CN103301442A

CN103301442A - Application of BST-2 protein in preparing medicaments for treating diseases caused by influenza A virus

Info

Publication number: CN103301442A
Application number: CN2012100591144A
Authority: CN
Inventors: 岑山
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2012-03-07
Filing date: 2012-03-07
Publication date: 2013-09-18

Abstract

The invention discloses an application of a BST-2 protein in preparing medicaments for treating diseases caused by influenza A virus. The BST-2 protein can inhibit IAV viral infection and prevent budding and releasing of the IAV virus from host cell surfaces through inhibiting expression of IAV virus M2 protein and interacting with the M2 protein. According to the invention, the BST-2 protein will become a new target for anti-influenza drugs, paves the way for research and development of the anti-influenza drugs in the future, and may even provide a potential therapeutic approach for treatment of the influenza.

Description

BST-2 albumen causes application in the medicine of disease at preparation treatment influenza A virus

Technical field

The invention belongs to biomedicine field, relate to BST-2 in the infection that suppresses influenza A virus and the application in discharging.

Background technology

Influenza A virus is common influenza virus, can cause the outburst of seasonal influenza and provincialism influenza.The host of influenza A virus is wider, except the people, also has pig, horse, leopard, marine mammal (sea dog, whale) and birds.Therefore surface antigen HA and the NA of influenza A virus then more easily morph, and easily cause popularly on a large scale, are caused by Influenza A1 virus (H1N1) such as spanish influenzas in 1918, cause that approximately 2,000 ten thousand people are dead.The global epidemic diseases epidemic situation that causes of influenza A virus (H1N1) in 2009 causes the whole world 1.85 ten thousand people dead.Therefore influenza A virus has consisted of serious threat to worldwide public health.

First type stream virus (Influenza A Virus, IAV) belong to orthomyxoviridae family, system contains 8 sections genomic enveloped viruses of strand RNA, and the genome total length is 14～15k nt approximately, altogether 11 kinds of albumen of codified: hemagglutinin (Hemagglutinin, HA), neuraminidase (Neuraminidase, NA), stromatin-1 (Matrix-1, M1), stromatin-2 (Matrix-2, M2 also claim ionophorous protein), nucleoprotein (Nucleoprotein, NP), and non-structural protein-1 (non-structural protein 1, NSP1), non-structural protein-2 (non-structural protein 2, NS2; Be also referred to as importin, nuclear export protein, NEP), polymerase acidic protein (polymerase acidic protein, PA), polymerase basic protein-1 (polymerase basic protein 1, PB1), polymerase basic protein-2 (polymerase basic protein 2, PB2) and polymerase basic protein-1-F2 albumen (polymerase basic protein 1-F2, PB1-F2).The biocycle of IAV virus comprises absorption and enters host cell, genome vRNPs transports into host cell nuclear, virus genomic transcribing in copying, and vRNPs transports out host cell nuclear, assemble on the host cell membrane with sprout, the stage such as release.Influenza virus sprouts from the cell membrane of host cell, and can to form spherical virus particle and the diameter that diameter is 100 nanometers after discharging be that 100 nanometers (nm) length is the fibrous virus of 2～20 microns (um).The peplos of virus is comprised of bilayer lipid membrane and HA, NA, the M2 albumen of host cell.To be that abundance is the highest on the bilayer lipid membrane be about 80% to HA albumen, and it is that 17%, M2 abundance is minimum that NA takes second place, and each virion only contains 16～20 molecules.

M2 albumen is the neccessary composition of viral infection.M2 albumen is formed a homotetramer and is become proton selectivity ion channel by 97 amino acid residue, divide for the terminal extracellular region of the N-that formed by 24 amino acid residues on the structure, the ion channel hole that the single span film district that 19 amino acid residues form forms and forms intracellular region tail end by 54 amino acid residues.M2 albumen has the selectivity ion channel activity, after virus is entered host cell, intracytoplasmic sour environment will activate this function of M2 albumen, make virion inside also reach sour environment, thereby so that the vRNPs in the virion separate final the copying of virus of being finished smoothly so that viral genome enters Cytoplasm fully from M1 albumen.

The assembling of influenza virus and sprouting is to finish at the Lipid Rafts of host cell membrane.Studies show that, the assembling of influenza virus and sprouting originates in the HA of Lipid Rafts mediation and NA albumen in the gathering on host cell membrane surface.The Lipid Rafts that is rich in phospholipid and cholesterol is dynamic diaphragm area.It can be used as the platform of protein aggregation, also is the main positions that many viruses are sprouted simultaneously, comprising influenza virus, and HIV-1 and Ebola virus.Chen etc. study discovery, and virus protein HA cross-film district is the important area that connects Lipid Rafts, and the disappearance in this cross-film district can reduce the formation that copies and suppress fine shape virus of virus.After virus protein HA and NA are positioned Lipid Rafts, cause the expansion with the Lipid Rafts zone of interconnecting between Lipid Rafts, thereby cause the outside deformation of cell membrane, and then start sprouting of influenza virus.Shown in Fig. 1-A, the born of the same parents of HA and NA inner (redness) and M1 albumen (purple) combination; Shown in Fig. 1-B, M1 albumen then with the NP protein-interacting, raise vRNPs complex (yellow spirillum).Simultaneously, M1 albumen by with the M2 protein-interacting, raise M2 albumen to virus the position of sprouting.Shown in Fig. 1-C, after virus had been packed enough vRNPs complexs, M2 albumen passes through its α spiral (AH) thereby regional conformation change changes host cell membrane curvature, and then finish the effect that film is sheared.At last,, cut off viral HA albumen and be connected with specificity between the host cell membrane receptor by its neuraminidase hydrolysis by NA albumen, finally finish Virus release.Whole IAV virus is sprouted model shown in Fig. 1-D.Therefore, the M2 albumen function that not only has an ionophorous protein also has finish the critical function that film is sheared when IAV virus is sprouted.

