Preparation and application of O / Cathay type foot and mouth disease virus monoclonal antibody and antibody variable region sequence

Abstract

The invention belongs to the technical field of biology, and particularly relates to a mouse O / Cathay type foot and mouth disease virus monoclonal antibody as well as preparation and application thereof. According to the invention, a monoclonal antibody 3G10 aiming at O / Cathay type foot and mouth disease virus is obtained; the prepared monoclonal antibody 3G10 can specifically react with the O / Cathay type foot-and-mouth disease virus, has the advantages of high specificity, sensitivity, low preparation cost and the like, provides a new material for serotype identification and quantification of the O / Cathay type foot-and-mouth disease virus, and also provides a new technical support for controlling epidemic situation transmission of the foot-and-mouth disease. The monoclonal antibody 3G10 provided by the invention can be used for recombination and transformation of a conventional gene engineering or protein engineering method, so that the long-term cryopreservation antibody loss of hybridoma cells is avoided, the optimization of the antibody on gene and protein levels is also facilitated, and the specificity and affinity of the antibody are further improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to preparation of a mouse-derived O / Cathay type foot-and-mouth disease virus monoclonal antibody 3G10 and application thereof in O / Cathay type foot-and-mouth disease virus typing and quantitative ELISA detection method. BACKGROUND

[0002] Foot-and-mouth disease virus (FMDV) belongs to the family of Picornaviridae, and is highly pathogenic and highly contagious in cloven-hoofed animals such as pigs, cattle, sheep, etc. There are 7 serotypes of foot-and-mouth disease virus, including O, A, Asia1, C, SAT1, SAT2 and SAT3, among which O, A and Asia1 are the most widely distributed FMDV serotypes reported by the World Organization for Animal Health (WOAH). O-type foot-and-mouth disease includes eleven topological types such as ME-SA, SEA and CATHAY. The global foot-and-mouth disease epidemic is generally becoming more complex, and there is no cross-protection between serotypes conferred by vaccination or previous infection. The ability to easily mutate is an inherent characteristic of RNA viruses, which enables them to evade antibody neutralization. The emergence of persistent viral mutations and epidemic variants makes it exceptionally challenging to eradicate FMDV. The O-type foot-and-mouth disease virus O-type (CATHAY) prevalent in China in recent years is very different from the virus prevalent previously, and the existing vaccine can no longer provide protection for the new CATHAY virus, so it is urgent to design and construct a new vaccine seed virus and mass-produce it to improve the protection effect. However, there is currently a lack of effective antigen quantification method for O / CATHAY strain, and after updating the O / CATHAY strain antigen in the vaccine components, it is not possible to quickly quantify the content of the old O-type antigen and the O-type (CATHAY) component in the vaccine in a short period of time, which makes it difficult to control the quality and evaluate the efficacy of the vaccine after production.

[0003] Foot-and-mouth disease virus typing refers to determining the specific type of the virus through laboratory detection methods, such as O-type, A-type, Asia-type, etc. Distinguishing O / CATHAY serotype from other types helps to discover foreign transmission, trace the source of the epidemic, and develop more targeted prevention and control strategies. Different types of foot-and-mouth disease virus have different antigenicity, and vaccines and control measures need to be designed for specific types. The distribution and changes of viral antigen epitopes have an important influence on vaccine efficacy, and through typing, it can be ensured that the vaccine is consistent with the prevalent virus type, and immune failure caused by type mismatch can be avoided.

