CN113866420A - ELISA antibody detection kit for neutralizing epitope protein fragment coated with porcine pseudorabies virus and vaccine composition containing ELISA antibody detection kit - Google Patents

ELISA antibody detection kit for neutralizing epitope protein fragment coated with porcine pseudorabies virus and vaccine composition containing ELISA antibody detection kit Download PDF

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CN113866420A
CN113866420A CN202010623161.1A CN202010623161A CN113866420A CN 113866420 A CN113866420 A CN 113866420A CN 202010623161 A CN202010623161 A CN 202010623161A CN 113866420 A CN113866420 A CN 113866420A
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protein
solution
porcine pseudorabies
enzyme
pseudorabies virus
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田克恭
谭菲菲
王莹
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Luoyang Pu Tai Biotechnology Co ltd
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Abstract

The invention provides an ELISA antibody detection kit for a porcine pseudorabies virus neutralizing antibody, which comprises a support medium coated with a protein fragment of a porcine pseudorabies virus gD protein neutralizing epitope, an enzyme-labeled secondary antibody reagent, a detection reagent for reacting the enzyme-labeled secondary antibody, a negative control and a positive control; the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is shown as SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5. The ELISA antibody detection kit can be used for evaluating the titer of the generated neutralizing antibody for vaccines prepared from different porcine pseudorabies virus strains, so as to evaluate the effect of the vaccines of different species after immunization, and is also helpful for timely formulating scientific immunization programs according to detection results. The invention also provides a vaccine composition containing the protein fragment of the neutralizing epitope of the gD protein of the porcine pseudorabies virus with immune quantity, which has a neutralizing effect on strains of PRV different types (including original vaccine strains, classical strains and variant strains).

Description

ELISA antibody detection kit for neutralizing epitope protein fragment coated with porcine pseudorabies virus and vaccine composition containing ELISA antibody detection kit
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an ELISA antibody detection kit coated with a protein fragment of a porcine pseudorabies virus neutralizing epitope, a vaccine composition containing the ELISA antibody detection kit and application of the ELISA antibody detection kit.
Background
Pseudorabies, also known as Aujeszky's disease, is an acute infectious disease of various domestic and wild animals such as pigs, cattle, sheep, dogs, cats, rabbits, mice, boars, minks, bears, and foxes caused by porcine herpesvirus 1strain (PRV) in the Alphaeoviridae, which is an acute infectious disease mainly manifested by fever, extreme itching (except pigs), and encephalomyelitis. The pseudorabies of the pigs widely exists in China and is seriously harmful, is one of main diseases restricting the production of large-scale pig farms, can cause abortion, dead fetus or mummy fetus of pregnant sows, and the piglets to have nervous symptoms, paralysis, 100 percent of death rate after infection within 20 weeks, respiratory symptoms of large and medium pigs and the like.
At present, more than 40 vaccine manufacturers are sold in the market, and in order to more accurately compare the immune effect, Guoshicheng and the like (a neutralization titer comparison test is carried out after the immunization of the pseudorabies virus Bartha strain and the variant vaccine, Guangdong veterinary science and technology of livestock husbandry, 2017,42(4):46-47) adopt a neutralization test to carry out quantitative detection on the titer of neutralizing antibodies of the pigs immunized by the vaccine, and the result shows that the neutralization titer after the immunization is still obviously higher than that of the original Bartha strain vaccine even if the immunization frequency of the PRV variant vaccine is less than that of the original Bartha vaccine. The Pracore company also has been studied with the prospect of 7 years, and the first approved inactivated vaccine (trade name: Pufajing) aiming at the porcine pseudorabies virus exotic strain is firstly developed in China, and the titer of the generated neutralizing antibody after the vaccine is immunized is 16-18 times that of the imported vaccine, 5-6 times that of the domestic live vaccine and 5-6 times that of the domestic inactivated vaccine, which are far higher than other existing products (https:// dy.163.com/v 2/article/detail/EFHKKL1051EHe9. html). Currently, the evaluation mode after immunization of vaccines prepared from variant strains is mainly a neutralization test, but the neutralization test takes about 5 days and must be completed in a laboratory with cell manipulation capability, so that the evaluation mode is not favorable for clinical application. In addition, the immune mechanism is not clear, and evaluation methods which can be clinically applied are also needed for later vaccine improvement.
Therefore, it is highly desirable to develop a rapid and simple detection method for detecting neutralizing antibodies generated by vaccines prepared from different porcine pseudorabies viruses, and to assign an immunization program according to the detection result.
In addition, a vaccine capable of neutralizing different types of porcine pseudorabies viruses is also needed in clinic to solve the practical clinical problem.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides an ELISA antibody detection kit of a protein fragment coated with a porcine pseudorabies virus neutralizing epitope, wherein the ELISA antibody detection kit comprises a support medium of the protein fragment coated with the porcine pseudorabies virus gD protein neutralizing epitope, an enzyme-labeled secondary antibody reagent, a detection reagent for reacting with the enzyme-labeled secondary antibody, a negative control and a positive control; the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is shown as SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.
The kit has the advantages that the detection result is consistent with the neutralization test result, the kit can replace the neutralization test which has complex test requirements, harsh environment and long consumed time (requiring 5 days), and not only can be used for evaluating the titer of the generated neutralizing antibody aiming at the vaccines prepared by the prior vaccine strain, the classical strain of the porcine pseudorabies virus and the vaccines prepared by the variant strain of the porcine pseudorabies virus, so as to evaluate the effects of different vaccines after immunization, but also be beneficial to timely formulating scientific immunization programs according to the detection result.
The protein containing the porcine pseudorabies virus neutralizing epitope is a polypeptide of porcine pseudorabies virus gD protein neutralizing epitope site 130-149 AA (shown as SEQ ID NO. 5), a polypeptide of porcine pseudorabies virus gD protein neutralizing epitope site 130-170 AA (shown as SEQ ID NO. 4), a polypeptide of porcine pseudorabies virus gD protein neutralizing epitope site 101-200 AA (shown as SEQ ID NO. 3) or porcine pseudorabies virus gD protein (porcine pseudorabies virus HN1201 strain gD protein).
The kit can coat porcine pseudorabies virus gD protein, protein fragments of neutralization epitopes of 101-200 AA, protein fragments of neutralization epitopes of 130-170 AA and protein fragments of neutralization epitopes of 130-149 AA, and the coated protein fragments or proteins can combine with neutralizing antibodies generated by different porcine pseudorabies virus strains and detect the porcine pseudorabies virus gD protein.
In one embodiment of the present invention, in the ELISA antibody detection kit, the negative control is serum with all PRV antigen antibodies negative, the positive control is serum of a porcine pseudorabies virus variant porcine pseudorabies vaccine immune pig, the enzyme-labeled secondary antibody is enzyme-labeled goat anti-pig IgG or enzyme-labeled rabbit anti-pig IgG, and the enzyme-labeled enzyme is horseradish peroxidase, alkaline phosphatase or β -D-galactoglycase.
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 mug/ml; the support medium is a microtiter plate; the enzyme-labeled reagent is a solution which is diluted by an enzyme-labeled diluent to a final volume of 0.05 percent V/V enzyme-labeled goat anti-pig IgG or enzyme-labeled rabbit anti-pig IgG; the enzyme-labeled diluent is phosphate buffer solution of 20% V/V newborn calf serum, 0.05% V/V ProClin300 and 0.05% Tween-20.
