Broad-spectrum neutralizing nano antibody for targeting coronavirus S protein and application of broad-spectrum neutralizing nano antibody

Abstract

The invention discloses a broad-spectrum neutralizing nano antibody targeting coronavirus S protein and application, and relates to the technical field of targeting coronavirus S protein. In order to facilitate research and development, hidden and conservative pathogenic sites which are difficult to recognize by conventional antibodies are recognized more effectively; the preparation method comprises the following steps: firstly, selecting a broad-spectrum neutralizing nano antibody 11D which is specifically combined with MERS-CoV, SARS-CoV and SARS-CoV-2 spike protein RBD, wherein the nano antibody is composed of a heavy chain variable region (VHH); according to the invention, through current mutant strain examples and structural biological analysis, a key site of broad-spectrum antiviral development is determined by taking an antigen with a stable structure after transformation as a basis and taking a natural nano antibody as a tool and combining multiple screening such as a cryoelectron microscope technology and experimental verification; and a new direction is provided for research and development of medicines for treating the high-pathogenicity coronavirus and prevention of new coronavirus possibly appearing in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of targeting coronavirus S protein, and in particular to a broad-spectrum neutralizing nano antibody targeting coronavirus S protein and an application thereof. Background Art

[0002] There is a kind of antibody without light chain in the serum of camelid animals, which is a heavy chain dimer structure, namely "heavy chain antibody". These heavy chain antibodies include two constant domains CH2 and CH3, a hinge region and an antigen binding / variable heavy chain domain (VHH), and still retain complete antigen binding ability. VHH is also called single domain antibody. Compared with the large molecular weight of conventional antibodies, VHH is only about 15KDa, with unique physical and chemical stability, higher thermal stability and solubility, small size and easy penetration into dense tissues and even blood-brain barrier. Based on the above advantages, it can bind Some conventional antibodies have hidden conservative pathogenic sites that are difficult to identify, and are easier to prepare and produce when highly expressed in prokaryotic or eukaryotic systems. The essence of phage display technology is a screening technology. Different exogenous genes are inserted into phage vectors. As the phage is propagated, the exogenous proteins will be displayed on the surface of the phage to form a phage library. The phage library is then screened with a specific protein to quickly obtain antibodies with a high affinity for the protein. The screened antibodies can be further used for biological function research;

[0003] Coronaviridase is a family of single-stranded positive-sense RNA viruses that mainly infect vertebrates. Its genome is relatively large among known RNA viruses. Coronaviruses are usually transmitted through the respiratory tract or fecal-oral route and can cause a variety of diseases, such as the common cold, bronchitis, pneumonia, gastroenteritis, and heart disease. Some coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2) have a high mortality rate and spread rapidly, which can cause serious social and public health problems. In addition, an increasing number of mutant strains have produced a variety of consequences, including accelerated transmission and changes in pathogenicity. Another direct consequence is that the protective effect of vaccines has decreased, and some monoclonal antibody drugs developed in the early stage have become ineffective or their neutralizing ability has been greatly reduced.

[0004] In view of this, researching and developing more effective broad-spectrum neutralizing nanoantibodies that can identify hidden conserved pathogenic sites that are difficult for conventional antibodies to identify and that target various mutant strains have important scientific significance and broad application prospects for the clinical treatment of diseases and the development of diagnostic reagents. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a broad-spectrum neutralizing nano antibody targeting coronavirus S protein and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] An application of a broad-spectrum neutralizing nanobody targeting the coronavirus S protein, first selecting a broad-spectrum neutralizing nanobody 11D that specifically binds to the MERS-CoV, SARS-CoV, and SARS-CoV-2 spike protein RBD, the nanobody is composed of a heavy chain variable region (VHH), and the heavy chain variable region (VHH) contains the following complementary determining regions:

[0008] The amino acid sequences are respectively the complementary determining region CDR1 at positions 31-35 of SEQ ID 11D, the complementary determining region CDR2 at positions 50-66 of SEQ ID 11D, and the complementary determining region CDR3 at positions 99-121 of SEQ ID 11D, and the application process thereof comprises the following steps:

[0009] S1: Antigenic modification;

[0010] S2: Antibody screening;

[0011] S3: Acquisition of nanobody genes;

[0012] S4: Recombinant construction of prokaryotic expression vector;

[0013] S5: Recombinant construction of eukaryotic expression vector;

[0014] S6: purified antibodies;

[0015] S7: pseudovirus neutralization assay;

[0016] S8: Use Elisa to measure the affinity of 11D to S, S1, RBD of MERS-CoV and SARS-CoV-2Omicron, and to SARS-CoVS and RBD proteins.

[0017] Preferably, in the antigen modification in the S1 step, two consecutive replacements of proline at residues V1060 and L1061 in MERS-CoV and at residues K986 and V987 in SARS-CoV-2Omicron increase the protein amount, and the introduction of two consecutive proline residues at the beginning of the central helix stabilizes the S protein in a highly active "pre-fusion conformation".

