ELISA antibody detection kit coated with neutralizing epitope protein fragment of pseudorabies virus, and vaccine composition containing the same

Abstract

The present invention provides an ELISA antibody detection kit for neutralizing antibodies of porcine pseudorabies virus, the kit comprising a support medium coated with a protein fragment of a neutralizing epitope of the gD protein of the porcine pseudorabies virus, an enzyme-labeled secondary antibody reagent, a detection reagent for reacting to the enzyme-labeled secondary antibody, a negative control and a positive control; the protein fragment of the neutralizing epitope of the gD protein of the porcine pseudorabies virus is shown in SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5. The ELISA antibody detection kit of the present invention can be used to evaluate the neutralizing antibody titer produced by vaccines prepared from different strains of porcine pseudorabies virus, so as to evaluate the effects of different vaccines after immunization, and also helps to formulate a scientific immunization program in a timely manner according to the test results. The present invention also provides a vaccine composition containing an immunizing amount of the protein fragment of the neutralizing epitope of the gD protein of the porcine pseudorabies virus, which has a neutralizing effect on strains of different types of PRV (including original vaccine strains, classical strains, and variant strains).

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an ELISA antibody detection kit coated with a protein fragment of a neutralizing epitope of a pseudorabies virus of porcine, a vaccine composition containing the same, and applications. Background Art

[0002] Pseudorabies, also known as Aujeszky's disease, is an acute infectious disease caused by the suid herpesvirus 1 strain (also known as pseudorabies virus, PRV) in the α subfamily of the Herpesviridae family, affecting a variety of domestic and wild animals such as pigs, cattle, sheep, dogs, cats, rabbits, mice, wild boars, minks, bears and foxes, with fever, itching (except pigs) and encephalomyelitis as the main symptoms. Pseudorabies in pigs is widely present in my country and is a serious hazard. It is one of the main diseases restricting the production of large-scale pig farms. It can cause abortion, stillbirth or mummification in pregnant sows, neurological symptoms and paralysis in piglets, especially 100% mortality after infection within 20 weeks of age, and respiratory symptoms in large and medium-sized pigs.

[0003] There are more than 40 vaccine manufacturers on the market. In order to more accurately compare the immunization effects, Guo Tiancheng et al. (Comparative study on neutralization titer of Bartha strain and variant strain vaccines of pseudorabies virus after immunization, Guangdong Animal Husbandry and Veterinary Science and Technology, 2017, 42(4):46-47) used a neutralization test to quantify the neutralizing antibody titer of pigs after vaccination. The results showed that even if the number of immunizations with the PRV variant vaccine was less than that of the original Bartha strain vaccine, the neutralization titer after immunization was still significantly higher than that of the original Bartha strain vaccine. It took 7 years for Pleco to conduct intensive research and develop the first approved inactivated vaccine for swine pseudorabies mutant strains in China (trade name: Puweijing). The neutralizing antibody titer produced by this vaccine after immunization is 16-18 times that of imported vaccines, 5-6 times that of domestic live vaccines, and 5-6 times that of domestic inactivated vaccines, which is much higher than other existing products (https: / / dy.163.com / v2 / article / detail / EFHKHKL10519EHE9.html). At present, the evaluation method for vaccines prepared from mutant strains after immunization is mainly neutralization test, but the neutralization test takes about 5 days and must be completed in a laboratory with cell manipulation capabilities, which is not conducive to clinical application. In addition, the immune mechanism is still unclear, and later vaccine improvements also require clinically applicable evaluation methods.

[0004] Therefore, there is an urgent need to develop a rapid and simple detection method that can detect the neutralizing antibodies produced by vaccines prepared from different types of porcine pseudorabies viruses and specify the immunization program based on the results.

[0005] In addition, there is an urgent need for a vaccine that can neutralize different types of porcine pseudorabies virus in order to solve real clinical problems. Summary of the invention

[0006] In order to solve the shortcomings of the prior art, the present invention provides an ELISA antibody detection kit coated with a protein fragment of a neutralizing epitope of a pseudorabies virus of porcine, wherein the ELISA antibody detection kit comprises a supporting medium coated with a protein fragment of a neutralizing epitope of a gD protein of a pseudorabies virus of porcine, an enzyme-labeled secondary antibody reagent, a detection reagent reacting to the enzyme-labeled secondary antibody, a negative control and a positive control; the protein fragment of the neutralizing epitope of a gD protein of a pseudorabies virus of porcine is shown as SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

[0007] The test results of the kit of the present invention are consistent with the results of the neutralization test, and can replace the neutralization test which has complex test requirements, harsh environment, and is time-consuming (requiring 5 days). It can be used to evaluate the neutralizing antibody titers produced by previous vaccine strains, vaccines prepared from classical strains of porcine pseudorabies virus, and vaccines prepared from variant strains of porcine pseudorabies virus, so as to evaluate the effects of different types of vaccines after immunization, and it is also helpful to formulate scientific immunization programs in a timely manner according to the test results.

[0008] The protein containing the neutralizing epitope of pseudorabies virus is a polypeptide of the neutralizing epitope site 130-149AA of the gD protein of pseudorabies virus (as shown in SEQ ID NO.5), a polypeptide of the neutralizing epitope site 130-170AA of the gD protein of pseudorabies virus (as shown in SEQ ID NO.4), a polypeptide of the neutralizing epitope site 101-200AA of the gD protein of pseudorabies virus (as shown in SEQ ID NO.3), or the gD protein of pseudorabies virus (gD protein of pseudorabies virus HN1201 strain).

[0009] The kit of the present invention can be coated with porcine pseudorabies virus gD protein, protein fragments of neutralizing epitopes of 101-200AA, protein fragments of neutralizing epitopes of 130-170AA, and protein fragments of neutralizing epitopes of 130-149AA. These coated protein fragments or proteins can bind to and detect neutralizing antibodies produced by different types of porcine pseudorabies virus strains.

[0010] As an embodiment of the present invention, in the ELISA antibody detection kit, the negative control is serum in which both PRV antigen and antibody are negative, the positive control is serum from pigs immunized with pseudorabies vaccine of a variant strain of pseudorabies virus, the enzyme-labeled secondary antibody is enzyme-labeled sheep anti-pig IgG or enzyme-labeled rabbit anti-pig IgG, and the enzyme-labeled enzyme is horseradish peroxidase, alkaline phosphatase or β-D-galactosidase.

[0011] As an embodiment of the present invention, in the ELISA antibody detection kit, the protein fragment coating concentration of the neutralizing epitope of the porcine pseudorabies virus gD protein is 0.1-2.0 μg / ml; the supporting medium is a microtiter plate; the enzyme labeling reagent is a solution diluted with an enzyme labeling diluent to a final volume of 0.05% V / V enzyme-labeled sheep anti-swine IgG or enzyme-labeled rabbit anti-swine IgG; the enzyme labeling diluent is a phosphate buffer solution of 20% V / V newborn calf serum, 0.05% V / V ProClin300, and 0.05% Tween-20.

[0012] The coating concentration is selected from 0.2μg / ml, 0.3μg / ml, 0.4μg / ml, 0.5μg / ml, 0.6μg / ml, 0.7μg / ml, 0.8μg / ml, 0.9μg / ml, 1.0μg / ml, 1.1μg / ml, 1.2μg / ml, 1.3μg / ml, 1.4μg / ml, 1.5μg / ml, 1.6μg / ml, 1.8μg / ml, 1.9μg / ml, 2.0μg / ml.

[0013] As a preferred embodiment of the present invention, the coating concentration of the porcine pseudorabies virus protein is 0.2-1.6 μg / ml.

[0014] As a more preferred embodiment of the present invention, the coating concentration of the porcine pseudorabies virus protein is 0.4-1.0 μg / ml.

