CN112225797B

CN112225797B - Monoclonal antibody for resisting SARS-CoV-2 nucleocapsid protein and application thereof

Info

Publication number: CN112225797B
Application number: CN202011019047.4A
Authority: CN
Inventors: 陆绍红; 孔庆明; 丁豪杰; 丁建祖; 高孟; 卓洵辉; 谢承作; 袁亚杰
Original assignee: Hangzhou Medical College
Current assignee: Hangzhou Medical College
Priority date: 2020-09-24
Filing date: 2020-09-24
Publication date: 2022-01-25
Anticipated expiration: 2040-09-24
Also published as: CN112225797A

Abstract

The invention discloses a monoclonal antibody of anti-SARS-CoV-2 nucleocapsid protein and its application. The monoclonal antibody comprises a light chain and a heavy chain, and is characterized in that the amino acid sequences of the variable region CDR1, CDR2 and CDR3 of the light chain are RASENIYSSLA, VATDLA and QHFWGTPWT respectively; the heavy chain variable region CDR1, CDR2 and CDR3 have amino acid sequences of TDHYII, EIYPGNGHTYNERFKG and SRYYGPFAYWG, respectively. The monoclonal antibody of the invention for resisting SARS-CoV-2 nucleocapsid protein is IgG1 subtype, and the affinity of the antibody can reach 5.28X 10^‑11The kit has the advantages of strong immunocompetence, good stability, high activity, strong affinity and the like, has strong detection specificity, high sensitivity and good accuracy, can be used in a kit of a full-automatic immunoassay analyzer, is also applied to immunochromatography test paper strips for detecting SARS-CoV-2 nucleocapsid protein, and can effectively promote the popularization and application of SARS-CoV-2 detection.

Description

Monoclonal antibody for resisting SARS-CoV-2 nucleocapsid protein and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a monoclonal antibody for resisting SARS-CoV-2 nucleocapsid protein and application thereof.

Background

The novel coronavirus pneumonia is a novel respiratory disease caused by severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2). SARS-CoV-2 is a seventh positive-stranded RNA virus capable of infecting humans with an envelope. The genomics identification of SARS-CoV-2 shows that it is a binuclear virus with 96% homology to RaTG13, but different from SARS-CoV. SARS-CoV-2 has a Receptor Binding Domain (RBD) structure similar to that of SARS-CoV. The functional Open Reading Frame (ORF) of SARS-CoV-2 comprises ORF1a and ORF1b, and the major structural proteins include spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N). According to prior studies, the M and E proteins are essential for viral assembly. The S protein is associated with viral adhesion to host cells, where the RBD structure of the S protein mediates the interaction with angiotensin enzyme 2. The S protein is localized to the surface of the viral particle and is highly immunogenic. One of the major structures of N protein viruses, involved in transcription and replication of viral RNA, functions to package the viral genome into virions and interfere with the cellular cycle. In many coronaviruses, including SARS-CoV, the N protein is highly immunogenic and is expressed in large quantities during infection. The S and N proteins are therefore potential antigens for COVID-19 serological diagnosis.

At present, COIVD-19 is mainly diagnosed by detecting viral RNA by real-time quantitative PCR (RT-PCR). Studies have shown that SARS-CoV-2 primarily infects the lower respiratory tract, as detected by nasopharyngeal swabs and samples of the bronchi. However, the collection of lower airway samples requires specialized equipment and skilled personnel. Previous studies show that the positive rate of sputum samples of patients confirmed by RT-PCR is 74.4% -88.9%; the positive rate of the nasal swab sample is 53.6-73.3%; pharyngeal swabs more than 8 days after onset have a very low positive rate. Since the main site of viral infection is in the lower respiratory tract, the different respiratory tract samples collected give rise to a high negative rate. Therefore, there is a strong need for an accurate and efficient detection method for evaluating patients with suspected symptoms.

