Monoclonal antibody 3B6 for resisting swine pox virus p35 protein and application of monoclonal antibody 3B6
By preparing monoclonal antibody 3B6 against porcine poxvirus p35 protein, the gap in porcine poxvirus diagnosis was solved, efficient and highly specific detection was achieved, and the prevention and control of the porcine poxvirus epidemic and the healthy development of the pig industry was supported.
Patent Information
- Application Number
- CN202510406835.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-07-11
AI Technical Summary
At present, there are no effective monoclonal antibodies for the diagnosis of pig pox virus, which has affected the prevention and control of pig pox virus epidemic and the healthy development of the pig industry.
It provides a monoclonal antibody 3B6, which is an anti-pig poxvirus p35 protein. The heavy chain and light chain each have three CDR subregions that constitute antigen binding sites. It is used to prepare an ELISA detection kit, an immunochromatography diagnostic kit or an immunohistochemistry kit for detecting pig poxvirus, and performs rapid detection of pig poxvirus through serological detection.
Monoclonal antibody 3B6 has high affinity, strong immunity and high specificity, and can detect pig pox virus with high sensitivity, support the prevention and control and purification of SWPV epidemics, and promote the healthy development of the pig industry.
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Figure CN120289625A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to a monoclonal antibody 3B6 against porcine poxvirus p35 protein and its application. Background Art
[0002] Swine pox is a mild, acute and typical skin disease caused by the infection of Swine pox virus (SWPV). It mainly causes skin damage, forming pox, vesicles, pustules, scabs and other symptoms, and is known as the "smallpox" in pigs. Swine pox is a relatively common viral disease in the process of pig breeding, and has been reported in many regions around the world, including the United States, Russia, Europe, India and Brazil. In China, it mainly shows sporadic occurrence. Although the mortality rate of infected pigs is not high, it has an important negative impact on the production performance of the body, and is likely to affect the economic benefits of farms.
[0003] Monoclonal antibody is an immunoglobulin designed against a specific epitope on an antigen and is an important tool for many molecular immunology studies. Monoclonal antibodies have become a key component of a large number of laboratory diagnostic tests. Monoclonal antibodies have the advantages of highly uniform physical and chemical properties, strong specificity, single biological activity, and ability to reproduce indefinitely. They have played a huge role in research such as disease prevention, quarantine, diagnosis, treatment and epidemiological investigation. However, there is currently no research application report on monoclonal antibodies against porcine poxvirus.
[0004] Therefore, if new diagnostic techniques can be found, it is of great significance for the prevention, control and purification of SWPV epidemic and the healthy development of the pig industry chain. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a monoclonal antibody 3B6 against porcine poxvirus p35 protein and its application.
[0006] To solve the technical problem, the solution of the present invention is:
[0007] Provide a monoclonal antibody 3B6 against porcine poxvirus p35 protein. Each of the heavy chain and light chain of the monoclonal antibody has three CDR sub-regions that form antigen-binding sites, wherein: the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
[0008] In the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.
[0009] In the present invention, the heavy chain variable region of the monoclonal antibody is encoded by the nucleic acid sequence shown in SEQ ID NO:9, and the light chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO:10.
[0010] In the present invention, the constant region of the heavy chain is of IgG2a type, and the constant region of the light chain is a κ chain.
[0011] The present invention further provides an application method of the aforementioned monoclonal antibody, which is to use the monoclonal antibody to prepare a kit for detecting swine pox virus.
[0012] The present invention also provides a kit for detecting swine pox virus, which comprises any one of the aforementioned swine pox virus monoclonal antibodies.
[0013] As a preferred embodiment of the present invention, the kit is an ELISA test kit, or any one of an immunochromatographic diagnostic kit and an immunohistochemical kit using an immunochromatographic test strip; when the kit is used, the detection method is a serological test. That is, the kit is used in a serological test, and the sample to be tested is venous blood.
[0014] As a preferred embodiment of the present invention, the kit comprises: an ELISA plate coated with rHis-p35 protein, the monoclonal antibody of claim 1 or 2 labeled with HRP, a positive control serum and a negative control serum.
[0015] As a preferred embodiment of the present invention, the kit further comprises PBS buffer, PBST buffer, blocking solution, antibody diluent, coating solution and stop solution.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] 1. The monoclonal antibody 3B6 provided by the present invention has high affinity, strong immune ability, good specificity and uniformity, and has the advantages of stability, high activity, strong affinity, etc.