[0015] As a most preferred embodiment of the present invention, the coating concentration of the porcine pseudorabies virus protein is 0.4 μg / ml.

[0016] As an embodiment of the present invention, in the ELISA antibody detection kit, the detection reagent for the enzyme-labeled secondary antibody reaction includes a color developing solution and a stop solution, the color developing solution includes a color developing solution A and a color developing solution B, the color developing solution A is a solution containing 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid, and 0.03% w / v urea peroxide, and the color developing solution B is a solution containing 0.02% w / v tetramethylbenzenediamine and 10% v / v anhydrous ethanol; the stop solution is a 2M H2SO4 solution; the ELISA antibody detection kit also includes a washing solution and a sample diluent, the washing solution is a phosphate buffer, and the sample diluent is a PBS solution containing 10% V / V fetal bovine serum, 0.1% V / V Tween 20, 1% W / V BSA, 0.05% to 0.5% W / V Casein, and 1% W / V Proclin300.

[0017] The present invention also provides a method for preparing the ELISA antibody detection kit, wherein the method comprises: step (1) expressing the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein, coating it on the supporting medium at a coating concentration of 0.1-2.0 μg / ml and 100 μl / well, wherein the supporting medium is a microtiter plate, coating at 2-8°C for 16-24 hours or at 37°C for 2 hours, and then washing, blocking and drying; step (2) blocking the microtiter plate coated in step (1) with a blocking solution, wherein the blocking solution contains 5% W / V sucrose, 20% V / V newborn calf serum, 0.05% V / V ProClin300 phosphate buffer, the blocking conditions are 2-8°C blocking for 16-24 hours or 37°C blocking for 2 hours; step (3) diluting the enzyme-labeled sheep anti-pig IgG or enzyme-labeled rabbit anti-pig IgG to a final volume of 0.05% V / V with an enzyme-labeled diluent to prepare an enzyme-labeled secondary antibody reagent, the enzyme-labeled diluent is 20% V / V newborn calf serum, 0.05% V / V ProClin300, and 0.05% Tween-20 phosphate buffer; step (4) respectively preparing a washing solution, a color developing solution, a stop solution, a positive control, a negative control, and a sample diluent; and step (5) assembling the microtiter plate prepared in step (2), the enzyme-labeled secondary antibody reagent in step (3), and the washing solution, color developing solution, stop solution, positive control, and negative control group prepared in step (4) to form the ELISA antibody detection kit.

[0018] The preparation method of the present invention optimizes various conditions in the preparation process of each step, thereby ensuring the sensitivity of the prepared kit.

[0019] As an embodiment of the present invention, the coating concentration of the porcine pseudorabies virus protein in the step (1) is 0.2-1.6 μg / ml, and 100 μl / well is coated.

[0020] As a preferred embodiment of the present invention, the coating concentration of the porcine pseudorabies virus protein in the step (1) is 0.4-1.0 μg / ml, and 100 μl / well is coated.

[0021] As a more preferred embodiment of the present invention, the coating concentration of the porcine pseudorabies virus protein in step (1) is 0.4 μg / ml, and 100 μl / well is coated.

[0022] As an embodiment of the present invention, the drying conditions in step (1) are drying for 3 to 6 hours at 18° C. to 26° C. and a relative humidity not higher than 30%.

[0023] As an embodiment of the present invention, the color developing solution in step (4) includes color developing solution A and color developing solution B, wherein the color developing solution A contains 14.7g of disodium hydrogen phosphate, 9.3g of citric acid, and 0.3g of urea peroxide per 1L of water, and the color developing solution B contains 0.2g of tetramethylbenzenediamine and 100ml of anhydrous ethanol per 1L of water; the positive control is the serum of pigs immunized with the variant strain pseudorabies vaccine, and the negative control is the serum in which both PRV antigen and antibody are negative; the washing solution is a phosphate buffer solution, and 1L of a 20-fold concentrated solution of the washing solution contains 160g of sodium chloride, 58g of disodium hydrogen phosphate, 4.8g of potassium dihydrogen phosphate, 4g of potassium chloride, and 10mL of Tween 20; the sample diluent contains 10% V / V fetal bovine serum, 0.1% V / V Tween 20, 1% W / V BSA, 0.05%-0.5% W / V Casein, and 1% W / V Proclin300 solution in PBS; and the stop solution is 2M H2SO4 solution.

[0024] The present invention also provides the use of the ELISA antibody detection kit in non-immune diagnosis, wherein the application of the non-immune diagnosis includes vaccine immunization program evaluation and epidemiological investigation of ex vivo serum.

[0025] The present invention also provides a vaccine composition, wherein the vaccine composition comprises an immunizing amount of a protein fragment of a neutralizing epitope of the gD protein of pseudorabies virus of porcine and a pharmaceutically acceptable carrier, wherein the protein fragment of the neutralizing epitope of the gD protein of pseudorabies virus of porcine is shown as SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

[0026] After immunizing pigs with the vaccine composition containing the neutralizing epitope of the gD protein of pseudorabies virus of the present invention, a high level of neutralizing antibodies is produced, which can neutralize strains of different types of PRV (including original vaccine strains, classical strains, and variant strains).

[0027] As an embodiment of the present invention, in the vaccine composition described in the present invention, the protein fragment content of the neutralizing epitope of the porcine pseudorabies virus gD protein is ≥15 μg / ml; the pharmaceutically acceptable carrier is an adjuvant, the adjuvant is 206 adjuvant, and the adjuvant content is 46 v / v%.

[0028] As a preferred embodiment of the present invention, in the vaccine composition of the present invention, the content of the protein fragment of the neutralizing epitope of the gD protein of pseudorabies virus is 15-100 μg / ml.

[0029] As a more preferred embodiment of the present invention, in the vaccine composition of the present invention, the content of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 20-100 μg / ml.

[0030] The present invention also provides the use of the vaccine composition in preparing a medicine for preventing and / or treating porcine pseudorabies virus infection.

[0031] The vaccine composition of the present invention can produce neutralizing antibodies against different types of porcine pseudorabies viruses, that is, it can prevent and / or treat different types of porcine pseudorabies virus infections.

[0032] The advantages of the present invention are:

[0033] The present invention unexpectedly discovered the distinctive neutralizing epitopes on the gD protein of pseudorabies virus of porcine through a large number of experiments.

[0034] The kit prepared by the present invention using a protein containing a neutralizing epitope of the gD protein of pseudorabies virus of porcine as a coating source has consistent results with neutralization tests conducted using mutant strains, and can replace neutralization tests that have complex test requirements, harsh environments, and are time-consuming (requiring 5 days), so as to be used to evaluate the effects of different vaccines after immunization, and to help formulate scientific immunization programs in a timely manner based on the test results; it can also be used to monitor maternal antibodies.

[0035] The vaccine composition prepared by the present invention produces relatively high neutralizing antibodies against different types of PRV strains (including original vaccine strains, classical strains, and variant strains). BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Comparison of the results of 14 samples tested by Kit A and the neutralization test. DETAILED DESCRIPTION

[0037] The term "epitope", also known as antigen determinant, is composed of some amino acid residues that participate in the formation of the bond between antigen and antibody molecules. It is the core component of the antigen and the specific region that can be recognized by the antibody molecule. These epitopes are the structural basis for the generation of antigenicity, and epitope vaccines are proposed on this basis. Epitope vaccines have the advantages of not requiring the separation of infectious pathogens, not spreading toxins, and reducing the mutual interference of different immunodominant antigen epitopes.

[0038] The term "neutralizing epitope", also known as "neutralizing antigen epitope", "neutralizing antigen determinant", "neutralizing antigen site", "neutralizing antigen determinant" or "neutralizing epitope", refers to a protein fragment or peptide segment in an antigen molecule that induces the production of neutralizing active antibodies.