The coating concentration is selected from the group consisting of 0.2. mu.g/ml, 0.3. mu.g/ml, 0.4. mu.g/ml, 0.5. mu.g/ml, 0.6. mu.g/ml, 0.7. mu.g/ml, 0.8. mu.g/ml, 0.9. mu.g/ml, 1.0. mu.g/ml, 1.1. mu.g/ml, 1.2. mu.g/ml, 1.3. mu.g/ml, 1.4. mu.g/ml, 1.5. mu.g/ml, 1.6. mu.g/ml, 1.8. mu.g/ml, 1.9. mu.g/ml, 2.0. mu.g/ml.
In a preferred embodiment of the invention, the coating concentration of the porcine pseudorabies virus protein is 0.2-1.6 mu g/ml.
In a more preferred embodiment of the present invention, the coating concentration of the porcine pseudorabies virus protein is 0.4-1.0 μ g/ml.
As a most preferred embodiment of the present invention, the porcine pseudorabies virus protein is coated at a concentration of 0.4. mu.g/ml.
In the ELISA antibody detection kit, the detection reagent for the enzyme-labeled secondary antibody comprises a developing solution and a stop solution, the developing solution comprises a developing solution A and a developing solution B, the 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 developing solution B is a solution containing 0.02% w/v tetramethyl biphenyl diamine and 10% v/v absolute ethyl alcohol; the stop solution is 2M H2SO4A solution; the ELISA antibody detection kit further comprises a washing solution and a sample diluent, wherein the washing solution is a phosphate buffer solution, and the sample diluent is a PBS solution containing 10% V/V fetal calf serum, 0.1% V/ V Tween 20, 1% W/V BSA, 0.05-0.5% W/V Casein and 1% W/VProclin 300.
The invention also provides a method for preparing the ELISA antibody detection kit, wherein the method comprises the following steps: expressing a protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein, coating the protein fragment on a support medium according to the coating concentration of 0.1-2.0 mu g/ml and 100 mu l/hole, wherein the support medium is a microtiter plate, coating the support medium at 2-8 ℃ for 16-24 hours or coating the support medium at 37 ℃ for 2 hours, and then washing, sealing and drying the coated support medium; sealing the microtiter plate coated in the step (1) by using a sealing solution, wherein the sealing solution is a phosphate buffer solution containing 5% W/V sucrose, 20% V/V newborn calf serum and 0.05% V/V ProClin300, and the sealing condition is sealing for 16-24 hours at the temperature of 2-8 ℃ or sealing for 2 hours at the temperature of 37 ℃; diluting enzyme-labeled goat anti-pig IgG or enzyme-labeled rabbit anti-pig IgG to a final volume of 0.05% V/V by using an enzyme-labeled diluent to prepare an enzyme-labeled secondary antibody reagent, wherein the enzyme-labeled diluent is phosphate buffer solution of 20% V/V newborn bovine serum, 0.05% V/VPROClin300 and 0.05% Tween-20; step (4) preparing a washing solution, a developing solution, a stop solution, a positive control, a negative control and a sample diluent respectively; and (5) assembling the microtiter plate prepared in the step (2), the enzyme-labeled secondary antibody reagent prepared in the step (3), and the washing solution, the developing solution, the stop solution, the positive control and the negative control prepared in the step (4) into the ELISA antibody detection kit.
The preparation method of the invention ensures the sensitivity of the prepared kit by optimizing various conditions in the preparation process of each step.
As an embodiment of the invention, the coating concentration of the porcine pseudorabies virus protein in the step (1) is 0.2-1.6 mu g/ml, and 100 mu l/hole coating is adopted.
As a preferable embodiment of the invention, the coating concentration of the porcine pseudorabies virus protein in the step (1) is 0.4-1.0 mu g/ml, and 100 mu l/hole coating.
As a more preferred embodiment of the present invention, the concentration of the porcine pseudorabies virus protein coated in step (1) is 0.4. mu.g/ml, and 100. mu.l/well of coating is used.
In one embodiment of the present invention, the drying in step (1) is performed at 18 to 26 ℃ and a relative humidity of not higher than 30% for 3 to 6 hours.
As one embodiment of the present invention, the color developing solution in the step (4) includes a color developing solution a and a color developing solution B, the color developing solution a contains 14.7g of disodium hydrogen phosphate, 9.3g of citric acid and 0.3g of carbamide peroxide per 1L of water, and the color developing solution B contains 0.2g of tetramethyldiphenyldiamine and 100ml of absolute ethyl alcohol per 1L of water; the positive control is serum of a pig immunized by the variant strain porcine pseudorabies vaccine, and the negative control is serum with all PRV antigen antibodies negative; the washing solution is phosphate buffer solution, and 1L of 20-time concentrated solution of the washing solution contains 160g of sodium chloride, 58g of disodium hydrogen phosphate, 4.8g of monopotassium phosphate, 4g of potassium chloride and 2010 mL of tween; the sample diluent is PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/VBSA, 0.05-0.5% W/V Casein and 1% W/V Proclin 300; and the stop solution is 2M H2SO4And (3) solution.
The invention also provides application of the ELISA antibody detection kit in non-immune diagnosis, wherein the application of the non-immune diagnosis comprises evaluation of vaccine immunization program and epidemiological investigation of isolated serum.
The invention also provides a vaccine composition, wherein the vaccine composition comprises an immunizing dose of protein fragments of the neutralizing epitope of the porcine pseudorabies virus gD protein and a pharmaceutically acceptable carrier, and the protein fragments of the neutralizing epitope of the porcine pseudorabies virus gD protein are shown as SEQ ID No.3, SEQ ID No.4 or SEQ ID No. 5.
After the vaccine composition containing the neutralizing epitope of the gD protein of the porcine pseudorabies virus is used for immunizing a pig, a higher neutralizing antibody is generated, and the neutralizing antibody can generate a neutralizing effect on strains of PRV of different types (including original vaccine strains, classical strains and variant strains).
As an embodiment of the invention, in the vaccine composition, the content of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is more than or equal to 15 mu g/ml; the pharmaceutically acceptable carrier is an adjuvant, the adjuvant is 206 adjuvant, and the adjuvant content is 46 v/v%.
As a preferable embodiment of the invention, in the vaccine composition, the content of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 15-100 mug/ml.
As a more preferable embodiment of the invention, in the vaccine composition, the content of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 20-100 mug/ml.
The invention also provides application of the vaccine composition in preparation of a medicament for preventing and/or treating porcine pseudorabies virus infection.
The vaccine composition can generate neutralizing antibodies for different types of porcine pseudorabies viruses, namely can prevent and/or treat different types of porcine pseudorabies virus infection.
The invention has the advantages that:
the invention discovers the differential neutralizing epitope on the porcine pseudorabies virus gD protein by a large number of experiments.
The kit prepared by using the protein containing the neutralizing epitope of the gD protein of the porcine pseudorabies virus as the coating antigen has the same neutralizing test result with the variant strain, can replace the neutralizing test with complex test requirements, harsh environment requirements and long time consumption (5 days), is used for evaluating the effect of different vaccines after immunization, and is beneficial to timely formulating scientific immunization programs according to the detection result; can also be used for monitoring maternal antibodies.
The vaccine composition prepared by the invention generates high neutralizing antibodies for different types of PRV strains (including original vaccine strains, classical strains and variant strains).
