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CN115093477A - Monoclonal antibody for resisting N terminal region of novel coronavirus nucleoprotein and application thereof - Google Patents

Monoclonal antibody for resisting N terminal region of novel coronavirus nucleoprotein and application thereof Download PDF

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CN115093477A
CN115093477A CN202210630134.6A CN202210630134A CN115093477A CN 115093477 A CN115093477 A CN 115093477A CN 202210630134 A CN202210630134 A CN 202210630134A CN 115093477 A CN115093477 A CN 115093477A
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CN115093477B (en
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方剑秋
冯晓燕
谭金凤
张贺秋
王维
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Zhejiang Orient Gene Biotech Co Ltd
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Abstract

The invention discloses a monoclonal antibody for resisting a novel coronavirus nucleoprotein N-terminal region and application thereof. In the monoclonal antibody, the amino acid sequences of a light chain CDR1, a CDR2 and a CDR3 are respectively shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4; the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively. The monoclonal antibody for resisting the N terminal region of the novel coronavirus nucleoprotein has good specificity and affinity, and can be applied to preparation of products for detecting the novel coronavirus and products for treating patients with the novel coronavirus.

Description

Monoclonal antibody for resisting N terminal region of novel coronavirus nucleoprotein and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to a monoclonal antibody for resisting a novel coronavirus nucleoprotein N-terminal region and application thereof.
Background
The detection of the specific antigen of the novel coronavirus aims at the self-synthesized antigen of the novel coronavirus and is direct evidence that a patient is infected by a pathogen. The nucleoprotein and spinous process glycoprotein of the novel coronavirus have strong antigenicity, are main antigens for inducing host immune response and are main targets for antigen detection. The conservation of the nucleoprotein amino acid sequence of the novel coronavirus is higher, so that the novel coronavirus is favored by researchers of antigen detection reagents. The existing novel coronavirus antigen detection is mainly based on a double-antibody sandwich principle, and the establishment of an antigen detection reagent needs a high-affinity and high-specificity paired antibody, so that the preparation of the high-affinity and high-specificity antibody is the key for research and development of the novel coronavirus antigen detection reagent.
In addition, the targeted therapy of diseases refers to that after entering the body, the therapeutic drug is specifically combined with a target organ, a target cell or a target pathogen through a specific guiding mechanism, so that the drug can more directly play a role to achieve the aim of targeted therapy. The targeted delivery of the drug is utilized for treatment, so that the concentration of the drug at the focus part can be greatly improved, the curative effect is improved, and the toxic and side effects are reduced. Targeted therapies mainly include biological targeting and physicochemical targeting. Biological targeted therapy is the drug delivery therapy of a specific target by coupling a targeting molecule with a drug. In recent years, the monoclonal antibody with high affinity and specificity is coupled with a therapeutic drug to obtain a targeting drug for treatment, which has good treatment effect and becomes a hotspot and a main field of biological targeting drug delivery treatment. Monoclonal antibodies with high affinity and specificity are key factors for the success of such biological targeted therapies. Therefore, obtaining monoclonal antibodies with high affinity and specificity is particularly important for biological targeted therapy.
At present, the research on the treatment of the novel coronavirus infection mainly focuses on oral drug therapy and neutralizing antibody therapy, and the research on biological targeted drug therapy by using a monoclonal antibody with high affinity and specificity for targeting the novel coronavirus pathogen is still lacked.
Disclosure of Invention
Therefore, the invention aims to provide a monoclonal antibody for resisting the N terminal region of the novel coronavirus nucleoprotein, which is prepared from the fused hybridoma cell strain, and the monoclonal antibody obtained through experiments has good specificity and affinity, and can be used for detecting the novel coronavirus nucleoprotein or preparing a product for treating novel coronavirus infection.
Accordingly, one aspect of the present invention relates to a monoclonal antibody or antigen-binding fragment thereof against a novel coronavirus nucleoprotein, comprising a light chain variable region comprising CDR1, CDR2 and CDR3 and a heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein,
the amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.2 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.2, or the amino acid sequence containing the sequence;
the amino acid sequence of the light chain CDR2 is the sequence shown in SEQ ID NO.3 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.3, or the amino acid sequence containing the sequence;
the amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.4 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.4, or the amino acid sequence containing the sequence;
the amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.6 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.6, or the amino acid sequence containing the sequence;
the amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.7 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.7, or the amino acid sequence containing the sequence;
the amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.8 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.8, or the amino acid sequence containing the sequence.
