WO2018129767A1 - A型流感病毒温度敏感性的关键磷酸化位点及其应用 - Google Patents

A型流感病毒温度敏感性的关键磷酸化位点及其应用 Download PDF

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WO2018129767A1
WO2018129767A1 PCT/CN2017/072063 CN2017072063W WO2018129767A1 WO 2018129767 A1 WO2018129767 A1 WO 2018129767A1 CN 2017072063 W CN2017072063 W CN 2017072063W WO 2018129767 A1 WO2018129767 A1 WO 2018129767A1
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plasmid
phh21
virus
protein
recombinant
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刘文军
郑伟楠
李晶
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中国科学院微生物研究所
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Definitions

  • the present invention relates to a key phosphorylation site for temperature sensitivity of influenza A virus and uses thereof.
  • Influenza A viruses contain eight segmented RNA fragments that can be used to encode 14 viral proteins. The replication and transcription of the viral genome needs to be accomplished by the functional unit of the RNP complex. The virus infects the host cell with a two-layer barrier and four shuttles.
  • the first barrier is that the virus enters the cell and needs to cross the plasma membrane. At this time, the virus uses hemagglutinin protein HA to bind to the sialic acid receptor on the cell surface, and undergoes endocytosis to invade the host cell.
  • Substrate M1 then releases the vRNP complex into the cytoplasm, which is required to occur in the nucleus due to genomic replication and transcription, at which point the vRNP complex faces a second barrier, the nuclear membrane.
  • vRNP utilizes the atypical bidirectional NLS of the N-terminus of the NP protein to bind to the nuclear transport receptor protein importin- ⁇ , allowing it to cross the nuclear pore complex and enter the nucleus to initiate replication and transcription.
  • the mRNA produced by transcription is translated in the cytoplasm, and the newly synthesized viral polymerase component will re-assemble into the RNP complex by using its own NLS to enter the nucleus.
  • the RNP complex will form a large complex with the M1 protein and the NEP protein using the NP protein, and then transport it to the cytoplasm using the cytoplasmic transport protein CRM1 protein to complete the shuttle barrier and assemble. Complete virus particles.
  • NP protein plays an important role in the process of two shuttles to the nuclear membrane.
  • the present invention first protects a recombinant virus, designated as virus WSN-Y385F, which is a codon that mutates the codon of the 385th tyrosine residue encoding the NP protein into the phenylalanine residue in the influenza A virus genome.
  • virus WSN-Y385F is a temperature-sensitive virus that can normally replicate and survive at 37 ° C, cannot replicate normally at 33 ° C and cannot survive.
  • the influenza A virus may specifically be a WSN virus A/WSN/1933 (H1N1) strain.
  • the NP protein is shown as Sequence 1 of the Sequence Listing.
  • the present invention also protects a protein, designated protein NP-Y385F, which is a protein obtained by mutating the 385th tyrosine residue of the NP protein to a phenylalanine residue.
  • the NP protein is shown as Sequence 1 of the Sequence Listing.
  • the gene encoding the protein NP-Y385F is also within the scope of the present invention.
  • the gene encoding the protein NP-Y385F was named gene NP-Y385F.
  • the gene NP-Y385F may specifically be as follows (a) or (b):
  • Recombinant plasmids containing the gene NP-Y385F are also within the scope of the invention.
  • the recombinant plasmid containing the gene NP-Y385F may specifically be the recombinant plasmid pHH21-NP-Y385F.
  • the recombinant plasmid pHH21-NP-Y385F is a recombinant plasmid obtained by inserting the gene NP-Y385F into the multiple cloning site of the vector pHH21 (for example, BsmBI cleavage site).
  • the recombinant plasmid containing the gene NP-Y385F may specifically be the recombinant plasmid pcDNA3.0-NP-Y385F.
  • the recombinant plasmid pcDNA3.0-NP-Y385F is a recombinant plasmid obtained by inserting the gene NP-Y385F into the multiple cloning site of the vector pcDNA3.0 (for example, between the KpnI and XhoI restriction sites).
  • the invention also protects a temperature-sensitive recombinant virus, the preparation method comprising the following steps:
  • Plasmid pHH21-PA Plasmid pHH21-PA, plasmid pHH21-PB1, plasmid pHH21-PB2, plasmid pHH21-HA, plasmid pHH21-NA, plasmid pHH21-M, plasmid pHH21-NS, plasmid pcDNA3.0-PA, plasmid pcDNA3.0-PB1
  • the recombinant plasmid obtained by co-transfection of the plasmid pcDNA3.0-PB2, the recombinant plasmid pHH21-NP-Y385F and the recombinant plasmid pcDNA3.0-NP-Y385F into isolated mammalian cells;
  • the plasmid pHH21-PA is a plasmid obtained by inserting a double-stranded DNA molecule shown in SEQ ID NO: 3 of the sequence listing at a multiple cloning site (for example, BsmBI cleavage site) of the vector pHH21;
  • the plasmid pHH21-PB1 is at the vector pHH21 a multiple cloning site (eg, a BsmBI cleavage site) is inserted into a plasmid obtained from the double-stranded DNA molecule shown in SEQ ID NO: 4 of the Sequence Listing;
  • the plasmid pHH21-PB2 may specifically be a multiple cloning site at the vector pHH21 (eg, BsmBI) Enzyme cleavage site) a plasmid obtained by the double-stranded DNA molecule shown in column 5;
  • the plasmid pHH21-HA is a double-stranded DNA molecule inserted in the cloning site
  • the plasmid pHH21-NA is a plasmid obtained by inserting a double-stranded DNA molecule shown in SEQ ID NO: 8 of the Sequence Listing at a multiple cloning site (for example, BsmBI cleavage site) of the vector pHH21;
  • the plasmid pHH21-M is A plasmid obtained by inserting a double-stranded DNA molecule shown in SEQ ID NO: 2 of the Sequence Listing at a multiple cloning site of the vector pHH21 (for example, a BsmBI cleavage site);
  • the plasmid pHH21-NS is a multiple cloning site at the vector pHH21 (for example, BsmBI cleavage site)
  • the plasmid pcDNA3.0-PA is a cloning site of the vector pcDNA3.0
  • the mammalian cell may specifically be HEK 293T/17 cells.
  • the culture conditions may specifically be 6-78 hours at 37 °C.
  • the present invention also protects a method for inhibiting phosphorylation of an NP protein of influenza A virus by mutating the amino acid residue at position 385 of the NP protein of influenza A virus from tyrosine to phenylalanine.
  • the present invention also protects a method for reducing the phosphorylation level of NP protein of influenza A virus by mutating the NP protein of influenza A virus from amino acid residue 385 at the N-terminus to phenylalanine from tyrosine.
  • the present invention also protects a method for inhibiting phosphorylation of an NP protein of influenza A virus by cleavage of a tyrosine codon encoding a codon of the NP protein from the N-terminal amino acid residue 385 in the influenza A virus genome. Is a phenylalanine codon.
  • the present invention also protects a method for reducing the phosphorylation level of NP protein of influenza A virus,
  • the codon encoding the amino acid residue at position 385 of the NP protein from the N-terminus of the influenza A virus genome is mutated from a tyrosine codon to a phenylalanine codon.
  • the NP protein is shown as Sequence 1 of the Sequence Listing.
  • the invention also protects the use of any of the recombinant viruses of any of the above in the preparation of influenza A virus vaccine.
  • the invention also protects the use of any of the recombinant viruses described above as a vaccine for influenza A virus.
  • the present invention also protects an influenza A virus vaccine, the active ingredient of which is any of the recombinant viruses described above.
  • the invention also protects a plasmid combination from plasmid pHH21-PA, plasmid pHH21-PB1, plasmid pHH21-PB2, plasmid pHH21-HA, plasmid pHH21-NA, plasmid pHH21-M, plasmid pHH21-NS, plasmid pcDNA3.0- PA, plasmid pcDNA3.0-PB1, plasmid pcDNA3.0-PB2, recombinant plasmid pHH21-NP-Y385F and recombinant plasmid pcDNA3.0-NP-Y385F.
  • Each plasmid may be packaged separately, or all plasmids may be mixed and packaged, or any of the plasmids in the combination may be mixed and packaged.
  • the invention also contemplates a kit for the preparation of the recombinant virus (virus WSN-Y385F), comprising the plasmid combination.
  • the kit can also include ex vivo mammalian cells.
  • the mammalian cell may specifically be HEK 293T/17 cells.
