CN115991765B - Anti-porcine delta coronavirus M protein monoclonal antibody, epitope peptide identified by same and application thereof - Google Patents
Anti-porcine delta coronavirus M protein monoclonal antibody, epitope peptide identified by same and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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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
Technical Field
The invention relates to an anti-coronavirus monoclonal antibody, in particular to an anti-porcine delta coronavirus M protein monoclonal antibody, an antigen epitope peptide identified by the same and a hybridoma cell strain secreting the monoclonal antibody, and also relates to application of the same in preparation of reagents or medicines for diagnosing or preventing porcine delta coronavirus, belonging to the field of anti-porcine delta coronavirus M protein monoclonal antibodies and application thereof.
Background
Since the SARS-CoV outbreak in 2003, coronaviruses have received widespread attention, and more new coronaviruses have been discovered, which severely threaten animal and human health and cause serious economic losses to various farming industries. Coronaviruses are single-stranded positive-strand RNA viruses and are divided into four genera of alpha, beta, gamma and delta, and porcine delta coronaviruses (Porcine Deltacoronavirus, PDCoV) are novel porcine enterocoronaviruses and belong to the genus delta coronaviruses. The pig delta coronavirus genome is about 25-30kb in size and encodes four structural proteins, namely a fiber (S) protein, a capsule (E) protein, a membrane (M) protein and a nucleocapsid (N) protein.
Pig delta coronavirus infection mainly affects piglets, and severe atrophic enteritis, severe diarrhea, emesis and other symptoms appear after infection, which can lead to death. Porcine delta coronavirus can also spread among different species, and researches show that calves are also susceptible to the porcine delta coronavirus, and a situation that one person infects the porcine delta coronavirus is recently reported for the first time. So far, little is known about the functional study of the M protein of porcine delta coronavirus. Therefore, the M protein of the porcine delta coronavirus provides a monoclonal antibody and a hybridoma cell line secreting the monoclonal antibody, which is particularly important for diagnosis or prevention of infection of the porcine delta coronavirus.
Disclosure of Invention
It is an object of the present invention to provide monoclonal antibodies against the M protein of porcine delta coronavirus.
The second object of the invention is to provide a hybridoma cell strain secreting the monoclonal antibody against the M protein of the porcine delta coronavirus.
The invention also provides an epitope peptide recognized by the monoclonal antibody against the M protein of the porcine delta coronavirus.
The invention also provides application of the monoclonal antibody of the anti-porcine delta coronavirus M protein or the identified epitope thereof in preparing a reagent or a medicament for diagnosing or treating diseases caused by the porcine delta coronavirus.
The above object of the present invention is achieved by the following technical solutions:
in one aspect of the invention, there is provided an anti-porcine delta coronavirus M protein monoclonal antibody having amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of SYTFSDYA, ISPYYGNS and A respectively; the amino acid sequences of CDR1, CDR2, 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-porcine delta coronavirus M protein monoclonal antibody is shown as SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 4; the amino acid sequence of the light chain variable region is shown as SEQ ID NO.3, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 5.
The monoclonal antibody against M protein of porcine delta coronavirus provided by the invention can only specifically recognize M protein of PDCoV, and has no cross reaction with other coronaviruses, so that the monoclonal antibody can be used as a detection antibody for diagnosing or detecting porcine delta coronavirus.
As a reference embodiment for detecting the porcine delta coronavirus by using the anti-porcine delta coronavirus M protein monoclonal antibody, the invention provides an ELISA detection kit for detecting the porcine delta coronavirus, which comprises the following components: primary antibody, enzyme-labeled secondary antibody, antibody diluent, washing solution, sealing solution and color development solution; wherein the primary antibody or the secondary antibody is the monoclonal antibody of the anti-porcine delta coronavirus M protein.
In another aspect, the present invention provides an epitope peptide recognized by a monoclonal antibody against M protein of porcine delta coronavirus, wherein the amino acid sequence of the epitope peptide is shown as SPESRL; correspondingly, the genes for encoding the epitope peptides, recombinant expression vectors and recombinant host cells containing the genes and the like also belong to the protection scope of the 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, a recombinant eukaryotic expression cell, a recombinant fungal cell or a recombinant yeast cell; the recombinant prokaryotic expression cell is preferably Escherichia coli.
