WO2021239086A1 - SARS-CoV-2假病毒及其检测样品中和SARS-CoV-2能力的方法 - Google Patents

SARS-CoV-2假病毒及其检测样品中和SARS-CoV-2能力的方法 Download PDF

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WO2021239086A1
WO2021239086A1 PCT/CN2021/096593 CN2021096593W WO2021239086A1 WO 2021239086 A1 WO2021239086 A1 WO 2021239086A1 CN 2021096593 W CN2021096593 W CN 2021096593W WO 2021239086 A1 WO2021239086 A1 WO 2021239086A1
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cov
sars
pseudovirus
protein
ace2
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PCT/CN2021/096593
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French (fr)
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贾文双
汪伟明
李恋曲
张娴
杨梦丽
刘传鑫
殷刘松
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南京蓬勃生物科技有限公司
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Priority to CN202310528005.0A priority Critical patent/CN116622641A/zh
Priority to CN202310524561.0A priority patent/CN116904405A/zh
Priority to CN202180038153.3A priority patent/CN115708418A/zh
Publication of WO2021239086A1 publication Critical patent/WO2021239086A1/zh

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Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a SARS-CoV-2 pseudovirus and a method for detecting the ability of a sample to neutralize the SARS-CoV-2 virus or its variants.
  • the SARS-CoV-2 virus is an RNA coronavirus.
  • COVID-19 novel coronavirus pneumonia
  • 2019 Novel Coronavirus 2019 Novel Coronavirus
  • SARS-CoV-2 virus is a type of RNA virus with an envelope and a linear single-stranded positive-stranded genome.
  • S protein transmembrane spike (S) glycoprotein (S protein).
  • S protein is divided into two subunits, S1 and S2.
  • the S1 subunit is responsible for binding to the host cell receptor, and the S2 subunit is responsible for the fusion of the viral membrane and the cell membrane.
  • S1 promotes viral infection by binding to the host receptor. It contains two domains, namely the N-terminal domain and the C-terminal receptor binding domain (RBD domain).
  • the C-terminal RBD domain is the interaction site with human ACE2 (Angiotensin Converting Enzyme 2) receptor, which plays an important role in the process of virus infection.
  • the present invention provides a method for detecting the ability of a sample to neutralize SARS-CoV-2 virus or its mutants, characterized in that the method includes the following steps:
  • the SARS-CoV-2 pseudovirus or variants thereof are constructed based on lentiviral vector system packaging.
  • the lentiviral vector system is selected from the second-generation lentiviral system or the third-generation lentiviral system.
  • the SARS-CoV-2 pseudovirus or variants thereof include a lentiviral backbone and the spike protein of SARS-CoV-2 or variants thereof.
  • the spike protein of SARS-CoV-2 or a variant thereof includes S1 subunit and S2 subunit.
  • the spike protein mutant of SARS-CoV-2 includes one or more amino acid substitutions, deletions and/or additions relative to the spike protein of wild-type SARS-CoV-2, preferably including at most 15 Amino acid substitutions, deletions and/or additions.
  • the SARS-CoV-2 pseudovirus or variants thereof comprise a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 2, 5-8, 21 or 22. In some specific solutions, the SARS-CoV-2 pseudovirus or variants thereof comprise at least 70%, at least 75%, at least 80%, and the amino acid sequence shown in SEQ ID NO: 2, 5-8, 21 or 22. A sequence that is at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical. In a specific embodiment, the SARS-CoV-2 pseudovirus or variants thereof comprise the amino acid sequence shown in SEQ ID NO: 2, 5-8, 21 or 22.
  • the amino acid sequence of the SARS-CoV-2 pseudovirus or a variant thereof is shown in SEQ ID NO: 2, 5-8, 21 or 22.
  • the SARS-CoV-2 pseudovirus comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, and the amino acid sequence shown in SEQ ID NO: 2 A sequence that is at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical.
  • the SARS-CoV-2 pseudovirus includes the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence of the SARS-CoV-2 pseudovirus is shown in SEQ ID NO: 2.
  • the SARS-CoV-2 pseudovirus variant comprises an amino acid sequence shown in SEQ ID NO: 5, 6, 7, 8, 21 or 22 at least 70%, at least 75%, at least 80%, A sequence that is at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical.
  • the SARS-CoV-2 pseudovirus variant comprises the amino acid sequence shown in SEQ ID NO: 5, 6, 7, 8, 21 or 22.
  • the amino acid sequence of the SARS-CoV-2 pseudovirus variant is shown in SEQ ID NO: 5, 6, 7, 8, 21 or 22.
  • the spike protein of SARS-CoV-2 or a variant thereof includes an extramembrane region, a transmembrane region, and an intramembrane region.
  • the transmembrane region includes a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 9, and the intramembrane region includes a sequence that is identical to SEQ ID NO: 10, 11, 12, 13, and 14. , 15 or 16 with at least 70% identity.
  • the transmembrane region comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89% of the amino acid sequence shown in SEQ ID NO: 9 , At least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequence
  • the intramembrane region comprises a sequence with SEQ ID NO: 10, 11, 12, 13, 14, 15 or 16. Shows the amino acid sequence is at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% Consistent sequence.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 9, and the intramembrane region comprises the amino acid sequence shown in SEQ ID NO: 10, 11, 12, 13, 14, 15 or 16. Amino acids.
  • the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 9, and the amino acid of the intramembrane region is shown in SEQ ID NO: 10, 11, 12, 13, 14, 15 or 16. Shown.
  • the SARS-CoV-2 pseudovirus or its variant in contact with the drug at an MOI of 0.04-300, preferably the pseudovirus or its variant is in contact with the drug at an MOI of 0.1-150
  • the MOI of is in contact with the drug, and more preferably, the virus is in contact with the sample at an MOI of 1-100.
  • the SARS-CoV-2 pseudovirus or variants thereof in step (1) has an MOI of 0.04, 0.08, 0.1, 1.0, 20, 25, 50, 100, 200, or 300 with the Drug exposure.
  • the SARS-CoV-2 pseudovirus or a variant thereof in step (1) is in contact with the drug at an MOI of 0.1-150.
  • the SARS-CoV-2 pseudovirus or a variant thereof is contacted with the drug at an MOI of 1-100.
  • the SARS-CoV-2 pseudovirus or a variant thereof is contacted with the drug at an MOI of 1.0.
  • the SARS-CoV-2 pseudovirus or a variant thereof is contacted with the drug at an MOI of 100.
  • the step of contacting the pseudovirus or its variant with the drug in step (1) includes mixing and incubating.
  • the incubation is 0.5-3 hours, and more preferably, the incubation is 1 hour.
  • the step of contacting the drug in step (1) includes mixing and incubation, and the incubation time is 1 hour.
  • the cell line overexpressing ACE2 in step (2) is selected from HEK293, Hela, Vero E6 or CHO, preferably HEK293T, Hela or CHO-K1.
  • the cell line is selected from HEK293 overexpressing ACE2, Hela overexpressing ACE2, or CHO-K1 overexpressing ACE2, preferably HEK293 overexpressing ACE2.
  • the cell line is HEK293 overexpressing ACE2 or Hela overexpressing ACE2.
  • the cell line is HEK293, Hela, Vero E6 or CHO-K1 expressing both ACE2 and TMPRSS2 (Transmembrane protease, serine 2, transmembrane serine protease 2), preferably expressing both ACE2 and TMPRSS2 HEK293.
  • the cell line is HEK293 expressing both ACE2 and TMPRSS2.
  • the cell line is Hela expressing both ACE2 and TMPRSS2.
  • the cell line is Vero E6 expressing both ACE2 and TMPRSS2.
  • the cell line is CHO-K1 that expresses both ACE2 and TMPRSS2.
  • the added amount of the cell line overexpressing ACE2 in step (2) is 0.2 ⁇ 10 4 -3 ⁇ 10 4 cells, preferably 2.0 ⁇ 10 4 cells.
  • the added amount of the ACE2 overexpressed HEK293 or ACE2 overexpressed Hela is 0.2 ⁇ 10 4 -3 ⁇ 10 4 cells.
  • the added amount of HEK293 overexpressed ACE2 or Hela overexpressed ACE2 is 2.0 ⁇ 10 4 cells.
  • the amount of HEK293, Hela, Vero E6 or CHO-K1 dual-expressing ACE2 and TMPRSS2 is 0.2 ⁇ 10 4 -3 ⁇ 10 4 cells.
  • the added amount of HEK293, Hela, Vero E6 or CHO-K1 expressing both ACE2 and TMPRSS2 is 2.0 ⁇ 10 4 cells.
  • the contact culture step in step (2) includes mixing the mixture of the pseudovirus and the drug with the ACE2 overexpressing cell line and culturing for 24-72 hours, then lysing the cells, preferably culturing 48 Cells were lysed after hours.
  • the contact culture step in step (2) includes mixing the mixture of the pseudovirus and the drug with the ACE2 overexpressing cell line, culturing for 48-72 hours, and then lysing the cells.
  • the contact culture step in step (2) includes mixing the mixture of the pseudovirus and the drug with the ACE2 overexpressing cell line, culturing for 48 hours, and then lysing the cells.
  • the genome of the SARS-CoV-2 pseudovirus or a variant thereof includes the reporter gene described in step (3).
  • the reporter gene may be selected from genes conventional in the art, which are expressed in cells, tissues/organs or individuals under specific conditions and allow them to produce traits that are easy to detect and are not originally produced by experimental materials.
  • the reporter gene is selected from a green fluorescent protein gene or a luciferase gene, preferably a luciferase gene.
  • the reporter gene is a luciferase gene.
  • the reporter gene is a green fluorescent protein gene.
  • the protein encoded by the luciferase gene includes a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO:1. In other embodiments, the protein encoded by the luciferase gene includes at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87% of the amino acid sequence shown in SEQ ID NO:1. , At least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequence. In a specific embodiment, the protein encoded by the luciferase gene comprises the amino acid sequence shown in SEQ ID NO:1. In another specific embodiment, the amino acid sequence of the protein encoded by the luciferase gene is shown in SEQ ID NO:1.
  • step (3) determining whether the sample has the ability to neutralize the SARS-CoV-2 virus or its mutants includes preparing a dose-response curve between the sample concentration and the reporter gene signal, and obtaining the test drug IC 50 , compare and confirm with the positive control.
  • the positive control is ACE2-Fc fusion protein.
  • the mixture of the pseudovirus and the sample is mixed with one of the cell lines and then the culture is continued. After the pseudovirus that has not been neutralized enters the cell, the luciferase is expressed through the luciferase and the substrate.
  • the reaction signal infers the amount of virus in the infected cell.
  • the obtained reaction signal is compared with the positive control group of the ACE2-Fc fusion protein and the negative control group without neutralization reaction to infer the neutralization ability of the test antibody or drug against the pseudovirus. .
  • the sample includes antibodies, polypeptides or small molecule compounds capable of acting on SARS-CoV-2 virus or its mutants, preferably neutralizing antibodies against SARS-CoV-2 virus or its mutants.
  • the sample is a neutralizing antibody against SARS-CoV-2 virus or a mutant thereof.
  • the samples in the present invention include, but are not limited to, serum, plasma, whole blood, pleural effusion, cerebrospinal fluid, tissue specimens or chemically prepared samples that act on SARS-CoV-2 virus or its mutants derived from animals or humans.
  • the compound reagent is preferably derived from serum, plasma and whole blood.
  • the present invention provides a SARS-CoV-2 pseudovirus or a variant thereof, the pseudovirus or a variant thereof includes the spike protein of SARS-CoV-2 or a variant thereof.
  • the SARS-CoV-2 pseudovirus or variants thereof provided by the present invention is characterized in that the pseudovirus is constructed based on the lentiviral vector system packaging and includes the lentiviral backbone and the spike of SARS-CoV-2 Protein or variants thereof.
  • the spike protein of SARS-CoV-2 or a variant thereof includes S1 subunit and S2 subunit.
  • the S1 subunit of the spike protein of SARS-CoV-2 or a variant thereof includes at least 70% identity with the amino acid sequence shown in SEQ ID NO: 3.
  • the S1 subunit of the spike protein of SARS-CoV-2 or a variant thereof includes at least 70%, at least 75%, at least 80%, or at least 83% of the amino acid sequence shown in SEQ ID NO: 3. , At least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequences.
  • the S1 subunit of the spike protein of SARS-CoV-2 or a variant thereof includes the amino acid sequence shown in SEQ ID NO: 3. In another specific embodiment, the amino acid sequence of the S1 subunit of the spike protein of SARS-CoV-2 or a variant thereof is shown in SEQ ID NO: 3.
  • the spike protein mutant of SARS-CoV-2 includes one or more amino acid substitutions, deletions or additions, preferably including up to 15 amino acid substitutions, deletions and/or additions, more preferably It includes substitutions and/or deletions of up to 10 amino acids. In other embodiments, the spike protein mutant of SARS-CoV-2 includes 1, 2, 3, 4, or 5 amino acid substitutions, deletions and/or additions.
  • the spike protein mutant of SARS-CoV-2 includes a group of amino acid substitutions selected from D364Y, V367F, W436R, D614G, E484K, and N501Y relative to the spike protein of wild-type SARS-CoV-2 One or more of them preferably comprise an amino acid substitution selected from D364Y, V367F, W436R or D614G.
  • the S1 subunit of the spike protein mutant of SARS-CoV-2 contains amino acid substitutions of D364Y, V367F, W436R or D614G relative to the spike protein of SARS-CoV-2.
  • the S1 subunit of the spike protein mutant of SARS-CoV-2 contains an amino acid substitution of D364Y relative to the spike protein of wild-type SARS-CoV-2.
  • the spike protein mutant of SARS-CoV-2 contains an amino acid substitution of V367F relative to the spike protein of wild-type SARS-CoV-2.
  • the spike protein mutant of SARS-CoV-2 contains an amino acid substitution of W436R relative to the spike protein of wild-type SARS-CoV-2.
  • the spike protein mutant of SARS-CoV-2 contains an amino acid substitution of D614G relative to the spike protein of wild-type SARS-CoV-2.
  • the spike protein mutant of SARS-CoV-2 contains an E484K amino acid substitution relative to the spike protein of wild-type SARS-CoV-2. In other specific embodiments, the spike protein mutant of SARS-CoV-2 contains N501Y amino acid substitutions relative to the spike protein of wild-type SARS-CoV-2.
  • the S1 subunit of the spike protein mutant of SARS-CoV-2 contains H69, V70 and Y144 amino acid deletions and N501Y relative to the S1 subunit of the spike protein of SARS-CoV-2. A570D, D614G, and P681H amino acid substitutions.
  • the S2 subunit of the spike protein mutant of SARS-CoV-2 includes T716I, S982A, and D1118H amino acid substitutions relative to the S2 subunit of the spike protein of SARS-CoV-2.
  • the S1 subunit of the spike protein mutant of SARS-CoV-2 relative to the S1 subunit of the spike protein of SARS-CoV-2 includes L242, A243 and L244 amino acid deletions and L18F , D80A, D215G, R246I, E484K, K417N, N501Y and D614G amino acid substitutions, the S2 subunit of the spike protein mutant of SARS-CoV-2 relative to the S2 subunit of the spike protein of SARS-CoV-2 includes A701V amino acid substitution.
  • the S2 subunit of the spike protein of SARS-CoV-2 or a variant thereof comprises a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 4. In other embodiments, the S2 subunit of the spike protein of SARS-CoV-2 or a variant thereof comprises at least 70%, at least 75%, at least 80%, at least the amino acid sequence shown in SEQ ID NO: 4 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequence. In a specific embodiment, the S2 subunit of the spike protein of SARS-CoV-2 or a variant thereof comprises the amino acid sequence shown in SEQ ID NO: 4. In another specific embodiment, the amino acid sequence of the S2 subunit of the spike protein of SARS-CoV-2 or a variant thereof is shown in SEQ ID NO: 4.
  • the spike protein of SARS-CoV-2 or a variant thereof comprises an amino acid sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 2, 5, 6, 7, 8, 21 or 22 sequence. In other embodiments, the spike protein of SARS-CoV-2 or a variant thereof comprises at least 70%, at least at least 70% of the amino acid sequence shown in SEQ ID NO: 2, 5, 6, 7, 8, 21 or 22 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequences.
  • the spike protein of SARS-CoV-2 or a variant thereof comprises the amino acid sequence shown in SEQ ID NO: 2, 5, 6, 7, 8, 21 or 22.
  • the amino acid sequence of the spike protein of SARS-CoV-2 or a variant thereof is shown in SEQ ID NO: 2, 5, 6, 7, 8, 21 or 22.
  • the SARS-CoV-2 pseudovirus comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, and the amino acid sequence shown in SEQ ID NO: 2 A sequence that is at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical.
  • the SARS-CoV-2 pseudovirus includes the amino acid sequence shown in SEQ ID NO: 2. In another specific embodiment, the amino acid sequence of the SARS-CoV-2 pseudovirus is shown in SEQ ID NO: 2. In some embodiments, the SARS-CoV-2 pseudovirus variant comprises an amino acid sequence shown in SEQ ID NO: 5, 6, 7, 8, 21 or 22 at least 70%, at least 75%, at least 80%, A sequence that is at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical. In a specific embodiment, the SARS-CoV-2 pseudovirus variant comprises the amino acid sequence shown in SEQ ID NO: 5, 6, 7, 8, 21 or 22. In another specific embodiment, the amino acid sequence of the SARS-CoV-2 pseudovirus variant is shown in SEQ ID NO: 5, 6, 7, 8, 21 or 22.
  • the spike protein of SARS-CoV-2 or a variant thereof includes an extramembrane region, a transmembrane region, and an intramembrane region.
  • the intramembrane region comprises a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 10, 11, 12, 13, 14, 15, or 16.
  • the intramembrane region comprises at least 70%, at least 75%, at least 80%, or at least 83% of the amino acid sequence shown in SEQ ID NO: 10, 11, 12, 13, 14, 15 or 16. , At least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequences.
  • the intramembrane region comprises the amino acid shown in SEQ ID NO: 10, 11, 12, 13, 14, 15 or 16. In another specific embodiment, the amino acid of the intramembrane region is as shown in SEQ ID NO: 10, 11, 12, 13, 14, 15 or 16.
  • the transmembrane region comprises a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO:9. In other embodiments, the transmembrane region comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89% of the amino acid sequence shown in SEQ ID NO: 9 , At least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequences. In a specific embodiment, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:9. In another specific embodiment, the amino acid sequence of the transmembrane region is shown in SEQ ID NO: 9.
  • the virus or variant thereof contains a reporter gene.
  • the reporter gene may be selected from genes conventional in the art, which are expressed in cells, tissues/organs or individuals under specific conditions and allow them to produce traits that are easy to detect and are not originally produced by experimental materials.
  • the reporter gene is selected from a green fluorescent protein gene or a luciferase gene, preferably a luciferase gene.
  • the reporter gene is a luciferase gene, preferably a firefly luciferase gene.
  • the reporter gene is a green fluorescent protein gene.
  • the present invention provides a method for preparing the above pseudovirus or a variant thereof, characterized in that the method comprises the following steps:
  • step (2) Prepare the packaging plasmid and transfer plasmid of the lentiviral vector expression system, mix with the envelope plasmid of the SARS-CoV-2 spike protein or its variant constructed in step (1), and then add it to the virus-producing cells to continue culturing;
  • the SARS-CoV-2 spike protein or its variant plasmid in step (1) contains and encodes the amino acid sequence shown in SEQ ID NO: 2, 5, 6, 7, 8, 21 or 22
  • the nucleotide sequence is at least 70% identical.
  • the SARS-CoV-2 pseudovirus or variants thereof comprise at least 70% of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5, 6, 7, 8, 21 or 22, A sequence that is at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical.