[0004] Currently, the main methods for the typing of foot-and-mouth disease virus are real-time fluorescent RT-PCR, colloidal gold immunochromatography (GICA), ELISA and other methods. Although RT-PCR and GICA methods have advantages in rapid detection, they have problems such as high requirements for reaction environment and certain false positive rate. ELISA has balanced advantages in sensitivity, specificity, quantitative ability, standardization and automation, can reflect the presence of virus particles, and is suitable for detection of large quantities of serum samples. Coating a universal capture antibody for capturing foot-and-mouth disease virus 146S antigen can improve the specific capture of effective antigen (i.e. 146S antigen). Monoclonal antibodies can accurately recognize and bind to trace amounts of antigens, have strong specificity and high sensitivity, and are uniform in properties, have strong repeatability in production process, and are suitable for large-scale production. The establishment of a typing quantitative ELISA method for foot-and-mouth disease virus O type (CATHAY) in inactivated foot-and-mouth disease vaccine using the two methods can improve the convenience of the detection method, make the method more accurate and rapid, and provide a reference for the quantitative determination of antigen content and evaluation of vaccine potency in the vaccine production process. SUMMARY

[0005] In view of the above technical problems, the present application screens a murine anti-foot-and-mouth disease virus monoclonal antibody 3G10, which has the characteristics of strong specificity, good biological activity and strong antigen binding capacity. Using the monoclonal antibody 3G10 and coating a universal capture rabbit antibody, a typing quantitative ELISA method for O / Cathay type is established, which can be used for the serotype diagnosis and accurate quantification of O / Cathay type foot-and-mouth disease virus, and provides a reference for the quality control and potency evaluation after vaccine production, and has important significance in the prevention and control of foot-and-mouth disease. Specifically, the following contents are included:

[0006] In a first aspect, the present application provides an O / Cathay type foot-and-mouth disease virus monoclonal antibody, which comprises an antibody heavy chain and an antibody light chain.

[0007] The variable region CDR of the antibody heavy chain comprises CDR1 as shown in SEQ ID No. 1, CDR2 as shown in SEQ ID No. 2 and CDR3 as shown in SEQ ID No. 3.

[0008] The variable region CDR of the antibody light chain comprises CDR1 as shown in SEQ ID No. 4, CDR2 as shown in SEQ ID No. 5 and CDR3 as shown in SEQ ID No. 6.

[0009] Preferably, the O / Cathay type foot-and-mouth disease virus monoclonal antibody comprises an antibody heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 7 and an antibody light chain variable region with an amino acid sequence as shown in SEQ ID No. 8.

[0010] In a second aspect, the present application provides a nucleic acid molecule encoding the O / Cathay type foot-and-mouth disease virus monoclonal antibody of the first aspect.

[0011] Preferably, the nucleic acid molecule comprises the nucleotide sequences as shown in SEQ ID No. 9 and SEQ ID No. 10.

[0012] In a third aspect, the present application provides an expression vector comprising the nucleic acid molecule of the second aspect.

[0013] In a fourth aspect, the present application provides a recombinant cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.

[0014] In a fifth aspect, the present application provides use of the O / Cathay type foot-and-mouth disease virus monoclonal antibody of the first aspect in the preparation of a product for quantitative detection of O / Cathay type foot-and-mouth disease virus typing.

[0015] In a sixth aspect, the present application provides use of the O / Cathay type foot-and-mouth disease virus monoclonal antibody of the first aspect in quantitative detection of O / Cathay type foot-and-mouth disease virus typing for purposes other than disease diagnosis.

[0016] In a seventh aspect, the present application provides an O / Cathay type foot-and-mouth disease virus typing quantitative detection kit comprising the O / Cathay type foot-and-mouth disease virus monoclonal antibody of the first aspect.

[0017] Preferably, the kit further comprises an enzyme-labeled plate, a blocking solution, a diluent, a washing solution, a color developing agent, and a termination solution.

[0018] In the eighth aspect, the application provides a preparation method of the O / Cathay type foot-and-mouth disease virus monoclonal antibody in the first aspect, and the method is: using PEG1500 to purify inactivated O / Cathay type foot-and-mouth disease virus; using the purified inactivated O / Cathay type foot-and-mouth disease virus as an antigen to immunize Balb / c mice, taking mouse spleen cells to fuse with SP2 / 0 cells to prepare hybridoma cells; screening positive clones by using supernatant of the cells to perform indirect ELISA verification; after three times of subcloning, injecting the hybridoma cells into mice to prepare ascites, and finally purifying the obtained ascites to obtain the O / Cathay type foot-and-mouth disease virus monoclonal antibody 3G10.

