CN115819563A

CN115819563A - Monoclonal antibody of porcine pseudorabies virus gE protein and application thereof

Info

Publication number: CN115819563A
Application number: CN202211552455.5A
Authority: CN
Inventors: 巩玉洁; 张琼林; 钟鸣; 赵荣茂; 陈娟; 杨晓霞; 赵方圆; 袁婷婷
Original assignee: Beijing Nabai Bio Tech Co ltd
Current assignee: Beijing Nabai Bio Tech Co ltd
Priority date: 2022-12-05
Filing date: 2022-12-05
Publication date: 2023-03-21

Abstract

The invention discloses a monoclonal antibody of anti-porcine pseudorabies virus gE protein and application thereof. The monoclonal antibody has the name V _H And having the designation V _L The light chain variable region of (1), said V _H And V _L Both consist of a complementarity determining region and a framework region; the complementarity determining region consists of CDR1, CDR2 and CDR 3; the V is _H The amino acid sequence of CDR1 is shown in SEQ ID No. 1; the V is _H The amino acid sequence of CDR2 is shown in SEQ ID No. 2; the V is _H The amino acid sequence of CDR3 of (1) is shown in SEQ ID No. 3; the V is _L The amino acid sequence of CDR1 of (1) is shown in SEQ ID No. 4; the V is _L The amino acid sequence of CDR2 is shown as SEQ ID No.5 is shown; the V is _L The amino acid sequence of CDR3 of (1) is shown in SEQ ID No. 6. The detection reagent prepared by the monoclonal antibody for resisting the porcine pseudorabies virus gE protein has high specificity, good sensitivity and good coincidence rate.

Description

Monoclonal antibody of porcine pseudorabies virus gE protein and application thereof

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to a monoclonal antibody for resisting porcine pseudorabies virus gE protein and application thereof.

Background

Pseudorabies virus (PR) belongs to herpesviridae, subfamily alpha-herpesviridae, genus varicella, and can cause male pig testicle swelling or atrophy, loss of fertility, abortion in pregnant sows, stillbirth, mummy, vomiting and diarrhea in young pigs, inappetence, and ultimately death due to systemic failure. The virus can be latent in a host body without causing diseases, and is easy to generate mixed infection with other bacteria and viruses. Data show that although the feeding management level of pig farms is improved in recent years and vaccines are widely used, the prevalence of wild PRV in swinery is still serious, and PRV has a great threat to the pig industry. There are a number of vaccines against this viral disease: attenuated live vaccines, inactivated vaccines, gene deletion vaccines, recombinant vector vaccines, subunit vaccines, nucleic acid vaccines and the like.

PRV is a double-stranded DNA virus, which is observed under electron microscope to be circular or elliptical, and consists of capsid, envelope and nucleolus, and the diameter of the particle is 150-180 nm. The genome length is 143 Kb, and the GC content is as high as about 72 percent. The porcine pseudorabies virus is mainly prevented by immunizing gE gene deletion vaccine, and distinguishing and identifying wild strain infected animals and vaccine immunized animals by using a gE antibody serodiagnosis method. Thus, diagnostic methods for detecting protein antibodies can determine the resistance of an animal to a viral strain. Meanwhile, by monitoring the generation of antibodies by the vaccine, the method can be used as an important means for selecting the vaccine, evaluating the rationality of an immunization program and mastering the health state of a population in daily monitoring, and can also be used for determining an important basis for the time point of vaccine injection.

At present, diagnostic reagents for detecting the disease are different, and the separation of PRV is considered to be the 'gold standard' for identifying the virus in both sensitivity and specificity, the method is only suitable for laboratory detection, and the separation of the virus fails due to errors in any links such as sample processing, cell inoculation and the like, and long detection time is required. The serum neutralization test has fast response, strong specificity but low sensitivity, the test result can be influenced by different virus titers, and the serum neutralization test has higher requirements on samples and is not suitable for clinical large-scale detection.

The enzyme-linked immunosorbent assay has the advantages of rapidness, simplicity, convenience, low price and automatic batch detection, but has the defects of weak specificity, high sensitivity, easy occurrence of false positive, need of enzyme-linked immunosorbent assay, and unsuitability for single sample detection. The immunofluorescence staining technique has the advantages of simplicity, convenience, high accuracy, rapid diagnosis and the like, is mainly used for diagnosing pseudorabies viruses, but has the defects of specificity cross and unsuitability for large-scale detection. The latex agglutination test is similar to the enzyme-linked immunosorbent assay, is multi-purpose for early infection detection, has high sensitivity and strong specificity, and can carry out rapid serum screening detection. The agar immunodiffusion test has the advantages of simple operation, high cost performance and wider application range, but the detection sensitivity is influenced by the reliability of the method for extracting the antigen and the positive serum, so that the detection is inconvenient.

