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CN109900903B - Porcine pseudorabies virus gE blocking ELISA antibody detection kit and application thereof - Google Patents

Porcine pseudorabies virus gE blocking ELISA antibody detection kit and application thereof Download PDF

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CN109900903B
CN109900903B CN201910249606.1A CN201910249606A CN109900903B CN 109900903 B CN109900903 B CN 109900903B CN 201910249606 A CN201910249606 A CN 201910249606A CN 109900903 B CN109900903 B CN 109900903B
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pseudorabies virus
porcine pseudorabies
enzyme
solution
chain variable
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CN109900903A (en
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董春娜
张蕾
李静
李玲
王飞
肖进
齐鹏
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China Animal Husbandry Industry Co Ltd
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Abstract

The invention discloses a porcine pseudorabies virus gE blocking ELISA antibody detection kit and application thereof. The kit comprises an enzyme label plate, positive control serum, negative control serum, an enzyme-labeled porcine pseudorabies virus monoclonal antibody, a sample diluent, a 20-time concentrated washing solution, a substrate solution A, a substrate solution B and a stop solution, wherein the enzyme label plate is coated with a porcine pseudorabies virus gE protein expressed by a baculovirus expression system and a cell suspension culture process, the porcine pseudorabies virus gE protein is purified and used as a coating antigen and an immunogen, and a specific monoclonal antibody of the pseudorabies virus gE protein is prepared and screened out and is labeled with peroxidase to be used as a blocking enzyme-labeled monoclonal antibody. The kit has high sensitivity, good specificity and convenient operation, adopts an S/N ratio method for result judgment, has high accuracy, and has a detection coincidence rate of more than 98 percent compared with an imported kit.

Description

Porcine pseudorabies virus gE blocking ELISA antibody detection kit and application thereof
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a porcine pseudorabies virus gE blocking ELISA antibody detection kit and application thereof, which are suitable for specific, rapid and accurate detection of a porcine pseudorabies virus gE antibody.
Background
Pseudorabies (Pseudorabies), also known as Aujeszky's Disease (AD), is an acute infectious disease caused by the Pseudorabies virus (PRV). The disease can affect various animals including pig, cattle, sheep, rabbit, fox, dog and cat, ferret, mouse, and mink. Pigs are the natural host and reservoir, and except pigs, other animals develop symptoms of itch, high fever and encephalomyelitis, and can even die. After PRV infection of pigs, pregnant sow abortion, stillbirth, mummy fetus and pig sterility are mainly caused, the mortality rate is high after suckling piglets have neurological symptoms, fattening pig respiratory symptoms are bred, latent infection is easily formed due to PRV virus infection, and long-term toxic carrying and external toxin expelling are caused; in addition, PRV is easily activated under stress conditions, causing repeated infections and virus shedding, and thus the decontamination and eradication of this disease is very important to the entire swine industry.
The PRV virus particle is an elliptic or spherical virus particle with the size of 150-180 nm, is a typical herpes virus structure and consists of a nucleoprotein core, an icosahedral nucleocapsid with 162 capsomeres, a protein coat and a lipid bilayer membrane, wherein gE is a non-envelope protein necessary for the replication of PRV, contains 577 amino acid residues, is a heterodimer membrane protein and is highly conserved in α herpes virus, mediates the fusion between PRV and infected animal cells, promotes the diffusion of PRV and the release of PRV, and promotes neurotropic properties.
In China, the prevention, control and eradication of pseudorabies is a difficult task at present, and most of porcine pseudorabies vaccines used in the current market are attenuated vaccines lacking a gE gene and a TK gene. After the vaccine is immunized, antibodies aiming at the gE cannot be detected in the animal body, and after the wild strain is infected, the gE antibodies can be detected in the animal body infected with the wild strain because the wild strain contains the gE gene, so that whether the animal is infected with the wild virus can be judged by detecting whether the gE antibodies exist in a serum sample.
At present, Enzyme-Linked immunosorbent assay (ELISA) is the mainstream immunoassay technology in the market, is widely applied to clinical detection, is rapid and convenient, has high sensitivity, does not need special instrument and equipment, and can be used for detecting the antibody of the porcine pseudorabies virus. In the current market, most of kits for detecting the porcine pseudorabies virus antibody are indirect ELISA antibody detection kits, the sensitivity and specificity of the method have certain limitations on the level of detecting the porcine pseudorabies virus antibody, the requirement on the purity of the coating antigen is higher, and otherwise, false positive and false negative are easy to occur.
The invention is a blocking ELISA antibody detection method based on a specific monoclonal antibody aiming at porcine pseudorabies virus gE, so that the kit has good sensitivity and specificity.
Disclosure of Invention
The invention aims to provide a blocking ELISA kit which has high sensitivity and strong specificity and can quickly, simply and conveniently detect the porcine pseudorabies virus gE antibody.
One of the advantages of the kit is that a monoclonal antibody marked by horseradish peroxidase (HRP) is used, so that the sensitivity and specificity of detection are improved.
Based on the purpose, the detection kit for the porcine pseudorabies virus gE blocking ELISA antibody comprises an enzyme-linked reaction plate and an enzyme-labeled antibody, wherein the enzyme-linked reaction plate takes porcine pseudorabies virus gE protein as a coating antigen; the enzyme-labeled antibody is an enzyme-labeled antibody prepared from a monoclonal antibody (PRV-Mc2) specifically bound with porcine pseudorabies virus gE protein. The enzyme-labeled antibody is preferably an antibody labeled by a horseradish-labeled enzyme, and the horseradish peroxidase can be crosslinked on the antibody by a glutaraldehyde method or a periodic acid method.
Preferably, the porcine pseudorabies virus gE protein is a porcine pseudorabies virus gE purified protein obtained by expression of a baculovirus expression system and an insect cell suspension culture process. The sequence of the porcine pseudorabies virus gE purified protein is a sequence 6 in a sequence table.
Preferably, the monoclonal antibody (PRV-Mc2) specifically binding to the gE protein of porcine pseudorabies virus comprises a heavy chain variable region PRV-Mc2-VHAnd the light chain variable region PRV-Mc2-VL(ii) a The PRV-Mc2-VHAnd PRV-Mc2-VLBoth consist of a determinant complementary region and a framework region; the PRV-Mc2-VHAnd said PRV-Mc2-VLEach of the determinant complementary regions of (a) consists of a CDR1, a CDR2 and a CDR 3; the PRV-Mc2-VHThe amino acid sequence of the CDR1 is shown as amino acids 25 to 34 of SEQ ID No. 1; the PRV-Mc2-VHThe amino acid sequence of the CDR2 is shown as amino acids 52-59 of SEQ ID No. 1; the PRV-Mc2-VHThe amino acid sequence of the CDR3 is shown as amino acids 94-108 of SEQ ID No. 1; the PRV-Mc2-VLThe amino acid sequence of the CDR1 is shown as amino acids 24-35 of SEQ ID No. 2; the PRV-Mc2-VLThe amino acid sequence of the CDR2 is shown as amino acids 50-59 of SEQ ID No. 2; the PRV-Mc2-VLThe amino acid sequence of CDR3 is shown as amino acids 94-100 of SEQ ID No. 2.