[0039] The term "microtiter plate" (ProPlate Microtiter Plates) refers to a well with a transparent bottom, and at least two adjacent wells have at least one physical deformation between them, wherein the physical deformation refers to a shape that can have, for example, a channel, a ridge, a hole, a slit or a step. The microtiter plate of the present invention includes but is not limited to a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, preferably a 96-well plate.

[0040] The terms "developer solution A" and "developer A solution" can be used interchangeably, and "developer solution B" and "developer B solution" can be used interchangeably.

[0041] The term "phosphate buffer" refers to a solution containing phosphoric acid or its salt and adjusted to an ideal pH value. It is the most widely used buffer in biochemical research. Generally, phosphate buffer is prepared from phosphoric acid or phosphates (including but not limited to sodium and potassium salts). Some phosphates are already known in the art, such as sodium dihydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate, sodium phosphate and potassium phosphate. It is known that phosphates exist in the form of salt hydrates. Due to the secondary dissociation of the buffer, the pH range of the buffer is very wide, such as a range of about 4-10, preferably a range of about 5-9, more preferably a range of about 6-8, and most preferably about 7.4. Further preferably, the phosphate buffer is a phosphate buffer containing sodium chloride and potassium chloride.

[0042] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0043] The phosphate buffer used in the embodiment of the present invention is PBS with a pH value of 7.4, and its 1L volume formula is: NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 2.9g, KH2PO4 0.2g, and ultrapure water is used to make up to 1L, but this implementation mode does not constitute a limitation of the present invention under any circumstances. The experimental methods described in the present invention are conventional methods unless otherwise specified; the biological materials described can be obtained from commercial channels unless otherwise specified.

[0044] Example 1 Preparation, purification and identification of monoclonal antibodies against pseudorabies virus in early stage

[0045] Five monoclonal antibodies against gD protein of pseudorabies virus (3G1, 3B6, 4A5, 4D9, 5G7, see Chinese patent CN109206509A for details), one monoclonal antibody against gB protein of pseudorabies virus (1H1, see Chinese patent CN106188280A for details), and four monoclonal antibodies against gC protein of pseudorabies virus (1B9, 1F2, 3D4, 4C7, see Chinese patent CN109212205A for details) prepared in the early stage were respectively used to infect the variant strain HN1201 of pseudorabies virus (PRV The HN1201 strain has a preservation number of CCTCCNO.V201311, see patent CN104004774A), the classic strain Fa strain (purchased from the China Veterinary Drug Administration), and the vaccine strain Bartha strain were subjected to neutralization tests. The results showed that only monoclonal antibodies 5G7 and 1H1 had non-complement-dependent neutralizing activity against the three types of PRV strains, and there was no significant difference in neutralization titer; the others did not have non-complement-dependent neutralizing activity against the three types of PRV strains. At present, these monoclonal antibodies to PRV cannot distinguish between the significant differences in neutralizing antibodies produced by vaccine strains prepared from early vaccine strains and late variant strains after immunizing pigs.

[0046] In addition, a large number of experimental studies conducted by the research and development team of the present invention found that the neutralizing antibodies of the pseudorabies virus variant strains gB and gC are not very high, and the neutralizing titer of gD is more than 1:20. The gD protein is the most important immune protection-related protein, which can better reflect the immune status after vaccination and can more accurately formulate the immunization program (https: / / www.zgyz001.com / news / enterprise_news / 123390.html). Based on this, the present inventors focused on the preparation of monoclonal antibodies against the gD protein of pseudorabies virus in pigs, especially the preparation of monoclonal antibodies that can recognize the neutralizing epitope sites of PRVgD protein.

[0047] Example 2 Preparation, purification and identification of monoclonal antibodies against pseudorabies virus gD protein

[0048] 2.1 Preparation and identification of pseudorabies virus gD protein

[0049] PRV HN1201 virus or its different generations of culture were inoculated on well-growing PK15 cells, and the virus fluid was collected. A small amount was used to extract PRV genomic DNA and prepare PRVgD protein according to patent CN105251000A. The content of PRVgD protein (abbreviated as PRVgD1) was determined by SDS-PAGE optical density method to be 200 μg / ml.

[0050] 2.2 Preparation and purification of monoclonal antibody against PRVgD protein

[0051] First, the PRV HN1201 strain virus liquid was inactivated by formaldehyde and emulsified with an equal volume of Freund's adjuvant, and then 10 mice were immunized once every 2 weeks. The indirect immunofluorescence (IFA) antigen plate prepared by the PRV HN1201 strain prepared in Example 1.1 was used to continuously detect the mouse sera after 3-6 immunizations by IFA. The results showed that the IFA titer of the mouse sera in each group was ≤1:640. One mouse was randomly selected for cell fusion according to the operating method of Harlow E et al. (Harlow E, Lane D. Antibodies: a laboratory manual. New York: Cold Spring Harbor Laboratory Press. 1998, 139-312). The positive rate after fusion was 0, and it was abandoned.

[0052] Secondly, the PRV HN1201 strain virus liquid was inactivated by formaldehyde and emulsified with an equal volume of Freund's adjuvant for the first immunization of 20 mice. After 2 weeks, the PRVgD protein was emulsified with an equal volume of Freund's adjuvant for the second immunization, and the immunization was repeated for 3 rounds. After the second round of immunization, 20 mice were blooded 9 days after each immunization and the serum was continuously tested by ELISA using the ELISA method coated with the PRVgD protein prepared in Example 1. After the mouse serum was immunized 4-6 times, the ELISA titer had an upward trend and only 2 mice with an ELISA titer of ≥1:12800 were subjected to cell fusion, and they were subjected to limiting dilution subclone screening by ELISA to obtain 6 positive hybridoma cells, with a positive rate of only 2%. PK-15 cells infected with PRV HN1201 strain were coated on microplates, and the supernatants of 6 positive hybridoma cells were evaluated by indirect immunofluorescence IFA. One strain was found to have non-specific adsorption and was discarded, and 5 positive hybridoma cells (3G1, 3B6, 4A5, 4D9, 5G7) were obtained. After preparing ascites from the 5 hybridoma cells, the neutralization titer was determined. The results showed that only monoclonal antibody 5G7 had non-complement-dependent neutralizing activity against the three types of PRV strains, and there was no significant difference in neutralization titer, while the rest had no non-complement-dependent neutralizing activity.

[0053] Again, the prepared PRVgD protein was emulsified with an equal volume of Freund's adjuvant and 30 mice were immunized for the first time. Two weeks later, the PRVgD protein was emulsified with an equal volume of Freund's adjuvant for the second immunization, and the immunization was repeated for three rounds. After the second round of immunization, blood was collected from 30 mice 9 days after each immunization and the serum was continuously tested by IFA using an indirect immunofluorescence antigen plate (i.e., IFA antigen plate) prepared by the PRV variant HN1201 strain. Results: After 4-6 times of immunization, the IFA titer of the mouse serum showed an upward trend and the IFA titer was ≥1∶6400 in only one mouse. The mouse serum was tested by ELISA using an ELISA plate coated with PRVgD protein. The results showed that the ELISA titer of only one mouse serum was ≥1∶128000. The mouse was subjected to cell fusion and subclone screening by limiting dilution method using IFA to obtain 15 positive hybridoma cells, with a positive rate of only 2%.