The polymerase chain reaction is suitable for amplification of PRV DNA and clinical detection of PRV latent infection, and can be used for rapid differential diagnosis of pseudorabies vaccine virus and wild virus. However, the detection needs a special detection instrument, is expensive, has high requirements on the detection environment, and is not very suitable for basic detection.

In summary, the detection method is widely applied to clinical detection of porcine pseudorabies virus gE gene, but various defects still exist, and further improvement is urgently needed. Therefore, how to prepare the porcine pseudorabies virus gE protein specific antibody, and establish a diagnostic reagent and a diagnostic method which are rapid, sensitive, strong in specificity, simple in operation, economical and practical, and have important significance for clinically and accurately detecting the porcine pseudorabies virus.

Disclosure of Invention

Therefore, the embodiment of the invention provides a monoclonal antibody for resisting porcine pseudorabies virus gE protein and application thereof.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

the embodiment of the invention provides a monoclonal antibody of anti-porcine pseudorabies virus gE protein or a functional fragment thereof,

the monoclonal antibody has the name V _H And having the designation V _L The light chain variable region of (1), said V _H And V _L Both consist of a complementarity determining region and a framework region;

the complementarity determining region consists of CDR1, CDR2 and CDR 3;

the V is _H The amino acid sequence of CDR1 of (A) is shown in SEQ ID No. 1;

the V is _H The amino acid sequence of CDR2 is shown in SEQ ID No. 2;

the V is _H The amino acid sequence of CDR3 of (1) is shown in SEQ ID No. 3;

the V is _L The amino acid sequence of CDR1 of (1) is shown in SEQ ID No. 4;

the V is _L The amino acid sequence of CDR2 of (1) is shown in SEQ ID No. 5;

the V is _L The amino acid sequence of CDR3 of (1) is shown in SEQ ID No. 6.

In one embodiment of the present invention, said V _H And V _L The framework regions were all derived from mice.

In one embodiment of the present invention, said V _H The amino acid sequence of (A) is shown as SEQ ID No.7 in the sequence table;

the V is _L The amino acid sequence of (A) is shown as SEQ ID No.8 in the sequence table.

In one embodiment of the invention, the monoclonal antibody is any one of the following:

(a) From the above-mentioned V _H And V as described above _L Linking the obtained single-chain antibody;

(b) A fusion antibody comprising the single chain antibody of (a);

(c) Containing the above-mentioned V _H And V as described above _L The Fab of (1);

(d) Containing the above-mentioned V _H And V as described above _L The whole antibody of (1).

The present invention also provides a biomaterial related to the above monoclonal antibody or a functional fragment thereof, which is any one of (A1) to (a 12):

(A1) Nucleic acid molecules encoding said monoclonal antibody or functional fragment thereof;

(A2) An expression cassette comprising the nucleic acid molecule of (A1);

(A3) A recombinant vector comprising the nucleic acid molecule of (A1);

(A4) A recombinant vector comprising the expression cassette of (A2);

(A5) A recombinant microorganism comprising the nucleic acid molecule of (A1);

(A6) A recombinant microorganism comprising the expression cassette of (A2);

(A7) A recombinant microorganism comprising the vector of (A3);

(A8) A recombinant microorganism comprising the vector of (A4);

(A9) A transgenic animal cell line comprising the nucleic acid molecule of (A1);

(A10) A transgenic animal cell line containing the expression vector of (A2);

(A11) A transgenic animal cell line comprising the vector of (A3);

(A12) A transgenic animal cell line comprising the vector of (A4).

In one embodiment of the present invention, the nucleic acid molecule of (A1) is a gene encoding the monoclonal antibody or a functional fragment thereof described above.

In one embodiment of the present invention, the gene is a DNA molecule as follows:

the V is _H The coding sequence of CDR1 of (1) is shown in SEQ ID No. 9;

the V is _H The coding sequence of CDR2 of (1) is shown in SEQ ID No. 10;

the V is _H The coding sequence of CDR3 of (1) is shown in SEQ ID No. 11;

the V is _L The coding sequence of CDR1 of (1) is shown in SEQ ID No. 12;

the V is _L The coding sequence of CDR2 of (1) is shown in SEQ ID No. 13;

the V is _L The coding sequence of CDR3 of (1) is shown in SEQ ID No. 14.

The invention also provides application of the monoclonal antibody or the functional fragment thereof or the biological material in preparation of a reagent for detecting porcine pseudorabies virus gE protein.

The embodiment of the invention has the following advantages:

the test proves that: the monoclonal antibody capable of combining with the porcine pseudorabies virus is prepared by utilizing the porcine pseudorabies virus gE protein expressed by insect cells, and has strong combining capacity with the virus, good specificity and strong stability.