In a further aspect, the invention also relates to a monoclonal antibody or an antigen binding fragment thereof, wherein the light chain variable region sequence is represented by SEQ ID No.1 and the heavy chain amino acid sequence is represented by SEQ ID No. 5.
The invention also relates to the monoclonal antibody or the antigen binding fragment thereof, wherein the antibody or the antigen binding fragment is Fab fragment, Fab 'fragment, F (ab') 2 Fragments, single chain antibodies or humanized antibodies, which antibodies or antigen binding fragments retain the variable regions of the light and heavy chains and are therefore capable of recognizing and binding to the N-terminal region of the novel coronavirus nucleoprotein.
Furthermore, the present invention relates to a nucleic acid molecule comprising a nucleic acid encoding the above-described antibody or antigen-binding fragment thereof, and an expression vector comprising the above-described nucleic acid molecule, said expression vector being capable of expressing the above-described antibody or antigen-binding fragment thereof. The invention also relates to a recombinant comprising the nucleic acid molecule or the expression vector, which can produce the antibody or the antigen-binding fragment thereof.
In another aspect, the invention relates to a monoclonal antibody hybridoma cell strain for resisting novel coronavirus nucleoprotein, wherein the monoclonal antibody hybridoma cell strain secretes the monoclonal antibody. Further, the invention relates to a monoclonal antibody hybridoma cell strain for resisting novel coronavirus nucleoprotein, wherein the monoclonal antibody hybridoma cell strain is a mouse hybridoma cell 2A998135 with the preservation number of CGMCC No. 45129.
In a further aspect, the present invention relates to the use of the monoclonal antibody or antigen-binding fragment thereof as described above for the preparation of a product for the detection of a novel coronavirus or for the treatment of a novel coronavirus infection. Further, the present invention relates to a kit for detecting a novel coronavirus, which comprises the above monoclonal antibody or an antigen-binding fragment thereof for recognizing and binding to the N-terminal region of a novel coronavirus nucleoprotein.
Biological material preservation instructions
The monoclonal antibody hybridoma of the invention: the mouse hybridoma cell 2A998135 is preserved in China general microbiological culture Collection center (CGMCC), the registration number of the preservation center is CGMCC No.45129, and the preservation date is as follows: 03 and 10 months in 2022. The addresses of the China general microbiological culture Collection center are as follows: west road No.1, north zhou, chaoyang, beijing, No.3, zip code 100101.
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FIG. 1 is an SDS-PAGE electrophoresis showing prokaryotic expression of the novel coronavirus nucleoprotein, wherein each symbol is: m is Marker; 1 is the N terminal region of the purified pColdi-NP-N recombinant plasmid expression nucleoprotein; 2 is purified pGEX-NP-N recombinant plasmid expression nucleoprotein N terminal region.
FIG. 2 is a graph showing the result of the subclass identification of the monoclonal antibody directed against the N-terminal region of the novel coronavirus nucleoprotein, and the identified antibody subclass is IgG 1.
FIG. 3 is a graph showing the results of N-terminal monoclonal antibody-ELISA immunoassay of nucleoprotein against novel coronavirus nucleoprotein, wherein NP is a novel coronavirus nucleoprotein full-length protein expressed by eukaryotic cells; TB is prokaryotic expression mycobacterium tuberculosis protein; EB is EB virus protein of prokaryotic expression; b19 is human parvovirus B19 protein expressed by pronucleus. The results show that the monoclonal antibody against the novel coronavirus nucleoprotein can specifically recognize the nucleoprotein of the novel coronavirus.
Detailed Description
The invention aims to provide a monoclonal antibody for resisting a novel coronavirus nucleoprotein N-terminal region, which is prepared by using a fused hybridoma cell strain.