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FIG. 1 is a comparison of the results of the test of 14 samples with the neutralization test using kit A.
Detailed Description
The term "epitope", also known as antigenic determinant, is composed of a portion of the amino acid residues involved in the formation of bonds between antigen and antibody molecules, is the core component of an antigen, and is a specific region of an antigen that can be recognized by an antibody molecule. These epitopes are the structural basis for antigenic generation, on which epitope vaccines have been proposed. The epitope vaccine has the advantages of no need of separating infectious pathogens, no toxicity dispersion, reduction of mutual interference of different immunodominant epitopes and the like.
The term "neutralizing epitope" also known as "neutralizing epitope", "neutralizing antigenic site", "neutralizing epitope" or "neutralizing epitope" refers to a protein fragment or peptide fragment of an antigenic molecule that induces the production of neutralizing antibodies.
The term "Microtiter plate" (protein Microtiter Plates) refers to wells having a transparent bottom with at least one physical deformation between at least two adjacent wells, wherein physical deformation refers to a shape that may have, for example, channels, ridges, holes, slits or steps. Microtiter plates of the invention include, but are not limited to, 6-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates, preferably 96-well plates.
The terms "color-developing liquid a" and "developer a liquid" are used interchangeably, and "color-developing liquid B" and "developer B liquid" are used interchangeably.
The term "phosphate buffer" refers to a solution containing phosphoric acid or a salt thereof and adjusted to a desired pH, and is one of the most widely used buffers in biochemical studies. Typically, phosphate buffers are prepared from phosphoric acid or phosphates (including but not limited to sodium and potassium salts). Some phosphates are known in the art, such as sodium and potassium dihydrogen phosphate, disodium and dipotassium hydrogen phosphate, sodium and potassium phosphate. Phosphate salts are known to exist as hydrates of salts. The buffered pH ranges widely, for example, from about 4 to about 10, preferably from about 5 to about 9, more preferably from about 6 to about 8, and most preferably about 7.4, due to secondary dissociation of the buffer. Further preferably, the phosphate buffer is a phosphate buffer containing sodium chloride and potassium chloride.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The phosphate buffer used in the examples of the present invention was PBS with pH 7.4, and its formulation volume of 1L was: NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 2.9g、KH2PO40.2g, and the volume is made 1L with ultrapure water, but this embodiment is not intended to limit the present invention in any case. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 preparation, purification and identification of early-stage porcine pseudorabies virus monoclonal antibody
Neutralizing tests are respectively carried out on the 5 strains (3G1, 3B6, 4A5, 4D9 and 5G7, which are prepared in the early stage and are detailed in Chinese patent CN109206509A) of the porcine pseudorabies virus gD protein monoclonal antibody, the 1strain (1H1, which is detailed in Chinese patent CN106188280A) of the porcine pseudorabies virus gB protein monoclonal antibody, the 4 strains (1B9, 1F2, 3D4 and 4C7, which is detailed in Chinese patent CN109212205A) of the porcine pseudorabies virus variant HN1201 strain (the preservation number of the PRV HN strain is CCTCC NO. V201311, which is referred to CN104004774A), the classical Fa strain (which is purchased from Chinese medicine inspection institute) and the vaccine strain Bartha strain, and results are as follows: only the monoclonal antibodies 5G7 and 1H1 have non-complement-dependent neutralizing activity on three types of PRV strains, and the neutralizing titer has no obvious difference; the other three types of PRV strains do not have complement independent neutralizing activity. At present, the monoclonal antibodies of PRV can not distinguish the obvious difference of neutralizing antibodies generated after the pigs are immunized by the vaccine strains prepared from early vaccine strains and later variant strains.
In addition, a great deal of experimental research conducted by the research and development team of the present invention finds that: the neutralizing antibodies of the porcine pseudorabies variant strains gB and gC are not very high, the neutralizing titer of gD is more than 1:20, the gD protein is the most important immunoprotection related protein, the immunity condition after the vaccination can be reflected, and the immunization program can be more accurately established (https:// www.zgyz001.com/news/enderprise _ news/123390. html). Based on the situation, the inventor emphatically prepares the monoclonal antibody of the gD protein of the porcine pseudorabies virus, and particularly prepares the monoclonal antibody capable of identifying the PRVgD protein neutralizing epitope site.
Example 2 preparation, purification and identification of porcine pseudorabies virus gD protein monoclonal antibody
2.1 preparation and identification of porcine pseudorabies virus gD protein
PRV HN1201 virus or cultures of different generations of PRV HN1201 virus are inoculated on PK15 cells which grow well, virus liquid is collected, a small amount of virus liquid is used for extracting PRV genome DNA, PRVgD protein is prepared according to patent CN105251000A, and the content of the PRVgD protein (abbreviated as PRVgD1) is determined to be 200 mu g/ml by an SDS-PAGE densitometry.
2.2 preparation and purification of PRVgD protein monoclonal antibody
Firstly, after PRV HN1201 strain virus liquid is inactivated by formaldehyde and emulsified with Freund's adjuvant in the same volume, 10 mice are immunized 1 time at 2 weeks, and the mouse serum after 3-6 times of immunization is subjected to IFA continuous detection by using an indirect Immunofluorescence (IFA) antigen plate prepared from PRVHN1201 strain prepared in example 1.1, and the result is: the serum IFA titer of each group of mice is less than or equal to 1:640, and optionally 1 cell fusion is carried out according to the operation 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 is 0, and the process is abandoned.
Secondly, after the PRV HN1201 strain virus liquid is inactivated by formaldehyde and emulsified with Freund's adjuvant in the same volume, 20 mice are immunized for the first time, after 2 weeks, PRVgD protein is emulsified with Freund's adjuvant in the same volume for the 2 nd immunization, and the immunization is repeated for 3 rounds. After the 2 nd round of immunization, 20 mice were bled 9 days after each immunization and the sera were subjected to continuous ELISA testing using the ELISA method coated with PRVgD protein prepared in example 1. After the mouse serum is immunized for 4-6 times, only 2 mice with the ELISA titer increasing trend and the ELISA titer being more than or equal to 1: 12800 are subjected to cell fusion, and are subjected to subcloning screening by using an ELISA limiting dilution method to obtain 6 strains of positive hybridoma cells, wherein the positive rate is only 2%. PK-15 cells infected with PRV HN1201 strain were coated on a microplate, and supernatants of 6 positive hybridoma cells were evaluated by indirect immunofluorescence IFA, and it was found that 1 positive hybridoma cell was discarded due to nonspecific adsorption, to thereby obtain 5 positive hybridoma cells (3G1, 3B6, 4A5, 4D9, and 5G 7). The neutralization titer is measured after ascites is prepared from 5 hybridoma cells, and the result is as follows: only monoclonal antibody 5G7 had complement-independent neutralizing activity against all three types of PRV strains, with no significant difference in neutralizing titers, and none of the others had complement-independent neutralizing activity.
