Sheep aphtha virus F1L protein monoclonal antibody as well as blocking ELISA (enzyme-linked immunosorbent assay) detection kit and application thereof

Abstract

The invention discloses an orf virus F1L protein monoclonal antibody as well as a blocking ELISA (enzyme-linked immunosorbent assay) detection kit and application thereof. Belongs to the technical field of biological detection. The kit comprises F1L protein expressed by baculovirus and an ORFV F1L protein monoclonal antibody marked by HRP (horse radish peroxidase). The ORFV F1L protein monoclonal antibody disclosed by the invention has the characteristics of strong specificity, high titer, stable property and the like, and is suitable for establishing an ORFV antibody blocking ELISA (Enzyme-Linked Immunosorbent Assay) kit. According to the invention, the HRP-labeled monoclonal antibody is used, so that non-specific reaction possibly introduced due to the use of an enzyme-labeled secondary antibody is reduced. The kit disclosed by the invention can be used for detecting a large batch of samples, has the advantages of low cost, simplicity, convenience, sensitivity, high specificity and the like, and provides an effective technical means for scientific prevention and control of the contagious pustular dermatitis of sheep.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more particularly to a monoclonal antibody against acanthosis stomatitis virus F1L protein and a blocking ELISA detection kit and application thereof. Background Art

[0002] Contagious pustular dermatitis of sheep, commonly known as sheep sore, is an acute, contact-transmitted infectious disease shared by ruminants (mainly sheep and goats) and humans, caused by infection with the orf virus (ORFV). The disease is currently listed as a Category III epidemic disease in the "Catalogue of Animal Pathogenic Microorganisms" and the "Catalogue of Pathogenic Microorganisms Transmitted by Humans". The disease is clinically characterized by the formation of papules, blisters, pustules and thick wart-like crusts at the site of infection, and generally does not form a systemic infection. At present, the disease is distributed worldwide. Since ORFV can survive for a long time in the environment and sheep that have recovered from the infection can be infected again, it poses a serious threat to the global sheep industry. In addition, the disease poses a certain public health threat to professionals related to the sheep industry, such as veterinarians, herders, slaughterhouse workers, and mutton sellers.

[0003] Due to the widespread prevalence of contagious pustular dermatitis in sheep and the widespread use of live attenuated vaccines, there is a significant market demand for serum antibody testing in naturally infected and vaccinated sheep. However, the currently reported method for detecting ORFV antibodies is primarily an indirect enzyme-linked immunosorbent assay (ELISA). However, because the antibody levels produced by ORFV infection are generally low and are affected by the heterogeneity of serum from different sheep, the specificity and sensitivity of the indirect ELISA test are relatively low, resulting in the actual detection rate not reflecting the true level.

[0004] In summary, how to provide an ORFV antibody detection method with high sensitivity and specificity is an urgent problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a monoclonal antibody against the F1L protein of the oropharyngeal canker sore virus and a blocking ELISA detection kit and application thereof.

[0006] The main immunogenic proteins of ORFV include B2L and F1L. B2L can stimulate cellular and humoral immune responses and secrete antibodies against B2L, making it one of the primary targets for ORFV serological diagnosis. Shang Youjun et al. developed an ORFV B2L protein antibody blocking ELISA assay (Publication No. CN118064375 A) using prokaryotically expressed B2L as a coating antigen and a horseradish peroxidase (HRP)-conjugated B2L monoclonal antibody as a competing antibody. F1L is the primary envelope protein of ORFV and an important protective antigen. Antibodies against F1L induced by ORFV infection have neutralizing activity. Previous studies have shown that ORFV-positive sera react more strongly with F1L than with B2L. Therefore, using F1L as a detection antigen has a relative advantage. Furthermore, the present invention uses eukaryotically expressed F1L as a detection antigen, which, compared to prokaryotically expressed proteins, has the advantage of mimicking the virus's native protein, theoretically resulting in better results.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A monoclonal antibody against the F1L protein of aphthous leukemia virus, comprising a light chain and a heavy chain, wherein the light chain comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3 with amino acid sequences as shown in SEQ ID No. 5 to SEQ ID No. 7; and the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 with amino acid sequences as shown in SEQ ID No. 8 to SEQ ID No. 10.

[0009] Furthermore, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 3, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No. 4.

[0010] The use of the above-mentioned monoclonal antibodies in the preparation of products for detecting ORFV antibodies.

[0011] Furthermore, the product is an ELISA detection kit.

[0012] An antibody blocking ELISA detection kit, comprising an enzyme-labeled antibody,

[0013] The enzyme-labeled antibody is the above-mentioned monoclonal antibody labeled with HRP.

[0014] Furthermore, it also includes F1L protein.

[0015] Furthermore, the F1L protein is prepared as follows: the F1L gene sequence (SEQ ID No. 2) is cloned into the baculovirus expression vector pFastBac-His to obtain the recombinant vector pFastBac-His-F1L. This recombinant vector is transformed into DH10Bac, and the recombinant baculovirus plasmid Bacmid-His-F1L is obtained through transposition. This bacmid is then used to transfect insect cells Sf9 to produce the first-generation recombinant baculovirus (P1). The baculovirus P1 is inoculated into Sf9 cells to mass-produce the second-generation recombinant baculovirus (P2). The P2-generation baculovirus is then inoculated into Sf9 cells to express and purify the target protein F1L in large quantities.