The detection reagent prepared by the monoclonal antibody for resisting the porcine pseudorabies virus gE protein has high specificity, good sensitivity and good coincidence rate.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so that those skilled in the art can understand and read the present invention, and do not limit the conditions for implementing the present invention, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the functions and purposes of the present invention, should still fall within the scope of the present invention.

FIG. 1 is a purity identification chart of Bacv-PRV-gE recombinant protein provided by the embodiment of the invention, and it can be seen that the size of a target protein band is 63 KD;

FIG. 2 is a diagram showing SDS polyacrylamide gel electrophoresis of the purified anti-PRV gE protein monoclonal antibody according to the results provided in the examples of the present invention;

FIG. 3 is a diagram showing the analysis of the homology of the nucleotide sequence of the heavy chain variable region of the anti-PRV gE protein monoclonal antibody provided in the example of the present invention;

FIG. 4 is a diagram showing the analysis of the homology of the nucleotide sequences of the variable regions of the light chains of the anti-PRV gE protein monoclonal antibodies provided in the examples of the present invention;

FIG. 5 is a diagram showing the analysis of the homology of the amino acid sequences of the heavy chain variable regions of the anti-PRV gE protein monoclonal antibody provided in the example of the present invention;

FIG. 6 is a diagram showing the analysis of the homology of the amino acid sequences of the light chain variable regions of the anti-PRV gE protein monoclonal antibody according to the present invention.

Detailed Description

The present invention is described in terms of specific embodiments, and other advantages and benefits of the present invention will become apparent to those skilled in the art from the following disclosure. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of porcine pseudorabies virus gE recombinant protein

The preparation method of the porcine pseudorabies virus gE recombinant protein provided by the embodiment comprises the following steps:

1. construction of recombinant shuttle vector Bacmid-PRV

Synthesizing a gE gene according to a PRV-gE sequence (AF 207700.1) logged in GenBank, connecting the gE gene to a pFastBac 1 vector, optimizing codons to adapt to the expression of insect cells, selecting Sma I and PvuII enzyme cutting sites, transforming the synthesized recombinant plasmid into DH10Bac engineering bacteria to obtain a recombinant transformed strain, coating the recombinant transformed strain on a screening culture medium plate (containing 50 mu g/mL kanamycin, 7 mu g/mL gentamycin, 10 mu g/mL tetracycline, 40 mu g/mL X-gal and 40 mu g/mL IPTG), after 48 hours, forming blue and white colonies on the plate, selecting the white colonies, streaking on the screening culture medium plate, purifying twice to obtain white colonies, screening and culturing the colonies, extracting the plasmid, namely obtaining a recombinant shuttle vector Bacmid-PRV gE plasmid vector, and transfecting the plasmid vector into the insect cells.

2. Preparation of recombinant baculovirus

Standing and culturing SF9 insect cell in 27 deg.C incubator until cell density reaches 8.0 × 10 ⁵ cells/mL, 1 μ g of Bacmid-PRV gE plasmid is transfected into SF9 insect cells through Cellffectin II Reagent. And observing no pollution within 24 hours after transfection, observing lesions after 72 hours, collecting the first generation recombinant baculovirus P1, and taking cells and supernatant to perform SDS-PAGE gel electrophoresis and Western blot detection so as to analyze and determine whether the target protein is normally expressed. Subsequently, the supernatant of SF9 insect cells was further infected with the P1 generation virus to obtain P2 generation recombinant baculovirus, and the obtained P2 generation recombinant baculovirus was frozen at-80 ℃.

3. Purity identification of recombinant protein

The purity of the Bacv-PRV-gE protein after purification is detected by protein SDS-polyacrylamide gel electrophoresis, and the specific steps are as follows: preparing 12% separation gel and 5% concentrated gel, injecting the separation gel into 1.5 mm glass plate gap with height of 10 cm, sealing with pure water, polymerizing at room temperature for 20 min, discarding the pure water, adding concentrated gel liquid with height of 1 cm, carefully inserting into comb to avoid air bubble in the gel, and polymerizing at room temperature for 10 min. 20. mu.L of the sample was mixed with a quarter volume of 5 Xloading buffer and heated at 100 ℃ for 10 min. Sampling 25 mu L of each hole, changing the procedure to 120V after 80V and 20 min, after 50 min, when bromophenol blue reaches the bottom of the separation gel, taking out the gel, putting the gel into Coomassie brilliant blue staining solution, oscillating and staining for 2h, washing the gel with clear water, removing redundant staining solution, then changing the decolorizing solution for decolorizing, changing the decolorizing solution every 30min until the protein band is clear, analyzing the protein band, identifying the protein band as a Bacv-PRV-gE protein band, identifying the purity of Bacv-PRV-gE recombinant protein as shown in figure 1, wherein the size of the target band can be seen to be 63 KD, the band is clear, no impurity band exists, and the purification standard is achieved.