[0054] The supernatants of 15 positive hybridoma cells were collected and used to prepare indirect immunofluorescence antigen plates (i.e., IFA antigen plates) using the classical strain Fa and the vaccine strain Bartha (purchased from). The results showed that only 10 hybridoma cells (strain numbers: 1G2, 2A7, 2F5, 2G10, 3C5, 3E6, 3H3, 4B2, 4H5, 5F6) secreted monoclonal antibodies in the supernatant that showed good reactions with PRV variants, classical strains, and vaccine strains (IFA titers were all ≥1:8), and the monoclonal antibodies secreted in the supernatant by the remaining 5 hybridoma cells reacted weakly with the PRV classical strain or vaccine strain (IFA titers were all <1:2) and were therefore discarded.

[0055] Mouse ascites were prepared from the 10 positive hybridoma cells, which were purified by caprylic acid-ammonium sulfate combined precipitation method and then identified by SDS-PAGE gel electrophoresis. The results showed that the purity of the 10 monoclonal antibodies was not less than 85%. Quantitative analysis was performed using the BCA protein quantification kit according to the instructions, and the results showed that the protein content of the 10 monoclonal antibodies was not less than 2.0 mg / ml.

[0056] 2.3 Determination of neutralization titer of monoclonal antibodies against pseudorabies virus

[0057] The neutralizing titers of the 10 monoclonal antibodies prepared in Example 2.2 were determined by the fixed virus dilution serum method described in the 2015 edition of the Chinese Veterinary Pharmacopoeia. Different dilutions of the monoclonal antibodies (1:1 V / V-1:2048 V / V) were first incubated with PK15 cells at 37°C and 5% CO2 for 1 hour, and then virus dilutions of the PRV vaccine strain Bartha, the classic strain Fa, and the variant strain HN1201 (all containing 100 TCID 50 / ml), and cultured at 37°C, 5% CO2 for 72-120 hours to observe the cytopathic effect and calculate the neutralization titer. The results are shown in Table 1.

[0058] Table 1 Summary of neutralization titers of 10 monoclonal antibodies

[0059]

[0060] The results showed that only monoclonal antibody 5F6 had a neutralizing reaction with different types of PRV strains (vaccine strains, classic strains, and variant strains), but the neutralizing activity was different from the reaction results of monoclonal antibody 5G7. The neutralizing titer against vaccine strains was significantly lower, and the neutralizing titer against clinical strains (classic strains and HN1201 strains) was significantly higher (at least 448 times higher than against vaccine strains). Monoclonal antibody 5F6 can be further used for PRVgD protein structure analysis, neutralization active sites, and the production of high-titer neutralizing antibodies after immunization with variant vaccine preparation.

[0061] 2.4 Evaluation of the monoclonal antibody 5F6 against pseudorabies virus

[0062] 2.4.1 Identification of monoclonal antibody subtypes

[0063] The subtype of monoclonal antibody 5F6 was identified using a monoclonal antibody subtype identification kit. Results: The heavy chain subtype of monoclonal antibody 5F6 was IgG1, and the light chain type was kappa.

[0064] 2.4.2 Specificity identification

[0065] Classical swine fever virus CSFV, porcine reproductive and respiratory syndrome virus PRRSV, porcine circovirus type 2 PCV2, porcine parvovirus PPV, pseudorabies virus PRV, porcine epidemic diarrhea virus PEDV, porcine transmissible gastroenteritis virus TGEV, porcine rotavirus PRoV and cultured PRV PK15 cells were fixed in microplates, and monoclonal antibody 5F6 ascites was used as primary antibody, and fluorescent-labeled rabbit anti-mouse secondary antibody was used as secondary antibody. The detection was carried out according to the indirect immunofluorescence method IFA. The results showed that monoclonal antibody 5F6 only reacted with PRV, and did not cross-react with other common porcine diseases and cultured cells.

[0066] PRVgBΔ148~546 protein, PRVgB protein (prepared according to patent CN10563827A), PRVgC protein (expressed by baculovirus vector according to PRVgC sequence shown in patent CN104004774A), and PRVgD protein prepared in Example 2.1 were coated in microplates respectively, monoclonal antibody 5F6 ascites was used as primary antibody, enzyme-labeled goat anti-mouse secondary antibody was used as secondary antibody, and ELISA was used for detection. The results showed that monoclonal antibody 5F6 only reacted with PRVgD protein, but not with other PRV proteins. It shows that monoclonal antibody 5F6 is a specific monoclonal antibody for PRVgD protein.

[0067] 2.4.3IFA evaluation

[0068] The titer of monoclonal antibody 5F6 was determined by immunofluorescence detection (IFA). IFA detection method: culture adherent PK-15 cells, inoculate with virus containing PRV HN1201 strain, Fa strain, Ma strain, Bartha strain at a virus infection multiplicity MOI of 0.005-0.1, and set up healthy cell control wells. After culturing for 48-72 hours at 37℃ and 5% CO2, discard the supernatant; fix with 80% acetone solution at 2-8℃ for 30 minutes, wash 3 times with PBS, add 100μl of monoclonal antibody 5F6 diluted 1:100 and then diluted 2 times to the virus inoculation well and cell control well, respectively, and add PRV mouse positive serum to the virus inoculation well as a positive control, and act at 37℃ for 60 minutes; wash 3 times with PBS and dry; add 1:500 diluted FITC-labeled goat anti-mouse IgG, act at 37℃ for 60 minutes; wash 3 times with PBS and dry; add 50μl PBS and observe under a fluorescence microscope. IFA titer determination: The maximum antibody dilution corresponding to the infected cell plate wells where yellow-green fluorescence can be observed is taken as the IFA titer of the antibody.

[0069] Results: The IFA titers of monoclonal antibody 5F6 against PRV HN1201, Fa, Ma and Bartha strains were 1:12800, 1:6400, 1:6400 and 1:6400, respectively.

[0070] 2.4.4 ELISA titer

[0071] PRVgD protein was coated on ELISA plate at 100 ng / well and 100 μl / well. Monoclonal antibody 5F6 was diluted 100 times with phosphate buffer and then diluted 2 times as primary antibody and incubated at 37°C for 1 hour. After washing, enzyme-labeled goat anti-mouse IgG was added and incubated at 37°C for 30 minutes. After washing, substrate was added for color development. After termination, OD was read on the microplate reader. 450nm The indirect ELISA reaction was performed and the monoclonal antibody 5G7 was used as a positive control. Results: The ELISA titer of the monoclonal antibody was 1:512000.

[0072] 2.4.5 Variable region sequencing

[0073] According to the sequence characteristics of mouse monoclonal antibodies, the heavy chain variable region primer sequences were designed: P1: 5'-ACTAGTCGACATGAAATGCTCGTGGRTYATSAACTT-3' P2: 5'-ACTAGTCGACATGAAATGCAGCTGGRTYAT-3'

[0074] Design light chain variable region primer sequences:

[0075] P3: 5'-ACTAGTCGACATGGTYGTYATVTCCTTGCT-3'

[0076] P4: 5'-ACTAGTCGACATGGGCWTCAAGATGRAGTCACAKW-3'

[0077] Hybridoma cells 5F6 were cultured and collected, RNA was extracted and reverse transcribed as a template, the variable region sequence was amplified using the above primers, and the amplified product was sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. Results: The nucleotide sequences of the heavy chain variable region and light chain variable region of monoclonal antibody 5F6 are shown in SEQ.ID No.1 and SEQ.ID No.2, respectively.