[0018] Further: the antibody screening in the S2 step includes phage amplification, rescue, adsorption, elution and infection.

[0019] Further: the nano antibody gene of the S3 step is obtained by subtracting the ElisaOD450 value of the phage coated with BSA protein from the ElisaOD450 value of the phage coated with MERS-CoV S protein and SARS-CoV-2Omicron S protein, and 20 strains are screened out. The corresponding bacterial solution is sent to a sequencing company for sequencing to obtain the sequence information of the qualified sequencing results, and the nano antibody gene sequence is obtained after removing the sequence of the vector part.

[0020] As a preferred solution of the present invention: the recombinant prokaryotic expression vector is constructed in the S4 step, 2B and 11D bacterial solutions are PCR amplified, primers are designed to recombinate them with the pet28a vector to obtain recombinant plasmids pet28a-2B-his and pet28a-11D-his, and the two prokaryotic expression vectors pet28a-2B-his and pet28a-11D-his are separately transformed into BL21 Escherichia coli competent cells, respectively inoculated into LB liquid culture medium containing kanamycin, and cultured at 37°C and 230rpm to OD600 of 0.5, and then IPTG with a final concentration of 0.1mM is added to the culture system, and the induction culture is continued at 18°C ​​and 110rpm for 16 hours, and the bacterial cells are harvested by centrifugation and stored at -80°C for later use.

[0021] As a further scheme of the present invention: the recombinant eukaryotic expression vector is constructed in the S5 step, 2B and 11D bacterial solutions are PCR amplified, primers are designed to recombine them with the plenti-hFc vector to obtain recombinant plasmids plenti-2B-hFc and plenti-11D-hFc, HEK293T cells are co-transfected with lentiviral packaging plasmid (psPAX2), envelope plasmid (pMD2.G) and plenti-2B-hFc and plenti-11D-hFc plasmids, and plenti-2B-hFc and plenti-11D-hFc lentiviral particles are prepared using HEK293T cells; HEK293F cells are infected with plenti-2B-hFc and plenti-11D-hFc lentiviral particles, and positive HEK293F cells infected with lentivirus and supernatant are screened with a culture medium containing puromycin, and stored at -80°C for purification.

[0022] As a further solution of the present invention: the purification step of the purified antibody in step S6 comprises:

[0023] (1) Lysis of bacteria: resuspend the obtained bacteria in 50 ml PBS, disrupt the bacteria with ultrasound, centrifuge at 12000 × g for 20 min, and transfer the supernatant to a new centrifuge tube;

[0024] (2) Affinity chromatography:

[0025] 1. Filter the sample and all solutions used in the purification process using a 0.22 μm filter.

[0026] 2. Pump the protein into the HisTrapHP prepacked column;

[0027] 3. Install the pre-packed column into the ATKAgo purifier and elute the desired protein;

[0028] (3) Concentrate the protein using a 50K ultrafiltration tube, centrifuge at 4000 rpm and 4°C, replace the eluate with PBS, repeat 3 times to make the final volume 1-2 ml, collect the protein into a clean EP tube, and freeze it in a -20°C refrigerator.

[0029] Based on the above scheme: the pseudovirus neutralization experiment in step S7 includes the following steps:

[0030] (1) 293T-ACE2 cells were inoculated into a 96-well plate and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 cell culture incubator for 12 h;

[0031] (2) Dilute the purified antibody with MEM from the initial concentration to the initial concentration (1 mg / ml or 100 μg / ml), perform 2-fold dilution, and dilute 9 concentration gradients in total; add 30 μl of pseudovirus suspension to each well, mix thoroughly, and add to a 96-well cell culture plate. Set up 3 replicate wells for each concentration gradient mixture and culture in a 37°C, 5% CO2 cell culture incubator for 24 h;

[0032] (3) After 12 h, 100 μl of 10% fetal bovine serum DMEM medium was added to each well and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0033] (4) 48 h after infection, the cell culture supernatant was aspirated, and 60 μl of cell lysis buffer was added to each well and lysed for 15 min;

[0034] (5) Transfer 40 μl of cell lysate to a 96-well ELISA plate, add 10 μl of luciferase, read the luciferase signal value, fit the curve using GraphPad Prism8, and calculate the IC values ​​of antibodies 11D and 2B. 50 .