[0019] In the ninth aspect, the application provides a preparation method of the O / Cathay type foot-and-mouth disease virus monoclonal antibody in the first aspect, and the method is: connecting the heavy chain variable region and the light chain variable region sequence of the O / Cathay type foot-and-mouth disease virus monoclonal antibody to the antibody heavy chain constant region and the antibody light chain constant region sequence respectively, and inserting into an expression vector to obtain antibody heavy chain and antibody light chain recombinant expression plasmids; co-transfecting the obtained recombinant expression plasmids into expression cells to obtain the O / Cathay type foot-and-mouth disease virus monoclonal antibody.

[0020] The application has the following beneficial effects: the application uses PEG1500 to purify inactivated O / Cathay type foot-and-mouth disease virus, uses the inactivated O / Cathay type foot-and-mouth disease virus as an immunization source to immunize mice, and successfully obtains a monoclonal antibody 3G10 against the O / Cathay type foot-and-mouth disease virus through cell fusion and subcell screening; the prepared monoclonal antibody 3G10 can specifically react with the O / Cathay type foot-and-mouth disease virus, has the advantages of high specificity, sensitivity and low preparation cost, provides new materials for serotype identification and quantification of the O / Cathay type foot-and-mouth disease virus, and provides new technical support for controlling the spread of the foot-and-mouth disease; the application also provides the sequence of the monoclonal antibody 3G10, which can be used for recombinant modification by using a conventional genetic engineering or protein engineering method, avoids loss of the antibody due to long-term storage of the hybridoma cells, is also conducive to optimization of the antibody at the genetic and protein levels, and further improves the specificity and affinity of the antibody. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of the provided drawings.

[0022] Figure 1 Purification results of the monoclonal antibody 3G10;

[0023] Figure 2 WB reaction results of monoclonal antibody 3G10 with inactivated O / Cathay type foot-and-mouth disease virus

[0024] Figure 3 Results of titer determination of prepared monoclonal antibody 3G10

[0025] Figure 4 Results of subtype identification of monoclonal antibody 3G10

[0026] Figure 5 Results of specificity identification of monoclonal antibody 3G10

[0027] Figure 6 Calculation method of antigen content in quantitative detection of O / Cathay type foot-and-mouth disease virus subtyping

[0028] Figure 7 Results of accuracy verification of quantitative detection method of O / Cathay type foot-and-mouth disease virus subtyping

[0029] Figure 8 Results of repeatability verification of quantitative detection method of O / Cathay type foot-and-mouth disease virus subtyping

[0030] Figure 9 Results of specificity verification of quantitative detection method of O / Cathay type foot-and-mouth disease virus subtyping DETAILED DESCRIPTION

[0031] The application will be described in detail below with specific examples, but the scope of protection of the application is not limited to the following examples. Any technical solution that can be thought of by those skilled in the art on the basis of the application and in combination with common knowledge in the art belongs to the scope of protection of the application. In addition, if the specific technical operation steps or conditions are not specified in the examples, they are performed according to the technology or conditions described in the general literature in the art or according to the product instructions. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0032] The light chain is divided into κ chain or λ chain according to the constant region. The subunit structure and three-dimensional configuration of different types of immunoglobulin are well known to those skilled in the art. In the present application, VH is the heavy chain variable region, VL is the light chain variable region, which is divided into κ type and λ type.

[0033] The materials involved in the following examples include: BALB / c mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; HRP Conjugation Kit-Lighting- The reagent kit was purchased from Abeam Company, protein G column filler, Freund's complete adjuvant and incomplete adjuvant were purchased from Sigma Company, mouse monoclonal antibody subtype identification kit was purchased from Proteintech Company; Trizol, DNA fragment recovery kit and plasmid extraction kit were OMEGA products; molecular biology reagents were from Sigma Company; other biochemical reagents were all domestic analytical pure.