The person skilled in the art can transform, transduce or transfect the recombinant vector into a host cell according to methods conventional in the art, such as calcium chloride chemical transformation, high voltage shock transformation, preferably shock transformation; the epitope peptide may be isolated and purified from the recombinant host cell using methods commonly used in the art. For example, the culture medium and recombinant host cells are centrifuged, the cells are broken up by high pressure homogenization, cell debris is removed by centrifugation, and the epitope peptide is purified by affinity chromatography. For the product of the epitope peptide obtained by separation and purification, purity identification can be performed using methods commonly used in the art.
The antigen epitope peptide identified by the monoclonal antibody for resisting the porcine delta coronavirus M protein provided by the invention has good specificity and immunogenicity, and can be used as a detection reagent for diagnosis or detection of the porcine delta coronavirus.
As a reference embodiment for detecting the porcine delta coronavirus by using the epitope peptide, the invention provides a detection kit for detecting the porcine delta coronavirus, which comprises the following components: coating antigen, primary antibody, enzyme-labeled secondary antibody, positive control serum, negative control serum, antibody diluent, washing solution and confining solution, and developing solution; wherein the coating antigen is the epitope peptide provided by the invention, and the primary antibody is the monoclonal antibody for resisting the M protein of the porcine delta coronavirus provided by the invention.
The invention further couples the monoclonal antibody with one or more of enzyme phase (such as horseradish peroxidase, alkaline phosphatase, etc.), radioisotope, fluorescent compound or chemiluminescent compound to obtain conjugates, and the conjugates can also be used for diagnosis or detection of pig delta coronavirus.
The third aspect of the invention provides a hybridoma cell strain PDCoV-M-24-A6 secreting the monoclonal antibody against porcine delta coronavirus M protein, which is deposited in China center for type culture collection, and has the following deposit address: the preservation number of the university of Wuhan in Chinese is: CCTCCNO: C2022187, the preservation time is: 2022, 6 and 20.
The invention further provides a method for constructing the hybridoma cell strain PDCoV-M-24-A6, which comprises the following steps:
(1) And cutting off the region with larger span according to the hydrophilicity and hydrophobicity of the M gene, selecting the region with smaller span for cutting off to obtain a cut-off porcine delta coronavirus M gene and TF carrier protein coding gene, fusing and constructing a prokaryotic recombinant expression vector, and carrying out expression purification to obtain recombinant proteins.
(2) The recombinant protein and Freund's adjuvant are mixed in equal volume to obtain immunogen, and the mice are immunized according to immunization program.
(3) And taking spleen cells of the immunized mice to fuse with myeloma (sp 2/0) cells, and finally obtaining hybridoma cells secreting monoclonal antibodies against the M protein of the porcine delta coronavirus through screening and subcloning after the fusion.
The truncated porcine delta coronavirus M gene is a nucleotide sequence corresponding to amino acids 80 to 217 of a protein sequence (the amino acid sequence of the M protein is SEQ ID NO. 1) of the truncated porcine delta coronavirus M gene.
The invention optimizes truncated M gene protein to form fusion recombinant protein which is easy to express and is beneficial to generating immunogenicity of mice in BL21 (DE 3) host escherichia coli with plasmid of pCold-TF carrying carrier protein, immunizing mice after SDS-PAGE electrophoresis gel cutting purification, and finally obtaining a monoclonal hybridoma cell strain secreting anti-porcine delta coronavirus M protein and monoclonal antibody secreted by the cell strain through cell fusion, screening and subcloning. The monoclonal antibody provided by the invention has the characteristics of high specificity and the like, and IFA and Western blot results show that the monoclonal antibody provided by the invention can specifically identify the membrane M protein of the porcine delta coronavirus and has no cross reaction with other coronaviruses. Therefore, the monoclonal antibody and the antigen epitope peptide recognized by the monoclonal antibody can be used for detecting or diagnosing the porcine delta coronavirus.
Definition of terms in connection with the present invention
The terms "heavy chain variable region" and "light chain variable region" are used to describe the light and heavy chain regions which comprise the hypervariable region HVR (hypervariable region) or Complementarity determining region CDR (Complementarity-determining region) and the FR-backbone regions within the variable region of the variable region (V) and constant region (C) by varying about 110 amino acid sequences over the near N-terminus of the H and L chains of an immunoglobulin, with the other portions of the amino acid sequences being relatively constant.