[0016] Furthermore, the purification is performed using a nickel column.

[0017] Furthermore, it also includes coating solution, blocking solution, washing solution, sample diluent, color developing solution and stop solution.

[0018] Furthermore, the coating solution is a carbonate buffer solution with a pH of 9.6;

[0019] The blocking solution is PBS containing 2% w / v BSA;

[0020] The washing solution is PBST at pH 7.4;

[0021] The sample diluent is PBS containing 2% w / v BSA;

[0022] The color developing solution is a single-component TMB color developing solution;

[0023] The stop solution is 2M H2SO4.

[0024] The use of the above-mentioned kit in the preparation of a detection reagent for ORFV antibodies.

[0025] The above-mentioned kit, when used to detect anti-ORFV F1L protein antibodies, comprises the following steps:

[0026] Purified F1L protein was diluted with carbonate buffer (pH 9.6) and coated on a 96-well ELISA plate at 4°C for 12 h; the plate was washed three times with PBST (pH 7.4) and blocked with blocking buffer; after blocking, the plate was washed three times and diluted serum to be tested was added at 100 μL / well and incubated at 37°C for 1.5 h. The plate was washed three times with PBST (pH 7.4). An enzyme-labeled antibody with an equal volume of serum to be tested was added and incubated at 37°C for 1 h. The plate was washed three times with PBST (pH 7.4). TMB color development solution was added at 100 μL / well and incubated at 37°C for 10 min. The plate was not washed and stop solution was added at 50 μL / well. The OD450 nm value was read immediately after color development.

[0027] Among them, preferably, the coating concentration of the purified F1L protein is 1 μg / mL, the coating conditions are 4°C, 12h; the dilution of the enzyme-labeled antibody is 1:2000; the blocking solution is 2% w / v BSA, blocked at 37°C for 1h; and the serum to be tested is diluted at a ratio of 1:4.

[0028] It can be seen from the above technical solutions that, compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The coating antigen F1L protein in the present invention is a eukaryotic protein and is soluble in expression. It carries a His tag for easy purification, has good reproducibility, and the purified F1L protein has high purity.

[0030] (2) The anti-ORFV F1L protein monoclonal antibody of the present invention has the characteristics of high titer, strong specificity and stable properties after being labeled with HRP.

[0031] (3) The present invention uses an HRP-labeled anti-ORFV F1L protein monoclonal antibody, eliminating the nonspecific reaction that may be introduced by using an enzyme-labeled secondary antibody.

[0032] (4) The anti-ORFV F1L protein monoclonal antibody prepared by the method of the present invention can be used to establish an ORFV antibody blocking ELISA detection method and has good application prospects.

[0033] (5) The method of the present invention is applicable to the detection of large quantities of samples and is low-cost, simple, sensitive and highly specific. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0035] Figure 1 The transmembrane region of the ORFV F1L protein in Example 1 of the present invention is predicted;

[0036] Figure 2Figure 1 is the prokaryotic expression and identification of the recombinant protein F1L in Example 1 of the present invention, wherein Figure A shows the expression of the recombinant protein F1L, M is a protein marker, 1 is the whole protein of the induced recombinant bacteria, 2 is the precipitate of the induced recombinant bacteria protein, 3 is the supernatant of the induced recombinant bacteria protein, and 4 is the whole protein of the induced negative control bacteria; Figure B shows the purification of the recombinant protein F1L, M is a protein marker, E1 is the protein eluted for the first time, E2 is the protein eluted for the second time, and E3 is the protein eluted for the third time; Figure C shows the Western blot identification of the purified recombinant protein F1L, E3 is the protein eluted for the third time, and 4 is the whole protein of the induced negative control bacteria;

[0037] Figure 3 This is an SDS-PAGE analysis of the anti-ORFV F1L protein monoclonal antibody purified in Example 2 of the present invention, wherein M is a protein marker, E1 is the monoclonal antibody eluted for the first time, E2 is the monoclonal antibody eluted for the second time, and E3 is the monoclonal antibody eluted for the third time;

[0038] Figure 4 The reactivity of the anti-ORFV F1L protein monoclonal antibody with ORFV was detected by Western blot in Example 2 of the present invention;

[0039] Figure 5 The reactivity of the anti-ORFV F1L protein monoclonal antibody with ORFV was detected by indirect immunofluorescence in Example 2 of the present invention;

[0040] Figure 6 This is the subtype detection of the anti-ORFV F1L protein monoclonal antibody in Example 2 of the present invention;

[0041] Figure 7 Figure 4 shows the baculovirus-expressed F1L protein and its purification and identification in Example 4 of the present invention, wherein Figure A shows the expression and identification of the eukaryotic F1L protein, M is a protein marker, 0h to 96h are whole protein samples at different times, 1 is the whole protein, 2 is the supernatant protein, and 3 is the precipitated protein; Figure B shows the SDS-PAGE results of the purified eukaryotic F1L protein, M is a protein marker, E1 is the first eluted protein, E2 is the second eluted protein, E3 is the third eluted protein, E4 is the fourth eluted protein, and E5 is the fifth eluted protein;