  • the SARS-CoV-2 pseudovirus or variants thereof comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5, 6, 7, 8, 21 or 22.
  • the amino acid sequence of the SARS-CoV-2 pseudovirus or a variant thereof is shown in SEQ ID NO: 2 or SEQ ID NO: 5, 6, 7, 8, 21 or 22
  • the SARS-CoV-2 spike protein or variant plasmid thereof in step (1) comprises a nucleotide sequence encoding the intramembrane region of the SARS-CoV-2 spike protein or variant thereof.
  • the nucleotide sequence encoding the intramembrane region of the SARS-CoV-2 spike protein or variants thereof contains the same as those encoding SEQ ID NO: 10, 11, 12, 13, 14, 15 or
  • the nucleotide sequence of the amino acid sequence shown in 16 is a nucleotide sequence with at least 70% identity.
  • the nucleotide sequence encoding the intramembrane region of the SARS-CoV-2 spike protein or variants thereof comprises the same as those encoding SEQ ID NO: 10, 11, 12, 13, 14, 15 or
  • the nucleotide sequence of the amino acid sequence shown in 16 is at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95% , A nucleotide sequence with at least 97% or at least 99% identity.
  • the nucleotide sequence encoding the intramembrane region of the SARS-CoV-2 spike protein or a variant thereof comprises encoding SEQ ID NO: 10, 11, 12, 13, 14, 15 or 16.
  • the step (1) includes synthesizing the gene sequence of the SARS-CoV-2 spike protein or its variants, ligating it with the plasmid vector pMD2.G or pcDNA3.1 eukaryotic expression vector after restriction digestion, and transforming Large intestine competent cells to obtain a plasmid of the SARS-CoV-2 spike protein or a variant thereof.
  • the transfer plasmid is selected from pLVX-CMV-Luciferase, and the packaging plasmid is selected from psPAX2.
  • the added amount of the packaging plasmid is 7-10 ⁇ g
  • the added amount of the envelope plasmid is 5-8 ⁇ g
  • the added amount of the transfer plasmid is 8-13 ⁇ g.
  • the virus-producing cell is selected from suspended HEK293 or adherent HEK293, preferably suspended HEK293. In a specific embodiment, the virus-producing cell is a suspension HEK293. In another specific embodiment, the virus-producing cell is adherent HEK293.
  • the culture time of the lentiviral system and the virus-producing cells in step (3) is 48-96 hours, preferably 48-72 hours. In a specific embodiment, the culture time of the lentiviral system and the virus-producing cells in step (3) is 48-72 hours.
  • the present invention provides the application of the above-mentioned SARS-CoV-2 pseudovirus or its variants in screening anti-SARS-CoV-2 or its variants.
  • the present invention provides a cell line double overexpressing ACE2 and TMPRSS2, the cell line comprising the ACE2 gene and the TMPRSS2 gene.
  • the cell line further includes a resistance gene.
  • the resistance gene is selected from puromycin resistance gene or hygromycin resistance gene.
  • the TMPRSS2 gene includes a nucleotide sequence that encodes a nucleotide sequence that is at least 70% identical to the nucleotide sequence of the amino acid sequence shown in SEQ ID NO:20.
  • the TMPRSS2 gene comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87% of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20 , At least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical nucleotide sequence.
  • the TMPRSS2 gene includes a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20.
  • the sequence of the TMPRSS2 gene is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:20.
  • the dual expression cell line is selected from HEK293, Hela, Vero E6 or CHO, preferably HEK293T or CHO-K1.
  • the cell line is HEK293 expressing both ACE2 and TMPRSS2.
  • the cell line is Hela expressing both ACE2 and TMPRSS2.
  • the cell line is Vero E6 expressing both ACE2 and TMPRSS2.
  • the cell line is CHO-K1 that expresses both ACE2 and TMPRSS2.
  • the present invention provides a method for preparing the above-mentioned dual-expression cell line, which includes the following steps:
  • step (3) Mix the plasmid obtained in step (2) with the envelope plasmid and the packaging plasmid and then add it to the virus production cell to produce the lentivirus;
  • step (3) The lentivirus obtained in step (3) is infected with the cell line of step (1) expressing TMPRSS2, resistance screening is carried out and a monoclonal cell line is selected.
  • the step (1) includes: a. Synthesize the DNA sequence of the ACE2 protein, and clone it into the pLVX vector after digestion; b. Mix the plasmid obtained in step a with the envelope plasmid and the packaging plasmid and then add the virus Infected cells to produce lentivirus; c. Infect target cells with the lentivirus obtained in step b, and select monoclonal cell lines after resistance screening.
  • the cell line in step (1) is selected from HEK293, Hela, Vero E6 or CHO, preferably HEK293 or CHO-K1.
  • the TMPRSS2 gene in step (2) includes a nucleotide sequence that encodes a nucleotide sequence that is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 20.
  • the TMPRSS2 gene comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87% of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20 , At least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical nucleotide sequence.
  • the TMPRSS2 gene includes a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 20.
  • the sequence of the TMPRSS2 gene is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:20.
  • step (3) the amount of plasmid obtained in step (2) is 9-13 ⁇ g, the amount of enveloped plasmid is 7-10 ⁇ g, and the amount of packaging plasmid is 5-8 ⁇ g.
  • the present invention provides the application of ACE2 single overexpression cell line or ACE2 and TMPRSS2 double overexpression cell line in infection with coronavirus.
  • Coronavirus especially SARS-CoV-2 or its variants can efficiently bind to the surface of the ACE2 single overexpression cell line or the ACE2 and TMPRSS2 double overexpression cell line to infect the cell line and synthesize the reporter gene in the cell.
  • the coronavirus is selected from SARS-CoV-2 or its variants or SARS-CoV or its variants, preferably SARS-CoV-2 or its variants.
  • the present invention provides a SARS-CoV-2 pseudovirus based on lentivirus, which can efficiently infect ACE2 single overexpression cell line or ACE2 and TMPRSS2 double overexpression cell line, using the pseudovirus and the cell line It is possible to develop and research samples that specifically and effectively neutralize SARS-CoV-2.
  • a SARS-CoV-2 pseudovirus constructed based on lentivirus packaging includes a lentiviral backbone and a SARS-CoV-2 coronavirus spike protein or a variant thereof.
  • the SARS-CoV-2 pseudovirus and its pseudovirus variants based on lentivirus packaging include the lentiviral backbone and the extramembrane region and transmembrane region of the S protein of SARS-CoV-2 And the area inside the membrane.
  • the lentiviral backbone includes a second-generation lentiviral packaging plasmid (Packaging plasmid), transfer plasmid (Transfer plasmid) and envelope plasmid (Envelope plasmid).
  • the packaging plasmid includes the protein required by the lentivirus to provide viral particles; the transfer plasmid includes a reporter gene luciferase (Luciferase) as shown in SEQ ID NO:1; the envelope plasmid includes an envelope protein, which is protected by SARS- S protein replacement of CoV-2.
  • the S protein includes an extramembrane region, a transmembrane region, and an intramembrane region as shown in SEQ ID NO: 2. Part of the extramembrane region of the S protein belongs to the S1 subunit of the S protein, including the receptor binding domain (RBD).
  • the extramembrane, transmembrane and intramembrane regions other than the S1 subunit of the S protein belong to the S2 subunit of the S protein.
  • the S1 subunit and S2 subunit sequences are shown in SEQ ID NOs: 3 and 4, respectively, and the S protein sequence including an amino acid substitution in the S1 subunit is shown in SEQ ID NO: 5-8, respectively; the S2 subunit
  • the sequence of the transmembrane region of the base is shown in SEQ ID NO: 9; the sequence of the intramembrane region of the S2 subunit is shown in SEQ ID NO: 10, or the replacement sequence of the intramembrane region of the S2 subunit is shown in SEQ ID NO : As shown in 11-16.
  • a type of ACE2 single overexpression cell line or ACE2 and TMPRSS2 double overexpression cell line cell line is provided.
  • the above pseudovirus can efficiently bind to the surface of the cell line ACE2 to infect the cell line and infect the cell line.
  • the reporter gene is synthesized in the cell.
  • the cell line includes a cell line established with Hela, HEK293, Vero E6 or CHO-K1 as a host that stably overexpresses the S protein receptor ACE2 and/or the accessory protein TMPRSS2.
  • the C-terminal tail of the ACE2 intramembrane region includes a tag (TAG) as shown in SEQ ID NO: 17 or no tag.
  • a method for detecting the ability of a sample to neutralize the S protein of SARS-CoV-2 and its variants based on the cell level, the sample including antibodies, polypeptides, small molecule compounds, and the like.
  • the method includes the following steps: 1) contacting the SARS-CoV-2 pseudovirus or its variant with the sample, and 2) determining whether the pseudovirus is prevented by the sample from entering the ACE2 single overexpression cell line or ACE2 and TMPRSS2 are doubled Express the cell line and express the reporter gene in the cell.
  • the cell line includes a cell line established with Hela, HEK293, Vero E6, Huh7 or CHO-K1 as a host that stably overexpresses the S protein receptor ACE2 and/or the accessory protein TMPRSS2.
  • the C-terminal tail of the ACE2 intramembrane region contains a flag as shown in SEQ ID NO: 17.
  • the sample to be tested is diluted at a single point and then mixed and cultured with the pseudovirus or the selected pseudovirus variant.
  • the culture medium is mixed with one of the cell lines and then continued to be cultured.
  • luciferase is expressed, and the amount of virus in the infected cell is estimated by the reaction signal of the luciferase and the substrate. Comparing the obtained reaction signal with the positive control group of ACE2-Fc fusion protein and the negative control group without neutralization reaction, the neutralization ability of the test sample to the pseudovirus can be inferred.
  • the method firstly mixes and cultures the pseudovirus or selected pseudovirus variants after the sample is diluted in equal proportions.
  • the culture medium is mixed with one of the cell lines and then continued to be cultured. After the unneutralized pseudovirus enters the cell, luciferase is expressed, and the amount of virus in the infected cell is estimated by the reaction signal of the luciferase and the substrate.
  • the present invention provides a method for detecting the ability of a sample to neutralize the SARS-CoV-2 virus.
  • the method can accurately and quickly detect SARS without involving the SARS-CoV-2 true virus.
  • This method can quickly, accurately and quickly detect that candidate vaccines are produced in serum or plasma after immunization And the effect of antibodies, in order to facilitate the evaluation of candidate vaccine effects.
  • This method can be used to detect the neutralizing effect of a variety of SARS-CoV-2 samples to facilitate the discovery of broad-spectrum drugs and vaccines.
  • coronavirus refers to a type of zoonotic RNA virus that spreads between animals and humans. Coronavirus can infect mammals and birds, causing digestive tract diseases in cattle and pigs or upper respiratory tract diseases in chickens. Common in nature, there are seven types of coronaviruses known to infect humans, which can cause respiratory infections in humans, and can cause common colds, and even Middle East Respiratory Syndrome (MERS-Cov) and Severe Acute Respiratory Syndrome (SARS-Cov), 2019 Coronavirus disease (COVID-19 or SARS-CoV-2) and other more serious diseases.
  • MERS-Cov Middle East Respiratory Syndrome
  • SARS-Cov Severe Acute Respiratory Syndrome
  • COVID-19 or SARS-CoV-2 2019 Coronavirus disease 2019 Coronavirus disease
  • the coronavirus in the present invention is selected from SARS-CoV-2 or its variants, SARS-CoV or its variants, or MERS-Cov or its variants.
  • the coronavirus is SARS-CoV or a variant thereof.
  • new coronavirus (SARS-CoV-2), also known as COVID-19, belongs to the ⁇ -coronavirus and has an envelope.
  • the virus particles are round or elliptical with a diameter of 60-140nm. Its genome is only 80% similar to that of SARS-CoV-1, while the similarity of the gene sequence to the coronavirus (Bat coronavirus RaTG13) isolated from Rhinolophus affinis is as high as 96%.
  • SARS-CoV-2 virus variants refer to a variety of new coronaviruses that have high sequence identity with SARS-CoV-2 virus genes, and viruses that have SARS-CoV-2 virus infection and pathogenicity, Such as hCoV-19/Portugal/PT0548/2020, hCoV-19/England/CAMB-1B2093/2020, hCoV-19/USA/VA-DCLS-0287/2020, hCoV-19/India/NIHSAD-1005-52/ 2020, hCoV-19/Spain/COV000664/2020, hCoV-19/Beijing/DT-travelGR01/2020.
  • a coronavirus isolated from Malayan pangolin by a research team has shown 100%, 98.2%, 96.7% and 90.4% amino acid identity with 2019-nCoV in the E, M, N, and S genes, respectively. .
  • the receptor binding domain of the S protein of pangolin coronavirus is actually the same as the receptor binding domain of the S protein of 2019-nCoV, with only one amino acid difference (https://www.biorxiv.org/content/10.1101/ 2020.02.17.951335v1).
  • the gene sequence of the SARS-CoV-2 virus variant described in the present invention and the gene sequence of the new coronavirus have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • SARS-CoV-2 virus antigen in the present invention refers to the antigen of the SARS-CoV-2 whole virus lysate or the recombinantly expressed SARS-CoV-2 antigen.
  • SARS-CoV-2 virus includes spike protein (S), envelope protein (E), matrix protein (M) and nucleocapsid protein (N) antigens, among which S protein is the largest structural protein of SARS-CoV-2.
  • S protein can be cleaved into S1 and S2 subunits under the action of host enzymes.
  • the S1 subunit contains the receptor binding region RBD and is the main target antigen.
  • the SARS-CoV-2 virus antigen is selected from spike protein (S), envelope protein (E), matrix protein (M) and/or nucleocapsid protein (N) antigen.
  • the SARS-CoV-2 virus antigen is SARS-CoV-2 S1 subunit or SARS-CoV-2 S RBD antigen.
  • the SARS-CoV-2 S RBD antigen described in the present invention can be produced by conventional recombinant expression methods, by constructing a plasmid expressing SARS-CoV-2 S RBD, such as pFastBac1, pTT5, and transfecting expression cells with an expression vector containing the target gene , Such as CHO cells and SF9 cells, express and purify SARS-CoV-2 S RBD recombinant protein.
  • the anti-SARS-CoV-2 virus antibody in the present invention refers to an antibody against the SARS-CoV-2 virus antigen, including neutralizing antibodies.
  • antibody refers to an immunoglobulin molecule composed of four polypeptide chains, including two heavy chains (H) and two light chains (L), connected by disulfide bonds to form a complete antibody molecule.
  • lentivirus refers to a gene carrier developed on the basis of HIV-1, which is a retrovirus and is infectious to both dividing and non-dividing cells.
  • the SARS-CoV-2 pseudovirus uses a lentivirus as its backbone, and replaces the envelope protein on the surface of the lentivirus with the S protein of SARS-CoV-2.
  • neutralization refers to the process by which the sample binds to the S protein of the SARS-CoV-2 pseudovirus to prevent it from adhering to the cell membrane, fusing and entering the cell.
  • neutralizing antibody refers to antibodies that prevent cells from being attacked by a certain antigen or source of infection by binding to virus molecules, and its principle is to inhibit or even neutralize their certain biochemical effects.
  • SARS-CoV-2 virus neutralizing antibody refers to an antibody that blocks the binding of the SARS-CoV-2 virus RBD to the human ACE2 receptor by binding to the SARS-CoV-2 virus RBD protein, and prevents the virus from binding to the human ACE2 receptor. Cell membranes adhere, fuse and enter cells.
  • SARS-CoV-2 virus RBD protein herein can be embodied as recombinant SARS-CoV-2 virus RBD protein, SARS-CoV-2 virus RBD protein with His tag, and SARS-CoV-2 with Fc tag Viral RBD protein, including molecules of SARS-CoV-2 virus RBD protein, such as SARS-CoV-2 virus S1 subunit and SARS-CoV-2 virus S protein.
  • TMPRSS2 transmembrane serine protease 2
  • SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus Type 2
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus Type 2
  • the transmembrane serine protease 2 inhibitors can effectively reduce the entry of coronaviruses such as SARS-CoV-2 into target cells and reduce their infectivity.
  • amino acid mutation refers to the substitution, deletion or insertion of one or more amino acids in the existing amino acid sequence.
  • amino acid substitution refers to the replacement of existing amino acid residues with different amino acid residues in the existing amino acid sequence, which is divided into conservative substitutions and radical substitutions.
  • Conservative substitution refers to the substitution between amino acid molecules with similar traits. The traits include the ionicity, hydrophobicity, and molecular weight of the molecule. Radical substitution refers to the mutual substitution of amino acids with very different physical and chemical properties.
  • Amino acid deletion refers to the deletion of one or more amino acids in the existing amino acid sequence.
  • the S1 subunit of the spike protein of SARS-CoV-2 contains one or more amino acid deletions, such as one or more amino acids at positions H69, V70, Y144, L242, A243, and L244 Delete.
  • the S1 subunit of the spike protein of SARS-CoV-2 includes amino acid deletions at positions H69, V70, and Y144.
  • the S1 subunit of the spike protein of SARS-CoV-2 includes amino acid deletions at positions L242, A243, and L244.
  • the "percent (%) amino acid sequence identity" of a peptide or polypeptide sequence is defined as comparing the sequences and introducing gaps when necessary to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Candidates The percentage of amino acid residues in the sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence. Sequence comparisons can be performed in a variety of ways within the skill of the art to determine percent amino acid sequence identity, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required to obtain the maximum comparison over the entire length of the sequence being compared.
  • IC 50 is also known as half maximal inhibitory concentration, refers to a drug capable of cell growth, viral replication inhibitory concentration required to produce 50%.
  • a SARS-CoV-2 pseudovirus constructed based on lentivirus packaging includes a lentiviral backbone and the S protein of SARS-CoV-2 or related variants thereof.
  • the variant of the S protein is derived from the SARS-CoV-2 S protein with amino acid substitutions, deletions, or additions, including RBD regions and non-RBD regions, and the substitutions can affect the binding affinity of the S protein to ACE2, Enhance the infectivity of SARS-CoV-2 pseudovirus.
  • the related variants of the S protein include the substitution of the S protein membrane region and/or the amino acid substitution of the S1 subunit to construct a new chimeric protein.
  • the intramembrane region of the S protein replaces or is derived from the S protein of SARS-CoV (NC_004718), the S protein of MERS-CoV (NC_019843), the membrane protein gp41 of HIV-1 (HXB2, NC_001802), and the membrane of VSV (J02428)
  • the sequences of protein G, the membrane protein of Marburg virus (NC_001608) and the membrane protein of Ebola virus (NC_002549) are shown in SEQ ID NO: 11-16, respectively.
  • a type of ACE2 single overexpression cell line or ACE2 and TMPRSS2 double overexpression cell line cell line is provided.
  • the above pseudovirus can efficiently bind to the surface of the cell line ACE2 to infect the cell line and infect the cell line.
  • the reporter gene is synthesized in the cell.
  • the ACE2 single overexpression cell line is transfected into target cells with a lentiviral vector to establish a cell pool overexpressing ACE2. From the cell pool overexpressing ACE2, further select monoclonal cells overexpressing ACE2 to establish a stable overexpression cell line.
  • the target cells include Hela, HEK293 or Vero E6.
  • the ACE2 and TMPRSS2 double overexpression cell lines are established on ACE2 single overexpression target cell lines.
  • the target cell line is established with Hela, HEK293 or Vero E6 as a host.
  • the target cell line was further transfected with a lentivirus to mediate the SARS-CoV-2 fusion helper protein TMPRSS2.
  • the obtained cell pool undergoes further monoclonal screening to establish a monoclonal cell line with stable double overexpression of ACE2 and TMPRSS2.