[0018] 2. The monoclonal antibody provided by the present invention has high sensitivity and strong specificity, and can be used in SWPV diagnostic kits; it can provide new technical support for the rapid detection of SWPV antibodies, which is of great significance for promoting the healthy development of the pig industry and the prevention, control and purification of SWPV epidemics. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is an analysis of serum antibody titers after mice were immunized with SWPV p35 protein in Example 1.
[0020] Figure 2 They are the cloned hybridoma cells in Example 1.
[0021] Figure 3 They are the Western blot results of monoclonal antibody specificity analysis in Example 2.
[0022] Figure 4 They are the indirect immunofluorescence results of monoclonal antibody specificity analysis in Example 2.
[0023] Figure 5 They are the results of detection by establishing a competitive ELISA system using the kit of the present invention. Specific implementation plan
[0024] Vaccinia virus, as the prototype virus of poxviruses, has been relatively well studied. Vaccinia virus P35 protein is an immunodominant antigen protein on the surface of the envelope of intracellular mature virus particles (IMV). It is the main protein that induces the production of antibodies, especially plays an important role in generating the secondary immune response, and is involved in the adsorption of the virus to host cells, and plays a major role in aspects such as binding, assembling mature virus particles, virus virulence, and immunogenicity. However, there has been no report on the function of swinepox virus P35 protein so far.
[0025] The R & D team of the applicant found during a long-term in-depth study that the P35 protein is a major structural protein on the surface of the envelope of intracellular mature virus particles (IMV) of swinepox virus. It is encoded by the 71st open reading frame, located at 63768bp - 64742bp of the genome, with a size of 975bp, encoding a total of 324 amino acids, and the protein size is about 37.8 kDa. Therefore, the applicant took this as the starting point of the innovative design scheme and selected this protein as the target protein to prepare monoclonal antibodies. The monoclonal antibodies obtained on this basis have good specificity, good homogeneity, and can be supplied infinitely, providing new technical support for the rapid detection of SWPV antigens and antibodies.
[0026] In order to better illustrate the technical solution of the present invention, the following will describe the technical solution in combination with examples and drawings, but not limited thereto.
[0027] Example 1 Preparation of hybridoma cells and screening of monoclonal antibodies
[0028] 1.1 Animal immunization
[0029] In the present invention, the purified swinepox virus rGST-p35 protein is used as an antigen. The recombinant protein rGST-p35 is expressed as inclusion bodies in the precipitate, and the recombinant protein is purified by protein gel cutting.
[0030] The rGST-p35 protein was emulsified with an equal volume of Freund's (in)complete adjuvant and then used to immunize SPF-grade female BALB / c mice aged 6-8 weeks by intraperitoneal and subcutaneous multi-point injection. The immunization process is shown in Table 1.
[0031] Table 1 Immunization process
[0032]
[0033] Figure 1 For the analysis of serum antibody titers in mice immunized with the SWPV p35 protein in Example 1, numbers 1-5 refer to serum samples from 5 different mice respectively.
[0034] 1.2 Preparation of hybridoma cells
[0035] One mouse with the best immunization effect was selected, bled, sacrificed by cervical dislocation, soaked and disinfected in 75% ethanol, and its splenocytes were fused with SP2 / 0 cells. The washed SP2 / 0 cells and splenocytes were added to a 15 mL centrifuge tube at a ratio of 1:10, 1640 culture medium was added to 10 mL, gently pipetted and mixed evenly, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cell pellet was gently flicked with a finger to disperse it, and then the cells were placed in a 37 °C water bath in a beaker. 1 mL of pre-warmed PEG cell fusion agent at 37 °C was slowly added and added completely within 1 min while gently shaking the centrifuge tube and pipette, and then left for 90 s. Subsequently, 1 mL of pre-warmed complete culture medium was added and added completely within 1 min while shaking, left standing for one minute, and repeated three times. Finally, the pellet was resuspended with 10 mL of 1640 medium, centrifuged at 800 rpm for 10 min, the supernatant was discarded, and the pellet was resuspended with HAT complete medium. First, resuspend with a small dose, gently disperse it, and then make up to 100 mL, and mix evenly. Spread it into a 96-well cell culture plate containing feeder cells, 200 μL per well, change the medium after 7 days, and observe the cell growth status.
