CN115991765B - Anti-porcine delta coronavirus M protein monoclonal antibody, epitope peptide identified by same and application thereof - Google Patents

Abstract

The invention discloses an anti-porcine delta coronavirus M protein monoclonal antibody, an epitope peptide identified by the same and application thereof. The invention makes it be favorable to form fusion protein which is easy to express and is favorable for mouse to produce immunogenicity in host colibacillus by truncating pig delta coronavirus M gene, and monoclonal hybridoma cell strain which secretes pig delta coronavirus M protein and monoclonal antibody which secretes pig delta coronavirus M protein are obtained by cell fusion, screening and subcloning. The monoclonal antibody provided by the invention can specifically recognize M protein of porcine delta coronavirus, and has no cross reaction with other coronaviruses. The invention further provides an epitope peptide identified by the monoclonal antibody, the amino acid sequence of which is shown as SPESRL, and the epitope peptide has good specificity. The monoclonal antibody or the antigen epitope peptide identified by the monoclonal antibody can be applied to detection or diagnosis of porcine delta coronavirus.

Description

Anti-swine delta coronavirus M protein monoclonal antibody and the antigenic epitope peptide it recognizes and application

Technical field

The present invention relates to anti-coronavirus monoclonal antibodies, in particular to anti-swine delta coronavirus M protein monoclonal antibodies and the antigenic epitope peptides they recognize and hybridoma cell lines secreting the monoclonal antibodies. The invention also relates to their use in the preparation of diagnostics. Or the application in reagents or medicines for preventing and treating porcine delta coronavirus, which belongs to the field of anti-porcine delta coronavirus M protein monoclonal antibodies and their applications.

Background technique

Since the outbreak of SARS-CoV in 2003, coronaviruses have attracted widespread attention. More and more new coronaviruses have been discovered, seriously threatening the health of animals and humans and causing serious economic losses to various breeding industries. Coronaviruses are single-stranded positive-strand RNA viruses, divided into four genera: α, β, γ, and δ. Porcine deltacoronavirus (PDCoV) is a new type of porcine enteric coronavirus, belonging to the genus deltacoronavirus. The genome size of porcine delta coronavirus is about 25-30kb, encoding four structural proteins: spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein.

Porcine delta coronavirus infection mainly affects piglets. After infection, severe atrophic enteritis occurs accompanied by severe diarrhea, vomiting and other symptoms, which can lead to death. Porcine delta coronavirus can also spread between different species, and studies have shown that calves are also susceptible to it. The first case of human infection with porcine delta coronavirus was recently reported. To date, little is known about the function of the porcine deltacoronavirus M protein. Therefore, providing a monoclonal antibody against the M protein of porcine delta coronavirus and a hybridoma cell line secreting its antibody are particularly important for the diagnosis or prevention of porcine delta coronavirus infection.

Contents of the invention

One of the purposes of the present invention is to provide anti-swine delta coronavirus M protein monoclonal antibodies.

The second object of the present invention is to provide a hybridoma cell strain secreting the anti-swine delta coronavirus M protein monoclonal antibody.

The third object of the present invention is to provide the antigenic epitope peptide recognized by the anti-swine delta coronavirus M protein monoclonal antibody.

The fourth object of the present invention is to apply the anti-porcine delta coronavirus M protein monoclonal antibody or the antigen epitope recognized by it in the preparation of reagents or drugs for diagnosing or treating diseases caused by porcine delta coronavirus.

The above objects of the present invention are achieved through the following technical solutions:

One aspect of the present invention provides an anti-swine delta coronavirus M protein monoclonal antibody. The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the monoclonal antibody are shown as SYTFSDYA, ISPYYGNS and A respectively; The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody are shown as KSVSTSGYSYL, VS, and QHIREL, respectively.

Further preferably, the amino acid sequence of the heavy chain variable region of the anti-swine delta coronavirus M protein monoclonal antibody is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4 ; The amino acid sequence of the light chain variable region is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5.