At occurring in nature, host cell exists the defense mechanism to virus, and these defense mechanisms are to be finished by the relevant alpha mediated host's factor of IFN of the complicated and diversified natural immunity in the host cell.Bone Marrow Stromal cell antigen2 (BST-2, again called after Tetherin/CD317/HN1.24) is the host's restriction factor by a kind of interferon (IFN α) mediation of recently finding.BST-2 albumen belongs to II type single span film AQP-CHIP, and BST-2 albumen can form dimer.BST-2 albumen is comprised of 180 amino acid residues, apparent molecular weight is in 28～45kDa, has special topological structure, comprise N-terminal (N-term), cross-film district (TM), the extracellular region that is positioned at born of the same parents and the C-terminal (as shown in Figure 2) that modifies with glycosyl-phosphatidyl inositol anchor (glycosyl-phosphatidylinositol (GPI) anchor), thereby be fixed on the Lipid Rafts of cell membrane by BST-2 albumen two ends.HIV (human immunodeficiency virus) (HIV-1) is that first is found can be by the enveloped virus that discharges from host cell surface at the new virion that produces of the BST-2 of cell surface severe inhibition (suppressing virus discharges more than 90%).Along with progress of research, the antiviral activity that it is found that BST-2 not only is confined to HIV-1, and to other retrovirus such as Ebola virus (Ebola), marburg virus (Marburg), Lassa virus (Lassa), kaposi's sarcoma virus (KSHV), murine leukemia virus (MLV), the Virus releases such as multiple enveloped virus such as porcine hepatocyte endogenous retrovirus (PERV), and then the copying of blocking virus.This is indicating that BST-2 has widely inhibition activity to the enveloped virus that discharges by sprouting.Influenza virus also belongs to enveloped virus, but has not yet to see the release of BST-2 infected by influenza granule and copy the report of impact.In addition, above-mentioned enveloped virus also can overcome by different virus proteins BST-2 to its inhibition of sprouting, such as the Vpu albumen of HIV-1, and the Env albumen of HIV-2, the Env/Nef albumen of SIV Virus Interaction, the GP albumen of Ebola virus and the K5 albumen of kaposi's sarcoma virus.Wherein, research more thorough is that the expression of Vpu albumen by downward modulation BST-2 of HIV-1 overcomes BST-2 to the HIV-1 Virus release.Since influenza virus also Lipid Rafts (lipid raft) as assembling and the platform that sprouts, so the sprouting and discharge of BST-2 certain influence influenza virus.

Summary of the invention

In order to address the above problem, the invention provides the application in the medicine of the disease that preparation treatment influenza A virus causes of BST-2 albumen or its active mutant.

In the present invention, the sequence preference of described BST-2 albumen is shown in SEQ ID No.1.The agedoite that the active mutant of described BST-2 albumen is preferably the agedoite of the 65th of N-terminal and the 92nd replaces with the BST-2 albumen of alanine.

In the present invention, the BST-2 albumen of heterogenous expression total length suppresses approximately 1000 times of IAV infection rates, and unexpectedly, and this mutain of BST-2 (N65A/N92A) is 10 times of total length BST-2 albumen suppression ratio to approximately 10000 times of the infection rates of IVA.

Among the present invention, the disease that described influenza A virus causes is viral influenza, is preferably influenza A.

The invention provides a kind of medicine for the treatment of the disease that influenza A virus causes, it contains BST-2 albumen or its active mutant of effective dose.

Among the present invention, the strain of described influenza A virus can be any pathogenic strain well known in the art, is preferably Influenza A virus/WSN/33 strain.

The present invention takes the kinds of experiments technological means by take Influenza A Virus/WSN/33 strain as experimental subject, and the phenomenon and the inhibition mechanism thereof that host's factor B ST-2 are suppressed influenza A virus disclose step by step.

1.BST-2 protein-specific ground suppresses the infectivity of influenza virus; And this inhibition is dose dependent to the BST-2 protein expression level.

2. can find out by the burnt experimental result of Electronic Speculum, ultracentrifugation and laser light copolymerization, BST-2 albumen can reduce the level that IAV virus is sprouted and discharged specifically.

3. learn by laser co-focusing, BRET and Co-IP experimental result, BST-2 albumen inhibition IAV viral infection and IAV virus are sprouted from the host cell membrane surface and are discharged by suppressing IAV virus protein M2 expression and finishing with its interaction.

The present invention makes BST-2 will become the novel targets of following Tamiflu, and paves the way for the research and development of following Tamiflu, even a potential orientation treatment may be provided for the treatment of influenza.

Description of drawings

Figure 1 shows that the IAV virus model that sprouts.

Figure 2 shows that the monomer structure feature of BST-2 albumen.

The infectivity (A) that Figure 3 shows that IAV reduces (50,100...1000ng be the amount of the Bst2 expression plasmid of transfection 293T cell) gradually with the increase of BST-2 expression (B).

Figure 4 shows that copying with appeal of IAV virus suppressed by BST-2 albumen in the mdck cell.A figure is 4-5 times that the fluorescent value of MDCK group is about the MDCK-BST-2 group; B figure is the expression that western blot detects BST-2 in the MDCK-BST-2 cell line.C figure is the relative virus infectivity of two cell lines of MDCK and MDCK-BST-2.

Figure 5 shows that and knock out BST-2 albumen in the Hela cell and to the inhibition of IAV virus replication.A expresses the level of Bst2 in Hela, Hela-CDS, Hela-UTR and the Hela-Vpu cell line; B, the expression of Bst2 in Hela, Hela-CDS, Hela-UTR and the Hela-Vpu cell line of infection IAV; C, the relative virus infectivity of Hela, Hela-CDS, Hela-UTR and Hela-Vpu cell line.

Figure 6 shows that the alpha mediated endogenous BST-2 of IFN protein expression, thereby suppress copying of IAV virus.Add the variation of interferon-induced rear Bst2 expression and the variation of the level that IAV copies in A, B:Hela, Hela-CDS, Hela-UTR and the Hela-Vpu cell line.Add the variation of interferon-induced rear Bst2 expression and the variation of the level that IAV copies in C, D:A549, A549-CDS and the A549-UTR cell line.

Figure 7 shows that the sudden change of BST-2 albumen and disappearance are for the impact of IAV viral infection.A, the schematic diagram of the deletion mutant of constructed BST-2 albumen; B, the artificial Bst2 that utilizes the albumen of structural similarity to be spliced; C, the Western blot testing result of the various deletion mutants of BST-2 albumen; D; The antiviral activity of the various deletion mutants of BST-2 albumen and artificial Bst2 detects.

Figure 8 shows that Electronic Speculum detects BST-2 albumen to the inhibition of IAV virus.A figure demonstration is compared with the MDCK group, and MDCK-BST-2 group cell surface has obvious influenza virus, and is fibrous in the majority; , B figure (B is the partial enlarged drawing in the A picture frame) shows that MDCK-BST-2 group cell virus granule then is the string shape and a viral end is connected with cell membrane, to discharge from cell surface fully.This shows that BST-2 albumen has suppressed the release of IAV to a certain extent; Figure C, transmission electron microscope observing MDCK group and MDCK-BST-2 group infect influenza virus that IAV virus result shows MDCK-BST-2 group cell surface and are and go here and there shape or link to each other in twos, have obvious BST-2 albumen to exist around the virus, mdck cell surface virus mostly is the single virus release of sprouting.

Figure 9 shows that detecting BST-2 albumen suppresses IAV virus release protein level design sketch.Infect respectively 293T cell (A) or MDCK (B) cell that has BST-2 to express with the IAVWSN/33 strain, after 36 hours, Western Blot detects the expression of various virus proteins in virion and the cell.