Through the analysis of the dominant epitope of the novel coronavirus by the inventor, the segment of the 20 th-215 th amino acid in the N terminal region of the novel coronavirus nucleoprotein is considered as the dominant epitope antigen segment, so that the segment is selected to express, immunize a mouse and screen a monoclonal antibody hybridoma cell strain. After screening, a mouse hybridoma cell expressing a monoclonal antibody with high specificity and affinity is obtained and named as 2A 998135. The cell is cultured in China general microbiological culture Collection center (CGMCC) at 3 months and 10 days in 2022, with the preservation number of CGMCC No.45129 and the preservation date of 2022, 03 months and 10 days. The preservation address is No.3 of Xilu No.1 of Beijing, Chaoyang, Beijing, Chao code 100101.
The inventor carries out sequencing on the monoclonal antibody generated by the strain of mouse hybridoma cell, wherein the sequence of the variable region of the monoclonal antibody light chain is 109 amino acids, and the sequence is as follows: DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGV PARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWSS (SEQ ID NO.1) in which the underlined sequences are CDR1, CDR2 and CDR3 in that order, wherein CDR1 is located between 27 and 36aa and the amino acid sequence is KSVSTSGYSY (SEQ ID NO. 2); CDR2 is located at 54-56aa, and the amino acid sequence is LVS (SEQ ID NO. 3); CDR3 is located at 93-100aa, and has the amino acid sequence QHIRELR (SEQ ID NO. 4). The heavy chain variable region amino acid sequence is 110 amino acids, and the sequence is as follows: VESGPELKKPGETVKISCKASGYTFTNYAMNWVKQAPGKGLKWMGYINTYTGEPTYTDD FKGRFAFSLETSASTAYLQINNLKNEDMATYFCARGGSYADWGQGTTLTVS (SEQ ID NO.5) in which the underlined sequences are CDR1, CDR2 and CDR3 in that order, wherein CDR1 is located between 22-29aa and the amino acid sequence is GYTFTNYA (SEQ ID NO. 6); CDR2 is located at 47-54aa, and has the amino acid sequence of INTYTGEP (SEQ ID NO. 7); CDR3 is located at 93-100aa, and has amino acid sequence of ARGGSYAD (SEQ ID NO. 8). The monoclonal antibody has high specificity and affinity to the new coronavirus nucleoprotein.
It is well known that antibody heavy and light chain CDR regions are important sequences that recognize and bind to the corresponding antigen, and that 1 or 2 conservative amino acid substitutions in the amino acid sequence do not generally alter or slightly alter the properties of the protein. Thus, monoclonal antibodies or antigen-binding fragments thereof obtained by conservative amino acid substitutions of 1 or 2 in the light chain CDR1 and/or light chain CDR2 and/or light chain CDR3 and/or heavy chain CDR1 and/or heavy chain CDR2 and/or heavy chain CDR3 are still capable of recognizing and binding to novel coronavirus nucleoproteins. Conservative amino acid substitution refers to the substitution of an amino acid in a protein by another chemically similar amino acid, such as the substitution of aromatic amino acids Phe, Trp and Tyr, the substitution of aliphatic amino acids Ala, Gly, Leu, Ile and Val, the substitution of polar amino acids Gln and Asn, the substitution of basic amino acids Lys, Arg and His, the substitution of acidic amino acids Asp and Glu, and the substitution of hydroxy amino acids Ser and Thr.
Furthermore, it is well known in the art that in an antibody or antigen-binding fragment thereof, a framework region is formed outside each CDR of a light chain or a heavy chain, and a small number of amino acids, for example 1 or 2 amino acids, are added between the CDR sequence and the framework region sequence, so that the steric structure of the antibody or antigen-binding fragment thereof is less affected, and thus the corresponding antigen can still be recognized and bound. Therefore, the light chain CDR1 sequence of the monoclonal antibody or antigen binding fragment thereof of the present invention can be a sequence comprising the above sequence in addition to the sequence shown in SEQ ID NO.2 or a sequence having 1 or 2 conservative amino acid substitutions compared thereto. Similarly, the light chain CDR2 sequence of the monoclonal antibody or antigen binding fragment thereof of the present invention can be a sequence comprising the above sequence in addition to the sequence shown in SEQ ID NO.3 or a sequence having 1 or 2 conservative amino acid substitutions compared thereto; the light chain CDR3 sequence of the monoclonal antibody or antigen binding fragment thereof of the invention can be a sequence containing the sequence as shown in SEQ ID NO.4 or a sequence with 1 or 2 conservative amino acid substitutions compared with the sequence; the heavy chain CDR1 sequence of the monoclonal antibody or antigen binding fragment thereof of the present invention can be a sequence comprising the above sequence in addition to the sequence shown in SEQ ID NO.6 or a sequence having 1 or 2 conservative amino acid substitutions compared thereto, and the heavy chain CDR2 sequence of the monoclonal antibody or antigen binding fragment thereof of the present invention can be a sequence comprising the above sequence in addition to the sequence shown in SEQ ID NO.7 or a sequence having 1 or 2 conservative amino acid substitutions compared thereto; the heavy chain CDR3 sequence of the monoclonal antibody or antigen binding fragment thereof of the present invention can be a sequence containing the above sequence in addition to the sequence shown in SEQ ID NO.8 or a sequence having 1 or 2 conservative amino acid substitutions compared with the sequence.