The term "amino acid sequence" refers to the order in which amino acids are linked to one another to form a peptide chain (or polypeptide), and the amino acid sequence can be read in only one direction. There are 100 different types of amino acids, 20 of which are commonly used, and the present invention does not exclude other substances on the amino acid chain, such as sugar, lipid, etc., and the present invention is not limited to the commonly used amino acids in 20.
The term "nucleotide sequence" or "polynucleotide sequence" refers to the arrangement of bases in DNA or RNA, i.e., A, T, G, C in DNA, or A, U, G, C in mRNA, including rRNA, tRNA, mRNA. It should be understood that the antibody genes claimed in the present invention encompass RNA (rRNA, tRNA, mRNA) and their complements in addition to DNA sequences.
The term "recombinant expression vector (Expression vectors)" refers to a vector in which expression elements (e.g., promoter, RBS, terminator, etc.) are added to the basic skeleton of a cloning vector so that a desired gene can be expressed. Expression vector four parts: a target gene, a promoter, a terminator and a marker gene. The invention includes, but is not limited to, prokaryotic expression vectors, eukaryotic expression vectors, or other cellular expression vectors.
The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used to insert to produce a recombinant host cell, such as direct uptake, transduction, or other methods known in the art.
Drawings
FIG. 1 shows the results of a hydrophilic-hydrophobic analysis of M gene; wherein the horizontal axis represents the number of amino acids, the vertical axis is bounded by 0, <0 represents hydrophilic, >0 represents hydrophobic.
FIG. 2 shows the results of recombinant protein expression and purification; wherein M is a relative molecular mass standard, and units kDa,1, 2, 3, 4, 5 and 6 respectively represent pCold-TF bacterial liquid (control), pCold-TF-M recombinant protein bacterial liquid, pCold-TF-M recombinant protein supernatant, pCold-TF-M recombinant protein precipitation, purified pCold-TF-M recombinant protein and pCold-TF-M recombinant protein bacterial liquid (control).
FIG. 3 is a graph showing the results of IFA detection of monoclonal antibodies; wherein, specific fluorescence appeared after the PDCOV inoculated LLC-PK1 cells (FIG. 3B), whereas no fluorescence appeared without the PDCOV inoculated cells (FIG. 3A); PEDV inoculated vero cells, with PEDV N protein mab stored in the present inventors laboratory as positive control, showed no specific fluorescence (fig. 3C), while PDCoV M protein mab was fluorescent (fig. 3D).
FIG. 4 is a Western blot diagram of monoclonal antibodies; wherein M is the relative molecular mass standard, unit 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 show that lane 1 shows a specific band of M protein, 24.6kDa in size, and no band appears in the control.
FIG. 5 is a Western blot result diagram of monoclonal antibody epitope identification; m is a relative molecular mass standard, and units kDa,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 respectively correspond to amino acid fragments in the left graph, and the result shows that the antigen epitope is finally positioned on the fragment 12, and the amino acid sequence of the antigen epitope is SPESRL.
FIG. 6 shows the results of the specificity identification of monoclonal antibodies; the identification result shows that the monoclonal antibody only specifically recognizes PDCoV.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Preparation of various solutions and Medium used in the present invention
1. Preparation of the solution
Phosphate Buffer (PBS): 8g of sodium chloride, 0.2g of potassium chloride, 1.42g of disodium hydrogen phosphate and 0.27g of monopotassium phosphate are weighed and dissolved in 800ml of distilled water, the pH is adjusted to 7.4, and the volume is fixed to 1L.
4% paraformaldehyde solution: 4g of paraformaldehyde powder are weighed out and dissolved in 100ml of PBS.
0.1% Triton X-100 solution: mu.l of Triton X-100 diluted in 10ml of 2% BSA in PBS was pipetted in: 2g of BSA powder was weighed and dissolved in 100ml PBS.
Reagent: 50% PEG1500 (Sigma Co.).
2. Preparation of culture Medium
LB medium: 10g of Tryptone, 5g of Yeast Extract and 10g of NaCl are weighed, dissolved in 800ml of distilled water and fixed to 1L.
HAT medium: 2% of 50 XHAT (Beijing Boolong Co.), 12% of fetal bovine serum (Sigma Co.), 1% of 100 XGreen streptomycin (Soxhobao Co.) and 85% of DMEM (Gibco Co.).