Example 2 preparation of monoclonal antibody against gE protein of porcine pseudorabies Virus

This example provides a preparation method of monoclonal antibody against gE protein of porcine pseudorabies virus, which comprises the following steps:

1. animal immunization

The porcine pseudorabies virus gE recombinant protein which is prepared and purified by recombinant baculovirus expression in example 1 is used as immunogen, is mixed with Freund's immunologic adjuvant in equal amount, is fully emulsified, BALB/C mice are immunized with 50 mug/mouse immunization dose for the first immunization, and then are mixed with Freund's incomplete adjuvant in equal amount and are emulsified in 50 mug/mouse immunization dose for subcutaneous multi-point immunization.

The immunization is carried out at intervals of 2 to 3 weeks. Collecting blood from mouse tail 7 days after 3 th immunization to detect immunization titer, standing at 4 deg.C for 30min to separate out serum, diluting the serum with PBS, and treating with blank mouse serum as control, and OD of experimental group and control group _450nm When the ratio is more than 2.1, the maximum serum dilution is the mouse serum titer. Mice with high titers were selected and boosted by intraperitoneal injection of 50 μ g of an emulsion of porcine pseudorabies virus gE recombinant protein antigen and incomplete freund's adjuvant in equal volumes of PBS (200 μ L). Mice were sacrificed 3 days later and cell fusion was performed.

2. Cell fusion

Blood was collected from the eyeballs of the mice that had completed the booster immunization, and serum was isolated as a positive control. After the neck of the mouse is cut off, the mouse is placed into 75% ethanol to be soaked for 10 min, and the mouse is fixed on an anatomical table in a super clean bench. The skin of the abdomen was lifted with sterilized tweezers, and small pieces of skin were cut from the lower part of the abdomen with sterile scissors, the skin and peritoneum were separated, other internal organs were carefully removed, the spleen was separated, gently removed, and placed in a petri dish containing 20 mL of incomplete DMEM medium. The spleen was punctured from the top of the spleen using a syringe filled with 20 mL of incomplete DMEM medium, passed through the spleen, the syringe was gently pushed, the spleen cells were blown out into the dish, and the process was repeated several times until the spleen did not change color any more, and the spleen cells were filtered with a filter.

Counting SP2/0 and splenocytes, mixing the splenocytes with SP2/0 according to the cell number ratio of 8; beating the deposited cells to be uniformly distributed at the bottom of the tube under the state of 37 ℃ water bath, standing for 1 min, adding 1 mL of PEG1450 into the centrifuge tube within 1 min, standing for 1 min, and adding 1 mL of 37 ℃ preheated incomplete DMEM culture solution along the tube wall within 30 s to terminate the cell fusion reaction; the gun head is stretched below the liquid level, 1 mL of incomplete DMEM is added after 1 min, and the step is repeated until 20 mL of incomplete DMEM culture solution is added; slowly adding 30 mL of incomplete DMEM medium, centrifuging at 800 rpm for 4 min, discarding the supernatant, adding 50 mL of incomplete DMEM medium, centrifuging at 800 rpm for 4 min, discarding the supernatant, adding HAT medium, gently blowing up cells, adding 96-well cell culture plates (200 muL/well), and marking; indirect ELISA identification was performed 7 days later.

3. Positive hybridoma cell for screening PRV gE protein resisting monoclonal antibody

Selecting hybridoma cell supernatant, screening positive hybridoma cells by indirect ELISA, and selecting OD _450nm Wells with higher values and only single cell clumps, discard medium, add 200 μ L of HT medium, blow-beat cells and count, half-pack about 200 cells into 96-well plates, passage the remaining cells to 48-well plates for continued expansion culture, and freeze-deposit. 7d later, ELISA identification was carried out on the monoclonal cells, subcloning was carried out again by the above method, and after 3 times of subcloning, OD was selected _450nm The higher value single cell clumps were re-cloned as described above, if there were any cells with well OD _450nm Higher value, cell-free clump well OD _450nm The value is not higher than that of the negative control, and the cell strain is regarded as a monoclonal cell strain capable of secreting the anti-PRV gE protein monoclonal antibody.