Various antibody fragments, i.e., antigen-binding fragments, such as, but not limited to, Fab ', F (ab') 2 . Fab fragments are the antigen-binding regions of the antibody structure, consisting of an intact variable VH and constant CH1 domains (Fd segment) of the light and heavy chains, both of which have a constant and a variable domain, with disulfide bonds between the light and heavy chains. Antigen binding fragments can be prepared, for example, by degrading antibody IgG into two Fab fragments and one Fc fragment (crystalline fragment) after digestion with papain. Degradation of antibody IgG to an F (ab') 2 Fragment and a pFC 'fragment, F (ab') 2 The fragments were further reduced to form two Fab' fragments. The antigen binding fragment can be applied to the preparation of products for detecting novel coronavirus nucleoprotein and therapeutic products for treating novel coronavirus patients.
Single chain antibodies (scFv) can also be prepared from the monoclonal antibodies of the invention by techniques known in the art. The single-chain antibody is formed by connecting an antibody heavy chain variable region and an antibody light chain variable region through a short peptide linker of a plurality of amino acids, has only one chain, and is an artificially synthesized antibody. The length and amino acid composition of the short peptide linker are well known in the art, and the short peptide linker that can be used against the monoclonal antibody of the present invention can be determined by simple repeated experiments. Single-chain antibodies can be expressed, for example, in E.coli by genetic engineering techniques. The single-chain antibody has the advantages of small molecular weight, strong penetrating power, weak antigenicity and the like, and can be applied to the detection of novel coronavirus nucleoprotein and the diagnosis and treatment products of novel coronavirus patients.
The light chain constant region and the heavy chain constant region of the monoclonal antibody of the invention can be replaced by the amino acid sequence of the human antibody by the prior art in the field, so that the mouse monoclonal antibody of the invention can be humanized and transformed into a humanized antibody for antibody therapy of human patients with new coronavirus, and the immune side reaction of the mouse antibody to the human body can be reduced. Therefore, the humanized monoclonal antibody of the invention can be applied to detection of novel coronavirus nucleoprotein, diagnosis and treatment products of novel coronavirus patients.
The skilled person can design and synthesize the nucleic acid molecule encoding the variable region of the monoclonal antibody against the novel coronavirus nucleoprotein based on the amino acid sequence of the variable region, and can insert the synthesized nucleic acid molecule into a nucleic acid vector to construct an expression vector, wherein the expression vector can express the monoclonal antibody or the antigen-binding fragment thereof against the novel coronavirus nucleoprotein. The skilled person can also introduce the synthesized nucleic acid molecule or the constructed expression vector into an organism such as cells, bacteria, yeast, etc. to obtain a recombinant, and express and produce the antibody or the antigen-binding fragment thereof of the present invention via the recombinant, and the antibody or the antigen-binding fragment thereof thus expressed can bind and recognize the novel coronavirus nucleoprotein, so that the nucleic acid molecule, the expression vector and the recombinant are within the scope of the claims of the present invention. And the above techniques are well known in the art and can be performed by those skilled in the art without inventive effort.
As described above, the antibody or antigen-binding fragment thereof of the present invention can recognize and bind to the novel coronavirus nucleoprotein, and thus can be used to prepare a kit for detecting the novel coronavirus nucleoprotein, which can be any kit that utilizes the binding reaction of the antibody or antigen-binding fragment thereof of the present invention with the novel coronavirus nucleoprotein, such as, but not limited to, colloidal gold immunochromatography, fluorescence immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, immunohistochemistry-type kits.
