the expression of σ A, σ NS, μ A, μ B and μ NS
Respectively after transfection 2h, 4h, 6h, 12h and 24h, collect the cell of σ A-pcAGEN, σ NS-pcAGEN, the Vero groups of cells of μ A-pcAGEN, μ B-pcAGEN and μ NS-pcAGEN transfection, empty carrier pcAGEN group and negative cells control group respectively, proceed as follows respectively:
(1) indirect immunofluorescene assay
The cell PBST that each group of each time point is collected is washed three times, adds-20 DEG C of cold acetones preserved (acetone: ethanol is 2:3 mixing by volume) according to 500uL/ hole and be fixed, room temperature effect 10min; Wash three times with PBST, add the ARV chicken positive serum doubly diluted with PBS 1:100 according to 200uL/ hole, 37 DEG C of effect lh; Wash three times with PBST, add the goat-anti chicken IgY marked with the Alexa fluor 488 of PBS 1:500 dilution according to 200uL/ hole, 37 DEG C of effect lh; Wash three times with PBST, add glycerol buffer (glycerine: PBS 1:1 mixing by volume) according to 100uL/ hole, cell is observed under inverted fluorescence microscope.
The indirect immunofluorescene assay result of each group of cell after transfection 6h as shown in Figure 2.
In Fig. 2, A is negative cells control group; B is empty carrier pcAGEN group; C is the Vero groups of cells of σ A-pcAGEN transfection; D is the Vero groups of cells of σ NS-pcAGEN transfection; E is the Vero groups of cells of μ A-pcAGEN transfection; F is the Vero groups of cells of μ B-pcAGEN transfection; G is the Vero groups of cells of μ NS-pcAGEN transfection.
(2) Western blot detects
By cell reference literature " the Xie Z that each group of each time point is collected, Qin C, Xie L, et al.Recombinantprotein-based ELISA for detection and differentiation of antibodies againstavian reovirus in vaccinated and non-vaccinated chickens [J] .J VirolMethods, 2010, 165 (1): 108-111. " method, to the target protein σ A of correspondence, σ NS, μ A, μ B and μ NS carries out Western blot detection, the ARV positive serum reference literature " Qin Chunxiang used in detection, Xie Zhixun, Xie Liji, Liu Jiabo, Pang Yaoshan, Deng Xianwen, Xie Zhiqin. utilize σ 3 and σ 2 recombinant protein to detect the foundation [J] of Avianreovirus antibody ELISA. southwestern agriculture journal, 2009, 02:492-496. " disclosed method preparation.
The Western blot detected result of the target protein of each group of cell after transfection 6h as shown in Figure 3.
In Fig. 3,1 is negative cells control group; 2 is empty carrier pcAGEN group; 3 is the Vero groups of cells of σ A-pcAGEN transfection; 4 is the Vero groups of cells of σ NS-pcAGEN transfection; 5 is the Vero groups of cells of μ A-pcAGEN transfection; 6 is the Vero groups of cells of μ B-pcAGEN transfection; 7 is the Vero groups of cells of μ NS-pcAGEN transfection.
Fig. 2 and Fig. 3 shows, transfection is after 6 hours, target protein σ A, σ NS, μ A, μ B and μ NS obtain good expression in the Vero groups of cells of the σ A-pcAGEN of correspondence, σ NS-pcAGEN, μ A-pcAGEN, μ B-pcAGEN and μ NS-pcAGEN transfection respectively, and empty carrier pcAGEN group and negative cells control group are feminine gender, without the expression of any target protein.
Five, flow cytometry and Western blot detect the expression of P-Akt
Protein kinase B (P-Akt) due to ARV activating phosphatase be occur in ARV infect early stage, and according to the detected result of step 4, get the cell after σ A-pcAGEN, σ NS-pcAGEN, the Vero groups of cells of μ A-pcAGEN, μ B-pcAGEN and μ NS-pcAGEN transfection, empty carrier pcAGEN group and the transfection of negative cells control group 4h, 6h and 12h respectively, carry out the detection of following flow cytometry and Western blot.
(1) Flow cytometry
The cell Cytofix that each group of each time point is collected is fixed/rupture of membranes test kit fixed cell 20 minutes, FACS washs 2 times, add the Phospho-Akt monoclonal antibody action 30 minutes with PBS 1:100 dilution, FACS washs 2 times, the goat anti-rabbit igg effect that the Alexa fluor 488 adding use PBS 1:500 dilution marks 30 minutes, FACS washs 1 time, add 300ul FACS resuspension cell, flow cytometer detects the expression amount of the protein kinase B (P-Akt) of phosphorylation, record analysis data.