The anti-porcine delta coronavirus M protein monoclonal antibody provided by the present invention can only specifically recognize the M protein of PDCoV and has no cross-reactivity with other coronaviruses. Therefore, it can be used as a detection antibody to diagnose or detect porcine delta coronavirus.

As a reference embodiment for detecting porcine delta coronavirus using the anti-porcine delta coronavirus M protein monoclonal antibody, the present invention provides an ELISA detection kit for detecting porcine delta coronavirus, including: primary antibody, enzyme label Secondary antibodies, antibody diluents, washing solutions, blocking solutions, and color developing solutions; wherein, the primary antibody or secondary antibody is the anti-porcine delta coronavirus M protein monoclonal antibody of the present invention.

Another aspect of the present invention is to provide an epitope peptide recognized by an anti-swine delta coronavirus M protein monoclonal antibody, the amino acid sequence of which is shown in SPESRL; correspondingly, the gene encoding the epitope peptide and the gene containing the gene Recombinant expression vectors and recombinant host cells also belong to the protection scope of the present invention; wherein, the recombinant expression vector can be a recombinant prokaryotic expression vector or a recombinant eukaryotic expression vector; the recombinant host cell is a recombinant prokaryotic expression cell, Recombinant eukaryotic expression cells, recombinant fungal cells or recombinant yeast cells; the recombinant prokaryotic expression cells are preferably Escherichia coli.

Those skilled in the art can transform, transduce or transfect the recombinant vector into host cells according to conventional methods in the art, such as chemical transformation by calcium chloride method, high-voltage electroporation transformation, preferably electroporation transformation; methods commonly used in the art can be used Methods Isolating and purifying the epitope peptide from recombinant host cells. For example, centrifugation separates culture medium and recombinant host cells, high-pressure homogenization disrupts cells, centrifugal filtration removes cell debris, and affinity chromatography purifies epitope peptides. For the separated and purified antigen epitope peptide products, purity identification can be carried out using methods commonly used in the art.

The antigen epitope peptide recognized by the anti-porcine delta coronavirus M protein monoclonal antibody provided by the invention has good specificity and immunogenicity, and can be used as a detection reagent for the diagnosis or detection of porcine delta coronavirus.

As a reference embodiment for detecting porcine delta coronavirus using the antigen epitope peptide, the present invention provides a detection kit for detecting porcine delta coronavirus, including: coating antigen, primary antibody, and enzyme-labeled secondary antibody , positive control serum, negative control serum, antibody diluent, washing solution, blocking solution, chromogenic solution; wherein, the coating antigen is the epitope peptide provided by the invention, and the primary antibody is the epitope peptide provided by the invention. Anti-porcine delta coronavirus M protein monoclonal antibody.

The present invention further couples the monoclonal antibody to one or more of an enzyme phase (such as horseradish peroxidase, alkaline phosphatase, etc.), a radioactive isotope, a fluorescent compound or a chemiluminescent compound to obtain a conjugated compound. Conjugates, these conjugates can also be used for the diagnosis or detection of porcine delta coronavirus.

The third aspect of the present invention is to provide a hybridoma cell strain PDCoV-M-24-A6 secreting the anti-porcine delta coronavirus M protein monoclonal antibody, which is deposited in the China Type Culture Collection Center at the deposit address: Wuhan University, Wuhan, China, preservation number: CCTCCNO:C2022187, preservation date: June 20, 2022.

The present invention further provides a method for constructing the hybridoma cell line PDCoV-M-24-A6, which method includes:

(1) According to the hydrophilicity and hydrophobicity of the M gene, the larger span region is truncated, and the smaller span region is selected for truncation to obtain the truncated porcine delta coronavirus M gene and the TF carrier protein coding gene fused to construct a prokaryotic recombinant expression vector. The recombinant protein was obtained after expression and purification.

(2) Mix the recombinant protein and Freund's adjuvant in equal volumes to prepare the immunogen, and immunize mice according to the immunization program.