4. Purification preparation of anti-PRV gE protein monoclonal antibody

Injecting the cell strain which is obtained by screening and secretes PRV gE protein antibody into the abdominal cavity of a mouse for culturing, and extracting ascites from the abdominal cavity of the mouse for purification. The specific operation steps are as follows:

mice were injected intraperitoneally with 500. Mu.L Freund's incomplete adjuvant, and after 24 hours, approximately 1X 10 cells were collected ⁷ Injecting the hybridoma cells into abdominal cavity of the mouse, and collecting ascites after 7 d. The antibody is purified by utilizing a commercial antibody purification kit, and the specific operation is as follows: centrifuging ascites at 10000 rpm for 10 min, collecting supernatant, and collecting 20 μ L to obtain sample; add 60 μ L of 1M Tris-HCl (pH = 9.0) to the centrifuge tube; filtering the Binding buffer and the Elution buffer by using a 0.45-micron filter for later use; ascites diluted twice with Binding buffer; sucking 10 mL Binding buffer with a syringe, connecting the buffer on a purification column, removing bubbles, slowly pushing a piston, and removing a storage solution; sucking 10 mL Binding buffer with flow rate of 1 mL/min, and balancing the column; the diluted ascites is sucked by a syringe, and the flow rate is 0.2 mL/min, so that the antibody is combined with the column; sucking 10 mL Binding buffer, washing away the unbound antibody until the effluent liquid is colorless; 5 mL of Elution buffer was aspirated, and the antibody bound to the column was eluted, and added dropwise to the above-mentioned Tris-HCl-added centrifuge tube, 8 drops/tube. Sampling 20 mu L of each tube to prepare a sample; and (3) analyzing the antibody after SDS-PAGE protein electrophoresis, coomassie brilliant blue staining and decoloring, identifying the purity of the monoclonal antibody, and dialyzing by PBS to obtain the anti-PRV gE protein monoclonal antibody. As shown in FIG. 2, the results show that the concentration of the purified anti-PRV gE protein monoclonal antibody is 17 mg/ml, and the SDS polyacrylamide gel electrophoresis analysis shows that clear immunoglobulin heavy chains and light chains are visible, no impurity band exists, and the purity of the antibody meets the expected requirements.

5. Verification of activity of anti-PRV gE protein monoclonal antibody

The PRV gE protein resisting monoclonal antibody is subjected to SDS-PAGE analysis, dialysis and concentration measurement, and finally the activity is verified by ELISA. ELISA detection step: coating the purified PRV-gE antibody 0.1. Mu.g/mL, 100. Mu.L/well; incubating at 37 ℃ for 2h by using 0.1% gelatin; wash plate 3 times with 250 μ L/well 0.1% PBST wash; diluted porcine pseudorabies virus 100 mu L/holeIncubation at 37 ℃ for 1 h (control group without inactivated virus, three replicates per group); wash plate 3 times with 250 μ L/well 0.1% PBST wash; diluting the porcine pseudorabies positive serum and the porcine pseudorabies negative serum by 100 times, incubating for 30min at 37 ℃ in a hole of 100 mu L; wash plate 3 times with 250 μ L/well 0.1% PBST wash; after drying, 100 mu L/hole of HRP enzyme-labeled rabbit anti-pig secondary antibody (diluted by PBS according to 1 10000) is added, and incubation is carried out for 30min at 37 ℃; washing the plate for 3 times again, beating to dry, adding commercial TMB color development liquid 100 μ L/hole, and developing for 10 min at room temperature; finally, 0.5M sulfuric acid was added at 50. Mu.L/well to terminate the reaction, and OD was measured with a microplate reader _450nm The value is obtained. The results of the activity test of the anti-PRV gE protein monoclonal antibody are shown in Table 1.

TABLE 1

Inactivated virus + positive pig serum	Inactivated virus + negative pig serum	Inactivated virus + PBS	Virus diluent and positive pig serum	Virus diluent and negative pig serum	Virus diluent + PBS
						2.594	0.056	0.035	0.068	0.064	0.049
2.567	0.061	0.039	0.073	0.067	0.046
						2.624	0.064	0.038	0.070	0.065	0.051

6. Sequencing of anti-PRV gE protein monoclonal antibody

Extracting total RNA of hybridoma cells, and designing specific upstream and downstream universal primers of a murine heavy chain antibody gene and a light chain antibody gene, wherein the primers are as follows:

heavy chain upstream primer: TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC;

heavy chain downstream primer: AGGTSMARCTGCAGGAGSAGTCGWGG;

light chain upstream primer 1: CCGTTTGATTTCCAGCTTGGTGCC;

light chain upstream primer 2: CCGTTTTATTTCCAGCTTGGTCCC;

light chain upstream primer 3: CCGTTTTATTTCCAACTTTGTCCC;

light chain upstream primer 4: CCGTTTCAGCTCCAGCTTGGTCCC;

light chain downstream primer: and (3) amplifying cDNA of a heavy chain and a light chain by using an RT-PCR kit by using GACATTGAGCTCACCCAGTCTCCTCCA, connecting the cDNA to a pMD18-T cloning vector, and performing gene sequencing to obtain a gene sequence of the PRV gE protein resisting monoclonal antibody.