[0035] Based on the above scheme: Elisa is used in step S8 to determine the affinity of 11D to S, S1, RBD of MERS-CoV and SARS-CoV-2Omicron, and to S and RBD proteins of SARS-CoV, and the steps are as follows:

[0036] (1) Dilute the protein with coating solution to a concentration of 1 μg / ml, add 50 μl / well to a 96-well ELISA plate, and incubate at 4°C overnight;

[0037] (2) The next day, wash the plate once with PBST and pat dry, add blocking solution (PBS containing 3% BSA), 100 μl per well, and incubate at 37°C for 1 h;

[0038] (3) Wash the plate once with PBST and pat dry;

[0039] (4) Take another 96-well dilution plate, dilute the antibody in equal proportions, and then transfer it to a 96-well ELISA plate, 50 μl / well, and incubate at 37°C for 45 min;

[0040] (5) Wash the plate three times with PBST and pat dry;

[0041] (6) Dilute the label antibody with blocking solution, add 50 μl / well to a 96-well ELISA plate, and incubate at 37°C for 30 min;

[0042] (7) Wash the plate three times with PBST and pat dry;

[0043] (8) Add TMB single-component colorimetric solution, 50 μl / well, and incubate at room temperature or 37°C for several minutes;

[0044] (9) When the color develops to an appropriate depth (usually 5-10 min), add 50 μl / well of stop solution and tap the plate gently to mix. Use an ELISA reader to read the absorbance at 450 nm.

[0045] A broad-spectrum neutralizing nanobody targeting coronavirus S protein, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1 is: positions 31-35 of SEQ ID 11D; CDR2 is: positions 50-66 of SEQ ID 11D; and CDR3 is: positions 99-121 of SEQ ID 11D.

[0046] The beneficial effects of the present invention are:

[0047] 1. A broad-spectrum neutralizing nanobody targeting coronavirus S protein and its application. Through current mutant strain examples and structural biology analysis, based on antigens with stable structures after modification, natural nanoantibodies are used as tools, combined with cryo-electron microscopy technology and experimental verification, and multiple screenings, the key sites for the development of broad-spectrum antiviral drugs are determined, providing new directions for the development of drugs for highly pathogenic coronaviruses and the prevention of new coronaviruses that may appear in the future.

[0048] 2. A broad-spectrum neutralizing nanoantibody targeting coronavirus S protein and its application. We have successfully obtained a broad-spectrum neutralizing antibody 11D that specifically binds to the spike protein RBD of MERS-CoV, SARS-CoV, and SARS-CoV-2. It has a significant inhibitory effect on MERS-CoV, SARS-CoV, and SARS-CoV-2. It is a neutralizing antibody with broad-spectrum effect, good broad spectrum and neutralizing activity, and provides a basis for constructing high-affinity, high-quality, and highly specific therapeutic neutralizing antibodies for coronaviruses. It has a high application value for the prevention, control, and treatment of the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of the application of a broad-spectrum neutralizing nano antibody targeting coronavirus S protein proposed by the present invention;

[0050] Figure 2 This is a schematic diagram of the phage adsorption-elution-amplification structure of the application of a broad-spectrum neutralizing nano-antibody targeting the coronavirus S protein proposed by the present invention;

[0051] Figure 3 The invention proposes a broad-spectrum neutralizing nanoantibody targeting the coronavirus S protein, and the antibody 11D-his is used to measure the affinity of SARS-CoV-1, MERS-CoV, and Omicron (EC 50 )picture;

[0052] Figure 4 The invention proposes a broad-spectrum neutralizing nano-antibody targeting coronavirus S protein, and the neutralizing activity test of antibodies 11D-His and 2B-His on pseudovirus (IC 50 ) Antibodies 11D-FC and 2B-FC were tested for neutralizing activity against pseudoviruses (1C 50 ) is a schematic diagram of the detection results. DETAILED DESCRIPTION

[0053] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0054] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0055] Embodiment 1:

[0056] Application of a broad-spectrum neutralizing nanobody targeting coronavirus S protein, such as Figure 1As shown, firstly, a broad-spectrum neutralizing nanobody 11D that specifically binds to the spike protein RBD of MERS-CoV, SARS-CoV, and SARS-CoV-2 is selected, wherein the nanobody is composed of a heavy chain variable region (VHH), and the heavy chain variable region (VHH) comprises a complementary determining region as follows:

[0057] The amino acid sequences are respectively the complementary determining region CDR1 at positions 31-35 of SEQ ID 11D, the complementary determining region CDR2 at positions 50-66 of SEQ ID 11D, and the complementary determining region CDR3 at positions 99-121 of SEQ ID 11D, and the application process thereof comprises the following steps:

[0058] S1: Antigenic modification

[0059] The protein amount was increased by replacing proline twice in residues V1060 and L1061 in MERS-CoV and K986 and V987 in SARS-CoV-2Omicron. The introduction of two consecutive proline residues at the beginning of the central helix stabilizes the S protein in a highly active "prefusion conformation". The introduction of two proline mutations in the protein greatly improves its stability and maintains a highly immunogenic prefusion state. At the same time, the addition of a trimer motif makes its structure relatively stable and more likely to expose some conservative pathogenic epitopes, which is conducive to screening.