HT medium: 2% of 50 XHT (Beijing Boolong Co.), 12% of fetal bovine serum, 1% of 100 XGreen streptomycin and 85% of DMEM.
LLC-PKI medium: 10% fetal bovine serum, 1% 1M HEPES solution with 100 Xof Streptomyces, 88% MEM.
Coronavirus medium: 10% Tryptone Phosphate Broth (TPB), 0.0002% pancreatin, 1% 100 Xgreen streptomycin and 89% DMEM.
EXAMPLE 1 construction of hybridoma cell lines and screening and identification of monoclonal antibodies against porcine delta coronavirus M protein
1. Preparation of antigens
Acquisition and preparation of recombinant proteins
Selection and synthesis of the porcine delta coronavirus M gene (the amino acid sequence of the porcine delta coronavirus M protein is shown as SEQ ID No. 1): according to the whole genome of a pig delta coronavirus strain CHN-GD16-05 in GenBank, searching an M gene sequence, cutting off a region with larger hydrophilic-hydrophobic span in the M gene, and selecting a region with smaller hydrophilic-hydrophobic span for cutting off to obtain a truncated pig delta coronavirus M gene and TF carrier protein coding gene, and fusing to construct a prokaryotic recombinant expression vector, wherein the specific method is as follows:
a pair of specific primers PDCoV-M-F and PDCoV-M-R are designed by selecting M gene sequences, and enzyme cutting sites EcoRI and HindIII are respectively inserted into the upstream and downstream of the primers.
PDCoV-M-F 5’-3’：atccgaattcatatcctgggccaagtac；
PDCoV-M-R 5’-3’：tcgacaagcttttacatatacttatacaggc (underlined as cleavage site).
Further, RT-PCR amplification is performed, wherein the genome of CHN-GD16-05 strain viral RNA is firstly reverse transcribed into cDNA, and further, M truncated genes are amplified according to designed primers.
The total volume of the PCR reaction system was 50. Mu.l:
ddH 2 O：21μl
Max DNA Polymerase：25μl
PDCoV-M-F：1μl
PDCoV-M-R：1μl
cDNA(PDCoV)：2μl
reaction conditions: pre-denaturation at 98℃for 3min; denaturation at 98℃for 15s, annealing at 55℃for 10s, elongation at 72℃for 30s, this step being carried out for 30 cycles; extending at 72℃for 10min. Further, PCR purification was performed by a PCR purification kit (Omga Co.), further, nucleic acid electrophoresis was performed, and nucleic acid was recovered by using a gel recovery kit (Omga Co.), and then, cleavage treatment was performed.
After the restriction enzyme (Bao Ri doctor technology Co.) of EcoRI and Hind III is cut by M gene, it is connected to pCold-TF plasmid vector with correspondent cutting site to construct prokaryotic expression vector, after the construction of PCR identification or enzyme cutting identification is successful, the recombinant plasmid is transferred into BL21 (DE 3) colibacillus strain, and single clone bacterial colony is selected and inoculated into LB culture medium, when OD value is up to about 0.5, IPTG is added to make induction expression, and placed in constant temp. box at 16 deg.C for continuous culture for 8-12 hr. And centrifugally collecting bacteria for ultrasonic disruption, collecting supernatant after disruption for SDS-PAGE analysis, wherein the protein has a specific band at a target size position, and finally purifying a large amount of target proteins by cutting gel.
2. Immunization of mice
After obtaining the PDCoV-M recombinant protein, 1:1 with Freund's complete adjuvant (Sigma company), placing two grinding beads into a vibration grinder, vibrating and grinding to obtain white viscous liquid, subcutaneously injecting BALB/c mice with 6-8 weeks age at 200 μl/80 μg protein dose, performing second immunization at interval of 14d, and recombining protein 1:1, mixing with Freund's incomplete adjuvant, and subcutaneously injecting 200 μl/80 μg protein at multiple points; at interval 14d, performing a third immunization, wherein the immunization strategy is the same as that of the second immunization; 7-10d, the orbital venous plexus of the mice is sampled, if the antibody titer reaches 1:1000 were given at 3 weeks intervals and were given an impact immunization, followed by direct intraperitoneal injection of 200. Mu.l/80. Mu.g of protein stock.