[0042] Figure 8 This is the titer determination result of the HRP-labeled anti-ORFV F1L protein monoclonal antibody in Example 5 of the present invention;

[0043] Figure 9 This is a statistical chart of the specificity test results of the blocking ELISA kit in Example 5 of the present invention. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] The reagents required for the present invention are conventional experimental reagents, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0046] Example 1

[0047] Preparation and identification of F1L prokaryotic protein

[0048] 1) When expressing the target protein in a prokaryotic system, the presence of a transmembrane region can affect the protein's expression level and form. To maximize soluble expression of the F1L protein, the online software TMHMM (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ) was used to predict the presence of a transmembrane region in the F1L protein (GenBank ID: OQ686990). Figure 1 The following is a prediction result of the F1L protein. The full length of the F1L protein is 342 amino acids (aa) (the amino acid sequence is shown in SEQ ID No. 1), and its C-terminus has two closely connected transmembrane regions, corresponding to the 282nd to 342nd aa of the protein. Therefore, the present invention uses the first 281aa (1 to 281aa) of the N-terminus of the F1L protein instead of the full-length F1L protein as the expression object. General Bio (Anhui) Co., Ltd. was commissioned to synthesize the F1L gene (the nucleotide sequence is shown in SEQ ID No. 2) and inserted it between the BamHI and XhoI restriction sites of the pET-28a vector to construct the recombinant expression plasmid pET-28a-F1L. pET-28a-F1L was transformed into BL21 (DE3) competent cells and coated on kanamycin-resistant LB solid agar plates.

[0049] MDPPEITAYIIGVAEGRGTKEVFPTLPYLVGLADDPPKPQPAPAPSPAPAPAPAPAPAPAPAPAPAPAPKPSPPAPHPKGDHVLKAVEWKDVDSKDYPHFFTDMCKSTCPKEMQRRAAHHLNLWESISAGTVSTKYSDNDFILVVDNDMAFRKPEMVKPLIEAMRTNGWYMAQLKETYMTGALATNVPGTGDPELMVYPGGYDVSLDAYIINVGGMKKLYDAIIKDGGLRSGLLTEVFTLEKRLSLARVVLSGAEQVVYPEYYIQVKTRLGGAPSLWSLLATWLARFWPGAIYFLTTPLFSFMGLFDVDVVDVFILAYLLVLVLLLPNSRLLWFIAGLLVTAIV, SEQ ID No. 1.

[0050] , SEQ ID No.2.

[0051] 2) Pick a single colony and inoculate it into kanamycin-resistant LB liquid medium, and culture it at 37°C overnight. The next day, take 1 mL of the bacterial solution as the inoculum, add it to 100 mL of kanamycin-resistant LB liquid medium, and culture it at 37°C with shaking. 600When the value reaches 0.6-0.8, the inducer IPTG is added to a working concentration of 0.5 mM. After induction at 18°C ​​for 24 hours, the cells are harvested by centrifugation. A control group containing the empty vector pET-28a is also established in parallel. After induction, cells from both the experimental and control groups are harvested by centrifugation and disrupted by sonication. Protein samples are prepared and subjected to SDS-PAGE electrophoresis to analyze F1L protein expression.

[0052] like Figure 2 As shown in Figure A, compared to the control group, the experimental group exhibited an additional protein band after induction, approximately 40 kDa in size, consistent with the expected size of the F1L protein. Solubility analysis revealed that at low temperature (18°C), the protein was primarily expressed as inclusion bodies, with no clear target protein band observed in the supernatant.

[0053] 3) Affinity purification of recombinant protein F1L. Follow the instructions for the Beyotime His-tag protein purification kit (denaturation-resistant formulation). The steps are as follows: Centrifuge the induced expression bacterial solution at 10,000 rpm at 4°C for 5 minutes to collect the bacterial pellet. Add 8 mL of denaturing lysis buffer to the bacterial pellet and fully resuspend the bacteria. Ultrasonicate the bacteria on ice using the following ultrasonic conditions: 200W, ultrasonic treatment for 2 seconds, 2 seconds interval, and a total of 15 minutes of ultrasonication. After ultrasonication, centrifuge at 10,000 rpm at 4°C for 10 minutes, collect the bacterial lysate supernatant, and place on ice. Place 2 mL of 50% BeyoGold His-tag purification resin (denaturation-resistant formulation) in a new EP tube and centrifuge at 1000 rpm at 4°C to discard the storage solution. Add one column volume of denaturing lysis buffer to the gel to equilibrate the resin. Centrifuge at 1000 rpm at 4°C to discard the liquid. Repeat the equilibration twice and discard the liquid. Mix the bacterial lysate supernatant collected above with the balanced resin, place it on a side-swing shaker, and incubate at 4°C for 2 hours to allow the His-tagged protein to fully bind to the nickel column. After incubation, load the mixture into a 15mL empty affinity chromatography column tube. Under the action of gravity, the protein that is not bound to the nickel column will be filtered out with the liquid flow. First, wash the column 5 times with denatured lysis buffer, adding 2mL of denatured lysis buffer each time. Then wash the column 5 times again, adding 2mL of special washing buffer each time. Finally, elute the target protein with 0.5mL of protein eluent each time, and collect the eluent to be the purified recombinant protein F1L. Analyze the purity of the purified protein by SDS-PAGE. Figure 2 B shows that the purified recombinant protein F1L has good purity, with a few impurities.