[0060] S2: Antibody Screening

[0061] Screening mainly includes phage amplification, rescue, adsorption, elution and infection;

[0062] First, the TG1 containing phagemid is amplified, then helper bacteria are added to assist in the packaging of phage antibodies, and then the phage antibodies are isolated from them. Specific steps:

[0063] (1) On the evening of the first day, the M13 single-chain filamentous phage displaying alpaca nanobody immune library was inoculated at a ratio of 1:100 in 100 ml of 2×YTAG liquid medium and cultured at 37°C with shaking until the bacterial solution OD600 was approximately 0.5;

[0064] (2) Add M13K07 helper phage, let stand at 37°C for 30 min, and then shake at the same temperature for 30 min;

[0065] (3) Transfer the bacterial solution to a centrifuge bottle and centrifuge (3300 × g, room temperature, 10 min), and discard the supernatant;

[0066] (4) Resuspend the bacterial pellet in 100 ml of 2×YTAK liquid medium and then incubate at 37°C with shaking overnight;

[0067] (5) On the next morning, centrifuge the bacterial solution (3300 × g, room temperature, 10 min), and transfer the supernatant to a new centrifuge bottle;

[0068] (6) Add 5 ml of PEG / NaCl solution to the supernatant, mix well, and place on ice for 70 min;

[0069] (7) After the ice bath is completed, centrifuge (12000×g, 4°C, 30 min). After the centrifugation, a small amount of white precipitate can be seen at the bottom. This precipitate is the bacteriophage.

[0070] (8) Discard the supernatant and centrifuge again (12,000 × g, 4°C, 2 min), discarding as much residual liquid as possible;

[0071] (9) Resuspend the pellet with 400 μl PBS, then transfer to a 1.5 ml centrifuge tube and continue centrifugation (6000 × g, 4°C, 5 min);

[0072] (10) Transfer the supernatant to a new centrifuge tube to rescue the phage and place it at -80°C for later use;

[0073] The second step is to adsorb the phage antibodies onto a plate coated with the MERS-CoV spike protein, wash to remove non-specific binding and low-affinity phage antibodies, then use a strong elution reagent to elute the specific binding and high-affinity phage antibodies, and use the latter to infect TG1. The specific steps are as follows:

[0074] (1) On the first day, the plate was coated with MERS-CoVS protein at a concentration of 1 μg / ml, 100 μl, and incubated at 4°C overnight;

[0075] (2) On the morning of the second day, discard the liquid in the plate and wash the plate three times with PBS;

[0076] (3) Add 5% skim milk to the Elisa plate and incubate at 4°C overnight;

[0077] (4) Pour off the milk in the plate, wash three times with PBS, add 100 μl of rescued phage, and incubate at 37°C for 2.5 h;

[0078] (5) Wash the plate 20 times with PBST and then 20 times with PBS;

[0079] (6) Add 100 μl of trypsin digestion solution to the plate and incubate at 37°C for 30 min.

[0080] (7) Transfer the eluted phage to a 15 ml centrifuge tube, add 10 ml of TG1 bacterial solution with an OD600 of about 0.5, and also add 100 μl of the bacterial solution to the plate;

[0081] (9) Place the centrifuge tube in a 37°C water bath for 30 min and place the plate in a 37°C incubator for 30 min.

[0082] (10) The two tubes of bacterial suspension were mixed, centrifuged (3300 × g, room temperature, 10 min) and the supernatant was discarded;

[0083] (11) Resuspend the bacterial pellet in 400 μl of 2×YT liquid medium, then evenly spread on three 2×YTAG solid culture plates and incubate at 30°C overnight;

[0084] Collect TG1 infected with phage antibodies, which is the antibody library used in the next round of screening. The specific steps are as follows:

[0085] (1) On the third day, dense colonies have grown on the solid culture plate to form a "biofilm". Add 2 ml of 2×YT liquid medium to the culture plate, scrape off the colonies with a cell scraper, and collect them in a 10 ml centrifuge tube;

[0086] (2) Add 2 ml of 2×YT liquid medium to the culture plate again, rinse the remaining colonies and collect them into the above centrifuge tube;

[0087] (3) Centrifuge the tube (3300 × g, room temperature, 10 min) and discard the supernatant;

[0088] (4) Resuspend the pellet in 2 ml of 2×YT liquid medium and then add glycerol at a 1:1 ratio;

[0089] (5) The bacterial solution was aliquoted and stored at -80°C for use in the next round of screening;

[0090] The subsequent rounds of screening steps are basically similar, with the following changes:

[0091] (1) The concentrations of the coating antigens in the second, third, and fourth rounds were adjusted to MERS-CoV spike 0.1 μg / ml, SARS-CoV-2O micron spike 1 μg / ml, and SARS-CoV-2O micron spike 0.1 μg / ml, respectively;