The invention with publication number CN111187354A discloses a test paper for detecting SARS-CoV-2 antibody by colloidal gold capture method, comprising a bottom plate, a sample pad, a combination pad, a coating film and an absorption pad, wherein the sample pad, the combination pad, the coating film and the absorption pad are fixed on the bottom plate in sequence along the flowing direction of a liquid sample to be detected; the binding pad is loaded with gold-labeled protein, wherein the gold-labeled protein comprises nCOVAg 01; the envelope film is provided with a detection area and a quality control area, the detection area is loaded with a mouse anti-human IgM/IgG antibody, and the quality control area is loaded with a goat anti-rabbit polyclonal antibody; nCOVAg01 includes the recombinant antigen.

The invention with the publication number of CN111426844A discloses a novel fluorescence immunochromatographic test strip for joint detection of coronavirus SARS-CoV-2 IgG-IgM antibodies, which comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially connected end to end and pasted on the bottom plate, the combination pad is coated with SARS-CoV-2 structural protein-marker and goat anti-rabbit IgG-marker, and the nitrocellulose membrane is provided with a detection line T1 coated with mouse anti-human IgG monoclonal antibodies, a detection line T2 coated with mouse anti-human IgM monoclonal antibodies and a quality control line C coated with rabbit IgG.

Both of the above-mentioned prior arts are used for detecting whether human serum contains an antibody against SARS-CoV-2, and the in vivo antibody level is not used as a real-time diagnostic means because SARS-CoV-2 has been infected before.

Serological diagnosis is an accurate and efficient method for screening pathogens and has the advantage of good sample consistency. The monoclonal antibody provides powerful support for serological diagnosis, has the advantages of good specificity and high sensitivity in the serological diagnosis, is used for detecting whether a sample contains SARS-CoV-2, and can provide an accurate and rapid diagnosis means for prevention and control of novel coronavirus pneumonia.

The invention with publication number CN111303254A discloses an antibody against SARS-CoV-2, which specifically binds to the antigen; the antibody is a monoclonal antibody; the protein composed of any amino acid sequence shown in SEQ ID NO.2-SEQ ID NO.11 is used as an antigen, and is mixed with an adjuvant to prepare immunogen, and the immunogen is immunized with an animal to obtain an antibody. However, no specific information on antibodies is provided throughout this document, i.e. no corresponding antibody has actually been prepared in this prior art.

Disclosure of Invention

The invention provides a monoclonal antibody of anti SARS-CoV-2 nucleocapsid protein and its application.

A monoclonal antibody against SARS-CoV-2 nucleocapsid protein comprises light chain and heavy chain, wherein the amino acid sequences of the variable region CDR1, CDR2 and CDR3 of the light chain are RASENIYSSLA, VATDLA and QHFWGTPWT respectively; the heavy chain variable region CDR1, CDR2 and CDR3 have amino acid sequences of TDHYII, EIYPGNGHTYNERFKG and SRYYGPFAYWG, respectively.

Preferably, the monoclonal antibody has the amino acid sequence of the light chain variable region shown in SEQ ID No.4 and the amino acid sequence of the heavy chain variable region shown in SEQ ID No. 5.

Preferably, the monoclonal antibody is a full length antibody, a Fab antibody or a F (ab')₂An antibody.

The invention also provides a gene encoding the monoclonal antibody. Preferably, the gene sequence encoding the variable region of the light chain is shown as SEQ ID number 2; the gene sequence for coding the heavy chain variable region is shown as SEQ ID number 3.

The invention also provides the application of the monoclonal antibody in preparing a kit for detecting SARS-CoV-2. Preferably, in the application, the detection mode is serological detection.

The invention also provides a kit for detecting SARS-CoV-2, comprising the monoclonal antibody. Preferably, the kit is an ELISA detection kit or an immunochromatography diagnostic kit using an immunochromatography test strip. More preferably, the sample to be tested is venous blood.