(3) Take spleen cells from immunized mice and fuse them with myeloma (sp2/0) cells. After fusion, screening and subcloning are performed to finally obtain hybridoma cells secreting anti-porcine delta coronavirus M protein monoclonal antibodies.

The truncated porcine delta coronavirus M gene described in the present invention is a nucleotide sequence corresponding to amino acids 80 to 217 of its protein sequence (the amino acid sequence of the M protein is SEQ ID NO. 1).

By optimizing the truncated M gene protein, the present invention is conducive to forming a fusion recombinant protein that is easy to express in BL21 (DE3) host E. coli and is conducive to the production of immunogenicity in mice with the pCold-TF plasmid carrying a carrier protein. After gel purification by SDS-PAGE electrophoresis, the mice were immunized, and then through cell fusion, screening and subcloning, a monoclonal hybridoma cell line secreting anti-porcine delta coronavirus M protein and a monoclonal hybridoma cell line secreted by the cell line were finally obtained. Cloning antibodies. The monoclonal antibody provided by the present invention has the characteristics of high specificity. The results of IFA and Western blot show that the monoclonal antibody provided by the present invention can specifically recognize the membrane M protein of porcine delta coronavirus and has no cross-reaction with other coronaviruses. Therefore, the monoclonal antibodies provided by the present invention and the antigen epitope peptides they recognize can be used to detect or diagnose porcine delta coronavirus.

Definitions of terms involved in the present invention

The terms "heavy chain variable region" and "light chain variable region" have great changes in the sequence of about 110 amino acids near the N-terminus of the H and L chains of immunoglobulins. The amino acid sequences of other parts are relatively constant. According to This can divide the light chain and heavy chain into variable region (V) and constant region (C). The variable region contains the hypervariable region HVR (hypervariable region) or complementarity-determining region CDR (Complementarity-determining region) and FR skeleton. district.

The term "amino acid sequence" refers to the order in which amino acids are connected to form a peptide chain (or polypeptide). The amino acid sequence can only be read in one direction. There are more than 100 different types of amino acids, of which 20 are commonly used. The present invention does not exclude the modification of other substances on the amino acid chain, such as sugars, lipids, etc., and the present invention is not limited to the 20 commonly used amino acids.

The term "nucleotide sequence" or "polynucleotide sequence" refers to the sequence of bases in DNA or RNA, that is, the sequence of A, T, G, and C in DNA, or A, U, The order of G and C also includes the order of bases in rRNA, tRNA, and mRNA. It should be understood that in addition to DNA sequences, the antibody genes claimed in the present invention also cover RNA (rRNA, tRNA, mRNA) and their complementary sequences.

The term "recombinant expression vectors" refers to vectors that add expression elements (such as promoters, RBS, terminators, etc.) to the basic skeleton of the cloning vector to enable the expression of the target gene. The expression vector consists of four parts: target gene, promoter, terminator, and marker gene. The present invention includes, but is not limited to, prokaryotic cell expression vectors, eukaryotic cell expression vectors or other cell expression vectors.

The term "host cell" or "recombinant host cell" means a cell containing a polynucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, or as known in the art Other methods.

Description of drawings

Figure 1 shows the analysis results of the hydrophilicity and hydrophobicity of the M gene; the horizontal axis represents the number of amino acids, and the vertical axis is bounded by 0. <0 represents hydrophilicity and >0 represents hydrophobicity.

Figure 2 shows the expression and purification results of the recombinant protein; among them, M is the relative molecular mass standard in kDa, and 1, 2, 3, 4, 5, and 6 represent pCold-TF bacterial liquid (control) and pCold-TF-M respectively. Recombinant protein bacterial liquid, pCold-TF-M recombinant protein supernatant, pCold-TF-M recombinant protein precipitation, purified pCold-TF-M recombinant protein, pCold-TF-M recombinant protein bacterial liquid (control).