Preferably, the PRV-Mc2-VHThe amino acid sequence of (A) is shown as 1 st to 123 th sites of SEQ ID No.1 in a sequence table; its PRV-Mc2-VLThe amino acid sequence of (A) is shown as 1 st to 117 th sites of SEQ ID No.2 in the sequence table.
The optimal coating preparation method and conditions of the enzyme-linked reaction plate are that porcine pseudorabies virus gE purified protein is dissolved in 100 mu l of carbonate solution with pH of 9.6, then the solution is added into a 96-hole polystyrene enzyme-linked reaction plate, 0.1 mu g-1 mu ggE purified protein is placed in each hole for 8-12 hours at the temperature of 2-8 ℃, so that coating antigen is fully combined with the enzyme-linked reaction plate, then PBS buffer solution containing 10mg/ml bovine serum albumin with pH of 7.4 is added into each hole according to 300 mu l, the solution is sealed for 2-3 hours at the temperature of 37 ℃, and after being dried, the enzyme-linked reaction plate is sealed and stored at the temperature of 4 ℃.
Preferably, the kit further comprises a positive control serum and a negative control serum, wherein the positive control serum is the porcine serum collected after the artificial infection of the porcine pseudorabies virus; the negative control serum was porcine serum free of specific pathogens (no infection with porcine pseudorabies virus pathogens and no vaccination).
Furthermore, the kit also comprises a sample diluent, a 20-time concentrated washing solution, a substrate solution A, a substrate solution B and a stop solution. The enzyme-linked reaction plate is a detachable 96-hole enzyme label plate. The sample diluent was a phosphate buffer solution containing 5mg/ml casein and having a value of 0.01M, pH of 7.4. The 20-time concentrated washing solution is 0.01M phosphate buffer solution with the pH value of 7.4 and contains 0.8-1.2% (ml/ml) of Tween-20. The substrate solution A is a citrate phosphate buffer solution containing 0.6mg/ml of urea hydrogen peroxide, and the substrate solution B is a tetramethylbenzidine solution containing 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when in use. The stop solution is a 2mol/L sulfuric acid solution.
The invention also claims a monoclonal antibody which can be specifically combined with the porcine pseudorabies virus and is any one of the following monoclonal antibodies:
1) comprises heavy chain variable region PRV-Mc2-VHAnd the light chain variable region PRV-Mc2-VL(ii) a The PRV-Mc2-VHAnd PRV-Mc2-VLBoth consist of a determinant complementary region and a framework region; the PRV-Mc2-VHAnd said PRV-Mc2-VLEach of the determinant complementary regions of (a) consists of a CDR1, a CDR2 and a CDR 3; the PRV-Mc2-VHThe amino acid sequence of the CDR1 is shown as amino acids 25 to 34 of SEQ ID No. 1; the PRV-Mc2-VHThe amino acid sequence of the CDR2 is shown as amino acids 52-59 of SEQ ID No. 1; the PRV-Mc2-VHThe amino acid sequence of the CDR3 is shown as amino acids 94-108 of SEQ ID No. 1; the PRV-Mc2-VLThe amino acid sequence of the CDR1 is shown as amino acids 24-35 of SEQ ID No. 2; the PRV-Mc2-VLThe amino acid sequence of the CDR2 is shown as amino acids 50-59 of SEQ ID No. 2; the PRV-Mc2-VLThe amino acid sequence of CDR3 is shown as amino acids 94-100 of SEQ ID No. 2.
2) The PRV-Mc2-VHThe amino acid sequence of (A) is shown as 1 st to 123 th sites of SEQ ID No.1 in a sequence table; its PRV-Mc2-VLThe amino acid sequence of (A) is shown as 1 st to 117 th sites of SEQ ID No.2 in the sequence table.
The heavy chain variable region and the light chain variable region sequences can be connected with animal-derived constant regions (such as heavy chain and light chain constant regions of a murine antibody) to prepare the monoclonal antibody capable of being specifically combined with the porcine pseudorabies virus.
The application of the enzyme-linked immunoassay kit in the detection of the gE blocking antibody of the porcine pseudorabies virus also belongs to the protection scope of the invention.
The application of the monoclonal antibody capable of being specifically combined with the porcine pseudorabies virus in the preparation of the kit for detecting the porcine pseudorabies virus is also within the protection scope of the invention. In particular to the application in the preparation of a kit for detecting the porcine pseudorabies virus antibody.
The porcine pseudorabies virus gE protein is a purified porcine pseudorabies virus gE protein obtained by expression of a baculovirus expression system and an insect cell suspension culture process, and can comprise the following steps:
1) extracting porcine pseudorabies virus total RNA: adding 750 μ l Trizol into 250 μ l virus solution, mixing, adding 200 μ l chloroform, mixing, and centrifuging at 4 deg.C and 12000rpm for 15 min. The supernatant was pipetted into a new 1.5ml EP tube, 600. mu.l of isopropanol was added, mixed well and centrifuged for 10 min. The isopropanol was discarded, washed with 75% DEPC ethanol and centrifuged. The ethanol was discarded, oven dried and the RNA was dissolved in 20. mu.l of RNase-free water.
2) Reverse transcription, PCR amplification and gene sequencing: carrying out reverse transcription by using an Invitrogen reverse transcription kit according to the instruction to obtain cDNA, designing a primer (F1: 5'-ATGCGGCCCTTTCTGCTGC-3'; R1: 5'-TTAAGCGGGGCGGGACAT-3') according to a GenBank report (M17321.1) sequence, amplifying a target fragment by using the primer, recovering the fragment after amplification, and then connecting a vector for sequence determination to obtain gE gene sequence information.