  • influenza A viruses described above may specifically be a WSN virus A/WSN/1933 (H1N1) strain.
  • Figure 1 is the result of step one of the first embodiment.
  • WSN virus A/WSN/1933 H1N1 strain: Neumann, G. et al., Generation of influenza A viruses entirely from cloned cDNAs. P Natl Acad Sci Usa 96 (16), 9345 (1999).
  • the WSN virus is the influenza virus.
  • the viral infection solution is used to adjust the virus concentration to achieve different doses of infection.
  • Vector pHH21 Neumann, G. et al., Generation of influenza A viruses entirely from cloned cDNAs. P Natl Acad Sci Usa 96 (16), 9345 (1999).
  • HEK 293T/17 cells (abbreviated as 293T cell-derived lines, human embryonic kidney cells): ATCC, CRL-11268.
  • Vector pcDNA3.0 Shanghai Hippi Biotechnology Co., Ltd., catalog number CPC030.
  • Escherichia coli DH5 ⁇ Shanghai Beinuo Biotechnology Co., Ltd.
  • A549 cells human lung adenocarcinoma cells
  • BALB/c mice Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • MDCK cells ATCC, CCL-34.
  • Cell lysate (pH 7.4): containing 150 mM sodium chloride, 20 mM HEPES, 10% by volume glycerol, 1 mM EDTA, 1 g/100 mL NP40, a protease inhibitor, and the balance being water.
  • Elution buffer The concentration of sodium chloride was 300 mM, and other cell lysates.
  • Viral infection solution trypsin containing 2 ⁇ g/ml TPCK (trypsin is added as trypsin mother liquor, trypsin mother liquor is a solution of trypsin concentration 0.25g/100mL prepared in PBS buffer), 100U/ Serum DMEM medium with ml penicillin and 100 U/ml streptomycin.
  • Alkaline phosphatase Takara, article number D2250.
  • a protease inhibitor was purchased from Roche.
  • SDM enzyme Beijing Saibaisheng Gene Technology Co., Ltd., article number: SDM-15.
  • the gel used in phos-tag SDS-PAGE was Phos-tag Acrylamide, available from Wako (Japan).
  • Anti-phosphotyrosine antibody murine monoclonal antibody, sc-508 was purchased from Santa Cruz. Pre-stained protein standards of known molecular weight were purchased from Thermo.
  • HEPES N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid
  • glycerin glycerin
  • EDTA ethylenediaminetetraacetic acid
  • NP40 Nonidet P-40
  • Pancreatin treated with TPCK was purchased from Sigma.
  • Bovine serum albumin (BSA) was purchased from Jiangchen Bio. Penicillin and streptomycin were purchased from Biyuntian Company.
  • SDS sodium dodecyl sulfate
  • low melting agarose were purchased from Amersco.
  • Monoclonal antibody against NP protein ie, murine monoclonal antibody against influenza A virus NP protein: MR Yu#, XL Liu#, Sh Cao, Zh-D Zhao, K Zhang, Q Xie, CW Chen, Sh- Y Gao, Y-H Bi, L Sun, X Ye, George F. Gao, W-J Liu*. 2012. Identification and Characterization of three novel nuclear export signals in influenza A virus nucleoprotein. Journal of Virology, 86(9): 4970-80.
  • Plasmid pHH21-PA, plasmid pHH21-PB1, plasmid pHH21-PB2, plasmid pHH21-HA, plasmid pHH21-NP, plasmid pHH21-NA, plasmid pHH21-M, plasmid pHH21-NS, plasmid pcDNA3.0-PA, plasmid pcDNA3 .0-PB1, plasmid pcDNA3.0-PB2 and plasmid pcDNA3.0-NP were co-transfected into HEK 293T/17 cells, and cultured to obtain WSN virus A/WSN/1933 (H1N1) strain.
  • the WSN virus A/WSN/1933 (H1N1) strain is also known as the wild type of WSN virus.
  • step 2 The cells harvested in step 1 were treated with cell lysate at 4 ° C for 30 minutes, centrifuged at 12,000 rpm for 15 min, and the supernatant was collected.
  • step 3 Add the anti-NP protein monoclonal antibody to the supernatant obtained in step 2, incubate at 4 ° C for 1 hour, then add protein G beads for 4 hours at 4 ° C, aspirate the supernatant, and buffer the beads with elution. The solution was washed 3 times at 4 ° C (10 minutes each time), and the NP protein was bound to the beads.
  • NP protein-bound beads obtained in step 3 were treated with alkaline phosphatase at 37 ° C for 2 hours (alkaline phosphatase acts to dephosphorylate the phosphorylated protein).
  • the NP protein before alkaline phosphatase treatment (obtained in step 3) and the alkaline phosphatase-treated NP protein (obtained in step 4) were respectively subjected to phosphorylated protein electrophoresis (phos-tag SDS-PAGE) and silver. Dyeing color.
  • lane 1 is an alkaline phosphatase-treated NP protein
  • lane 2 is an alkaline phosphatase-treated NP protein.
  • the alkaline phosphatase-treated NP protein was used as the standard NP protein, and the NP protein before alkaline phosphatase treatment showed a slower and more alkaline phosphatase-sensitive band on the gel, which was phosphorylated.
  • NP strips The results showed that the NP protein obtained in step 3 was a phosphorylated protein, and alkaline phosphatase could cause dephosphorylation.
  • the phosphorylated NP band was excised from the gel and sent to a large instrument platform of the Institute of Zoology of the Chinese Academy of Sciences for sample processing and mass spectrometry (Nano-LC MS/MS, LCQ DECA XP PLUS Thermo).
  • the double-stranded DNA molecule shown in SEQ ID NO: 3 of the Sequence Listing was inserted into the BsmBI cleavage site of the vector pHH21 to obtain plasmid pHH21-PA.
  • the double-stranded DNA molecule shown in SEQ ID NO: 4 of the Sequence Listing was inserted into the BsmBI cleavage site of the vector pHH21 to obtain plasmid pHH21-PB1.
  • the double-stranded DNA molecule shown in SEQ ID NO: 5 of the Sequence Listing was inserted into the BsmBI cleavage site of the vector pHH21 to obtain plasmid pHH21-PB2.
  • the double-stranded DNA molecule shown in SEQ ID NO: 6 of the Sequence Listing was inserted into the BsmBI cleavage site of the vector pHH21 to obtain plasmid pHH21-HA.
  • the double-stranded DNA molecule shown in SEQ ID NO: 7 of the Sequence Listing was inserted into the BsmBI cleavage site of the vector pHH21 to obtain plasmid pHH21-NP.
  • the double-stranded DNA molecule shown in SEQ ID NO: 8 of the Sequence Listing was inserted into the BsmBI cleavage site of the vector pHH21 to obtain plasmid pHH21-NA.
  • the double-stranded DNA molecule shown in SEQ ID NO: 9 of the Sequence Listing was inserted into the BsmBI cleavage site of the vector pHH21 to obtain plasmid pHH21-NS.
  • a double-stranded DNA molecule shown in SEQ ID NO: 3 of the Sequence Listing was inserted between the KpnI and XhoI cleavage sites of the vector pcDNA3.0 to obtain plasmid pcDNA3.0-PA.
  • a double-stranded DNA molecule shown in SEQ ID NO: 4 of the Sequence Listing was inserted between the KpnI and XhoI restriction sites of the vector pcDNA3.0 to obtain plasmid pcDNA3.0-PB1.
  • the double-stranded DNA molecule shown in SEQ ID NO: 5 of the Sequence Listing was inserted between the KpnI and XhoI restriction sites of the vector pcDNA3.0 to obtain plasmid pcDNA3.0-PB2.
  • a double-stranded DNA molecule shown in SEQ ID NO: 7 of the Sequence Listing was inserted between the KpnI and XhoI restriction sites of the vector pcDNA3.0 to obtain plasmid pcDNA3.0-NP.
  • a primer pair consisting of NP-Y385A-F and NP-Y385A-R was subjected to PCR amplification to obtain a PCR amplification product (mutation plasmid).
  • step (3) The product of the step (2) was transformed into competent cells of Escherichia coli DH5 ⁇ to obtain a recombinant strain Y385A-I (i.e., Escherichia coli containing the recombinant plasmid pHH21-NP-Y385A).
  • a recombinant strain Y385A-I i.e., Escherichia coli containing the recombinant plasmid pHH21-NP-Y385A.