[0054] 4) Western blotting of recombinant proteins. Purified recombinant F1L protein was subjected to SDS-PAGE electrophoresis and then transferred to a nitrocellulose (NC) membrane. The membrane was blocked with 5% skim milk for 2 hours at room temperature, incubated with a His-tagged primary antibody for 2 hours at room temperature, and incubated with an HRP-conjugated goat anti-mouse secondary antibody for 1 hour at room temperature. After blocking, primary antibody incubation, and secondary antibody incubation, the NC membrane was washed three times with PBST (10 minutes each). Finally, ECL working solution was added for color development.

[0055] Western Blot results ( Figure 2 C) shows that the His-tag antibody reacts with the purified recombinant protein F1L, forming a specific band near 40 kDa, while the His-tag antibody does not react with the control sample, indicating that the purified protein is indeed the recombinant protein F1L and has good reactogenicity.

[0056] Example 2

[0057] Preparation and purification of monoclonal antibodies against ORFV F1L protein

[0058] 1) Mouse immunization and antibody titer determination

[0059] Purified recombinant protein F1L was thoroughly mixed with Freund's adjuvant in a 1:1 volume ratio and emulsified. BALB / c mice were subcutaneously injected with a dose of 100 μL per mouse, containing 50 μg of F1L protein antigen. Complete adjuvant was used for the initial emulsification, while incomplete adjuvant was used for the booster. A 21-day interval was allowed between immunizations. Two weeks after the third immunization, mice were tail-cuffed and blood was collected for serum antibody titer determination using an indirect ELISA.

[0060] 2) Cell fusion and hybridoma screening

[0061] One week before fusion, SP2 / 0 cells were revived and passaged two to three times before being used for cell fusion. Three days before fusion, selected mice with the highest titer were boosted with 50 μg of F1L protein antigen injected intraperitoneally. Three days after this booster, spleens were aseptically removed from the mice, and splenocytes were collected and counted. SP2 / 0 cells were mixed with splenocytes and centrifuged at 1500 rpm for 3 minutes at room temperature, and the supernatant was discarded. SP2 / 0 cells and splenocytes were fused using 50% PEG 1450. The fused cells were then plated into 96-well cell culture plates containing hybridoma culture supplements (Biolong) using HAT medium and cultured in a 5% CO2, 37°C incubator. Cell status was monitored regularly. Positive wells were screened for antibody secretion using indirect ELISA. Positive wells were cloned and purified using limiting dilution. After three subcloning cycles, the hybridoma clone with the highest titer (designated ORF059-A4B9) was selected for expansion and cryopreservation.

[0062] 3) Preparation and purification of anti-ORFV F1L protein monoclonal antibody ascites

[0063] SPF female BALB / c mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Ten days later, the positive hybridoma cells ORF059-A4B9 were collected and counted, and the cells were diluted to 1×10 7 Each mouse was injected intraperitoneally with 0.5 mL of the antibody. Starting from the 7th day after injection, the mice's condition, especially their abdominal circumference, should be observed daily. When their abdomen is noticeably distended and their mental state deteriorates, ascites is extracted from their abdominal cavity using a syringe. The ascites is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is the prepared ascites antibody. Antibody purification was performed using a Protein A+G affinity chromatography column from Beyotime Biotechnology Co., Ltd. The antibody purification procedures were performed according to the product instructions and are not detailed here.

[0064] like Figure 3 As shown, after SDS-PAGE separation, the purified anti-ORFV F1L monoclonal antibody showed the heavy and light chains at 50 kDa and 25 kDa, respectively, with virtually no contaminating bands, demonstrating excellent antibody purification. UV microspectrophotometry determined the purified antibody concentration to be 1.1 μg / μL.

[0065] 4) Specificity detection and subtype identification of monoclonal antibodies

[0066] (1) Western blot (WB) test to test antibody specificity: ORFV-infected goat endometrial epithelial cells (GEEC) and uninfected GEEC were used to prepare protein samples for WB analysis. Figure 4) showed that the anti-ORFV F1L protein monoclonal antibody could recognize the F1L protein produced by viral replication, but did not react with blank cells, indicating that the antibody specifically recognized the ORFV F1L protein.

[0067] (2) Indirect immunofluorescence assay (IFA) to identify antibody specificity: a. GEEC were plated at 2×10 4 / well of a 24-well plate, wait until the cells grow to 80% to 90%, inoculate 0.5MOI of the ORFV attenuated strain, and culture in a 37°C, 5% CO2 incubator for 48 hours. At this time, cell lesions can be seen under an optical microscope. b. Discard the culture medium, wash 3 times with PBST, and then fix the cells with 4% paraformaldehyde and fix them at room temperature for 30 minutes. Discard the fixative, wash once with PBST, add 0.5mL of 5% skim milk, and block at 37°C for 1 hour. c. Discard the blocking solution, wash 3 times with PBST, add 1:1000 diluted ascites monoclonal antibody, and incubate at room temperature for 1 hour. d. Discard the primary antibody, wash 3 times with PBST, add FITC-labeled goat anti-mouse IgG (1:2000), and incubate at room temperature in the dark for 1 hour. d. Discard the secondary antibody, wash 3 times with PBST, add 250μL / well of DAPI staining solution, and incubate at room temperature for 10 minutes. e. After staining the nucleus, wash 3 times with PBST and observe under a fluorescence microscope. Figure 5 As shown, the anti-ORFV F1L protein monoclonal antibody can specifically recognize ORFV-infected cells and show green fluorescence, while blank cells have no fluorescent signal.