3) Synthesis of gE gene sequence, construction of shuttle vector, screening and extracting of recombinant Bacmid, and rescue of recombinant baculovirus ① gene sequence, namely, according to the measured gE ectodomain gene sequence, adding His label, synthesizing the gene sequence by Beijing Zhongmeitai company, and optimizing insect cell codon, ② construction of shuttle vector, according to gE sequence information and pFastBacdual vector sequence information, design of corresponding primer (F2: 5' -CTGCCTTTGCGGCGGAT)GAATTCATGAGGCCTTTCCTGCTCA-3’；R2：5’-CTAGTACTTCTCGACAAGCTTTTAGTGATGATGATGATGATGCAGACCTCCTGGA-3'), amplifying gE ectodomain fragment, recovering glue, connecting into pFastBacdual carrier PH promoter by homologous recombination method, connecting into carrier, sequencing to ensure sequence accuracy, screening and extracting ③ recombinant Bacmid, transforming constructed shuttle carrier into DH10Bac competence, then coating three-resistance plate (kanamycin, gentamicin, tetracycline), culturing for 48h in 37 deg.C incubator, picking up white spot, identifying by primer, selecting positive clone target fragment with size of 1308bp, selecting positive clone shake bacteria, extracting Bacmid by isopropanol precipitation method after 12h, then utilizing Nanodrop to determine concentration, rescuing ④ recombinant baculovirus, wherein density is 2 × 10 before transfection6And (2) spreading SF9 cells on a six-hole plate, transfecting recombinant Bacmid according to the amount of 5 mu g and 2.5 mu g, wherein the using amount of a transfection reagent is 8 mu l, changing the liquid after 4-6 h of transfection, culturing at 28 ℃, harvesting and amplifying P2 generation virus after 72h, and amplifying the P3 generation virus by adopting the same method. The amplification of the P4 generation virus adopts shake flask amplification, and the inoculation ratio of the virus is 1: 100.
4) expression and purification of gE protein: inoculating the P4 generation virus at a ratio of 1:5 with a density of 2 × 106Hi5 cells, cultured at 28 ℃, harvested after 48h, centrifuged at 8000r/min for 1h to obtain the supernatant, and then filtered through a 0.22 μm filter for later use. And (3) balancing a His column by using a 20mmol/L Tris50mmol/L NaCl PH8.0 solution, then hanging the cell culture supernatant on the column, eluting by using a 20mmol/L Tris50mmol/L NaCl PH8.0300mmol/L imidazole solution, and carrying out affinity chromatography purification and identification to obtain the purified porcine pseudorabies virus gE protein.
The method for obtaining the monoclonal antibody capable of being specifically combined with the porcine pseudorabies virus gE protein comprises the following steps: screening the porcine pseudorabies virus specific monoclonal cell strain according to a conventional method known in the field, determining the gene sequence of the specific monoclonal cell strain by adopting a gene sequencing method, and preparing a stably expressed monoclonal antibody as the enzyme-labeled monoclonal antibody by utilizing a method of gene synthesis and construction of a recombinant expression vector. Specifically, the method for obtaining the specific monoclonal antibody of the porcine pseudorabies virus can comprise the following steps:
1) the gE protein expressed and purified by a baculovirus expression system is taken as immunogen, the purity is not lower than 80%, and the antigen concentration is adjusted to 100 mug/ml;
2) continuously immunizing for 4 times, each time at an interval of 14 days, adopting a multipoint subcutaneous immunization mode for the first 3 times, and adopting an intraperitoneal injection immunization mode for the 4 th time, wherein each time is 10 mu g/animal;
3) separating splenocytes from immune animals, fusing the splenocytes with myeloma cells, screening hybridoma cells by using HAT selective culture medium, and screening specific positive clones from the supernatant of the hybridoma cells by using an indirect ELISA method; when the serum antibody level of the immunized animal is detected by indirect ELISA with a titer exceeding 1:50000, spleen cells of the animal can be isolated and prepared into a single cell suspension, and fused with myeloma cells (preferably mouse myeloma cells SP2/0) under induction of a suitable fusing agent (such as polyethylene glycol) to form hybridomas; it was determined that a monoclonal cell strain preferably secreting PRV-Mc1 specifically reacts with porcine pseudorabies virus gE protein.
4) Extracting total RNA of the specific positive clone hybridoma cell strain: taking 250 ul of hybridoma cell suspension, adding 750 ul of Trizol, turning upside down and mixing, adding 200 ul of chloroform, mixing, and centrifuging at 12000rpm at 4 ℃ for 15 min. The supernatant was pipetted into a new 1.5ml EP tube, 600. mu.l of isopropanol was added, mixed well and centrifuged for 10 min. The isopropanol was discarded, washed with 75% DEPC ethanol and centrifuged. The ethanol was discarded, oven dried and the RNA was dissolved in 20. mu.l of RNase-free water.
5) Reverse transcription, PCR amplification and gene sequencing: reverse transcription was performed using the Invitrogen reverse transcription kit as per the instructions to obtain cDNA for hybridoma cells. For the heavy chain (V)H-1：5’-GTGAATTCATGCAGGTGCAGCTGTTGGAGTCTGG-3’；VH-2: 5'-ATGTCGACTGAGGAGACGGTGACCAGGGTGCC-3') and light chain (V)L-1：5’-GTGAATTCATGGACATTGTGATGACCCAGTCTCC-3’；VL-2: 5'-CAGTCGACTTACGTTTGATCTCCAGCTTGGTCCC-3') designing universal primers for the variable regions, and amplifying the target fragments by using the amplification primersAnd (3) amplifying, recovering fragments from the gel after amplification, and then connecting the fragments with a vector for sequence determination to obtain sequence information of heavy chain and light chain variable regions of the monoclonal antibody.
6) Synthesis of gene sequence of specific monoclonal antibody, construction of shuttle vector, screening and extraction of recombinant Bacmid, rescue of recombinant baculovirus ① gene sequence, which is to supplement the sequences of the heavy chain and light chain constant regions of mouse antibody to the variable region part according to the measured sequences of the heavy chain and light chain variable regions of monoclonal antibody PRV-Mc2, then send the sequences to Meitai company in Beijing, and optimize insect cell codons ② shuttle vector construction, which is to design corresponding primers according to the sequence information of the heavy chain and light chain and the sequence information of pFastBacdeal vector, and aim at the heavy chain (Mc 2-HF: 5' -TCATACATCTACGCGGCC)GCTAGCGAAGTTCAGCTGCAA-3’；Mc2-HR：5’-TCCCCCATCTCCCGGTACCCTTGCCAGGAGAGAG-3') and a light chain (Mc 2-LF: 5' -CTGCCTTTGCGGCGGATGAATTCCAATCATATCTCACACAAC-3’；Mc2-LR：5’-CTAGTACTTCTCGACAAGCTTTGAGCATTCGGTTGG-3'), amplifying fragments of heavy chain and light chain full length, connecting into pFastBacduoal vector by homologous recombination after recovery of glue, wherein the pFastBacduoal vector contains two promoters, namely PH promoter and P10 promoter, and performing sequence determination to ensure sequence accuracy after connecting into the vector, screening and extracting ③ recombinant Bacmid, transforming constructed shuttle vector into DH10Bac competence, then coating three-resistance plate (kanamycin, gentamycin and tetracycline), picking up white spot after culturing in 37 ℃ incubator for 48h, identifying, selecting 4600bp and 300bp fragments of positive clone target, selecting clone shake bacteria without 300bp band, extracting Bacmid by isopropanol precipitation method after 12h, then determining concentration by using Nanodrop, saving ④ recombinant virus, namely, selecting 2 × 10 bp rod density before transfection6And (2) spreading SF9 cells on a six-hole plate, transfecting recombinant Bacmid according to the amount of 5 mu g and 2.5 mu g, wherein the using amount of a transfection reagent is 8 mu l, changing the liquid after 4-6 h of transfection, culturing at 28 ℃, harvesting and amplifying P2 generation virus after 72h, and amplifying the P3 generation virus by adopting the same method. The amplification of the P4 generation virus adopts shake flask amplification, and the inoculation ratio of the virus is 1: 100.