Figure 3 shows the IFA detection results of monoclonal antibodies; among them, specific fluorescence appeared after PDCoV was inoculated into LLC-PK1 cells (Figure 3B), while cells without PDCoV showed no fluorescence (Figure 3A); PEDV was inoculated into vero cells. Using the PEDV N protein monoclonal antibody stored in the inventor's laboratory as a positive control, no specific fluorescence appeared (Fig. 3C), while the PDCoV M protein monoclonal antibody showed fluorescence (Fig. 3D).

Figure 4 is a Western blot diagram of a monoclonal antibody; where M is the relative molecular mass standard in kDa, lane 1 is the total protein of LLC-PK1 infected with PDCoV, and lane 2 is the total protein of LLC-PK1 not infected with PDCoV (control) . The results showed that a specific band of M protein appeared in lane 1, with a size of 24.6kDa, and no band appeared in the control.

Figure 5 shows the Western blot results of monoclonal antibody epitope identification; M is the relative molecular mass standard, in kDa, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 respectively correspond to the amino acid fragments in the left picture. The results show that the epitope is finally located in fragment 12, and its amino acid sequence is SPESRL.

Figure 6 shows the specific identification results of the monoclonal antibody; the identification results show that the monoclonal antibody only specifically recognizes PDCoV.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

Preparation of various solutions and culture media used in the present invention

1. Preparation of solution

Phosphate buffer solution (PBS): Weigh 8g sodium chloride, 0.2g potassium chloride, 1.42g disodium hydrogen phosphate and 0.27g potassium dihydrogen phosphate, dissolve in 800ml distilled water, adjust the pH to 7.4, and dilute to 1L .

4% paraformaldehyde solution: Weigh 4g of paraformaldehyde powder and dissolve it in 100 ml of PBS.

0.1% Triton X-100 solution: pipette 10 μl of Triton X-100 and dilute it in 10 ml of PBS. 2% BSA solution: weigh 2 g of BSA powder and dissolve it in 100 ml of PBS.

Reagents: 50% PEG1500 (Sigma).

2. Preparation of medium

LB medium: Weigh 10g of Tryptone, 5g of Yeast Extract and 10g of NaCl, dissolve in 800ml of distilled water, and adjust the volume to 1L.

HAT medium: 2% 50×HAT (Beijing Boaolong Company), 12% fetal calf serum (Sigma Company), 1% 100×penicillin-streptomycin (Solebao Company) and 85% DMEM ( Gibco Corporation).

HT medium: 2% 50×HT (Beijing Boaolong Company), 12% fetal calf serum, 1% 100× penicillin and streptomycin and 85% DMEM.

LLC-PKⅠ medium: 10% fetal calf serum, 1% 1M HEPES solution, 100× penicillin-streptomycin, and 88% MEM.

Coronavirus culture medium: 10% tryptone phosphate broth (TPB), 0.0002% trypsin, 1% 100× penicillin-streptomycin, and 89% DMEM.

Example 1 Construction of hybridoma cell lines and screening and identification of monoclonal antibodies against M protein of porcine delta coronavirus

1. Preparation of Antigen

Obtaining and Preparing Recombinant Proteins

Selection and synthesis of porcine delta coronavirus M gene (the amino acid sequence of porcine delta coronavirus M protein is shown in SEQ ID No. 1): Search the M gene sequence based on the whole genome of porcine delta coronavirus strain CHN-GD16-05 in GenBank , cut off the region with a large hydrophobicity span in the M gene, and select the region with a smaller span for truncation to obtain the truncated porcine delta coronavirus M gene and the TF carrier protein encoding gene to fuse to construct a prokaryotic recombinant expression vector. Specific methods as follows:

A pair of specific primers PDCoV-M-F and PDCoV-M-R were selected from the M gene sequence, and the enzyme cutting sites EcoRⅠ and HindⅢ were inserted upstream and downstream of them respectively.

PDCoV-MF 5'-3': atcc gaattc atatcctgggccaagtac;

PDCoV-MR 5'-3': tcgac aagctt ttacatatacttatacaggc (underlined is the restriction site).