The monoclonal antibody of the invention for resisting SARS-CoV-2 nucleocapsid protein is IgG1 subtype, and the affinity of the antibody can reach 5.28X 10^-11The kit has the advantages of strong immunocompetence, good stability, high activity, strong affinity and the like, has strong detection specificity, high sensitivity and good accuracy, can be used in a kit of a full-automatic immunoassay analyzer, is also applied to an immunochromatography test strip for detecting SARS-CoV-2 nucleocapsid protein, and can effectively promote the detection of SARS-CoV-2Popularization and application.

Drawings

FIG. 1 is a SDS-PAGE analysis of the SARS-CoV-2-N protein induced in example 1; m: and (3) protein Marker.

FIG. 2 is an SDS-PAGE analysis of the purified SARS-CoV-2-N protein of example 1.

FIG. 3 is an SDS-PAGE electrophoresis of the monoclonal antibody of example 2; m: protein Marker, 1: n10, 2: n15.

FIG. 4 is a graph showing the titer detection of monoclonal antibodies in example 3.

FIG. 5 is a graph showing the subtype identification of monoclonal antibodies in example 4.

FIG. 6 is a graph showing the affinity assay of the antibody in example 5.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out under conventional conditions. In the examples of the present invention, unless otherwise specified, reagents and consumables used therein are commercially available.

Example 1: preparation of SARS-CoV-2-N recombinant protein

The gene sequence of the N protein is shown as SEQ ID NO: 1, designing primers as follows:

N-F: 5'-GCCGGATCCATGTCTGATAATGGACCCCAAAA-3' (with)BamHI cleavage site);

N-R: 5'-GCCGTCGACAGGCCTGAGTTGAGTCAGCAC-3' (with)SalI enzyme cutting site),

the primers are adopted, PCR is carried out by taking artificially synthesized N protein gene as a template, and the product is usedBamHI andSalafter the restriction enzyme I is subjected to double enzyme digestion, the DNA fragment is inserted into a pET-28a (+) prokaryotic expression vector which is subjected to the same two enzyme digestion treatments, and a recombinant plasmid pET28a-SARS-CoV-2-N inserted with an N protein gene sequence is obtained.

The correct plasmid of pET28a (+) -SARS-CoV-2-N expression vector for sequencing verification was transformed into E.coli BL21, spread on LB plate containing 100. mu.g/ml kanamycin sulfate, cultured overnight at 37 ℃, single colony was picked up, cultured with 300ml LB medium containing the same concentration of kanamycin sulfate at 37 ℃ until OD600 reached about 0.6, and induced to express with IPTG at final concentration of 0.4-1 mM at 25-37 ℃ and 250rpm for 5-8 h. After induction, the culture was centrifuged at 4 ℃ and 7000rpm for 10min to collect the cells. Centrifuging 1mL of culture solution, adding a sample buffer solution, boiling for 10min, and centrifuging to remove bacterial debris. SDS-PAGE analysis is carried out on the supernatant, the SARS-CoV-2-N fusion protein is expressed in the form of inclusion bodies, and the result is shown in figure 1, which shows that the fusion protein with higher purity is obtained.

The bacterial precipitation is dissolved by inclusion body combined buffer solution, protein purification nickel column is used for purifying recombinant SARS-CoV-2-N protein, urea solutions with different concentrations are used for elution and collection, SDS-PAGE detection shows that polyacrylamide gel electrophoresis is carried out when 300mM imidazole is used for elution, and the result is shown in figure 2, and a single target protein band with higher concentration appears at a 45KDa position.

The purified recombinant protein was dialyzed with dialysis buffer at 4 ℃ and the dialysate was changed every 12 hours. Taking out dialyzed protein solution, concentrating with polyethylene glycol-20000, and storing at-20 deg.C.