Since the monoclonal antibody or antigen-binding fragment thereof of the present invention is capable of specifically binding to a neocoronavirus nucleoprotein, the antibody or antigen-binding fragment thereof or humanized antibody can be used for the preparation of a product for the treatment of a neocoronavirus infection, such as a medicament. The therapy may be, for example and without limitation, targeted therapy in which the monoclonal antibody or antigen-binding fragment thereof or humanized antibody of the present invention is conjugated to a novel coronavirus therapeutic drug, and the conjugated drug is targeted to the novel coronavirus through the antibody or antigen-binding fragment thereof or humanized antibody of the present invention, thereby enhancing the therapeutic effect.
In order to explain the technical content, the achieved objects and effects of the technical solution in detail, the following description is given with reference to the specific embodiments.
Example 1: preparation of antigens for immunization and antigens for screening
According to the novel coronavirus nucleoprotein amino acid sequence disclosed in Genebank and 419 amino acids in the total length, BIOSUN bioinformatics software is adopted to analyze the nucleoprotein amino acid sequence, firstly, the full-length sequence is input, then, B cell epitope and signal peptide analysis functions are utilized, according to the strength of hydrophobicity and the existence of signal peptide for analyzing the distribution condition of epitope, a stronger hydrophobic region exists at the position of amino acid 220-230 through analysis, and in addition, the analysis result shows that 1-19 amino acids are signal peptide sequences. Therefore, we judge the 20-215aa segment of the novel coronavirus nucleoprotein as the dominant epitope antigen segment, and therefore choose to prepare the 20-215aa dominant epitope antigen segment as an immunogen in this example.
The encoding gene of the nucleoprotein 20-215aa dominant epitope antigen is synthesized by Beijing Nosai biotechnology limited, the encoding gene of the 20-215aa dominant epitope antigen is respectively inserted into pColdi plasmid and pGEX-4T-1 which are also subjected to double enzyme digestion by BamHI and EcoRI by adopting BamHI and EcoRI enzyme digestion sites through double enzyme digestion, and the pColdi-NP-N recombinant plasmid and pGEX-NP-N recombinant plasmid are obtained.
Transforming the recombinant expression plasmid with correct sequencing into BL21 competent cells, selecting a single colony in 3mL LB liquid culture medium containing ampicillin sodium, carrying out shake culture at 37 ℃ overnight, inoculating the single colony in 250mL fresh LB liquid culture medium the next day, carrying out culture at 37 ℃ and 160rpm for 4h to logarithmic phase, adding 150. mu.l of 1mol/L IPTG induction liquid, and inducing at 15 ℃ for 12-14 h. Centrifuging at 4 deg.C and 6000rpm for 10min to collect induced thallus; resuspending the thallus with 25mmol/L Tris-HCl (pH8.5), and performing ice bath ultrasound; the supernatant was collected by centrifugation at 12000rpm for 10min at 4 ℃ and stored. SDS-PAGE gel electrophoresis is carried out, and the dominant epitope antigens of the 20-215aa of the nuclear protein are mainly expressed in the supernatant in a soluble form.
The pColdi-NP-N recombinant plasmid expression antigen is purified by a Ni column, the pGEX-NP-N recombinant plasmid expression antigen is purified by a GST column, protein peaks are respectively collected, SDS-PAGE electrophoresis is carried out to analyze and purify products, the expression of the purified target protein is verified, and the result is shown in figure 1. The molecular weight and purity of the purified nucleoprotein 20-215aa dominant epitope antigen are analyzed by using Gel-ProR Analyzer Version 3.0 software, the molecular weight of the nucleoprotein 20-215aa dominant epitope antigen expressed by the pColdi-NP-N recombinant plasmid is 28.10kDa, the molecular weight is consistent with that shown by an electrophoresis chart, the purity is 96.23 percent, and the requirement of immunogen purity is met. The molecular weight of the pGEX-NP-N recombinant plasmid expression nucleoprotein 20-215aa dominant epitope antigen is 49.80kDa, the molecular weight is consistent with that shown by an electrophoretogram, the purity is 95.76 percent, and the pGEX-NP-N recombinant plasmid expression nucleoprotein dominant epitope antigen can be used as an antigen for detection.