[0034] The foot-and-mouth disease virus involved in the following examples includes: O-GXCX-CHA-2018-S strain (GenBank accession number MH791316), O / BY / 2010 (GenBank accession number JN998085), O / MYA98 (GenBank accession number JN998086), OZK / 93 (commercial vaccine virus, reference literature: Bai Xingwen. Molecular basis of phenotype difference of O panasia 1 foot-and-mouth disease virus in China [D]. Chinese Academy of Agricultural Sciences, 2012.), AF / 72 (GenBank accession number MT442614), A / GDMM / 2013 (GenBank accession number KF450794), A / WH / 09 (GenBank accession number JF792355).

[0035] The O / Cathay type foot-and-mouth disease virus described in the following examples takes O-GXCX-CHA-2018-S as an example.

[0036] Example 1 Preparation of mouse-derived O / Cathay type foot-and-mouth disease virus monoclonal antibody

[0037] 1.1 Immunization of mice

[0038] PEG1500 was used to purify inactivated O / Cathay type foot-and-mouth disease virus as inactivated antigen, and the purified O / Cathay type foot-and-mouth disease virus inactivated antigen was diluted to 200 μg / mL with PBS, and emulsified with equal amount of Freund's complete adjuvant. 6 six-week-old female BALB / c mice were taken, and 200 μL of emulsified antigen was injected subcutaneously in multiple points on the back. Two weeks after the first immunization, two booster immunizations were performed with Freund's incomplete adjuvant, and the method was the same as before. One week after the third immunization, tail vein blood was collected to measure the antibody. Mice with OD 450nm >2.0, 0.5 mL of antigen without adjuvant was injected intraperitoneally three days before fusion.

[0039] 1.2 Cell fusion

[0040] The BALB / c mice were bled to prepare positive serum 3 days after the booster immunization. The mice were sacrificed and the spleen was taken aseptically after sterilization and cleaned and the fat was removed. The spleen was cut and ground in incomplete RPMI-1640 and the spleen cells were collected. The spleen cells were mixed with Sp2 / 0 cells at 1:5-1:10, centrifuged, and PEG2000 was added to promote fusion, and then diluted with incomplete RPMI-1640. The fused cells were resuspended in HAT selection medium and inoculated in 96-well plates and cultured at 37°C, 5% CO2.

[0041] 1.3 Screening and subcloning of positive hybridoma cells

[0042] The fused cells were half-changed every 2-4 days (100 μL was discarded and 100 μL of new HAT medium was added). After 7-8 days of observation, the surviving wells were marked. When the hybridoma cells grew to more than 1 / 10 of the bottom of the well, the supernatant was screened for positive wells by ELISA. The cells in the positive wells were transferred to 24-well plates, and after they were fully grown, they were counted, diluted to 10 cells / mL, inoculated into 96-well plates containing feeder cells, and the screening was repeated. At the same time, part of the cells were frozen. Subcloning was performed continuously for 3 times until a single monoclonal hybridoma cell with good reactivity was screened. The monoclonal hybridoma cell with high titer and good reactivity was expanded and frozen.

[0043] 1.4 Preparation of monoclonal antibody and identification of reactivity

[0044] BALB / c female mice of 10-12 weeks were used for pre-stimulation by intraperitoneal injection of 0.5 mL Freund's incomplete adjuvant, and 3x10 6 cells were immunized into the mice 7 days later. The ascites were collected when the mice had abdominal swelling 7-10 days after immunization, and purified using a protein G column. The purification results are shown in Figure 1 , lanes 1, 2, and 3 are mouse ascites, purified flow-through, and purified wash, respectively, and lanes 4-6 are the purified products, i.e., the prepared monoclonal antibody against O-type foot-and-mouth disease inactivated virus, named monoclonal antibody 3G10.

[0045] After electrophoresis of the purified O / Cathay type foot-and-mouth disease virus inactivated antigen, it was transferred to a PVDF membrane and subjected to immunoblotting reaction with the cell supernatant of monoclonal antibody 3G10 to verify the reactivity. The results are shown in Figure 2 , monoclonal antibody 3G10 specifically reacted with the 146s protein of O / Cathay type foot-and-mouth disease inactivated virus.