Figure 10 shows that BST-2 albumen and IAV virus M2 protein-interacting: by (A) Coip, (B) Bret and (C) confocal microscope detect common location proof IAV virus M2 protein-interacting between M2 and the Bst-2.

The specific embodiment

Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.

Embodiment 1

Test material

1. key instrument

2. plasmid

1) influenza A virus (IAV) reverse genetic eight pUC pUCs are according to document (Hoffmann, E., et al., A DNA transfection system for generation of influenza A virus from eight plasmids.Proc Natl Acad Sci U S A, 2000.97 (11): p.6108-13) prepare, be respectively: pHW181-PB2, pHW182-PB1, pHW183-PA, pHW184-HA, pHW185-NP, pHW186-NA, pHW187-M, pHW188-NS.This plasmid system used carrier is pHW2000, and viral cDNA is inserted between rna plymerase i (pol I) promoter and the terminator sequence; The transcript unit of pol I then is positioned between rna plymerase ii (pol II) promoter and the polyA sequence.Can synthesize viral strand RNA and mRNA respectively in cell, synthetic virus protein subsequently finally is assembled into and has infective influenza virus particles.

2) the shRNA plasmid is available from Sigma company

3) plasmid pHH-Gluc is according to the article of delivering (the de Vries of Dr.Erik de Vries, E, et al., Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway.PLoS Pathog.7 (3): p.e1001329.) preparation.

4) bioanalysis light energy resonance transfer (Bioluminescence Resonance Energy Transfer, BRET) system's plasmid (available from BioSignal Packard).

3. cell strain

1) dog renal epithelial cell (Madin-Darby canine kidney, MDCK):

Number:CCL-34 ^TM

2) people's alveolar epithelium cancerous cell (A549): Number:CCL-185 ^TM

3) human embryonic kidney epithelial cells (293T):

Number:CRL-11268 ^TM

4) cervical cancer cell (Hela):

Number:CCL-2

4. cell culture medium

1) the DMEM-high glucose medium is available from GIBCO: adding final concentration is 100U/ml PS and 10% inactivated fetal bovine serum (GBICO-FBS), is used for the cultivation of 293T cell, A549 cell and Hela cell.

2) α-MEM high glucose medium is available from GIBCO: adding final concentration is 100U/ml PS and 10% inactivated fetal bovine serum (GBICO-FBS), is used for the cultivation of mdck cell.

5. viral maintenance medium

1) the DMEM-high glucose medium is available from GIBCO: adding final concentration is 2% inactivated fetal bovine serum (GBICO-FBS), is used for IAV copying at 293T cell, A549 cell and Hela cell.

2) α-MEM high glucose medium is available from GIBCO: adding final concentration is 2% inactivated fetal bovine serum (GBICO-FBS), is used for IAV copying at mdck cell.

6. test kit

1) the little extraction purification kit of plasmid Easy Pure Mini Plasmid Purification Kit: be ShiJi Co., Ltd available from health.

2) the large extraction reagent kit Hi-Speed of plasmid Plasmid Maxi Kit: available from QIAGEN company

3) plasmid transfection test kit LipofectAMINETM2000 Reagent: available from Invitrogen company.

4) glue reclaims test kit QIA quick Gel Extraction kit: available from QIAGEN company.

5) NeonTM Transfection System 100 μ l Kit: available from Invitrogen company.

6) enhanced chemical luminescent solution (ECL): available from Millpore company

7. reagent

7.1 antibiotic

1) ampicillin (Sigma): 100mg/mL, 1g Ampicillin dissolves in the 10mL distilled water, 0.22 μ m membrane filtration degerming, packing ,-20 ℃ of preservations.

2) G418 (Gibco): 100mg/ml, 100mg G418 dissolves in the 1mL DDW, 0.22 μ m membrane filtration degerming, packing ,-20 ℃ of preservations.

3) kanamycin (Amresco) 25mg/ml, 1g kanamycin dissolves in the 40mL distilled water, 0.22 μ m membrane filtration degerming, packing ,-20 ℃ of preservations.

4) puromycin (Invitrogen): final concentration is 1mg/100 μ l ,-20 ℃ of preservations.

5) HYG (Sigma): 25mg/ml, 4 ℃ of preservations.

7.2 immunofluorescence dyeing reagent

1) fixative 4% paraformaldehyde: available from the green skies, Haimen, Jiangsu biotechnology company.

2) Mounting Medium with DAPI: available from China fir Golden Bridge company limited in Beijing.

3) immunostaining primary antibodie diluent: available from the green skies, Haimen, Jiangsu biotechnology company.

4) immunostaining two anti-diluents: available from the green skies, Haimen, Jiangsu biotechnology company.

5) immunostaining confining liquid: available from the green skies, Haimen, Jiangsu biotechnology company.

6) immunostaining mountant: available from the green skies, Haimen, Jiangsu biotechnology company.

7) immune-gold labeled goat antirabbit-10nm gold: available from Beijing Wan Huashi company.

7.3 antibody

1) primary antibodie

The anti-human GFP polyclonal antibody of rabbit is available from Santa Cruz;

Mouse anti human β-actin monoclonal antibody is available from Santa Cruz;

The anti-rabbit HA-probe of mice (Y-11) monoclonal antibody is available from Santa Cruz;

The anti-mice Flu-HA of rabbit monoclonal antibody is available from Santa Cruz;

The anti-mice Flu-M1 of rabbit monoclonal antibody is available from Santa Cruz;

The anti-mice Flu-M2 of rabbit monoclonal antibody is available from Santa Cruz;

The anti-mice Flu-NP of rabbit monoclonal antibody is available from Santa Cruz;

The anti-mice Flu-PB1 of rabbit monoclonal antibody is available from Santa Cruz;

The anti-rabbit Flu-PB2 of mice monoclonal antibody is available from Santa Cruz;

The anti-mice Flu-NA of rabbit monoclonal antibody is available from Sino Biological Inc.;

The anti-BST-2 monoclonal antibody of rabbit is received in America NI H.

2) two is anti-

Horseradish peroxidase (HRP) labelling goat anti-rabbit igg;

Horseradish peroxidase (HRP) labelling mountain sheep anti-mouse igg;

FITC labelling goat anti-mouse IgG;

TRITC labelling goat anti-mouse IgG;

The anti-rabbit igg of TRITC labelling mice;

The anti-rabbit igg of FITC labelling mice;

7.4 organic solvent

1) methanol: available from the Beijing Chemical Plant

2) isopropyl alcohol: available from the Beijing Chemical Plant

3) ethanol: available from the Beijing Chemical Plant

4) acetone: available from the Beijing Chemical Plant

5) ethyl acetate: available from the Beijing Chemical Plant

6) DMSO: available from Solarbio

7.5 other consumptive materials

1) 96 porocyte culture plates, 24 porocyte culture plates, 6 porocyte culture plates, 60mm Tissue Culture Dish,

Tissue Culture Dish, 2ml evolute cell cryopreservation tube etc.: available from Costar company

2) the uciferase activity analysis is with the opaque blank in 96 holes: available from Costar company.