Thirdly, 30 mice are immunized for the first time after the prepared PRVgD protein and Freund's adjuvant are emulsified in equal volume, and after 2 weeks, the PRVgD protein and Freund's adjuvant are emulsified in equal volume for the 2 nd immunization, and the immunization is repeated for 3 rounds. After the 2 nd round of immunization, 30 mice were bled 9 days after each immunization and the sera were subjected to continuous IFA assay using an indirect immunofluorescence antigen plate (i.e., IFA antigen plate) prepared from the PRV variant HN1201 strain. As a result: after the mouse serum is immunized for 4-6 times, the IFA titer is increased, and the IFA titer is more than or equal to 1: 6400, only 1 mouse is used, and the mouse serum is subjected to ELISA detection by using an ELISA plate coated by PRVgD protein, and the result is that: the ELISA titers of only 1 mouse serum were > 1: 128000. The mice are subjected to cell fusion and are subjected to subclone screening by an IFA (fluorescence induced fluorescence) method to obtain 15 strains of positive hybridoma cells, and the positive rate is only 2%.
Supernatants from 15 positive hybridoma cells were collected and used for indirect immunofluorescence antigen plates (i.e., IFA antigen plates) prepared from classical strain Fa, vaccine strain Bartha (purchased from) and results: the monoclonal antibody secreted into the supernatant by only 10 strains (the strain numbers are 1G2, 2A7, 2F5, 2G10, 3C5, 3E6, 3H3, 4B2, 4H5 and 5F6 in sequence) of hybridoma cells shows good reaction with PRV variant strains, classical strains and vaccine strains (the IFA titer is more than or equal to 1: 8), and the monoclonal antibody secreted into the supernatant by the other 5 strains of hybridoma cells has weak reaction with PRV classical strains or vaccine strains (the IFA titer is less than 1: 2), and the titer is discarded.
Respectively preparing mouse ascites from 10 positive hybridoma cells, purifying by using an octanoic acid-ammonium sulfate combined precipitation method, and identifying by using SDS-PAGE gel electrophoresis, wherein the result is as follows: the purity of 10 monoclonal antibodies is not lower than 85%; quantitative analysis was performed with BCA protein quantification kit according to the instructions, respectively, and the results were: the protein content of 10 monoclonal antibodies is not less than 2.0 mg/ml.
2.3 determination of neutralizing potency of monoclonal antibody against porcine pseudorabies virus
The neutralizing titer of the 10 monoclonal antibodies prepared in example 2.2 was determined by the fixed virus dilution serum method described in the Chinese veterinary pharmacopoeia 2015 edition by mixing the monoclonal antibodies at different dilution ratios (1:1V/V-1:2048V/V) with PK15 cells at 37 ℃ and 5% CO2Incubating for 1 hr under the condition, adding PRV vaccine strain Bartha strain, classical strain Fa strain and variant strain HN1201 strain virus diluent (both containing 100 TCID)50/ml), standing at 37 ℃ and 5% CO2The cells were cultured for 72-120 hours under the conditions for observation of cytopathic effect and the neutralization titer was calculated, and the results are shown in Table 1.
TABLE 110 neutralizing titer of monoclonal antibody
Figure BDA0002563768320000111
The results show that: only the monoclonal antibody 5F6 has a neutralization reaction with different types of PRV strains (vaccine strain, classical strain and variant strain), but the neutralization activity is different from the reaction result of the monoclonal antibody 5G7, the neutralization potency of the neutralizing agent for the vaccine strain is obviously low, and the neutralization potency of the neutralizing agent for clinical strains (classical strain and HN1201 strain) is obviously high (at least 448 times higher than that of the vaccine strain). Further, the monoclonal antibody 5F6 can be used for the research of PRVgD protein structure analysis, active site neutralization and generation of high-titer neutralizing antibodies after vaccine preparation of variant strains.
2.4 evaluation of porcine pseudorabies Virus monoclonal antibody 5F6
2.4.1 identification of monoclonal antibody subtypes
And (3) identifying the subtype of the monoclonal antibody 5F6 by using a monoclonal antibody subtype identification kit. As a result: the heavy chain subtype of monoclonal antibody 5F6 was IgG1 and the light chain types were all kappa.
2.4.2 specific identification
The detection method comprises the following steps of respectively fixing CSFV, PRRSV, PCV2, PCV2, PPV, PRV, PEDV, TGEV, PRoV and culture PRV in a microporous plate by PK15 cells, using monoclonal antibody 5F6 ascites as a primary antibody, using a fluorescence-labeled rabbit anti-mouse secondary antibody as a secondary antibody, and detecting according to an indirect immunofluorescence method IFA, wherein the detection results comprise the following steps: monoclonal antibody 5F6 reacted only with PRV and did not cross-react with other common porcine disease and culture cells.
PRVgB Δ 148 to 546 proteins, PRVgB protein (prepared according to patent CN 10563827A), 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 a microplate, respectively, and monoclonal antibody 5F6 ascites was used as a primary antibody, and an enzyme-labeled goat-mouse secondary antibody was used as a secondary antibody, and the detection was performed by ELISA, with the results: monoclonal antibody 5F6 reacted only with PRVgD protein and not with other PRV proteins. Shows that: monoclonal antibody 5F6 is a specific monoclonal antibody to the PRVgD protein.
2.4.3IFA evaluation
Use and examineThe monoclonal antibody 5F6 was titer-determined by immunofluorescence assay (IFA). An IFA detection method comprises the following steps: culturing adherent PK-15 cells, inoculating the virus seeds containing PRV HN1201 strain, Fa strain, Ma strain and Bartha strain at a dose of 0.005-0.1 MOI, respectively, setting healthy cell control holes at 37 deg.C and 5% CO2Culturing for 48-72 hours under the condition, and then removing the supernatant; fixing with 80% acetone solution at 2-8 deg.C for 30min, washing with PBS for 3 times, adding 100 μ l monoclonal antibody 5F6 diluted at 1: 100 ratio into virus inoculation hole and cell control hole, respectively, adding PRV mouse positive serum into virus inoculation hole as positive control, and reacting at 37 deg.C for 60 min; washing with PBS for 3 times and drying; adding FITC-labeled goat anti-mouse IgG diluted at a ratio of 1: 500, and allowing the mixture to act at 37 ℃ for 60 minutes; washing with PBS for 3 times and drying; after addition of 50. mu.l PBS, observation was carried out under a fluorescence microscope. And (3) determining IFA titer: the maximum dilution of the antibody corresponding to the well of the cytotoxic cell plate in which yellow-green fluorescence was observed was used as the IFA titer of the antibody.
As a result: the IFA titer of the monoclonal antibody 5F6 to PRV HN1201 strain, Fa strain, Ma strain and Bartha strain is 1: 12800, 1: 6400 and 1: 6400 in sequence.
2.4.4ELISA titers
Coating PRVgD protein on an ELISA plate according to 100 ng/hole and 100 mul/hole, diluting monoclonal antibody 5F6 by 100 times with phosphate buffer solution, then diluting by 2 times, taking the diluted monoclonal antibody as a primary antibody, incubating for 1 hour at 37 ℃, adding enzyme-labeled goat anti-mouse IgG after washing, incubating for 30 minutes at 37 ℃, adding substrate after washing, developing, reading OD on an enzyme-labeling instrument after stopping450nmValue, indirect ELISA reaction was performed and monoclonal antibody 5G7 was used as a positive control. As a result: the ELISA titer of the monoclonal antibody was 1: 512000.