3. Cell fusion
Preparation of feeder cells: the mice were de-necked and lethal, soaked in 75% alcohol for 5min, the abdominal skin was cut in a biosafety cabinet, the abdominal cavity was exposed, 8ml HAT medium was sucked with a sterilization syringe, the abdominal cavity was injected into the mice, the abdomen was gently massaged several times down, the liquid was drawn back, the total volume was mixed with HAT medium to be 36ml, and the mixture was spread on 3 24 well plates with 500ul each.
Preparation of spleen cells: after 3d of impact immunization, spleens of mice were aseptically handled in 2ml DMEM medium, spleens were pricked with syringe needles multiple times until all cells of spleens were released, and the collected liquid was fed into a 50ml centrifuge tube to 20ml centrifuge to collect spleen cells.
Preparation of myeloma (sp 2/0) cells: myeloma (sp 2/0) cells were collected into 50ml centrifuge tubes and washed once by centrifugation with DMEM.
The culture medium, reagents, etc. used before the start of cell fusion were preheated in a 37℃incubator.
Cell fusion: myeloma (sp 2/0) cells and spleen cells were counted at 1:5, fully mixing the mixture in a 50ml centrifuge tube, centrifuging to discard the supernatant, only reserving cells, gently beating the bottom of the tube wall to uniformly disperse the cells around the bottom of the tube wall, placing the centrifuge tube in a beaker at 37 ℃, slowly rotating the tube wall, dropwise adding 1ml of 50% PEG1500, and finishing the addition within 1 min; continuing to rotate the pipe wall 20s; 2ml of DMEM was added dropwise, over 2 minutes; DMEM was added to 20ml, centrifuged at 1000r/min for 10min, the supernatant was discarded, the bottom cells were resuspended in HAT medium and added to 36ml, and plated in 3 24 well plates with feeder cells added in advance, 500 μl per well. Placing at 37deg.C, and containing 5% CO 2 Is cultured in a cell culture incubator.
IFA screening and subcloning
Half-changing liquid is carried out on the 5 th day and the 7 th day by using HAT culture medium, and IFA detection positive holes are carried out on the 8 th day and the 9 th day when cells grow to 2/3 of the hole bottoms. After positive wells were determined, the wells were expanded with HT medium and subcloned. The subcloning step was cell counting and diluting about 500 cells to 20ml of HT medium pre-loaded with feeder cells, mixing well, and plating 100. Mu.l per well in 2 96-well plates. When the cells grew to 50% of the bottom area of the wells, 50. Mu.l of the supernatant of the individual cloning wells was aspirated for IFA detection. Positive wells were subcloned again in one round as above.
5. Preparation of ascites
After the monoclonal positive Kong Zajiao tumor cells were cultured in an expanded manner, about 2.5X10 cells were collected 6 Cells were resuspended in 500. Mu.l DMEM and injected into the peritoneal cavity of mice that had been previously challenged with liquid paraffin. After 7d, the abdomen of the mice is obviously enlarged, the ascites is collected by needle puncture in a 10ml centrifuge tube, 1500g is centrifuged for 10min, and the supernatant is reserved and split charging is carried out and then stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃.
6. IFA detection of monoclonal antibodies
The PDCoV-M-24-A6 monoclonal antibodies are respectively reacted with the coated antigens after each time of multiple dilution, and then goat anti-mouse IgG secondary antibodies (Abcam company) marked by Dylight 488 are added to observe results under an inverted fluorescence microscope, and the results are shot and stored through a computer fluorescence acquisition system. The result shows that the antibody has the best fluorescent effect when diluted within 1:2000 times, and the highest dilution times can reach 1:16000 times, and the negative control group has no fluorescent display.
The collected ascites antibodies were applied to IFA as follows:
cell plating: LLC-PKI cells were plated in 96-well plates at 37℃with 5% CO 2 Culturing for 12-24h in an incubator until the cells grow to the bottom of the wells.
Virus access: the medium supernatant in 96-well plates was discarded, 100. Mu.l PBS was washed twice per well, and 50. Mu.l of coronavirus medium pre-diluted with virus stock was added per well and placed at 37℃in 5% CO 2 Cells were cultured in the incubator for 12-24 hours with about half of death, and plates were harvested.