  • the recombinant plasmid pHH21-NP-Y385A was structurally described as follows: The codon "tac" encoding the NP protein from the N-terminal 385th tyrosine in the plasmid pHH21-NP was mutated to the alanine codon. "GCG”.
  • PCR amplification was carried out using primer pairs consisting of NP-Y385A-F and NP-Y385A-R to obtain a PCR amplification product (mutation plasmid).
  • the product was amplified by PCR with the SDM enzyme at 37 ° C for 2 hours (digestion template plasmid).
  • the product of the step (5) was transformed into competent cells of Escherichia coli DH5 ⁇ to obtain a recombinant strain Y385A-II (i.e., Escherichia coli containing the recombinant plasmid pcDNA3.0-NP-Y385A).
  • a recombinant strain Y385A-II i.e., Escherichia coli containing the recombinant plasmid pcDNA3.0-NP-Y385A.
  • the recombinant plasmid pcDNA3.0-NP-Y385A was structurally described as follows: The codon "tac" encoding the NP protein from the N-terminal 385th tyrosine in the plasmid pcDNA3.0-NP was mutated to alanine. The acid codon "GCG”.
  • PCR amplification was carried out using primer pairs consisting of NP-Y385F-F and NP-Y385F-R to obtain a PCR amplification product (mutation plasmid).
  • the product of the step (8) was transformed into competent cells of Escherichia coli DH5 ⁇ to obtain a recombinant strain Y385F-I (i.e., Escherichia coli containing the recombinant plasmid pHH21-NP-Y385F).
  • a recombinant strain Y385F-I i.e., Escherichia coli containing the recombinant plasmid pHH21-NP-Y385F.
  • the recombinant plasmid pHH21-NP-Y385F was structurally described as follows: The codon "tac" encoding the NP protein from the N-terminal 385th tyrosine in the plasmid pHH21-NP was mutated to the phenylalanine code.
  • the sub-"TTC" that is, the double-stranded DNA molecule shown in SEQ ID NO: 10 of the Sequence Listing was inserted into the Bs
  • the product of the step (11) was transformed into competent cells of Escherichia coli DH5 ⁇ to obtain a recombinant strain Y385F-II (i.e., Escherichia coli containing the recombinant plasmid pcDNA3.0-NP-Y385F).
  • a recombinant strain Y385F-II i.e., Escherichia coli containing the recombinant plasmid pcDNA3.0-NP-Y385F.
  • the recombinant plasmid pcDNA3.0-NP-Y385F was structurally described as follows: The codon "tac" encoding the NP protein from the N-terminal 385th tyrosine in the plasmid pcDNA3.0-NP was mutated to phenylpropanoid.
  • TTC a double-stranded DNA molecule represented by SEQ ID NO: 10 of the Sequence Listing was inserted between the KpnI and XhoI cleavage sites of the vector pcDNA3.0.
  • PCR amplification was carried out using primer pairs consisting of NP-Y385E-F and NP-Y385E-R to obtain a PCR amplification product (mutation plasmid).
  • the product was amplified by PCR with the SDM enzyme at 37 ° C for 2 hours (digestion template plasmid).
  • the product of the step (14) was transformed into competent cells of Escherichia coli DH5 ⁇ to obtain a recombinant strain Y385E-I (i.e., Escherichia coli containing the recombinant plasmid pHH21-NP-Y385E).
  • a recombinant strain Y385E-I i.e., Escherichia coli containing the recombinant plasmid pHH21-NP-Y385E.
  • the recombinant plasmid pHH21-NP-Y385E was structurally described as follows: The codon "tac" encoding the NP protein from the N-terminal 385th tyrosine in the plasmid pHH21-NP was mutated to the codon of glutamic acid. "GAG”.
  • PCR amplification was carried out using primer pairs consisting of NP-Y385E-F and NP-Y385E-R to obtain a PCR amplification product (mutation plasmid).
  • the product of the step (17) was transformed into competent cells of Escherichia coli DH5 ⁇ to obtain a recombinant strain Y385E-II (i.e., Escherichia coli containing the recombinant plasmid pcDNA3.0-NP-Y385E).
  • a recombinant strain Y385E-II i.e., Escherichia coli containing the recombinant plasmid pcDNA3.0-NP-Y385E.
  • the recombinant plasmid pcDNA3.0-NP-Y385E was structurally described as follows: The codon "tac" encoding the NP protein from the N-terminal 385th tyrosine in the plasmid pcDNA3.0-NP was mutated to glutamine. The acid codon "GAG”.
  • liposome Lipofectamine 2000 Invitrogen
  • the medium in which the cells of the step (1) were replaced was a virus-infected solution, and the cells were harvested after incubation at 37 ° C for 72 hours.
  • the cells harvested in the step (2) were treated with the cell lysate at 4 ° C for 30 minutes, centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected.
  • the recombinant plasmid pHH21-NP-Y385F was replaced with the plasmid pHH21-NP and the recombinant plasmid pcDNA3.0-NP-Y385F was replaced with the plasmid pcDNA3.0-NP.
  • the other step 1 was the binding of beads to the beads.
  • the proteins obtained in steps 1 and 2 were subjected to western blot, and the primary antibody used was an anti-phosphotyrosine antibody, and the secondary antibody was an HRP-labeled goat anti-mouse IgG.
  • HEK 293T/17 cells were seeded in 60 mm dishes, 1 ⁇ 10 6 cells per dish, and cultured for 12 hours.
  • the HEK 293T/17 cells are grouped as follows:
  • the first group plasmid pHH21-PA, plasmid pHH21-PB1, plasmid pHH21-PB2, plasmid pHH21-HA, recombinant plasmid pHH21-NP-Y385A, plasmid pHH21-NA, plasmid pHH21-M, plasmid pHH21-NS, plasmid pcDNA3 .0-PA, plasmid pcDNA3.0-PB1, plasmid pcDNA3.0-PB2 And 0.5 ⁇ g of each recombinant plasmid pcDNA3.0-NP-Y385A was co-transfected into HEK 293T/17 cells by liposome Lipofectamine 2000 (Invitrogen), and cultured at 37 ° C for 6 hours, then the medium was changed to virus infection solution, and the culture was continued for 72 hours. cell.
  • Lipofectamine 2000 Invitrogen
  • the second group differs from the first group only in that the recombinant plasmid pHH21-NP-Y385F was used in place of the recombinant plasmid pHH21-NP-Y385A and the recombinant plasmid pcDNA3.0-NP-Y385F was used in place of the recombinant plasmid pcDNA3.0-NP-Y385A.
  • the third group differs from the first group only in that the recombinant plasmid pHH21-NP-Y385E was used in place of the recombinant plasmid pHH21-NP-Y385A and the recombinant plasmid pcDNA3.0-NP-Y385E was used in place of the recombinant plasmid pcDNA3.0-NP-Y385A.
  • the fourth group differs from the first group only in that the recombinant plasmid pHH21-NP-Y385A was replaced with the plasmid pHH21-NP and the recombinant plasmid pcDNA3.0-NP-Y385A was replaced with the plasmid pcDNA3.0-NP.
  • each group separately harvests the culture supernatant.
  • the culture supernatant of the fourth group contained the wild type of WSN virus, so the culture supernatant was named WSN-WT virus solution.
  • the culture supernatant of the first group contains the WSN virus mutant (the codon in the mutant virus genome encoding the NP protein from the N-terminal 385th tyrosine is a codon for the alanine, and the mutant virus is named WSN. -Y385A virus), so the culture supernatant was named WSN-Y385A virus solution.
  • the culture supernatant of the second group contains a WSN virus mutant (a codon mutation in the mutated viral genome encoding the NP protein from the N-terminal 385th tyrosine to the phenylalanine codon, and the mutant virus is named WSN-Y385F virus), so the culture supernatant was named WSN-Y385F virus solution.
  • WSN virus mutant a codon mutation in the mutated viral genome encoding the NP protein from the N-terminal 385th tyrosine to the phenylalanine codon, and the mutant virus is named WSN-Y385F virus
  • the culture supernatant of the third group contains a WSN virus mutant (a codon mutated in the mutated viral genome encoding the NP protein from the N-terminal 385th tyrosine to the glutamic acid codon, and the mutant virus is named WSN -Y385E virus), so the culture supernatant was named WSN-Y385E virus solution.
  • WSN virus mutant a codon mutated in the mutated viral genome encoding the NP protein from the N-terminal 385th tyrosine to the glutamic acid codon, and the mutant virus is named WSN -Y385E virus
  • step 4 Take the virus liquid obtained in step 3 and detect the virus titer by plaque identification.