3) 0.45 μ M PDGF film; 0.22 μ M microporous filter membrane: available from Millipore company

4) dye in advance the molecular weight of albumen standard: New England Biolabs.

5) dna molecular amount standard DL2000: available from TaKaRa company.

6) protease inhibitor: available from the Roche bio tech ltd.

7) RIPA buffer: available from the green skies, Haimen, Jiangsu biotechnology company.

8) NP-40 lysate: available from the green skies, Haimen, Jiangsu biotechnology company.

Test method

1. plasmid construction

1.1BRET system's plasmid construction

Make up pEYFP-N1-HA (flu) plasmid: take plasmid pHW184-HA as template, pcr amplification ha gene inserts in the pEYFP-N1 plasmid (available from BioSignal Packard) (restriction enzyme site is BamH I and Kpn I), and primer sees Table 3.

Table 3 makes up the primer of pEYFP-N1-HA (flu)

Make up pEYFP-C1-NA (flu) plasmid: take plasmid pHW186-NA as template, pcr amplification na gene inserts in the pEYFP-C1 plasmid (restriction enzyme site is HindIII and BamH I), and primer sees Table 4.

Table 4 makes up the primer of pEYFP-N1-NA (flu)

Make up pEYFP-N1-M1 (flu) plasmid: take plasmid pHW187-M as template, pcr amplification ml gene inserts in the pEYFP-N1 plasmid (restriction enzyme site is HindIII and BamH I), and primer sees Table 5.

Table 5 makes up the primer of pEYFP-N1-M1 (flu)

Make up pEYFP-N1-M2 (flu) plasmid: because coding m2 gene is at influenza M fragment (26-51, has jumping characteristic 740-1007nt), at first take plasmid pHW187-M as template, M2-A and M2-S1 are approximately 740-1007nt part of primer PCR amplification m2 gene; And then take this fragment as template, M2-A and M2-S2 (primer is brought M gene 26-51nt into) pcr amplification total length m2 gene, insert at last (restriction enzyme site is HindIII and BamH I) in the pEYFP-N1 plasmid, primer sees Table 6.

Table 6 makes up the primer of pEYFP-N1-M2 (flu)

1.2 the structure of influenza virus protein expression vector

Make up pcDNA3.1-HA (flu) plasmid: take plasmid pHW184-HA as template, pcr amplification ha gene inserts in the pcDNA3.1 plasmid (restriction enzyme site is HindIII and Kpn I), and primer sees Table 7.

Table 7 makes up the primer of pcDNA3.1-HA (flu)

Make up pcDNA3.1-NA (flu) plasmid: take plasmid pHW186-NA as template, pcr amplification na gene inserts in the pcDNA3.1 plasmid (restriction enzyme site is HindIII and Kpn I), and primer sees Table 8.

Table 8 makes up the primer of pcDNA3.1-NA (flu)

Make up pcDNA3.1-M1 (flu) plasmid: take plasmid pHW187-M as template, pcr amplification ml gene inserts in the pcDNA3.1 plasmid (restriction enzyme site is Nhe I and Kpn I), and primer sees Table 9.

Table 9 makes up the primer of pcDNA3.1-M1 (flu)

Make up pcDNA3.1-M2 (flu) plasmid: because coding m2 gene is at influenza M fragment (26-51, have jumping characteristic 740-1007nt), at first take plasmid pHW187-M as template, M2-A and M2-S1 be the about 740-1007nt part of primer PCR amplification m2 gene; And then take this fragment as template, M2-A and M2-S2 (primer is brought M gene 26-51nt into) pcr amplification total length m2 gene, insert at last (restriction enzyme site is HindIII and Kpn I) in the pcDNA3.1 plasmid, primer sees Table 10.

Table 10 makes up the primer of pcDNA3.1-M2 (flu)

2. the preparation of influenza A virus (IAV)

Inoculation 2ml concentration is 2.5～3.0 * 10 in every hole of six orifice plates ⁵The 293T cell of individual/well and mdck cell (1: 1);

After cultivating 24h, transfection IAV reverse genetic eight pUC pUCs, each plasmid consumption are 0.4 μ g/ hole, and transfection reagent is Lipofectamine 2000, and according to operation instructions, 10 μ l are used in every hole.

Behind the transfection 6h, be replaced by complete DMEM culture medium.

Every hole adds 20 μ l pancreatin behind the 12h.

Receive poison behind the 36h, after supernatant was collected, centrifugal 3000g * 10min received supernatant ,-80 ℃ of preservations.

3. the titration of virus infectivity

3.1TCID ₅₀Mensuration

Half cell culture infective dose TCID ₅₀(50%tissue culture infective dose) can reflect virus infectivity exactly.

In 96 orifice plates, virus liquid is carried out 10 times of gradient dilutions, from 10 ^-1～10 ^-10

Then its correspondence is inoculated in another 96 orifice plate, each dilution factor one tandem is totally 8 holes, and 100 μ l are inoculated in every hole.Add 2～3 * 10 in every hole ⁵/ ml mdck cell suspension 100 μ l.

If the normal cell contrast, two tandems (100 μ l growth-promoting medias+100 μ l cell suspension) are made in the normal cell contrast.

Day by day observation of cell pathological changes and record the result generally needs to observe 5～7 days.

The result calculates, and presses Reed-Muench Liang Shi method or Karber method.

3.2 utilize the relative virus infectivity of luciferase (Gaussia luciferase, Gluc) fast detecting IAV

Inoculation 293T cell is in 24 orifice plates, and concentration is every hole 1 * 10 ⁵/ ml, 37 ℃, 5%CO ₂Cultivate.

Behind the 24h, with plasmid pHH-Gluc (25ng/ hole) Lipofection 2000 transfection reagent transfections.

Behind the transfection 24h, more renew the DMEM culture medium postoperative infection virus of 10%FBS, viral volume≤50 μ l.

After infecting 24h, collect supernatant, every hole adds cleer and peaceful 1 μ l luciferase substrate Coelenterazine-h solution on the 100 μ l, behind the mixing, uses microplate reader to detect in the 480nm place fast.Acquisition time is 5～10sec.When virus infectivity is higher, the luciferase amount is larger, and the value of reading is also higher.

4. cell culture

1) mdck cell

Mdck cell is incubated in the α that contains 100U/ml penicillin, streptomycin (PS) and 10% hyclone (FBS)-MEM culture medium.The mdck cell system of stably express BST-HA is incubated at and contains 800 μ g/ml neomycin (G418), in the α of 100U/mlPS and 10%FBS-MEM culture medium.Place 5%CO ₂, cultivate in 37 ℃ of incubators.