7) expression and purification of specific monoclonal antibodies: inoculating the P4 generation virus at a ratio of 1:5 with a density of 2 × 106Hi5 cells, cultured at 28 ℃, harvested after 48h, centrifuged at 8000r/min for 1h to obtain the supernatant, and then filtered through a 0.22 μm filter for later use. And (3) balancing the ProteinA prepacked column by using a solution with a Na3PO4pH value of 7.0, balancing the volume of 3-5 columns, then combining the cell supernatant with the ProteinA prepacked column, and eluting by using an eluent with a Glycine-HCL pH value of 3.0 after the sample is combined, thus obtaining the purified porcine pseudorabies virus gE specific monoclonal antibody PRV-Mc 2.
The detection program of the kit of the invention is as follows:
1) balancing: taking out the kit from the refrigeration environment, and standing at room temperature for balancing for 30min for later use; the liquid reagents were mixed well before use.
2) Preparing liquid: diluting the concentrated washing solution by 20 times of distilled water or deionized water to obtain a washing buffer solution;
3) sample dilution: the serum to be detected is diluted 2 times by using a sample diluent in a serum dilution plate, and the negative and positive control serum is diluted and can be directly used.
4) Sample adding: and taking out the required laths, filling the rest laths into an aluminum foil bag, sealing, and storing at 2-8 ℃ for later use. And adding the diluted serum to be detected, the negative control serum and the positive control serum into an antigen coated plate at 100 mu l/hole. Each serum to be detected is provided with 1 hole, the negative control and the positive control are respectively provided with 2 holes, and the time span of the sample adding process is required to be as short as possible. Loading as shown in figure 1: n: indicating the addition of negative control serum; p: represents the addition of positive control serum; s1, S2, S3, S4 and the like indicate that each serum to be tested is added.
5) And (3) incubation: shaking and mixing evenly, placing in an incubator at 37 ℃ and reacting for 60 min.
6) Washing the plate: discarding the reaction solution, adding 300 μ l of diluted washing buffer solution into each well, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
7) Adding an enzyme: mu.l of enzyme-labeled secondary antibody was added to each well.
8) And (3) incubation: the reaction mixture was placed in an incubator at 37 ℃ and reacted for 30 min.
9) Washing the plate: discarding the reaction solution, adding 300 mu l of diluted washing buffer solution into each hole, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
10) Adding 100 μ l of substrate working solution (substrate working solution is obtained by mixing substrate solution A and substrate solution B in equal amount, and is prepared at present), shaking, mixing, placing in 37 deg.C incubator, and reacting for 15min in dark.
11) 50 μ l of color-developing stopping solution is added into each hole, the reaction is stopped by shaking and mixing, and the result is measured within 15 minutes.
12) The test is satisfied under the conditions: negative control OD450nmThe values should all be greater than or equal to 1.0. The S/N value of the positive control hole is less than or equal to 0.5.
13) And (3) judging: the OD of each well was measured on a microplate reader450nmThe value is obtained. S/N ═ sample OD450nmValue/negative control OD450nmThe value is obtained. The presence or absence of the antibody was determined by calculating the S/N value of each sample. Negative: S/N is more than or equal to 0.7; suspicious S/N is more than 0.6 and less than 0.7; positive S/N is less than or equal to 0.6.
The invention has the positive effects that: the invention provides an enzyme-linked immunoassay kit for detecting a gE antibody of porcine pseudorabies virus. The kit is an enzyme-linked immune antibody detection kit prepared by adopting porcine pseudorabies virus gE purified protein and a blocking method aiming at a gE specific monoclonal antibody, can determine the content of the specific antibody of the porcine pseudorabies virus gE in a sample by detecting the signal change generated by an enzyme catalytic substrate, and does not have cross reaction with other pathogens such as porcine foot-and-mouth disease virus O type, porcine foot-and-mouth disease virus A type, porcine circovirus, porcine reproductive and respiratory syndrome virus positive serum at present.
In conclusion, the kit adopts the porcine pseudorabies virus gE purified protein and the blocking method enzyme-linked immune antibody detection kit prepared aiming at the gE specific monoclonal antibody, has high sensitivity and strong specificity, can effectively detect the content of the specific antibody of the porcine pseudorabies virus gE in a sample, has the coincidence rate with the imported kit of more than 98 percent, and has wide market prospect and good economic and social benefits.
Drawings
FIG. 1 is a schematic view of the loading of an enzyme-linked immunosorbent assay plate of the kit of the invention.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biomaterials used are wide and any biomaterials available without violating laws and ethical ethics can be used instead as suggested in the examples.
The embodiments are provided in order to provide detailed embodiments and specific procedures, which will help understanding of the present invention, but the scope of the present invention is not limited to the following embodiments.
Example 1 preparation of porcine pseudorabies virus gE purified protein
The method comprises the following steps:
1) adding 750 μ l Trizol into 250 μ l virus solution, mixing, adding 200 μ l chloroform, mixing, and centrifuging at 4 deg.C and 12000rpm for 15 min. The supernatant was pipetted into a new 1.5ml EP tube, 600. mu.l of isopropanol was added, mixed well and centrifuged for 10 min. The isopropanol was discarded, washed with 75% DEPC ethanol and centrifuged. The ethanol was discarded, oven dried and the RNA was dissolved in 20. mu.l of RNase-free water.
2) Reverse transcription was performed using the Invitrogen reverse transcription kit as described in the specification to obtain cDNA, and primers were designed based on GenBank report (AF207700.1) sequence (F1: 5'-ATGCGGCCCTTTCTGCTGC-3', respectively; r1: 5'-TTAAGCGGGGCGGGACAT-3'), using the primer to amplify the target segment, recovering the segment after amplification, and then connecting the vector to perform sequence determination to obtain gE gene sequence information.
3) According to the determined gene sequence of the gE ectodomain, a His label is added, the obtained product is sent to Beijing Zhongmeitai and company for gene sequence synthesis, insect cell codon optimization is carried out, and the nucleotide sequence of the gE protein is shown as SEQ ID No.3 (the full-length sequence is the coding sequence) in the sequence table.
