CN113416245A

CN113416245A - Neutralizing antibody capable of combining SARS-CoV-2 virus RBD protein and application thereof

Info

Publication number: CN113416245A
Application number: CN202110660889.6A
Authority: CN
Inventors: 武鹏程; 马晓飞; 武雷; 孔双泉; 金丽珠; 张立帆; 尹长城
Original assignee: Beijing Protein Innovation Co ltd
Current assignee: Beijing Protein Innovation Co ltd
Priority date: 2021-06-15
Filing date: 2021-06-15
Publication date: 2021-09-21

Abstract

The invention discloses a monoclonal antibody 21336-98 capable of specifically Binding the Receptor Binding Domian (RBD) of SARS-CoV-2 virus spinous process protein (Spike, S protein), its preparation method and 6 antigenic determinant sequences (CDR) of heavy chain and light chain variable regions, said monoclonal antibody is secreted by hybridoma cell strain 21336-98, can specifically recognize SARS-Cov-2 virus S protein, and can block the Binding of S protein and its human host Receptor protein ACE 2. The antigen for preparing the antibody is the receptor binding domain RBD protein of SARS-CoV-2 virus S protein expressed by mammalian cells, the finally obtained antibody belongs to IgG2b subtype, the sequence of the coding variable region is obtained by gene cloning, and the antibody can be used for establishing the neutralizing antibody titer of the Spike protein generated aiming at SARS-CoV-2 in human body.

Description

Neutralizing antibody capable of combining SARS-CoV-2 virus RBD protein and application thereof

Technical Field

The present invention relates to the field of biomedicine. More specifically, the present invention relates to a monoclonal antibody specifically recognizing RBD in the S protein receptor binding region of SARS-CoV-2 virus, which can block the binding of the S protein to ACE2, a human host receptor protein, and the antibody of the present invention can be used for assembling the neutralizing ability and titer of the antibody generated against SARS-CoV-2 in human body, and can be further developed into an antibody preparation for relieving symptoms after infection.

Background

After vaccination, the immune system of the human body will generate antibodies against antigens, and the inactivated virus vaccine, recombinant protein vaccine, adenovirus vaccine or nucleic acid vaccine inoculated with new coronavirus aims to generate various antibodies against viral proteins, and the antibodies against RBD region may play an important role in protection in view of the currently known mechanism of infection with new coronavirus. Neutralizing antibodies are immunoglobulins (antibodies) secreted by plasma cells in vivo after immune cells in vivo are stimulated by an immunogen (or pathogen), wherein a part of the antibodies can bind to the pathogen to block the pathogen from invading the body to cause infection, and the part of the antibodies is neutralizing antibodies, which are special antibodies capable of inactivating the virus by binding to the virus, but not all antibodies generated against the antigen have a neutralizing effect.

According to past experience, neutralizing antibodies account for 1% of the total antibodies. To better monitor infection rates, population and protective immunity, and to evaluate vaccine efficacy during clinical trials and after mass vaccination, a detection method that can detect neutralizing antibodies to SARS-CoV-2 is needed. Conventional methods for detecting neutralizing antibodies require the treatment of the SARS-CoV-2 live virus in a biosafety tertiary protection laboratory, which can take 2-4 days to complete. The virus neutralization test using the pseudovirus can be operated in a biosafety secondary laboratory as an alternative scheme, but a live virus and cell culture process is still needed, so that the large sample test is difficult to perform, the period is long, and the cost is high.

The recombinant virus spinous process protein receptor binding domain-Fc fusion protein and the recombinant human ACE2 receptor protein can be used for simulating the binding effect of virus proteins and receptors on an enzyme-linked immunosorbent assay plate, the effect can be inhibited by specific neutralizing antibodies in serum of a patient, the mode is similar to the neutralizing assay principle of real viruses or pseudoviruses, and the titer of the neutralizing antibodies in the human serum aiming at RBD can be evaluated by using competitive neutralizing antibodies. The invention patent CN202110344480 discloses a method and a kit for detecting a high-sensitivity SARS-CoV-2 neutralizing antibody, which can enhance the specificity of detecting the neutralizing antibody by a sandwich method by capturing a non-neutralizing antibody in advance. Thereby realizing the detection of the SARS-CoV-2 neutralizing antibody. The invention patent 'SARS-CoV-2 neutralizing antibody detection kit' (CN202010560947) discloses a method for detecting neutralizing antibody by using a chemiluminescence immunoassay analyzer or a colloidal gold test strip mode by adopting a principle of combining a competition method with a magnetic particle chemiluminescence technology and a colloidal gold chromatography competition method. The invention adopts a qualitative detection method, and can not clearly and clearly evaluate the content or titer of the neutralizing antibody.