2) A549 cell

The A549 cell culture is in the DMEM culture medium that contains 100U/ml PS and 10%FBS.The A549 cell line of stably express shRNA-CDs, shRNA-UTR is incubated at and contains 2 μ g/ml puromycins, in the DMEM culture medium of 100U/ml PS and 10%FBS.Place 5%CO ₂, cultivate in 37 ℃ of incubators.

3) 293T cell

The 293T cell culture is in the DMEM culture medium that contains 100U/ml PS and 10%FBS.Place 5%CO ₂, cultivate in 37 ℃ of incubators.

4) Hela cell

The Hela cell culture is in the DMEM culture medium that contains 100U/ml PS and 10%FBS.The Hela cell line of stably express shRNA-CDs, shRNA-UTR5 is incubated at and contains 2 μ g/ml puromycins, in the DMEM culture medium of 100U/ml PS and 10%FBS.The Hela cell line of stably express Vpu albumen is incubated at and contains 400 μ g/ml neomycin (Neomycin), in the DMEM culture medium of 100U/ml PS and 10%FBS.Place 5%CO ₂, cultivate in 37 ℃ of incubators.

5. ultracentrifugation purification IAV is viral

Be after virus liquid is collected the 15ml centrifuge tube with the cell culture fluid supernatant that infects the IAV certain hour, after the PBS trim, in 4 ℃, the centrifugal 20min of 3000rpm.

Get viral supernatant liquid, it is added in the 5ml volume ultracentrifugation pipe (REF:326819) that has added in advance 30% sucrose pad (0.5ml) (can omit as not needing this step of sucrose pad purified virus), the PBS trim (error≤0.01g), in 4 ℃, the centrifugal 3h of 250000g (rotor: Beckman MSL50).

Carefully abandon supernatant, adding PBS transfers in the EP pipe after repeatedly blowing and beating for several times.

Detect as directly carrying out Western Blot, add RIPA buffer (50mmolL ^-1Tris-HCl (pH 7.4), 150molL ^-1NaCl, 1%NP-40,0.1%SDS, Roche protease inhibitor cocktail) 40 μ l, repeatedly after the piping and druming for several times, transfer in the new EP pipe, add 10 μ l, 5 * Loading Buffer.

After boiling 10min, carry out Western Blot detection in-20 ℃ of preservations or as described in method 7.

6.IAV Viral infection

Before infecting virus cell culture fluid is replaced by viral maintenance medium (culture medium that contains 2%FBS).

Needs according to concrete experiment are the virus stock solution used that infection multiplicity (Multiplicity Of Infection, MOI) adds certain volume according to virus and cell number ratio, and add an amount of pancreatin.

To change the new virus maintenance medium again behind the 1h, place 5%CO ₂, cultivate in 37 ℃ of incubators.

7. detected by Western blot (Western Blot)

1) the centrifugal 5min collecting cell of 2000g, total protein of cell is extracted in cell pyrolysis liquid RIPA buffer cracking.

2) SDS-PAGE electrophoretic separation protein sample.

3) be transferred on the pvdf membrane.

4) film is sealed 1h in 5% defatted milk powder;

5) add 4 ℃ of overnight incubation of primary antibodie (configuration of 2% defatted milk powder), or room temperature 3h (vibration).

6) use PBST (0.1%Tween 20in PBS) to wash 3 times, each 5min.

7) hatch again two anti-(configuration of 2% defatted milk powder) 1h.

8) re-use PBST and wash 3 times, each 5min.

9) use the colour developing of ECL test kit, gel imaging system is collected data.

8. laser co-focusing (Confocal Microscopy)

The cell that 1) will be inoculated on the burnt ware of copolymerization (Confocal plate) uses fixedly 10min of 4% paraformaldehyde.

2) wash 3 times each 5min with immunostaining cleaning mixture (Immunol Staining Wash Buffer).

3) use confining liquid (containing 2%FBS) sealing 2h.

4) the primary antibodie diluent of adding destination protein, 4 ℃ of overnight incubation.

5) use immunostaining cleaning mixture washing 3 times, each 5min.

6) add corresponding two anti-diluents in shaking table or the static 2h of hatching.

7) washing is 3 times, each 5min.

8) adding is with the immunostaining mountant of DAPI.

9) use Volocity software operation Germany Lycra TCM-SP2 to carry out image collection.

9. scanning electron microscope (Scanning Electron Microscope)

1) with the cell climbing sheet for preparing (about 1cm ²) discard culture medium, use the PBS of pre-cooling to wash 2 times, each 5min.

2) place fixative (4% paraformaldehyde+2.5% glutaraldehyde) 10min.

3) the PBS washing is 3 times.Each 10min.

4) fixing (1% osmic acid) 2h after.

5) isoamyl acetate 15min or tert-butyl alcohol 15min are changed in dehydration (ethanol 50%, 70%, 80%, 90%, 95% each 15min).

6) use vacuum evaporating instrument JEE-420 to carry out plated film after the lyophilization.

7) use the desk-top ultramicroscope of Hitachi TM-1000 to observe and image taking.

10. co-immunoprecipitation (Co-Immunoprecipitation, Co-IP)

1) cultured cell is abandoned culture medium, with cell in 5ml pre-cooling PBS piping and druming and the collection Ф 10cm Tissue Culture Dish.

2) 4 ℃, the centrifugal 5min of 2000g abandon supernatant.

3) 1ml pre-cooling PBS is resuspended, transfers in the 1.5ml EP pipe.The centrifugal 5min of 2000g abandons supernatant.

4) add 0.5ml NP-40 lysate (50mM Tris-HCl (pH 7.4), 150mM NaCl, 1%NP-40, Roche protease inhibitor) cracking, process 1h on ice, every 10min vortex vibration once.

5) 4 ℃, the centrifugal 10min of 12000g.Get supernatant, namely obtain total protein of cell.

6) take out 40 μ l, add 5 * albumen sample-loading buffer, 10 μ l.Boiling water bath 10min.The Pre-IP protein sample that obtains is stored in-20 ℃ or-80 ℃.

7) the residual protein sample replenishes volume to 0.5ml with above-mentioned NP-40 lysate, adds the anti-BST-2 antibody of rabbit (1: 400).

8) 1h is hatched in 4 ℃ of mixing.

9) add 20 μ l Protein A sepharose 4Bs, 4h is hatched in 4 ℃ of mixing.

10) 4 ℃, the centrifugal 5min of 1000g abandon supernatant.The PBS of 1ml pre-cooling on ice cleans 4 times.

11) 4 ℃, the centrifugal 5min of 1000g abandon supernatant, add 5 * albumen sample-loading buffer, 40 μ l, boiling water bath 3min.

12) the centrifugal 3min of 1000g gets supernatant.

13) Western Blot detects.