V _H Heavy chain variable region nucleotide gene sequence (SEQ ID No. 15):

gaggtgcagctgcaggagtctgcacatgagctcgtgaagcctggggcttcagtgaagataccctgcaaggcttctggatacacattcactgactacaacatgcactgggtgaagcagagcctccgaaagagccttgagtggattggagatattcatcctaacaatggtggtactatctacaaccagaagtgcatgggcaaggccacattgactgtagacaagtcctccagcacagccgacatggagctccgcagcctgacatctgacgacactgcagtctattcctgtgcaagagaggggattcattgctacggtagttgctacgtacggtacgacttggactactgccgccaaggcaccactctcacagtctcctca。

V _H the coding sequence of CDR1 of (1) is shown in SEQ ID No. 9: gactacaacatgcac;

V _H the coding sequence of CDR2 of (1) is shown in SEQ ID No. 10:

gatattcatcctaacaatggtggtactatctacaaccagaagtgcatgggc；

V _H the coding sequence of CDR3 of (1) is shown in SEQ ID No. 11:

gaggggattcattgctacggtagttgctacgtacggtacgacttggactac。

V _H heavy chain variable region amino acid sequence (SEQ ID No. 7):

EVQLQESAHELVKPGASVKIPCKASGYTFTDYNMHWVKQSLRKSLEWIGDIHPNNGGTIYNQKCMGKATLTVDKSSSTADMELRSLTSDDTAVYSCAREGIHCYGSCYVRYDLDYCRQGTTLTVSS。

V _L light chain variable region nucleotide gene sequence (SEQ ID No. 16):

gatactgtgctgacccagagctcggcgagcctggctgtgagcctgtgccagcgcgcgaccattagctgcaaagcgagccagagcgtggatcatgatggcgatagctatatgaactggtctcagcagagaccgagccagagctcgacactgctgatttatgcggcgagcaacctggaaagcggcattcctgcgcgctttagctgcagcggcagccgcaccgattttaccctgaacattcatcctgtggaagaagaagatgcggcgacctattattgccagcagacc。

V _L the coding sequence of CDR1 of (1) is shown in SEQ ID No. 12:

aaagcgagccagagcgtggatcatgatggcgatagctatatgaac；

V _L the coding sequence of CDR2 of (1) is shown in SEQ ID No. 13: gcggcgagcaacctggaaaagc;

V _L the coding sequence of CDR3 of (1) is shown in SEQ ID No.14, cagcagcag.

Light chain variable region amino acid sequence (SEQ ID No. 8):

DTVLTQSSASLAVSLCQRATISCKASQSVDHDGDSYMNWSQQRPSQSSTLLIYAASNLESGIPARFSCSGSRTDFTLNIHPVEEEDAATYYCQQT。

7. antibody nucleotide homology analysis

Inputting the heavy chain and light chain variable region nucleotides of the anti-PRV gE protein monoclonal antibody into an NCBI database for comparison, and analyzing homology, wherein the results are shown in figure 3 and figure 4, figure 3 is the nucleotide Sequence homology analysis of the heavy chain variable region of the anti-PRV gE protein monoclonal antibody, figure 4 is the nucleotide Sequence homology analysis of the light chain variable region of the anti-PRV gE protein monoclonal antibody, and the analysis result shows that the gene Sequence of the heavy chain variable region of the monoclonal antibody has the highest homology with the heavy chain variable region of the mouse immunoglobulin (Sequence ID: KU 256551.1), the homology is 361/379, and the homology percentage is 95%; the anti-PRV gE protein monoclonal antibody light chain variable region gene Sequence has the highest homology with a mouse immunoglobulin light chain variable region (Sequence ID: KM 393286.1), the homology is 272/285, and the homology percentage is 95%.

8. Antibody amino acid homology analysis

Inputting the amino acids of the heavy chain and light chain variable regions of the anti-PRV gE protein monoclonal antibody into an NCBI database for comparison, and analyzing homology, wherein the results are shown in figure 5 and figure 6, figure 5 is the amino acid Sequence homology analysis of the heavy chain variable region of the anti-PRV gE protein monoclonal antibody, figure 6 is the amino acid Sequence homology analysis of the light chain variable region of the anti-PRV gE protein monoclonal antibody, and the analysis results show that the amino acid Sequence of the heavy chain variable region of the anti-PRV gE protein monoclonal antibody has the highest homology with the mouse immunoglobulin heavy chain variable region (Sequence ID: AML 31200.1), the homology is 110/126, and the homology percentage is 87%; the amino acid Sequence of the light chain variable region of the anti-PRV gE protein monoclonal antibody has the highest homology with the mouse immunoglobulin light chain variable region (Sequence ID: AKH 14756.1), wherein the homology is 85/95, and the homology percentage is 89%.