[0078] 2.5 Identification of the antigenic epitope recognized by monoclonal antibody 5F6

[0079] 2.5.1 Preliminary identification of neutralizing epitope sites in PRVgD protein

[0080] Peptides were designed based on the amino acid sequences encoded by the nucleotide sequences of the gD protein of PRV HN1201 strain (NCBI accession number: KP722022.1) and the gD protein of Bartha strain (NCBI accession number: JF797217.1) (see Table 2). Each peptide was directly delivered to GenScript Biotech Co., Ltd. for synthesis and diluted with carbonate coating buffer (pH 9.6) and coated on ELISA plates at 500 ng / well and 100 μl per well. After blocking and drying, monoclonal antibodies 5F6, 5G7, 1H1, and 3B6 were diluted 100 times with phosphate buffer and used as primary antibodies for incubation at 37°C for 1 hour. After washing, enzyme-labeled goat anti-mouse IgG was added and incubated at 37°C for 30 minutes. After washing, substrate was added for color development. After termination, OD was read on a microplate reader. 450nm Values ​​were used to perform indirect ELISA reactions. At the same time, the PRVgD protein prepared in Example 2.1 was coated on an ELISA plate at 100 ng / well and 100 μl / well, and monoclonal antibodies 5F6, 5G7, 1H1, and 3B6 were diluted 16,000 times with phosphate buffer and used as primary antibodies for indirect ELISA reactions as controls. Results (see Table 2): Preliminary results show that the antigenic site of the monoclonal antibody 5F6 that recognizes the PRVgD protein is located at 101-200AA, as shown in SEQ ID No.3.

[0081] Table 2 Summary of the reaction results between PRVgD protein peptides and monoclonal antibodies (OD values)

[0082]

[0083] Note: AA stands for amino acid.

[0084] 2.5.2 Further identification of neutralizing epitope sites in PRVgD protein

[0085] According to the amino acid sequence encoded by the nucleotide sequence of the gD protein of PRV HN1201 strain (NCBI accession number: KP722022.1) and the gD protein of Bartha strain (NCBI accession number: JF797217.1), the peptides were designed. The 101-200AA was divided into three peptides (see Table 3) and directly submitted to GenScript Biotech Co., Ltd. for synthesis. After diluting with carbonate coating buffer (pH 9.6), the ELISA plates were coated at 500ng / well and 100μl / well. The monoclonal antibodies 5F6, 5G7, 1H1, and 3B6 were diluted 100 times with phosphate buffer and used as primary antibodies for indirect ELISA reaction. At the same time, the PRVgD protein peptide 101-200AA prepared in Example 4.1 was used as a control. Results (see Table 3): It was further determined that the antigenic site of the monoclonal antibody 5F6 recognizing the PRVgD protein was located at 130-170AA, as shown in SEQ ID No.4.

[0086] Table 3 Summary of the reaction results between PRVgD protein peptides and monoclonal antibodies (OD values)

[0087]

[0088] Note: AA stands for amino acid.

[0089] 2.5.3 Re-identification of the neutralizing epitope site of PRVgD protein

[0090] According to the amino acid sequence encoded by the nucleotide sequence of the gD protein of PRV HN1201 strain (NCBI accession number: KP722022.1) and the gD protein of Bartha strain (NCBI accession number: JF797217.1), the peptides were designed. The 130-170AA was divided into two peptides (see Table 4) and directly handed over to GenScript Biotech Co., Ltd. for synthesis. After diluting with carbonate coating buffer (pH 9.6), they were coated on ELISA plates at 500ng / well and 100μl / well. Monoclonal antibodies 5F6, 5G7, 1H1, and 3B6 were diluted 100 times with phosphate buffer and used as primary antibodies for indirect ELISA reaction. At the same time, the PRVgD protein peptide 101-200AA prepared in Example 2.5.1 was used as a positive control. Results (see Table 4): It can be determined that the antigenic site of the monoclonal antibody 5F6 recognizing the PRVgD protein is located at 130-149AA, and the amino acid sequence is shown in SEQ ID No.5.

[0091] Table 4 Summary of the reaction results between PRVgD protein peptides and monoclonal antibodies (OD values)

[0092]

[0093] Note: AA stands for amino acid.

[0094] Example 3 Preparation, detection method and evaluation of ELISA kit

[0095] The coating source of the antigen coating plate in the kit of the present invention is any one or more combinations of the polypeptide of the neutralizing epitope site 130-149AA of the pseudorabies virus gD protein in Example 2.5.3, the polypeptide of the neutralizing epitope site 130-170AA of the pseudorabies virus gD protein in Example 2.5.2, the polypeptide of the neutralizing epitope site 101-200AA of the pseudorabies virus gD protein in Example 2.5.1, and the pseudorabies virus gD protein, and the detection effect is equivalent. In order to facilitate the concise description of the embodiments of the present invention, only the polypeptide of the neutralizing epitope site 130-149AA of the pseudorabies virus gD protein in Example 2.5.3 is used as the coating source as an example.

[0096] 3.1 Preparation of the kit

[0097] Antigen coated plate: Dilute the polypeptide of the neutralizing epitope site 130-149AA of the gD protein of the pseudorabies virus in Example 2.5.3 to the preferred final concentration with carbonate buffer (pH 9.6, 0.05 mol / L) for coating, 100 μl / well, coat at 2-8°C for 16-24 hours, wash with washing solution, add blocking solution (phosphate buffer containing 5% W / V sucrose, 20% V / V newborn calf serum, 0.05% V / V ProClin300) at 150 μl / well, block at 2-8°C for 16-24 hours or at 37°C for 2 hours, discard the blocking solution and dry at 18-26°C and relative humidity not higher than 30% for 3-6 hours, seal and store at 2-8°C for use.

[0098] Enzyme labeling reagent: Mix 20% V / V newborn calf serum, 0.05% V / V ProClin300, and 0.05% Tween-20 in phosphate buffer to prepare enzyme labeling diluent. Dilute the commercial goat anti-swine IgG-HRP or rabbit anti-swine IgG-HRP with enzyme labeling diluent to a final volume of 0.05% V / V, mix well, filter through 0.22μm, and dispense aseptically.

[0099] Positive control: Variant strain of pseudorabies inactivated vaccine (HN1201-ΔgE strain, from Pleco Biotech Co., Ltd.) was used to immunize healthy susceptible pigs with negative PRV antigen and antibody in the neck muscle (2ml / head). After 28 days, blood was collected and serum was separated. The collected serum was the positive serum. 200ml of newborn calf serum, 10ml of positive serum, and 0.5ml of ProClin-300 were mixed and phosphate buffer was added to make the volume 1000mL. After filtering, the mixture was sterilely packaged.

[0100] Negative control: Select healthy susceptible pigs with negative PRV antigen and antibody, collect blood from the carotid artery and separate serum. The collected serum is negative serum. Mix 200ml of newborn calf serum, 10ml of negative serum, and 0.5ml of ProClin-300, add phosphate buffer to make the volume 1000mL, filter and package aseptically.

[0101] Sample diluent: PBS solution containing 10% V / V fetal bovine serum, 0.1% V / V Tween 20, 1% W / V BSA, 0.05%-0.5% W / V Casein, 1% W / V Proclin300, 0.22 μm filtered and sterile packaged.

[0102] 20× concentrated washing solution: Take 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, 800mL ultrapure water, and 10mL Tween 20. After complete dissolution, add ultrapure water to make up to 1000mL, filter with a 0.22μm filter membrane, and dispense aseptically. Dilute 20 times with distilled water before use.

[0103] Color developing solution: Disodium hydrogen phosphate 14.7g, citric acid 9.3g, urea peroxide 0.3g, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution A. Tetramethylbenzidine (TMB) 0.2g, anhydrous ethanol 100ml, dissolved in purified water, dilute to 1000mL, mix, filter, and aseptically dispense to obtain color developing solution B.

[0104] Stop solution: 2M H2SO4 solution.

[0105] 3.2 Establishment of detection method

[0106] All reagents should be equilibrated to room temperature (about 30 minutes) before use. Liquid reagents should be gently shaken to mix before the experiment and immediately sealed and stored at 2-8°C after use.