Antigen coating:
discarding the virus medium;
4% paraformaldehyde is fixed for 15min, 50 μl per well;
0.1% TritonX-100 was allowed to pass through for 30min, 50 μl per well;
2% BSA was blocked for 30min, 50 μl per well.
Incubation resistance: 50 μl of PBS or 1:2000 dilution of PDCoV-M-24-A6 mab was added to each well and incubated for 2h at room temperature.
Secondary antibody incubation: 50 μl of 1:2000 dilution of Dyight 488-labeled goat anti-mouse IgG as secondary antibody was added to each well and incubated for 1h at room temperature.
After each step is finished, PBS is required to be washed for 2 times, each time is required to be 2 minutes, the drying is performed for 1 time, and the observation result is photographed under an inverted fluorescence microscope, and the result is shown in figure 3.
As can be seen from the results of FIG. 3, LLC-PKI cells vaccinated with PDCoV exhibited specific fluorescence (FIG. 3B) while cells not vaccinated with PDCoV exhibited no fluorescence (FIG. 3A) using PDCoV-M-24-A6 mab as primary antibody; the PEDV N protein monoclonal antibody preserved in the laboratory of the present invention is used as a primary antibody (positive control), the vero cells inoculated with PEDV do not show specific fluorescence (figure 3C), the vero cells inoculated with PEDV have fluorescence, which shows that the monoclonal antibody prepared by the present invention only specifically recognizes the PDCoV, does not have cross reaction with the PEDV, and has good specificity.
Western blot detection of monoclonal antibody of Experimental example 1
Samples of the prepared porcine delta virus infected and uninfected cells were taken separately and subjected to SDS-PAGE protein electrophoresis. After the electrophoresis, the bands on the gel were transferred to a PVDF membrane. Blocking with 5% nonfat dry milk solution for 2 hours; adding 1:1000 times diluted monoclonal antibody secreted by the PDCoV-M-24-A6 hybridoma cell strain, and incubating overnight at 4 ℃; adding goat anti-mouse IgG secondary antibody (Abcam company) marked by horseradish peroxidase at a ratio of 1:8000, and incubating for 1h at room temperature; after each step, the solution is washed by TBST for 2 times for 10min each time. Finally, adding a luminescent solution (New Saimei corporation) and developing color in a gel imaging system through a computer to photograph and store, wherein the Western blot detection result is shown in figure 4.
As can be seen from the detection results of FIG. 4, lane 1 shows a specific band of the M protein, which is 24.6kDa, and the control shows no band, which proves that the monoclonal antibody prepared by the invention can specifically recognize the PDCoV M protein.
Test example 2 epitope identification of monoclonal antibody and light and heavy chain variable region sequence determination
1 epitope identification of monoclonal antibodies
Cutting 80 th-217 th amino acid residues of PDCoV M protein into 5 polypeptide fragments, wherein polypeptide fragment 1 is 80 th-115 th amino acid residues, polypeptide fragment 2 is 94 th-142 th amino acid residues, polypeptide fragment 3 is 123 th-168 th amino acid residues, polypeptide fragment 4 is 150 th-197 th amino acid residues, and polypeptide fragment 5 is 177 th-217 th amino acid residues; the coding gene fragments of the 5 polypeptide fragments are obtained through step-by-step amplification by a PCR method, are constructed into eukaryotic expression plasmid pEGFP-C3 by an enzyme digestion connection method, 293T cells are transfected after the construction is successful, cell proteins are collected for 24 hours, and Western blot analysis is carried out after SDS-PAGE (monoclonal antibodies identified in example 1 are used as primary antibodies, and HRP coat Anti-Mouse IgG is used as secondary antibodies).