The expression of results of the Flow cytometry P-Akt of each group of cell after transfection 6h as shown in Figure 4.
In Fig. 4, A is negative cells control group; B is empty carrier pcAGEN group; C is the Vero groups of cells of σ A-pcAGEN transfection; D is the Vero groups of cells of σ NS-pcAGEN transfection; E is the Vero groups of cells of μ A-pcAGEN transfection; F is the Vero groups of cells of μ B-pcAGEN transfection; G is the Vero groups of cells of μ NS-pcAGEN transfection.
Transfection 4,6, negative cells control group after 12h, empty carrier pcAGEN group, σ A-pcAGEN, σ NS-pcAGEN transfection Vero groups of cells to detect the positive rate result of P-Akt as shown in table 2.
(2) detection of Western blot
The method of the cell reference literature " Wang X; Zhang H; Abel AM; et al.Roleof phosphatidylinositol 3-kinase (PI3K) and Akt1kinase in porcinereproductive and respiratory syndrome virus (PRRSV) replication [J] .ArchVirol; 2014,159 (8): 2091-2096. " collected by each group of each time point carries out Western blot detection to the P-Akt in Vero cell.
Western blot after transfection 6h detects the expression of results of P-Akt as shown in Figure 5.
In Fig. 5,1 is negative cells control group; 2 is empty carrier pcAGEN group; 3 is the Vero groups of cells of σ A-pcAGEN transfection; 4 is the Vero groups of cells of σ NS-pcAGEN transfection; 5 is the Vero groups of cells of μ A-pcAGEN transfection; 6 is the Vero groups of cells of μ B-pcAGEN transfection; 7 is the Vero groups of cells of μ NS-pcAGEN transfection.
Flow cytometry and Western blot detected result show, σ A-pcAGEN and σ NS-pcAGEN is at transfected Vero cells 4h, after 6h and 12h, the positive rate of P-Akt is all higher than negative cells control group and empty carrier pcAGEN group (P<0.01), show transfection σ A-pcAGEN, the expression amount of the Vero cell P-Akt of σ NS-pcAGEN is apparently higher than negative cells control group and empty carrier pcAGEN group (as shown in table 2), (4h in the different time of σ A-pcAGEN and σ NS-pcAGEN transfection, 6h and 12h), the expression amount difference of Vero cell P-Akt is little.
And after μ A-pcAGEN, μ B-pcAGEN and μ NS-pcAGEN transfectional cell, the positive rate of P-Akt and negative cells control group and empty carrier pcAGEN group difference little (P>0.05), show that the expression amount of P-Akt does not produce considerable change.
Table 2 FCM detects the expression level of P-Akt
In table 2, represent difference not significantly (P>0.05) with same letter in a line, different letter represents difference extremely significantly (P<0.01).
Six, the detection of PI3K approach is depended on
With reference to Lipofectamine
tMthe specification sheets of 2000 test kits, σ A-pcAGEN, σ NS-pcAGEN and pcAGEN respectively transfection 6 orifice plate will grow up to the Vero cell of individual layer, correspondingly respectively obtain σ A-pcAGEN, the Vero groups of cells of σ NS-pcAGEN transfection and empty carrier pcAGEN group, set up negative cells control group (above-mentioned plasmid is replaced with isopyknic ultrapure water, and all the other steps are constant) simultaneously.
Before σ A-pcAGEN, σ NS-pcAGEN transfection 6 orifice plate grow up to the Vero cell of individual layer, 1h adds PI3K specific inhibitor LY294002 respectively in Vero cell, and make its final concentration be 20 μMs, all the other steps are identical with above-mentioned experimental procedure, obtain the Vero groups of cells of LY294002+ σ A-pcAGEN transfection and the Vero groups of cells of LY294002+ σ NS-pcAGEN transfection respectively.
According to step 5, adopt flow cytometry and Western blot method to detect the expression of the P-Akt of each group, after analyzing transfection σ A-pcAGEN, σ NS-pcAGEN, whether the rising that Vero cell P-Akt expresses depends on the activation of PI3K.
Flow cytometry result shows, the Vero cell of transfection LY294002+ σ A-pcAGEN and the Vero cell of transfection LY294002+ σ NS-pcAGEN, the expression amount of P-Akt be all obviously less than transfection σ A-pcAGEN Vero cell and the Vero cell (P<0.05) of transfection σ NS-pcAGEN, and with the expression amount difference little (P>0.05) of the P-Akt of empty carrier pcAGEN group and negative cells control group, the rising that cell P-Akt expresses is described, depends on σ A albumen, σ NS albumen activates PI3K.The detected result of Western blot is consistent with Flow cytometry result.