4) According to gE sequence information and pfastbacudual vector sequence information, a corresponding primer (F2: 5' -CTGCCTTTGCGGCGGATGAATTCATGAGGCCTTTCCTGCTCA-3’；R2：5’-CTAGTACTTCTCGACAAGCTTTTAGTGATGATGATGATGATGCAGACCTCCTGGA-3'), amplifying the gE ectodomain fragment, recovering the gel, connecting into pFastBacdual carrier PH promoter by homologous recombination, and determining the sequence after connecting into the carrier to ensure the accuracy of the sequence.
5) Transforming DH10Bac competence by the constructed shuttle vector, coating a three-resistance plate (kanamycin, gentamicin and tetracycline), culturing for 48h in an incubator at 37 ℃, picking out white spots, identifying by using primers, selecting positive clone shake bacteria with the target fragment size of 1308bp, extracting Bacmid by using an isopropanol precipitation method after 12h, and then determining the concentration by using Nanodrop.
6) Rescue of recombinant baculovirus: density was 2X 10 before transfection6And (2) spreading SF9 cells on a six-hole plate, transfecting recombinant Bacmid according to the amount of 5 mu g and 2.5 mu g, wherein the using amount of a transfection reagent is 8 mu l, changing the liquid after 4-6 h of transfection, culturing at 28 ℃, harvesting and amplifying P2 generation virus after 72h, and amplifying the P3 generation virus by adopting the same method. The amplification of the P4 generation virus adopts shake flask amplification, and the inoculation ratio of the virus is 1: 100.
7) inoculating the P4 generation virus at a ratio of 1:5 with a density of 2 × 106Hi5 cells, cultured at 28 ℃, harvested after 48h, centrifuged at 8000r/min for 1h to obtain the supernatant, and then filtered through a 0.22 μm filter for later use. And (3) balancing a His column by using a 20mmol/L Tris50mmol/L NaCl PH8.0 solution, then hanging the cell culture supernatant on the column, eluting by using a 20mmol/L Tris50mmol/L NaCl PH8.0300mmol/L imidazole solution, and carrying out affinity chromatography purification and identification to obtain the purified porcine pseudorabies virus gE protein.
Example 2 screening of porcine pseudorabies virus gE protein-specific hybridoma cell lines
The screening of the porcine pseudorabies virus gE protein specific hybridoma cell strain comprises the following steps:
1) the gE protein expressed and purified by the baculovirus expression system obtained in the example 1 is used as immunogen, the purity is not lower than 80%, and the antigen concentration is adjusted to 100 mu g/ml;
2) the immune animal is BALB/c mouse (purchased from Beijing Wittingle laboratory animal technology Co., Ltd.), the continuous immunization is carried out for 4 times, each time is 14 days, the multipoint subcutaneous immunization mode is adopted for the first 3 times, the intraperitoneal injection immunization mode is adopted for the 4 th time, and each mouse is injected with gB protein 10 mu g each time;
3) 7 days after the last immunization, separating serum from tail blood of the mouse, detecting by indirect ELISA (enzyme-linked immunosorbent assay), when the titer is more than 1:50000, separating splenocytes of the immunized animal, fusing the splenocytes with myeloma cells SP2/0 with good growth state, and screening by using HAT selective culture medium to obtain hybridoma cells;
4) screening specific positive clones from the hybridoma cell supernatant by an indirect ELISA method, and detecting to obtain a monoclonal cell strain secreting PRV-Mc2 capable of reacting with porcine pseudorabies virus gE protein specifically. The method comprises the following specific operation steps: dissolving porcine pseudorabies virus gB protein in 100 mu l of carbonate solution with the pH value of 9.6 to dilute the solution to the concentration of 2 mu g/ml, then adding the solution to a 96-hole polystyrene enzyme-linked reaction plate, placing the plate at the temperature of 2-8 ℃ for 8-12 hours, enabling the specific monoclonal antibody to be fully combined with the enzyme-linked reaction plate, then adding PBS buffer solution containing 10mg/ml bovine serum albumin with the pH value of 7.4 into the plate according to 300 mu l/hole, sealing the plate at the temperature of 37 ℃ for 2-3 hours, drying the plate, sealing the plate with aluminum foil paper after drying, and placing the plate at the temperature of 2-8 ℃ for storage.
Adding cell culture supernatant into an enzyme label plate coated with virus antigen, reacting for 30 minutes at 37 ℃, washing the plate for 4 times by using a washing solution (0.01M phosphate buffer solution with 0.8-1.2% (ml/ml) of Tween-20 and the pH value of 7.4, diluting 20 times by using double distilled water when in use), after drying, adding a rabbit anti-mouse IgG-HRP marker (purchased from Sigma company in America) diluted by 1:5000 into each hole, reacting for 30 minutes at 37 ℃, using the washing solution for 4 times, after drying, adding 50 mul of substrate working solution A (citrate phosphate buffer solution containing 0.6mg/ml of urea hydrogen peroxide) and substrate solution B (tetramethylbenzidine solution containing 0.2 mg/ml) into each hole, and reacting for 15 minutes at 37 ℃ in a dark place. Mu.l of stop solution (2mol/L sulfuric acid solution) was added to each well, and the reaction was terminated by shaking and mixing. OD per well was measured within 15 minutes450nmThe value is obtained. The determination of the absorbance value > negative control (i.e. washing plate culture medium). times.2.1 times is used as the positive determination standardThe titer of the specific monoclonal antibody in the serum is finally obtained, 1 strain of specific cell clone is finally obtained, and the strain has strong signal reaction with porcine pseudorabies virus gE protein, and is numbered as PRV-Mc 2.
Example 3 Gene sequencing of specific hybridoma cell line of porcine pseudorabies virus gE, establishment of monoclonal antibody recombinant expression System, and purification of monoclonal antibody
The gene sequencing of the specificity hybridoma cell strain of the porcine pseudorabies virus gE, the establishment of a monoclonal antibody recombinant expression system and the purification of a monoclonal antibody comprise the following steps:
1) extracting total RNA of a specific positive clone hybridoma cell strain, performing reverse transcription, performing PCR (polymerase chain reaction) and performing sequence determination:
① Total RNA extraction comprises collecting hybridoma cell suspension 250 μ l, adding 750 μ l Trizol, mixing, adding 200 μ l chloroform, mixing, centrifuging at 4 deg.C and 12000rpm for 15min, sucking supernatant into new 1.5ml EP tube, adding 600 μ l isopropanol, mixing, centrifuging for 10min, discarding isopropanol, washing with 75% DEPC ethanol, centrifuging, discarding ethanol, oven drying, and dissolving RNA with 20 μ l non-RNA enzyme water.