[0107] The steps are as follows:

[0108] (1) Add sample diluent: Add 100 μl of sample diluent to the corresponding wells of the microtiter plate. At the same time, set up negative control wells (add 100 μl of negative control / well), positive control wells (add 100 μl of positive control / well), and blank control wells (add 100 μl of PBS buffer).

[0109] (2) Add the sample to be tested: Add 10 μl of the sample to be tested to the corresponding wells except the negative control well, positive control well, and blank control well. Seal the plate and incubate at 37°C for 30 minutes.

[0110] (3) Washing: Wash 5 times with detergent and dry for the last time.

[0111] (4) Add enzyme-labeled reagent: Add 100 μl of enzyme-labeled reagent to each well except the blank control well, seal the plate and incubate at 37°C for 30 min.

[0112] (5) Washing: Wash 5 times with detergent and dry for the last time.

[0113] (6) Color development: Add 50 μl of color development solution A and 50 μl of color development solution B to each well, mix well, and develop color at 37°C or room temperature for 15 min in the dark.

[0114] (7) Stop and detect: Add 50 μl of stop solution to each well and measure the A value of each well using an ELISA reader within 10 minutes: OD 450nm Value (measured after adjusting the blank control well to zero), or OD 450nm -OD 600nm～650nm The value is determined according to the following result judgment criteria.

[0115] When the positive control well A value OD 450nm The test is established when the A value of the negative control well is ≥0.2 and the A value of the negative control well is ≤0.1, otherwise it is invalid. When the test is established, the S / P value is calculated as: sample A value / positive control A value average. Result judgment:

[0116] Those with an S / P value ≥ 0.5 are positive for pseudorabies virus antibodies.

[0117] Those with an S / P value of <0.5 were negative for pseudorabies virus antibodies.

[0118] 3.3 Selection of coating concentration

[0119] The polypeptides of the neutralizing epitope sites 130-149AA of the gD protein of pseudorabies virus in Example 2.5.3 were diluted to final concentrations of 0.1, 0.2, 0.4, 0.8, 1.0, 1.6, and 2.0 μg / ml respectively with carbonate buffer (pH 9.6, 0.05 mol / L) for coating to prepare antigen-coated plates. The positive control and negative control were tested according to the detection method, and the dilution with the larger P / N value (mean OD value of the positive control wells / mean OD value of the negative control wells) was taken. is the coating concentration, the results (see Table 5): when the coating concentration is 0.2-1.6μg / ml, the P / N values ​​are all >90, which are significantly higher than the P / N values ​​when the coating concentration is 0.1 and 2.0μg / ml (all <50), and can effectively distinguish between negative samples and positive samples; in particular, when the coating concentration is 0.4-1.0μg / ml, the P / N value is better than the P / N value when the coating concentration is 0.2 and 1.6μg / ml (all <70), and the P / N value is the highest when the coating concentration is 0.4μg / ml.

[0120] Table 5 Selection of coating concentration of pseudorabies virus gD protein peptide segment

[0121]

[0122] According to the above results, the prepared antigen-coated plates were coated with the coating concentration of 0.4 μg / ml, and the kits were prepared according to the preparation method of the kit in Example 3.1, and were named as Kit A.

[0123] 3.4 Application of the kit

[0124] 3.4.1 Sensitivity

[0125] 25 piglets at 21 days old with negative PRV antigen and antibody were randomly divided into 5 groups, 5 piglets / group. Group 1 was immunized with live pseudorabies vaccine (Bartha strain), Group 2 was immunized with live pseudorabies vaccine (HB-98 strain), Group 3 was immunized with inactivated pseudorabies vaccine (E A strain), Group 4 was immunized with variant pseudorabies vaccine (HN1201-ΔgE strain), i.e., Puweijing, and Group 5 was injected with an equal volume of DMEM medium as a control group. Blood was collected from each group at 2 weeks, 4 weeks, 8 weeks, and 16 weeks after immunization. The above sera were all tested with Kit A, and neutralizing antibodies were determined using the variant HN1201 strain according to the serum neutralization test method of GB / T18641-2002. The test results showed a consistent trend, and the data of one pig in each group were used as an example for explanation, as shown in Table 6. This showed that the test results of Kit A were consistent with the results of the neutralization test, and could replace the neutralization test, which has complex test requirements, harsh environment, and is time-consuming (requires 5 days). It can be used to evaluate the neutralizing antibody titer produced by previous vaccine strains, vaccines prepared from classical strains of porcine pseudorabies virus, and vaccines prepared from variant strains of porcine pseudorabies virus, so as to evaluate the effects of different types of vaccines after immunization, and it is also helpful to formulate scientific immunization programs in a timely manner based on the test results.

[0126] Table 6 Antibody detection results at different times after immunization with different vaccines

[0127]

[0128] Note: Neutralization titer <1:2 is negative, and ≥1:2 is positive.

[0129] At the same time, 14 samples were taken to compare the two detection methods. The results are shown in Figure 1 ,from Figure 1 The analysis found that the detection effect of Kit A was consistent with the detection trend of the classic detection method (neutralization test), the compliance rate was also high, and the positive boundary (the S / P value was 0.5) was well correlated with immune protection, further indicating that Kit A can be used to evaluate the effectiveness of vaccine immunity.

[0130] 3.4.2 Specificity

[0131] Five confirmed pseudorabies virus antibody negative sera, one porcine circovirus type 2 (PCV2) positive serum, one porcine parvovirus (PPV) positive serum, one porcine reproductive and respiratory syndrome virus (PRRSV) positive serum, one classical swine fever virus (CSFV) positive serum, one porcine Japanese encephalitis virus (JEV) positive serum, one Haemophilus parasuis (HPs) positive serum, one porcine atrophic rhinitis (Bb) positive serum, one Escherichia coli (BL21) positive serum, one Mycoplasma hyopneumoniae (Mhp) positive serum, one porcine epidemic diarrhea virus (PEDV) positive serum, one porcine transmissible gastroenteritis (TGEV) positive serum, and one porcine rotavirus (PRoV) positive serum were tested using the kit A prepared in Example 3, the neutralization test, and the commercial kit BioChek PRVgB antibody kit, respectively. The results showed that the test results of the kit A were consistent with the neutralization test and the commercial kit test results, all of which were negative.

[0132] The sera of mice immunized with PRVgB protein, PRVgC protein and PRVgE protein were detected using Kit A, and the results were all negative.

[0133] The above results show that Kit A has no cross reaction with positive sera of other viruses and has good specificity.

[0134] 3.4.3 Repeatability

[0135] The PRVgD-positive serum and PRVgD-negative serum identified by the neutralization test were tested using ELISA kit A from different batches and different ELISA kits from the same batch. The results showed that the test results of kit A were consistent with the results of the neutralization test, and the intra-batch and inter-batch coefficients of variation were both <15%, indicating that kit A has good repeatability.

[0136] 3.4.4 Shelf life

[0137] Three batches of kit A were prepared and stored at 2-8°C, and samples were taken at 3, 6, 9, 12, 15, and 18 months. Sensitivity, specificity, and repeatability were tested with reference to Example 3.4. Results: At different storage times, the results of negative samples tested by kit A were negative, and the results of positive samples tested were all positive, with coefficients of variation of <15%. The inventor unexpectedly found that kit A still had good sensitivity, specificity, and repeatability when stored at 2-8°C for 18 months.

[0138] 3.4.5 Clinical application

[0139] According to the preparation method of Example 3.1, the gD protein of the pseudorabies virus of Example 2 was used as the antigen plate to prepare the PRVgD protein ELISA kit, which was called Kit B. 10 sera from pigs in a pig farm that had not yet been immunized with pseudorabies vaccine were collected (numbered 1# to 10#), and the kits A, B and neutralization tests were used to test them respectively. The results showed that the neutralization titers of the sera of pigs 1# to 10# were all <1:2, and the tests of Kits A and B were all negative. The pig farm needs to be immunized with pseudorabies vaccine as soon as possible. It shows that Kits A and B are helpful to formulate immunization programs in a timely manner.