  • a method for detecting the ability of a sample to neutralize the S protein of SARS-CoV-2 and its variants based on the cell level includes the following steps: 1) Combining SARS-CoV-2 The pseudovirus or its variant is contacted with the sample, and 2) it is determined whether the pseudovirus is prevented by the sample from entering the ACE2 single overexpression cell line or the ACE2 and TMPRSS2 double overexpression cell line and express the reporter gene in the cell.
  • the sample to be tested is diluted at a single point and then mixed with the SARS-CoV-2 pseudovirus and/or SARS-CoV-2 pseudovirus variant and then cultured.
  • the unneutralized pseudovirus enters the cell, it expresses luciferase, and the reaction between luciferase and the substrate The signal infers the amount of virus that infects the cell.
  • the obtained reaction signal is compared with the positive control group of the ACE2-Fc fusion protein and the negative control group without neutralization reaction to infer the neutralizing ability of the test sample against the pseudovirus and/or pseudovirus variants.
  • the method firstly dilutes the sample to be tested in equal proportions and then mixes and cultures the SARS-CoV-2 pseudovirus and/or SARS-CoV-2 pseudovirus variants. Mix the culture medium with the ACE2 single overexpression cell line or the ACE2 and TMPRSS2 double overexpression cell line and continue the culture. After the unneutralized pseudovirus enters the cell, it expresses luciferase, and the reaction between luciferase and the substrate The signal infers the amount of virus that infects the cell.
  • Figure 1 shows the FACS detection of the expression level of ACE2 in the HEK293/ACE2 single overexpression cell line
  • Figure 1A shows the polyclonal pool of ACE2 overexpression
  • Figure 1B- Figure 1F shows the ACE2 overexpression single clone
  • Figure 2 shows the ACE2 expression level of clone 7 screened by FACS detection of Hela/ACE2 single overexpression cell line
  • Figure 3 shows the FACS detection of the TMPRSS2 expression level of clone 8 screened by the HEK293/ACE2/TMPRSS2 double overexpression cell line;
  • Figure 4 shows the test of the ability of SARS-CoV-2 pseudovirus and its variants to infect cells
  • Figure 4A shows the test of the ability of SARS-CoV-2 S protein pseudovirus to infect cells at different MOIs
  • Figure 4B shows the SARS-CoV-2 S mosaic Test of the ability of protein pseudovirus to infect cells
  • Figure 5 is a graph showing the effective neutralization of SARS-CoV-2 pseudovirus by ACE2-Fc fusion protein
  • Figure 5A is the dose-effect curve of ACE2-Fc fusion protein neutralizing pseudovirus
  • Figure 5B is the effect of ACE2-Fc fusion protein on pseudovirus Inhibition curve of the ability to infect cells
  • Figure 6 is a graph showing the influence of ACE2 and TMPRSS2 dual-expressing cell lines on the ability of SARS-CoV-2 to infect cells.
  • Figure 6A shows the SARS-CoV-2 pseudovirus and the concentration of 100 ⁇ g/ml (concentration 1) and 2 ⁇ g/ml (concentration).
  • FIG. 6B shows the SARS-CoV-2 pseudovirus and the concentration of 100 ⁇ g/ml (concentration 1) and 2 ⁇ g/ After incubation with ml (concentration 2) of ACE2-Fc protein, HEK293/ACE2/TMPRSS2 was infected with pseudovirus data
  • Figure 6C shows the SARS-CoV-2 pseudovirus and the concentration of 100 ⁇ g/ml (concentration 1) and 2 ⁇ g/ After incubation with ml (concentration 2) of ACE2-Fc protein, HEK293/ACE2 was infected with pseudovirus data.
  • SARS-CoV-2 pseudovirus incubated with buffer is a negative control, and there is no virus infection and no antibody added. Cells served as a blank control.
  • Figure 7 is a graph showing the ability of the ACE2-Fc fusion protein to neutralize the South African mutant strain and the British mutant strain using the ACE2 and TMPRSS2 dual expression cell line.
  • Figure 7A is a dose-effect curve of the ACE2-Fc fusion protein neutralizing the South African mutant strain and the UK mutant strain
  • Figure 7B is the inhibition rate curve of the ACE2-Fc fusion protein against the South African mutant strain and the UK mutant strain.
  • Figure 8 is a graph showing the ability of the anti-spike protein antibody (A02087) to neutralize wild-type SARS-CoV-2 pseudoviruses tested by ACE2 and TMPRSS2 dual expressing cell lines.
  • Figure 8 is the dose-effect curve of anti-spike protein antibody (A02087) neutralizing wild-type SARS-CoV-2 pseudovirus
  • Figure 8B is the anti-spike protein antibody (A02087) neutralizing wild-type SARS-CoV-2 pseudovirus Inhibition rate curve.
  • the DNA sequence of the ORF of the S protein of SARS-CoV-2 (the encoded amino acid sequence is shown in SEQ ID NO: 2). After gene synthesis, the gene sequence with the sticky end after the digestion with the sticky end is combined with the plasmid vector using CloneEZ technology (GenScript) The fragments of pMD2.G (synthesized by GenScript, sequence sourced from Addgene plasmid#12259) were ligated and transformed into competent E. coli cells to obtain plasmid pMD.2G-SARS-CoV-2-Spike.
  • the DNA sequence of the ORF of the S protein of SARS-CoV-2 (the encoded amino acid sequence is shown in SEQ ID NO: 2).
  • the gene sequence with the sticky end after the digestion with the sticky end is combined with the plasmid vector using CloneEZ technology (GenScript) pcDNA3.1 (GenScript self-synthesized, sequence sourced from Addgene Plasmid#2093) fragments were ligated and transformed into competent E. coli cells to obtain plasmid pcDNA3.1-SARS-CoV-2-Spike.
  • the inner region of the membrane protein S of SARS-CoV uses DNA sequence for gene synthesis, and then uses CloneEZ technology (GenScript) to connect the gene sequence with sticky ends after digestion with pMD2.G-SARS-CoV-2-Spike plasmid vector fragment , So as to replace the intramembrane region of the S protein (as shown in SEQ ID NO: 10) to the intramembrane region of the S protein of SARS-CoV. Replace sequence 1 (as shown in SEQ ID NO: 11), and then transform E. coli competent cells , The plasmid pMD.2G-SARS-CoV-2-Spike-SARS-CoV-Chimera was obtained.
  • the inner region of the membrane protein S of MERS-CoV uses DNA sequence for gene synthesis, and then uses CloneEZ technology (GenScript) to connect the gene sequence with sticky ends after digestion with pMD2.G-SARS-CoV-2-Spike plasmid vector fragment , So as to replace the intramembrane region of the S protein (as shown in SEQ ID NO: 10) into the membrane protein S intramembrane region of MERS-CoV. Replace sequence 2 (as shown in SEQ ID NO: 12), and then transform E. coli competent Cells to obtain the plasmid pMD.2G-SARS-CoV-2-Spike-MERS-CoV-Chimera.
  • the intramembrane region of the membrane protein of HIV-1-gp41 uses DNA sequence for gene synthesis, and then uses CloneEZ technology (GenScript) to cut the gene sequence with sticky ends and pMD2.G-SARS-CoV-2-Spike plasmid vector fragment Connect to replace the intramembrane region of the S protein (as shown in SEQ ID NO: 10) to the membrane protein intramembrane region of HIV-1-gp41. Replace sequence 3 (as shown in SEQ ID NO: 13), and then transform E. coli Competent cells to obtain the plasmid pMD.2G-SARS-CoV-2-Spike-HIV-1-gp41-Chimera.
  • VSV-G's membrane protein G membrane region uses DNA sequence for gene synthesis, and then uses CloneEZ technology (GenScript) to connect the gene sequence with sticky ends after digestion with pMD2.G-SARS-CoV-2-Spike plasmid vector fragment , So as to replace the intramembrane region of the S protein (as shown in SEQ ID NO: 10) to replace the sequence 4 (as shown in SEQ ID NO: 14) of the membrane protein G intramembrane region of VSV, and then transform E. coli competent cells, The plasmid pMD.2G-SARS-CoV-2-Spike-VSV-G-Chimera was obtained.
  • the inner region of Marburg membrane protein uses DNA sequence for gene synthesis and then uses CloneEZ technology (GenScript) to ligate the gene sequence with sticky ends after digestion with pMD2.G-SARS-CoV-2-Spike plasmid vector fragment to replace
  • the intramembrane region of the S protein (shown in SEQ ID NO: 10) is the inner region of the membrane protein of Marburg (shown in SEQ ID NO: 15), and then the competent cells of E. coli are transformed to obtain the plasmid pMD.2G-SARS -CoV-2-Spike-Marburg-Chimera.
  • the inner region of Ebola's membrane protein uses DNA sequence for gene synthesis, and then uses CloneEZ technology (GenScript) to connect the gene sequence with sticky ends after digestion with pMD2.G-SARS-CoV-2-Spike plasmid vector fragment to replace
  • the intramembrane region of the S protein (shown in SEQ ID NO: 10) is the inner region of the membrane protein of Ebola (shown in SEQ ID NO: 16), and then transformed E. coli competent cells to obtain the plasmid pMD.2G-SARS -CoV-2-Spike-Ebola-Chimera.
  • Opti-MEM serum-free medium containing PEI 300 ⁇ l of Opti-MEM serum-free medium containing plasmids, and let stand at room temperature for 8 minutes, and add 600 ⁇ l of the mixture to HEK293T cells. 12-16 hours after transfection, replace the medium containing the transfection mixture with 10 ml of fresh DMEM complete medium. After 48-72 hours of transfection, the culture supernatant containing the pseudovirus was collected, filtered with a 0.45 ⁇ m pore size filter, and the virus supernatant was frozen with 1-10% sucrose or 1-5% human serum protein added. The virus supernatant can also be concentrated by ultracentrifugation, resuspended in PBS containing 1-10% sucrose or 1-5% human serum protein, and then frozen.
  • HEK293T cells Inoculate 6-10 ⁇ 10 6 HEK293T cells in a 10 cm cell culture dish.
  • prepare the plasmids required for packaging of pseudovirus take 5-8 ⁇ g of each SARS-CoV-2-Spike-Variant plasmid of SARS-CoV-2 coronavirus S protein, and then mix with 7-10 ⁇ g psPAX2 and 8-13 ⁇ g respectively
  • the pLVX-CMV-Luciferase plasmids were mixed and added to 300 ⁇ l Opti-MEM serum-free medium, while 200-400 ⁇ l of 10mM PEI was added to 300 ⁇ l Opti-MEM serum-free medium.
  • Opti-MEM serum-free medium containing PEI 300 ⁇ l of Opti-MEM serum-free medium containing plasmids, and add 600 ⁇ l of the mixture to HEK293T cells. After 12-16 hours of transfection, the medium containing the transfection mixture was replaced with 10 ml of fresh DMEM complete medium. After 48-72 hours of transfection, the culture supernatant containing the pseudovirus was collected, filtered with a 0.45 ⁇ m pore size filter, and the virus supernatant was frozen with 1-10% sucrose or 1-5% human serum protein added. The virus supernatant can also be concentrated by ultracentrifugation, resuspended in PBS containing 1-10% sucrose or 1-5% human serum protein, and then frozen.
  • Adherent HEK293T produces SARS-CoV-2 S chimeric protein pseudovirus
  • Opti-MEM serum-free medium containing PEI 300 ⁇ l of Opti-MEM serum-free medium containing plasmids, and add 600 ⁇ l of the mixture to HEK293T cells. 12-16 hours after transfection, replace the medium containing the transfection mixture with 10 ml of fresh DMEM complete medium. After 48-72 hours of transfection, the culture supernatant containing the pseudovirus was collected, filtered with a 0.45 ⁇ m pore size filter, and the virus supernatant was frozen with 1-10% sucrose or 1-5% human serum protein added. The virus supernatant can also be concentrated by ultracentrifugation, resuspended in PBS containing 1-10% sucrose or 1-5% human serum protein, and then frozen.
  • plasmids needed for pseudovirus packaging. 5 ⁇ g plasmids expressing SARS-CoV-2 coronavirus S protein, 5-8 ⁇ g pMD.2G-SARS-CoV-2-Spike, 7-10 ⁇ g psPAX2 and 8-13 ⁇ g pL -Mix CMV-Luciferase plasmids, add 300 ⁇ l Opti-MEM serum-free medium, and add 200-400 ⁇ l of 10mM PEI into 300 ⁇ l Opti-MEM serum-free medium.
  • Opti-MEM serum-free medium containing PEI 300 ⁇ l of Opti-MEM serum-free medium containing plasmids, and add 600 ⁇ l of the mixture to HEK293T cells. 12-16 hours after transfection, replace the medium containing the transfection mixture with 10 ml of fresh DMEM complete medium. After 48-72 hours of transfection, the culture supernatant containing the pseudovirus was collected, filtered with a 0.45 ⁇ m pore size filter, and the virus supernatant was frozen with 1-10% sucrose or 1-5% human serum protein added. The virus supernatant can also be concentrated by ultracentrifugation, resuspended in PBS containing 1-10% sucrose or 1-5% human serum protein, and then frozen.
  • the DNA sequence of human ACE2 protein (the encoded ACE2 amino acid sequence is shown in SEQ ID NO: 18) after gene synthesis, the plasmid vector pLVX-Puro (Clontech, Cat.No.632164) was simultaneously digested with the same restriction enzymes After digestion, the ORF DNA fragment of ACE2-Flag protein and the plasmid vector fragment with sticky ends were ligated using CloneEZ (Genscript) to transform E. coli competent cells to obtain plasmid pLVX-Puro-ACE2.
  • HEK293T cells were trypsinized, resuspended in DMEM with 10% FBS, and plated with 6-10 ⁇ 10 6 HEK293T/plate (10 cm). Transfection can be carried out the next day. Each plate was transfected with 7-10 ⁇ g psPAX2, 5-8 ⁇ g PMD2.G-VSV-G and 9-13 ⁇ g pLVX-Puro-ACE2. Lipofectamine 3000 (Thermo Fisher, Cat. No. L3000001) and plasmid were mixed and added to the plate. The virus supernatant was collected 48-56 hours after transfection, filtered through a 0.45 ⁇ m filter, and ultracentrifuged. The virus pellet was resuspended in 500 ⁇ l of fresh medium and stored at -80°C.
  • Infect target cells spread HEK293 cells on a 12-well plate, and the number of cells should reach 50% the next day, and culture overnight. Before infection, remove and thaw the virus, aspirate the original medium of the cells, add 1/2 volume of fresh medium, and then add the virus stock to the cells and mix well. Infect a small volume at 37°C for 4-6 hours, then fill up the medium to a normal volume. On the second day after infection (about 24 hours), the culture medium containing the virus was aspirated, replaced with fresh complete culture medium, and the culture was continued at 37°C.
  • Puromycin (puromycin) resistance screening add 1-3 ⁇ g/ml Puromycin to the cell culture medium, and change the complete culture medium containing Puromycin once every 2-3 days, until the cells in the screening control group are not infected and killed by Puromycin. Continue to screen until stable cell lines are obtained.
  • FACS detection of ACE2 expression take a part of the obtained stable cells into a FACS tube and centrifuge to remove the supernatant. Add SARS-CoV-2 Spike-ECD (spike protein extracellular domain, GenScript, Cat. No. Z03481) and incubate at 4°C for 30 minutes. After 30 minutes, remove the supernatant, add a secondary antibody (GenScript, Cat. No. A01802), and incubate at 4°C for 30 minutes. After 30 minutes, the supernatant was washed off, resuspended in FACS buffer, and the ACE2 expression level was detected on the machine. As shown in Figure 1, FACS detected multiple HEK293/ACE2 monoclonal cell lines overexpressing ACE2, and the expression level was high.
  • Monoclonal selection limit the dilution of the cell pool to a 96-well plate, observe the 96-well plate under the microscope 7 days later, and mark the wells with a single clone. Monoclonal cells were transferred to a 24-well plate and then expanded to a 6-well plate.
  • the plasmid vector pLVX-Puro (Clontech, Cat.No.632164) was simultaneously digested with the same restriction enzymes, and the ORF DNA fragment (amino acid The sequence is shown in SEQ ID NO: 18) and the plasmid vector fragment with sticky ends are connected using CloneEZ (Genscript) to transform E. coli competent cells to obtain plasmid pLVX-Puro-ACE2.
  • HEK293T cells were trypsinized, resuspended in DMEM with 10% FBS, and plated with 6-10 ⁇ 10 6 HEK293T/culture dish (10 cm). Each dish was transfected with 7-10 ⁇ g psPAX2, 5-8 ⁇ g PMD2.G-VSV-G and 9-13 ⁇ g pLVX-Puro-ACE2. Lipofectamine 3000 (Thermo Fisher, Cat. No. L3000001) and plasmid were mixed and added to the petri dish. The virus supernatant was collected 48-56 hours after transfection, filtered through a 0.45 ⁇ m filter, and ultracentrifuged. The virus pellet was resuspended in 500 ⁇ l of fresh medium and stored at -80°C.
  • Infect the target cells spread Hela cells on a 12-well plate, the cell number should reach 50% the next day, and culture overnight. Before infection, remove and thaw the virus from the refrigerator, aspirate the original medium of the cells, add 1/2 volume of fresh medium, and then add the virus stock to the cells and mix well. On the second day after infection (about 24 hours), the culture medium containing the virus was aspirated, replaced with fresh complete culture medium, and the culture was continued at 37°C.
  • Puromycin resistance screening Add 1-3 ⁇ g/ml Puromycin to the cell culture medium, and change the complete culture medium containing Puromycin once every 2-3 days, until the cells in the selection control group are not infected and killed by Puromycin. Continue to screen until stable cell lines are obtained.
  • FACS detection of ACE2 expression take a part of the obtained stable cells into a FACS tube and centrifuge to remove the supernatant. Add 4% paraformaldehyde and fix for 20 minutes at room temperature. After 20 minutes, remove the supernatant, add BD Fixation/Permeabilization (BD Biosciences, Cat. No. 554714) and incubate at 4°C for 20 minutes. After 20 minutes, wash off the supernatant, add PE anti-DYKDDDDK Tag Antibody (Biolegend, Cat. No. 637310) and incubate for 30 minutes. After 30 minutes, the supernatant was washed off, resuspended in the FACS buffer, and the ACE2 expression level was detected on the machine. As shown in Figure 2, a monoclonal cell line with high ACE2 expression was screened. The single clone with high expression of ACE2 was expanded and cultivated until a stable cell line was established.
  • Monoclonal selection limit the dilution of the cell pool to a 96-well plate, observe the 96-well plate under the microscope 7 days later, and mark it. Note the wells with single clones. Monoclonal cells were transferred to a 24-well plate and then expanded to a 6-well plate.
  • Example 3.1 prepare a stable cell line of the HEK293 cell line overexpressing human ACE2 for use.
  • the PGKpromoter+Hygromycin B (PGK promoter and hygromycin B) gene (shown in SEQ ID NO: 19) was synthesized and the above fragment was cloned into the pLVX-Puro (Clontech, Cat. No. 632164) vector, using enzymes The cutting sites are xba I and MscI to obtain the vector pLVX-Hygro.
  • the plasmid vector pLVX-Hygro was digested with the same restriction enzymes at the same time, and the ORF DNA fragment of TMPRSS2 protein obtained after digestion (the encoded amino acid sequence is shown in SEQ ID NO: 20 ) And plasmid vector fragments with sticky ends were ligated with CloneEZ (GenScript) to transform E. coli competent cells to obtain plasmid pLVX-Hygro-TMPRSS2.