Example 2: establishment of hybridoma cell strain
Taking the purified pColdi-NP-N recombinant plasmid expression nucleoprotein 20-215aa dominant epitope antigen as an immunogen, and adopting an 8-week BALB/c female mouse, wherein the dominant epitope antigen is added with equivalent Freund complete adjuvant back and an intraperitoneal injection mouse (50 mu g/mouse); the same dose was administered at 2 nd and 3 rd week, and splenocytes were taken 3 days later for fusion.
SP20 myeloma cells were revived and cultured until they were in logarithmic growth phase. Taking immune BALB/c mouse, removing eyeball to collect blood and supply positive control serum, killing mouse at the same time of cervical dislocation, disinfecting body surface with 75% alcohol for 3-5min, taking spleen, and preparing spleen cell suspension.
Taking the spleen cells and myeloma cells according to the ratio of 5: 1, mixing uniformly in a serum-free DMEM medium, centrifuging at 1500rpm for 5 minutes, fully absorbing supernatant, lightly shaking the bottom of a centrifuge tube, vibrating cells, adding 1mL of preheated 50% PEG fused cells within 45-60 seconds, gently shaking uniformly while adding, standing for 90 seconds after adding, adding the serum-free DMEM medium to stop fusion (1 mL in the first minute, 2mL in the second minute and 8mL in the third minute), standing for 10min at 37 ℃, centrifuging at 1500rpm for 5 minutes, and settlingThe starch was suspended in HAT medium, and the suspension was dispensed into 96-well feeder cells-containing cell plates and cultured in a 5% CO2 cell culture chamber at 37 ℃. After 5 days of culture in a cell culture box, the HAT culture medium is used for changing the culture medium once, the HAT culture medium is used for changing the culture medium on the 10 th day, and when the fused cells cover 10% -50% of the bottom of the hole, the indirect ELISA method is adopted for screening positive clones. Carbonate coating buffer solution is used for diluting pGEX-NP-N recombinant plasmid expression nucleoprotein 20-215aa dominant epitope antigen, the concentration is 2.5 mu g/ml, each hole is coated with 100 mu l, and the temperature is kept overnight at 4 ℃; washing the plate with washing solution for 2 times, 200. mu.l/well; adding 110 mu l/hole sealing liquid, sealing for 6 hours at room temperature; the plate was washed 5 times with 200. mu.l/well of wash solution. ② after adding 200 mul sample diluent into each hole, respectively adding 10 mul cell culture supernatant, incubating for 30min at room temperature, and discarding the solution. ③ wash the plate with 1 Xwash solution, 300. mu.L per well, and repeat the washing 5 times. And (3) placing the washed ELISA plate on water-absorbing paper upside down, and beating forcefully to remove redundant washing liquid. And fourthly, adding 100 mul/hole HRP marked anti-mouse IgG antibody, and incubating for 20min at room temperature. Fifthly, washing the plate, and the operation is the same as the step 3. Sixthly, adding the substrate solution prepared freshly, 100 mu l/hole, and incubating for 10 minutes at room temperature in a dark place. Adding 2M H 2 SO 4 The reaction was stopped with 50. mu.L/well of stop solution. And determining OD value of each hole by setting the detection wavelength of 450nm by using an enzyme-labeling instrument, and reading within 10 minutes after termination.
A total of 17 positive clones were obtained, and among them, the clone with the highest detection value (OD450nm ═ 3.522) was selected for subsequent experiments and was designated mouse hybridoma cell 2a 998135.
Example 3: monoclonal antibody preparation and subtype analysis thereof
2A998135 hybridoma cells, using 10% fetal bovine serum in 1640 medium culture. Each BALB/c male mouse was intraperitoneally injected with 0.5mL of liquid paraffin. After 10 days, the cells were harvested and resuspended in 10mL of physiological saline, and 0.5mL (cell density approximately 1X 107 cells/mL) was injected intraperitoneally into each mouse. After 2 weeks, ascites was collected.
Antibody Purification was carried out using a Melon Gel Monoclonal IgG Purification Kit (Thermo Co.), and the purified antibody was stored at-20 ℃ after being dispensed.