2.4.5 variable region sequencing
Designing a heavy chain variable region primer sequence according to the sequence characteristics of the mouse-derived monoclonal antibody: p1: 5 '-ACTAGTCGACATGAAATGCTCGTGGRTYATSAACTT-3' P2: 5 '-ACTAGTCGACATGAAATGCAGCTGGRTYAT-3'
Design of light chain variable region primer sequence:
P3：5’-ACTAGTCGACATGGTYGTYATVTCCTTGCT-3’
P4：5’-ACTAGTCGACATGGGCWTCAAGATGRAGTCACAKW-3’
culturing and collecting hybridoma cell 5F6, extracting RNA, reverse transcribing to obtain template, amplifying the variable region sequence with the primer, and sequencing the amplified product to Suzhou Jinzhi biotechnology. As a result: the nucleotide sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 5F6 are respectively shown in SEQ.ID No.1 and SEQ.ID No. 2.
2.5 identification of epitope recognized by monoclonal antibody 5F6
2.5.1 preliminary identification of neutralizing epitope sites of PRVgD protein
Designing polypeptide (see table 2) according to the amino acid sequence coded by the nucleotide sequence of PRV HN1201 strain (NCBI accession number: KP722022.1) gD protein and Bartha strain (NCBI accession number: JF797217.1) gD protein, directly crossing each peptide segment synthesized by Kinry Biotech limited, diluting with carbonate coating buffer solution (pH9.6), coating on ELISA plate according to 500 ng/hole and 100 mul hole, blocking, drying, diluting monoclonal antibodies 5F6, 5G7, 1H1 and 3B6 with phosphate buffer solution 100 times, incubating as primary antibody at 37 deg.C for 1 hr, washing, adding enzyme-labeled goat anti-mouse IgG, incubating at 37 deg.C for 30min, washing, adding substrate, developing color, stopping, and reading OD on enzyme-labeling apparatus450nmValue to perform an indirect ELISA reaction. Meanwhile, the PRVgD protein prepared in example 2.1 was coated on ELISA plates at 100 ng/well and 100 μ l/well, and monoclonal antibodies 5F6, 5G7, 1H1, and 3B6 were diluted 16000-fold with phosphate buffer solution, respectively, and used as a control for indirect ELISA reaction. Results (see table 2): the primary result shows that the antigenic site of the PRVgD protein recognized by the monoclonal antibody 5F6 is located at 101-200 AA, and is shown as SEQ ID No. 3.
TABLE 2 summary of the results of the PRVgD protein peptide and monoclonal antibody reactions (OD values)
Figure BDA0002563768320000141
Note: AA represents an amino acid.
2.5.2 further identification of neutralizing epitope sites of PRVgD protein
An amino acid sequence coded by a PRV HN1201 strain (NCBI accession number: KP722022.1) gD protein and a Bartha strain (NCBI accession number: JF797217.1) gD protein is designed into polypeptide, 3 peptide segments (shown in table 3) at 101-200 AA positions are directly handed to Jinsrie biological technology limited company for synthesis, are diluted by carbonate coating buffer solution (pH9.6) and coated on an ELISA plate according to 500 ng/hole and 100 mul/hole, and monoclonal antibodies 5F6, 5G7, 1H1 and 3B6 are respectively diluted by 100 times by phosphate buffer solution to serve as primary antibodies for indirect ELISA reaction. Meanwhile, PRVgD protein peptide fragments 101-200 AA prepared in example 4.1 are used as a control. Results (see table 3): further judging that the antigenic site of the monoclonal antibody 5F6 for recognizing the PRVgD protein is positioned at 130-170 AA, and is shown as SEQ ID No. 4.
TABLE 3 summary of the results of the PRVgD protein peptide and monoclonal antibody reactions (OD values)
Figure BDA0002563768320000151
Note: AA represents an amino acid.
2.5.3 Re-identification of neutralizing epitope sites of PRVgD protein
An amino acid sequence coded by a PRV HN1201 strain (NCBI accession number: KP722022.1) gD protein and a Bartha strain (NCBI accession number: JF797217.1) gD protein is designed into polypeptide, 2 peptide segments (shown in table 4) at positions 130-170 AA are directly handed to Jinsrie biological technology limited company for synthesis, carbonate coating buffer solution (pH9.6) is used for diluting, then 500 ng/hole and 100 mul/hole are coated on an ELISA plate, and monoclonal antibodies 5F6, 5G7, 1H1 and 3B6 are respectively diluted by 100 times by phosphate buffer solution and used as primary antibodies for indirect ELISA reaction. Meanwhile, PRVgD protein peptide fragments 101-200 AA prepared in example 2.5.1 are used as positive control. Results (see table 4): the monoclonal antibody 5F6 can be judged to identify the antigenic site of the PRVgD protein, the antigenic site is located at 130-149 AA, and the amino acid sequence is shown as SEQ ID No. 5.
TABLE 4 summary of the results of the PRVgD protein peptide reaction with monoclonal antibodies (OD values)
Figure BDA0002563768320000152
Note: AA represents an amino acid.
Example 3 preparation, detection method and evaluation of ELISA kit
The antigen of the antigen coating plate in the kit is any one or a plurality of combinations of the polypeptide of the neutralizing epitope site 130-149 AA of the gD protein of the porcine pseudorabies virus in the embodiment 2.5.3, the polypeptide of the neutralizing epitope site 130-170 AA of the gD protein of the porcine pseudorabies virus in the embodiment 2.5.2, the polypeptide of the neutralizing epitope site 101-200 AA of the gD protein of the porcine pseudorabies virus in the embodiment 2.5.1, and the gD protein of the porcine pseudorabies virus, and the detection effect is equivalent. For the sake of simplicity and conciseness, the embodiment of the invention only takes the single polypeptide component of the neutralizing epitope site 130-149 AA of the gD protein of the porcine pseudorabies virus in the embodiment 2.5.3 as an example of the coating antigen.
3.1 preparation of the kit
Antigen coated plate: diluting the polypeptide of the neutralizing epitope site 130-149 AA of the gD protein of the porcine pseudorabies virus in example 2.5.3 to a preferred final concentration by using a carbonate buffer solution (pH value is 9.6, 0.05mol/L) for coating, coating for 16-24 hours at the temperature of 2-8 ℃ by 100 mul/hole, washing by using a washing solution, adding a sealing solution (a phosphate buffer solution containing 5% W/V sucrose, 20% V/V newborn calf serum and 0.05% V/V ProClin 300) according to 150 mul/hole, sealing for 16-24 hours at the temperature of 2-8 ℃ or sealing for 2 hours at the temperature of 37 ℃, discarding the sealing solution, drying for 3-6 hours at the temperature of 18-26 ℃ under the condition that the relative humidity is not higher than 30%, and storing for later use at the temperature of 2-8 ℃.
Enzyme labeling reagent: and uniformly mixing 20% of V/V newborn calf serum, 0.05% of V/V ProClin300 and 0.05% of Tween-20 in a phosphate buffer solution to prepare the enzyme-labeled diluent. Diluting commercial goat anti-pig lgG-HRP or rabbit anti-pig lgG-HRP with enzyme-labeled diluent according to 0.05% V/V of the final volume, mixing well, filtering with 0.22 μm, and packaging under aseptic condition.
Positive control: the variant porcine pseudorabies inactivated vaccine (HN 1201-delta gE strain, from Poleco bioengineering GmbH) is a healthy susceptible pig (2 ml/head) with negative neck muscle immune PRV antigen antibodies, blood is collected 28 days later, serum is separated, and the collected serum is positive serum. And (3) taking 200mL of newborn bovine serum, 10mL of positive serum and 0.5mL of ProClin-300, uniformly mixing, supplementing phosphate buffer solution to a constant volume of 1000mL, filtering, and performing sterile subpackaging.