[0036] 1.3 Screening of hybridoma cells
[0037] After each indirect ELISA detection, positive cell wells were picked out, and the cells were serially diluted according to the following steps to screen for positive monoclonal hybridoma cells:
[0038] (1) Take a 96-well cell culture plate, add 100 μL of HAT cell culture medium to each well, and add 150 μL to well A1; take a 48-well cell culture plate and add 1 mL of HAT cell culture medium to each well according to the number of positive cell wells;
[0039] (2) According to the iELISA test results, observe and mark the positive cell wells under the microscope. Discard the supernatant in the wells, wash twice with PBS, add 200 μL of cell culture medium, and gently blow down the cells at the bottom of the wells. Take 50 μL of the cell suspension and add it to well A1 of the above 96-well cell culture plate, and mix gently. Add the remaining suspension to the above 48-well cell culture plate, and successively passage and culture until the 6-well cell culture plate and then freeze for standby to prevent the loss of positive cells during the screening process;
[0040] (3) Take 100 μL of the cell suspension from well A1 above and add it to well B1. After mixing, successively add it to wells C1-H1 for 2-fold serial dilution, and make up the culture medium to 200 μL in each well. Use an 8-channel pipette to separately aspirate 100 μL of the cell suspension from wells A1-H1 in the first row and add it to the second row. After mixing, successively add it to rows 3-12 for 2-fold serial dilution, and make up the culture medium to 200 μL in each well, and culture under the conditions of 37 °C and 5% CO2;
[0041] (4) After culturing for 3 days, mark the monoclonal cell wells (only one cell cluster in each well) under the inverted microscope. Discard 100 μL of the cell culture supernatant from each well, and add an equal amount of HT cell culture medium for half-medium change. After 6 days, take 110 μL of the cell culture supernatant for standby, and make up with 120 μL of HT culture medium. Perform iELISA detection on the cell culture supernatant and perform the second detection after 2 days;
[0042] (5) Take the monoclonal cell wells with both detections being positive, having a higher OD450 value, and good cell growth status, and perform the second subcloning according to the above steps. Each positive hybridoma cell is subcloned 3-4 times until all the cell wells in the plate are positive and in a stable state;
[0043] The selected hybridoma cells are successively passaged from the 96-well cell culture plate to the 48-well, 24-well, 12-well, and 6-well plates and then transferred to a cell culture dish for expansion culture. During this period, gradually replace the HT culture medium with 20% FBS cell culture medium, and freeze the cells after the state is stable.
[0044] Figure 2 They are the cloned hybridoma cells in Example 1.
[0045] Specificity analysis of monoclonal antibodies in Example 2
[0046] 2.1 Western blot analysis (WB) of monoclonal antibodies
[0047] (1) Sample preparation: Take the rGST-p35, rGST, rHis-p35 or rHis protein solution, add 4xSDS-PAGE Loading Buffer and mix well, and keep it at 100 °C in a water bath for 10 min for standby.
[0048] (2) Electrophoresis: Perform 12% SDS-PAGE gel electrophoresis with a sample loading volume of 10 μL per well.
[0049] (3) Membrane transfer: After electrophoresis, transfer the gel to a PVDF membrane.
[0050] (4) Blocking: Block with 5% skim milk for 2 h, and wash the membrane 3 times with TBST, 10 min each time.
[0051] (5) Primary antibody: Dilute the supernatant of hybridoma cells 1000-fold with TBST, incubate with the PVDF membrane at room temperature for 1 h, and wash the membrane 3 times with TBST, 10 min each time.
[0052] (6) Secondary antibody: Dilute the enzyme-labeled secondary antibody 5000-fold, incubate with the PVDF membrane at room temperature for 1 h, and wash the membrane 3 times with TBST, 10 min each time.
[0053] (7) Exposure: Drop an appropriate amount of ECL chemiluminescent substrate on the membrane and perform exposure analysis using a gel imaging system.
[0054] Figure 3 This is the Western blot result for the specificity analysis of the monoclonal antibody in Example 2. Among them, 1 is the rGST-p35 recombinant protein, 2 is rGST, 3 is the rHis-p35 recombinant protein, and 4 is rHis.
[0055] 2.2 Indirect immunofluorescence analysis (IFA) of monoclonal antibody
[0056] (1) Transfection: Seed 293T cells in a 24-well plate. When they reach 60 - 70% confluence, transfection can begin. Prepare 50 μL of jet PRIME buffer, 0.5 μg of pEE-p35 plasmid, and 1 μL of jet PRIME Reagent solution according to the kit. Mix well and let stand for 10 min, then slowly add the mixture dropwise to the corresponding wells. After thorough mixing, incubate in a 37°C incubator.
[0057] (2) Fixation: After 24 h of transfection, discard the culture medium, add 200 μL of 4% paraformaldehyde, incubate at room temperature for 30 min, and wash 3 times with PBS, 5 min each time.
[0058] (3) Permeabilization: Permeabilize the fixed cells with 0.5% Triton X-100, wash 3 times with PBS at room temperature, 10 min each time.