  • HEK293T cells were trypsinized, resuspended in DMEM with 10% FBS, and plated with 6-10 ⁇ 10 6 HEK293T/culture dish (10 cm). Each dish was transfected with 7-10 ⁇ g psPAX2, 5-8 ⁇ g PMD2.G-VSV-G and 9-13 ⁇ g pLVX-Puro-ACE2. Lipofectamine 3000 (Thermo Fisher, Cat. No. L3000001) and plasmid were mixed and added to the petri dish. The virus supernatant was collected 48-56 hours after transfection, filtered through a 0.45 ⁇ m filter, and ultracentrifuged. The virus pellet was resuspended in 500 ⁇ l of fresh medium and stored at -80°C.
  • Infect the target cells spread the HEK293/ACE2 cells prepared in 4.1 on a 12-well plate, the cell number should reach 50% the next day, and culture overnight. Before infection, remove and thaw the virus from the refrigerator, aspirate the original medium of the cells, add 1/2 volume of fresh medium, and then add the virus stock to the cells and mix well. On the second day after infection (about 24 hours), the culture medium containing the virus was aspirated, replaced with fresh complete culture medium, and the culture was continued at 37°C.
  • Hygromycin (Hygromycin) resistance screening add 100-200 ⁇ g/ml Hygromycin B to the cell culture medium, change the complete culture medium containing Hygromycin B once in 2-3 days, until the cells in the selection control group are not infected and killed by Hygromycin B. Continue to screen until stable cell lines are obtained.
  • Monoclonal selection limit the dilution of the cell pool to a 96-well plate, observe the 96-well plate under the microscope 7 days later, and mark it. Note the wells with single clones. Monoclonal cells were transferred to a 24-well plate and then expanded to a 6-well plate.
  • FACS detection of TMPRSS2 expression take a part of the obtained stable cells into a FACS tube, centrifuge to remove the supernatant. Add 4% paraformaldehyde and fix for 20 minutes at room temperature. After 20 minutes, remove the supernatant, add BD Fixation/Permeabilization (BD Biosciences, Cat. No. 554714), and incubate at 4°C for 20 minutes. After 20 minutes, wash off the supernatant, add TMPRSS2 polyclonal antibody (THERMOFISHER, PA5-14264) and incubate for 30 minutes.
  • TMPRSS2 polyclonal antibody THERMOFISHER, PA5-14264
  • Example 2 First use p24 ELISA kit to determine the titer of SARS-CoV-2 pseudovirus in Example 2. Dilute the virus titer with EMEM complete medium to 1.65 ⁇ 10 7 , 3.3 ⁇ 10 at MOI of 25, 50, 100 and 200 7 ,6.6 ⁇ 10 7 ,and 13.2 ⁇ 10 7 IFU/ml. Resuspend the Trypsinized Hela-ACE2 cells prepared in Example 3.2 in EMEM complete medium at a cell concentration of 400,000 cells/ml, and inoculate 20,000 Hela-ACE2 cells per well in a 96-well plate (that is, 50 ⁇ l cell suspension). liquid). Add the cells to the corresponding cell wells in sequence according to the MOI, with 3 replicates in each group.
  • SARS-CoV-2 S chimeric protein S-VSV-G chimeric protein pseudovirus ability to infect cells
  • the HEK293/ACE2/TMPRSS2 cells prepared in Example 4 were resuspended in DMEM complete medium at a cell concentration of 400 000 cells/ml, and 20,000 HEK293/ACE2/TMPRSS2 cells were added to each well.
  • the SARS-CoV-2 S chimeric protein (S-VSV-G chimeric protein) pseudovirus and SARS-CoV-2 S protein pseudovirus prepared in Example 2 were added to the cells, and the multiplicity of infection (MOI) was set to 25 , After mixing uniformly, place it in an incubator for cultivation.
  • MOI multiplicity of infection
  • the SARS-CoV-2S chimeric protein (S- VSV-G chimeric protein) pseudovirus and SARS-CoV-2 S protein pseudovirus can significantly infect target cells, which proves that changes in the chimeric region do not affect the function of SARS-CoV-2 S protein, nor does it affect SARS-CoV-2 The infectivity of S protein pseudovirus.
  • the sample to be tested (antibody) and ACE2-Fc fusion protein are diluted according to the concentration gradient and configured into a series of samples with different concentrations.
  • the virus is mixed with antibodies of different concentrations according to the MOI of 100, and the virus is mixed with ACE2-
  • the Fc fusion protein incubated was used as a positive control, the virus that was not incubated with the antibody was used as a negative control, and the cells without virus infection and no antibody added were used as a blank control. Each group had 3 replicate wells and incubated at room temperature for 1 hour.
  • the neutralization ability of the test sample against pseudovirus and/or pseudovirus variants is inferred.
  • the dose-effect diagram of the positive control ACE2-Fc is shown in Figure 5A. As the concentration of ACE2-Fc increases, it neutralizes SARS-CoV. -2 The number of pseudoviruses gradually increases, so the luciferase signal produced by pseudovirus-infected cells gradually decreases.
  • the neutralization ability is the infection inhibition rate curve of the positive control ACE2-Fc. As the concentration of ACE2-Fc increases, the number of neutralized SARS-CoV-2 pseudoviruses gradually increases and the infection of cells is lost. Ability, until it is completely suppressed.
  • ACE2-Fc fusion protein Mix the ACE2-Fc fusion protein and the virus at a concentration of 100 ⁇ g/ml and 2 ⁇ g/ml at an MOI of 1.0.
  • the virus that is not incubated with the fusion protein is used as a negative control, and the cells without virus infection and no fusion protein are used as a blank control. 3 replicate wells in each group, incubated for 1 hour at room temperature.
  • ACE2 and TMPRSS2 dual-expressing cell lines are more susceptible to SARS-CoV-2 infection than ACE2 single overexpression cell lines, indicating that TMPRSS2 can significantly enhance SARS -The ability of CoV-2 to infect host cells.
  • Example 8 ACE2 and TMPRSS2 double overexpression target cells verify the ability of the ACE2-Fc fusion protein to neutralize the South African mutant strain and the British mutant strain pseudovirus
  • the fusion protein is configured into a series of samples of different concentrations (100 ⁇ g/ml, 5-fold dilution, 8 concentration points) according to the concentration gradient, the South African mutant strain pseudovirus or the British mutant strain pseudovirus and different concentrations of ACE2-Fc fusion protein Mix, the virus that was not incubated with the fusion protein was used as a negative control, and the cells without virus infection and no fusion protein were used as a blank control. Each group had 3 replicate wells and incubated for 1 hour at room temperature.
  • Example 9 ACE2 and TMPRSS2 double overexpression target cells to verify the ability of anti-neovirus spike protein neutralizing antibody to neutralize wild-type pseudovirus
  • the anti-Spike protein neutralizing antibody (GenScript, Cat. No. A02087) and the wild-type SARS-CoV-2 pseudovirus were taken out in advance.
  • the neutralizing antibody is diluted according to the concentration gradient and configured into a series of samples with different concentrations (80 000 U/ml, 2 times dilution, 16 concentration points).
  • the wild-type pseudovirus is mixed with different concentrations of neutralizing antibody, but not incubated with neutralizing antibody Virus was used as a negative control, and cells without virus infection and no neutralizing antibody were used as blank control. Three replicate wells for each group were incubated for 1 hour at room temperature.
  • SEQ ID NO: 1 reporter gene encoding protein contained in the lentiviral transfer plasmid
  • SEQ ID NO: 2 SARS-CoV-2[Wuhan-1]S protein amino acid sequence
  • SEQ ID NO: 3 SARS-CoV-2[Wuhan-1] S protein sequence S1 subunit amino acid sequence
  • SEQ ID NO: 4 SARS-CoV-2[Wuhan-1] S protein sequence S2 subunit amino acid sequence
  • SEQ ID NO: 5 SARS-CoV-2[Wuhan-1] S protein sequence S1 subunit amino acid substitution D364Y
  • SEQ ID NO: 6 SARS-CoV-2[Wuhan-1] S protein sequence S1 subunit amino acid substitution V367F
  • SEQ ID NO: 7 SARS-CoV-2[Wuhan-1] S protein sequence S1 subunit amino acid substitution W436R
  • SEQ ID NO: 8 SARS-CoV-2[Wuhan-1] S protein sequence S1 subunit amino acid substitution D614G
  • SEQ ID NO: 9 SARS-CoV-2[Wuhan-1]S protein transmembrane region sequence
  • SEQ ID NO: 10 SARS-CoV-2[Wuhan-1]S protein intramembrane region sequence
  • SEQ ID NO: 11 SARS-CoV-2[Wuhan-1] S protein intramembrane region replacement sequence 1: SARS-CoV
  • SEQ ID NO: 12 SARS-CoV-2[Wuhan-1] S protein membrane region replacement sequence 2: MERS-CoV
  • SEQ ID NO: 13 SARS-CoV-2[Wuhan-1] S protein intramembrane region replacement sequence 3: HIV-1 gp41
  • SEQ ID NO: 14 SARS-CoV-2[Wuhan-1] S protein intramembrane region replacement sequence 4: VSV-G
  • SEQ ID NO: 15 SARS-CoV-2[Wuhan-1] S protein intramembrane region substitution sequence 5: Marburg
  • SEQ ID NO: 16 SARS-CoV-2[Wuhan-1] S protein intramembrane region replacement sequence 6: Ebola
  • SEQ ID NO: 17 (Flag sequence of ACE2 intramembrane region)
  • SEQ ID NO: 21 South African mutant strain S protein sequence, B.1.351, the bold and underlined amino acids represent the substituted amino acids, the middle underlined amino acids represent the deleted amino acids
  • SEQ ID NO: 22 S protein sequence of the British mutant strain, B.1.1.7, the bold and underlined amino acids represent the substituted amino acids, and the middle underlined amino acids represent the deleted amino acids