[0092] (2) The library bacteria used in the next round of screening are the library bacteria collected from the solid culture plate and aliquoted and frozen in the previous round;

[0093] (3) During the fourth round of screening, the bacterial solution in the centrifuge tube and the plate was mixed, and then the bacterial solution was diluted ten-fold in a 2×YT liquid culture medium. Four dilutions of 10-3-10-6 were selected. Two 50 μl portions of each dilution were evenly spread on two 2×YTAG solid culture plates, inverted and incubated overnight at 30°C for subsequent single clone picking and identification;

[0094] After the fourth round of screening, it is necessary to pick monoclonal colonies for the preparation of monoclonal phages for subsequent Elisa identification. The specific steps are as follows:

[0095] (1) In the fourth round of screening, after the solid culture plate was cultured overnight, the density of colonies on the culture plates with different dilutions was different. The culture plates with relatively sparse colonies were randomly selected, and relatively isolated monoclonal colonies on the culture plates were selected. 192 colonies were randomly picked and inoculated into two 96-well deep-well plates. 500 μl 2×YTAG liquid culture medium was pre-added to each well and cultured at 37°C for 8 h with shaking. On the same evening of the same day when the monoclonal was picked, MERS-CoVS, SARS-CoV-2OmicronS and BSA proteins were used to coat 96-well ELISA plates, respectively, with a coating concentration of 1 μg / ml, 100 μl per well, and left to stand at 4°C overnight.

[0096] (2) Take 50 μl of bacterial solution from each well and transfer it to a new 96-well deep-well plate. 450 μl of 2×YTAG liquid medium has been added to each well and cultured at 37°C with shaking;

[0097] (3) Add 400 μl of glycerol to the original 96-well deep-well plate and freeze at -80°C;

[0098] (4) After shaking the new 96-well deep-well plate for 2.5 h, add M13K07 helper phage, 50 μl per well;

[0099] (5) First, keep the culture at 37°C for 30 min, then shake culture at 37°C for 30 min;

[0100] (6) The deep-well plate was centrifuged (1800 × g, room temperature, 10 min), the supernatant was discarded, 400 μl of 2×YTAK liquid medium was added to each well to resuspend the bacterial pellet, and the culture was shaken at 30°C overnight; the 96-well Elisa plate was blocked with 5% skim milk at 4°C overnight;

[0101] (7) On the second day, the deep-well plate was centrifuged (1800 × g, room temperature, 15 min), 50 μl of supernatant was aspirated from each well and transferred to a new Elisa plate; incubated at 37°C for 3 h, washed twice with PBS, added with antibody and incubated at 37°C for 2 h, and washed three times with PBS;

[0102] (8) Add 50 μl of color development solution to each well and incubate at 37°C for about 20 min;

[0103] (9) Add 50 μl of stop solution to each well and read OD450 on a microplate reader;

[0104] The principle of phage display technology is to insert a foreign gene into the appropriate position of the phage coat protein structural gene. When the reading frame is normal and does not affect the normal function of the coat protein, the foreign gene will be expressed along with the expression of the coat protein, so that the polypeptide or protein is displayed on the phage surface in the form of a fusion protein. The displayed protein can maintain a relatively independent spatial structure and biological activity, which is conducive to the binding of the target protein. Therefore, the target protein can be used to quickly screen the phage display library. After the display library is constructed, the target protein is used as the stationary phase and incubated with the display library for a period of time, the unbound phage is washed away, and the adsorbed phage is eluted with a competitive receptor. The eluted phage infects the host bacteria for reproduction and amplification, and then the next round of elution is carried out. After 3-5 rounds of "adsorption-elution-amplification" (for some antibodies with weak affinity, more rounds of elution are required), a high enrichment of phages that can specifically bind to the target protein can be obtained. The specific process is as follows Figure 2 As shown;

[0105] S3: Acquisition of Nanobody Genes

[0106] The ElisaOD450 value of the phage coated with MERS-CoV S protein and SARS-CoV-2Omicron S protein was subtracted from the ElisaOD450 value of the phage coated with BSA protein, and 20 strains were screened out. The corresponding bacterial liquid was sent to a sequencing company for sequencing. The sequence information of the qualified sequencing results was obtained, and the sequence of the vector part was removed to obtain the nanobody gene sequence;

[0107] After the above screening, the positive clones were used to amplify the complete reading frame of the antibody by high-fidelity PCR to obtain the encoding nucleic acid molecules of 11D and 2B and the amino acid sequences encoded by them.