② reverse transcription was carried out using Invitrogen reverse transcription kit according to the instructions to obtain cDNA from hybridoma cells.
③ PCR reaction and sequencing of clones of its products general primers were designed for the heavy and light chain variable regions with the following sequence information:
TABLE 1 Universal primers for heavy and light chain variable regions
Name (R) Sequence (5 '-3')
VH-1 (Forward) GTGAATTCATGCAGGTGCAGCTGTTGGAGTCTGG
VH-2 (reverse) ATGTCGACTGAGGAGACGGTGACCAGGGTGCC
VL-1 (Forward) GTGAATTCATGGACATTGTGATGACCCAGTCTCC
VL-2 (reverse) CAGTCGACTTACGTTTGATCTCCAGCTTGGTCCC
And amplifying the target fragment by using an amplification primer, recovering the fragment after amplification, and then connecting a vector for sequence determination to obtain sequence information of heavy chain and light chain variable regions of the monoclonal antibody.
Monoclonal antibody PRV-Mc2 contains heavy chain variable region PRV-Mc2-VHAnd light chain variable region PRV-Mc2-VLIts PRV-Mc2-VHThe amino acid sequence of (A) is shown as 1 st to 123 th sites of SEQ ID No.1 in a sequence table; its PRV-Mc2-VLThe amino acid sequence of (A) is shown as 1 st to 117 th sites of SEQ ID No.2 in the sequence table.
The heavy chain variable region is PRV-Mc2-VHAnd the light chain variable region PRV-Mc2-VLBoth consist of a determinant complementary region and a framework region; the PRV-Mc2-VHAnd said PRV-Mc2-VLEach of the determinant complementary regions of (a) consists of a CDR1, a CDR2 and a CDR 3; the PRV-Mc2-VHThe amino acid sequence of the CDR1 is shown as amino acids 25 to 34 of SEQ ID No. 1; the PRV-Mc2-VHThe amino acid sequence of the CDR2 is shown as amino acids 52-59 of SEQ ID No. 1; the PRV-Mc2-VHThe amino acid sequence of the CDR3 is shown as amino acids 94-108 of SEQ ID No. 1; the PRV-Mc2-VLThe amino acid sequence of the CDR1 is shown as amino acids 24-35 of SEQ ID No. 2; the PRV-Mc2-VLThe amino acid sequence of the CDR2 is shown as amino acids 50-59 of SEQID No. 2; the PRV-Mc2-VLThe amino acid sequence of CDR3 is shown as SEQ IDAmino acids 94 to 100 of No. 2.
2) Synthesis of gene sequence of specific monoclonal antibody and establishment of recombinant expression system
① Synthesis of Gene sequence according to the sequence of the variable region of the heavy and light chains of the monoclonal antibody PRV-Mc2, the constant region sequences of the heavy and light chains of the murine antibody are supplemented in the variable region part, then sent to Meitai and company of Beijing, to synthesize the gene sequence, and carry out insect cell codon optimization, the nucleotide sequence of the PRV-Mc2 heavy chain is shown as SEQ ID No.4 in the sequence table (the full-length sequence is the coding sequence), and the nucleotide sequence of the light chain is shown as SEQ ID No.5 in the sequence table (the full-length sequence is the coding sequence).
② construction of shuttle vector based on the sequence information of heavy and light chains and the sequence information of pFastBacDual (available from ThermoFisher company, Cat. No. 10712024) vector, designing corresponding primers (see the sequence in Table 2 below), amplifying the full-length fragments of heavy and light chains, and joining into pFastBacdial vector by homologous recombination after recovery of glue, wherein the pFastBacdual vector contains two promoters, namely PH promoter and P10 promoter, and contains GP67 signal peptide sequence information behind the PH promoter sequence, and HDM signal peptide sequence information behind the P10 promoter sequence, and after joining into the vector, sequencing to ensure the sequence accuracy.
TABLE 2 expression vector construction primer sequence information
Name (R) Sequence (5 '-3')
PRV-Mc2-HF TCATACATCTACGCGGCCGCTAGCGAAGTTCAGCTGCAA
PRV-Mc2-HR TCCCCCATCTCCCGGTACCCTTGCCAGGAGAGAG
PRV-Mc2-LF CTGCCTTTGCGGCGGATGAATTCCAATCATATCTCACACAAC
PRV-Mc2-LR CTAGTACTTCTCGACAAGCTTTGAGCATTCGGTTGG
③ screening and extracting recombinant Bacmid, namely transforming DH10Bac competence by the constructed shuttle vector, then coating a three-resistant plate (kanamycin, gentamicin and tetracycline), culturing for 48h in an incubator at 37 ℃, picking white spots, identifying by using an M13 primer, selecting a 4600bp positive cloning target fragment and 300bp negative cloning target fragment, selecting clone shake bacteria without a 300bp strip completely, extracting Bacmid by using an isopropanol precipitation method after 12h, and then determining the concentration by using Nanodrop.
④ rescue of recombinant baculovirus the density was 2X 10 before transfection6And (2) spreading SF9 cells on a six-hole plate, transfecting recombinant Bacmid according to the amount of 5 mu g and 2.5 mu g, wherein the using amount of a transfection reagent is 8 mu l, changing the liquid after 4-6 h of transfection, culturing at 28 ℃, harvesting and amplifying P2 generation virus after 72h, and amplifying the P3 generation virus by adopting the same method. The amplification of the P4 generation virus adopts shake flask amplification, and the inoculation ratio of the virus is 1: 100.
3) expression and purification of specific monoclonal antibodies: inoculating the P4 generation virus at a ratio of 1:5 with a density of 2 × 106Hi5 cells, cultured at 28 ℃, harvested after 48h, centrifuged at 8000r/min for 1h to obtain the supernatant, and then filtered through a 0.22 μm filter for later use. With Na3PO4And (3) balancing the ProteinA prepacked column by using a solution with the pH value of 7.0, balancing the volume of 3-5 columns, then combining the cell supernatant with the ProteinA prepacked column, and eluting with an eluent with the pH value of 3.0 of Glycine-HCL after the sample is combined, thereby obtaining the purified porcine pseudorabies virus gB specific monoclonal antibody PRV-Mc 2. OD measurement with UV spectrophotometer280nmValue using the OD280nmThe value divided by an empirical factor of 1.48 is the concentration of the monoclonal antibody in mg/ml. The results showed that PRV-Mc1 secreted a monoclonal antibody concentration of 2.45 mg/ml.