[0140] 100 sera (11#~110#) of newborn piglets (0-3 days) in a piglet farm were tested by kit A and neutralization test, respectively. The results showed that the S / P values ​​of 10 pigs detected by kit A were 0.179~0.426 (corresponding to neutralization titers of <1∶2), which were negative, and the results of kit B were also negative; the neutralization test of 90 pigs was positive (neutralization titers were ≥1∶2), the positive rate of kit A was 94% (85 / 90), and the positive rate of kit B was 86% (77 / 90), and the neutralization titers of 8 sera not detected by kit B were 1∶2~1∶22.39. This shows that kit A has high accuracy in detecting serum samples with low neutralization titers, which is closer to the results of neutralization test. In addition, the experimental results show that the same litter of pigs that are negative in all three detection methods in the pig farm need to be immediately immunized with pseudorabies vaccine. If the ELISA titer does not increase significantly 14 days after immunization, a booster immunization is required. The same litter of pigs that are positive in the neutralization test but negative in the test kit A also needs to be boosted at least once. The same litter of pigs that are positive in the neutralization test and positive in the test kit A only need to be immunized according to the routine immunization procedures of piglets in the pig farm. This shows that the kit can be used to monitor maternal antibodies.

[0141] 50 samples of sera (111#~160#) from pigs immunized with pseudorabies vaccine (Bartha strain) in a pig farm and 50 samples of sera (161#~210#) from pigs immunized with pseudorabies vaccine (Puweijing) in another pig farm were tested with kit A, kit B and neutralization test respectively. The results showed that the S / P values ​​of sera from pigs 111#~160# were 0.511~0.756 (corresponding to neutralization titers of 1∶2~1∶13.5) detected by kit A, which were weakly positive. The results of Kit B were all negative, indicating that Kit A can detect weakly positive neutralizing antibody titers, while Kit B cannot detect them, and the immunization program needs to be supplemented with a variant strain of pseudorabies vaccine; the S / P values ​​of the sera of pigs from 161# to 210# were 1.756 to 2.988 (corresponding to neutralization titers of 1:32 to 1:707.94), which were strongly positive, and Kit B tested positive, indicating that both Kit A and Kit B can detect strongly positive neutralizing antibody titers. This shows that Kit A can be used for antibody monitoring of pigs after vaccination.

[0142] Example 4 Preparation and identification of a vaccine composition containing a neutralizing epitope of the gD protein of pseudorabies virus

[0143] 4.1 Preparation of vaccine composition

[0144] The expressed polypeptide of the neutralizing epitope sites 130-149AA of the gD protein of pseudorabies virus in Example 2.5.3 was mixed with the 206 adjuvant in proportion, stirred at 120 rpm for 15 minutes at 30°C, and stored at 2-8°C to obtain a vaccine composition of the neutralizing epitope of the gD protein of pseudorabies virus. Vaccines 1 to 3 were prepared using the content of the polypeptide containing the neutralizing epitope of the gD protein in Table 7 as an example.

[0145] Table 7 Vaccine ratios for neutralizing epitopes of porcine pseudorabies virus gD protein

[0146] Components Vaccine 1 Vaccine 2 Vaccine 3 gD protein neutralizing epitope (μg / ml) 15 20 100 206 adjuvant (V / V%) 46 46 46

[0147] 4.2 Identification of vaccine composition

[0148] Twelve 21-day-old PRV antigen-antibody negative piglets were randomly divided into 4 groups, 3 piglets / group. Group 1, Group 2, and Group 3 were injected intramuscularly with vaccine 1, vaccine 2, and vaccine 3 prepared in Example 4.1, respectively, at 2 ml / head for subcutaneous immunization, and the control group in Group 4 was injected with DMEM medium 2 ml / head. Blood was collected 28 days after immunization to determine the ELISA titer and neutralization titer of the immunized pig serum. The ELISA titer was determined using Kit B, and the detection was performed according to the detection method in Example 3.2.

[0149] The neutralization test was carried out according to the method of serum neutralization test in GB / T18641-2002 to determine the neutralizing antibody titer against different strains of pseudorabies virus in pigs, including the PRV variant strain HN1201, the classic strains Fa and Ma, and the early vaccine strain Bartha. The test results showed a consistent trend, and the data of one pig in each group were used as an example to illustrate the results. The results are shown in Table 8: The data of the first to third groups detected by kit A and kit B were all strongly positive, and the S / P value increased with the increase of the antigen content in the vaccine. This indicates that after the polypeptide vaccine containing the neutralizing epitope of the gD protein of pseudorabies virus in pigs was immunized, a higher neutralizing antibody was produced, which can neutralize different types of PRV strains (including the original vaccine strain, the classic strain, and the variant strain).

[0150] Table 8 Summary of neutralization test results of different strains

[0151]

[0152] Note: Neutralization titer <1:2 is negative, and ≥1:2 is positive.

[0153] In summary, the kit prepared by the present invention is consistent with the results of the neutralization test, and can replace the neutralization test which has complex test requirements, harsh environment, and takes a long time (requires 5 days), so as to evaluate the effects of different vaccines after immunization, and help to formulate scientific immunization programs in a timely manner according to the test results; it can also be used for monitoring maternal antibodies. The vaccine composition prepared by the present invention produces high neutralizing antibodies against different types of PRV strains (including original vaccine strains, classical strains, and variant strains).

[0154] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention. SEQUENCE LISTING <110> Luoyang Putai Biotechnology Co., Ltd. <120> ELISA antibody detection kit coated with neutralizing epitope protein fragment of pseudorabies virus, and vaccine composition containing the same <160> 5 <170> PatentIn version 3.3 <210> 1 <211> 363 <212> DNA <213> Hybridoma cell <400> 1 gaggttcagc tgcagcagtc tggggcagaa cttgtgaagc caggggcctc agtcaagttg 60 tcctgcacag cttctggctt caacattaaa gacacttata ttcactgggt gaatcagagg 120 cctgaacagg gcctggagtg gattggaaag attgatcctg cgaatggtaa tactgaatat 180 gacccgaagt tccaggacaa ggccactatc acagcagaca catcctccaa cacagcctac 240 ctgcagctca ccagcctgac atctgaggac actgccgtct attactgtgc tagtcctttc 300 tacggtagta ggggcccctt ctttgcttac tggggccaag ggactctggt cactgtctct 360 gca 363 <210> 2 <211> 321 <212> DNA <213> Hybridoma cell <400> 2 actattgtga tgacccagac tcccaaattc ctgcttgtat cagcaggaga caggtttacc 60 ataacctgca aggccagtca gagtgtaagt aatgatgtag cttggtacca acagaagcca 120 gggcagtctc ctaaactgct gatatactat gcatccaatc gctacactgg agtccctgat 180 cgcttcactg gcagtggata tgggacggat ttcactttca ccatcagcac tgtgcaggct 240 gaagacctgg cagtttattt ctgtcagcag gattatagct ctcctccgac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 3 <211> 100 <212> PRT <213> Porcine pseudorabies virus <400> 3 Ile Ala Asp Gly Cys Ala His Leu Leu Tyr Phe Ile Glu Tyr Ala Asp 1 5 10 15 Cys Asp Pro Arg Gln Ile Phe Gly Arg Cys Arg Arg Arg Thr Thr Pro 20 25 30 Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro Thr Glu Asp Glu 35 40 45 Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe Asn Glu Gly Gln Tyr 50 55 60 Arg Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu Thr Asp Phe Met 65 70 75 80 Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala Arg Val Asp Gln 85 90 95 His Arg Thr Tyr 100 <210> 4 <211> 41 <212> PRT <213> Pseudorabies virus <400> 4 Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro Thr 1 5 10 15 Glu Asp Glu Leu Gly Leu Leu Met Val Ala Pro Gly Arg Phe Asn Glu 20 25 30 Gly Gln Tyr Arg Arg Leu Val Ser Val 35 40 <210> 5 <211> 20 <212> PRT <213> Pseudorabies virus <400> 5 Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro Thr 1 5 10 15 Glu Asp Glu Leu 20