[0068] (3) The antibody subtype was identified using a monoclonal antibody subtype identification kit (Proteintech product). The results showed that the heavy chain of the monoclonal antibody was IgG1 and the light chain was a Kappa chain ( Figure 6 ).

[0069] Example 3

[0070] Sequencing of anti-ORFV F1L protein monoclonal antibodies

[0071] Cultured hybridoma cells ORF059-A4B9 were centrifuged at 1000 rpm for 5 minutes, and the cell pellet was collected. Total cellular RNA was extracted using Trizol and then reverse-transcribed into cDNA. The heavy and light chain variable region sequences were amplified using mouse monoclonal antibody variable region primers (purchased from a commercial company), and the PCR products were sequenced to obtain the heavy and light chain variable region sequences of the monoclonal antibody.

[0072] The amino acid sequence of the light chain variable region is:

[0073] DIVMTQSTASLAVSLGQRATISCRASEQVDNYPFSFLNWFQQKPGQPP KLLIYAASGQNSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQTKEVP PTFGGGTKLEIK, SEQ ID No. 3.

[0074] The amino acid sequence of the heavy chain variable region is:

[0075] QVQLEESGAEVVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLE WIGRIDPVNGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCA RQFDYWGQGTTLTVSS, SEQ ID No. 4.

[0076] Light chain CDR1: RASEQVDNYPFSFLN, SEQ ID No. 5.

[0077] Light chain CDR2: AASGQNS, SEQ ID No. 6.

[0078] Light chain CDR3: QTKEVPPTF, SEQ ID No. 7.

[0079] Heavy chain CDR1: TYMHWV, SEQ ID No. 8.

[0080] Heavy chain CDR2: DPVNGNTKYGPKFQGKA, SEQ ID No. 9.

[0081] Heavy chain CDR3: RQFD, SEQ ID No.10.

[0082] Example 4

[0083] Expression and purification of eukaryotic F1L protein

[0084] (1) Construction of the eukaryotic expression vector pFastBac-His-F1L

[0085] First, the baculovirus expression vector pFastBac was modified, and a tag containing 8 histidines (His) was inserted between its BmaHI and EcoRI restriction sites to construct the recombinant vector pFastBac-His to facilitate subsequent protein purification. Then, the F1L gene (nucleotide sequence is shown in SEQ ID No. 2) was inserted between the EcoRI and HindIII restriction sites of the recombinant vector pFastBac-His. To this end, two pairs of primers were designed (Table 1): primers BamHI-His-U and EcoRI-His-L contain sticky ends for the nucleases BamHI and EcoRI. This pair of primers can be directly used in the ligation reaction after denaturation and annealing. Primers EcoRI-F1L-U and HindIII-F1L-L amplify the F1L gene.

[0086] Table 1 Primer information

[0087]

[0088] The pFastBac vector was double-digested with restriction endonucleases BamHI and EcoRI, and the digestion product was recovered using a DNA recovery kit. The pFastBac vector was then ligated with the His-tag fragment using the following ligation system: 1 μL of T4 DNA ligase, 1 μL of buffer, 6 μL of the His-tag fragment, and 2 μL of the digested pFastBac vector. The ligation was performed in a 20°C thermostated metal bath for 2 hours. The ligation product was transformed into Top10 competent cells, evenly plated on LB plates containing ampicillin / gentamicin, and incubated in an inverted incubator at 37°C. Single white colonies were selected, shaken, and sent to a biotechnology company for sequencing verification. The correct bacterial liquid verified by sequencing was expanded and cultured. 3 mL of LB liquid medium containing ampicillin / gentamicin was taken and cultured overnight at 37°C in a shaker at 220 rpm. The recombinant plasmid was extracted using the rapid plasmid extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the recombinant vector pFastBac-His containing 8 His in the multiple cloning site region.

[0089] Using pET-28a-F1L as a template, primers EcoRI-F1L-U and HindIII-F1L-L were used to amplify the F1L gene with restriction sites EcoRI and HindIII. The amplified F1L gene and the pFastBac-His plasmid were double-digested with restriction endonucleases EcoRI and HindIII, respectively. After digestion products were recovered, the DNA concentration was measured and ligated using T4 DNA ligase at the appropriate ratio, using the same ligation system and conditions as previously described. The ligation product was transformed into Top10 competent cells, evenly plated on LB plates containing ampicillin / gentamicin, and incubated in an inverted state at 37°C. Once white colonies appeared on the plates, single clones were selected for PCR verification. Positive clones were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The clones that were sequenced correctly were expanded and the recombinant plasmid pFastBac-His-F1L was extracted.