  • MDCK cells were seeded in a 12-well plate, about 1 ⁇ 10 5 cells per well, cultured overnight at 37 ° C, 5% CO 2 incubator; (2) washed with PBS buffer The medium on the cell surface was removed, and the virus solution to be tested was diluted with the virus infection solution and added to each well.
  • the well plate was inverted and inverted and cultured in a 37 ° C incubator.
  • the cell lesion was observed under a microscope and cultured for 3 days (in practical application, 2-4 days). After all, the 12-well plate was taken out of the incubator and the number of plaques was counted.
  • the titer of WSN-WT virus solution was 6.512 log 10 PFU/ml.
  • the titer of WSN-Y385F virus solution was 7.179 log 10 PFU/ml.
  • the titer of WSN-Y385A virus solution is 0, that is, MDCK cannot produce plaque.
  • the titer of the WSN-Y385E virus solution is 0, that is, the plaque cannot be caused by MDCK.
  • step 2 the cells were harvested in each group, and the cells were disrupted and subjected to western blot (detection of expression of each major viral protein).
  • Example 4 Differences in virus growth curves at different temperatures at the cell level
  • A549 cells were seeded in 10 cm dishes, 1 ⁇ 10 8 cells per dish, and cultured for 12 hours.
  • step 1 the A549 cells are grouped as follows:
  • the first group The WSN-WT virus solution prepared in Example 3 (the virus dose was 10 6 PFU) was inoculated into A549 cells, and the solution was changed to virus infection solution 1 hour after inoculation; the culture was carried out at 37 ° C, and inoculated at 12, 24, 36 respectively. After 48, 60, and 72 hours, the supernatant was collected, and the virus titer was detected by plaque identification.
  • the second group The WSN-Y385F virus solution prepared in Example 3 (the virus dose was 10 6 PFU) was inoculated into A549 cells, and the solution was changed to virus infection solution 1 hour after inoculation; cultured at 37 ° C, respectively, inoculation 12, 24, 36 After 48, 60, and 72 hours, the supernatant was collected, and the virus titer was detected by plaque identification.
  • the third group the difference from the first group is only that the culture temperature is changed from 37 ° C to 33 ° C.
  • the fourth group the difference from the second group is only that the culture temperature is changed from 37 ° C to 33 ° C.
  • the method of plaque identification was the same as in Example 3.
  • the WSN-WT virus could replicate normally, and the virus titer remained relatively stable and slowly rising.
  • the WSN-Y385F virus titer was 0, ie the WSN-Y385F virus could not replicate at 33 °C.
  • both WSN-WT virus and WSN-Y385F virus could replicate normally, and the virus titer remained relatively stable and slowly rising. The results showed that the WSN-Y385F virus is a temperature sensitive virus.
  • Example 5 Differences in virus growth curves at different temperatures at the animal level
  • mice weighing approximately 17 g were randomly divided into three groups after anesthesia with ether, and 12 rats in each group were treated as follows:
  • the first group inhalation of 50 ⁇ l of the WSN-WT virus solution prepared in Example 3 by a nasal inhalation method (virus titer of 10 4 PFU / ml);
  • the second group inhaled 50 ⁇ l of the WSN-Y385F virus solution prepared in Example 3 (virus titer of 10 4 PFU / ml);
  • Group 3 50 ⁇ l of sterilized PBS buffer was inhaled by nasal inhalation.
  • mice were dissected on the first day, the third day, the fifth day, and the seventh day (three mice per group at each time point) to obtain the mouse lung and turbinate bone (nose).
  • the temperature of the oracle bone and lung is different, the temperature of the turbinates is lower, about 33 ° C, and the temperature of the lung is higher, about 37 ° C).
  • the virus titer in the supernatant was detected by plaque identification (the plaque identification method was the same as in Example 3).