9. CDR region analysis of anti-PRV gE protein monoclonal antibody

The CDR regions of the heavy chain variable region and the CDR regions of the light chain variable region of the anti-PRV gE protein monoclonal antibody are analyzed in https:// www.novopro.cn/tools/CDR/cdr.html, so as to obtain the CDR regions of the heavy chain and the light chain of the anti-PRV gE protein monoclonal antibody, and the analysis results are shown in Table 2 as the amino acid sequences of the CDR regions of the heavy chain and the light chain variable region, and Table 3 as the amino acid sequences of the CDR regions of the light chain variable region.

TABLE 2

Heavy chain light chain variable region	Serial number	Amino acid sequence
			CDR-H1（CDR1）	SEQ ID No. 1	DYNMH
CDR-H2（CDR2）	SEQ ID No. 2	DIHPNNGGTIYNQKCMG
			CDR-H3（CDR3）	SEQ ID No. 3	EGIHCYGSCYVRYDLDY

TABLE 3

Light chain variable region	Serial number	Amino acid sequence
			CDR-L1（CDR1）	SEQ ID No. 4	KASQSVDHDGDSYMN
CDR-L2（CDR2）	SEQ ID No. 5	AASNLES
			CDR-L3（CDR3）	SEQ ID No. 6	QQ

Example 3 application of anti-PRV gE protein monoclonal antibody in preparation of enzyme-linked immunosorbent assay reagent for detecting porcine pseudorabies virus gE antibody

1. The embodiment provides an application of an enzyme-linked immunosorbent assay kit for detecting a porcine pseudorabies virus gE antibody, which is prepared from a PRV gE monoclonal antibody, wherein the detection process of the detection kit comprises the following steps:

the antigen coated plate is taken out from the kit, the serum to be detected is diluted by 2 times of the sample diluent on the serum dilution plate (60 mul of the sample diluent is mixed with 60 mul of the serum to be detected), 100 mul of the mixed solution is added into the enzyme label plate, and 2 holes are respectively added for the positive serum and the negative serum, and each hole is 100 mul.

The samples in each well were gently shaken, covered with a sealing plate film, and incubated at 37 ℃ for 30min.

The solution in the wells was discarded and 250. Mu.l of working wash was added to each well and repeated 3 times. And thoroughly drying the liquid after the plate is washed for the last time.

Mu.l of enzyme-labeled antibody was added to each well, the coated plate was covered with a sealing plate film, and incubated at 37 ℃ for 30min.

Add 100. Mu.l of substrate solution to each well, cover the coated plate with a sealing plate membrane, incubate for 10 min at room temperature.

The reaction was stopped by adding 50. Mu.l of stop solution to each well.

Determination of the absorbance (OD) at 450 nm for the samples and controls _450nm ）。

Interpretation criteria: IN = (negative control OD) _450nm OD of sample _450nm ) Negative control OD _450nm . IN is more than or equal to 0.45 and is positive; IN < 0.45 is negative.

2. Specificity of enzyme-linked immunosorbent assay (ELISA) detection reagent for porcine pseudorabies virus gE antibody

The standard antibody positive serum and the porcine pseudorabies antibody negative serum of porcine pseudorabies, swine fever, porcine foot and mouth disease (O type), porcine circovirus type 2, porcine reproductive and respiratory syndrome and the like are detected by using a porcine pseudorabies virus ELISA gE antibody detection kit.

The detection results are shown IN table 4, except that the IN value of the PRV standard antibody positive serum is significantly greater than 0.45, the IN values of the rest sera are less than 0.45, and the detection kit conforms to the judgment standard of negative sera, thereby indicating that the specificity of the detection kit is good.

TABLE 4

Item	PRV	CSFV	FMD-O	PCV-2	PRRSV	Negative control
							IN	0.836	0.084	0.122	0.074	0.105	0.020
OD _450nm	0.261	1.456	1.395	1.472	1.422	1.589

3. Sensitivity test of enzyme-linked immunosorbent assay (ELISA) detection reagent for porcine pseudorabies virus gE antibody

PRV standard antibody positive serum is respectively diluted by 2, 4, 8, 16, 32 and 64 times, and the detection kit for the ELISA gE antibody of the purchased porcine pseudorabies virus are used for detecting simultaneously. The results are shown in table 5, which shows the sensitive serum detection results of the detection kit of the present invention, the detection kit of the present invention can detect PRV standard antibody positive serum diluted 32 times, and the externally purchased kit can also detect PRV standard antibody positive serum diluted 16 times, indicating that the established porcine pseudorabies virus ELISA gE antibody detection kit of the present invention has good sensitivity.