Example 4 preparation of porcine pseudorabies virus gE blocking ELISA antibody detection kit
1) Preparing an antigen coated plate by using the porcine pseudorabies virus gE purified protein: diluting gE purified protein into 1 mu g/ml coating working solution by using a carbonate solution with the pH value of 9.6, then adding the coating working solution into a 96-hole polystyrene enzyme-linked reaction plate, placing the solution at 100 mu l/hole for 8-12 hours at the temperature of 2-8 ℃ to ensure that the coating antigen is fully combined with the enzyme-linked reaction plate, then adding PBS buffer solution containing 10mg/ml bovine serum albumin (pH7.4) into the solution according to 300 mu l/hole, sealing the solution at the temperature of 37 ℃ for 2-3 hours, drying the solution by spin drying, and sealing and storing the dried enzyme-linked reaction plate at the temperature of 2-8 ℃.
2) Preparation of horse radish peroxidase-labeled porcine pseudorabies virus gE specific monoclonal antibody
Coupling the monoclonal antibody specific to the porcine pseudorabies virus gE with horseradish peroxidase (HRP) by a glutaraldehyde oxidation method, fully dialyzing with PBS buffer solution with pH7.4, adding equal amount of high-quality glycerol, and storing at the temperature of-20 ℃. The method comprises the following specific steps:
① dissolving 5mg HRP in 0.2ml PBS buffer solution containing 1.25% glutaraldehyde and having pH of 6.8 of 0.1mol/L, coupling at room temperature for 18 hours, and dialyzing to remove excess glutaraldehyde;
② adding physiological saline to 1ml, then adding 2.5mg of the specific monoclonal antibody of the porcine pseudorabies virus gE and 0.1ml of 1mol/L carbonate buffer solution with the pH value of 9.6, placing at 2-8 ℃ for 24 hours;
③ adding 0.1ml of 0.3mol/L lysine solution, and standing at room temperature for 2 hours;
④ dialyzing thoroughly with PBS buffer solution of pH7.4, removing precipitate by centrifugation, the supernatant is enzyme conjugate, diluting with enzyme marker diluent at a certain ratio to obtain working solution (0.5ug/ml) of enzyme marker.
3) Positive control serum: porcine serum is collected after artificial infection of porcine pseudorabies virus and is used as positive control serum (1 tube, 1.5 ml/tube) of the kit.
4) Negative control serum: is Specific Pathogen Free (SPF) pig serum as negative control serum for the kit (1 tube, 1.5 ml/tube).
5) The sample dilutions were prepared as 1 vial (24 ml/vial) of 0.01M, pH value 7.4 phosphate buffer containing 5mg/ml casein.
6) Substrate solution A was prepared as citrate phosphate buffer containing 0.6mg/ml urea hydrogen peroxide (1 vial, 12 ml/vial)
7) Substrate solution B was prepared as a 0.2mg/ml solution of Tetramethylbenzidine (TMB) (1 vial, 12 ml/vial).
8) The 20-fold concentrated washing solution was prepared as 0.01M phosphate buffer (50 ml/vial, 2 vials) containing Tween-20 at a concentration of 0.8% to 1.2% (ml/ml) and a pH of 7.4.
9) Preparation of stop solution A2 mol/L sulfuric acid solution (1 vial, 12 ml/vial) was prepared.
10) If necessary, the kit may also contain a sample dilution plate (2, 96 wells/block) for sample dilution.
Example 5 application method of porcine pseudorabies virus gE blocking ELISA antibody detection kit
1) Balancing: taking out the kit from the refrigeration environment, and standing at room temperature for balancing for 30min for later use; the liquid reagents were mixed well before use.
2) Preparing liquid: diluting the concentrated washing solution by 20 times of distilled water or deionized water to obtain a washing buffer solution;
3) sample dilution: the serum to be detected is diluted 2 times by using a sample diluent in a serum dilution plate, and the negative and positive control serum is diluted and can be directly used.
4) Sample adding: and (3) taking an antigen coated plate, and adding the diluted serum to be detected, the negative control serum and the positive control serum into the antigen coated plate at a concentration of 100 mu l/hole. Each serum to be detected is provided with 1 hole, the negative control and the positive control are respectively provided with 2 holes, and the time span of the sample adding process is required to be as short as possible. Loading as shown in figure 1: n: indicating the addition of negative control serum; p: represents the addition of positive control serum; s1, S2, S3, S4 and the like indicate that each serum to be tested is added.
5) And (3) incubation: shaking and mixing evenly, placing in an incubator at 37 ℃ and reacting for 60 min.
6) Washing the plate: discarding the reaction solution, adding 300 μ l of diluted washing buffer solution into each well, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
7) Adding an enzyme: to each well, 100. mu.l of the working solution of the enzyme label prepared in example 4 was added.
8) And (3) incubation: the reaction mixture was placed in an incubator at 37 ℃ and reacted for 30 min.
9) Washing the plate: discarding the reaction solution, adding 300 mu l of diluted washing buffer solution into each hole, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
10) Adding 100 μ l of substrate working solution (substrate working solution is obtained by mixing substrate solution A and substrate solution B in equal amount, and is prepared at present), shaking, mixing, placing in 37 deg.C incubator, and reacting for 15min in dark.
11) 50 μ l of color-developing stopping solution is added into each hole, the reaction is stopped by shaking and mixing, and the result is measured within 15 minutes.
12) The test is satisfied under the conditions: negative control OD450nmThe values should all be greater than or equal to 1.0. The S/N value of the positive control hole is less than or equal to 0.5.
13) And (3) judging: the OD of each well was measured on a microplate reader450nmThe value is obtained. S/N ═ sample OD450nmValue/negative control OD450nmThe value is obtained. The presence or absence of the antibody was determined by calculating the S/N value of each sample. Negative: S/N is more than or equal to 0.7; suspicious S/N is more than 0.6 and less than 0.7; positive S/N is less than or equal to 0.6.
Example 6 sensitivity test
The results of the experiment using 3 batches of porcine pseudorabies virus gE blocking ELISA antibody detection kits (batches ZM2018001, ZM2018002 and ZM2018003) prepared according to the method of example 4 and 32 porcine pseudorabies virus wild virus infected porcine sera detected according to the method of example 5 are shown in Table 3, the kit of the invention detects 31 positive samples in total and 1 undetected sample, and the results show that the sensitivity of the kit to 30 wild virus infected sera is 96.9%.
TABLE 3 results of sensitivity test
Kit batch number Detection rate Sensitivity of the composition
ZM2018001 31/32 96.9％
ZM2018002 31/32 96.9％
ZM2018003 31/32 96.9％
Example 7 specificity test
Using 3 batches of porcine pseudorabies virus gE blocking ELISA antibody detection kits (batches ZM2018001, ZM2018002, ZM2018003) prepared according to the method of example 4, 50 healthy swine serum, 20 batches of porcine pseudorabies virus gE deletion attenuated vaccine immune serum, 2 batches of porcine foot and mouth disease virus type O (FMD-O) positive serum, 2 batches of porcine foot and mouth disease virus type a (FMD-a) positive serum, 2 batches of porcine circovirus positive serum (PCV2), 2 batches of porcine reproductive and respiratory syndrome positive serum (PRRS) were respectively detected according to the method of example 5.