[0059] (4) Blocking: Add the blocking solution and incubate at 37°C for 30 min, wash 3 times with PBS, 5 min each time.
[0060] (5) Incubate the primary antibody: Dilute the monoclonal antibody at 1:200 and add it to the wells, and incubate at 37 °C for 1 h. At the same time, set up a positive control (his antibody) and a negative control (pEE empty vector);
[0061] (6) Incubate the secondary antibody: Discard the primary antibody and wash 3 times with PBST. Dilute the FITC-labeled goat anti-mouse IgG at 1:2000, add 200 μL to each well, and incubate at 37 °C for 1 h;
[0062] (7) DAPI staining: Add 200 μL of DAPI staining solution and stain at room temperature for 10 min. Discard the DAPI staining solution and wash 3 times with PBST, 5 min each time;
[0063] (8) Observe the results: Observe the results under an inverted fluorescence microscope.
[0064] Figure 4 It is the indirect immunofluorescence result of the monoclonal antibody specificity analysis in Example 2.
[0065] Identification of Hybridoma Cell 3B6 in Example 3
[0066] 3.1 Determination of Antibody Variable Region Sequence
[0067] Collect the cultured cells, add 1 mL of Trizol reagent, repeatedly pipette to lyse the cells, and then extract the total RNA according to the Trizol reagent instruction manual. Use a multifunctional microplate reader to detect the concentration and purity of the extracted RNA. Take 1 μg of RNA and perform reverse transcription using HISCRIPT III All-in-one RT SuperMix Perfect for qPCR from Novoprotein. Take 2 μL of cDNA as the template and perform PCR amplification to obtain the target gene fragment using a high-fidelity enzyme (2xKOD plus Neo). The primer sequences are shown in Table 2 below. PCR reaction conditions: After pre-denaturation at 98 °C for 3 min, perform 30 cycles of amplification. Each cycle condition is: denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s, extension at 68 °C for 5 s, and finally complete extension at 68 °C for 5 min. Take the PCR product for 1% agarose gel electrophoresis.
[0068] Purify the PCR product with a size of about 300 bp according to the agarose gel recovery / PCR purification kit instruction manual. Use pMD18-T as the cloning vector and follow the TA cloning kit pMD TMThe instruction manual of the 18-T Vector Cloning Kit ligates the target genes of the heavy and light chain variable regions to pMD18-T respectively. The recombinant products are transformed into DH5α competent cells, and then spread on LB plates containing 100 μg / ml ampicillin, and incubated overnight at 37°C. 5-10 monoclonal colonies of the heavy and light chains are picked from the plates with fresh sterilized pipette tips and placed into 1 mL LB medium (containing 50 μg / mL ampicillin), and cultured at 37°C with 300 rpm for 8-12 h, and then the bacterial liquid is sent for sequencing with the universal primers M13F and M13R.
[0069] Table 2 Primer Sequence Table
[0070]
[0071]
[0072] The nucleotide sequence SEQ ID NO:9 of the heavy chain variable region and the nucleotide sequence SEQ ID NO:10 of the light chain variable region encoding the monoclonal antibody 3B6 are obtained by sequencing. After converting the sequences into amino acid sequences, the amino acid sequence SEQ ID NO:7 of the heavy chain variable region and the amino acid sequence SEQ ID NO:8 of the light chain variable region of the monoclonal antibody 3B6 are obtained. Through analysis, the sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and the sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively. It is determined that the monoclonal antibody 3B6 belongs to the κ-type Ig antibody.
[0073] 3.2 Identification of the Linear Epitope Recognized by the Antibody
[0074] The p35 protein is truncated step by step and cloned into the pGEX-6P-1 vector. Using the prepared monoclonal antibody 3B6 as the primary antibody, the antigen epitope region recognized by the p35 monoclonal antibody is identified by Western blot. First, three truncated p35 recombinant fragments p35 1-94aa (p1), p35 85-187aa (p2), p35 178-282aa (p3) are designed, which have 10 amino acid overlaps and are expressed in the form of short peptide GST fusion proteins. At the same time, the expression of each fusion protein is analyzed by Western blot. The results show that 3B6 reacts with the truncated body p2. Based on this preliminary result, in order to further determine the antigen epitope recognized by the 3B6 monoclonal antibody, 4 truncated bodies p35 are constructed by gradually adding 16 amino acids on the basis of the truncated body p35 178-282aa (p3)114-282aa (p7), p35 130-282aa (p6), p35 146-282aa , p35 162-282aa , identified by Western blot reactivity with the 3B6 monoclonal antibody, the results showed that the antibody reacted with the truncated p35 114-282aa (p7).