[0090] (2) Preparation and identification of baculovirus

[0091] ① Transformation of recombinant plasmids and blue-white screening. The recombinant plasmid pFastBac-His-F1L was transformed into DH10Bac competent cells. The transformed bacterial solution was spread onto LB plates containing three antibiotics (kanamycin 50μg / mL, gentamicin 7μg / mL, tetracycline 10μg / mL, Bluo-gal 100μg / mL, IPTG 40μg / mL). The plates were inverted and incubated overnight at 37°C for at least 30 hours. White colonies were picked and transferred to 3mL of LB medium containing three antibiotics (kanamycin, gentamicin, and tetracycline) and incubated overnight at 37°C with shaking at 200 rpm. The recombinant baculovirus plasmid Bacmid-His-F1L was extracted using the Bacmid extraction kit from Beyotime Biotechnology according to the manufacturer's instructions.

[0092] ② Preparation and identification of P1 and P2 generation baculovirus

[0093] First, Sf9 cells were revived and cultured. A portion of the Sf9 cells were cultured horizontally in a 27°C incubator for transfection with Bacmid-His-F1L. A portion of the Sf9 cells were placed in a shaker flask and cultured at 27°C in a shaker at 150 rpm for later inoculation and collection of P2 generation virus. Once the horizontally cultured Sf9 cells reached approximately 70% confluency, a mixture of transfection reagent (6 μL Lipofectamine 3000) and Bacmid DNA (2 μg Bacmid-His-F1L) was prepared. The mixture was allowed to stand at room temperature for 15 minutes before being added to the wells of a 6-well plate and gently mixed. After transfection, the cells were cultured for 4-5 days, and the cells were observed daily for cellular status (e.g., enlargement, levitation, etc.) and for the presence of intracellular green fluorescence. If the cells exhibited fluorescence, were significantly enlarged, and partially levitated, this indicated that the cells were producing P1 generation virus. The cell supernatant was then collected, representing the P1 generation baculovirus. Sf9 cells were inoculated with P1 baculovirus at an MOI of 0.1 and cultured on a shaker. After 24 hours of infection, 1 ml of cells were harvested every 12 hours, centrifuged at 6000 rpm for 2 minutes, resuspended in 120 μL of PBS, and boiled in 30 μL of 5× Loading Buffer for 5 minutes to prepare protein samples. Protein samples were then frozen at -20°C and stored until ready for use. At 96 hours of harvest, the remaining cells in the shaker flask were centrifuged at 6000 rpm for 3 minutes, and the supernatant was harvested as the P2 baculovirus. The cell pellet was resuspended in an appropriate amount of PBS and sonicated. 40 μL of total cell protein was collected, followed by centrifugation at 12000 rpm for 5 minutes, and 40 μL of supernatant protein was collected. Finally, the protein pellet was resuspended in the same proportions and 40 μL of the total cell protein, supernatant protein, and precipitated protein were collected, respectively. Protein samples were prepared by boiling each protein with 10 μL of 5× Loading Buffer for 5 minutes. The protein samples collected at each time phase previously were subjected to SDS-PAGE and Coomassie Brilliant Blue staining analysis together with the final protein sample, and were identified by Western blot using F1L monoclonal antibody as the primary antibody.

[0094] like Figure 7 As shown in Figure A, F1L protein expression gradually increased with culture time from 36 to 84 hours after inoculation with P1 baculovirus, but decreased slightly at 96 hours. Solubility analysis showed that the eukaryotic F1L protein was primarily present as inclusion bodies, although a small amount of F1L protein was also expressed soluble.

[0095] (3) Large-scale expression and purification of eukaryotic F1L protein

[0096] Take 200 ml of serum-free SFM medium, add appropriate amount of Sf9 cells, and culture on a shaker at 27°C. When the cell density reaches 1×10 6 / ml, inoculated with P2 baculovirus at an MOI of 0.1. After inoculation, cells were cultured on a shaker until the maximum expression of the F1L protein was achieved. The cell pellet was harvested by centrifugation, resuspended and washed once with PBS, and the supernatant discarded. The cell pellet was resuspended in 30 mL of Binding Buffer and placed in a 50 mL centrifuge tube. Ultrasonic disruption was performed for 30 minutes, with a 5-second sonication interval and a 5-second pause.

[0097] Considering that soluble proteins are easier to purify, Ni column affinity purification was used to purify the target protein F1L from the cell lysate supernatant. The specific purification steps were referred to the instructions of the His tag protein purification kit of Biyuntian Biotechnology Co., Ltd. SDS-PAGE showed that the purified eukaryotic F1L protein was of high purity and had no obvious impurities ( Figure 7 B).

[0098] Example 5

[0099] Establishment of a blocking ELISA method based on anti-ORFV F1L protein monoclonal antibody

[0100] 1. Horseradish peroxidase (HRP)-labeled anti-ORFV F1L protein monoclonal antibody

[0101] ① Label the purified antibody with HRP

[0102] The purified anti-ORFV F1L protein monoclonal antibody was taken and horseradish peroxidase was coupled to the purified monoclonal antibody using a simple sodium periodate method.