Further, RT-PCR amplification was performed, and the viral RNA genome of the CHN-GD16-05 strain was first reverse-transcribed into cDNA. Further, the M truncated gene was amplified according to the designed primers.

The total volume of the PCR reaction system is 50 μl:

ddH ₂ O: 21 μl

Max DNA Polymerase: 25μl

PDCoV-M-F: 1μl

PDCoV-M-R: 1μl

cDNA(PDCoV): 2μl

Reaction conditions: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 15 seconds, annealing at 55°C for 10 seconds, and extension at 72°C for 30 seconds. This step was performed for 30 cycles; extension at 72°C for 10 minutes. Further PCR purification was performed using a PCR purification kit (Omga Company), nucleic acid electrophoresis was further performed, and a gel recovery kit (Omga Company) was used to recover the nucleic acid, and enzyme digestion was performed.

After the M gene was digested with EcoRⅠ and HindⅢ restriction endonucleases (Baori Medical Biotechnology Co., Ltd.), it was connected to the pCold-TF plasmid vector with corresponding restriction sites to construct a prokaryotic expression vector. It was identified by PCR or enzyme After successful construction and identification, the recombinant plasmid was transferred into BL21 (DE3) E. coli strain, and a single clone colony was picked and inoculated into LB medium. When the OD value reached about 0.5, IPTG was added to induce expression and placed at 16 °C incubator for 8-12 hours. Bacteria were collected by centrifugation and subjected to ultrasonic disruption. After disruption, the supernatant was collected for SDS-PAGE analysis. The protein showed a specific band at the target size position. Finally, a large amount of the target protein was purified through gel cutting.

2. Mouse immunization

After obtaining the PDCoV-M recombinant protein, mix it with Freund's Complete Adjuvant (Sigma Company) in an equal volume of 1:1, add two grinding beads, and vibrate and grind in a vibrating grinder to turn it into a white viscous liquid, 200 μl/80 μg. The protein dose was subcutaneously injected into 6-8 week old BALB/c mice at multiple points, with an interval of 14 days, for the second immunization. The recombinant protein was mixed with Freund's incomplete adjuvant in an equal volume of 1:1, and the protein dose was 200 μl/80 μg subcutaneously. Multi-point injection; 14 days apart, the third immunization is carried out, and the immunization strategy is the same as the second time; 7-10 days, blood is collected from the orbital venous plexus of the mice. If the antibody titer reaches 1:1000, shock immunization will be carried out after an interval of 3 weeks, directly Inject 200μl/80μg protein stock solution intraperitoneally.

3. Cell fusion

Preparation of feeder cells: Kill the mouse by cervical dislocation, soak it in 75% alcohol for 5 minutes, cut the abdominal skin in a biological safety cabinet to expose its abdominal cavity, use a sterile syringe to absorb 8ml of HAT culture medium, and inject it into the abdominal cavity of the mouse. Gently massage the abdomen a few times, withdraw the liquid, mix it evenly with the HAT culture medium to a total volume of 36ml, and spread it on three 24-well plates, with 500ul in each well.

Preparation of spleen cells: 3 days after the shock immunization, aseptically remove the spleen of the mouse in 2 ml DMEM culture medium, prick the spleen multiple times with a syringe needle until all the cells in the spleen are released, collect the liquid and replenish it in a 50 ml centrifuge tube. Add DMEM to 20 ml and centrifuge to collect splenocytes.

Preparation of myeloma (sp2/0) cells: Collect myeloma (sp2/0) cells into a 50ml centrifuge tube and centrifuge and wash once with DMEM.

Before the start of cell fusion, all culture media and reagents used are placed in a 37°C incubator to preheat.