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Abstract

本发明属于生物技术领域，具体涉及SARS-CoV-2假病毒及其检测样品中和SARS-CoV-2能力的方法。本发明提供了一种检测样品中和SARS-CoV-2病毒或其突变体的能力的方法，包括如下步骤：(1)将SARS-CoV-2假病毒或其变体与所述样品接触，(2)将所述假病毒与样品的混合物与ACE2过表达的细胞系接触培养，和(3)通过检测所述细胞系表达报告基因的情况确定所述样品是否具有中和所述SARS-CoV-2病毒或其突变体的能力。

Description

SARS-CoV-2假病毒及其检测样品中和SARS-CoV-2能力的方法 技术领域
本发明属于生物技术领域，具体涉及一种SARS-CoV-2假病毒及其检测样品中和SARS-CoV-2病毒或其变体能力的方法。
背景技术
新型冠状病毒肺炎(COVID-19)病原体SARS-CoV-2病毒，又称2019新型冠状病毒(2019Novel coronavirus,2019-nCoV)，是一种RNA冠状病毒。目前全球已有220多个国家和地区累计报告逾500万名确诊病例，逾30万名患者死亡。2019冠状病毒病疫情逐渐变成一场全球性大瘟疫。SARS-CoV-2病毒是一类具有囊膜、基因组为线性单链正链的RNA病毒。SARS-CoV-2病毒进入宿主细胞是由跨膜刺突(S)糖蛋白(S蛋白)介导的。S蛋白分为S1和S2两个亚基，其中S1亚基负责与宿主细胞受体结合，S2亚基负责病毒膜和细胞膜融合。S1通过结合宿主受体促进病毒感染。它包含两个结构域，即N端结构域和C端受体结合域(RBD结构域)。C端RBD结构域是与人ACE2(血管紧张素转化酶2)受体的相互作用位点，在病毒的传染过程中发挥着重要作用。
全球至今尚未出现用于SARS-CoV-2病毒疾病的疫苗和特效药。作为天然适应性免疫系统的一部分，中和抗体(neutralizing antibody)在人体抵抗病毒感染过程中扮演了不可缺少的作用。目前许多研发团队都在积极开发针对新型冠状病毒的中和抗体。荷兰乌得勒支大学的47D11是来自于同时用六种人类冠状病毒S抗原免疫的和铂医药H2L2人源化平台产生的单克隆抗体，体外中和实验表明该抗体对新冠病毒有中和活性(Wang,Chunyan,et al.“A human monoclonal antibody blocking SARS-Cov-2-infection.”Nature communications11.1(2020):1-6)。也有研究表明，在新型冠状病毒肺炎患者治疗过程中，康复期病人血浆治疗取得了较好的疗效，显示出中和抗体在新冠病毒肺炎治疗方面的潜力。然而，如何有效的检测目前开发的新冠病毒中和抗体和抗新冠病毒其他化学药物的中和性能也是研发人员亟需解决的问题。鉴于新型冠状病毒的感染性强和传播速度快，采用新型冠状病毒检测抗病毒的药物在前期筛选风险较高，对实验条件要求高，同时会存在扩散的危险。因此，这也亟待研发人员开发出安全性高、适应性强的检测抗病毒药物的方法，以便更快的筛选到有效的新型冠状病毒药物。
发明概述
为解决上述问题，本发明提供了一种检测样品中和SARS-CoV-2病毒或其突变体能力的方法，其特征在于，所述方法包括如下步骤：
(1)将SARS-CoV-2假病毒或其变体与所述药物接触，
(2)将所述假病毒与药物的混合物与ACE2过表达的细胞系接触培养，和
(3)通过检测所述细胞系表达报告基因的情况确定所述药物是否具有中和所述SARS-CoV-2病毒或其突变体的能力。
在一些实施方案中，所述SARS-CoV-2假病毒或其变体是基于慢病毒载体系统包装构建的。其中，所述慢病毒载体系统选自二代慢病毒系统或三代慢病毒系统。
在一些实施方案中，所述SARS-CoV-2假病毒或其变体包括慢病毒骨架及SARS-CoV-2的刺突蛋白或其变体。其中，所述SARS-CoV-2的刺突蛋白或其变体包括S1亚基和S2亚基。优选地，所述SARS-CoV-2的刺突蛋白突变体相对于野生型SARS-CoV-2的刺突蛋白包括一个或多个氨基酸的替换，删除和/或增加，优选为包括至多15个氨基酸的替换，删除和/或增加。
在一些实施方案中，所述SARS-CoV-2假病毒或其变体包含与SEQ ID NO:2、5-8、21或22所示氨基酸序列至少70％一致性的序列。在一些具体方案中，所述SARS-CoV-2假病毒或其变体包含与SEQ ID NO:2、5-8、21或22所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述SARS-CoV-2假病毒或其变体包含SEQ ID NO:2、5-8、21或22所示的氨基酸序列。在另一个具体实施方案中，所述SARS-CoV-2假病毒或其变体的氨基酸序列如SEQ ID NO:2、5-8、21或22所示。在一些实施方案中，所述SARS-CoV-2假病毒包含与SEQ ID NO:2所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述SARS-CoV-2假病毒包含SEQ ID NO:2所示的氨基酸序列。在另一个具体实施方案中，所述SARS-CoV-2假病毒的氨基酸序列如SEQ ID NO:2所示。在一些实施方案中，所述SARS-CoV-2假病毒变体包含与SEQ ID NO:5、6、7、8、21或22所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述SARS-CoV-2假病毒变体包含SEQ ID NO:5、6、7、8、21或22所示的氨基酸序列。在另一个具体实施方案中，所述SARS-CoV-2假病毒变体的氨基酸序列如SEQ ID NO:5、6、7、8、21或22所示。
在一些实施方案中，所述SARS-CoV-2的刺突蛋白或其变体包括膜外区域、跨膜区域以及膜内区域。在一些实施方案中，所述跨膜区包含与SEQ ID NO:9所示氨基酸序列至少70％一致性的序列，所述膜内区域包含与SEQ ID NO:10、11、12、13、14、15或16所示氨基酸序列至少70％一致性的序列。在另一些实施方案中，所述跨膜区包含与SEQ ID NO:9所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列，所述膜内区域包含与SEQ ID NO:10、11、12、13、14、15或16所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述跨膜区包含SEQ ID NO:9所示的氨基酸序列，所述膜内区域包含SEQ ID NO:10、11、12、13、14、15或16所示的氨基酸。在另一个具体实施方案中，所述跨膜区的氨基酸序列如SEQ ID NO:9所示，所述膜内区域的氨基酸如SEQ ID NO:10、11、12、13、14、15或16所示。
在一些实施方案中，步骤(1)中所述SARS-CoV-2假病毒或其变体以0.04-300的MOI与所述药物接触，优选为所述假病毒或其变体以0.1-150的MOI与所述药物接触，更优选为， 病毒以1-100的MOI与所述样品接触。在另一些实施方案中，步骤(1)中所述SARS-CoV-2假病毒或其变体以0.04、0.08、0.1、1.0、20、25、50、100、200或300的MOI与所述药物接触。优选地，步骤(1)中所述SARS-CoV-2假病毒或其变体以0.1-150的MOI与所述药物接触。更优选地，步骤(1)中所述SARS-CoV-2假病毒或其变体以1-100的MOI与所述药物接触。在一个具体实施例中，所述SARS-CoV-2假病毒或其变体以1.0的MOI与所述药物接触。在另一个具体实施方案中，所述SARS-CoV-2假病毒或其变体以100的MOI与所述药物接触。
在一些实施方案中，步骤(1)中所述假病毒或其变体与所述药物的接触步骤包括混合和孵育。优选为孵育0.5-3小时，更优选为孵育1小时。在一个具体实施方案中，步骤(1)中所述药物的接触步骤包括混合和孵育，孵育时间为1小时。
在一些实施方案中，步骤(2)中所述ACE2过表达的细胞系选自HEK293、Hela、Vero E6或CHO，优选为HEK293T、Hela或CHO-K1。在一些具体实施方案中，所述细胞系选自ACE2过表达的HEK293、ACE2过表达的Hela或ACE2过表达的CHO-K1，优选为ACE2过表达的HEK293。在一些优选地实施方案中，所述细胞系为ACE2过表达的HEK293或ACE2过表达的Hela。在另一些实施方案中，所述细胞系为ACE2和TMPRSS2(Transmembrane protease,serine 2，跨膜丝氨酸蛋白酶2)双表达的HEK293、Hela、Vero E6或CHO-K1，优选为ACE2和TMPRSS2双表达的HEK293。在一些具体方案中，所述细胞系为ACE2和TMPRSS2双表达的HEK293。在另一些具体实施方案中，所述细胞系为ACE2和TMPRSS2双表达的Hela。在一些具体实施方案中，所述细胞系为ACE2和TMPRSS2双表达的Vero E6。在另一些具体实施方案中，所述细胞系为ACE2和TMPRSS2双表达的CHO-K1。
在一些实施方案中，步骤(2)中所述ACE2过表达的细胞系加入量为0.2x10 4-3x10 4个细胞，优选为2.0x10 4个细胞。在一个具体实施方案中，所述ACE2过表达的HEK293或ACE2过表达的Hela加入量为0.2x10 4-3x10 4个细胞。优选地，所述ACE2过表达的HEK293或ACE2过表达的Hela加入量为2.0x10 4个细胞。在另一个具体实施方案中，所述ACE2和TMPRSS2双表达的HEK293、Hela、Vero E6或CHO-K1加入量为0.2x10 4-3x10 4个细胞。优选地，所述ACE2和TMPRSS2双表达的HEK293、Hela、Vero E6或CHO-K1加入量为2.0x10 4个细胞。
在一些实施方案中，步骤(2)中所述接触培养步骤包括将所述假病毒与药物的混合物与ACE2过表达的细胞系混匀后培养24-72小时，再裂解细胞，优选为培养48小时后裂解细胞。在一些具体实施方案中，步骤(2)中所述接触培养步骤包括将所述假病毒与药物的混合物与ACE2过表达的细胞系混匀后培养48-72小时，再裂解细胞。在另一些具体实施方案中，步骤(2)中所述接触培养步骤包括将所述假病毒与药物的混合物与ACE2过表达的细胞系混匀后培养48小时，再裂解细胞。
在一些实施方案中，所述SARS-CoV-2假病毒或其变体的基因组包括步骤(3)中所述报告基因。所述报告基因可选自本领域常规的，在细胞、组织/器官或个体处于特定情况下会表达并使得他们产生易于检测、且实验材料原本不会产生的性状的基因。在一些实施方案中，所述报告基因选自绿色荧光蛋白基因或荧光素酶基因，优选为荧光素酶基因。在一些具 体实施方案中，所述报告基因为荧光素酶基因。在另一些实施方案中，所述报告基因为绿色荧光蛋白基因。
在一些实施方案中，所述荧光素酶基因编码的蛋白包括与SEQ ID NO:1所示氨基酸序列至少70％一致性的序列。在另一些实施方案中，所述荧光素酶基因编码的蛋白包括与SEQ ID NO:1所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述荧光素酶基因编码的蛋白包含SEQ ID NO:1所示的氨基酸序列。在另一个具体实施方案中，所述荧光素酶基因编码蛋白的氨基酸序列如SEQ ID NO:1所示。
在一些实施方案中，步骤(3)确定所述样品是否具有中和所述SARS-CoV-2病毒或其突变体的能力包括制作样品浓度与报告基因信号的量效曲线，获取待测药物的IC 50，与阳性对照进行比对确认。所述阳性对照为ACE2-Fc融合蛋白。在一个具体实施方案中，将所述假病毒与样品的混合物与所述细胞系之一混合后继续培养，未被中和的假病毒进入细胞后表达荧光素酶，通过荧光素酶与底物的反应信号推测出感染细胞的病毒量，所获反应信号与ACE2-Fc融合蛋白的阳性对照组以及无中和反应的阴性对照组比较，推断出待测抗体或药物对假病毒的中和能力。
在一些实施方案中，所述样品包括能作用于SARS-CoV-2病毒或其突变体的抗体、多肽或小分子化合物，优选为抗SARS-CoV-2病毒或其突变体的中和抗体。在一些实施方案中，所述样品为抗SARS-CoV-2病毒或其突变体的中和抗体。本发明中所述样品包括但不限于来源于动物或人的血清、血浆、全血、胸腹腔积液、脑脊液、组织标本或化学方法制备的作用于SARS-CoV-2病毒或其突变体的化合物试剂，优选地，来源于血清、血浆和全血。
另一方面，本发明提供了一种SARS-CoV-2假病毒或其变体，所述假病毒或其变体包括SARS-CoV-2的刺突蛋白或其变体。具体地，本发明提供的SARS-CoV-2假病毒或其变体，其特征在于，所述假病毒是基于慢病毒载体系统包装构建的，包括慢病毒骨架和SARS-CoV-2的刺突蛋白或其变体。
在一些实施方案中，所述SARS-CoV-2的刺突蛋白或其变体包括S1亚基和S2亚基。其中，所述SARS-CoV-2的刺突蛋白或其变体的S1亚基包括与SEQ ID NO:3所示氨基酸序列至少70％的一致性。在一些实施方案中，所述SARS-CoV-2的刺突蛋白或其变体的S1亚基包括与SEQ ID NO:3所示氨基酸序列至少70％、至少75％至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述SARS-CoV-2的刺突蛋白或其变体的S1亚基包括SEQ ID NO:3所示氨基酸序列。在另一个具体实施方案中，所述SARS-CoV-2的刺突蛋白或其变体的S1亚基的氨基酸序列如SEQ ID NO:3所示。
在一些实施方案中，所述SARS-CoV-2的刺突蛋白突变体包括一个或多个氨基酸的替换、删除或增加，优选为包括至多15个氨基酸的替换、删除和/或增加，更优选为包括至多10个氨基酸的替换和/或删除。在另一些实施方案中，述SARS-CoV-2的刺突蛋白突变体包括1个、2个、3个、4个或5个氨基酸的替换、删除和/或增加。在一些实施方案中，所述 SARS-CoV-2的刺突蛋白突变体相对于野生型SARS-CoV-2的刺突蛋白包括选自D364Y、V367F、W436R、D614G、E484K和N501Y一组氨基酸替换中的一个或多个，优选为包含选自D364Y、V367F、W436R或D614G的氨基酸替换。在另一些实施方案中，所述SARS-CoV-2的刺突蛋白突变体的S1亚基相对于SARS-CoV-2的刺突蛋白包含D364Y、V367F、W436R或D614G的氨基酸替换。在一个具体实施方案中，所述SARS-CoV-2的刺突蛋白突变体的S1亚基相对于野生型SARS-CoV-2的刺突蛋白包含D364Y的氨基酸替换。在另一个具体实施方案中，所述SARS-CoV-2的刺突蛋白突变体相对于野生型SARS-CoV-2的刺突蛋白包含V367F的氨基酸替换。在一个具体实施方案中，所述SARS-CoV-2的刺突蛋白突变体相对于野生型SARS-CoV-2的刺突蛋白包含W436R的氨基酸替换。在另一个具体实施方案中，所述SARS-CoV-2的刺突蛋白突变体相对于野生型SARS-CoV-2的刺突蛋白包含D614G的氨基酸替换。在一些具体实施方案中，所述SARS-CoV-2的刺突蛋白突变体相对于野生型SARS-CoV-2的刺突蛋白包含E484K的氨基酸替换。在另一些具体实施方案中，所述SARS-CoV-2的刺突蛋白突变体相对于野生型SARS-CoV-2的刺突蛋白包含N501Y的氨基酸替换。
在一个具体实施方案中，所述SARS-CoV-2的刺突蛋白突变体的S1亚基相对于SARS-CoV-2的刺突蛋白的S1亚基包含H69、V70和Y144氨基酸删除以及N501Y,A570D,D614G,P681H氨基酸替换，所述SARS-CoV-2的刺突蛋白突变体的S2亚基相对于SARS-CoV-2的刺突蛋白的S2亚基包括T716I,S982A,D1118H氨基酸替换。在另一个具体实施方案中，所述SARS-CoV-2的刺突蛋白突变体的S1亚基相对于SARS-CoV-2的刺突蛋白的S1亚基包括L242,A243和L244氨基酸删除以及L18F,D80A,D215G,R246I,E484K,K417N,N501Y和D614G氨基酸替换,所述SARS-CoV-2的刺突蛋白突变体的S2亚基相对于SARS-CoV-2的刺突蛋白的S2亚基包括A701V氨基酸替换。
在一些实施方案中，所述SARS-CoV-2的刺突蛋白或其变体的S2亚基包含与SEQ ID NO:4所示氨基酸序列至少70％一致性的序列。在另一些实施方案中，所述SARS-CoV-2的刺突蛋白或其变体的S2亚基包含与SEQ ID NO:4所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述SARS-CoV-2的刺突蛋白或其变体的S2亚基包含SEQ ID NO:4所示氨基酸序列。在另一个具体实施方案中，所述SARS-CoV-2的刺突蛋白或其变体的S2亚基的氨基酸序列如SEQ ID NO:4所示。
在一些实施方案中，所述SARS-CoV-2的刺突蛋白或其变体包含与SEQ ID NO:2、5、6、7、8、21或22所示氨基酸序列至少70％一致性的序列。在另一些实施方案中，所述SARS-CoV-2的刺突蛋白或其变体包含与SEQ ID NO:2、5、6、7、8、21或22所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述SARS-CoV-2的刺突蛋白或其变体包含SEQ ID NO:2、5、6、7、8、21或22所示氨基酸序列。在另一个实施方案中，所述SARS-CoV-2的刺突蛋白或其变体的氨基酸序列如SEQ ID NO:2、5、6、7、8、21或22所示。在一些实施方案中，所述SARS-CoV-2假病毒包含与SEQ ID NO:2所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。 在一个具体实施方案中，所述SARS-CoV-2假病毒包含SEQ ID NO:2所示的氨基酸序列。在另一个具体实施方案中，所述SARS-CoV-2假病毒的氨基酸序列如SEQ ID NO:2所示。在一些实施方案中，所述SARS-CoV-2假病毒变体包含与SEQ ID NO:5、6、7、8、21或22所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述SARS-CoV-2假病毒变体包含SEQ ID NO:5、6、7、8、21或22所示的氨基酸序列。在另一个具体实施方案中，所述SARS-CoV-2假病毒变体的氨基酸序列如SEQ ID NO:5、6、7、8、21或22所示。
在一些实施方案中，所述SARS-CoV-2的刺突蛋白或其变体包括膜外区域、跨膜区域以及膜内区域。在一些实施方案中，所述膜内区域包含与SEQ ID NO:10、11、12、13、14、15或16所示氨基酸序列至少70％一致性的序列。在另一些实施方案中，所述膜内区域包含与SEQ ID NO:10、11、12、13、14、15或16所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述膜内区域包含SEQ ID NO:10、11、12、13、14、15或16所示的氨基酸。在另一个具体实施方案中，所述膜内区域的氨基酸如SEQ ID NO:10、11、12、13、14、15或16所示。
在一些实施方案中，所述跨膜区包含与SEQ ID NO:9所示氨基酸序列至少70％一致性的序列。在另一些实施方案中，所述跨膜区包含与SEQ ID NO:9所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一个具体实施方案中，所述跨膜区包含SEQ ID NO:9所示的氨基酸序列。在另一个具体实施方案中，所述跨膜区的氨基酸序列如SEQ ID NO:9所示。
在一些实施方案中，所述述病毒或其变体包含报告基因。所述报告基因可选自本领域常规的，在细胞、组织/器官或个体处于特定情况下会表达并使得他们产生易于检测、且实验材料原本不会产生的性状的基因。在一些实施方案中，所述报告基因选自绿色荧光蛋白基因或荧光素酶基因，优选为荧光素酶基因。在一些具体实施方案中，所述报告基因为荧光素酶基因，优选为萤火虫荧光素酶基因。在另一些实施方案中，所述报告基因为绿色荧光蛋白基因。
又一方面，本发明提供了一种制备上述假病毒或其变体的方法，其特征在于，所述方法包括如下步骤：
(1)构建表达SARS-CoV-2刺突蛋白或其变体的包膜质粒；
(2)制备慢病毒载体表达系统的包装质粒、转移质粒，与步骤(1)构建的SARS-CoV-2刺突蛋白或其变体的包膜质粒混合后，加入至病毒生产细胞继续培养；
(3)培养一段时间后，收集培养液上清，获得所述假病毒或其变体。
在一些实施方案中，步骤(1)中所述SARS-CoV-2刺突蛋白或其变体质粒包含与编码SEQ ID NO:2、5、6、7、8、21或22所示氨基酸序列的核苷酸至少70％一致性的序列。在一些具体方案中，所述SARS-CoV-2假病毒或其变体包含与SEQ ID NO:2或SEQ ID NO:5、6、7、8、21或22所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序 列。在一个具体实施方案中，所述SARS-CoV-2假病毒或其变体包含SEQ ID NO:2或SEQ ID NO:5、6、7、8、21或22所示的氨基酸序列。在另一个具体实施方案中，所述SARS-CoV-2假病毒或其变体的氨基酸序列如SEQ ID NO:2或SEQ ID NO:5、6、7、8、21或22所示