[0103] ② Determination of the titer of monoclonal antibodies against ORFV F1L protein

[0104] The titer of monoclonal antibodies against the ORFV F1L protein was determined using an indirect ELISA method. First, the purified eukaryotic F1L protein was diluted to a concentration of 1 μg / ml in carbonate buffer (pH 9.6). 100 μL / well of the ELISA plate was added and coated overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed three times with PBST (pH 7.4). 100 μL of PBS containing 2% BSA was added to each well, blocked at 37°C for 1 hour, and washed three times with PBST. HRP-labeled anti-ORFV F1L protein monoclonal antibody and negative control (goat anti-mouse IgG-HRP) were diluted with PBS to 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, and 1:204800 times, and added to the ELISA plate at 100 μL / well. Incubate at 37°C for 1 hour and wash 3 times with PBST. Add 100 μL / well of single-component TMB colorimetric solution and incubate at 37°C for 5 minutes. Add 50 μL of 2M H2SO4 to each well to stop the reaction, and measure the OD value with a microplate reader. 450 The titer of HRP-labeled anti-ORFV F1L protein monoclonal antibody was determined using a P / N value of ≥ 2.1.

[0105] The results show that ( Figure 8 ): The highest ELISA titer of HRP-labeled anti-ORFV F1L protein monoclonal antibody was 1:5.12×10 4 .

[0106] 2. Establishment of an anti-ORFV F1L protein monoclonal antibody blocking ELISA assay

[0107] ①Optimal coating concentration of antigen and optimal dilution of enzyme-labeled monoclonal antibody

[0108] The matrix titration method was used to determine the optimal antigen concentration and enzyme-labeled monoclonal antibody dilution factor. The purified eukaryotic F1L protein was diluted to 4μg / mL, 2μg / mL, 1μg / mL, and 0.5μg / mL using the coating solution, 100μL / well, and coated overnight at 4°C. Two columns were coated for each concentration; PBS solution containing 2% BSA was blocked at 37°C for 1h; ORFV negative serum and positive serum were diluted 1:2 with PBS containing 2% BSA, 100μL / well, and incubated at 37°C for 1h; the plate was washed 3 times with PBST, and HRP-labeled anti-ORFV F1L protein monoclonal antibody was diluted with PBS at a ratio of 1:1000, 1:2000, 1:4000, and 1:8000, 100μL / well, overlapped with ORFV negative and positive serum, and incubated at 37°C for 1h; the remaining steps were the same as the above basic operating procedures. According to OD 450 The blocking rate (PI) is calculated using the following formula:

[0109] Percent of inhibition (PI) = (negative OD 450 - Positive OD 450 ) / negative OD 450 ×100%

[0110] The reaction conditions with the highest blocking rate were used as the optimal coating concentration of antigen and the optimal dilution of enzyme-labeled monoclonal antibody.

[0111] Table 2 Optimal antigen coating concentration and monoclonal antibody dilution

[0112]

[0113]

[0114] The results are shown in Table 2. The highest PI was 92.8%. At this time, the optimal coating concentration of eukaryotic F1L protein was 1 μg / mL, and the optimal dilution of HRP-labeled anti-ORFV F1L protein monoclonal antibody was 2000-fold.

[0115] ②Optimization of other reaction conditions

[0116] Based on the determined antigen coating concentration and enzyme-labeled monoclonal antibody dilution, similar methods are used to determine the antigen coating conditions, blocking solution and blocking conditions, test serum dilution and reaction conditions, sample diluent, enzyme-labeled monoclonal antibody reaction conditions, and substrate reaction conditions.

[0117] The optimal reaction conditions for blocking ELISA are summarized in Table 3.

[0118] Table 3 Optimal reaction conditions for blocking ELISA

[0119]

[0120] 3. Determination of critical value

[0121] According to the determined optimal reaction conditions, 50 ORFV-negative sera were tested, the average blocking rate PI of negative sera was calculated, and the critical values ​​A and B were calculated according to the formula.

[0122] Critical value A = average blocking rate of negative serum (PI value) + 3 × standard deviation (SD value);

[0123] Critical value B = average blocking rate of negative serum (PI value) + 2 × standard deviation (SD value).

[0124] If the blocking rate (PI) of the sample to be tested is greater than the critical value A, it is judged as ORFV antibody positive; if the blocking rate (PI) of the sample to be tested is less than the critical value B, it is judged as ORFV antibody negative; if the critical value A is greater than the blocking rate (PI) of the sample to be tested and greater than the critical value B, it is judged as suspicious and requires re-examination. If the re-examination is still suspicious, it is judged as ORFV antibody negative.

[0125] According to the experimental data, the average PI of 50 negative sera was 10.26%, the standard deviation was 9.07%, the critical value A = 37.47%, and the critical value B = 28.40%.

[0126] 4. Sensitivity evaluation

[0127] The experiment was conducted under the determined optimal conditions, and 100 samples of ORFV vaccine-immune sera were tested by blocking ELISA and indirect immunofluorescence assay (IFA). The IFA test for immune sera here is basically the same as the aforementioned IFA method, but the primary antibody is vaccine-immune sera and the corresponding FITC-labeled rabbit anti-goat secondary antibody. The blocking ELISA determines whether the serum is negative or positive by calculating the PI value and comparing it with the IFA results to determine the sensitivity of the blocking ELISA method.