11. transmission electron microscope (Transmission Electron Microscope)

1) immuno-gold labeling (10nm-gold)

After cell behind the viral infection is resuspended, the centrifugal 5min of 800rpm, abandon supernatant, add 1ml PBS resuspended, the centrifugal 5min of 800rpm abandons and adds 4 ℃ of 1ml 4% paraformaldehydes behind the supernatant and place 10min, the centrifugal 5min of 800rpm, adding 1ml PBST is resuspended after abandoning supernatant, and the centrifugal 5min of 800rpm repeats 3 times.After adding the primary antibodie diluent, hatch 3h or spend the night for 4 ℃, the centrifugal 5min of 800rpm reclaims primary antibodie, adds PBST resuspended, and the centrifugal 5min of 800rpm repeats 3 times.Abandon and add two anti-(labelling 10nm Gold, working concentration 1: 10) diluents behind the supernatant, hatch the centrifugal 5min of 800rpm behind 1～2h, reclaim two anti-diluents, add PBS resuspended, the centrifugal 5min of 800rpm repeats 3 times, abandons at last supernatant.

2) dehydration

After chemistry is fixing, cell is dipped in ethanol, the acetone and other organic solvent to remove the free water of tissue.Ethanol 50%, 70%, 80%, 90%, 95% each 15min.

3) soak into

Use embedding medium, progressively replace the dehydrant in the cell, until resin is impregnated in cyto-architectural all spaces equably.

4) embedding

After soaking into, cell is put in the mould, injects the mixture such as epoxy monomer and sclerosing agent, make resin polymerization become hard solid by methods such as heating.

5) section

The diamant that the preparation ultrathin section will use special ultramicrotome Leica EM UC6 and make with the glass plate of fracture.With containing the part of biomaterial in the resin embedding piece, under stereoscopic microscope, be trimmed to tiny pyramid with blade first, again with ultramicrotome be cut into thickness moderate (

About) superthin section, section should be coupled to each other the formation slicing band successively.Slicing band floats on the water surface in the tank that is contained on the microtome.Section with the special 100 order copper mesh that are covered with thin supporting film, is fished for from the water surface usually.

6) section statining

The copper mesh that is loaded with section is floating or be immersed in the dyeing liquor and dye.The general employed stain of section statining is the double staining of metal uranium salt and lead salt.

7) transmission electron microscope Microscopic observation utilizes OSIS MEGAVIEW G2 side-plug-in CCD camera to adopt picture.

12. fluorescence quantitative PCR method

12.1 cell total rna extracts

1) gets 1.5ml EP pipe, with PBS piping and druming and collecting cell, clean once the rear centrifugal 5min of 800rpm, remove PBS, add 5-10 times of volume Trizol liquid, mixing.

2) room temperature is placed 5min, then adds the 0.2ml ratio with every 1ml Trizol liquid and adds chloroform, acutely sways 15sec.

3) room temperature is placed 10min, in 4 ℃, and the centrifugal 15min of 13000rpm.Get the supernatant water and manage in new EP, the ratio that adds 0.5ml in every 1ml Trizol liquid adds isopropyl alcohol, and room temperature is placed 10min, the centrifugal 10min of 12000rpm.

4) abandoning supernatant adds at least ratio 75% ethanol of 1ml, mixing, 4 ℃, the centrifugal 5min of 7500rpm in every ml Trizol liquid.

5) repeating step 4.

6) careful abandoning supernatant, then room temperature is placed 5-10min.

7) RNA (white) is dissolved in the DEPC water, places 10min.-70 ℃ of preservations.

12.2 viral RNA extracts

1) gets 25 μ l E.C. 3.4.21.64s in 1.5ml EP pipe, add 400 μ l virus liquids.

2) add 400 μ l BufferAL, concussion 15sec, 56 ℃ of water-bath 15min.

3) add 500 μ l dehydrated alcohol, concussion 15sec transfers to solution on the MinElute column, and the centrifugal 1min of 6000g discards solution;

4) add 500 μ l BufferAW1, the centrifugal 1min of 6000g discards solution.

5) add 500 μ l BufferAW2, the centrifugal 1min of 6000g discards solution.

6) add 500 μ l, 75% ethanol, the centrifugal 1min of 6000g discards solution, the centrifugal 1min of 20000g.

7) column is placed on the new EP pipe, add 50 μ l RNase-free water, room temperature 1min, the centrifugal 1min of 20000g namely gets viral RNA.

12.3Real-time RT-PCR detects

Use Bio-Rad test kit iScript ^TM(included random primer) comes reverse transcription cDNA ,-20 ℃ of preservations.

Use Bio-Rad test kit SsofastTest ^TMCarry out Real-time PCR and detect (PCR condition: 95 ℃ of 30sec; 95 ℃ of 2sec; 60 ℃ of 30sec; 40 circulations).Primer sequence such as table 11.

Table 11 is used for the primer of Real-Time RT-PCR

13. the bioluminescence resonance energy shifts (BRET) and detects protein-interacting

2 * 10 ⁵/ well293T cell is inoculated 6 orifice plates.

Behind the 24h, by carrying out cotransfection such as grouping in the table 12

The amount in the every hole of table 12BRET plasmid

Behind the transfection 48h, cell is collected to 1.5ml EP pipe with the PBS of 1ml pre-cooling piping and druming in every hole, dispel cell after, from each EP pipe, get 100 μ l cell suspension to 96 orifice plates (special-purpose 96 orifice plates of microplate reader), every hole adds 1 μ l substrate survey BRET.

Result of the test

1.BST-2 albumen suppresses the IAV infectivity and is dose dependent

With the BST-2 plasmid according to 0,50,100,200,500, the dosage of 1000ng/ hole (six orifice plates) and IAV eight pUC pUC cotransfections are in the 293T cell, collect the upper cleer and peaceful cell sample of virus behind the 48h, carry out respectively the detection of virus sample titre and cell protein immunoblotting and detect.As shown in Figure 3, the IAV virus titer reduces gradually with the increase of BST-2 expression.When BST-2 plasmid amount was 50～100ng, the relative virus titer of IAV was about about 40%.When BST-2 plasmid amount was 500ng, the relative virus titer of IAV was about about 3%.When BST-2 plasmid amount was 1000ng, the relative virus titer of IAV was about about 0.2% (result is three independent experiment gained).