[0108] S4: Recombinant construction of prokaryotic expression vector

[0109] 2B and 11D bacterial solutions were amplified by PCR, and primers were designed to recombinant them with pet28a vector to obtain recombinant plasmids pet28a-2B-his and pet28a-11D-his. The two prokaryotic expression vectors pet28a-2B-his and pet28a-11D-his were transformed into BL21 Escherichia coli competent cells, respectively, and inoculated into LB liquid culture medium containing kanamycin, and cultured at 37°C and 230rpm until OD600 was 0.5. Then, IPTG with a final concentration of 0.1mM was added to the culture system, and the induction culture was continued at 18°C ​​and 110rpm for 16 hours. The cells were harvested by centrifugation and stored at -80°C for later use.

[0110] S5: Recombinant construction of eukaryotic expression vector

[0111] PCR amplification was performed on 2B and 11D bacterial suspensions, and primers were designed to recombinate them with the plenti-hFc vector to obtain recombinant plasmids plenti-2B-hFc and plenti-11D-hFc, and HEK293T cells were co-transfected with lentivirus packaging plasmid (psPAX2), envelope plasmid (pMD2.G) and plenti-2B-hFc and plenti-11D-hFc plasmids, and plenti-2B-hFc and plenti-11D-hFc lentiviral particles were prepared using HEK293T cells; HEK293F cells were infected with plenti-2B-hFc and plenti-11D-hFc lentiviral particles, and positive HEK293F cells and supernatants infected with lentivirus were screened with a culture medium containing puromycin, and stored at -80°C for purification;

[0112] S6: Purify the antibody, the purification step comprising:

[0113] (1) Lysis of bacteria: resuspend the obtained bacteria in 50 ml PBS, disrupt the bacteria with ultrasound, centrifuge at 12000 × g for 20 min, and transfer the supernatant to a new centrifuge tube;

[0114] (2) Affinity chromatography:

[0115] 1. Filter the sample and all solutions used in the purification process using a 0.22 μm filter.

[0116] 2. Pump the protein into the HisTrapHP prepacked column;

[0117] 3. Install the pre-packed column into the ATKAgo purifier and elute the desired protein;

[0118] (3) Concentrate the protein using a 50K ultrafiltration tube, centrifuge at 4000 rpm and 4°C, replace the eluate with PBS, repeat 3 times to make the final volume 1-2 ml, collect the protein into a clean EP tube, and freeze it in a -20°C refrigerator;

[0119] The expression and purification of Fc fragment antibodies and affinity chromatography process include:

[0120] 1. Filter the sample and all solutions used in the purification process using a 0.22 μm filter;

[0121] 2. Pump the supernatant containing plenti-2B-hFc and plenti-11D-hFc into the protein A pre-packaged chromatography column;

[0122] 3. Wash the non-specific binding products with PBS; elute the products with 0.1 M citric acid and neutralize with Tris-HCl (PH = 9);

[0123] 4. Use UV spectrophotometer to detect the concentration and purity of the purified protein;

[0124] S7: Pseudovirus Neutralization Experiment

[0125] (1) 293T-ACE2 cells were inoculated into a 96-well plate and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 cell culture incubator for 12 h;

[0126] (2) Dilute the purified antibody with MEM from the initial concentration to the initial concentration (1 mg / ml or 100 μg / ml), perform 2-fold dilution, and dilute 9 concentration gradients in total; add 30 μl of pseudovirus suspension to each well, mix thoroughly, and add to a 96-well cell culture plate. Set up 3 replicate wells for each concentration gradient mixture and culture in a 37°C, 5% CO2 cell culture incubator for 24 h;

[0127] (3) After 12 h, 100 μl of 10% fetal bovine serum DMEM medium was added to each well and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0128] (4) 48 h after infection, the cell culture supernatant was aspirated, and 60 μl of cell lysis buffer was added to each well and lysed for 15 min;

[0129] (5) Transfer 40 μl of cell lysate to a 96-well ELISA plate, add 10 μl of luciferase, read the luciferase signal value, fit the curve using GraphPad Prism8, and calculate the IC values ​​of antibodies 11D and 2B. 50 ;

[0130] S8: Elisa was used to determine the affinity of 11D to S, S1, RBD of MERS-CoV and SARS-CoV-2Omicron, and to SARS-CoVS and RBD proteins. The steps are as follows:

[0131] (1) Dilute the protein with coating solution to a concentration of 1 μg / ml, add 50 μl / well to a 96-well ELISA plate, and incubate at 4°C overnight;

[0132] (2) The next day, wash the plate once with PBST and pat dry, add blocking solution (PBS containing 3% BSA), 100 μl per well, and incubate at 37°C for 1 h;

[0133] (3) Wash the plate once with PBST and pat dry;

[0134] (4) Take another 96-well dilution plate, dilute the antibody in equal proportions, and then transfer it to a 96-well ELISA plate, 50 μl / well, and incubate at 37°C for 45 min;

[0135] (5) Wash the plate three times with PBST and pat dry;

[0136] (6) Dilute the label antibody with blocking solution, add 50 μl / well to a 96-well ELISA plate, and incubate at 37°C for 30 min;

[0137] (7) Wash the plate three times with PBST and pat dry;

[0138] (8) Add TMB single-component colorimetric solution, 50 μl / well, and incubate at room temperature or 37°C for several minutes;

[0139] (9) When the color develops to an appropriate depth (usually 5-10 min), add 50 μl / well of stop solution and tap the plate gently to mix. Use an ELISA reader to read the absorbance at 450 nm.