[0046] Purified O / Cathay type foot-and-mouth disease virus inactivated antigen was coated into ELISA plates at 300 ng per well and incubated overnight at 4°C. After washing with PBST, commercial blocking buffer was added and the plates were blocked overnight. The previously prepared monoclonal antibody 3G10 was serially diluted and added to the coated plates, incubated at 37°C for 1 h, and washed 4 times with PBST. HRP-labeled goat anti-mouse IgG (1:15000 dilution) was added, incubated at 37°C for 1 h, and washed 4 times with PBST. TMB was developed for 15 min, and then stop buffer was added. OD values ​​were read using a microplate reader. 450nm The titer of the monoclonal antibody 3G10 was determined. The results are as follows: Figure 3 As shown, the titer of monoclonal antibody 3G10 is greater than 1:128000.

[0047] The isotype of monoclonal antibody 3G10 was determined using a commercially available monoclonal antibody isotype identification kit. Results are as follows: Figure 4 As shown, the heavy chain of monoclonal antibody 3G10 is of type IgG2a, and the light chain is of type κ.

[0048] Foot-and-mouth disease virus (FMD) of types O / Cathay (O-GXCX-CHA-2018-S), O / MYA98, O / BY / 2010, OZK / 93, AF / 72, A / GDMM / 2013, and A / WH / 09 were coated onto ELISA plates, and the specificity of monoclonal antibody 3G10 was detected using an indirect ELISA method. Results are as follows: Figure 5 As shown, the monoclonal antibody 3G10 described in this application can specifically bind to foot-and-mouth disease virus O-GXCX-CHA-2018-S. This indicates that the monoclonal antibody 3G10 described in this application specifically reacts with O / Cathay type foot-and-mouth disease virus and has high specificity.

[0049] Example 2: Application of monoclonal antibodies in the quantitative detection of O / Cathay type foot-and-mouth disease virus typing

[0050] 2.1 Establishment of a quantitative detection method for foot-and-mouth disease virus (FMD) type O / Cathay

[0051] 2.1.1 Optimization of working conditions for the detection method

[0052] According to HRP Conjugation Kit-Lighting- The kit procedure involves HRP labeling the purified 3G10 capture antibody. The immobilized enzyme-labeled monoclonal antibody 3G10 is diluted 1:15000. The optimal operating conditions for the ELISA method were determined using checkerboard titration: 100 ng of capture antibody protein coating per well and a 3G10 dilution ratio of 1:30000.

[0053] 2.1.2 Detection of standard curve and result calculation method

[0054] Standard curve establishment: The O / Cathay type 146S antigen was diluted by 2 times gradient to prepare standard solution with concentration range of 1 to 600 μg per milliliter, ELISA detection was carried out under the above-mentioned screening conditions, the standard solution was repeatedly detected 6 times by different time and different personnel, the measured OD 450nm value was fitted with the reference product dilution theoretical concentration, the coefficient of determination (R2) was calculated, R2>0.99, the fitting effect was good, and the standard curve was drawn, the selected range of the standard curve was 0.3-2.3, the OD 450nm value was taken as the abscissa x, and the standard corresponding content was taken as the ordinate y, the standard regression curve and equation were drawn: the data area was selected by left mouse button, the chart wizard was inserted by clicking "insert" at the top of Excel, and "scatter plot xy (only with data marker)" was inserted, and "confirm" was clicked. Click the standard point in the chart "add trend line" with the right mouse button, check "polynomial, display formula, display R square value". Thus, the regression curve and equation are obtained in the graph. In order to ensure that the detection OD 450nm of the positive standard after gradient dilution is within the specified range, the antigen concentration range (0.1875 μg / ml-3 μg / ml) corresponding to the selected range of the standard curve is determined, and the initial concentration of the positive standard is 6 μg / ml.

[0055] Result calculation method: the OD 450nm value of the sample between 1.0-2.0 (i.e. x value) is substituted into the regression equation, and the y value of the sample with different dilution is obtained, then the sample concentration C is obtained by multiplying the y value by the corresponding dilution multiple, and finally the average value of the selected sample C value is the antigen concentration of the sample, and the result calculation method is shown in Figure 6 .