Cell fusion: Mix myeloma (sp2/0) cells and splenocytes thoroughly in a 50ml centrifuge tube at a ratio of 1:5. Centrifuge and discard the supernatant, leaving only the cells. Gently tap the bottom of the tube wall to disperse them evenly. Go around the bottom of the tube wall, place the centrifuge tube in a 37°C beaker, slowly rotate the tube wall and add 1ml 50% PEG1500 dropwise, and finish the addition within 1 minute; continue to rotate the tube wall for 20s; add 2ml DMEM dropwise, and finish the addition within 2 minutes. ; Add DMEM to 20ml, centrifuge at 1000r/min for 10min, discard the supernatant, gently resuspend the bottom cells in HAT medium and add to 36ml, spread on three 24-well plates with feeder cells added in advance, 500μl per well . Place in a 37°C cell culture incubator containing 5% _CO2 .

4.IFA screening and subcloning

On the 5th and 7th days, use HAT medium for half-change of medium. On the 8th and 9th days, wait until the cells have grown to cover 2/3 of the bottom of the wells, and conduct IFA-positive holes. After determining the positive wells, use HT medium to expand the cells in the wells and conduct subcloning. The subcloning step is to count cells and dilute approximately 500 cells into 20 ml of HT medium that has been previously added with feeder cells, mix well, and spread 100 μl per well into two 96-well plates. When the cells have grown to 50% of the bottom area of the well, 50 μl of the supernatant from a single clone well was taken for IFA detection. The positive wells were subjected to another round of subcloning using the same method as above.

5. Preparation of ascites

After the monoclonal positive well hybridoma cells were expanded and cultured, approximately 2.5×10 ⁶ cells were collected, resuspended in 500 μl DMEM, and injected into the peritoneal cavity of mice that had been injected with liquid paraffin in advance. After 7 days, the abdomen of the mice was obviously enlarged. The ascites was collected by needle puncture in a 10 ml centrifuge tube, centrifuged at 1500 g for 10 min, and the supernatant was retained and aliquoted and stored in a -80°C ultra-low temperature refrigerator.

6. IFA detection of monoclonal antibodies

The PDCoV-M-24-A6 monoclonal antibody was diluted each time and reacted with the coated antigen respectively, and then the goat anti-mouse IgG secondary antibody labeled with Dylight 488 (Abcam Company) was added to observe the results under an inverted fluorescence microscope. The results are captured and saved through a computerized fluorescence acquisition system. The results showed that the antibody had the best fluorescence effect when diluted within 1:2000 times, and the highest dilution factor could reach 1:16000 times. The negative control group showed no fluorescence.

The collected ascitic fluid antibodies were used for IFA as follows:

Cell plating: Plate LLC-PKⅠ cells on a 96-well plate and culture them in an incubator containing 5% _CO2 at 37°C for 12-24 hours until the cells cover the bottom of the well.

Virus access: Discard the original culture medium supernatant in the 96-well plate, wash twice with 100 μl PBS per well, add pre-diluted coronavirus stock solution, 50 μl per well, and place in 37°C with 5% _CO2 After culturing for 12-24 hours in the incubator, about half of the cells will die and the plates will be collected.

Antigen coating:

Discard viral culture medium;

Fix with 4% paraformaldehyde for 15 min, 50 μl per well;

Permeabilize with 0.1% TritonX-100 for 30 minutes, 50 μl per well;

Block with 2% BSA for 30 min, 50 μl per well.

Primary antibody incubation: Add 50 μl of PBS or 1:2000 diluted PDCoV-M-24-A6 monoclonal antibody to each well as the primary antibody, and incubate at room temperature for 2 hours.

Secondary antibody incubation: Add 50 μl of 1:2000 diluted Dylight 488-labeled goat anti-mouse IgG to each well as secondary antibody, and incubate at room temperature for 1 hour.

After each of the above steps, it is necessary to wash twice with PBS, 2 minutes each time, pat dry the last time, and take pictures under an inverted fluorescence microscope to observe the results. The results are shown in Figure 3.