The subtype of the antibody is identified by adopting a Pierce pad Isotyping Kit-Mouse Kit, firstly, the antibody is diluted to be 100ng/mL by using a sample diluent, then, 150 mu l of diluted antibody is added into each hole, and the result is observed and recorded after 5-10 min. The results showed that this monoclonal antibody was a mouse IgG1 subtype, as shown in fig. 2.
Example 4: monoclonal antibody variable region sequencing
Culturing mouse hybridoma 2A998135, extracting hybridoma total RNA by Trizol method, reverse transcribing cDNA by High Capacity cDNA Rever Transcription Kit of Thermo Fisher company, designing and synthesizing heavy and light chain primer of the antibody by Beijing Populaceae biotechnology limited according to mouse monoclonal antibody primer sequence in recombinant antibody (science publishing Co., Shenbei Prateness edition, published 2005), PCR amplifying (amplification program: preheating at 95 ℃ for 1min, performing 30 cycles (30 sec at 95 ℃, 30 sec at 58 ℃, 45 sec at 72 ℃) and finally extending at 72 ℃ for 5min), connecting PMD18-T vector, expressing Escherichia coli HB109, selecting positive clone and sequencing. The determined sequences were aligned on the BLAST website (https:// www.ncbi.nlm.nih.gov/igblast /) to analyze the mouse-derived monoclonal antibody CDR region sequences.
After sequence analysis, the amino acid sequence of the light chain variable region is 109 amino acids, and the sequence is as follows: DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGV PARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWSS (SEQ ID NO.1) with the underlined sequences being CDR1, CDR2 and CDR3 in that order, wherein CDR1 is located at 27-36aa and the amino acid sequence is KSVSTSGYSY (SEQ ID NO. 2); CDR2 is located at 54-56aa, and the amino acid sequence is LVS (SEQ ID NO. 3); CDR3 is located at 93-100aa, and has the amino acid sequence QHIRELR (SEQ ID NO. 4).
The heavy chain variable region amino acid sequence is 110 amino acids, and the sequence is as follows: VESGPELKKPGETVKISCKASG YTFTNYAMNWVKQAPGKGLKWMGYINTYTGEPTYTDD FKGRFAFSLETSASTAYLQINNLKNEDMATYFCARGGS YADWGQGTTLTVS (SEQ ID NO.5) in which the underlined sequences are CDR1, CDR2 and CDR3 in that order, wherein CDR1 is located between 22-29aa and the amino acid sequence is GYTFTNYA (SEQ ID NO. 6); CDR2 is located at 47-54aa, and has the amino acid sequence of INTYTGEP (SEQ ID NO. 7); CDR3 is located at 93-100aa, and has amino acid sequence of ARGGSYAD (SEQ ID NO: 1)ID NO.8)。
Example 5: immunodetection of novel coronavirus nucleoproteins by monoclonal antibodies
The immunodetection performance of the prepared monoclonal antibody on the novel coronavirus nucleoprotein is detected by adopting an indirect enzyme-linked immunosorbent assay method. In order to examine the specificity and affinity of the monoclonal antibody assay of the present invention, the novel coronavirus NP protein (cat HDS-5056), Mycobacterium tuberculosis TB protein (cat HDS-A5040), EB virus protein (cat HDS-M1001) and human parvovirus B19 protein (cat HDS-M1009) from Handson Biotech, Inc., Qingdao were simultaneously assayed at the time of the experiment.
Diluting each protein to a concentration of 2.5. mu.g/mL with carbonate coating buffer, coating each well with 100. mu.l, and standing overnight at 4 deg.C; washing the plate with washing solution 2 times, 200. mu.l per well; adding 100 mul of sealing liquid into each hole, and sealing for 6 hours at room temperature; washing the plate with washing solution 5 times, 200. mu.l per well; diluting the novel coronavirus NP protein monoclonal antibody to the concentrations of 100, 50, 25, 12.5, 6.25, 1.0, 0.5, 0.25, 0.125, 0.0625 and 0.03125ng/mL by using an antibody diluent, loading 100 mu l of the antibody diluent in each well and setting a blank well (100 mu l of the antibody diluent); incubating at 37 ℃ for 30 min; washing the plate with washing solution 5 times, 200. mu.l per well; incubating goat anti-mouse secondary antibody marked by HRP for 20min at 37 ℃; washing the plate with washing solution 5 times, 200. mu.l per well; adding a freshly prepared substrate solution, incubating at 37 ℃ for 10 minutes in 100. mu.l per well; add 50. mu.l of 2M H per well 2 SO 4 The reaction was terminated, and the absorbance of each well was measured at a wavelength of 450nm using a microplate reader, the results of which are shown in FIG. 3. FIG. 3 shows that the prepared monoclonal antibody can specifically recognize novel coronavirus nucleoprotein, and does not recognize other proteins, which indicates that the prepared monoclonal antibody has very high specificity. And the minimum effective concentration of the prepared monoclonal antibody is 0.03125ng/mL, which shows that the prepared monoclonal antibody has very high affinity to the novel coronavirus nucleoprotein.