[0140] Embodiment 2:

[0141] A broad-spectrum neutralizing nanobody targeting coronavirus S protein, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1 is: positions 31-35 of SEQ ID 11D; CDR2 is: positions 50-66 of SEQ ID 11D; and CDR3 is: positions 99-121 of SEQ ID 11D.

[0142] Embodiment 3:

[0143] Application of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein. In order to verify the test results, this example introduces ELISA experiments and pseudovirus neutralization tests in 293T cells:

[0144] (1) ELISA experiment 11D-his binding to ectodomain, S1, and RBD of SARS-CoV-1, MERS-CoV, and SARS-CoV-2Omicron Serially diluted antibodies were used to bind to ectodomain, S1, and RBD of SARS-CoV-1, MERS-CoV, and variant SARS-CoV-2Omicron. 50 The values ​​are the mean ± SD of three independent results. The experiments were repeated three times with similar results, and a representative curve is shown. Figure 3 As shown;

[0145] (2) Neutralization test of pseudoviruses in 293T cells. Evaluation of the neutralization performance of 11D, 11D-Fc and 2B, 2B-FC against pseudoviruses. Serially diluted antibodies were used to neutralize the pseudovirus variants Wuhan-Hu-1, Delta, SARS-CoV-2Omicron, as well as MERS-CoV and SARS-CoV-1. The IC values ​​of pseudoviruses were 50 The values ​​are the mean ± SD of three independent results. The experiments were repeated three times with similar results, and a representative curve is shown. Figure 4 shown.

[0146] The above is a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the field within the technical scope disclosed by the present invention in combination with the prior art or public common sense, within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.

Claims

1. An application of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein, characterized in that: First, a broad-spectrum neutralizing nanobody 11D that specifically binds to the spike protein RBD of MERS-CoV, SARS-CoV, and SARS-CoV-2 is selected. The nanobody is composed of a heavy chain variable region (VHH), and the heavy chain variable region (VHH) contains the following complementary determining regions: The amino acid sequences are respectively the complementary determining region CDR1 at positions 31-35 of SEQ ID 11D, the complementary determining region CDR2 at positions 50-66 of SEQ ID 11D, and the complementary determining region CDR3 at positions 99-121 of SEQ ID 11D, and the application process thereof comprises the following steps: S1: Antigenic modification; S2: Antibody screening; S3: Acquisition of nanobody genes; S4: Recombinant construction of prokaryotic expression vector; S5: Recombinant construction of eukaryotic expression vector; S6: purified antibodies; S7: pseudovirus neutralization assay; S8: Use Elisa to measure the affinity of 11D to S, S1, RBD of MERS-CoV and SARS-CoV-2Omicron, and to SARS-CoV S and RBD proteins.

2. The use of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein according to claim 1, characterized in that: In the antigen modification in the S1 step, two consecutive replacements of proline at residues V1060 and L1061 in MERS-CoV and at residues K986 and V987 in SARS-CoV-2Omicron increased the protein amount, and the introduction of two consecutive proline residues at the beginning of the central helix stabilized the S protein in the highly active "prefusion conformation".

3. The use of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein according to claim 2, characterized in that: The antibody screening in the S2 step includes phage amplification, rescue, adsorption, elution and infection.

4. The use of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein according to claim 3, characterized in that: The nanobody gene of the S3 step is obtained by subtracting the ElisaOD450 value of the phage coated with BSA protein from the ElisaOD450 value of the phage coated with MERS-CoV S protein and SARS-CoV-2Omicron S protein, and 20 strains are screened out. The corresponding bacterial solution is sent to a sequencing company for sequencing to obtain the sequence information of qualified sequencing results, and the nanobody gene sequence is obtained after removing the sequence of the vector part.

5. The use of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein according to claim 4, characterized in that: In the recombinant construction of the prokaryotic expression vector in the S4 step, 2B and 11D bacterial solutions are PCR amplified, primers are designed to recombinate them with the pet28a vector to obtain recombinant plasmids pet28a-2B-his and pet28a-11D-his, and the two prokaryotic expression vectors pet28a-2B-his and pet28a-11D-his are separately transformed into BL21 Escherichia coli competent cells, respectively inoculated into LB liquid culture medium containing kanamycin, and cultured at 37°C and 230rpm until OD600 is 0.5, and then IPTG with a final concentration of 0.1mM is added to the culture system, and the induction culture is continued at 18°C ​​and 110rpm for 16 hours, and the bacterial cells are harvested by centrifugation and stored at -80°C for later use.