在一些实施方案中，步骤(1)中所述SARS-CoV-2刺突蛋白或其变体质粒包含编码SARS-CoV-2刺突蛋白或其变体的膜内区域的核苷酸序列。在另一些实施方案中，所述编码SARS-CoV-2刺突蛋白或其变体的膜内区域的核苷酸序列包含与编码SEQ ID NO:10、11、12、13、14、15或16中所示氨基酸序的核苷酸序列至少70％一致性的核苷酸序列。在一些具体实施方案中，所述编码SARS-CoV-2刺突蛋白或其变体的膜内区域的核苷酸序列包含与编码SEQ ID NO:10、11、12、13、14、15或16中所示氨基酸序的核苷酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的核苷酸序列。在一个具体实施方案中，所述编码SARS-CoV-2刺突蛋白或其变体的膜内区域的核苷酸序列包含编码SEQ ID NO:10、11、12、13、14、15或16中所示氨基酸序的核苷酸序列。
在一些实施方案中，所述步骤(1)包括合成SARS-CoV-2刺突蛋白或其变体的基因序列，酶切后与质粒载体pMD2.G或pcDNA3.1真核表达载体连接，转化大肠感受态细胞，获得所述SARS-CoV-2刺突蛋白或其变体的质粒。
在一些实施方案中，所述转移质粒选自pLVX-CMV-Luciferase，包装质粒选自psPAX2。在一个具体实施方案中，所述包装质粒加入量为7-10μg，所述包膜质粒加入量为5-8μg，所述转移质粒加入量为8-13μg。
在一些实施方案中，所述病毒生产细胞选自悬浮HEK293或贴壁HEK293，优选为悬浮HEK293。在一个具体实施方案中，所述病毒生产细胞为悬浮HEK293。在另一个具体实施方案中，所述病毒生产细胞为贴壁HEK293。
在一些实施方案中，步骤(3)中慢病毒系统与病毒生产细胞的培养时间为48-96小时，优选为48-72小时。在一个具体实施方案中，步骤(3)中慢病毒系统与病毒生产细胞的培养时间为48-72小时。
又一方面，本发明提供了上述SARS-CoV-2假病毒或其变体在筛选抗SARS-CoV-2或其变体的药物中的应用。
又一方面，本发明提供了一种ACE2和TMPRSS2双过表达的细胞系，所述细胞系包括ACE2基因和TMPRSS2基因。在一些实施方案中，所述细胞系进一步包括抗性基因。优选地，所述抗性基因选自嘌呤霉素抗性基因或潮霉素抗性基因。其中，所述TMPRSS2基因包含编码与SEQ ID NO:20所示氨基酸序列的核苷酸序列至少70％一致性的核苷酸序列。在一些实施方案中，所述TMPRSS2基因包含与编码SEQ ID NO:20所示氨基酸序列的核苷酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的核苷酸序列。在一个具体实施方案中，所述TMPRSS2基因包含编码SEQ ID NO:20所示氨基酸序列的核苷酸序列。在另一个具体实施方案中，所述TMPRSS2基因的序列为编码SEQ ID NO:20所示氨基酸序列的核苷酸序列。
在一些实施方案中，所述双表达细胞系选自HEK293、Hela、Vero E6或者CHO，优选为HEK293T或者CHO-K1。在一些具体方案中，所述细胞系为ACE2和TMPRSS2双表达的HEK293。在另一些具体实施方案中，所述细胞系为ACE2和TMPRSS2双表达的Hela。在一些具体实施方案中，所述细胞系为ACE2和TMPRSS2双表达的Vero E6。在另一些具体实施方案中，所述细胞系为ACE2和TMPRSS2双表达的CHO-K1。
再一方面，本发明提供了一种制备上述双表达细胞系的方法，包括如下步骤：
(1)构建ACE2单过表达的细胞系；
(2)将合成的TMPRSS2基因酶切后克隆至pLVX载体转化大肠杆菌感受态细胞，获得包含TMPRSS2基因的质粒；
(3)将步骤(2)得到的质粒与包膜质粒、包装质粒混合后加入病毒生产细胞生产慢病毒；
(4)步骤(3)得到的慢病毒感染步骤(1)的细胞系表达TMPRSS2，进行抗性筛选并挑取单克隆细胞株。
在一些实施方案中，所述步骤(1)包括：a.合成ACE2蛋白的DNA序列，酶切后克隆至pLVX载体；b.将步骤a得到的质粒与包膜质粒、包装质粒混合后加入病毒感染细胞生产慢病毒；c.将步骤b获得的慢病毒感染靶细胞，进行抗性筛选后挑取单克隆细胞株。
在另一些实施方案中，步骤(1)所述细胞系选自HEK293、Hela、Vero E6或者CHO，优选为HEK293或者CHO-K1。
在一些实施方案中，步骤(2)中所述TMPRSS2基因包含编码与SEQ ID NO:20所示氨基酸序列的核苷酸序列至少70％一致性的核苷酸序列。在一些实施方案中，所述TMPRSS2基因包含与编码SEQ ID NO:20所示氨基酸序列的核苷酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的核苷酸序列。在一个具体实施方案中，所述TMPRSS2基因包含编码SEQ ID NO:20所示氨基酸序列的核苷酸序列。在另一个具体实施方案中，所述TMPRSS2基因的序列为编码SEQ ID NO:20所示氨基酸序列的核苷酸序列。
在一些实施方案中，步骤(3)中加入步骤(2)得到的质粒量为9-13μg、包膜质粒加入量为7-10μg和包装质粒加入量为5-8μg。
又一方面，本发明提供了ACE2单过表达细胞系或ACE2和TMPRSS2双过表达细胞系在感染冠状病毒中的应用。冠状病毒，特别是SARS-CoV-2或其变体能高效地与该ACE2单过表达细胞系或ACE2和TMPRSS2双过表达细胞系表面ACE2结合进而感染该细胞系并在该细胞内合成报告基因。其中，所述冠状病毒选自SARS-CoV-2或其变体或SARS-CoV或其变体，优选为SARS-CoV-2或其变体。
本发明提供一种基于慢病毒的SARS-CoV-2假病毒，该假病毒能高效地感染ACE2单过表达细胞系或ACE2和TMPRSS2双过表达的细胞系，使用该假病毒和所述细胞系可以开发研究特异和有效中和SARS-CoV-2的样品。
在本发明的一方面，提供一种基于慢病毒包装构建的SARS-CoV-2假病毒，所述假病毒包括慢病毒骨架以及SARS-CoV-2的冠状病毒刺突蛋白或其变体。
在本发明的一个实施方案中，所述基于慢病毒包装的SARS-CoV-2假病毒及其假病毒变体包括慢病毒骨架以及SARS-CoV-2的S蛋白的膜外区域、跨膜区域和膜内区域。所述慢病毒骨架包括第二代慢病毒包装质粒(Packaging plasmid)、转移质粒(Transfer plasmid)以及包膜质粒(Envelope plasmid)。其中所述包装质粒包括慢病毒提供病毒颗粒的所需蛋白；转移质粒包括一个报告基因荧光素酶(Luciferase)如SEQ ID NO:1所示；包膜质粒包括一个包膜蛋白，其被SARS-CoV-2的S蛋白替换。所述S蛋白如SEQ ID NO:2所示包括膜外区域、跨膜区域和膜内区域。其中S蛋白部分膜外区域属于S蛋白S1亚基(S1 subunit),包括受体结合区域(RBD)。S蛋白的S1亚基以外的膜外区域、跨膜区域以及膜内区域属于S蛋白S2亚基。所述S1亚基和S2亚基序列分别如SEQ ID NO:3和4所示，在S1亚基包括一个氨基酸替换的S蛋白序列分别如SEQ ID NO:5-8所示；所述S2亚基的跨膜区域序列如SEQ ID NO:9所示；所述S2亚基的膜内区域序列如SEQ ID NO:10所示，或所述S2亚基的膜内区域替换序列如SEQ ID NO:11-16所示。
在本发明的另一方面，提供一类ACE2单过表达细胞系或ACE2和TMPRSS2双过表达细胞系细胞系，上述假病毒能高效地与该细胞系表面ACE2结合进而感染该细胞系并在该细胞内合成报告基因。在本发明的一个实施案例中，所述细胞系包括以Hela、HEK293、Vero E6或CHO-K1为宿主建立的稳定过表达S蛋白受体ACE2和/或辅助蛋白TMPRSS2的细胞系。在本发明的一个实施案例中，所述ACE2膜内区域C端尾部包含一个标记(TAG)如SEQ ID NO:17所示或无标记。
在本发明的再一个方面，提供一种基于细胞水平检测样品中和SARS-CoV-2的S蛋白及其变体能力的方法，所述样品包括抗体、多肽、小分子化合物等。所述方法包括如下步骤：1)将SARS-CoV-2假病毒或其变体与样品接触，和2)确定所述假病毒是否被样品阻止进入ACE2单过表达细胞系或ACE2和TMPRSS2双过表达细胞系并在细胞内表达报告基因。
在本发明的又一个实施案例中，所述细胞系包括以Hela、HEK293、Vero E6、Huh7或CHO-K1为宿主建立的稳定过表达S蛋白受体ACE2和/或辅助蛋白TMPRSS2的细胞系。所述ACE2膜内区域C端尾部包含一个标记(Flag)如SEQ ID NO:17所示。
在本发明的再一个实施案例中，所述方法中待测样品单点稀释后与所述假病毒或选定的假病毒变体混合培养。将培养液与所述细胞系之一混合后继续培养，未被中和的假病毒进入细胞后表达荧光素酶，通过荧光素酶与底物的反应信号推测出感染细胞的病毒量。所获反应信号与ACE2-Fc融合蛋白的阳性对照组以及无中和反应的阴性对照组比较，推断出待测样品对假病毒的中和能力。
在本发明的再一个实施案例中，所述方法首先待样品等比稀释后分别与所述假病毒或选定的假病毒变体混合培养。将培养液与所述细胞系之一混合后继续培养，未被中和的假病毒进入细胞后表达荧光素酶，通过荧光素酶与底物的反应信号推测出感染细胞的病毒量。将所述待测样品的浓度或稀释度作为X，与对应浓度或稀释度测出的反应信号作为Y作出待测样品的量效曲线，获得所测样品的IC 50，与同理获得的ACE2-Fc的阳性对照组比较，推断出待测样品对假病毒的中和能力。感染抑制率通过以下方程得出：感染抑制率(％)＝[1–(检测样品信号值–基底信号)/(阴性对照组–基底信号)]×100。所述待测样品的浓度或稀释度作为X，抑制率作为Y作出感染抑制率曲线进而所述样品的中和滴度IC 50，推断出待测样品对假病毒的中和能力。
本发明提供了一种检测样品中和SARS-CoV-2病毒能力的方法，特别地，该方法能够在不涉及致病的SARS-CoV-2真病毒的情况下，准确、快速的检测出SARS-CoV-2病毒中和抗体或其他分子，以阻止假病毒进入靶细胞的能力，以利于治疗药物的发现；该方法能够快速准确、快速的检测出候选疫苗免疫后，血清或者血浆中产生中和抗体的效果，以利于候选疫苗效果评价。此方法可用于检测样品对多种SARS-CoV-2的中和效果，以利于发现广谱药物及疫苗。
发明详述
除非另有说明，本发明所用的技术和科学术语具有与本发明所属领域的普通技术员通常所理解的含义。
本发明中“冠状病毒”是指是一类在动物与人类之间传播的人畜共患的RNA病毒。冠状病毒可感染哺乳动物、鸟类，引起牛和猪的消化道疾病或鸡的上呼吸道疾病。自然界常见，已知可感染人类的冠状病毒共有七种，会引起人类的呼吸道感染，可引发普通感冒，乃至中东呼吸综合征(MERS-Cov)和严重急性呼吸综合征(SARS-Cov)、2019年冠状病毒疾病(COVID-19或SARS-CoV-2)等较严重疾病。本发明中所述冠状病毒选自SARS-CoV-2或其变体、SARS-CoV或其变体或者MERS-Cov或其变体。优选地，所述冠状病毒为SARS-CoV或其变体。
术语“新型冠状病毒”(SARS-CoV-2)，亦称为COVID-19，其属于β属冠状病毒，有包膜，病毒颗粒呈圆形或椭圆形，直径为60-140nm。其基因组与非典(SARS-CoV-1)的基因组相似度仅为80％，而与从中菊头蝠(Rhinolophus affinis)分离得到的冠状病毒(Bat coronavirus RaTG13)基因序列相似度高达96％。
本发明中“SARS-CoV-2病毒变体”是指多种与SARS-CoV-2病毒基因有高度序列一致性新型冠状病毒，具有SARS-CoV-2病毒的感染和致病性的病毒，如如hCoV-19/Portugal/PT0548/2020，hCoV-19/England/CAMB-1B2093/2020，hCoV-19/USA/VA-DCLS-0287/2020，hCoV-19/India/NIHSAD-1005-52/2020，hCoV-19/Spain/COV000664/2020，hCoV- 19/Beijing/DT-travelGR01/2020。目前已有研究团队从马来亚穿山甲中分离出的一种冠状病毒在E，M，N和S基因中分别与2019-nCoV表现出100％，98.2％，96.7％和90.4％的氨基酸一致性。特别地，穿山甲冠状病毒的S蛋白的受体结合域实际上与2019-nCoV的S蛋白的受体结合域相同，仅具有一个氨基酸差异(https://www.biorxiv.org/content/10.1101/2020.02.17.951335v1)。本发明中所述SARS-CoV-2病毒变体的基因序列与新型冠状病毒的基因序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列一致性。
本发明中“SARS-CoV-2病毒抗原”是指SARS-CoV-2全病毒裂解液的抗原或者重组表达的SARS-CoV-2抗原。SARS-CoV-2病毒包括刺突蛋白(S)、包膜蛋白(E)、基质蛋白(M)和核衣壳蛋白(N)抗原，其中S蛋白是SARS-CoV-2最大的结构蛋白。S蛋白在宿主酶的作用下能裂解为S1和S2亚单位，其中S1亚单位含有受体结合区RBD，是主要的靶抗原。本发明中，所述SARS-CoV-2病毒抗原选自刺突蛋白(S)、包膜蛋白(E)、基质蛋白(M)和/或核衣壳蛋白(N)抗原。优选地，所述SARS-CoV-2病毒抗原为SARS-CoV-2 S1亚基或SARS-CoV-2 S RBD抗原。本发明中所述SARS-CoV-2 S RBD抗原可采用常规重组表达的方法生产，通过构建表达SARS-CoV-2 S RBD的质粒，如pFastBac1、pTT5，包含目的基因的表达载体转染表达细胞，如CHO细胞、SF9细胞，表达纯化得到SARS-CoV-2 S RBD重组蛋白。本发明中的抗SARS-CoV-2病毒抗体是指针对所述SARS-CoV-2病毒抗原的抗体，包括中和抗体。
术语“抗体”是指由四条多肽链组成的免疫球蛋白分子，包括两条重链(H)和两条轻链(L)，通过二硫键连接成为一个完整的抗体分子。
术语“慢病毒”是指以HIV-1为基础发展起来的基因载体，属于反转录病毒，对分裂细胞和非分裂细胞均具有感染力。SARS-CoV-2假病毒是以慢病毒为骨架，将慢病毒表面的包膜蛋白替换为SARS-CoV-2的S蛋白。
术语“中和”是指样品与SARS-CoV-2假病毒的S蛋白结合阻止其与细胞膜黏附，融合和进入细胞的过程。本文所用的术语“中和抗体”指通过与病毒分子结合防止细胞被某种抗原或感染源侵害的抗体，其原理是通过抑制乃至中和它们的某种生化作用。本文所用的术语“SARS-CoV-2病毒中和抗体”是指通过与SARS-CoV-2病毒RBD蛋白结合来阻断SARS-CoV-2病毒RBD与人ACE2受体结合的抗体，阻止病毒与细胞膜黏附，融合和进入细胞。
本文术语“SARS-CoV-2病毒RBD蛋白”，可以体现为重组SARS-CoV-2病毒RBD蛋白，带有His标签的SARS-CoV-2病毒RBD蛋白，带有Fc标签的SARS-CoV-2病毒RBD蛋白，包含有SARS-CoV-2病毒RBD蛋白的分子，比如SARS-CoV-2病毒S1亚基和SARS-CoV-2病毒S蛋白。
本文中“TMPRSS2(跨膜丝氨酸蛋白酶2)”属于丝氨酸蛋白酶家族。包含II型跨膜蛋白结构域、A类受体结构域、清道夫受体富含半胱氨酸结构域和蛋白酶结构域。有些冠状病毒(比如严重急性呼吸道综合征冠状病毒(SARS-CoV)和严重急性呼吸相同综合症冠状病毒2型(SARS-CoV-2))在与ACE2结合后，冠状病毒表面的S蛋白会被跨膜丝氨酸蛋白酶2 激活并促进冠状病毒进入靶细胞，从而增强病毒的侵染力。而跨膜丝氨酸蛋白酶2抑制剂能有效地降低冠状病毒如SARS-CoV-2进入靶细胞，降低其侵染力。
术语“氨基酸突变”指在已有的氨基酸序列中，存在一个或多个氨基酸的替换、删除或插入。其中“氨基酸替换”，指在已有的氨基酸序列中，用不同的氨基酸残基代替现有的氨基酸残基，分为保守性替换和激进性替换。保守性替换是指性状相近的氨基酸分子之间的替换，其中的性状包含分子的离子性、疏水性和分子量等。而激进替换是指物理化学性质非常不同的氨基酸所产生的相互替换。“氨基酸删除”是指在已有的氨基酸序列中存在一个或多个氨基酸的删除，删除后整个氨基酸序列变短，可能伴随氨基酸序列功能的增强或减弱，也可能没有明显功能变化。在一些实施方案中，所述SARS-CoV-2的刺突蛋白S1亚基包含一个或多个氨基酸的删除，如在H69、V70、Y144、L242、A243和L244位点包含一个或多个氨基酸的删除。在一个具体实施方案中，所述SARS-CoV-2的刺突蛋白S1亚基包含H69、V70和Y144位点的氨基酸删除。在另一个具体实施方案中，所述SARS-CoV-2的刺突蛋白S1亚基包含L242、A243和L244位点的氨基酸删除。
关于肽或多肽序列的“百分比(％)氨基酸序列一致性”定义为对比序列并在必要时引入缺口以获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分率。可以本领域技术范围内的多种方式进行序列对比以测定百分比氨基酸序列同一性，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定测量对比的适宜参数，包括对所比较的序列全长获得最大对比所需的任何算法。
术语“IC 50”亦称为半数抑制浓度，是指一种药物能将细胞生长、病毒复制等抑制50％所需的浓度。
除非另外特别说明，否则单数的使用包括复数。除非另外特别说明，否则词语“一个(a)”或“一个(an)”意指“至少一个”。除非另外说明，否则“或”的使用意指“和/或”。短语“至少一个”的含义等同于短语“一个或多个”的含义。此外，术语“包括(including)”以及其他形式诸如“包括(includes)”和“包括(included)”的使用不是限制性的。此外，除非另外特别说明，否则术语诸如“要素”或“组分”包括包含一个单元的元素或组分以及包含多于一个单元的元素和组分。
SARS-CoV-2假病毒构建
在本发明的一方面，提供一种基于慢病毒包装构建的SARS-CoV-2假病毒，所述假病毒包括慢病毒骨架以及SARS-CoV-2的S蛋白或其相关变体。在一些实施方案中，所述S蛋 白的变体来自氨基酸替换、删除或增加的SARS-CoV-2 S蛋白，包括RBD区域和非RBD区域，所述替换可影响S蛋白与ACE2结合的亲和力，增强SARS-CoV-2假病毒的感染力。
在一些实施方案中，所述S蛋白的相关变体包括S蛋白膜内区域替换，和/或S1亚基的氨基酸替换进而构建一种新的嵌合蛋白。所述S蛋白的膜内区域替换或来源SARS-CoV(NC_004718)的S蛋白、MERS-CoV(NC_019843)的S蛋白、HIV-1(HXB2,NC_001802)的膜蛋白gp41、VSV(J02428)的膜蛋白G、Marburg virus(NC_001608)的膜蛋白以及Ebola virus(NC_002549)的膜蛋白，其序列分别如SEQ ID NO:11-16所示。
细胞系构建
在本发明的另一方面，提供一类ACE2单过表达细胞系或ACE2和TMPRSS2双过表达细胞系细胞系，上述假病毒能高效地与该细胞系表面ACE2结合进而感染该细胞系并在该细胞内合成报告基因。在本发明的一些实施案例中，所述ACE2单过表达细胞系通过慢病毒载体转染靶细胞，建立过表达ACE2的一个细胞池。在过表达ACE2的细胞池上，进一步挑选过表达ACE2的单克隆细胞，建立稳定的过表达细胞系。所述靶细胞包括Hela、HEK293或Vero E6。
在本发明的另一些实施案例中，所述ACE2和TMPRSS2双过表达细胞系建立在ACE2单过表达的靶细胞系。所述的靶细胞系以Hela、HEK293或Vero E6为宿主建立。该靶细胞系进一步用慢病毒转染介导SARS-CoV-2融合的辅助蛋白TMPRSS2。所获得的细胞池经历进一步单克隆筛选，建立ACE2和TMPRSS2稳定双过表达的单克隆细胞系。
SARS-CoV-2假病毒中和试验
在本发明的再一个方面，提供一种基于细胞水平检测样品中和SARS-CoV-2的S蛋白及其变体的能力的方法，所述方法包括如下步骤：1)将SARS-CoV-2假病毒或其变体与样品接触，和2)确定所述假病毒是否被样品阻止进入ACE2单过表达细胞系或ACE2和TMPRSS2双过表达细胞系并在细胞内表达报告基因。
在本发明的一些实施案例中，所述方法中待测样品单点稀释后与所述SARS-CoV-2假病毒和/或SARS-CoV-2假病毒变体混合后培养。将培养液与所述ACE2单过表达细胞系或ACE2和TMPRSS2双过表达细胞系混合后继续培养，未被中和的假病毒进入细胞后表达荧光素酶，通过荧光素酶与底物的反应信号推测出感染细胞的病毒量。所获反应信号与ACE2-Fc融合蛋白的阳性对照组以及无中和反应的阴性对照组比较，推断出待测样品对假病毒和/或假病毒变体的中和能力。
在本发明的又一些实施案例中，所述方法首先待测样品等比稀释后分别与所述SARS-CoV-2假病毒和/或SARS-CoV-2假病毒变体混合培养。将培养液与所述ACE2单过表达细 胞系或ACE2和TMPRSS2双过表达细胞系混合后继续培养，未被中和的假病毒进入细胞后表达荧光素酶，通过荧光素酶与底物的反应信号推测出感染细胞的病毒量。将所述待测样品的浓度或稀释度作为X，与对应浓度或稀释度测出的反应信号作为Y作出待测样品的量效曲线，获得所测样品的IC 50，与同理获得的ACE2-Fc融合蛋白的阳性对照组比较，推断出待测样品对假病毒和/或假病毒变体的中和能力。感染抑制率通过以下方程得出：感染抑制率(％)＝[1–(检测样品信号值–基底信号)/(阴性对照组–基底信号)]×100。所述待测样品的浓度或稀释度作为X，抑制率作为Y作出感染抑制率曲线进而所述样品的中和滴度IC 50，推断出待测样品对假病毒和/或假病毒变体的中和能力。