Sequence listing
<110> Zhejiang Oriental Gene biologicals GmbH
<120> monoclonal antibody for resisting N terminal region of novel coronavirus nucleoprotein and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 109
<212> PRT
<213> mice (Mus musculus)
<400> 1
Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg
85 90 95
Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Ser Ser
100 105
<210> 2
<211> 10
<212> PRT
<213> mice (Mus musculus)
<400> 2
Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr
1 5 10
<210> 3
<211> 3
<212> PRT
<213> mice (Mus musculus)
<400> 3
Leu Val Ser
1
<210> 4
<211> 8
<212> PRT
<213> mice (Mus musculus)
<400> 4
Gln His Ile Arg Glu Leu Thr Arg
1 5
<210> 5
<211> 110
<212> PRT
<213> mouse (Mus musculus)
<400> 5
Val Glu Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile
1 5 10 15
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Ala Met Asn Trp
20 25 30
Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Tyr Ile Asn
35 40 45
Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe Lys Gly Arg Phe
50 55 60
Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn
65 70 75 80
Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys Ala Arg Gly Gly
85 90 95
Ser Tyr Ala Asp Trp Gly Gln Gly Thr Thr Leu Thr Val Ser
100 105 110
<210> 6
<211> 8
<212> PRT
<213> mice (Mus musculus)
<400> 6
Gly Tyr Thr Phe Thr Asn Tyr Ala
1 5
<210> 7
<211> 8
<212> PRT
<213> mice (Mus musculus)
<400> 7
Ile Asn Thr Tyr Thr Gly Glu Pro
1 5
<210> 8
<211> 8
<212> PRT
<213> mice (Mus musculus)
<400> 8
Ala Arg Gly Gly Ser Tyr Ala Asp
1 5

Claims (10)

1. A monoclonal antibody or an antigen-binding fragment thereof against a novel coronavirus nucleoprotein, comprising a light chain variable region comprising CDR1, CDR2 and CDR3 and a heavy chain variable region comprising CDR1, CDR2 and CDR3,
the amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.2 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.2, or the amino acid sequence containing the sequence;
the amino acid sequence of the light chain CDR2 is the sequence shown in SEQ ID NO.3 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.3, or the amino acid sequence containing the sequence;
the amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.4 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.4, or the amino acid sequence containing the sequence;
the amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.6 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.6, or the amino acid sequence containing the sequence;
the amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.7 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.7, or the amino acid sequence containing the sequence;
the amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.8 or the amino acid sequence with 1 or 2 conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.8, or the amino acid sequence containing the sequence.
2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region sequence is represented by SEQ ID No.1 and the heavy chain amino acid sequence is represented by SEQ ID No. 5.
3. The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab 'fragment, a F (ab') 2 A fragment, a single chain antibody or a humanized antibody.
4. A nucleic acid molecule comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 3.
5. An expression vector comprising the nucleic acid molecule of claim 4.
6. A recombinant comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.
7. A monoclonal antibody hybridoma cell strain against a novel coronavirus nucleoprotein, characterized in that said monoclonal antibody hybridoma cell strain secretes the monoclonal antibody of claim 1 or 2.
8. The monoclonal antibody hybridoma cell strain of claim 7, wherein the monoclonal antibody hybridoma cell strain is a mouse hybridoma cell 2A998135 with a collection number of CGMCC No. 45129.
9. Use of a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 for the preparation of a product for the detection of a novel coronavirus or for the treatment of a novel coronavirus infection.
10. A kit for detecting a novel coronavirus, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.
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