Claims

1. An ELISA antibody detection kit coated with a protein fragment of a neutralizing epitope of pseudorabies virus, wherein: The ELISA antibody detection kit comprises a support medium coated with a protein fragment of a neutralizing epitope of the gD protein of pseudorabies virus of porcine, an enzyme-labeled secondary antibody reagent, a detection reagent that reacts to the enzyme-labeled secondary antibody, a negative control and a positive control; the protein fragment of the neutralizing epitope of the gD protein of pseudorabies virus of porcine is shown in SEQ ID NO.

5.

2. The ELISA antibody detection kit according to claim 1, wherein The negative control is serum in which both PRV antigen and antibody are negative, the positive control is serum from pigs immunized with pseudorabies vaccine of a variant strain of pseudorabies virus, the enzyme-labeled secondary antibody is enzyme-labeled sheep anti-pig IgG or enzyme-labeled rabbit anti-pig IgG, and the enzyme-labeled enzyme is horseradish peroxidase, alkaline phosphatase or β-D-galactosidase.

3. The ELISA antibody detection kit according to claim 1, wherein The coating concentration of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 0.1-2.0 μg / ml; the supporting medium is a microtiter plate; the enzyme-labeled secondary antibody is a solution diluted with an enzyme-labeled diluent to a final volume of 0.05% V / V enzyme-labeled sheep anti-swine IgG or enzyme-labeled rabbit anti-swine IgG; the enzyme-labeled diluent is a phosphate buffer solution of 20% V / V newborn calf serum, 0.05% V / V ProClin300, and 0.05% Tween-20.

4. The ELISA antibody detection kit according to claim 3, wherein The coating concentration of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 0.2-1.6 μg / ml.

5. The ELISA antibody detection kit according to claim 4, wherein The coating concentration of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 0.4-1.0 μg / ml.

6. The ELISA antibody detection kit according to claim 5, wherein The coating concentration of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 0.4 μg / ml.

7. The ELISA antibody detection kit according to claim 1, wherein The detection reagent for the enzyme-labeled secondary antibody reaction includes a color developing solution and a stop solution, wherein the color developing solution includes a color developing solution A and a color developing solution B, wherein the color developing solution A is a solution containing 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid, and 0.03% w / v urea peroxide, and the color developing solution B is a solution containing 0.02% w / v tetramethylbenzidine and 10% v / v anhydrous ethanol; the stop solution is a 2M H2SO4 solution; The ELISA antibody detection kit also includes a washing solution and a sample diluent. The washing solution is a phosphate buffer solution, and the sample diluent is a PBS solution containing 10% V / V fetal bovine serum, 0.1% V / V Tween 20, 1% W / V BSA, 0.05%-0.5% W / V Casein, and 1% W / V Proclin 300.

8. A method for preparing the ELISA antibody detection kit according to claim 1, wherein: The method comprises: Step (1) expressing the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein, coating the protein fragment on the support medium at a coating concentration of 0.1-2.0 μg / ml and 100 μl / well, wherein the support medium is a microtiter plate, coating at 2-8° C. for 16-24 hours or at 37° C. for 2 hours, then washing, sealing and drying; Step (2) blocking the microtiter plate coated in step (1) with a blocking solution, wherein the blocking solution is a phosphate buffer containing 5% W / V sucrose, 20% V / V newborn calf serum, and 0.05% V / V ProClin300, and the blocking conditions are blocking at 2-8° C. for 16-24 hours or blocking at 37° C. for 2 hours; Step (3) diluting the enzyme-labeled goat anti-swine IgG or enzyme-labeled rabbit anti-swine IgG to a final volume of 0.05% V / V with an enzyme-labeled diluent to prepare an enzyme-labeled secondary antibody reagent, wherein the enzyme-labeled diluent is a phosphate buffer solution containing 20% ​​V / V newborn calf serum, 0.05% V / V ProClin300, and 0.05% Tween-20; Step (4) respectively prepare a washing solution, a color developing solution, a stop solution, a positive control, a negative control, and a sample diluent; Step (5) comprises the microtiter plate prepared in step (2), the enzyme-labeled secondary antibody reagent prepared in step (3), and the washing solution, color developing solution, stop solution, positive control solution, and negative control solution prepared in step (4) to form the ELISA antibody detection kit.

9. The method according to claim 8, wherein: The coating concentration of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein in step (1) is 0.2-1.6 μg / ml; the drying condition is drying for 3-6 hours at 18° C.-26° C. and a relative humidity not higher than 30%; The color developing solution in step (4) includes color developing solution A and color developing solution B, wherein the color developing solution A contains 14.7 g of disodium hydrogen phosphate, 9.3 g of citric acid, and 0.3 g of urea peroxide per 1 L of water, and the color developing solution B contains 0.2 g of tetramethylbenzenediamine and 100 ml of anhydrous ethanol per 1 L of water; The positive control is the serum of pigs immunized with the variant strain pseudorabies vaccine, and the negative control is the serum with negative PRV antigen and antibody; The washing solution is a phosphate buffer solution, and 1L of the 20-fold concentrated solution of the washing solution contains 160g of sodium chloride, 58g of disodium hydrogen phosphate, 4.8g of potassium dihydrogen phosphate, 4g of potassium chloride, and 10mL of Tween 20; The sample diluent is a PBS solution containing 10% V / V fetal bovine serum, 0.1% V / V Tween 20, 1% W / V V BSA, 0.05% to 0.5% W / V Casein, and 1% W / V Proclin 300; and The stop solution is 2M H2SO4 solution.

10. The method according to claim 9, wherein: In the step (1), the coating concentration of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 0.4-1.0 μg / ml.

11. The method according to claim 10, wherein: The coating concentration of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein in the step (1) is 0.4 μg / ml.

12. Use of the ELISA antibody detection kit according to claims 1 to 7 in non-immune diagnosis, wherein the application of non-immune diagnosis includes vaccine immunization program evaluation.

13. A vaccine composition, wherein: The vaccine composition comprises an immunizing amount of a protein fragment of a neutralizing epitope of the gD protein of pseudorabies virus of porcine and a pharmaceutically acceptable carrier. The protein fragment of a neutralizing epitope of the gD protein of pseudorabies virus of porcine is shown as SEQ ID NO.

5.

14. The vaccine composition according to claim 13, wherein The protein fragment content of the neutralizing epitope of the porcine pseudorabies virus gD protein is ≥15 μg / ml; The pharmaceutically acceptable carrier is an adjuvant, the adjuvant is 206 adjuvant, and the adjuvant content is 46 v / v%.

15. The vaccine composition according to claim 14, wherein The protein fragment content of the neutralizing epitope of the porcine pseudorabies virus gD protein is 15-100 μg / ml.

16. The vaccine composition according to claim 15, wherein The protein fragment content of the neutralizing epitope of the porcine pseudorabies virus gD protein is 20-100 μg / ml.

17. Use of the vaccine composition according to claims 13 to 16 in the preparation of a medicament for preventing and / or treating porcine pseudorabies virus infection.

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