  • WSN-WT virus can replicate normally, and the virus titer remains relatively stable and slowly declines.
  • the WSN-Y385F virus titer was 0, that is, the WSN-Y385F virus could not be replicated at 33 °C.
  • both WSN-WT virus and WSN-Y385F virus can replicate normally, and the virus titer remained relatively stable and slowly decreased. The results showed that the WSN-Y385F virus is a temperature sensitive virus.
  • the invention has great value for analyzing the mechanism of influenza virus infection and preventing and treating influenza virus.

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Abstract

提供一种重组病毒，该重组病毒是将A型流感病毒基因组中编码NP蛋白的第385位酪氨酸残基的密码子突变为苯丙氨酸残基的密码子而得到的。还提供一种抑制A型流感病毒的NP蛋白发生磷酸化的方法，是将A型流感病毒的NP蛋白自N末端第385位氨基酸残基由酪氨酸突变为苯丙氨酸。

Description

A型流感病毒温度敏感性的关键磷酸化位点及其应用 技术领域
本发明涉及一种A型流感病毒温度敏感性的关键磷酸化位点及其应用。
背景技术
A型流感病毒包含8个分节段的RNA片段，利用这些RNA片段，共能编码14种病毒蛋白。而病毒基因组的复制和转录需要由RNP复合物这一功能单元来完成。病毒感染宿主细胞面临着2层屏障，4次穿梭。
第一个屏障是病毒进入细胞需要穿过细胞质膜，这时病毒利用血凝素蛋白HA和细胞表面的唾液酸受体结合，进行发生内吞作用入侵到宿主细胞内部。
之后基质蛋白M1会释放vRNP复合物到细胞质中，而由于基因组复制和转录需要在细胞核内进行，此时的vRNP复合物面临的第二道屏障，即细胞核膜。vRNP会利用NP蛋白N端的非典型双向NLS同细胞核运输受体蛋白importin-α结合，从而得以穿过核孔复合物，进入到细胞核内部开始复制和转录。
转录产生的mRNA在细胞质得到翻译，新合成的病毒聚合酶组分又会分别利用自身的NLS进入到细胞核重新组装成RNP复合物。
待病毒基因组复制完毕后，RNP复合物会利用NP蛋白同M1蛋白和NEP蛋白形成一个大的复合物，再利用细胞质运输蛋白CRM1蛋白将之运输到细胞质中，完成再一次的穿梭屏障，然后组装成完整的病毒粒子。
NP蛋白在两次穿梭核膜的过程中，发挥着重要的作用。
发明公开
本发明的目的是提供一种A型流感病毒温度敏感性的关键磷酸化位点及其应用。
本发明首先保护一种重组病毒，命名为病毒WSN-Y385F，是将A型流感病毒基因组中编码NP蛋白的第385位酪氨酸残基的密码子突变为苯丙氨酸残基的密码子得到的重组病毒。病毒WSN-Y385F为温度敏感型病毒，在37℃可以正常复制并存活，在33℃不能正常复制并且不能存活。
所述A型流感病毒具体可为WSN病毒A/WSN/1933(H1N1)毒株。
所述NP蛋白如序列表的序列1所示。
本发明还保护一种蛋白质，命名为蛋白质NP-Y385F，是NP蛋白的第385位酪氨酸残基突变为苯丙氨酸残基得到的蛋白质。
所述NP蛋白如序列表的序列1所示。
编码蛋白质NP-Y385F的基因也属于本发明的保护范围。编码蛋白质NP-Y385F的基因命名为基因NP-Y385F。
基因NP-Y385F具体可为如下(a)或(b)：
(a)编码区如序列表的序列10自5’末端第26-1522位核苷酸所示的DNA分子；
(b)序列表的序列10所示的DNA分子。
含有基因NP-Y385F的重组质粒也属于本发明的保护范围。
含有基因NP-Y385F的重组质粒具体可为重组质粒pHH21-NP-Y385F。重组质粒pHH21-NP-Y385F为：在载体pHH21的多克隆位点(例如BsmBI酶切位点)插入基因NP-Y385F得到的重组质粒。
含有基因NP-Y385F的重组质粒具体可为重组质粒pcDNA3.0-NP-Y385F。重组质粒pcDNA3.0-NP-Y385F为：在载体pcDNA3.0的多克隆位点(例如KpnI和XhoI酶切位点之间)插入基因NP-Y385F得到的重组质粒。
本发明还保护一种温度敏感型重组病毒，其制备方法包括如下步骤：
将质粒pHH21-PA、质粒pHH21-PB1、质粒pHH21-PB2、质粒pHH21-HA、质粒pHH21-NA、质粒pHH21-M、质粒pHH21-NS、质粒pcDNA3.0-PA、质粒pcDNA3.0-PB1、质粒pcDNA3.0-PB2、重组质粒pHH21-NP-Y385F和重组质粒pcDNA3.0-NP-Y385F共转染离体哺乳动物细胞后进行培养得到的重组病毒；
所述质粒pHH21-PA为在载体pHH21的多克隆位点(例如BsmBI酶切位点)插入序列表的序列3所示的双链DNA分子得到的质粒；所述质粒pHH21-PB1为在载体pHH21的多克隆位点(例如BsmBI酶切位点)插入序列表的序列4所示的双链DNA分子得到的质粒；所述质粒pHH21-PB2具体可为在载体pHH21的多克隆位点(例如BsmBI酶切位点)插入序列表的序 列5所示的双链DNA分子得到的质粒；所述质粒pHH21-HA为在载体pHH21的多克隆位点(例如BsmBI酶切位点)插入序列表的序列6所示的双链DNA分子得到的质粒；所述质粒pHH21-NA为在载体pHH21的多克隆位点(例如BsmBI酶切位点)插入序列表的序列8所示的双链DNA分子得到的质粒；所述质粒pHH21-M为在载体pHH21的多克隆位点(例如BsmBI酶切位点)插入序列表的序列2所示的双链DNA分子得到的质粒；所述质粒pHH21-NS为在载体pHH21的多克隆位点(例如BsmBI酶切位点)插入序列表的序列9所示的双链DNA分子得到的质粒；所述质粒pcDNA3.0-PA为在载体pcDNA3.0的多克隆位点(例如KpnI和XhoI酶切位点之间)插入序列表的序列3所示的双链DNA分子得到的质粒；所述质粒pcDNA3.0-PB1为在载体pcDNA3.0的多克隆位点(例如KpnI和XhoI酶切位点之间)插入序列表的序列4所示的双链DNA分子得到的质粒；所述质粒pcDNA3.0-PB2为在载体pcDNA3.0的多克隆位点(例如KpnI和XhoI酶切位点之间)插入序列表的序列5所示的双链DNA分子得到的质粒；所述重组质粒pHH21-NP-Y385F为在载体pHH21的多克隆位点(例如BsmBI酶切位点)插入基因NP-Y385F得到的质粒；所述重组质粒pcDNA3.0-NP-Y385F为在载体pcDNA3.0的多克隆位点(例如KpnI和XhoI酶切位点之间)插入基因NP-Y385F得到的质粒。
所述哺乳动物细胞具体可为HEK 293T/17细胞。
所述培养的条件具体可为37℃培养6-78小时。
本发明还保护一种抑制A型流感病毒的NP蛋白发生磷酸化的方法，是将A型流感病毒的NP蛋白自N末端第385位氨基酸残基由酪氨酸突变为苯丙氨酸。
本发明还保护一种降低A型流感病毒的NP蛋白磷酸化水平的方法，是将A型流感病毒的NP蛋白自N末端第385位氨基酸残基由酪氨酸突变为苯丙氨酸。
本发明还保护一种抑制A型流感病毒的NP蛋白发生磷酸化的方法，是将A型流感病毒基因组中编码NP蛋白自N末端第385位氨基酸残基的密码子由酪氨酸密码子突变为苯丙氨酸密码子。
本发明还保护一种降低A型流感病毒的NP蛋白磷酸化水平的方法， 是将A型流感病毒基因组中编码NP蛋白自N末端第385位氨基酸残基的密码子由酪氨酸密码子突变为苯丙氨酸密码子。
所述NP蛋白如序列表的序列1所示。
本发明还保护以上任一所述重组病毒在制备A型流感病毒疫苗中的应用。
本发明还保护以上任一所述重组病毒作为A型流感病毒疫苗的应用。
本发明还保护一种A型流感病毒疫苗，其活性成分为以上任一所述重组病毒。
本发明还保护一种质粒组合，由质粒pHH21-PA、质粒pHH21-PB1、质粒pHH21-PB2、质粒pHH21-HA、质粒pHH21-NA、质粒pHH21-M、质粒pHH21-NS、质粒pcDNA3.0-PA、质粒pcDNA3.0-PB1、质粒pcDNA3.0-PB2、重组质粒pHH21-NP-Y385F和重组质粒pcDNA3.0-NP-Y385F组成。各个质粒可以单独包装，也可以将所有质粒混合包装，也可以将组合中的任意几个质粒混合包装。
本发明还保护一种用于制备所述重组病毒(病毒WSN-Y385F)的试剂盒，包括所述质粒组合。所述试剂盒还可包括离体的哺乳动物细胞。所述哺乳动物细胞具体可为HEK 293T/17细胞。
以上任一所述A型流感病毒具体可为WSN病毒A/WSN/1933(H1N1)毒株。
附图说明
图1为实施例1的步骤一的结果。
图2为实施例1的步骤二的结果。
图3为实施例2的结果。