Example 2: preparation of SARS-CoV-2-N monoclonal antibody

The recombinant antigen SARS-CoV-2-N protein is used as immunogen, and when it is first immunized, 50-100 microgram of recombinant antigen SARS-CoV-2-N protein and equal volume of complete Freund's adjuvant are co-emulsified, and then the multipoint injection immunization is implemented under the subcutaneous skin of BALB/c mouse. The second time and the third time, 50-100 mug/recombinant antigen SARS-CoV-2-N protein is emulsified together with equal volume of incomplete Freund's adjuvant, and the immune interval is 15 days. After three immunizations, blood was collected from the tail vein of the mice, and the sera were separated and assayed for titer against the recombinant antigen SARS-CoV-2-N protein by indirect ELISA.

50-100 mu g/recombinant antigen SARS-CoV-2-N protein was injected into the abdominal cavity of mice 7d before cell fusion for boosting. Preparing feeder layer cells one day before fusion, taking one healthy mouse, separating spleen, preparing cell suspension, uniformly spreading the cell suspension in a cell plate, standing and culturing overnight to allow the cell to adhere to the wall. Spleen of immunized mice was isolated in sterile environment on the day of fusion, and cell suspension was prepared and mixed well in IMDM medium. The well-conditioned SP2/0 cells were compared with mouse splenocytes 1: 5-1: 10 mix, wash the mixed cells 3 times in serum-free IMDM medium preheated at 37 deg.C, discard the wash, add PEG1500 at 37 deg.C to fuse the cells. After centrifugation, 80mL of culture medium (IMDM 62mL, HAT 2mL, fetal bovine serum 16 mL) was added to the fused cells, mixed gently and mixed, and then added to a 96-well plate with feeder cells, and cultured in an incubator. In 11d fusion, titer detection was performed on the supernatants of the culture wells in which hybridoma cells grew by indirect ELISA, and two hybridoma cell lines with the highest positive values, N10 and N15, were obtained. The ELISA positive wells were subcloned 3 times by limiting dilution, and hybridoma cell lines were expanded and stored in liquid nitrogen.

Injecting paraffin oil into the abdominal cavity of BALB/c mouse of 8-10 weeks old, inoculating the recovered hybridoma cells after one week, wherein the inoculation amount is 1 × 10⁶A/only. When the abdomen of the mouse is obviously enlarged, the ascites of the mouse is extracted by a sterile syringe, and the ascites is collected after centrifugation. The monoclonal antibody is precipitated and crude extracted by saturated ammonium sulfate solution, and SDS-PAGE electrophoresis is carried out to identify the purity of the antibody, the result is shown in figure 3, two monoclonal antibodies with numbers N10 and N15 clearly present two target bands at 55KDa and 25KDa, which shows that the monoclonal antibody against SARS-CoV-2-N with higher purity is successfully prepared.

Example 3: indirect ELISA method for detecting monoclonal antibody titer

The SARS-CoV-2-N protein purified in example 1 was coated at 1. mu.g/ml and 100. mu.l/well on an ELISA plate and incubated overnight at 4 ℃. The ELISA plates were washed with PBST, 200. mu.l/well of 5% nonfat dry milk was added, and blocked at 37 ℃ for 2 h. After washing, the monoclonal antibody of example 2 was added at a dilution of concentration by two at 1: 500, 1: 1000, 1: 2000, 1: 4000, 1: 8000, 1: 16000, 1: 32000, 1: 64000, 1: 128000, 1: 256000, 1: 512000, 1: 1024000, 100. mu.l/well, and incubated at 37 ℃ for 1 hour. After washing the plate, the appropriate dilution of horseradish peroxidase-labeled goat anti-mouse antibody was added and incubated at 37 ℃ for 1 h. Adding 100 mul/hole TMB chromogenic substrate for reaction for 10min, adding 50 mul 2mol/L sulfuric acid stop solution for stopping reaction, and reading OD value of each hole at 450nm by an enzyme-labeling instrument. The detection result is shown in figure 4, the titer of the anti-SARS-CoV-2-N protein monoclonal antibodies with the numbers N10 and N15 can reach 512000 and 1024000 respectively, and the antibody sensitivity is high.