2.MDCK BST-2 albumen suppresses copying of IAV virus in the cell

To be in the mdck cell system of mdck cell and the stably express BST-2 albumen of exponential phase: MDCK-BST-2 is inoculated in the burnt ware of copolymerization in right amount, 12h postoperative infection IAV virus (MOI=0.1), change fresh viral maintenance medium behind the 1h, carrying out laser co-focusing behind the 24h described in experimental technique 7 detects, primary antibodie is Anti-HA, two anti-are sheep anti mouse FITC, detect the distribution situation of HA in MDCK and MDCK-BST-2 cell, thereby judge the copy situation of IAV virus in two cells.Use Image J software that 5 field-of-view images of every group of cell are carried out the statistical computation of fluorescent value, get at last 5 field-of-view image fluorescent values and get meansigma methods.Shown in Fig. 4 A, the fluorescent value of MDCK group is about 4～5 times of MDCK-BST-2 group.Fig. 4 B is Western Blot checking MDCK-BST-2 stable expression cell line BST-2 protein expression level.Fig. 4 C is for infecting WSN (MOI=0.1) in MDCK and two cell lines of MDCK-BST-2, collect viral supernatant behind the 24hpi (hour post infection) by the relative virus infectivity of detection as described in method 2.2, the result shows, when mdck cell was expressed BST-2, appeal decline was about about 40% under the same terms.To sum up the result shows, BST-2 albumen also can suppress copying of IAV virus in mdck cell.

3.Hela BST-2 albumen reduces IAV Viral infection and viral yield in the cell

3.1Hela cytotostatic is expressed shRNA-CDS/shRNA-UTR

The Hela cell can endogenous ground be expressed BST-2 albumen, knocks out BST-2 albumen in the Hela cell by shRNA, its expression is reduced or does not express.Each cell line BST-2 protein expression level of Hela, Hela-CDS, Hela-UTR and Hela-Vpu is shown in Fig. 5 A.Compare with the Hela cell, the expression of Hela-CDS is minimum.The result shows, in the Hela cell, shRNA-CDS/shRNA-UTR can specificly knock out the BST-2 protein expression.After infecting IAV, the expression of BST-2 does not have significant change (Fig. 5 B) in each cell line.

3.2Hela BST-2 albumen can reduce the IAV Viral infection in the cell

IAV viral infection (MOI=5) Hela, Hela-CDS, Hela-UTR and each cell line of Hela-Vpu behind the 24hpi, are collected and are processed cell and use Western Blot method to detect the expression of BST-2.Virus infectivity is then according to method 2.2 described detections.Experimental result is shown in Fig. 5 C, and appeal is the highest relatively to be knocked the IAV virus of Hela-CDS groups of cells of BST-2, is about 5 times more than of Hela groups of cells; The Hela-UTR group is organized approximately about 3 times so the relative appeal of its IAV also is lower than the Hela-CDS group and is higher than Hela because the BST-2 expressing quantity is higher than Hela-CDS, is lower than Hela.The result shows, the appeal that the BST-2 albumen of endogenous expression in the Hela cell can specific inhibition IAV.

4.IFN alpha mediated endogenous BST-2 albumen suppresses copying of IAV virus

4.1IFN α can increase BST-2 protein expression amount in Hela and the A549 cell

Behind Hela and A549 cell use 1000U/ml IFN α processing 48h, collect cell, and extract total protein of cell and carry out Western Blot detection BST-2 expressing quantity.Use Image J software that each protein band among the Western Blot result is carried out statistical analysis, thereby calculate albumen relative expression level.The result is shown in Fig. 6 A, 6C, and the cell BST-2 albumen relative expression level (being the ratio of BST-2 and β-actin expression) after IFN α processes is apparently higher than non-processed group cell.Wherein, processing Hela cell BST-2 albumen relative expression level value through IFN α is higher than without IFN α and processes Hela cell approximately 0.21.The result who processes with IFN α in A549, A549-CDS and the A549-UTR cell is similar to the Hela cell: the BST-2 relative expression level of A549-CDS, A549-UTR cell of processing through IFN α has rebound significantly with cell without IFN α processing.

4.2IFN alpha mediated endogenous BST-2 albumen can suppress copying of IAV virus

Behind Hela and A549 cell use 1000U/ml IFN α processing 12h, infect IAV virus (MOI is respectively 0.1,1,10), 48hpi collects viral supernatant, uses the relative virus infectivity of luciferase Gaussia luciferase (Gluc) fast detecting IAV.The result shown in Fig. 6 B, through IFN α process Hela cell IAV virus relatively infectious (Relative viral infectivity) be starkly lower than without IFN α and process the Hela groups of cells; And process the relatively infectious not significantly variation of IAV virus in Hela-CDS, Hela-UTR, the Hela-VPN cell with IFN α.Illustrate that IFN α suppresses copying of IAV virus by the endogenous BST-2 albumen that mediates.Through IFN α process A549-CDS, A549-UTR groups of cells IAV virus relatively infectivity also be higher than through IFN α and process the A549 groups of cells.This result also shows, the alpha mediated endogenous BST-2 albumen of IFN can suppress copying of IAV virus.

5.BST-2 the sudden change of albumen is for the impact of IAV virus replication

The coded sequence of BST-2 protein mutant is inserted in the polyclone district of carrier for expression of eukaryon pcDNA3.1 and obtains following BST-2 protein mutant expression plasmid: pBST-2 (Δ TM), pBST-2 (3Cys), pBST-2 (N65A/N92A), pBST-2 (Δ CC), pBST-2 (Δ GPI), pBST-2 (artTethrin), concrete preparation method is referring to list of references David Perez-Caballero et al, Tetherin inhibits HIV-1 release by directly tethering virions to cells.Cell.2009.139 (3): 499-511.

To the 293T cell, collect cell and viral supernatant liquid according to eight plasmids (as described in the method 2) cotransfection of 200ng/ hole (six orifice plates) and runoff yield Influenza Virus behind the 48h.Wherein, Δ TM is for knocking out the cross-film district; Δ GPI is for knocking out the glycosyl-phosphatidyl inositol site; 3Cys is the C-terminal 53,63 that knocks out BST-2, two sulfur connection site of 91 cysteine; N65A/N92A makes it lose glycosylated modification mutant for BST-2 the 62nd, 92 agedoite are become alanine; Δ CC is for knocking out BST-2 albumen coiled-coil zone.PBST-2 (artTeth) can express artificial constructed class BST-2 albumen: artTethrin, this albumen is all identical in size, topological structure and post translational modification with BST-2 albumen, but its major function district is all from the different same merit albumen of various aminoacid sequences.Cell detects according to the method 7 described Western Blot that carry out.Virus liquid is described according to method 3.1, measures viral TCID ₅₀BST-2 protein mutation disappearance pattern is such as figure as a result shown in the 7A.Each lacks protein expression level shown in Fig. 7 C, does not wherein detect Δ CC protein expression.Each lack albumen on the impact of IAV Viral infection shown in Fig. 7 D, compare with matched group, total length BST-2 albumen can suppress approximately 1000 times of IAV appeal.In the BST-2 mutain, BST-2 (Δ TM), BST-2 (3lys), BST-2 (Δ CC), BST-2 (Δ GPI) and art Bst-2 are for the infective impact of IAV and little; And BST-2 (N65A/N92A) group is compared with the antiviral activity of wild type Bst-2, and antiviral activity improves nearly 10 times.This result shows, the integrity of BST-2 primary structure is most important to its antiviral functions, and its secondary structure does not play a decisive role; Be 65 and 92 asparagine mutations alanine, make its forfeiture glycosylation function, can increase substantially its antiviral activity.