图4为实施例3的结果。
图5为实施例4的结果。
图6为实施例5的结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施 例中的定量试验，均设置三次重复实验，结果取平均值。
WSN病毒A/WSN/1933(H1N1)毒株：Neumann,G.et al.,Generation of influenza A viruses entirely from cloned cDNAs.P Natl Acad Sci Usa 96(16),9345(1999)。WSN病毒即流感病毒。实施例中，采用病毒感染液调整病毒浓度从而实现不同剂量的感染。
载体pHH21：Neumann,G.et al.,Generation of influenza A viruses entirely from cloned cDNAs.P Natl Acad Sci Usa 96(16),9345(1999)。
HEK 293T/17细胞(简称293T细胞衍生系，人胚肾细胞)：ATCC,CRL-11268。载体pcDNA3.0：上海希匹吉生物技术有限公司,产品目录号CPC030。大肠杆菌DH5α：上海北诺生物科技有限公司。A549细胞(人肺腺癌细胞)：上海拜力生物科技有限公司。BALB/c小鼠：北京维通利华实验动物技术有限公司。MDCK细胞：ATCC,CCL-34。
细胞裂解液(pH7.4)：含150mM氯化钠、20mM HEPES、10％(体积比)甘油、1mM EDTA、1g/100mL NP40、蛋白酶抑制剂(cocktail)，其余为水。
洗脱缓冲液：氯化钠的浓度为300mM，其它同细胞裂解液。
病毒感染液：含2μg/ml TPCK处理的胰酶(胰酶是以胰酶母液的方式加入的，胰酶母液为用PBS缓冲液配制的胰酶浓度为0.25g/100mL的溶液)、100U/ml青霉素和100U/ml链霉素的无血清DMEM培养基。
碱性磷酸酶：Takara，货号D2250。蛋白酶抑制剂(cocktail)购自Roche公司。SDM酶：北京赛百盛基因技术有限公司，货号:SDM-15。phos-tag SDS-PAGE中所用的凝胶为Phos-tag Acrylamide，购自Wako(日本)。抗磷酸化酪氨酸抗体(鼠单克隆抗体，sc-508)购自Santa Cruz。具有已知分子量的预先染色的蛋白标准品购自Thermo。氯化钠、N-(2-羟乙基)哌嗪-N’-2-乙烷磺酸(简称HEPES)、甘油、乙二胺四乙酸(简称EDTA)、Nonidet P-40(简称NP40)和TPCK处理的胰酶均购自Sigma。牛血清白蛋白(BSA)购自江晨生物。青霉素、链霉素购自碧云天公司。十二烷基硫酸钠(简称SDS)、低熔点琼脂糖购自Amersco公司。
抗NP蛋白的单抗(即抗A型流感病毒NP蛋白的鼠源单克隆抗体)：M-R Yu#,X-L Liu#,Sh Cao,Zh-D Zhao,K Zhang,Q Xie,C-W Chen,Sh-Y  Gao,Y-H Bi,L Sun,X Ye,George F.Gao,W-J Liu*.2012.Identification and Characterization of three novel nuclear export signals in influenza A virus nucleoprotein.Journal of Virology,86(9):4970-80。
将质粒pHH21-PA、质粒pHH21-PB1、质粒pHH21-PB2、质粒pHH21-HA、质粒pHH21-NP、质粒pHH21-NA、质粒pHH21-M、质粒pHH21-NS、质粒pcDNA3.0-PA、质粒pcDNA3.0-PB1、质粒pcDNA3.0-PB2和质粒pcDNA3.0-NP共转染HEK 293T/17细胞，培养后得到WSN病毒A/WSN/1933(H1N1)毒株。WSN病毒A/WSN/1933(H1N1)毒株又称WSN病毒野生型。
实施例1、磷酸化NP蛋白的获得以及磷酸化位点的鉴定
一、磷酸化NP蛋白的获得
1、将A/WSN/1933(H1N1)毒株以MOI＝0.1的剂量感染HEK 293T/17细胞，37℃培养12-16小时后收获细胞。
2、将步骤1收获的细胞用细胞裂解液4℃处理30分钟，12000rpm离心15min，收集上清液。
3、在步骤2得到的上清液中加入抗NP蛋白的单抗，4℃孵育1小时，然后加入protein G beads 4℃孵育3小时，吸弃上清，将珠子(beads)用洗脱缓冲液在4℃下洗3次(每次10分钟)，与珠子(beads)结合的即为NP蛋白。
4、将步骤3获得的结合有NP蛋白的珠子用碱性磷酸酶37℃处理2小时(碱性磷酸酶的作用为：使磷酸化蛋白去磷酸化)。
5、将碱性磷酸酶处理前的NP蛋白(步骤3得到的)和碱性磷酸酶处理后的NP蛋白(步骤4得到的)分别进行磷酸化蛋白电泳(phos-tag SDS-PAGE)并银染显色。
结果见图1。图1中，泳道1为碱性磷酸酶处理后的NP蛋白，泳道2为碱性磷酸酶处理前的NP蛋白。将碱性磷酸酶处理后的NP蛋白作为标准NP蛋白，碱性磷酸酶处理前的NP蛋白在胶上出现比标准NP蛋白迁移速率慢且对碱性磷酸酶敏感的条带,即为磷酸化NP条带。结果表明，步骤3获得的NP蛋白为磷酸化蛋白，碱性磷酸酶可以使其发生去磷酸化反应。
二、质谱鉴定磷酸化位点
将磷酸化的NP条带从胶上切下，送样至中国科学院动物研究所大型仪器平台进行样品处理及质谱鉴定(Nano-LC MS/MS,LCQ DECA XPPLUS Thermo)。
结果见图2。B2与b3的核质比差值显示Y385被磷酸化修饰。鉴定结果显示，该条带为A型流感病毒的NP蛋白，且在385位酪氨酸残基处具有磷酸化修饰。
实施例2、突变蛋白的制备和磷酸化鉴定
一、构建重组质粒
1、构建质粒pHH21-PA
在载体pHH21的BsmBI酶切位点插入序列表的序列3所示的双链DNA分子，得到质粒pHH21-PA。
2、构建质粒pHH21-PB1
在载体pHH21的BsmBI酶切位点插入序列表的序列4所示的双链DNA分子，得到质粒pHH21-PB1。
3、构建质粒pHH21-PB2
在载体pHH21的BsmBI酶切位点插入序列表的序列5所示的双链DNA分子，得到质粒pHH21-PB2。
4、构建质粒pHH21-HA
在载体pHH21的BsmBI酶切位点插入序列表的序列6所示的双链DNA分子，得到质粒pHH21-HA。
5、构建质粒pHH21-NP
在载体pHH21的BsmBI酶切位点插入序列表的序列7所示的双链DNA分子，得到质粒pHH21-NP。
6、构建质粒pHH21-NA
在载体pHH21的BsmBI酶切位点插入序列表的序列8所示的双链DNA分子，得到质粒pHH21-NA。
7、构建质粒pHH21-M
在载体pHH21的BsmBI酶切位点插入序列表的序列2所示的双链DNA 分子，得到质粒pHH21-M。
8、构建质粒pHH21-NS
在载体pHH21的BsmBI酶切位点插入序列表的序列9所示的双链DNA分子，得到质粒pHH21-NS。
9、构建质粒pcDNA3.0-PA
在载体pcDNA3.0的KpnI和XhoI酶切位点之间插入序列表的序列3所示的双链DNA分子，得到质粒pcDNA3.0-PA。
10、构建质粒pcDNA3.0-PB1
在载体pcDNA3.0的KpnI和XhoI酶切位点之间插入序列表的序列4所示的双链DNA分子，得到质粒pcDNA3.0-PB1。
11、构建质粒pcDNA3.0-PB2
在载体pcDNA3.0的KpnI和XhoI酶切位点之间插入序列表的序列5所示的双链DNA分子，得到质粒pcDNA3.0-PB2。
12、构建质粒pcDNA3.0-NP
在载体pcDNA3.0的KpnI和XhoI酶切位点之间插入序列表的序列7所示的双链DNA分子，得到质粒pcDNA3.0-NP。
13、构建重组质粒
NP-Y385A-F:
5’-ctgagaagcagaGCGtgggccataaggaccagaagtggag-3’；
NP-Y385A-R:
5’-ccttatggcccaCGCtctgcttctcagttcaagggtacttg-3’。
NP-Y385F-F:
5’-ctgagaagcagaTTCtgggccataaggaccagaagtggag-3’；
NP-Y385F-R:
5’-ccttatggcccaGAAtctgcttctcagttcaagggtacttg-3’。
NP-Y385E-F:
5’-ctgagaagcagaGAGtgggccataaggaccagaagtggag-3’；
NP-Y385E-R:
5’-ccttatggcccaCTCtctgcttctcagttcaagggtacttg-3’。
使用Newpep点突变试剂盒(Cat.No.80111-01,北京诺派生物科技有限 公司)按试剂盒说明书构建各种重组质粒。
(1)以质粒pHH21-NP为模板，用NP-Y385A-F和NP-Y385A-R组成的引物对进行PCR扩增，得到PCR扩增产物(突变质粒)。
(2)用SDM酶在37℃酶切步骤(1)的PCR扩增产物2小时(消化模板质粒)。
(3)将步骤(2)的产物转化大肠杆菌DH5α的感受态细胞，得到重组菌Y385A-Ⅰ(即含有重组质粒pHH21-NP-Y385A的大肠杆菌)。根据测序结果，对重组质粒pHH21-NP-Y385A进行结构描述如下：将质粒pHH21-NP中的编码NP蛋白自N末端第385位酪氨酸的密码子“tac”突变为了丙氨酸的密码子“GCG”。