TABLE 5

4. The coincidence rate of the enzyme-linked immunosorbent assay reagent of the porcine pseudorabies virus gE antibody

30 serum samples are simultaneously detected by using the detection kit and the externally purchased porcine pseudorabies virus ELISA gE antibody detection kit, the results are compared with sample coincidence rates shown in a table 6, the coincidence rate of the detection kit and the externally purchased kit is 97%, and the detection kit and the control kit have good correspondence.

TABLE 6

TABLE 7

The sequence of the porcine pseudorabies virus gE antibody is different from the sequence of the known gE antibody, the monoclonal antibody has strong affinity with the virus and high sensitivity and specificity, and a detection kit prepared by applying the antibody has the advantages of high specificity, good sensitivity and high coincidence rate.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A monoclonal antibody or a functional fragment thereof against the gE protein of porcine pseudorabies virus, which is characterized in that,

the monoclonal antibody has the name V _H And the heavy chain variable region of (1) and the name V _L The light chain variable region of (1), said V _H And V _L Both consist of a complementarity determining region and a framework region;

the complementarity determining region consists of CDR1, CDR2 and CDR 3;

the V is _H The amino acid sequence of CDR1 is shown in SEQ ID No. 1;

the V is _H The amino acid sequence of CDR2 is shown in SEQ ID No. 2;

the V is _H The amino acid sequence of CDR3 of (1) is shown in SEQ ID No. 3;

the V is _L The amino acid sequence of CDR1 of (1) is shown in SEQ ID No. 4;

the V is _L The amino acid sequence of CDR2 of (1) is shown in SEQ ID No. 5;

the V is _L The amino acid sequence of CDR3 of (1) is shown in SEQ ID No. 6.

2. The monoclonal antibody against porcine pseudorabies virus gE protein or a functional fragment thereof according to claim 1,

the V is _H And V _L The framework regions were all derived from mice.

3. The monoclonal antibody against porcine pseudorabies virus gE protein or a functional fragment thereof according to claim 1,

the V is _H The amino acid sequence of (A) is shown as SEQ ID No.7 in the sequence table;

4. The monoclonal antibody against porcine pseudorabies virus gE protein or a functional fragment thereof according to claim 1 or 2, wherein said monoclonal antibody is any one of the following:

(a) V as claimed in claim 1 or 2 _H And V as claimed in claim 1 or 2 _L Linking the obtained single-chain antibody;

(b) A fusion antibody comprising the single-chain antibody of (a);

(c) Comprising V according to claim 1 or 2 _H And V as claimed in claim 1 or 2 _L The Fab of (1);

(d) Comprising V according to claim 1 or 2 _H And V as claimed in claim 1 or 2 _L The whole antibody of (1).

5. A biomaterial related to the monoclonal antibody or the functional fragment thereof according to any one of claims 1 to 4, which is any one of (A1) to (A12):

(A1) A nucleic acid molecule encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1-4;

(A2) An expression cassette comprising the nucleic acid molecule of (A1);

(A3) A recombinant vector comprising the nucleic acid molecule of (A1);

(A4) A recombinant vector comprising the expression cassette of (A2);

(A5) A recombinant microorganism comprising the nucleic acid molecule of (A1);

(A6) A recombinant microorganism comprising the expression cassette of (A2);

(A7) A recombinant microorganism comprising the vector of (A3);

(A8) A recombinant microorganism comprising the vector of (A4);

(A10) A transgenic animal cell line containing the expression vector of (A2);

(A11) A transgenic animal cell line comprising the vector of (A3);

(A12) A transgenic animal cell line comprising the vector of (A4).

6. The biomaterial according to claim 5, wherein (A1) said nucleic acid molecule is a gene encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1 to 4.

7. The biomaterial of claim 6, wherein the gene is a DNA molecule as follows:

the V is _H The coding sequence of CDR1 of (1) is shown in SEQ ID No. 9;

the V is _H The coding sequence of CDR2 of (1) is shown in SEQ ID No. 10;

the V is _H The coding sequence of CDR3 of (1) is shown in SEQID No. 11;

the V is _L The coding sequence of CDR1 of (1) is shown in SEQ ID No. 12;

the V is _L The coding sequence of CDR2 of (1) is shown in SEQ ID No. 13;

the V is _L The coding sequence of CDR3 of (1) is shown in SEQ ID No. 14.

8. Use of the monoclonal antibody or functional fragment thereof according to any one of claims 1-4 or the biomaterial according to any one of claims 5-7 in the preparation of a reagent for detecting porcine pseudorabies virus gE protein.