附图说明
图1为FACS检测HEK293/ACE2单过表达细胞系单克隆的ACE2的表达水平，图1A为ACE2过表达的多克隆池，图1B-图1F是ACE2过表达的单克隆；
图2为FACS检测Hela/ACE2单过表达细胞系筛选的克隆7的ACE2表达水平；
图3为FACS检测HEK293/ACE2/TMPRSS2双过表达细胞系筛选的克隆8的TMPRSS2表达水平；
图4为SARS-CoV-2假病毒及其变体感染细胞能力测试，图4A为不同MOI时SARS-CoV-2 S蛋白假病毒感染细胞能力测试，图4B为SARS-CoV-2 S嵌合蛋白假病毒感染细胞能力测试；
图5为ACE2-Fc融合蛋白有效中和SARS-CoV-2假病毒的曲线图，图5A为ACE2-Fc融合蛋白中和假病毒的量效曲线，图5B为ACE2-Fc融合蛋白对假病毒感染细胞能力的抑制曲线；
图6为ACE2和TMPRSS2双表达细胞系对SARS-CoV-2感染细胞的能力影响图，图6A为SARS-CoV-2假病毒与浓度分别为100μg/ml(浓度1)和2μg/ml(浓度2)的ACE2-Fc蛋白孵育后，HEK293/ACE2/TMPRSS2与HEK293/ACE2被假病毒感染试验对比；图6B为SARS-CoV-2假病毒与浓度分别为100μg/ml(浓度1)和2μg/ml(浓度2)的ACE2-Fc蛋白孵育后，HEK293/ACE2/TMPRSS2被假病毒感染程度数据图；图6C为SARS-CoV-2假病毒与浓度分别为100μg/ml(浓度1)和2μg/ml(浓度2)的ACE2-Fc蛋白孵育后，HEK293/ACE2被假病毒感染程度的数据图，其中SARS-CoV-2假病毒与缓冲液孵育的为阴性对照，没有病毒感染也没有加抗体的细胞作为空白对照。
图7为用ACE2和TMPRSS2双表达细胞系验证ACE2-Fc融合蛋白中和南非突变毒株和英国突变毒株能力的曲线图。图7A为ACE2-Fc融合蛋白中和南非突变毒株和英国突变毒株的量效曲线，图7B为ACE2-Fc融合蛋白对南非突变毒株和英国突变毒株的抑制率曲线。
图8为ACE2和TMPRSS2双表达细胞系测试抗刺突蛋白抗体(A02087)中和野生型SARS-CoV-2假病毒能力的曲线图。图8为抗刺突蛋白抗体(A02087)中和野生型SARS-CoV-2假病毒的量效曲线，图8B为抗刺突蛋白抗体(A02087)中和野生型SARS-CoV-2假病毒的抑制率曲线。
具体实施方式
除非另有说明，本发明所用的技术和科学术语具有与本发明所属领域的普通技术员通常所理解的含义。本发明通过以下实施例作进一步举例说明，这些实施例不应解释为对本发明的限制。凡在本发明的精神和原则之内所做的任何修改、等同替换和改进，均应包含在本发明的保护范围之内。
实施例1冠状病毒S蛋白的质粒构建
1.1表达SARS-CoV-2的S蛋白质粒pMD2.G-SARS-CoV-2-Spike的构建方法
SARS-CoV-2的S蛋白ORF的DNA序列(编码的氨基酸序列如SEQ ID NO:2所示)进行基因合成后使用CloneEZ技术(GenScript)将酶切后带有粘性末端的基因序列与质粒载体pMD2.G(GenScript自主合成，序列来源自Addgene plasmid#12259)片段连接，转化大肠杆菌感受态细胞，获得质粒pMD.2G-SARS-CoV-2-Spike。
1.2表达SARS-CoV-2的S蛋白质粒pcDNA3.1-SARS-CoV-2-Spike的构建方法
SARS-CoV-2的S蛋白ORF的DNA序列(编码的氨基酸序列如SEQ ID NO:2所示)进行基因合成后使用CloneEZ技术(GenScript)将酶切后带有粘性末端的基因序列与质粒载体pcDNA3.1(GenScript自主合成，序列来源自Addgene plasmid#2093)片段连接，转化大肠杆菌感受态细胞，获得质粒pcDNA3.1-SARS-CoV-2-Spike。
1.3冠状病毒S蛋白S1亚基氨基酸替换变体的质粒构建
表达SARS-CoV-2的S蛋白质粒氨基酸替换pMD2.G-SARS-CoV-2-Spike-Variant的构建方法
在已合成的SARS-CoV-2的S蛋白ORF的DNA序列上进行突变，使用CloneEZ技术(GenScript)将酶切后带有粘性末端的基因序列与质粒载体片段使用连接，转化大肠杆菌感受态细胞，获得pMD2.G-SARS-CoV-2-Spike-Variant质粒。通过该方法共构建4种pMD2.G-SARS-CoV-2-Spike-Variant质粒,其中Spike-variant的氨基酸序列分别如SEQ ID NO:5-8所示。
1.4冠状病毒S嵌合蛋白的质粒构建
(1)表达SARS-CoV-2的S嵌合蛋白质粒pMD2.G-SARS-CoV-2-Spike-SARS-CoV-Chimera的构建方法
SARS-CoV的膜蛋白S膜内区域使用DNA序列进行基因合成后使用CloneEZ技术(GenScript)将酶切后带有粘性末端的基因序列与pMD2.G-SARS-CoV-2-Spike质粒载体片段连接，从而替换S蛋白的膜内区域(如SEQ ID NO:10所示)为SARS-CoV的S蛋白膜内区 域替换序列1(如SEQ ID NO:11所示)，然后转化大肠杆菌感受态细胞，获得质粒pMD.2G-SARS-CoV-2-Spike-SARS-CoV-Chimera。
(2)表达SARS-CoV-2的S嵌合蛋白质粒pMD2.G-SARS-CoV-2-Spike-MERS-CoV-Chimera的构建方法
MERS-CoV的膜蛋白S膜内区域使用DNA序列进行基因合成后使用CloneEZ技术(GenScript)将酶切后带有粘性末端的基因序列与pMD2.G-SARS-CoV-2-Spike质粒载体片段连接，从而替换S蛋白的膜内区域(如SEQ ID NO:10所示)为MERS-CoV的膜蛋白S膜内区域替换序列2(如SEQ ID NO:12所示)，然后转化大肠杆菌感受态细胞，获得质粒pMD.2G-SARS-CoV-2-Spike-MERS-CoV-Chimera。
(3)表达SARS-CoV-2的S嵌合蛋白质粒pMD2.G-SARS-CoV-2-Spike-HIV-1-gp41-Chimera的构建方法
HIV-1-gp41的膜蛋白膜内区域使用DNA序列进行基因合成后使用CloneEZ技术(GenScript)将酶切后带有粘性末端的基因序列与pMD2.G-SARS-CoV-2-Spike质粒载体片段连接，从而替换S蛋白的膜内区域(如SEQ ID NO:10所示)为HIV-1-gp41的膜蛋白膜内区域替换序列3(如SEQ ID NO:13所示)，然后转化大肠杆菌感受态细胞，获得质粒pMD.2G-SARS-CoV-2-Spike-HIV-1-gp41-Chimera。
(4)表达SARS-CoV-2的S嵌合蛋白质粒pMD2.G-SARS-CoV-2-Spike-VSV-G-Chimera的构建方法
VSV-G的膜蛋白G膜内区域使用DNA序列进行基因合成后使用CloneEZ技术(GenScript)将酶切后带有粘性末端的基因序列与pMD2.G-SARS-CoV-2-Spike质粒载体片段连接，从而替换S蛋白的膜内区域(如SEQ ID NO:10所示)为VSV的膜蛋白G膜内区域替换序列4(如SEQ ID NO:14所示)，然后转化大肠杆菌感受态细胞，获得质粒pMD.2G-SARS-CoV-2-Spike-VSV-G-Chimera。
(5)表达SARS-CoV-2的S嵌合蛋白质粒pMD2.G-SARS-CoV-2-Spike-Marburg-Chimera的构建方法
Marburg的膜蛋白膜内区域使用DNA序列进行基因合成后使用CloneEZ技术(GenScript)将酶切后带有粘性末端的基因序列与pMD2.G-SARS-CoV-2-Spike质粒载体片段连接，从而替换S蛋白的膜内区域(如SEQ ID NO:10所示)为Marburg的膜蛋白膜内区域(如SEQ ID NO:15所示)，然后转化大肠杆菌感受态细胞，获得质粒pMD.2G-SARS-CoV-2-Spike-Marburg-Chimera。
(6)表达SARS-CoV-2的S嵌合蛋白质粒pMD2.G-SARS-CoV-2-Spike-Ebola-Chimera的构建方法
Ebola的膜蛋白膜内区域使用DNA序列进行基因合成后使用CloneEZ技术(GenScript)将酶切后带有粘性末端的基因序列与pMD2.G-SARS-CoV-2-Spike质粒载体片段连接，从而替换S蛋白的膜内区域(如SEQ ID NO:10所示)为Ebola的膜蛋白膜内区域(如SEQ ID NO:16所示)，然后转化大肠杆菌感受态细胞，获得质粒pMD.2G-SARS-CoV-2-Spike-Ebola-Chimera。
实施例2假病毒生产
2.1贴壁HEK293T生产SARS-CoV-2假病毒
在10cm细胞培养皿中接种6-10×10 6HEK293T细胞。第二天准备假病毒包装所需质粒，将5-8μg表达SARS-CoV-2冠状病毒S蛋白的质粒pMD2.G-SARS-CoV-2-Spike或pcDNA3.1-SARS-CoV-2-Spike、7-10μg psPAX2(GenScript自主合成，序列来源自Addgene plasmid#12260)与8-13μg pLVX-CMV-Luciferase质粒(GenScript自主合成，通过基因合成CMV-Luciferase，将CMV-Luc以CloneEZ的方法亚克隆至PLVX-Hygromycin B载体中，构建成pLVX-CMV-Luciferase)混合，加入300μl Opti-MEM无血清培养基中，同时将200-400μl of 10mM PEI分别加入300μl Opti-MEM无血清培养基中，室温静置5分钟。将含有PEI的300μl Opti-MEM无血清培养基与300μl含有质粒的Opti-MEM无血清培养基混合，室温静置8分钟，将600μl混合物加入HEK293T细胞中。转染后12-16小时用10ml新鲜DMEM完全培养基替换含有转染混合物的培养基。转染48-72小时后收集含有假病毒的培养上清，用0.45μm孔径的滤器过滤后，病毒上清加入1-10％的蔗糖或1-5％的人血清蛋白后冻存。病毒上清亦可经超速离心浓缩后用含有1-10％蔗糖或1-5％的人血清蛋白的PBS重悬后冻存。
2.2贴壁HEK293T生产SARS-CoV-2-Spike(刺突蛋白)Variant(变体)假病毒
在10cm细胞培养皿中接种6-10×10 6HEK293T细胞。第二天准备假病毒包装所需质粒，取每个SARS-CoV-2冠状病毒S蛋白的SARS-CoV-2-Spike-Variant质粒各5-8μg，然后各自与7-10μg psPAX2和8-13μg pLVX-CMV-Luciferase质粒混合，加入300μl Opti-MEM无血清培养基中，同时将200-400μl of 10mM PEI分别加入300μl Opti-MEM无血清培养基中。将含有PEI的300μl Opti-MEM无血清培养基与300μl含有质粒的Opti-MEM无血清培养基混合，将600μl混合物加入HEK293T细胞中。转染12-16小时后用10ml新鲜DMEM完全 培养基替换含有转染混合物的培养基。转染48-72小时后收集含有假病毒的培养上清，用0.45μm孔径的滤器过滤后，病毒上清加入1-10％的蔗糖或1-5％的人血清蛋白后冻存。病毒上清亦可经超速离心浓缩后用含有1-10％蔗糖或1-5％的人血清蛋白的PBS重悬后冻存。
2.3贴壁HEK293T生产SARS-CoV-2的S嵌合蛋白假病毒
在10cm细胞培养皿中接种6-10×10 6HEK293T细胞。第二天准备假病毒包装所需质粒，取1.3中构建的每个SARS-CoV-2冠状病毒S嵌合蛋白的质粒各5-8μg，然后各自与7-10μg psPAX2和8-13μg pLVX-CMV-Luciferase质粒混合，加入300μl Opti-MEM无血清培养基中，同时将200-400μl of 10mM PEI分别加入300μl Opti-MEM无血清培养基中。将含有PEI的300μl Opti-MEM无血清培养基与300μl含有质粒的Opti-MEM无血清培养基混合，将600μl混合物加入HEK293T细胞中。转染后12-16小时用10ml新鲜DMEM完全培养基替换含有转染混合物的培养基。转染48-72小时后收集含有假病毒的培养上清，用0.45μm孔径的滤器过滤后，病毒上清加入1-10％的蔗糖或1-5％的人血清蛋白后冻存。病毒上清亦可经超速离心浓缩后用含有1-10％蔗糖或1-5％的人血清蛋白的PBS重悬后冻存。
2.4悬浮HEK293T生产SARS-CoV-2假病毒
在摇瓶中接种6-10×10 6HEK293T细胞。第二天准备假病毒包装所需质粒，将5μg表达SARS-CoV-2冠状病毒S蛋白的质粒、5-8μg pMD.2G-SARS-CoV-2-Spike、7-10μg psPAX2与8-13μg pL-CMV-Luciferase质粒混合，加入300μl Opti-MEM无血清培养基中，同时将200-400μl of 10mM PEI分别加入300μl Opti-MEM无血清培养基中。将含有PEI的300μl Opti-MEM无血清培养基与300μl含有质粒的Opti-MEM无血清培养基混合，将600μl混合物加入HEK293T细胞中。转染后12-16小时用10ml新鲜DMEM完全培养基替换含有转染混合物的培养基。转染48-72小时后收集含有假病毒的培养上清，用0.45μm孔径的滤器过滤后，病毒上清加入1-10％的蔗糖或1-5％的人血清蛋白后冻存。病毒上清亦可经超速离心浓缩后用含有1-10％蔗糖或1-5％的人血清蛋白的PBS重悬后冻存。
实施例3 ACE2单过表达细胞系构建
3.1 ACE2单过表达HEK293细胞系构建
人ACE2蛋白的DNA序列(编码的ACE2氨基酸序列如SEQ ID NO:18所示)进行基因合成后，同时利用相同限制性内切酶酶切质粒载体pLVX-Puro(Clontech，Cat.No.632164)酶切后获得的ACE2-Flag蛋白ORF DNA片段和带有粘性末端的质粒载体片段使用CloneEZ(Genscript)连接，转化大肠杆菌感受态细胞，获得质粒pLVX-Puro-ACE2。
慢病毒生产：将HEK293T细胞用胰酶消化，重悬在加入10％FBS的DMEM中，铺6-10×10 6HEK293T/平皿(10cm)。第二天可进行转染。每个平皿按照7-10μg psPAX2,5-8μg PMD2.G-VSV-G和9-13μg pLVX-Puro-ACE2转染。将Lipofectamine 3000(Thermo Fisher,Cat.No.L3000001)与质粒混合加入平皿中。转染后48-56小时收集病毒上清，收集后以0.45μm滤器过滤，超速离心。将病毒沉淀用500μl新鲜培养基重悬，置于-80℃保存。
感染靶细胞：将HEK293细胞铺至12孔板，细胞数以第二天达到50％为宜，培养过夜。感染前，取出并融化病毒，吸去细胞原有培养基，加入1/2体积新鲜培养基，再将病毒原液加入细胞中混匀。37℃小体积感染4-6小时，然后补齐培养基至正常体积。感染后第二天(约24小时)，吸去含病毒的培养液，换上新鲜的完全培养液，继续37℃培养。
Puromycin(嘌呤霉素)抗性筛选：细胞培养基中加入1-3μg/ml Puromycin，2-3天换含Puromycin的完全培养液一次，至不感染筛选对照组细胞被Puromycin杀光。连续筛选至获得稳定细胞株。
FACS检测ACE2表达：取一部分获得的稳定细胞至FACS管中，离心去掉上清。加入SARS-CoV-2 Spike-ECD(刺突蛋白胞外域，GenScript,Cat.No.Z03481)4℃孵育30分钟。30分钟后去掉上清，加入二抗(GenScript,Cat.No.A01802)，4℃孵育30分钟。30分钟后洗去上清，重悬在FACS缓冲液，上机检测ACE2表达水平，如图1所示，FACS检测到多株HEK293/ACE2单克隆细胞系过表达ACE2，且表达水平较高。
单克隆挑选：将细胞池极限稀释至96孔板中，7天后显微镜下观察96孔板，并标记出有单克隆的孔。单克隆细胞转移至24孔板中，随后扩大至6孔板。
3.2 ACE2单过表达Hela细胞系构建
人ACE2蛋白的DNA序列进行基因合成后，同时利用相同限制性内切酶酶切质粒载体pLVX-Puro(Clontech，Cat.No.632164)，酶切后获得的ACE2-Flag蛋白ORF DNA片段(氨基酸序列如SEQ ID NO:18所示)和带有粘性末端的质粒载体片段使用CloneEZ(Genscript)连接，转化大肠杆菌感受态细胞，获得质粒pLVX-Puro-ACE2。
慢病毒生产：将HEK293T细胞用胰酶消化，重悬在加入10％FBS的DMEM中，铺6-10×10 6HEK293T/培养皿(10cm)。每个培养皿按照7-10μg psPAX2，5-8μg PMD2.G-VSV-G和9-13μg pLVX-Puro-ACE2转染。将Lipofectamine 3000(Thermo Fisher,Cat.No.L3000001)与质粒混合加入培养皿中。转染后48-56小时收集病毒上清，收集后以0.45μm滤器过滤，超速离心。将病毒沉淀用500μl新鲜培养基重悬，置于-80℃保存。
感染靶细胞：将Hela细胞铺至12孔板，细胞数以第二天达到50％为宜，培养过夜。感染前，从冰箱取出并融化病毒，吸去细胞原有培养基，加入1/2体积新鲜培养基，再将病毒 原液加入细胞中混匀。感染后第二天(约24小时)，吸去含病毒的培养液，换上新鲜的完全培养液，继续37℃培养。
Puromycin抗性筛选：细胞培养基中加入1-3μg/ml Puromycin，2-3天换含Puromycin的完全培养液一次，至不感染筛选对照组细胞被Puromycin杀光。连续筛选至获得稳定细胞株。
FACS检测ACE2表达：取一部分获得的稳定细胞至FACS管中，离心去掉上清。加入4％多聚甲醛室温固定20分钟。20分钟后去上清，加入BD Fixation/Permeabilization(BD Biosciences,Cat.No.554714)4℃孵育20分钟。20分钟后洗掉上清，加入PE anti-DYKDDDDK Tag Antibody(Biolegend,Cat.No.637310)孵育30分钟。30分钟后洗去上清，重悬在FACS buffer，上机检测ACE2表达水平，如图2所示，筛选到ACE2高表达的单克隆细胞株。高表达ACE2的单克隆扩大培养直至建立稳定的细胞株。
单克隆挑选：将细胞池极限稀释至96孔板中，7天后显微镜下观察96孔板，并标。记出有单克隆的孔。单克隆细胞转移至24孔板中，随后扩大至6孔板。
实施例4 ACE2和TMPRSS2双过表达细胞系构建
4.1 ACE2单过表达HEK293细胞系构建
参照实施例3.1制备人ACE2单过表达HEK293细胞系的稳定细胞株待用。
4.2 ACE2和TMPRSS2双过表达细胞系的构建
将PGKpromoter+Hygromycin B(PGK启动子和潮霉素B)基因(如SEQ ID NO:19所示)合成并将上述片段克隆到pLVX-Puro(Clontech，Cat.No.632164)载体中，使用酶切位点为xba I和MscI，得到载体pLVX-Hygro。人TMPRSS2蛋白的DNA序列进行基因合成后，同时利用相同限制性内切酶酶切质粒载体pLVX-Hygro，酶切后获得的TMPRSS2蛋白ORF DNA片段(编码的氨基酸序列如SEQ ID NO:20所示)和带有粘性末端的质粒载体片段使用CloneEZ(GenScript)连接，转化大肠杆菌感受态细胞，获得质粒pLVX-Hygro-TMPRSS2。
慢病毒生产：将HEK293T细胞用胰酶消化，重悬在加入10％FBS的DMEM中，铺6-10×10 6HEK293T/培养皿(10cm)。每个培养皿按照7-10μg psPAX2，5-8μg PMD2.G-VSV-G和9-13μg pLVX-Puro-ACE2转染。将Lipofectamine 3000(Thermo Fisher,Cat.No.L3000001)与质粒混合加入培养皿中。转染后48-56小时收集病毒上清，收集后以0.45μm滤器过滤，超速离心。将病毒沉淀用500μl新鲜培养基重悬，置于-80℃保存。
感染靶细胞：将4.1制备的HEK293/ACE2细胞铺至12孔板，细胞数以第二天达到50％为宜，培养过夜。感染前，从冰箱取出并融化病毒，吸去细胞原有培养基，加入1/2体积新 鲜培养基，再将病毒原液加入细胞中混匀。感染后第二天(约24小时)，吸去含病毒的培养液，换上新鲜的完全培养液，继续37℃培养。
Hygromycin(潮霉素)抗性筛选：细胞培养基中加入100-200μg/ml Hygromycin B，2-3天换含Hygromycin B的完全培养液一次，至不感染筛选对照组细胞被Hygromycin B杀光。连续筛选至获得稳定细胞株。
单克隆挑选：将细胞池极限稀释至96孔板中，7天后显微镜下观察96孔板，并标。记出有单克隆的孔。单克隆细胞转移至24孔板中，随后扩大至6孔板。
FACS检测TMPRSS2表达：取一部分获得的稳定细胞至FACS管中，离心去掉上清。加入4％多聚甲醛室温固定20分钟。20分钟后去上清，加入BD Fixation/Permeabilization(BD Biosciences,Cat.No.554714)，4℃孵育20分钟。20分钟后洗掉上清，加入TMPRSS2多克隆抗体(THERMOFISHER,PA5-14264)孵育30分钟。30分钟后洗去上清，重悬细胞后加入Goat Anti-Rabbit羊抗兔lgG H&L(Alexa
Figure PCTCN2021096593-appb-000001