In addition to vaccine studies, 234 companies or institutions from 26 countries are developing 164 antibody drug programs against new coronavirus infections, of which 54 have reached the clinical stage. Of these antibody candidates, three immediate use drugs have been granted for neutralizing antibodies directed against new coronavirus infection, including the biologies' neutralizing antibody BamHanivimab (LY-CoV555) monotherapy, the combination therapy-BamHanivimab (LY-CoV555)700mg and etesevimab (JS016 or LY-CoV016)1400mg, and the Kurarin Schekin New crown antibody drug Sotrovimab (VIR-7831, S309). The three neutralizing antibodies are strong and neutralizing monoclonal antibodies aiming at SARS-CoV-2 spike protein.

S309 binds to a highly conserved epitope in the antibody binding domain (RBD) of the spike protein, and exhibits the ability to neutralize not only SARS-CoV-1, but also the novel coronavirus and a variety of other coronaviruses. In 3 months 2021, published in nature, a number of corona neutralizing antibodies, only a few of which, such as VIR-7831(S309), retained a high degree of neutralizing activity against the south African mutant B.1.351 and the British mutant B.1.1.7. In the same year, 3 months, an interim data analysis showed that sotrovimab reduced the risk of hospitalization or death of the early high risk COVID9 patient by 85% (p 0.002).

The novel coronavirus (SARS-CoV-2) has four major structural proteins: spike protein (S protein), Nucleocapsid protein (N protein), Membrane protein (M protein), Envelope protein (E protein). The novel coronavirus spike protein S protein has two subunits: s1 and S2, the receptor binding site (RBD) is located on the S1 subunit. It constitutes a spike on the outer membrane surface of the virion in the form of a trimer, whose main function is to recognize host cell surface receptors and mediate fusion with host cells. S2 may act as a fusion protein of a class I virus to mediate fusion of the virion and the cell membrane. The protein is currently known to have at least three conformational states: the native state before fusion, the intermediate state before hairpin and the hairpin state after fusion. During fusion of the viral and target cell membranes, the coiled helical region (heptad repeat) adopts a hairpin trimer structure, positioning the fusion peptide near the C-terminal region of the ectodomain.

According to the study, neutralizing antibodies can be classified into four categories:

1) directly blocking the combination of ACE2 of a human host and a virus Receptor Binding Domain (RBD) in an up conformation state, breaking the fusion of virus host membranes, and further inhibiting the infection capacity of the virus;

2) the neutralizing antibody which can be combined with the virus Receptor Binding Domain (RBD) in the conformation state of up/down can not only compete with ACE2, but also utilize epitope to contact the adjacent virus Receptor Binding Domain (RBD) to change the trimerization conformation of the spinous process protein;

3) a neutralizing antibody targeting a non-ACE 2 binding domain;

4) an antibody that binds to the neo-corona RBD binding domain, but has no neutralizing effect. One of the main reasons for the failure of the neutralization of the above four classes of antibodies is the point mutation of the spinous process protein.

Neutralizing antibodies currently under development are primarily targeted to the Receptor Binding Domain (RBD) of the new coronally-derived spinous process protein (SPIKE); by blocking the binding of ACE2 of the human host to the viral receptor binding domain, the membrane fusion ability of the virus is inhibited.

It is noted that some of the spinous process protein point mutations have developed broad spectrum resistance to neutralizing antibodies: most mutants in the Receptor Binding Domain (RBD) were found to have less impact on the ability of the virus to infect, but RBD mutants a475V, L452R, V483A and F490L were resistant to certain neutralizing antibodies. Novel neutralizing antibodies recognizing more conserved epitopes still have development prospects.

Disclosure of Invention

The first purpose of the present invention is to provide a monoclonal antibody which can specifically recognize RBD in the S protein receptor binding region of SARS-CoV-2 virus.

It is another object of the present invention to provide an RBD-neutralizing antibody that blocks the binding of the S protein to its human receptor ACE 2.