[0056] 2.2 Verification of O / Cathay type foot-and-mouth disease virus O type quantitative detection method

[0057] Accuracy verification: the foot-and-mouth disease virus O / Cathay type 146S antigen was diluted into high (600 μg / ml), medium (60 μg / ml) and low (6 μg / ml) three concentrations with PBS, and the established method was used for detection, each concentration of reference was repeatedly detected 3 times, and 2 duplicate holes were set each time. The recovery rate was calculated by the formula [recovery rate / %=(detection value / true value) x 100%]. The results are shown in Figure 7 The detection recovery rate of the two antigens by the method is more than 95%, and the detection accuracy is high.

[0058] Repeatability verification: Take 4 different batches of enzyme-labeled plates, dilute O / Cathay type 146S antigen into high (600 μg / ml), medium (60 μg / ml) and low (6 μg / ml) three concentrations with PBS, respectively, and detect them with the established method, each sample is repeated 4 times, batch repeatability is performed on the same enzyme-labeled plate, and batch repeatability is performed between different enzyme-labeled plates, and the coefficient of variation is calculated by the measured antigen content value. The results are shown in Figure 8 The batch and batch variation coefficients of the two antigens detected by the method are less than 10%, indicating that the repeatability of the method is good.

[0059] Specificity verification: The established detection procedure is used to detect the antigen content in Re-O / MYA98, Re-O-GXCX-CHA-2018-S, AF / 72, A / GDMM / 2013 and A / WH / 09 foot-and-mouth disease viruses, and the specificity of the method is verified. The results are shown in Figure 9 The O / Cathay type foot-and-mouth disease virus antigen quantitative detection method of the application has no cross reaction with other topological type O type and A type foot-and-mouth disease virus antigens, indicating that the method has good specificity.

[0060] Example 3 Sequencing of variable region of monoclonal antibody

[0061] 3.1 Extraction of total mRNA of positive hybridoma cells

[0062] The foregoing frozen monoclonal hybridoma cells are resuscitated, and after 2-3 generations of culture, a cell suspension is prepared. Add 1 mL of TRizol and mix well, and place at 4°C for 5 min. Add 250 μL of chloroform, mix well, and then place at 4°C for 10 min, and centrifuge at 12000 r / min for 15 min. Absorb 450 μL of supernatant, add an equal amount of isopropanol, and place at -20°C for 30 min, and centrifuge as above. Discard the supernatant, and wash the precipitate with 1 mL of 75% ethanol, and centrifuge for 5 min. Dry the precipitate, and dissolve it with 25 μL of RNase-free water to obtain total mRNA.

[0063] 3.2 Synthesis of double-stranded cDNA and purification

[0064] Add the components shown in Table 1 to 0.2 mL of PCR amplification tube in turn, mix well, and then place the PCR amplification tube in a PCR instrument, and use the reaction program shown in Table 2 for amplification. The obtained product is the whole genome cDNA, which is stored at -20°C for standby use.

[0065] Table 1 Reverse transcription reagent system

[0066]

[0067] 3.3 Sequencing of variable region of monoclonal antibody

[0068] 3.2.1 Primer synthesis

[0069] Reference (von Boehmer L, Liu C, Ackerman S, Gitlin AD, Wang Q, Gazumyan A, Nussenzweig MC. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc. 2016 Oct; 11(10): 1908-1923. doi: 10.1038 / nprot.2016.102. Epub 2016 Sep 15. PMID: 27658009.) designed the primers shown in SEQ ID Nos. 11-40, which were synthesized by Beijing Geneseeq Biotech Co., Ltd. According to the literature, each group of primer premix was prepared, and the PCR amplification program was designed to perform the PCR amplification reaction. The specific amplification primers are as follows:

[0070] First round of amplification primers:

[0071] (1) Amplification of VH sequence primers:

[0072] I: 5'-AGGAACTGCAGGTGTCC-3' (SEQ ID No. 11);