6. The use of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein according to claim 5, characterized in that: In the recombinant construction of the eukaryotic expression vector in step S5, 2B and 11D bacterial solutions are PCR amplified, primers are designed to recombinate them with the plenti-hFc vector to obtain recombinant plasmids plenti-2B-hFc and plenti-11D-hFc, and HEK293T cells are co-transfected with lentivirus packaging plasmid (psPAX2), envelope plasmid (pMD2.G) and plenti-2B-hFc and plenti-11D-hFc plasmids to prepare plenti-2B-hFc and plenti-11D-hFc lentivirus particles using HEK293T cells; HEK293F cells are infected with plenti-2B-hFc and plenti-11D-hFc lentivirus particles, and positive HEK293F cells and supernatants infected with lentivirus are screened with a culture medium containing puromycin, and stored at -80°C for purification.

7. The use of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein according to claim 6, characterized in that: The purified antibody in step S6 comprises: (1) Lysis of bacteria: resuspend the obtained bacteria in 50 ml PBS, disrupt the bacteria with ultrasound, centrifuge at 12000 × g for 20 min, and transfer the supernatant to a new centrifuge tube; (2) Affinity chromatography:

1. Filter the sample and all solutions used in the purification process using a 0.22 μm filter.

2. Pump the protein into the HisTrapHP prepacked column; 3. Install the pre-packed column into the ATKAgo purifier and elute the desired protein; (3) Concentrate the protein using a 50K ultrafiltration tube, centrifuge at 4000 rpm and 4°C, replace the eluate with PBS, repeat 3 times to make the final volume 1-2 ml, collect the protein into a clean EP tube, and freeze it in a -20°C refrigerator.

8. The use of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein according to claim 7, characterized in that: The pseudovirus neutralization experiment in step S7 comprises the following steps: (1) 293T-ACE2 cells were inoculated into a 96-well plate and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 cell culture incubator for 12 h; (2) Dilute the purified antibody with MEM from the initial concentration to the initial concentration (1 mg / ml or 100 μg / ml), perform 2-fold dilution, and dilute 9 concentration gradients in total; add 30 μl of pseudovirus suspension to each well, mix thoroughly, and add to a 96-well cell culture plate. Set up 3 replicate wells for each concentration gradient mixture and culture in a 37°C, 5% CO2 cell culture incubator for 24 h; (3) After 12 h, 100 μl of 10% fetal bovine serum DMEM medium was added to each well and cultured in a 37°C, 5% CO2 incubator for 24 h; (4) 48 h after infection, the cell culture supernatant was aspirated, and 60 μl of cell lysis buffer was added to each well and lysed for 15 min; (5) Transfer 40 μl of cell lysate to a 96-well ELISA plate, add 10 μl of luciferase, read the luciferase signal value, fit the curve using GraphPad Prism8, and calculate the IC values ​​of antibodies 11D and 2B. 50 .

9. The use of a broad-spectrum neutralizing nanoantibody targeting coronavirus S protein according to claim 8, characterized in that: In the S8 step, Elisa is used to determine the affinity of 11D with S, S1, RBD of MERS-CoV and SARS-CoV-2Omicron, and with SARS-CoV S and RBD proteins, and the steps are as follows: (1) Dilute the protein with coating solution to a concentration of 1 μg / ml, add 50 μl / well to a 96-well ELISA plate, and incubate at 4°C overnight; (2) The next day, wash the plate once with PBST and pat dry, add blocking solution (PBS containing 3% BSA), 100 μl per well, and incubate at 37°C for 1 h; (3) Wash the plate once with PBST and pat dry; (4) Take another 96-well dilution plate, dilute the antibody in equal proportions, and then transfer it to a 96-well ELISA plate, 50 μl / well, and incubate at 37°C for 45 min; (5) Wash the plate three times with PBST and pat dry; (6) Dilute the label antibody with blocking solution, add 50 μl / well to a 96-well ELISA plate, and incubate at 37°C for 30 min; (7) Wash the plate three times with PBST and pat dry; (8) Add TMB single-component colorimetric solution, 50 μl / well, and incubate at room temperature or 37°C for several minutes; (9) When the color develops to an appropriate depth (usually 5-10 min), add 50 μl / well of stop solution and tap the plate gently to mix. Use an ELISA reader to read the absorbance at 450 nm.

10. A broad-spectrum neutralizing nanobody targeting coronavirus S protein, characterized in that: It comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein CDR1 is: positions 31-35 of SEQ ID 11D; CDR2 is: positions 50-66 of SEQ ID 11D; and CDR3 is: positions 99-121 of SEQ ID 11D.

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