It is still another object of the present invention to provide a method for preparing hybridoma secreting the above specific antibody.

In a first aspect of the present invention, there is provided a monoclonal antibody consisting of (a) a light chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 01, or a CDR-L1 region of the amino acid sequence shown in seq id No. 01.

(ii) Comprises the amino acid sequence of SEQ ID NO: 02, or a CDR-L2 region of the amino acid sequence shown in SEQ ID NO.

(iii) Comprises the amino acid sequence of SEQ ID NO: 03, and the CDR-L3 region.

(b) Heavy chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 04, or a CDR-H1 region of the amino acid sequence shown in 04.

(ii) Comprises the amino acid sequence of SEQ ID NO: 05 of the amino acid sequence shown in SEQ ID NO. 05.

(iii) Comprises the amino acid sequence of SEQ ID NO: 06, or a CDR-H3 region of the amino acid sequence shown in SEQ ID NO.

In a second aspect of the invention, there is provided a DNA molecule encoding a protein selected from the group consisting of antibody heavy chain sequences and antibody light chain sequences taken from the six CDR regions described above and combinations thereof.

In a third aspect of the present invention, there is provided a vector comprising the above-mentioned DNA encoding the antibody of the present invention, and a host cell transformed or transduced with the vector or a host cell directly transformed or transduced with the above-mentioned DNA. Technical means for solving the technical problems

The invention obtains a hybridoma cell strain secreting anti-SARS-CoV-2-RBD antibody by large-scale cell fusion and screening, and the preparation method of the hybridoma cell strain comprises the following steps:

1) analyzing according to the published SARS-CoV-2 virus S protein coding sequence, selecting receptor binding region RBD for recombination expression according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure on cell membrane, in order to promote soluble expression of recombinant protein, utilizing eukaryotic cell to make secretion expression, respectively fusing Fc fragment of rabbit antibody and Fc fragment of mouse antibody at its C end to make secretion expression and easy purification, cloning into expression plasmid vector pcDNA3.4 after gene synthesis, transfecting Expi293 cell to make culture, separating and purifying expression product from culture supernatant as immunogen and antibody screening.

2) The spleen cells of the immune qualified mice are taken aseptically as antigen-sensitized B cells, the B cells are fused with myeloma cells SP2/0 strain according to a conventional method, and then the B cells are screened by a conventional fused cell HAT screening method, so as to obtain the fused cell growth clone.

3) Antibody preparation and screening for S-RBD protein that specifically recognizes SARS-Cov-2: the S protein of SARS-CoV-2 binds to their common receptor angiotensin converting enzyme 2 (ACE2) via the Receptor Binding Domain (RBD) and mediates viral entry into host cells. The S protein comprises two functional subunits, S1 and S2, where S1 is responsible for binding to host cell receptors and the S2 subunit is responsible for viral membrane and cell membrane fusion. During infection, the S protein is cleaved by a host protease (such as TMPRSS2) into an N-terminal S1 subunit and a C-terminal S2 subunit, and is converted from a pre-fusion state to a post-fusion state. S1 and S2 are composed of an extracellular domain (ECD) and a single transmembrane helix, mediating receptor binding and membrane fusion, respectively. S1 consists of an N-terminal domain (NTD) and a Receptor Binding Domain (RBD) and is critical in determining tissue tropism and host range. The Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein is immunodominant and is also the target for 90% of the neutralizing antibodies present in SARS-CoV-2 immune serum. The invention utilizes the eukaryotic expression S protein RBD segment to obtain phosphorylation and glycosylation modification similar to natural samples. Meanwhile, in order to enhance the solubility of the recombinant protein and facilitate the purification, an Fc segment of the antibody is fused at the C end of the RBD. In addition, in order to improve the expression yield and reduce the cross reaction of later detection, the Fc sections of mouse IgG and rabbit IgG are respectively fused with the Fc sections for expression, two antigens are used for cross immunization, and rabbit Fc fusion protein RBD-rFc with high expression quantity and small cross reaction with an enzyme-labeled secondary antibody is used as a screening protein, so that a high-quality antibody with high affinity, high specificity and neutralizing activity is obtained.