Example 4: monoclonal anti-SARS-CoV-2-N protein subtype assay

The types of the monoclonal antibodies were identified using an ELISA rapid qualitative kit (reagent No. 062020, Jiangsu Bora enzyme immunoassay Biotech Co., Ltd.), and the results are shown in FIG. 5, and the anti-SARS-CoV-2-N monoclonal antibodies of Nos. N10 and N15 were determined by the heavy chain identification, and both monoclonal antibodies were of the IgG1 subclass.

Example 5: antibody affinity assay

Diluting recombinant antigen SARS-CoV-2-N protein, labeling with biotin, desalting with desalting column, wetting the end of SA sensor, and curing in the above antigen. The monoclonal antibody against SARS-CoV-2-N to be detected is diluted with PBS to the concentration of 0.3125nmol/L, 0.625nmol/L, 1.25nmol/L, 2.5nmol/L, 5nmol/L, 10nmol/L, 20nmol/L, and the total volume of each group of samples is 200 muL. Placing the diluted anti-SARS-CoV-2-N monoclonal antibody sample to be detected and blank control in a 96-well plate, and adopting ForteBio Octet Red biomolecule interaction instrument to detect the sample to be detected. The obtained data were analyzed by using Octet data analysis software CFR Part 11 Version 6.x, and affinity constants KD of the anti-SARS-CoV-2-N monoclonal antibodies with numbers N10 and N15 were obtained as 3.39X 10, respectively, after data fitting^-11And 5.28X 10^-11(FIG. 6).

Example 6: sequence analysis of monoclonal antibody encoding gene

The hybridoma cell RNA of the anti-SARS-CoV-2-N monoclonal antibody with the high affinity number N15 is extracted and reverse transcribed into cDNA. 2 pairs of degenerate primers are designed according to the constant regions of the heavy chain and the light chain of the antibody, PCR amplification is respectively carried out, and the target fragment is recovered and sequenced. The amino acid sequence of the light chain variable region obtained by analysis is shown as SEQ ID number 2, and the gene sequence of the coded light chain variable region is shown as SEQ ID number 4; the amino acid sequence of the heavy chain variable region is shown as SEQ ID number 3, and the gene sequence for coding the heavy chain variable region is shown as SEQ ID number 5.

Sequence listing

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Claims

1. A monoclonal antibody against SARS-CoV-2 nucleocapsid protein, comprising a light chain and a heavy chain, characterized in that the amino acid sequences of the variable region CDR1, CDR2 and CDR3 of the light chain are RASENIYSSLA, VATDLA and QHFWGTPWT respectively; the heavy chain variable region CDR1, CDR2 and CDR3 have amino acid sequences of TDHYII, EIYPGNGHTYNERFKG and SRYYGPFAYWG, respectively.

2. The monoclonal antibody of claim 1, wherein the light chain variable region amino acid sequence is set forth in SEQ ID No.4 and the heavy chain variable region amino acid sequence is set forth in SEQ ID No. 5.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a full length antibody, a Fab antibody, or a F (ab')₂An antibody.

4. A gene encoding the monoclonal antibody according to any one of claims 1 to 3.

5. The gene of claim 4, wherein the gene encoding the light chain variable region has the sequence shown in SEQ ID No. 2; the gene sequence of the coding heavy chain variable region is shown in SEQ ID NO. 3.

6. Use of the monoclonal antibody of any one of claims 1 to 3 in the preparation of a kit for detecting SARS-CoV-2.

7. The use of claim 6, wherein the assay format is a serological assay.

8. A kit for detecting SARS-CoV-2, comprising the monoclonal antibody according to any one of claims 1 to 3.

9. The kit of claim 8, wherein the kit is an ELISA detection kit or an immunochromatographic diagnostic kit using an immunochromatographic test strip.

10. The kit of claim 9, wherein the sample to be tested is venous blood.