[0075] To accurately determine the epitope of the p35 protein recognized by the monoclonal antibody, the truncated p35 protein p35 114-129aa (p7) was used. And Western blot was performed with the mAb. The results showed that the deletion of the 114th amino acid residue (K) from the C-terminus of 3B6 significantly affected the reactivity; the deletion of the 119th amino acid (K) from the N-terminus also prevented the recognition by the mAb. Therefore, it can be concluded that 114KDPEKK119 is the minimum linear epitope bound by the 3B6 mAb.
[0076] Example 4 Kit and Application Method
[0077] 4.1 Prepare the reagents for the ELISA kit according to the following formula:
[0078] (1) PBS buffer, 1.44 g of disodium hydrogen phosphate heptahydrate, 0.24 g of potassium dihydrogen phosphate, 8 g of sodium chloride, 0.2 g of potassium chloride, add 800 m of deionized water and dissolve thoroughly, transfer to a volumetric flask and make up to 1 L (PH = 7.4).;
[0079] (2) PBST buffer, add 1 mL of Tween-20 to 1 L of PBS buffer;
[0080] (3) Blocking solution, add 1 g of bovine serum albumin to 100 mL of PBST buffer;
[0081] (4) Antibody diluent, which is PBST buffer;
[0082] (5) Coating solution, 0.75 g of sodium carbonate, 1.46 g of sodium bicarbonate, make up to 500 mL with ddHO;
[0083] (6) Stop solution, slowly add 5.4 mL of 98% concentrated sulfuric acid to 50 mI.
[0084] 4.2 Use the ELISA kit according to the following method:
[0085] (1) Coating: Dilute the antigen protein with the coating solution, 50 ng / well, coat at 37 °C for 2 h;
[0086] (2) Blocking: Block with 1% bovine serum albumin, 300 μL / well, block at 37 °C for 1 h, discard the blocking solution, wash with PBST 3 times, 6 min each time;
[0087] (3) Incubation of serum with HRP-labeled antibody: dilute HRP-labeled monoclonal antibody 1:1000 with PBST buffer, dilute serum 1:5, mix the two 1:1, and incubate at 100 μL / well at 37°C for 2 h. Set up negative and positive control wells at the same time, and wash with PBST three times, 6 min each time.
[0088] (4) Color development: TMB color development solution 100 μL / well, incubate at 37°C in the dark for 12 min;
[0089] (5) Termination: Add reaction termination solution, 100 μL / well, mix well and measure OD immediately 450 nm value.
[0090] Take 5 pig sera (including PEDV PRRSV ASFV CSFV FMDV strain infection), 1 negative pig serum (Negative) and 1 positive pig serum (Positive).
[0091] The competitive ELISA system established above was used for detection, and the results were as follows Figure 5 When PI% is less than 27.71%, it is considered negative; when it is greater than 43.29%, it is considered positive; when PI% is between 27.71% and 43.29%, it is considered suspicious.
[0092] The above results indicate that the SWPV competitive ELISA system established in the present invention can specifically detect SWPV.
[0093] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.
[0094]
[0095]
[0096]
[0097]
Claims
1. A monoclonal antibody 3B6 against porcine poxvirus p35 protein, characterized in that, The heavy chain and light chain of the monoclonal antibody each have three CDR sub-regions that constitute the antigen-binding site, where: the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively.
2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:
8.
3. The monoclonal antibody according to claim 2, wherein The heavy chain variable region of the monoclonal antibody is encoded by the nucleic acid sequence shown in SEQ ID NO:9, and the light chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO:
10.
4. The monoclonal antibody according to claim 1 or 2, characterized in that, The constant region of the heavy chain is of the IgG2a type, and the constant region of the light chain is a κ chain.
5. The application method of any one of the monoclonal antibodies described in claim 1 or 2, characterized in that, It is to use the monoclonal antibody to prepare a kit for detecting swinepox virus.
6. A kit for detecting swinepox virus, characterized in that, The kit includes any one of the monoclonal antibodies against swinepox virus described in claim 1 or 2.
7. The kit according to claim 6, wherein The kit is an ELISA detection kit, or any one of an immunochromatographic diagnostic kit using an immunochromatographic test strip and an immunohistochemistry kit; when using the kit, the detection method is serological detection.
8. The kit according to claim 6, wherein The kit includes: an enzyme-labeled plate coated with rHis-p35 protein, the monoclonal antibody described in claim 1 or 2 labeled with HRP, positive control serum, and negative control serum.
9. The kit according to claim 8, characterized in that, The kit further includes PBS buffer, PBST buffer, blocking solution, antibody diluent, coating solution, and termination solution.