488)Secondary Antibody(羊抗兔lgG H&L二抗，abcam,ab150077)。30分钟后洗去上清，重悬在FACS缓冲液，上机检测TMPRSS2表达水平，如图3所示，筛选到TMPRSS2高表达的单克隆细胞株。高表达ACE2的单克隆扩大培养直至建立稳定的细胞株。
实施例5假病毒感染细胞能力测试
5.1 SARS-CoV-2假病毒感染细胞能力测试
首先用p24 ELISA kit确定实施例2中SARS-CoV-2假病毒的滴度，按MOI为25、50、100和200将病毒滴度用EMEM完全培养基稀释为1.65×10 7,3.3×10 7,6.6×10 7,and 13.2×10 7IFU/ml。将胰酶处理好的实施例3.2制备的Hela-ACE2细胞重悬在EMEM完全培养基，细胞浓度为400000cells/ml，并在96孔板中每孔接种20000个Hela-ACE2细胞(即50μl细胞悬液)。按照MOI依次将细胞加入对应的细胞孔里，每组3个重复。另留一组细胞不添加假病毒。将96孔板放入培养箱培养。感染48小时后，吸去细胞培养上清，加入50μl细胞裂解液，在室温孵育25分钟后，吸取30μl细胞裂解液转移至Luciferase(荧光素酶)检测96孔板中，加入30μl Luciferase反应底物，放入Luciferase荧光微孔板读板仪读取数据，如图4A所示假病毒的感染细胞的能力随着感染滴度的增加呈线性增强趋势。
5.2 SARS-CoV-2 S嵌合蛋白(S-VSV-G嵌合蛋白)假病毒感染细胞能力测试
将实施例4制备的HEK293/ACE2/TMPRSS2细胞重悬在DMEM完全培养基，细胞浓度为400 000cells/ml，每个孔中加入20 000个HEK293/ACE2/TMPRSS2细胞。将实施例2中 制备的SARS-CoV-2 S嵌合蛋白(S-VSV-G嵌合蛋白)假病毒和SARS-CoV-2 S蛋白假病毒加入细胞中，感染复数(MOI)设为25，混合均匀后，置于培养箱中培养。
48小时后，吸去细胞培养上清，加入50μl Luciferase反应底物，放入Luciferase荧光微孔板读板仪读取数据，如图4B所示，SARS-CoV-2 S嵌合蛋白(S-VSV-G嵌合蛋白)假病毒与SARS-CoV-2 S蛋白假病毒均能显著感染靶细胞，证明嵌合区域改变不影响SARS-CoV-2 S蛋白功能，也不影响SARS-CoV-2 S蛋白假病毒的感染能力。
实施例6 ACE2单过表达靶细胞的假病毒中和试验
6.1样品中和SARS-CoV-2假病毒的量效曲线
将待测样品(抗体)和ACE2-Fc融合蛋白(GenScript,Cat.No.Z03484)按照浓度梯度稀释配置成一系列不同浓度的样品，病毒按照100的MOI与不同浓度的抗体混合，病毒与ACE2-Fc融合蛋白孵育的作为阳性对照，没有与抗体孵育的病毒作为阴性对照，没有病毒感染也没有加抗体的细胞作为空白对照，每个组别3个复孔，室温孵育1小时。
抗体孵育时间结束前半个小时准备细胞接种：将胰酶消化过的Hela-ACE2细胞重悬在EMEM完全培养基，细胞浓度为400000cells/ml，并在96孔板中每孔接种20000个Hela-ACE2细胞(即50μl细胞悬液)，将细胞均匀铺板。
待抗体孵育结束，加入上述抗体病毒混合液、阳性、阴性和空白对照至已铺Hela-ACE2的96孔板，每个样品感染3个复孔。使抗体病毒混合液混合均匀后，置于培养箱中培养。
48小时后，吸去细胞培养上清，加入50μl细胞裂解液，在室温孵育25分钟后，吸取30μl细胞裂解液转移至Luciferase检测96孔板中，加入30μl Luciferase反应底物，放入Luciferase荧光微孔板读板仪读取数据。
将抗体浓度或稀释度作为X，与对应浓度的反应信号作为Y作出待测抗体的量校曲线，获得所测样品的IC 50，与同理获得的ACE2-Fc融合蛋白的阳性对照组比较，推断出待测样品对假病毒和/或假病毒变体的中和能力，其中阳性对照ACE2-Fc的量效图如图5A所示，随着ACE2-Fc浓度的增加，其中和SARS-CoV-2假病毒的数量逐渐增多，因而假病毒感染细胞产生的荧光素酶信号逐渐降低。
感染抑制率通过以下方程得出：感染抑制率(％)＝[1–(检测样品信号值–基底信号)/(阴性对照组–基底信号)]×100。所述待测样品的浓度或稀释度作为X，抑制率作为Y作出感染抑制率曲线进而所述样品的中和滴度IC 50，推断出待测样品对假病毒和/或假病毒变体的中和能力，如图5B所示为阳性对照ACE2-Fc的感染抑制率曲线，随着ACE2-Fc浓度的 增加，被中和的SARS-CoV-2假病毒的数量逐渐增多并丧失感染细胞的能力，直至被完全抑制。
实施例7 ACE2单过表达靶细胞与ACE2和TMPRSS2双过表达靶细胞的假病毒中和试验对比
将浓度分别为100μg/ml和2μg/ml ACE2-Fc融合蛋白和病毒按照1.0的MOI混合，没有与融合蛋白孵育的病毒作为阴性对照，没有病毒感染也没有加融合蛋白的细胞作为空白对照，每个组别3个复孔，室温孵育1小时。
融合蛋白孵育时间结束前半个小时准备细胞接种：将胰酶消化过的实施例3.1制备的HEK293/ACE2细胞或实施例4制备的HEK293/ACE2/TMPRSS2细胞重悬在EMEM或DMEM完全培养基，细胞浓度为400 000cells/ml，并在96孔板中每孔接种20 000个HEK293/ACE2细胞或HEK293/ACE2/TMPRSS2细胞(即50μl细胞悬液)，将细胞均匀铺板。
待孵育结束，加入上述ACE2-Fc融合蛋白和病毒混合液、阴性和空白对照至已铺HEK293/ACE2细胞或HEK293/ACE2/TMPRSS2细胞的96孔板，每个样品感染3个复孔。混合均匀后，置于培养箱中培养。
48小时后，吸去细胞培养上清，加入50μl细胞裂解液，在室温孵育25分钟后，吸取30μl细胞裂解液转移至Luciferase检测96孔板中，加入30μl Luciferase反应底物，放入Luciferase荧光微孔板读板仪读取数据，如图6A-C所示，相比于ACE2单过表达细胞系，ACE2和TMPRSS2双表达细胞系更易受到SARS-CoV-2感染，从而说明TMPRSS2能显著增强SARS-CoV-2感染宿主细胞的能力。在施加不同浓度的ACE2-Fc后，SARS-CoV-2感染ACE2单过表达或ACE2和TMPRSS2双过表达细胞系的能力都显著下降，在两个细胞系呈现统一趋势，说明ACE2和TMPRSS2双过表达细胞系检测SARS-CoV-2假病毒中和能力的可靠性。
实施例8 ACE2和TMPRSS2双过表达靶细胞验证ACE2-Fc融合蛋白中和南非突变毒株和英国突变毒株假病毒的能力
将ACE2-Fc融合蛋白和南非突变毒株B.1.351假病毒(S1亚基和S2亚基多点突变，如SEQ ID NO:21所示，参照实施例2的方法制备)及英国突变毒株B.1.1.7假病毒(S1亚基和S2亚基多点突变，如SEQ ID NO:22所示，参照实施例2的方法制备)提前取出。将融合蛋白按照浓度梯度稀释配置成一系列不同浓度的样品(100μg/ml,5倍稀释，8浓度点)，南非突变毒株假病毒或英国突变毒株假病毒与不同浓度的ACE2-Fc融合蛋白混合，没有与 融合蛋白孵育的病毒作为阴性对照，没有病毒感染也没有加融合蛋白的细胞作为空白对照，每个组别3个复孔，室温孵育1小时。
融合蛋白孵育时间结束前半个小时准备细胞接种：将实施例4制备的HEK293/ACE2/TMPRSS2细胞重悬在DMEM完全培养基，细胞浓度为600 000cells/ml。待孵育结束，在ACE2-Fc融合蛋白和病毒混合液、阴性和空白对照孔中加入30 000个HEK293/ACE2/TMPRSS2细胞。混合均匀后，置于培养箱中培养。
48小时后，吸去细胞培养上清，加入50μl Luciferase反应底物，放入Luciferase荧光微孔板读板仪读取数据，如图7A-B所示，随着ACE2-Fc融合蛋白浓度增加，南非突变毒株假病毒和英国突变毒株假病毒侵染细胞的信号逐步降低(抑制率逐步增加)，说明在中和检测实验系统中，ACE2和TMPRSS2双过表达细胞系能用于验证样品中和SARS-CoV-2假病毒及其变体的能力。
实施例9 ACE2和TMPRSS2双过表达靶细胞验证抗新冠病毒刺突蛋白中和抗体中和野生型假病毒的能力
将抗Spike蛋白中和抗体(GenScript,Cat.No.A02087)和野生型SARS-CoV-2假病毒提前取出。将中和抗体按照浓度梯度稀释配置成一系列不同浓度的样品(80 000U/ml,2倍稀释，16浓度点)，野生型假病毒与不同浓度的中和抗体混合，没有与中和抗体孵育的病毒作为阴性对照，没有病毒感染也没有加中和抗体的细胞作为空白对照，每个组别3个复孔，室温孵育1小时。
抗体孵育时间结束前半个小时准备细胞接种：将实施例4制备的HEK293/ACE2/TMPRSS2细胞重悬在DMEM完全培养基，细胞浓度为400 000cells/ml。待孵育结束，在中和抗体和病毒混合液、阴性和空白对照孔中加入20 000个HEK293/ACE2/TMPRSS2细胞。混合均匀后，置于培养箱中培养。
48小时后，吸去细胞培养上清，加入50μl Luciferase反应底物，放入Luciferase荧光微孔板读板仪读取数据，如图8A-B所示，随着中和抗体浓度增加，野生型假病毒侵染细胞的信号逐步降低(抑制率逐步增加)，说明在中和检测实验系统中，ACE2和TMPRSS2双过表达细胞系能用于验证中和抗体中和SARS-CoV-2假病毒的能力。
序列信息：
SEQ ID NO:1(慢病毒转移质粒包含的报告基因编码蛋白)
Figure PCTCN2021096593-appb-000002
Figure PCTCN2021096593-appb-000003
SEQ ID NO:2(SARS-CoV-2[Wuhan-1]S蛋白氨基酸序列)
Figure PCTCN2021096593-appb-000004
SEQ ID NO:3(SARS-CoV-2[Wuhan-1]S蛋白序列S1亚基氨基酸序列)
Figure PCTCN2021096593-appb-000005
SEQ ID NO:4(SARS-CoV-2[Wuhan-1]S蛋白序列S2亚基氨基酸序列)
Figure PCTCN2021096593-appb-000006
Figure PCTCN2021096593-appb-000007
SEQ ID NO:5(SARS-CoV-2[Wuhan-1]S蛋白序列S1亚基氨基酸替换D364Y)
Figure PCTCN2021096593-appb-000008
SEQ ID NO:6(SARS-CoV-2[Wuhan-1]S蛋白序列S1亚基氨基酸替换V367F)
Figure PCTCN2021096593-appb-000009
SEQ ID NO:7(SARS-CoV-2[Wuhan-1]S蛋白序列S1亚基氨基酸替换W436R)
Figure PCTCN2021096593-appb-000010
Figure PCTCN2021096593-appb-000011
SEQ ID NO:8(SARS-CoV-2[Wuhan-1]S蛋白序列S1亚基氨基酸替换D614G)
Figure PCTCN2021096593-appb-000012
SEQ ID NO:9(SARS-CoV-2[Wuhan-1]S蛋白跨膜区域序列)
Figure PCTCN2021096593-appb-000013
SEQ ID NO:10(SARS-CoV-2[Wuhan-1]S蛋白膜内区域序列)
Figure PCTCN2021096593-appb-000014
SEQ ID NO:11(SARS-CoV-2[Wuhan-1]S蛋白膜内区域替换序列1:SARS-CoV)
Figure PCTCN2021096593-appb-000015
SEQ ID NO:12(SARS-CoV-2[Wuhan-1]S蛋白膜内区域替换序列2:MERS-CoV)
Figure PCTCN2021096593-appb-000016
SEQ ID NO:13(SARS-CoV-2[Wuhan-1]S蛋白膜内区域替换序列3:HIV-1 gp41)
Figure PCTCN2021096593-appb-000017
SEQ ID NO:14(SARS-CoV-2[Wuhan-1]S蛋白膜内区域替换序列4:VSV-G)
Figure PCTCN2021096593-appb-000018
SEQ ID NO:15(SARS-CoV-2[Wuhan-1]S蛋白膜内区域替换序列5:Marburg)
Figure PCTCN2021096593-appb-000019
SEQ ID NO:16(SARS-CoV-2[Wuhan-1]S蛋白膜内区域替换序列6:Ebola)
Figure PCTCN2021096593-appb-000020
SEQ ID NO:17(ACE2膜内区域Flag序列)
Figure PCTCN2021096593-appb-000021
SEQ ID NO:18(ACE2-Flag序列)
Figure PCTCN2021096593-appb-000022
SEQ ID NO:19(PGK promoter+Hygromycin B序列)
Figure PCTCN2021096593-appb-000023
Figure PCTCN2021096593-appb-000024
SEQ ID NO:20(TMPRSS2序列)
Figure PCTCN2021096593-appb-000025
SEQ ID NO:21(南非突变毒株S蛋白序列，B.1.351，加粗加下划线的氨基酸表示替换后的氨基酸，中间划线的氨基酸表示删除的氨基酸)
Figure PCTCN2021096593-appb-000026
SEQ ID NO:22(英国突变毒株S蛋白序列，B.1.1.7，加粗加下划线的氨基酸表示替换后的氨基酸，中间划线的氨基酸表示删除的氨基酸)
Figure PCTCN2021096593-appb-000027
Figure PCTCN2021096593-appb-000028

Claims (37)

  1. 一种检测样品中和SARS-CoV-2病毒或其突变体能力的方法，其特征在于，所述方法包括如下步骤：
    (1)将SARS-CoV-2假病毒或其变体与所述样品接触，
    (2)将所述假病毒与样品的混合物与ACE2过表达的细胞系接触培养，和
    (3)通过检测所述细胞系表达报告基因的情况确定所述样品是否具有中和所述SARS-CoV-2病毒或其突变体的能力。
  2. 根据权利要求1所述的方法，所述SARS-CoV-2假病毒或其变体是基于慢病毒载体系统包装构建的。
  3. 根据权利要求1或2所述的方法，所述SARS-CoV-2假病毒或其变体包括慢病毒骨架及SARS-CoV-2的刺突蛋白或其变体。
  4. 根据权利要求3所述的方法，所述SARS-CoV-2的刺突蛋白突变体在野生型SARS-CoV-2的刺突蛋白基础上包括一个或多个氨基酸的替换、删除和/或增加。
  5. 根据权利要求3或4所述的方法，所述SARS-CoV-2的刺突蛋白或其变体包括与SEQ ID NO:2、5-8、21或22所示氨基酸序列至少80％一致性的序列。
  6. 根据权利要求3-5中任一项所述的方法，所述SARS-CoV-2的刺突蛋白或其变体包括膜外区域、跨膜区域以及膜内区域。
  7. 根据权利要求6所述的方法，所述跨膜区包含与SEQ ID NO:9所示氨基酸序列至少70％一致性的序列，所述膜内区域包含与SEQ ID NO:10、11、12、13、14、15或16所示氨基酸序列至少70％一致性的序列。
  8. 根据权利要求1-7中任一项所述的方法，步骤(1)中所述假病毒以0.04-300的MOI与所述样品接触，优选为假病毒以0.1-150的MOI与所述样品接触。
  9. 根据权利要求1-8中任一项所述的方法，步骤(1)中所述假病毒或其变体与所述样品的接触步骤包括混合和孵育步骤。
  10. 根据权利要求1-9中任一项所述的方法，步骤(2)中所述ACE2过表达的细胞系选自HEK293、Hela、Vero E6或CHO，优选为HEK293、Hela或CHO-K1。
  11. 根据权利要求10所述的方法，所述ACE2过表达的细胞系为HEK293或Hela。
  12. 根据权利要求10或11所述的权利要求，所述细胞系为ACE2和TMPRSS2双表达的HEK293、Hela、Vero E6或CHO-K1，优选为ACE2和TMPRSS2双表达的HEK293。
  13. 根据权利要求1-12中任一项所述的方法，步骤(2)中所述接触培养步骤包括将所述假病毒与样品的混合物与ACE2过表达的细胞系混匀后培养24-72小时，再裂解细胞。
  14. 根据权利要求1-13中任一项所述的方法，所述SARS-CoV-2假病毒或其变体的基因组包括步骤(3)中所述报告基因。
  15. 根据权利要求14所述的方法，所述报告基因选自绿色荧光蛋白基因或荧光素酶基因，优选为荧光素酶基因。
  16. 根据权利要求15所述的方法，所述荧光素酶基因编码的蛋白包括与SEQ ID NO:1所示氨基酸序列至少70％一致性的序列。
  17. 根据权利要求1-16中任一项所述的方法，步骤(3)确定所述样品是否具有中和所述SARS-CoV-2病毒或其突变体的能力包括制作样品浓度与报告基因信号的量效曲线，获取待测样品的IC 50，与阳性对照进行比对确认。
  18. 根据权利要求17所述的方法，所述阳性对照为ACE2-Fc蛋白。
  19. 一种SARS-CoV-2假病毒或其变体，其特征在于，所述假病毒是基于慢病毒载体系统包装构建的，包括慢病毒骨架和SARS-CoV-2的刺突蛋白或其变体。
  20. 根据权利要求19所述的假病毒或其变体，所述SARS-CoV-2的刺突蛋白突变体包括一个或多个氨基酸的替换、删除和/或增加。
  21. 根据权利要求19或20所述的假病毒或其变体，所述SARS-CoV-2的刺突蛋白突变体相对于野生型SARS-CoV-2的刺突蛋白包含选自D364Y、V367F、W436R、D614G、E484K和N501Y一组氨基酸替换中的一个或多个，优选为包含选自V367F、W436R或D614G的氨基酸替换。
  22. 根据权利要求19-21中任一项所述的假病毒或其变体，所述SARS-CoV-2的刺突蛋白或其变体包含与SEQ ID NO:2、5、6、7、8、22或23所示氨基酸序列至少70％一致性的序列。
  23. 根据权利要求19-22中任一项所述的假病毒或其变体，其特征在于，所述病毒或其变体包含报告基因。
  24. 根据权利要求23中所述的假病毒或其变体，所述报告基因选自绿色荧光蛋白基因或荧光素酶基因，优选为荧光素酶基因。
  25. 一种制备权利要求19-24任一项所述假病毒或其变体的方法，其特征在于，所述方法包括如下步骤：
    (1)构建表达SARS-CoV-2刺突蛋白或其变体的包膜质粒；
    (2)制备慢病毒载体表达系统的包装质粒、转移质粒，与步骤(1)构建的SARS-CoV-2刺突蛋白或其变体的包膜质粒混合后，加入至病毒生产细胞继续培养；
    (3)培养一段时间后，收集培养液上清，获得所述假病毒或其变体。
  26. 根据权利要求25所述的制备方法，所述步骤(1)包括合成SARS-CoV-2刺突蛋白或其变体的基因序列，酶切后与质粒载体pMD2.G或pcDNA3.1真核表达载体连接，转化大肠感受态细胞，获得所述SARS-CoV-2刺突蛋白或其变体的质粒。
  27. 根据权利要求25或26所述的制备方法，所述包膜质粒包括报告基因。
  28. 根据权利要求25-27中任一项所述的制备方法，所述转移质粒选自pLVX-CMV-Luciferase-2PLuciferase，包装质粒选自psPAX2。
  29. 根据权利要求25-28中任一项所述的制备方法，所述病毒生产细胞选自悬浮HEK293或贴壁HEK293，优选为悬浮HEK293。
  30. 根据权利要求25-29中任一项所述的制备方法，步骤(3)中慢病毒系统与病毒生产细胞的培养时间为48-96小时，优选为48-72小时。
  31. 权利要求19-24中任一项所述假病毒或其变体在筛选抗SARS-CoV-2或其变体的药物中的应用。
  32. 一种ACE2和TMPRSS2双过表达的细胞系，所述细胞系包括ACE2基因和TMPRSS2基因。
  33. 根据权利要求32所述的双表达细胞系，所述细胞系进一步包括抗性基因，所述抗性基因选自嘌呤霉素抗性基因或潮霉素抗性基因。
  34. 根据权利要求32或33所述的双表达细胞系，所述细胞系选自HEK293、Hela、Vero E6或者CHO，优选为HEK293T或者CHO-K1。
  35. 一种制备权利要求32-34中任一项所述双表达细胞系的方法，包括如下步骤：
    (1)构建ACE2单过表达的细胞系；
    (2)将合成的TMPRSS2基因酶切后克隆至pLVX载体转化大肠杆菌感受态细胞，获得包含TMPRSS2基因的质粒；
    (3)将步骤(2)得到的质粒与包膜质粒、包装质粒混合后加入病毒生产细胞生产慢病毒；
    (4)步骤(3)得到的慢病毒感染步骤(1)的细胞系表达TMPRSS2，进行抗性筛选并挑取单克隆细胞株。
  36. 根据权利要求35所述的制备方法，所述步骤(1)包括：a.合成ACE2蛋白的DNA序列，酶切后克隆至pLVX载体；b.将步骤a得到的质粒与包膜质粒、包装质粒混合后加入病毒感染细胞生产慢病毒；c.将步骤b获得的慢病毒感染靶细胞，进行抗性筛选后挑取单克隆细胞株。
  37. 权利要求32-34中所述的ACE2和TMPRSS2双过表达细胞系在感染SARS-CoV-2或其变体中的应用。
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