Negative control: selecting healthy susceptible pigs with negative PRV antigen antibodies, collecting blood from carotid artery, separating serum, and collecting serum to obtain negative serum. And (3) taking 200mL of newborn bovine serum, 10mL of negative serum and 0.5mL of ProClin-300, uniformly mixing, supplementing phosphate buffer solution to a constant volume of 1000mL, filtering, and performing sterile subpackaging.
Sample diluent: PBS solution containing 10% V/V fetal calf serum, 0.1% V/ V Tween 20, 1% W/V BSA, 0.05% -0.5% W/V Casein and 1% W/V Proclin300, filtering at 0.22 μm, and aseptically packaging.
20 × concentrated washing solution: taking 160g of sodium chloride, 58g of disodium hydrogen phosphate, 4.8g of potassium dihydrogen phosphate, 4g of potassium chloride, 800mL of ultrapure water and 2010 mL of tween, completely dissolving, then using the ultrapure water to fix the volume to 1000mL, filtering by using a 0.22 mu m filter membrane, and carrying out sterile subpackaging. It is diluted 20 times with distilled water.
Color development liquid: dissolving 14.7g of disodium hydrogen phosphate, 9.3g of citric acid and 0.3g of carbamide peroxide in purified water, metering to 1000mL, mixing uniformly, filtering, and performing aseptic packaging to obtain color developing solution A. Taking 0.2g of Tetramethylbenzidine (TMB) and 100mL of absolute ethyl alcohol, dissolving in purified water to a constant volume of 1000mL, mixing uniformly, filtering, and packaging aseptically to obtain a color developing solution B.
Stopping liquid: 2M H2SO4And (3) solution.
3.2 establishment of detection method
All reagents should be balanced to room temperature (about 30 minutes) before use, liquid reagents are gently shaken and uniformly mixed before experiments, and the liquid reagents are immediately sealed and placed back to 2-8 ℃ for storage after use.
The operation steps are as follows:
(1) adding a sample diluent: 100 mul of sample diluent is added into corresponding wells of the microplate, and a negative control well (100 mul/well of negative control), a positive control well (100 mul/well of positive control) and a blank control well (100 mul of PBS buffer) are arranged at the same time.
(2) Adding a sample to be detected: the samples to be detected are respectively added into corresponding holes except for a negative control hole, a positive control hole and a blank control hole by 10 mul. The plates were incubated at 37 ℃ for 30 minutes.
(3) Washing: washing with washing solution for 5 times, and drying.
(4) Adding an enzyme standard reagent: mu.l of enzyme-labeled reagent was added to each well except for the blank control well, and the plate was sealed and incubated at 37 ℃ for 30 minutes.
(5) Washing: washing with washing solution for 5 times, and drying.
(6) Color development: adding 50 mul of color development liquid A and color development liquid B into each hole, mixing uniformly, and developing for 15 minutes at 37 ℃ or at room temperature in a dark place.
(7) Terminating and detecting: adding 50 mul of stop solution into each well, and measuring the A value of each well by using a microplate reader within 10 minutes: OD450nmValue (measured after zeroing blank control wells), or OD450nm－OD600nm～650nmThe values were determined according to the following result determination criteria.
When the positive control well is in the A value OD450nmThe test is established when the value is more than or equal to 0.2 and the value of the negative control hole A is less than or equal to 0.1, otherwise, the test is invalid. When the test is established, calculating an S/P value: sample a value/positive control a value mean. And (4) judging a result:
the pig pseudorabies virus antibody is positive when the S/P value is more than or equal to 0.5,
the pig pseudorabies virus antibody is negative when the S/P value is less than 0.5.
3.3 selection of coating concentration of coating sources
The polypeptides of neutralizing epitope sites 130-149 AA in gD protein of porcine pseudorabies virus of example 2.5.3 were diluted with carbonate buffer (pH9.6, 0.05mol/L) to final concentrations of 0.1, 0.2, 0.4, 0.8, 1.0, 1.6, 2.0. mu.g/ml to prepare antigen coated plates, positive control and negative control were detected according to the detection method, and the dilution with the larger P/N value (positive control well OD value mean/negative control well OD value mean) was taken as the coating concentration, and the results were obtained (see Table 5): when the coating concentration is 0.2-1.6 mug/ml, the P/N values are all more than 90 and are obviously higher than the P/N values (all less than 50) when the coating concentration is 0.1 and 2.0 mug/ml, and negative samples and positive samples can be effectively distinguished; particularly, the P/N value is better than that of the coating concentration of 0.2 and 1.6 mu g/ml (both are less than 70) when the coating concentration is 0.4-1.0 mu g/ml, and the P/N value is the highest when the coating concentration is 0.4 mu g/ml.
TABLE 5 selection of peptide fragment coating concentration of porcine pseudorabies virus gD protein
Figure BDA0002563768320000181
According to the above results, the prepared antigen-coated plates were coated with a coating concentration of 0.4. mu.g/ml, and the kits were prepared according to the method for preparing the kit of example 3.1 and named kit A, respectively.
3.4 applications of the kit
3.4.1 sensitivity
25 piglets, all of which were negative for 21-day-old PRV antigen antibodies, were randomly divided into 5 groups, 5/group. The vaccine comprises a group 1 immune porcine pseudorabies live vaccine (Bartha strain), a group 2 immune porcine pseudorabies live vaccine (HB-98 strain), a group 3 immune porcine pseudorabies inactivated vaccine (Hu A strain), a group 4 immune variant strain porcine pseudorabies inactivated vaccine (HN 1201-delta gE strain) which is promethazine, and a group 5 injected with DMEM medium with the same volume as the control group. Each group was bled at 2, 4, 8, and 16 weeks post immunization. The above sera were all tested with kit A, and the neutralizing antibody was determined with variant strain HN1201 strain according to the method for serum neutralization test of GB/T18641-2002. The detection results have consistent trends, data of 1 pig in each group are taken as an example for explanation, and the data are shown in Table 6 in detail, so that the detection result of the kit A is consistent with the neutralization test result, the kit A can replace the neutralization test with complex test requirements, harsh environment and long time consumption (5 days), and the vaccines prepared aiming at the prior vaccine strains, classical strains of porcine pseudorabies viruses and variant strains of porcine pseudorabies viruses can be used for evaluating the titer of the generated neutralizing antibody so as to evaluate the effects of different vaccines after immunization, and the kit A is favorable for timely formulating scientific immunization programs according to the detection results.
TABLE 6 antibody detection results at different times after immunization with different vaccines
Figure BDA0002563768320000191
Note: neutralization potency is less than 1:2, negative, more than or equal to 1:2, positive.
14 samples were taken simultaneously and the results of 2 detection methods were compared as shown in FIG. 1, and it was found from the analysis in FIG. 1 that: the detection effect of the kit A is consistent with the detection trend of a classical detection method (neutralization test), the coincidence rate is high, the positive boundary (the boundary node is when the S/P value is 0.5) has good correlation with immune protection, and the kit A can be further used for evaluating the immune effect of the vaccine.