[0128] The results (Table 4) are as follows: Of the 100 samples, IFA detected 92 positive sera and 8 negative sera. Blocking ELISA detected 94 positive sera and 6 negative sera. Using the serum background as the standard, the blocking ELISA method had a compliance rate of 94%, while the IFA method had a compliance rate of 92%. The blocking ELISA method established in the present invention was slightly more sensitive than the IFA method. Using the blocking ELISA results as the standard, the relative compliance rate of the two methods was 97.87% (92 / 94).

[0129] Table 4 Coincidence between blocking ELISA and indirect IFA

[0130]

[0131] Four sera positive for high PI values ​​detected by blocking ELISA were selected and serially diluted two-fold. The titers of the serum samples were determined using both the blocking ELISA and IFA methods. The results (Table 5) show that the highest titers of these four sera measured by the blocking ELISA method were 128, 64, 64, and 32, while the corresponding titers measured by IFA were 64, 64, 32, and 16, indicating that the blocking ELISA method established in this invention is 1 to 2 times more sensitive than the indirect IFA method.

[0132] Table 5 Sensitivity test results

[0133]

[0134] 5. Specificity evaluation

[0135] The blocking ELISA established by the present invention was used to detect positive sera of foot-and-mouth disease virus (FMDV), peste des petits ruminants virus (PPRV), goat pox virus (GTPV) and oropharyngeal follicle virus (ORFV) stored in the laboratory, as well as negative serum controls, to evaluate the specificity of the blocking ELISA method.

[0136] The results are as follows ( Figure 9 ): After testing, only the ORFV positive serum tested positive, while other virus serum samples and negative serum controls were all negative, indicating that this method has no cross-reaction with other common sheep virus antibodies and has good specificity.

[0137] 6. Repeatability evaluation

[0138] In order to verify the stability of the blocking ELISA method of the present invention, 10 serum samples were tested using ELISA plates coated with the same batch and ELISA plates coated with different batches. Each sample was tested 3 times, and the blocking rate PI and its coefficient of variation were calculated 3 times. Test the stability of this method.

[0139] The results showed (Table 6) that the coefficient of variation (CV) values ​​of the intra-batch and inter-batch repeated tests were both less than 10%, indicating that the blocking ELISA method of the present invention has good repeatability.

[0140] Table 6 Batch stability test results

[0141]

[0142] Example 6

[0143] Assembly of anti-ORFV F1L protein monoclonal antibody blocking ELISA kit

[0144] The kit comprises the following components:

[0145] 1. 96-well ELISA plate.

[0146] 2. An HRP-labeled monoclonal antibody against the ORFV F1L protein, secreted by the hybridoma cell line ORF059-A4B9. The light chain variable region sequences of the monoclonal antibody are SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7. The heavy chain variable region sequences of the monoclonal antibody are SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10.

[0147] 3. The baculovirus-expressed F1L protein was prepared according to the method of Example 4.

[0148] 4. Coating solution: carbonate buffer (pH 9.6).

[0149] 5. Blocking solution: PBS (pH 7.4) containing 2% w / v BSA.

[0150] 6. Sample diluent: PBS (pH 7.4) containing 2% w / v BSA.

[0151] 7. Washing solution: PBST (0.05% Tween-20, pH 7.4).

[0152] 8. Color developing solution: single-component TMB color developing solution.

[0153] 9. Stop solution: 2 mol / L H2SO4.

[0154] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0155] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A monoclonal antibody against the F1L protein of oropharyngeal canker sore virus, characterized in that: The monoclonal antibody includes a light chain and a heavy chain, wherein the light chain includes a light chain CDR1, a light chain CDR2 and a light chain CDR3 as shown in the amino acid sequences of SEQ ID No. 5 to SEQ ID No. 7; and the heavy chain includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 as shown in the amino acid sequences of SEQ ID No. 8 to SEQ ID No.

10.

2. The monoclonal antibody according to claim 1, wherein The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 3, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No.

4.

3. Use of the monoclonal antibody according to any one of claims 1 or 2 in the preparation of a product for detecting ORFV antibodies.

4. The use according to claim 3, characterized in that The product is an ELISA detection kit.

5. An antibody blocking ELISA detection kit, characterized in that: Including enzyme-labeled antibodies, The enzyme-labeled antibody is the monoclonal antibody according to any one of claims 1 or 2 labeled with HRP.

6. The kit according to claim 5, wherein Also included is the F1L protein.

7. The kit according to claim 6, wherein It also includes coating solution, blocking solution, washing solution, sample diluent, color developing solution and stop solution.

8. The kit according to claim 7, wherein The coating solution is a carbonate buffer solution with a pH of 9.6; The blocking solution is PBS containing 2% w / v BSA; The washing solution is PBST at pH 7.4; The sample diluent is PBS containing 2% w / v BSA; The color developing solution is a single-component TMB color developing solution; The stop solution is 2M H2SO4.

9. Use of the kit according to any one of claims 5 to 8 in the preparation of an ORFV antibody detection reagent.

Images