(4)以pcDNA3.0-NP为模板，用NP-Y385A-F和NP-Y385A-R组成的引物对进行PCR扩增，得到PCR扩增产物(突变质粒)。
(5)用SDM酶在37℃酶切步骤(4)的PCR扩增产物2小时(消化模板质粒)。
(6)将步骤(5)的产物转化大肠杆菌DH5α的感受态细胞，得到重组菌Y385A-Ⅱ(即含有重组质粒pcDNA3.0-NP-Y385A的大肠杆菌)。根据测序结果，对重组质粒pcDNA3.0-NP-Y385A进行结构描述如下：将质粒pcDNA3.0-NP中的编码NP蛋白自N末端第385位酪氨酸的密码子“tac”突变为了丙氨酸的密码子“GCG”。
(7)以质粒pHH21-NP为模板，用NP-Y385F-F和NP-Y385F-R组成的引物对进行PCR扩增，得到PCR扩增产物(突变质粒)。
(8)用SDM酶在37℃酶切步骤(7)的PCR扩增产物2小时(消化模板质粒)。
(9)将步骤(8)的产物转化大肠杆菌DH5α的感受态细胞，得到重组菌Y385F-Ⅰ(即含有重组质粒pHH21-NP-Y385F的大肠杆菌)。根据测序结果，对重组质粒pHH21-NP-Y385F进行结构描述如下：将质粒pHH21-NP中的编码NP蛋白自N末端第385位酪氨酸的密码子“tac”突变为了苯丙氨酸的密码子“TTC”；也就是说，在载体pHH21的BsmBI酶切位点插入序列表的序列10所示的双链DNA分子。
(10)以质粒pcDNA3.0-NP为模板，用NP-Y385F-F和NP-Y385F-R组 成的引物对进行PCR扩增，得到PCR扩增产物(突变质粒)。
(11)用SDM酶在37℃酶切步骤(10)的PCR扩增产物2小时(消化模板质粒)。
(12)将步骤(11)的产物转化大肠杆菌DH5α的感受态细胞，得到重组菌Y385F-Ⅱ(即含有重组质粒pcDNA3.0-NP-Y385F的大肠杆菌)。根据测序结果，对重组质粒pcDNA3.0-NP-Y385F进行结构描述如下：将质粒pcDNA3.0-NP中的编码NP蛋白自N末端第385位酪氨酸的密码子“tac”突变为了苯丙氨酸的密码子“TTC”；也就是说，在载体pcDNA3.0的KpnI和XhoI酶切位点之间插入了序列表的序列10所示的双链DNA分子。
(13)以质粒pHH21-NP为模板，用NP-Y385E-F和NP-Y385E-R组成的引物对进行PCR扩增，得到PCR扩增产物(突变质粒)。
(14)用SDM酶在37℃酶切步骤(13)的PCR扩增产物2小时(消化模板质粒)。
(15)将步骤(14)的产物转化大肠杆菌DH5α的感受态细胞，得到重组菌Y385E-Ⅰ(即含有重组质粒pHH21-NP-Y385E的大肠杆菌)。根据测序结果，对重组质粒pHH21-NP-Y385E进行结构描述如下：将质粒pHH21-NP中的编码NP蛋白自N末端第385位酪氨酸的密码子“tac”突变为了谷氨酸的密码子“GAG”。
(16)以质粒pcDNA3.0-NP为模板，用NP-Y385E-F和NP-Y385E-R组成的引物对进行PCR扩增，得到PCR扩增产物(突变质粒)。
(17)用SDM酶在37℃酶切步骤(16)的PCR扩增产物2小时(消化模板质粒)。
(18)将步骤(17)的产物转化大肠杆菌DH5α的感受态细胞，得到重组菌Y385E-Ⅱ(即含有重组质粒pcDNA3.0-NP-Y385E的大肠杆菌)。根据测序结果，对重组质粒pcDNA3.0-NP-Y385E进行结构描述如下：将质粒pcDNA3.0-NP中的编码NP蛋白自N末端第385位酪氨酸的密码子“tac”突变为了谷氨酸的密码子“GAG”。
二、突变蛋白的制备
1、Y385F突变蛋白的制备
(1)将质粒pHH21-PA、质粒pHH21-PB1、质粒pHH21-PB2、质粒 pHH21-HA、重组质粒pHH21-NP-Y385F、质粒pHH21-NA、质粒pHH21-M、质粒pHH21-NS、质粒pcDNA3.0-PA、质粒pcDNA3.0-PB1、质粒pcDNA3.0-PB2和重组质粒pcDNA3.0-NP-Y385F以等质量的配比通过脂质体Lipofectamine2000(Invitrogen)共转染HEK 293T/17细胞，37℃培养6小时。
(2)更换步骤(1)的细胞的培养基为病毒感染液，37℃培养72小时后收获细胞。
(3)将步骤(2)收获的细胞用细胞裂解液4℃处理30分钟，12000rpm离心15min，收集上清液。
(4)在步骤(3)得到的上清液中加入抗NP蛋白的单抗，4℃孵育1小时，然后加入protein G beads 4℃孵育3小时，吸弃上清，将珠子(beads)用洗脱缓冲液在4℃下洗3次(每次10分钟)，与珠子(beads)结合的即为Y385F突变蛋白。
2、NP蛋白的制备
用质粒pHH21-NP代替重组质粒pHH21-NP-Y385F并且用质粒pcDNA3.0-NP代替重组质粒pcDNA3.0-NP-Y385F，其它同步骤1，与珠子(beads)结合的即为NP蛋白。
3、Western Blot检测
将步骤1和步骤2得到的蛋白分别进行western blot，采用的一抗为抗磷酸化酪氨酸抗体，采用的二抗为HRP标记的羊抗鼠IgG。
结果见图3。结果表明，与NP蛋白相比Y385F突变蛋白的磷酸化水平显著下降，即NP蛋白第385位的酪氨酸残基为主要的磷酸化位点。
实施例3、病毒拯救
1、将HEK 293T/17细胞接种于60mm平皿，每皿1×106个细胞，培养12小时。
2、完成步骤1后，对HEK 293T/17细胞进行分组处理如下：
第一组：将质粒pHH21-PA、质粒pHH21-PB1、质粒pHH21-PB2、质粒pHH21-HA、重组质粒pHH21-NP-Y385A、质粒pHH21-NA、质粒pHH21-M、质粒pHH21-NS、质粒pcDNA3.0-PA、质粒pcDNA3.0-PB1、质粒pcDNA3.0-PB2 和重组质粒pcDNA3.0-NP-Y385A各0.5μg通过脂质体Lipofectamine2000(Invitrogen)共转染HEK 293T/17细胞，37℃培养6小时后更换培养基为病毒感染液，继续培养72小时后收获细胞。
第二组：与第一组的差异仅在于用重组质粒pHH21-NP-Y385F代替重组质粒pHH21-NP-Y385A并且用重组质粒pcDNA3.0-NP-Y385F代替重组质粒pcDNA3.0-NP-Y385A。
第三组：与第一组的差异仅在于用重组质粒pHH21-NP-Y385E代替重组质粒pHH21-NP-Y385A并且用重组质粒pcDNA3.0-NP-Y385E代替重组质粒pcDNA3.0-NP-Y385A。
第四组：与第一组的差异仅在于用质粒pHH21-NP代替重组质粒pHH21-NP-Y385A并且用质粒pcDNA3.0-NP代替重组质粒pcDNA3.0-NP-Y385A。
3、完成步骤2后，各组分别收获培养上清。
第四组得到的培养上清中含WSN病毒野生型，因此该将培养上清命名为WSN-WT病毒液。
第一组得到的培养上清中含有WSN病毒突变型(突变病毒基因组中编码NP蛋白自N末端第385位酪氨酸的密码子突变为了丙氨酸的密码子，将该突变病毒命名为WSN-Y385A病毒)，因此该将培养上清命名为WSN-Y385A病毒液。
第二组得到的培养上清中含有WSN病毒突变型(突变病毒基因组中编码NP蛋白自N末端第385位酪氨酸的密码子突变为了苯丙氨酸的密码子，将该突变病毒命名为WSN-Y385F病毒)，因此该将培养上清命名为WSN-Y385F病毒液。
第三组得到的培养上清中含有WSN病毒突变型(突变病毒基因组中编码NP蛋白自N末端第385位酪氨酸的密码子突变为了谷氨酸的密码子，将该突变病毒命名为WSN-Y385E病毒)，因此该将培养上清命名为WSN-Y385E病毒液。
4、取步骤3得到的各个病毒液，通过噬斑鉴定检测病毒滴度。
噬斑鉴定的方法：(1)将MDCK细胞接种于12孔板中，每孔约1×105个细胞，37℃、5％CO2培养箱中培养过夜；(2)用PBS缓冲液洗去细胞表 面的培养基，将待测的病毒液用病毒感染液梯度稀释后分别加入各孔中，每个稀释度设置三个重复孔，37℃孵育1小时；(3)吸弃上清并用PBS缓冲液清洗细胞，每孔加入1毫升混合溶液(混合溶液的制备方法：将1体积份融化后降温至37℃左右的3％低熔点琼脂糖与1体积份预热到37℃的无酚红DMEM培养基等体积混合，且混合物中加入TPCK处理的胰酶、青霉素和链霉素，使得胰酶浓度为2μg/ml，青霉素和链霉素的浓度均为100U/ml)；(4)将12孔板4℃放置15分钟以上，待琼脂凝固后，将孔板翻转过来倒置在37℃培养箱中培养，在显微镜下观察细胞病变情况，培养3天(实际应用中，2-4天均可)后，将12孔板从培养箱中取出，计数空斑数。
WSN-WT病毒液的滴度为6.512log10PFU/ml。WSN-Y385F病毒液的滴度为7.179log10PFU/ml。WSN-Y385A病毒液的滴度为0，即不能使MDCK产生噬斑。WSN-Y385E病毒液的滴度为0，即不能使MDCK产生噬斑。
5、完成步骤2后，各组分别收获细胞，将细胞破碎后进行western blot(检测各主要病毒蛋白的表达)。