6.BST-2 albumen can suppress the IAV Virus release

6.1 Electronic Speculum (Electron Microscope) detects the inhibition that BST-2 albumen discharges IAV virus

6.1.1 scanning electron microscope (SEM) detects the inhibition that BST-2 albumen discharges IAV virus

With IAV viral infection (MOI=1) cell climbing sheet MDCK and MDCK-BST-2, collect cell during 8hpi described according to method 9, it is carried out scanning electron microscopic observation.The result compares with the MDCK group shown in Fig. 8 A, and MDCK-BST-2 group cell surface has obvious influenza virus, and is fibrous in the majority; Virion then is string shape (B be A picture frame in partial enlarged drawing) as can be seen from Figure 8B, and a viral end is connected with cell membrane, discharging from cell surface fully.

6.1.2 transmission electron microscope (TEM) detects the inhibition that BST-2 albumen discharges IAV virus

With IAV viral infection (MOI=1) cell climbing sheet MDCK and MDCK-BST-2, collect cell during 8hpi described according to method 10, it is carried out transmission electron microscope observing.The result compares with the MDCK group shown in Fig. 8 C, and the influenza virus of MDCK-BST-2 group cell surface is the string shape or links to each other in twos, and has obvious BST-2 albumen to have (arrow indication among Fig. 8 C) around the virus; Mdck cell surface virus then mostly is the single virus release of sprouting.This shows, BST-2 albumen has suppressed the IAV Virus release.

6.2 detecting BST-2, protein level suppresses the IAV Virus release

Plasmid pcDNA3.1-BST-2 and each 200ng transfection of pBST-2 (Δ TM) are done contrast with pcDNA3.1 to the 293T cell, 24h postoperative infection IAV virus (MOI=0.1) is collected cell and viral supernatant liquid during 24hpi.Cell is according to method 7 the above processing and carry out Western Blot detection; The virus supernatant is then described according to method 4, and at first then ultracentrifugation virus carried out Western Blot and detected.Experimental result is shown in Fig. 9 A, and the BST-2 group is compared with other experimental grouies, and virus protein N P and M1 expression do not change in the cell, and the M2 expressing quantity then has obvious decline; Virus (Virion) albumen NP expressing quantity does not change in the supernatant, and M1 albumen has obvious minimizing in BST-2 group and BST-2 (Δ TM), and M2 albumen then only obviously reduces in the BST-2 group.This shows, BST-2 (Δ TM) albumen only with suppress M1 albumen in the viral supernatant granule distribution but can not affect the IAV Virus release; BST-2 albumen then can suppress the expression of M2 albumen in cell, thereby further has influence on sprouting and release of virus.

In addition, with IAV viral infection (MOI=1) cell MDCK and MDCK-BST-2, collect cell and viral supernatant during 48hpi, carry out the above-mentioned experiment such as 293T, also obtained identical result (such as Fig. 9 B).

6.3RNA horizontal detection BST-2 suppresses the IAV Virus release

Plasmid pcDNA3.1-BST-2 and each 200ng transfection of pBST-2 (Δ TM) are done contrast with pcDNA3.1 to the 293T cell, 24h postoperative infection IAV virus (MOI=1) is collected cell and viral supernatant liquid during 24hpi.Carry out quantitative Real-Time PCR and detect in the cell and the viral RNA content in the viral supernatant liquid according to method 12 is described.The result is shown in Fig. 9 B, and viral RNA all is starkly lower than matched group in BST-2 group cell and the supernatant; PBST-2 (Δ TM) group is then without significant change.This shows, BST-2 has suppressed the IAV Virus release.

7.BST-2 albumen and IAV virus M2 protein-interacting affect assembling and the release of influenza virus

7.1 confocal laser scanning microscope BST-2 and M2 protein-interacting

After being laid on the Hela cell on the laser co-focusing plate, behind infection IAV virus (MOI=5) 24h, use laser confocal microscopes to observe according to method 8.The result is shown in Figure 10-A, and the Hela cell BST-2 albumen fluorescence (red) that infects IAV is bright Hela cell in not infecting obviously, this and as a result 3.1Western Blot testing result match; IAV virus protein M2 (green) locates (orange) altogether with BST-2 albumen (red).Figure 10 B is that 10A amplifies and the 3D sectional view, can find among the figure, and the common location of M2 albumen and BST-2 albumen only occurs in the interior but not surface of cell membrane of cell; In addition, the Hela cell surface of infection IAV has obvious M2 protein aggregation.This may be virus cell surface do not sprout fully discharge due to, this and Electronic Speculum result are able to mutual checking.The result shows, BST-2 interacts with M2 in cell and affects sprouting and release of IAV virus.

7.2 co-immunoprecipitation method checking BST-2 albumen and M2 protein-interacting

MDCK and MDCK-BST-2 cell are laid on the φ 10cm Tissue Culture Dish, during 6h postoperative infection IAV virus (MOI=0.1) 24hpi, use co-immunoprecipitation method checking BST-2 albumen and M2 protein-interacting according to method 10.The result obtains including IAV virus protein M1 and M2 in the co-immunoprecipitation sample by BST-2 albumen shown in Figure 10 C.This shows, BST-2 albumen and M1 and M2 protein-interacting in cell.

7.3BRET method checking BST-2 albumen and M2 protein-interacting

With plasmid pRluc-C3-BST2 and pEGFP-N1-M2 and pRluc-C3-BST2 and pEGFP-N1-Vpu respectively cotransfection in the 293T cell, collect cell behind the 24h, identify the interactions of BST-2 albumen and M2 albumen according to method 13 described use BRET.The result is shown in Figure 10 D, and positive is that the BRET value of BST-2 and Vpu albumen is about 0.47; The BRET value of BST-2 albumen and M1, NA, HA albumen all approaches with negative findings; The BRET value of BST-2 albumen and M2 albumen then is 0.48, and this shows that BST-2 albumen and M2 albumen combine in cell.

To sum up the result shows that BST-2 suppresses sprouting with release by reducing M2 protein expression level and finishing with the M2 protein-interacting of virus in the cell that has infected IAV virus.

The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims

1.BST-2 albumen or its active mutant application in the medicine of the disease that preparation treatment influenza A virus causes.

2. application as claimed in claim 1 is characterized in that, the active mutant of described BST-2 albumen is the albumen that the agedoite of the 65th of N-terminal and the 92nd 's agedoite replaces with alanine.

3. application as claimed in claim 1 or 2 is characterized in that, described disease is viral influenza.

4. application as claimed in claim 1 or 2 is characterized in that, the strain of described influenza A virus is Influenza A virus/WSN/33 strain.

5. medicine for the treatment of the disease that influenza A virus causes, it contains BST-2 albumen or its active mutant of effective dose.