Western blot analysis results show that the fragments are in one-to-one correspondence with lanes (figure 5), specific bands are shown in lanes 1 and 2, but the positions of the antigen epitope at the overlapping positions of the polypeptide fragment 1 and the polypeptide fragment 2 (namely, amino acid residues at positions 94-115 of the polypeptide fragment 6) are proved, the polypeptide fragment 6 is further deleted from N end and C end and is divided into a polypeptide fragment 7 (amino acid residues at positions 99-115) and a polypeptide fragment 8 (amino acid residues at positions 94-108), the specific bands are shown in the two lanes, the antigen epitope is proved to be the overlapping part of the polypeptide fragment 7 and the polypeptide fragment 8 (namely, the polypeptide fragment 9, the amino acid residues at positions 99-108), the polypeptide fragment 9 is divided into a polypeptide fragment 10 (amino acid residues at positions 101-108) and a polypeptide fragment 11 (amino acid residues at positions 99-107) according to the method, the antigen epitope is positioned at the polypeptide fragment 10, the polypeptide fragment 10 is respectively deleted from two amino acids and three amino acids form a polypeptide fragment 12 (amino acid residues 103-108) from N end and the polypeptide fragment 8 (amino acid residues at positions 104-108), the antigen epitope is positioned at positions 103-108, and the final antigen epitope is determined to be the final antigen epitope of the polypeptide fragment is the final polypeptide fragment which is positioned at positions of the polypeptide fragment 103-108.
2 determination of the light and heavy chain variable region sequences of monoclonal antibodies
Total RNA of the hybridoma cell line secreting the anti-PDCoV M protein is extracted by an RNA kit, and is further reversely transcribed into cDNA by a reverse transcription kit.
Designing a light chain variable region primer sequence:
VLF 5’-3’：atggagacagacacactcctgctat
VLR 5’-3’：ggatacagttggtgcagcatcagcccgttt
designing a heavy chain variable region primer sequence:
VHF 5’-3’：atggratgsagctgkgtmatsctct
VHR 5’-3’：tggggstgtygttttggctgmrgagacrgtga
PCR reaction 50. Mu.l:
ddH 2 O：39μl
10×PCR Buffer：5μl
rTaq：0.25μl
dNTP Mix：2.75μl
an upstream primer: 0.5 μl
A downstream primer: 0.5 μl
cDNA：2μl
PCR reaction procedure: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 55℃for 15s, elongation at 72℃for 30s, 33 cycles; extending at 72℃for 5min.
Further, the PCR product was purified by the kit and ligated with pMD19-T vector (Takara Co.), and the corresponding positive clone plasmid was picked up for sequencing. The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3. The nucleotide sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5.
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 amino acid sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the monoclonal antibody prepared in example 1 are shown as KSVSTSGYSYL, VS and QHIREL, respectively.
Test example 3 specific identification of monoclonal antibodies
To identify the specificity of the monoclonal antibodies prepared in example 1, western blot analysis was performed on porcine delta coronavirus strain PDCoV-GD16-05, porcine delta coronavirus strain PDCoV-GX2018, porcine delta coronavirus strain PDCoV-GDHK, porcine acute diarrhea syndrome coronavirus (SADS), porcine epidemic diarrhea coronavirus (PEDV), getavirus (GETV), type A saint virus (SVV), porcine Sapelo Virus (PSV) using the monoclonal antibodies. And collecting cell infection samples of different strains and other strains of the PDCoV, using a monoclonal antibody PDCoV-M-24-A6 as a primary antibody, and using HRP coat Anti-Mouse IgG as a secondary antibody to carry out Western blot identification.
The Western blot identification result is shown in FIG. 6. The identification result shows that the monoclonal antibody prepared in the example 1 only specifically recognizes PDCoV and has no specific reaction with other viruses, and shows that the monoclonal antibody provided by the invention or the epitope recognized by the monoclonal antibody (SPESRL) has good specificity and only specifically recognizes PDCoV, and the monoclonal antibody provided by the invention or the epitope recognized by the monoclonal antibody can be prepared into various diagnostic reagents (such as detection antibodies or diagnostic antigens) for identifying or detecting pig delta coronaviruses.
Claims (5)
1. The monoclonal antibody against the M protein of the porcine delta coronavirus is characterized in that the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 3.
2. A hybridoma cell strain secreting monoclonal antibodies against porcine delta coronavirus M protein is characterized by having a preservation number of CCTCC NO. C2022187.
3. A conjugate obtained by coupling the monoclonal antibody of claim 1 to one or more of an enzyme phase, a radioisotope, or a chemiluminescent compound.
4. A detection kit for detecting porcine delta coronavirus, comprising a detection antibody; wherein the detection antibody is the monoclonal antibody of claim 1.
5. Use of the monoclonal antibody of claim 1, the hybridoma cell line of claim 2 or the conjugate of claim 3 for the preparation of a diagnostic porcine delta coronavirus agent.
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