The monoclonal antibody is prepared from the hybridoma cell strain, and the preparation method comprises the following two steps:

1) in vitro, the hybridoma cells are cultured in a serum-free culture medium, and the culture supernatant is harvested and separated by immunoaffinity chromatography to purify the required monoclonal antibody.

2) 21336-98 hybridoma cells are inoculated in the abdominal cavity of the animal, and the required monoclonal antibody is separated and purified after the ascites of the animal is harvested. The invention relates to a receptor competitive monoclonal antibody aiming at an RBD structural domain, which is obtained by purifying an antibody prepared by a ascites method through Protein A/G column affinity chromatography.

Advantages and advantageous effects of the invention

The monoclonal antibody secreted by the hybridoma 21336-98 obtained by the invention can specifically recognize SARS-CoV-2 virus S protein, can block the specific combination of the S protein and a human receptor ACE2 thereof, can be used as a positive control of serum neutralizing antibodies, and is used for evaluating the titer of the neutralizing antibodies generated in serum. The target medicine antibody for treating the new coronary pneumonia.

Description of the drawings:

FIG. 1: expression region design of SARS-Cov-2 Spike protein

FIG. 2: the RBD recombinant protein of SARS-Cov-2 virus cultured and purified in Expi293 cells in serum-free form, wherein M is a molecular weight marker, the left panel is a RBD fusion rFc purified protein, the right panel is a RBD fusion mFc purified protein, the theoretical molecular weight of the expressed product is about 52kDa, the molecular weight is shown as 60kDa, and the post-translational modification of individual sites is presumed to be included, and the protein is separated by using 12% SDS-PAGE gel.

FIG. 3: the RBD monoclonal antibody 21336-98 can block the combination of RBD-rFc and ACE2-hFc, and the RBD-rFc recombinant protein is used as a coating antigen, and the recombinant human ACE2-hFc protein and the RBD monoclonal antibody 21336-98 compete to bind to RBD, so that the result shows that NO. 98 has neutralization and inhibition activity, the inhibition rate reaches 50% of antibody concentration, and the IC50 is 1.55 nM.

Detailed Description

The present invention is further described with reference to the drawings and the detailed description, so that the technical solutions of the present invention can be more clearly understood by those skilled in the art, and the present invention is not limited thereto.

EXAMPLE 1 preparation of RBD protein immunogen of recombinant SARS-Cov-2 Virus

The open reading frame region of RBD (319-544aa) was synthesized with reference to the nucleotide sequence encoding the S protein in the novel coronavirus genome sequence numbered MN908947.3 in GenBank and cloned into a plasmid vector pUC57 (Nanjing Kingsry Biotech Co., Ltd.), EcoRI cutting points are respectively introduced at the upstream and XhoI cutting sites are respectively introduced at the downstream by gene synthesis, and after EcoRI/XhoI double cutting and recovery, the EcoRI/XhoI double cutting is respectively cloned into eukaryotic expression vectors pBPI-RD58-mFc and pBPI-RD59-rFc (all purchased from Beijing Huada protein research and development center, Ltd.) which are subjected to the same double cutting, and pBPI-RD58-mFc and pBPI-RD59-rFc vectors respectively carry Fc fragments (mFc) of mouse IgG and rabbit IgG at the downstream of an expression frame, and carry an IL-2 signal peptide sequence which is conveniently secreted into a culture medium at the upstream. After sequencing and identification, plasmid preparation was carried out, according to Expi293^TMExpi293 cells were transfected and cultured in Expression System Kit (Seimer Feissuer science and technology (China) Co., Ltd., product No. A14635) instructions to maintain cell viability at 70% or more, and purified after 120 hours of culture. Because the recombinant RBD protein has an Fc tag,affinity purification was performed using Protein A column, and FIG. 2 shows the purification results of recombinant proteins fused with rabbit antibody and mouse antibody Fc, respectively. The purity of the recombinant RBD-rFc and RBD-mFc proteins is more than 90%, the concentration is 0.5mg/mL, and the requirements of immune animals and antibody screening and identification can be met.