3.4.2 specificity
5 parts of confirmed porcine pseudorabies virus antibody negative serum, 1 part of porcine circovirus type 2 (PCV2) positive serum, 1 part of Porcine Parvovirus (PPV) positive serum, 1 part of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) positive serum, 1 part of swine fever virus (CSFV) positive serum, 1 part of porcine Japanese Encephalitis Virus (JEV) positive serum, 1 part of Haemophilus Parasuis (HPs) positive serum, 1 part of porcine atrophic rhinitis (Bb) positive serum, 1 part of escherichia coli (BL21) positive serum, 1 part of porcine mycoplasma pneumoniae (Mhp) positive serum, 1 part of Porcine Epidemic Diarrhea Virus (PEDV) positive serum, 1 part of porcine Transmissible Gastroenteritis (TGEV) positive serum and 1 part of porcine rotavirus (PRoV) positive serum, the detection was carried out using kit a, the neutralization assay and the commercial kit BioChek PRVgB antibody kit, respectively, prepared in example 3, and as a result: the detection result of the kit A is consistent with the detection results of a neutralization test and a commercialized kit, and the detection results are negative.
The test kit A is used for detecting the serum of mice immunized by PRVgB protein, PRVgC protein and PRVgE protein respectively, and the results are as follows: all were negative.
The results show that the kit A has no cross reaction with positive serum of other viruses and has good specificity.
3.4.3 repeatability
The PRVgD positive serum and the PRVgD negative serum identified by the neutralization test are respectively detected by different batches of ELISA kits A, and the result is as follows: the detection result of the kit A is consistent with the neutralization test result, and the variation coefficients in batches and among batches are less than 15%, which shows that the kit A has good repeatability.
3.4.4 shelf life
The 3 batches of kit A were prepared and stored at 2-8 ℃ and sampled at 3, 6, 9, 12, 15 and 18 months respectively. Reference example 3.4 was used for sensitive, specific and reproducible assays. As a result: and in different storage times, the result of the kit A for determining the negative sample is negative, the result of the kit A for determining the positive sample is positive, and the coefficient of variation is less than 15%. The inventors unexpectedly found that the kit A still has good sensitivity, specificity and repeatability when stored at 2-8 ℃ for 18 months.
3.4.5 clinical applications
The PRVgD protein ELISA kit, called kit B, was prepared according to the preparation method of example 3.1 using the porcine pseudorabies virus gD protein of example 2 as an antigen plate. 10 parts of pig serum (number 1# -10 #) of a porcine pseudorabies vaccine which is not immunized temporarily in a certain pig farm is collected, and the detection is respectively carried out by using a kit A, a kit B and a neutralization test, and the result is as follows: neutralizing titer of 1# to 10# pig serum is less than 1:2, and detection of the kit A and the kit B is negative. The pig farm needs to immunize the porcine pseudorabies vaccine as soon as possible. Indicating that kit A, B facilitates timely formulation of immunization programs.
100 parts of serum (11# -110 #) of newborn piglets (0-3 days) in a certain piglet farm are respectively detected by using a kit A and a neutralization test, and the result is as follows: the kit A detects that the S/P value of 10 pigs is 0.179-0.426 (corresponding to neutralization titer less than 1: 2), and is negative, and the kit B detects that the kit B is negative; wherein the neutralization test of 90 pigs is positive (the neutralization titer is more than or equal to 1: 2), the positive rate detected by the kit A is 94% (85/90), the positive rate detected by the kit B is 86% (77/90), and the neutralization titer of 8 parts of serum not detected by the kit B is 1: 2-1: 22.39. The result shows that the kit A has high accuracy in detecting the serum sample with low neutralization titer and is closer to the detection result of the neutralization test. In addition, from the experimental results, it can be seen that: the three detection methods in the pig farm detect that the same pig with all negative pigs needs to be immunized with the porcine pseudorabies vaccine immediately, and if the ELISA titer rising trend is not obvious after immunization for 1 time, the immunization needs to be strengthened; the same litter pig which is positive in the detection of the neutralization test and negative in the detection of the kit A also needs to be boosted at least 1 time, and the same litter pig which is positive in the detection of the neutralization test and positive in the detection of the kit A only needs to be immunized according to the routine immunization program of piglets in a pig farm. The kit can be used for monitoring the maternal antibody.
50 parts of pig serum (111# -160 #) of a pig farm immune porcine pseudorabies vaccine (Bartha strain) and 50 parts of pig serum (161# -210 #) of another pig farm immune porcine pseudorabies vaccine (Pupseudo-Net) are respectively detected by a kit A, a kit B and a neutralization test, and the results are as follows: the S/P value of 111# -160 # pig serum detected by the kit A is 0.511-0.756 (corresponding to neutralization titer is 1: 2-1: 13.5), the pig serum is weak positive, the kit B is negative, the result shows that the kit A can detect weak positive neutralization antibody titer, the kit B can not detect, and the variant porcine pseudorabies vaccine needs to be supplemented on the immunization program; the S/P value of 161# to 210# pig serum detected by the kit A is 1.756-2.988 (corresponding to neutralization titer is 1: 32-1: 707.94), the pig serum is strong positive, the kit B is positive, and the result shows that the kit A and the kit B can detect the titer of the neutralization antibody of the strong positive. The result shows that the kit A can be used for antibody monitoring of pigs after vaccine immunization.
Example 4 preparation and characterization of vaccine compositions containing neutralizing epitope of gD protein of porcine pseudorabies virus
4.1 preparation of vaccine compositions
The expressed polypeptide of the porcine pseudorabies virus gD protein neutralizing epitope site 130-149 AA in the example 2.5.3 is mixed with 206 adjuvant in proportion, stirred for 15 minutes at the temperature of 30 ℃ at 120 rpm, and stored at the temperature of 2-8 ℃, so that the vaccine composition of the porcine pseudorabies virus gD protein neutralizing epitope is obtained, and the vaccine 1-3 is prepared by taking the content of the polypeptide containing the gD protein neutralizing epitope in the table 7 as an example.
TABLE 7 vaccine proportioning for neutralizing epitope of porcine pseudorabies virus gD protein
Components Vaccine 1 Vaccine 2 Vaccine 3
gD protein neutralizing epitope (μ g/ml) 15 20 100
206 adjuvant (V/V%) 46 46 46
4.2 identification of vaccine compositions
12 piglets with 21-day-old PRV antigen-antibody negative are randomly divided into 4 groups and 3 piglets per group, the 1st group, the 2 nd group and the 3 rd group are respectively immunized by intramuscular injection of the vaccine 1, the vaccine 2 and the vaccine 3 prepared in the example 4.1 according to the volume of 2ml per scalp, and the 4 th group is injected with the DMEM culture medium of 2ml per head. Blood is collected 28 days after immunization, and ELISA titer and neutralization titer of the pig serum after immunization are determined. ELISA titers were determined using kit B and detected according to the detection method of example 3.2.
Neutralization test the neutralizing antibody titer to different strains of porcine pseudorabies virus is determined by referring to a method of a GB/T18641-2002 method serum neutralization test, wherein the different strains comprise a PRV variant HN1201 strain, a classical strain Fa strain, a Ma strain and an early vaccine strain Bartha strain. The trends of the detection results are consistent, and the data of 1 pig in each group is taken as an example for illustration, and the results are shown in table 8: the data of the 1st group to the 3 rd group detected by the kit A and the kit B are strong positive, and the S/P value is increased along with the increase of the antigen content in the vaccine. The polypeptide vaccine containing the neutralizing epitope of the gD protein of the porcine pseudorabies virus is shown to generate higher neutralizing antibody after immunizing a pig, and can generate neutralizing effect on strains of PRV of different types (including original vaccine strains, classical strains and variant strains).