[0073] II: 5'-CAGCTACAGGTGTCCACTCC-3' (SEQ ID No. 12);

[0074] III: 5'-TGGCAGCARCAGCTACAGG-3' (SEQ ID No. 13);

[0075] IV: 5'-CTGCCTGGTGACATTCCCA-3' (SEQ ID No. 14);

[0076] V: 5'-CCAAGCTGTGTCCTGTC-3' (SEQ ID No. 15);

[0077] VI: 5'-TTTTAAAAGGTGTCCAGKGT-3' (SEQ ID No. 16);

[0078] VII: 5'-CCTGTCAGTAACTRCAGGTGTCC-3' (SEQ ID No. 17);

[0079] VIII: 5'-TTTTAAAAGGGGTCCAGTGT-3' (SEQ ID No. 18);

[0080] IX: 5'-CAGTTCCTGTTTCTGTTARTGCTCTGG-3' (SEQ ID No. 31);

[0081] X: 5'-ATGAAGTTGTGGYTRAACTGG-3' (SEQ ID No. 20);

[0082] XI: 5'-TGTTGGGGCTKAAGTGGG-3' (SEQ ID No. 21);

[0083] XII: 5'-AGAAGGTGTGCACACCGCTGGAC-3' (SEQ ID No. 22).

[0084] (2) VL sequence amplification primers:

[0085] I: 5'-RGTGCAGATTTTCAGCTTCCTGCT-3' (SEQ ID No. 23);

[0086] II: 5'-TGGACATGAGGGCYCCTGCTCAGT-3' (SEQ ID No. 24);

[0087] III: 5'-CTSTGGTTGTCTGGTGTTGAYGGA-3' (SEQ ID No. 25);

[0088] IV: 5'-GTTGCTGCTGCTGTGGCTTACA-3' (SEQ ID No. 26);

[0089] V: 5'-GTATCTGGTACCTGTGG-3' (SEQ ID No. 27);

[0090] VI: 5'-TGCCTGTTAGGCTGTTGGTGCT-3' (SEQ ID No. 28);

[0091] VII: 5'-GCTCAGTTCCTTGGTCTCCTGTTGC-3' (SEQ ID No. 29);

[0092] VIII: 5'-TGGGTGCTGCTGCTCTGGGT-3' (SEQ ID No. 30);

[0093] IX: 5'-CAGTTCCTGTTTCTGTTARTGCTCTGG-3' (SEQ ID No. 31);

[0094] X: 5'-TGCTCTGGTTATATGGTGCTGATGGG-3' (SEQ ID No. 32);

[0095] XI: 5'-ACTGAGGCACCTCCAGATGTT-3' (SEQ ID No. 33).

[0096] Second round amplification primer:

[0097] I: 5'-GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG-3' (SEQ ID No. 34); II: 5'-GCTCAGGGAARTAGCCCTTGAC-3' (SEQ ID No. 35);

[0098] III: 5'-GAYATTGTGMTSACMCARWCTMCA-3' (SEQ ID No. 36);

[0099] IV: 5'-TGGGAAGATGGATACAGTT-3' (SEQ ID No. 37);

[0100] V: 5'-CAGGCTGTTGTGACTCAG-3' (SEQ ID No. 38);

[0101] VI: 5'-CAACTTGTGCTCACTCAG-3' (SEQ ID No. 39);

[0102] VII: 5'-CTCYTCAGRGGAAGGTGGRAACA-3' (SEQ ID No. 40).

[0103] 3.2.2 First round amplification

[0104] The following Table 3, Table 4 components were added in order to 0.2 mL PCR amplification tube, using Table 5 reaction program for PCR amplification. The amplification product was stored at -20°C.

[0105] Table 3 Antibody VH gene PCR amplification system

[0106]

[0107] Table 4 Antibody VL gene PCR amplification system

[0108]

[0109] Table 5 Antibody VH / VL amplification program

[0110]

[0111] 3.2.3 Second round of amplification

[0112] The VH / VL product of the first round of amplification procedure described above was amplified again using the amplification system of Table 6, according to the reaction procedure of Table 7, respectively. The amplified product was stored at -20°C.