The present invention confirms the σ A albumen of ARV first, σ NS albumen can activate PI3K/Akt signal path, and the expression amount of cell P-Akt is raised.
Claims (10)
1. the σ A albumen of Avianreovirus or its relevant biological material improve the application in the product of the expression amount of the protein kinase B of Intracellular phosphorylation in preparation;
The relevant biological material of the σ A albumen of described Avianreovirus is following B1) to B9) in any one:
B1) to encode the nucleic acid molecule of σ A albumen of described Avianreovirus;
B2) containing B1) expression cassette of described nucleic acid molecule;
B3) containing B1) recombinant vectors of described nucleic acid molecule;
B4) containing B2) recombinant vectors of described expression cassette;
B5) containing B1) recombinant microorganism of described nucleic acid molecule;
B6) containing B2) recombinant microorganism of described expression cassette;
B7) containing B3) recombinant microorganism of described recombinant vectors;
B8) containing B4) recombinant microorganism of described recombinant vectors;
B9) reconstitution cell of Avianreovirus σ A albumen is expressed.
2. the σ A albumen of Avianreovirus or its relevant biomaterial are preparing the application in activating cells in PI3K/Akt signal path product;
The relevant biological material of the σ A albumen of described Avianreovirus is following B1) to B9) in any one:
B1) to encode the nucleic acid molecule of σ A albumen of described Avianreovirus;
B2) containing B1) expression cassette of described nucleic acid molecule;
B3) containing B1) recombinant vectors of described nucleic acid molecule;
B4) containing B2) recombinant vectors of described expression cassette;
B5) containing B1) recombinant microorganism of described nucleic acid molecule;
B6) containing B2) recombinant microorganism of described expression cassette;
B7) containing B3) recombinant microorganism of described recombinant vectors;
B8) containing B4) recombinant microorganism of described recombinant vectors;
B9) reconstitution cell of Avianreovirus σ A albumen is expressed.
3. express the application in the cell model of reconstitution cell material of Avianreovirus σ A protein-active in the cell of preparation screening suppression infection Avianreovirus of Avianreovirus σ A albumen.
4. suppress the material of Avianreovirus σ A protein-active preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes.
5. suppress the application of material in the preparation suppression growth of Avianreovirus and/or the product of propagation of Avianreovirus σ A protein-active.
6. suppress the material of Avianreovirus σ A protein expression preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes.
7. suppress the application of material in the preparation suppression growth of Avianreovirus and/or the product of propagation of Avianreovirus σ A protein expression.
8. suppress the material of Avianreovirus σ A genetic expression preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes.
9. suppress the application of material in the preparation suppression growth of Avianreovirus and/or the product of propagation of Avianreovirus σ A genetic expression.
10. the material being action target spot with Avianreovirus σ A albumen is preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes;
Or,
The material being action target spot with Avianreovirus σ A albumen suppresses the application in the growth of Avianreovirus and/or the product of propagation in preparation;
Or,
The material being action target spot with Avianreovirus σ A gene is preparing the application prevented and/or treated in the product of the disease that Avianreovirus causes;
Or,
The material being action target spot with Avianreovirus σ A gene suppresses the application in the growth of Avianreovirus and/or the product of propagation in preparation.
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| CN113832113A (en) * | 2021-09-14 | 2021-12-24 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Novel duck reovirus attenuated strain and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107384942A (en) * | 2017-07-20 | 2017-11-24 | 佛山科学技术学院 | A kind of method for expressing novel duck reovirus s σ B recombinant proteins |
| CN113527463A (en) * | 2021-08-20 | 2021-10-22 | 广西壮族自治区兽医研究所 | Application of IFITM3 protein-related substance in preparation of medicines for treating diseases caused by avian reovirus |
| CN113527463B (en) * | 2021-08-20 | 2023-03-07 | 广西壮族自治区兽医研究所 | Application of IFITM3 protein related substance in preparation of medicine for treating diseases caused by avian reovirus |
| CN113832113A (en) * | 2021-09-14 | 2021-12-24 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Novel duck reovirus attenuated strain and application thereof |
| CN113832113B (en) * | 2021-09-14 | 2023-09-22 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Novel duck reovirus attenuated strain and application thereof |
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