Example 2 establishment of hybridoma cell lines and antibody screening

Animal immunization

Six female Balb/c mice (purchased from Beijing Wintolite laboratory animals technologies, Inc.) of 4-6 weeks old, numbered sequentially 21336A-21336F, were immunized with the recombinant RBD-rFc and protein of example 1 emulsified with Freund's complete adjuvant (purchased from Sigma). The dose per mouse was 60. mu.g/mouse by abdominal subcutaneous injection. The booster was administered once every 14 days, the booster antigen was RBD-mFc, and emulsified with Freund's incomplete adjuvant (Sigma Co.) at a dose of 30. mu.g/mouse. 7 days after 3 times of booster immunization, the multi-antibody titer of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, the antigen is uniformly heated by physiological saline, and the dosage is 50 mu g/mouse.

Second, cell fusion

A suspension of mouse splenocytes up to the immune standard was prepared aseptically, mixed with mouse myeloma cells sp2/0(ATCC) at a ratio of 5:1, centrifuged at 1500rpm for 5min, the supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1mL of PEG1500 (Roche) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10mL of serum-free IMDM (Sigma Co.) was added, and the mixture was mixed, centrifuged at 1000rpm, and after 5min the supernatant was discarded, 10mL of serum (PAA Co.) was added to gently blow up the cells, and 5mL of thymocytes mixed with 10 XHAT (Sigma Co.) were added and mixed. Then, 25mL of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and then poured into 20 cell culture dishes uniformly. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

Screening and preserving positive hybridoma

The cell mass is moderate in size and density 7 days after fusion, and round, solid and large cell masses are sucked and injected under a dissecting mirrorCulturing in 96-well culture plate prepared with culture medium at 37 deg.C in an incubator with CO₂The concentration was 5%.

Fourth, ELISA screening positive hybridoma cell

After 3 days of hybridoma culture, the amount of cells accounted for approximately floor area 2/3, and 200. mu.l of complete medium containing feeder cells and 1% HT (Sigma) was added. And performing second ELISA screening after two days to obtain hybridoma with OD value higher than 0.5 detected by indirect ELISA of immunogen as positive clone for subsequent culture. These positive clones were transferred to 24-well plate cultures prepared in advance with medium (containing feeder cells and HT). Five days later 100. mu.l of the supernatant was used for a third ELISA screening. The screening process is as follows, 100 μ l of supernatant is taken to take eukaryotic expression SARS-Cov-2 virus RBD-rFc protein as coating antigen to carry out the specificity screening of ELISA method. For screening, the antigen protein was diluted to 2. mu.g/ml with PBS buffer. Adding 100 mu L of antigen into each hole, coating for 12 hours at 4 ℃, discarding the solution, washing the plate for 3 times by using a washing solution (PBS (phosphate buffered saline) containing 0.05 percent Tween), draining, adding 300 mu L of 2 percent (v/w) bovine serum albumin serving as a sealing solution into each hole, incubating for 3 hours at 37 ℃, repeatedly washing the plate for 3 times, adding cell culture supernatant to be identified, adding a sample to be detected into the hole of the ELISA plate after finishing the sealing after incubation and screening, incubating for 1 hour at 37 ℃, washing the plate for 3 times, adding 1: mu.l HRP-labeled goat anti-mouse antibody (purchased from Takara Shuzo) diluted 5000 times, incubated at 37 ℃ for 1H, washed for 3 times, drained, added 100. mu.L of TMB color development buffer per well, incubated at room temperature for 8 min, and added 50. mu.l of H per well₂SO₄(2M) the reaction was stopped and the absorbance at 450nm was read on a microplate reader.

Six mice are preliminarily screened to obtain 62 strains of hybridoma cells which can specifically recognize RBD protein but not recognize Fc labels, and 12 strains of positive hybridoma cells with the light absorption value of 450nm higher than 1.0 are selected for ascites preparation and antibody purification for later neutralization activity identification.

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵1ml of suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. Taken out ofAscites were centrifuged at 4000rpm at 4 ℃ and the intermediate ascites were carefully aspirated for 10min and collected in centrifuge tubes for purification. Purification of the antibody was carried out according to the instructions of HiTrap rProtein AFF (available from GE, cat. No. 17-5079-02).