[0113] Table 6 Amplification system of the second round of PCR of the antibody

[0114]

[0115] Table 7 Amplification procedure of the second round of PCR of the antibody

[0116]

[0117] 3.3 Alignment of gene sequences

[0118] After amplification, the target fragment was ligated into pMD-19T vector to construct sequencing plasmid, which was sent to Beijing GenScript Biotech Co., Ltd. for sequence determination. The variable region sequences obtained by sequencing were aligned with the mouse antibody heavy chain variable region sequences, light chain Lambda chain, and light chain Kappa chain published on the NCBI website using NCBI and IMGT gene libraries.

[0119] The sequencing results showed that the sequences obtained by amplification were the complementarity determining region (CDR) sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody as shown in Table 8; the amino acid sequences of the light chain and the heavy chain variable region as shown in Table 9; and the gene sequences encoding the light chain and the heavy chain variable region as shown in Table 10.

[0120] Table 8 Complementarity determining region sequences of the antibody variable region

[0121]

[0122] Table 9 Amino acid sequences of the antibody variable region

[0123]

[0124] Table 10 Base sequences of the antibody variable region

[0125]

[0126] In summary, the application provides a monoclonal antibody 3G10 of O / Cathay type foot-and-mouth disease virus, which can specifically recognize and combine with the 146S region of the O / Cathay type foot-and-mouth disease virus, has the advantages of high specificity, sensitivity, low preparation cost and the like. The specific embodiment case provides an application of the monoclonal antibody 3G10 in a typing and quantitative detection method of the O / Cathay type foot-and-mouth disease virus, provides a reference in post-vaccine production quality control and potency evaluation, and provides new technical support for controlling the spread of the foot-and-mouth disease epidemic; the monoclonal antibody sequence provided by the application can be used for recombination and modification by a conventional genetic engineering or protein engineering method, avoids loss of the antibody due to long-term storage of the hybridoma cells, is also beneficial to optimization of the antibody at the genetic and protein levels, and further improves the specificity and affinity of the antibody.

[0127] The above-described embodiments are only part of the embodiments of the application, and are not intended to limit the implementation scope of the application, so equivalent changes or modifications made according to the structure, features and principles described in the patent scope of the application should be included in the patent scope of the application.

Claims

1. A monoclonal antibody against O / Cathay type foot-and-mouth disease virus, characterized in that, The O / Cathay type foot-and-mouth disease virus monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3; The variable region CDR of the antibody light chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 4, CDR2 shown in SEQ ID No. 5, and CDR3 shown in SEQ ID No.

6.

2. The O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1, characterized in that, The O / Cathay type foot-and-mouth disease virus monoclonal antibody includes an antibody heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 7 and an antibody light chain variable region with an amino acid sequence as shown in SEQ ID No.

8.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The nucleic acid molecule includes the nucleotide sequences shown in SEQ ID No. 9 and SEQ ID No.

10.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 3 or 4.

6. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecules of claim 3 or 4, or the expression vector of claim 5.

7. The application of the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2 in the preparation of products for the quantitative detection of O / Cathay type foot-and-mouth disease virus typing.

8. The application of the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2 in the quantitative detection of O / Cathay type foot-and-mouth disease virus for non-disease diagnosis purposes.

9. A quantitative detection kit for O / Cathay type foot-and-mouth disease virus typing, characterized in that, The kit includes the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2.

10. The method for preparing the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2, characterized in that, The method is as follows: the heavy chain variable region and light chain variable region sequences of the O / Cathay type foot-and-mouth disease virus monoclonal antibody are linked to the antibody heavy chain constant region and light chain constant region sequences, respectively, and inserted into the expression vector to obtain the antibody heavy chain and antibody light chain recombinant expression plasmid; the obtained recombinant expression plasmid is co-transfected into expression cells, and O / Cathay type foot-and-mouth disease virus monoclonal antibody is obtained by culturing.

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