TABLE 8 neutralization test results for different strains
Figure BDA0002563768320000231
Note: neutralization potency is less than 1:2, negative, more than or equal to 1:2, positive.
In conclusion, the kit prepared by the invention is consistent with the result of a neutralization test, can replace the neutralization test which has complex test requirements, harsh environment and long time consumption (5 days), is used for evaluating the effect of different vaccines after immunization, and is beneficial to timely formulating scientific immunization programs according to the detection result; can also be used for monitoring maternal antibodies. The vaccine composition prepared by the invention generates high neutralizing antibodies for different types of PRV strains (including original vaccine strains, classical strains and variant strains).
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Luoyang Putai Biotech Ltd
<120> ELISA antibody detection kit for coating porcine pseudorabies virus neutralizing epitope protein fragment, and vaccine composition containing same
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 363
<212> DNA
<213> hybridoma cell (hybridoma)
<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 (hybridoma)
<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 (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
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Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala Arg Val Asp Gln
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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> porcine pseudorabies virus (pseudorabies virus)
<400> 5
Thr Thr Pro Met Trp Trp Thr Pro Ser Ala Asp Tyr Met Phe Pro Thr
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Glu Asp Glu Leu
20

Claims (10)

1. An ELISA antibody detection kit of a protein fragment coated with a porcine pseudorabies virus neutralizing epitope, wherein the ELISA antibody detection kit comprises a support medium of the protein fragment coated with the porcine pseudorabies virus gD protein neutralizing epitope, an enzyme-labeled secondary antibody reagent, a detection reagent for reacting with the enzyme-labeled secondary antibody, a negative control and a positive control; the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is shown as SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.
2. The ELISA antibody detection kit of claim 1, wherein the negative control is serum with PRV antigen antibodies negative, the positive control is serum of porcine pseudorabies virus variant porcine pseudorabies vaccine immune pigs, the enzyme-labeled secondary antibody is enzyme-labeled goat anti-pig IgG or enzyme-labeled rabbit anti-pig IgG, and the enzyme-labeled enzyme is horse radish peroxidase, alkaline phosphatase or beta-D-galactoglycase.
3. The ELISA antibody detection kit of claim 1, wherein the protein fragment coating concentration of the neutralizing epitope of porcine pseudorabies virus gD protein is 0.1-2.0 μ g/ml, preferably 0.2-1.6 μ g/ml, more preferably 0.4-1.0 μ g/ml, and most preferably 0.4 μ g/ml; the support medium is a microtiter plate; the enzyme-labeled reagent is a solution which is diluted by an enzyme-labeled diluent to a final volume of 0.05 percent V/V enzyme-labeled goat anti-pig IgG or enzyme-labeled rabbit anti-pig IgG; the enzyme-labeled diluent is phosphate buffer solution of 20% V/V newborn calf serum, 0.05% V/V ProClin300 and 0.05% Tween-20.
4. According to claimThe ELISA antibody detection kit of 1, wherein the detection reagent for the reaction of the enzyme-labeled secondary antibody comprises a developing solution and a stop solution, the developing solution comprises a developing solution A and a developing solution B, the 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 developing solution B is a solution containing 0.02% w/v tetramethyl biphenyl diamine and 10% v/v absolute ethyl alcohol; the stop solution is 2M H2SO4A solution;
the ELISA antibody detection kit further comprises a washing solution and a sample diluent, wherein the washing solution is a phosphate buffer solution, and the sample diluent is a PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.05-0.5% W/V Casein and 1% W/V Proclin 300.
5. A method of making the ELISA antibody detection kit of claim 1, wherein the method comprises:
expressing a protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein, coating the protein fragment on a support medium according to the coating concentration of 0.1-2.0 mu g/ml and 100 mu l/hole, wherein the support medium is a microtiter plate, coating the support medium at 2-8 ℃ for 16-24 hours or coating the support medium at 37 ℃ for 2 hours, and then washing, sealing and drying the coated support medium;
sealing the microtiter plate coated in the step (1) by using a sealing solution, wherein the sealing solution is a phosphate buffer solution containing 5% W/V sucrose, 20% V/V newborn calf serum and 0.05% V/V ProClin300, and the sealing condition is sealing for 16-24 hours at the temperature of 2-8 ℃ or sealing for 2 hours at the temperature of 37 ℃;
diluting enzyme-labeled goat anti-pig IgG or enzyme-labeled rabbit anti-pig IgG to a final volume of 0.05% V/V by using an enzyme-labeled diluent to prepare an enzyme-labeled secondary antibody reagent, wherein the enzyme-labeled diluent is phosphate buffer solution of 20% V/V newborn bovine serum, 0.05% V/V ProClin300 and 0.05% Tween-20;
step (4) preparing a washing solution, a developing solution, a stop solution, a positive control, a negative control and a sample diluent respectively;
and (5) assembling the microtiter plate prepared in the step (2), the enzyme-labeled secondary antibody reagent prepared in the step (3), and the washing solution, the developing solution, the stop solution, the positive control and the negative control prepared in the step (4) into the ELISA antibody detection kit.
6. The method according to claim 5, wherein the protein fragment coating concentration of the neutralizing epitope of the porcine pseudorabies virus gD protein in the step (1) is 0.2-1.6 μ g/ml, preferably 0.4-1.0 μ g/ml, and more preferably 0.4 μ g/ml; the drying condition is that the drying is carried out for 3 to 6 hours at the temperature of between 18 and 26 ℃ and under the condition that the relative humidity is not higher than 30 percent;
the color developing solution in the step (4) comprises a color developing solution A and a 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 carbamide peroxide in each 1L of water, and the color developing solution B contains 0.2g of tetramethyl biphenyl diamine and 100ml of absolute ethyl alcohol in each 1L of water;
the positive control is serum of a pig immunized by the variant strain porcine pseudorabies vaccine, and the negative control is serum with all PRV antigen antibodies negative;
the washing solution is phosphate buffer solution, and 1L of 20-time concentrated solution of the washing solution contains 160g of sodium chloride, 58g of disodium hydrogen phosphate, 4.8g of monopotassium phosphate, 4g of potassium chloride and 2010 mL of tween;
the sample diluent is PBS solution containing 10% V/V fetal calf serum, 0.1% V/V Tween 20, 1% W/V BSA, 0.05-0.5% W/V Casein and 1% W/V Proclin 300; and
the stop solution is 2M H2SO4And (3) solution.
7. Use of the ELISA antibody detection kit of claims 1-4 for non-immunodiagnostic applications including vaccine immunization program evaluation.
8. A vaccine composition comprises an immunizing amount of protein fragments of porcine pseudorabies virus gD protein neutralizing epitopes and a pharmaceutically acceptable carrier, wherein the protein fragments of the porcine pseudorabies virus gD protein neutralizing epitopes are shown as SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.
9. The vaccine composition according to claim 8, wherein the content of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is more than or equal to 15 μ g/ml; preferably, the content of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 15-100 mu g/ml; more preferably, the content of the protein fragment of the neutralizing epitope of the porcine pseudorabies virus gD protein is 20-100 mu g/ml;
the pharmaceutically acceptable carrier is an adjuvant, the adjuvant is 206 adjuvant, and the adjuvant content is 46 v/v%.
10. Use of the vaccine composition according to claims 8-9 for the preparation of a medicament for the prevention and/or treatment of porcine pseudorabies virus infection.
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