Western Blot中：用于检测NP蛋白的一抗购自Thermo Scientific公司，产品目录号：PA5-32242；用于检测M1蛋白的一抗为抗M1蛋白的单抗。
结果见图4。各组重组系统中流感病毒的两种重要蛋白(NP蛋白、M1蛋白)均能够正常表达。
实施例4、细胞水平下不同温度下病毒生长曲线的差异
1、将A549细胞接种于10cm平皿，每皿1×108个细胞，培养12小时。
2、完成步骤1后，对A549细胞进行分组处理如下：
第一组：将实施例3制备的WSN-WT病毒液(病毒剂量为106PFU)接种A549细胞，接种后1小时换液为病毒感染液；37℃培养，分别于接种12、24、36、48、60、72小时后收取上清液，通过噬斑鉴定检测病毒滴度。
第二组：将实施例3制备的WSN-Y385F病毒液(病毒剂量为106PFU)接种A549细胞，接种后1小时换液为病毒感染液；37℃培养，分别于接种12、24、36、48、60、72小时后收取上清液，通过噬斑鉴定检测病毒 滴度。
第三组：与第一组的差异仅在于培养温度由37℃改为33℃。
第四组：与第二组的差异仅在于培养温度由37℃改为33℃。
噬斑鉴定的方法同实施例3。
每组设置10个重复处理，结果取平均值。
结果见图5。图5中，A为第三组和第四组的结果，B为第一组和第二组的结果。
33℃培养过程中，WSN-WT病毒均能正常复制，病毒滴度保持相对稳定、缓慢上升的趋势。33℃培养24小时后，WSN-Y385F病毒滴度为0，即WSN-Y385F病毒在33℃不能复制。37℃培养过程中，WSN-WT病毒和WSN-Y385F病毒均能正常复制，病毒滴度均保持相对稳定、缓慢上升的趋势。结果表明，WSN-Y385F病毒为温度敏感性病毒。
实施例5、动物水平下不同温度下病毒生长曲线的差异
36只体重约17g的6-8周龄BALB/c小鼠经过乙醚麻醉后随机分为三组，每组12只，分别进行如下处理：
第一组：利用鼻吸法吸入50μl实施例3制备的WSN-WT病毒液(病毒滴度为104PFU/ml)；
第二组：利用鼻吸法吸入50μl实施例3制备的WSN-Y385F病毒液(病毒滴度为104PFU/ml)；
第三组：利用鼻吸法吸入50μl灭菌后的PBS缓冲液。
完成上述处理后开始计时，分别于第1天、第3天、第5天、第7天时解剖小鼠(每个时间点每组取3只小鼠)，获得小鼠肺脏和鼻甲骨(鼻甲骨和肺的温度不同，鼻甲骨温度较低、约为33℃，肺部温度较高、约为37℃)。
取0.1g鲜重的肺脏或鼻甲骨，加入1ml冰浴的pH7.2的PBS缓冲液，采用QIAGEN TissueLyser II进行组织匀浆(匀浆参数：30循环/s,共计4min)，然后5000g离心10min，收集上清液。
通过噬斑鉴定检测上清液中的病毒滴度(噬斑鉴定方法同实施例3)。
结果见图6。图6中，A为肺脏结果，B为鼻甲骨的结果。
鼻甲骨中，WSN-WT病毒均能正常复制，病毒滴度保持相对稳定、缓慢下降的趋势。鼻甲骨中，WSN-Y385F病毒滴度为0，即WSN-Y385F病毒在33℃不能复制。肺脏中，WSN-WT病毒和WSN-Y385F病毒均能正常复制，病毒滴度均保持相对稳定、缓慢下降的趋势。结果表明，WSN-Y385F病毒为温度敏感性病毒。
工业应用
本发明对于流感病毒侵染的机理分析，流感病毒的防治等具有重大价值。

Claims (15)

  1. 一种重组病毒，是将A型流感病毒基因组中编码NP蛋白的第385位酪氨酸残基的密码子突变为苯丙氨酸残基的密码子得到的重组病毒。
  2. 如权利要求1所述的重组病毒，其特征在于：所述NP蛋白如序列表的序列1所示。
  3. 一种蛋白质，是将NP蛋白的第385位酪氨酸残基突变为苯丙氨酸残基得到的蛋白质。
  4. 编码权利要求3所述蛋白质的基因。
  5. 含有权利要求4所述基因的重组质粒。
  6. 一种重组病毒，其制备方法包括如下步骤：
    将质粒pHH21-PA、质粒pHH21-PB1、质粒pHH21-PB2、质粒pHH21-HA、质粒pHH21-NA、质粒pHH21-M、质粒pHH21-NS、质粒pcDNA3.0-PA、质粒pcDNA3.0-PB1、质粒pcDNA3.0-PB2、重组质粒pHH21-NP-Y385F和重组质粒pcDNA3.0-NP-Y385F共转染离体哺乳动物细胞后进行培养得到的重组病毒；
    所述质粒pHH21-PA为在载体pHH21的多克隆位点插入序列表的序列3所示的双链DNA分子得到的质粒；所述质粒pHH21-PB1为在载体pHH21的多克隆位点插入序列表的序列4所示的双链DNA分子得到的质粒；所述质粒pHH21-PB2具体可为在载体pHH21的多克隆位点插入序列表的序列5所示的双链DNA分子得到的质粒；所述质粒pHH21-HA为在载体pHH21的多克隆位点插入序列表的序列6所示的双链DNA分子得到的质粒；所述质粒pHH21-NA为在载体pHH21的多克隆位点插入序列表的序列8所示的双链DNA分子得到的质粒；所述质粒pHH21-M为在载体pHH21的多克隆位点插入序列表的序列2所示的双链DNA分子得到的质粒；所述质粒pHH21-NS为在载体pHH21的多克隆位点插入序列表的序列9所示的双链DNA分子得到的质粒；所述质粒pcDNA3.0-PA为在载体pcDNA3.0的多克隆位点插入序列表的序列3所示的双链DNA分子得到的质粒；所述质粒pcDNA3.0-PB1为在载体pcDNA3.0的多克隆位点插入序列表的序列4所示的双链DNA分子得到的质粒；所述质粒pcDNA3.0-PB2为在载体pcDNA3.0的多克隆位点 插入序列表的序列5所示的双链DNA分子得到的质粒；所述重组质粒pHH21-NP-Y385F为在载体pHH21的多克隆位点插入权利要求4所述基因得到的质粒；所述重组质粒pcDNA3.0-NP-Y385F为在载体pcDNA3.0的多克隆位点插入权利要求4所述基因得到的质粒。
  7. 一种抑制A型流感病毒的NP蛋白发生磷酸化的方法，是将A型流感病毒的NP蛋白自N末端第385位氨基酸残基由酪氨酸突变为苯丙氨酸。
  8. 一种降低A型流感病毒的NP蛋白磷酸化水平的方法，是将A型流感病毒的NP蛋白自N末端第385位氨基酸残基由酪氨酸突变为苯丙氨酸。
  9. 一种抑制A型流感病毒的NP蛋白发生磷酸化的方法，是将A型流感病毒基因组中编码NP蛋白自N末端第385位氨基酸残基的密码子由酪氨酸密码子突变为苯丙氨酸密码子。
  10. 一种降低A型流感病毒的NP蛋白磷酸化水平的方法，是将A型流感病毒基因组中编码NP蛋白自N末端第385位氨基酸残基的密码子由酪氨酸密码子突变为苯丙氨酸密码子。
  11. 权利要求1或2或6所述重组病毒在制备A型流感病毒疫苗中的应用。
  12. 权利要求1或2或6所述重组病毒作为A型流感病毒疫苗的应用。
  13. 一种A型流感病毒疫苗，其活性成分为权利要求1或2或6所述重组病毒。
  14. 一种质粒组合，由权利要求6中的质粒pHH21-PA、质粒pHH21-PB1、质粒pHH21-PB2、质粒pHH21-HA、质粒pHH21-NA、质粒pHH21-M、质粒pHH21-NS、质粒pcDNA3.0-PA、质粒pcDNA3.0-PB1、质粒pcDNA3.0-PB2、重组质粒pHH21-NP-Y385F和重组质粒pcDNA3.0-NP-Y385F组成。
  15. 一种用于制备权利要求1或2所述重组病毒的试剂盒，包括权利要求14所述质粒组合。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104592367A (zh) * 2014-12-24 2015-05-06 中国科学院微生物研究所 流感病毒np蛋白突变体及其编码基因与应用
WO2016172588A1 (en) * 2015-04-24 2016-10-27 Andrew Cox Attenuated influenza vaccines and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101186637B (zh) * 2007-11-14 2011-09-14 中国科学院微生物研究所 抑制流感病毒感染的方法及其药物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104592367A (zh) * 2014-12-24 2015-05-06 中国科学院微生物研究所 流感病毒np蛋白突变体及其编码基因与应用
WO2016172588A1 (en) * 2015-04-24 2016-10-27 Andrew Cox Attenuated influenza vaccines and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, Z.: "Mutational Analysis of Conserved Amino Acids in the Influenza A Virus Nucleoprotein", JOURNAL OF VIROLOGY, vol. 83, no. 9, 1 May 2009 (2009-05-01), XP055508458, ISSN: 0022-538X *

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