Example 4 anti-RBD antibodies block binding of RBD to hACE2

Antibodies were tested for their ability to block RBD binding to its human host receptor hACE2 by competition ELISA. Recombinant RBD-rFc was diluted to 1. mu.g/mL in carbonate buffer at pH 9.6, 100. mu.L per well in 96-well plates, and coated overnight at 4 ℃. After washing the plates with PBST, 300. mu.L of 1% (w/v) BSA blocking solution per well was added and incubated at 37 ℃ for 2 hours. After washing, 50. mu.L of recombinant human ACE2-hFc (purchased from Abcam, cat # ab273687) (1. mu.g/mL) and 50. mu.L of anti-RBD antibody (2-fold serial dilution, final concentration range 33nM to 0.53nM) were added to each well and incubated at 37 ℃ for 1 hour. After washing the plate, 100. mu.L of goat anti-human IgG labeled with HRP (1: 10000) was added to each well, and incubated at 37 ℃ for 1 hour. After washing the plates, 100. mu.L of TMB chromogenic substrate was added to each well, incubated at room temperature for 10 minutes, followed by the addition of 50. mu.L of stop solution (2N H)₂SO₄) The reaction was terminated. It was preliminarily found that antibodies 19, 98, and 8 may have neutralizing inhibitory activity, antibodies 71 and 184 have no competitive activity, and weakly neutralizing antibodies are antibodies 21, 26, and 46 (see table 1). After the initial screening, the conditions are optimized, and screening and identification are carried out continuously, the results are shown in table 2, and the antibodies No. 98 and No. 19 are proved to have neutralizing activity and can be used for detecting and identifying the human neutralizing antibody. Among them, antibody 98 has a stronger inhibitory activity, and the concentration of the antibody reaching 50% inhibition rate is 1.55nM, and is preferably selected as a candidate antibody for subclass determination and sequence determination.

TABLE 1 competitive ELISA assay results for each antibody

TABLE 2 inhibition efficiency results of the respective antibodies

EXAMPLE 5 monoclonal antibody subclass identification and affinity determination

Subclass identification, goat anti-mouse IgG (Cunninghamia tsugae Biotech Co., Ltd., Beijing) coated with 100mM PBS (pH7.4) was diluted to 0.5. mu.g/

m

1, 100. mu.l was added to each well, the liquid was emptied overnight at 4 ℃, washed 3 times with PBS (PBS-T) containing 0.05% Tween, 200. mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. Mu.l of hybridoma culture supernatant or purified antibody was added to each well, incubated at 37 ℃ for 1h, the liquid was emptied, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse (kappa, lambda) antibody was diluted with a blocking solution 1:1000 or HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech Co.) was added to each well of the antibody sample to be tested in a volume of 100. mu.l per well, incubated at 37 ℃ for 1 hour, the solution was emptied, and washed 3 times with PBS-T. 50 μ l of 0.15% ABTS (Southern Biotech Co., cat. 0202-1) and 0.03% H was added to each well₂O₂The citric acid buffer (pH4.0) was developed, and the absorbance at a wavelength of 405nm was measured within 10 min. The results show that the monoclonal antibodies 21336-98 of the invention are of the IgG2b subtype, and the light chain is a kappa light chain.

Second, determination of affinity constant

Recombinant N protein was coated at a concentration of 2. mu.g/

m

1, 100. mu.1/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 4 was prepared from 1:200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody was diluted 1:20000 per well, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The reaction solution was developed in the citric acid phosphate buffer for 10min, and 50. mu.l of a 0.5M sulfuric acid solution was added to terminate the reaction. Measuring absorbance at 450nm with microplate reader, and plotting antibody dilution concentration and OD using GraphPad Prism v8 as data analysis plotting software₄₅₀Calculating affinity according to the curve to obtain 21336-98 antibody with affinity constant of 5.6 × 10¹⁰M^-1。

Example 5 determination of variable region sequences of antibodies

Culturing fresh hybridoma cells, taking the supernatant, verifying the antigen binding properties of the cell line used for cloning, confirming that the cell line can indeed secrete the required antibody, and collecting 10 by centrifugation⁶The hybridoma cells described above. Total RNA from hybridoma cells was extracted by Trizol method, and 9. mu.l of total RNA was added to 2.5. mu.l of oligo (dT) 12-18 primer (10 mM) and 5. mu.l of dNTPs, mixed well, incubated at 70 ℃ for 5 minutes and then placed on ice for 5 minutes, or denatured according to the reverse transcriptase used. Then, 5. mu.L of RT buffer (5X), 2.5. mu.L of DTT (0.1M) and 1. mu.L of reverse transcriptase were added and reacted at 42 ℃ for 1 hour. The reaction was terminated by incubation at 70 ℃ for 15 minutes, and the obtained cDNA was stored at-20 ℃. The obtained first strand cDNA was subjected to PCR amplification, and 25pmol each of primers was added to a 50. mu.L reaction system, and the sequences of the primers for amplification of the heavy chain variable region and the light chain variable region are shown in Table 3