According to the results in Figure 3, it can be seen that using PDCoV-M-24-A6 monoclonal antibody as the primary antibody, LLC-PKⅠ cells inoculated with PDCoV showed specific fluorescence (Figure 3B), while cells that were not inoculated with PDCoV showed no fluorescence (Figure 3A ); using the PEDV N protein monoclonal antibody stored in the inventor's laboratory as the primary antibody (positive control), the PEDV-inoculated vero cells did not show specific fluorescence (Figure 3C), while using the PDCoV-M-24-A6 monoclonal antibody as the primary antibody (positive control) The primary antibody, Vero cells inoculated with PEDV, showed fluorescence (Figure 3D), indicating that the monoclonal antibody prepared in the present invention only specifically recognized PDCoV, had no cross-reaction with PEDV, and had good specificity.

Test Example 1 Western blot detection of monoclonal antibodies

Take the prepared samples of porcine delta virus-infected and uninfected cells and perform SDS-PAGE protein electrophoresis. After electrophoresis, the bands on the gel were transferred to PVDF membrane. Block with 5% skimmed milk powder solution for 2 hours; add 1:1000-fold diluted monoclonal antibody secreted by the PDCoV-M-24-A6 hybridoma cell line and incubate at 4°C overnight; add 1:8000-fold diluted horseradish Peroxidase-labeled goat anti-mouse IgG secondary antibody (Abcam Company), incubate for 1 hour on a shaker at room temperature; after each of the above steps, wash twice with TBST solution, 10 minutes each time. Finally, a luminescent solution (New Semiconductor Company) was added, and the color was developed by computer, photographed and saved in the gel imaging system. The Western blot detection results are shown in Figure 4.

According to the detection results in Figure 4, it can be seen that a specific band of M protein appeared in lane 1, with a size of 24.6 kDa. In the control, no band appeared, proving that the monoclonal antibody prepared by the present invention can specifically recognize the PDCoV M protein.

Test Example 2 Antigen epitope identification of monoclonal antibodies and light and heavy chain variable region sequence determination

1 Antigen epitope identification of monoclonal antibodies

Amino acid residues 80-217 of PDCoV M protein are cut into 5 polypeptide fragments, among which polypeptide fragment 1 is amino acid residues 80-115, polypeptide fragment 2 is amino acid residues 94-142, and polypeptide fragment 3 is the amino acid residues 123-168, polypeptide fragment 4 is the amino acid residues 150-197, and polypeptide fragment 5 is the amino acid residues 177-217; the above five polypeptides are obtained by step-by-step amplification by PCR. The coding gene fragment of the fragment was constructed into the eukaryotic expression plasmid pEGFP-C3 by enzyme digestion and ligation. After successful construction, it was transfected into 293T cells. Cell proteins were collected for 24 hours. After SDS-PAGE, Western blot analysis was performed (to implement The monoclonal antibody identified in Example 1 is the primary antibody, and HRP Goat Anti-Mouse IgG is the secondary antibody).

The results of Western blot analysis (Figure 5) show that the fragments correspond to the lanes one by one. There are specific bands in lanes 1 and 2, but not in lanes 3, 4, and 5, which proves that the antigen epitope is in the polypeptide fragment 1. Overlapping with polypeptide fragment 2 (i.e., polypeptide fragment 6, amino acid residues 94-115), polypeptide fragment 6 was further deleted from the N-terminus and C-terminus by several amino acids respectively, and divided into polypeptide fragment 7 (amino acids 99-115). residues), polypeptide fragment 8 (amino acid residues 94-108), both lanes showed specific bands, proving that the antigenic epitope is the overlapping portion of polypeptide fragment 7 and polypeptide fragment 8 (i.e., polypeptide fragment 9, Amino acid residues 99-108), polypeptide fragment 9 is divided into polypeptide fragment 10 (amino acid residues 101-108), polypeptide fragment 11 (amino acid residues 99-107) according to the above method, and the antigen epitope Locate the polypeptide fragment 10, and delete two and three amino acids from the N-terminus of the polypeptide fragment 10 to form polypeptide fragment 12 (amino acid residues 103-108) and polypeptide fragment 13 (amino acid residues 104-108), It was finally determined that the antigenic epitope was finally located in polypeptide fragment 12, which is amino acid SPESRL at positions 103 to 108 of the M gene.