TABLE 3 amplification primers for antibody variable regions in mouse hybridomas

The rest dNTPs and buffer are added according to the conventional method, and finally 1 mu L of cDNA template and 1U of hot start Taq DNA polymerase are added. Setting PCR amplification program as 94 deg.c for 40 sec, 52 deg.c for 40 sec, 72 deg.c for 40 sec, 25 cycles of amplification, final extension at 72 deg.c for 3 min, and setting the product at 4 deg.c for use or direct electrophoresis. Taking 20 muL PCR product to carry out electrophoretic analysis, separating and cutting gel on 1.5% agarose gel for recovery, cloning the obtained heavy chain variable region and light chain variable region to pMD18T plasmid vector (TaKaRa) respectively, sequencing, analyzing the framework region, antigenic determinant (CDR) sequence and deduced amino acid sequence of the obtained DNA sequence of the heavy chain variable region of the antibody by using an on-line analysis tool, adding the constant regions of the heavy chain and the light chain into the sequence of the variable region of the antibody respectively at the downstream by using a gene cloning mode, obtaining an expression vector capable of recombining and expressing the antibody fragment by using a genetic recombination mode to express a recombined complete antibody or fragment in eukaryotic cells, and connecting the heavy chain variable region and the light chain variable region by using a coding sequence of a segment of interchain connecting peptide (such as GGSGGGGSGGGGS) to construct a single chain antibody to express the antibody fragment in proper host cells.

Sequence listing

<110> Beijing Huada protein research & development center, Ltd

<120> a neutralizing antibody capable of binding SARS-CoV-2 virus RBD protein and its application

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Ser Thr Ser

1

<210> 3

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Gln Gln Phe Ser Gly Tyr Pro Leu Thr

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Gly Tyr Thr Phe Thr Ser Tyr Trp

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Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

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Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

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Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

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Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

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210 215 220

Phe Asn

225

Claims

1. A monoclonal antibody capable of specifically binding to a receptor binding domain RBD of S protein of SARS-Cov-2 virus, which comprises:

(a) light chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 01, or a CDR1 region of the amino acid sequence shown in seq id no.

(ii) Comprises the amino acid sequence of SEQ ID NO: 02, or a CDR2 region of the amino acid sequence shown in SEQ ID NO.

(iii) Comprises the amino acid sequence of SEQ ID NO: 03, and a CDR3 region.

(b) Heavy chain variable region

(i) Comprises the amino acid sequence of SEQ ID NO: 04, or a CDR1 region of the amino acid sequence shown in SEQ ID No.

(ii) Comprises the amino acid sequence of SEQ ID NO: 05 of the amino acid sequence shown in seq id No. 2.

(iii) Comprises the amino acid sequence of SEQ ID NO: 06, or a CDR3 region of the amino acid sequence shown in SEQ ID NO.

2. It is characterized by being secreted by a mouse hybridoma cell line 21336-98.

3. The monoclonal antibody of claim 1, wherein the antigen for immunizing a mouse has the amino acid sequence shown in SEQ ID No. 7, and the antigen is recombinantly expressed and purified from mammalian cells.

4. The monoclonal antibody of claim 1, characterized as a mouse IgG2b subtype monoclonal antibody.

5. The monoclonal antibody of claim 1, wherein the heavy chain variable region gene sequence is fused to the heavy chain constant region coding sequence of human immunoglobulin and the light chain variable region gene sequence is fused to the light chain constant region coding sequence of human immunoglobulin by genetic recombination to construct a murine-human chimeric antibody, and the murine-human chimeric antibody is recombinantly expressed by transfecting cells.

6. The monoclonal antibody of claim 1, which is used as a quality control substance for the detection of neutralizing antibodies by constructing a mouse-human chimeric antibody for an immune colloidal gold test strip and a competitive enzyme-linked immunosorbent assay.

7. The monoclonal antibody of claim 1, which can block the binding of the S protein to ACE2 protein, a human host receptor, and can be used for preparing a target drug for treating new coronary pneumonia.