2 Sequence determination of light and heavy chain variable regions of monoclonal antibodies

Total RNA from hybridoma cell lines secreting anti-PDCoV M protein was extracted using an RNA kit, and further reverse transcribed into cDNA using a reverse transcription kit.

Design the light chain variable region primer sequence:

VLF 5’-3’:atggagacagacacactcctgctat

VLR 5’-3’:ggatacagttggtgcagcatcagcccgttt

Design heavy chain variable region primer sequences:

VHF 5’-3’:atggratgsagctgkgtmatsctct

VHR 5’-3’: tggggstgtygttttggctgmrgagacrgtga

50μl PCR reaction system:

ddH ₂ O: 39 μl

10×PCR Buffer: 5μl

rTaq: 0.25μl

dNTP Mix: 2.75μl

Upstream primer: 0.5μl

Downstream primer: 0.5μl

cDNA: 2μl

PCR reaction program: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 30 seconds, 33 cycles of this step; extension at 72°C for 5 minutes.

Further, the PCR product was purified by a kit and ligated with the pMD19-T vector (Takara Company), and the corresponding positive clone plasmid was selected for sequencing. The amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody were determined as shown in SEQ ID NO. 2 and SEQ ID NO. 3 respectively. The nucleotide sequences of its heavy chain variable region and light chain variable region are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.

The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody prepared in Example 1 are shown as SYTFSDYA, ISPYYGNS, and A respectively; the light chain variable region of the monoclonal antibody prepared in Example 1 is shown in The amino acid sequences of CDR1, CDR2, and CDR3 are shown as KSVSTSGYSYL, VS, and QHIREL, respectively.

Test Example 3 Specific Identification of Monoclonal Antibodies

In order to identify the specificity of the monoclonal antibody prepared in Example 1, the monoclonal antibody was used to detect porcine delta coronavirus strain PDCoV-GD16-05, porcine delta coronavirus strain PDCoV-GX2018, porcine delta coronavirus strain PDCoV-GDHK, porcine delta coronavirus strain PDCoV-GDHK, and porcine delta coronavirus strain PDCoV-GD16-05. Western blot analysis was performed on acute diarrhea syndrome coronavirus (SADS), porcine epidemic diarrhea coronavirus (PEDV), Getta virus (GETV), Seneca virus type A (SVV), and porcine Sapelo virus (PSV). Cell infection samples of different strains of the above-mentioned PDCoV and other virus strains were collected, and the monoclonal antibody PDCoV-M-24-A6 was used as the primary antibody and HRP Goat Anti-Mouse IgG was used as the secondary antibody for Western blot identification.

The Western blot identification results are shown in Figure 6. The identification results show that the monoclonal antibody prepared in Example 1 only specifically recognizes PDCoV and has no specific reaction with other viruses, indicating that the monoclonal antibody provided by the invention or the antigen epitope (SPESRL) it recognizes has good specificity. It only specifically recognizes PDCoV. The monoclonal antibodies provided by the invention or the antigenic epitopes recognized by them can be prepared into various diagnostic reagents (such as detection antibodies or diagnostic antigens, etc.) for identifying or detecting porcine delta coronavirus.

Claims (5)

1. Anti-swine delta coronavirus M protein monoclonal antibody, characterized in that the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.2; the amino acid sequence of its light chain variable region is shown in SEQ ID NO.3 Show. 2. A hybridoma cell line secreting monoclonal antibodies against the M protein of porcine delta coronavirus, characterized in that its deposit number is CCTCC NO.C2022187. 3. A conjugate, characterized in that it is obtained by coupling the monoclonal antibody of claim 1 with one or more of an enzyme phase, a radioactive isotope or a chemiluminescent compound. 4. A detection kit for detecting porcine delta coronavirus, including a detection antibody; characterized in that the detection antibody is the monoclonal antibody of claim 1. 5. Application of the monoclonal antibody of claim 1, the hybridoma cell line of claim 2 or the conjugate of claim 3 in the preparation of reagents for diagnosing porcine delta coronavirus.