The specific detection results of the kit are shown in the following table (table 4), and the detection results of 50 healthy pig sera are all negative, and the specificity of 3 kits is 100.0%; the detection results of 20 gE deletion attenuated vaccine immune sera are all negative, and the specificity of 3 kits is 100.0%; the detection results of 2 parts of swine foot and mouth disease virus type O (FMD-O) positive serum, 2 parts of swine foot and mouth disease virus type A (FMD-A) positive serum, 2 parts of porcine circovirus positive serum (PCV2) and 2 parts of swine reproduction and respiratory syndrome positive serum (PRRS) are all shown to be negative, so that the detection specificity of the 8 parts of related pathogen positive serum by 3 batches of the kit is 100%.
TABLE 4 specificity test results of porcine pseudorabies virus gE blocking ELISA antibody test kit
Figure BDA0002012016770000171
Example 8 compliance testing
30 parts of healthy pig serum, 15 parts of virus infection serum and 20 parts of gE deletion attenuated vaccine immune serum are simultaneously detected by adopting the porcine pseudorabies virus gE antibody detection kit of the American imported kit IDEXX and the kit of the invention, and the coincidence rate of the detection results of the 2 kits is compared.
Method of operation of the U.S. import kit (in "short-term incubation mode"):
1) add 100. mu.l of diluted negative control (2-fold dilution) to the appropriate duplicate wells.
2) Add 100. mu.l of diluted positive control (2-fold dilution) to the appropriate duplicate wells.
3) Add 100. mu.l of diluted sample (2-fold dilution) to the corresponding well.
4) The serum or plasma sample may be incubated for 60 minutes (+ -5 minutes) at 18-26 ℃ ("short-term incubation mode").
5) The microwells were washed 3-5 times with approximately 300. mu.l of wash solution per well. After each wash, the liquid in the wells was spun off. Between each wash and before addition of enzyme-labeled antibody, plate wells were prevented from drying out. After the plate is thrown for the last time, the plate is buckled on the water absorbing material, and the residual liquid is thoroughly removed.
6) Add 100. mu.l of enzyme-labeled antibody to each well.
7) And (3) incubating for 20 minutes (+ -1 minute) at the temperature of 18-26 ℃.
8) And 6, repeating the step.
9) Add 100. mu.l TMB substrate solution to each well and incubate at 18-26 ℃ for 15min (+ -1 min).
10) The reaction was stopped by adding 50. mu.l of stop solution to each well, and the absorbance A of the recorded sample and control was measured at 650nm (650).
11) And (3) calculating the result: and respectively calculating the average values of the negative control and the positive control, wherein the difference between the negative control average value and the positive control average value is required to be more than or equal to 0.3. Short-term incubation mode: the sample S/N is more than 0.7, and the sample is judged to be negative; S/N is more than 0.6 and less than or equal to 0.7, the result is judged to be suspicious, and S/N is less than or equal to 0.6, the result is judged to be positive.
The detection results of the porcine pseudorabies virus gE antibody detection kit of the kit and the American imported kit IDEXX on 30 parts of healthy pig serum, 15 parts of virus infection serum and 20 parts of gE deletion attenuated vaccine immune serum are shown in Table 5, the parts of the serum which is positive when the kit and the porcine pseudorabies virus gE antibody detection kit of the American imported kit IDEXX are 13 parts, and the parts of the serum which is negative when the kit and the American imported kit IDEXX are 51 parts. 65 parts of serum to be detected, 64 parts of serum with consistent detection results of the two kits, and the coincidence rate of 98.5 percent.
TABLE 5 results of compliance testing
Figure BDA0002012016770000181
Sequence listing
<110> Zhongmu industries GmbH
<120> porcine pseudorabies virus gE blocking ELISA antibody detection kit
<130>WHOI190021
<160>6
<170>SIPOSequenceListing 1.0
<210>1
<211>123
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Glu Val Gln Leu Gln Glu Trp Gly Ala Gly Ser Ser Lys Pro Ser Leu
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Thr Trp Ser Leu Thr Cys Val Tyr Ser Pro Arg Thr Arg Tyr Tyr Gly
20 25 30
Tyr Gln Trp Tyr Arg Gln Gly Ser Gly Lys Gly Ser Glu Trp Ile Glu
35 40 45
Ser Ile Asn Ser SerAla Lys Leu Tyr Gln Trp Pro Gly Leu Lys Ser
50 55 60
Arg Val Thr Pro Ser Val Asp Thr Ser Asp Tyr Gln Phe Ser Trp Lys
65 70 75 80
Leu Ser Val Thr Ala Gly Asp Thr Ala Gln Tyr Cys Ala Ser Gln Ser
85 90 95
Leu Thr Ile Val Tyr Pro Trp Ser Thr Asp Arg Arg Gln Tyr Gln Leu
100 105 110
Thr Leu Val Thr Pro Ser Arg Ala Ser Lys Gly
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<210>2
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<213> Artificial Sequence (Artificial Sequence)
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Gln Ser Tyr Leu Thr Gln Pro Glu Ser Val Pro Tyr Ala Thr Gly Gln
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Arg Trp Thr Ile Ser Cys Thr Trp Gln Ser Glu Asn Tyr Gly Trp Pro
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Phe Gln Arg Trp Tyr Pro Gln Leu Trp Tyr Gly Thr Ala Pro Lys Ser
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Leu Ile Tyr Val Asn Phe Trp Val Pro Asp Ser Val Pro Trp Arg Phe
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Ser Gly Ser Tyr Ser Gly Thr Ser Ala Gln Leu Ile Pro Ala Gly Leu
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Gln Ala Phe Arg Glu Thr Asp Tyr Trp Cys Gln Ser Gly Thr Ala Lys
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Gly Leu Arg Tyr Val Val Phe Gly Arg Gly Gly Arg Leu Thr Arg Leu
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Tyr Gln Pro Thr Ala
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atgaggcctt tcctgctcag agccgctcaa ttgctggctc tgttggcact ggccttgtct 60
actgaagctc ccagcctgag cgctgagaca actcctggac ccgtgaccga ggtgccctct 120
ccttcagccg aggtctggga cctgtcaacc gaggcagatg atgacgacct gaacggcgat 180
ctggacggcg acgacagaag ggcaggattt ggctctgctc tggcatcact gcgcgaggct 240
cctcctgccc acctggtcaa cgtgtccgag ggagcaaact ttaccctcga cgctagaggt 300
gatggcgctg ttctggctgg aatctggaca tttctcccag tgagaggttg tgatgccgtc 360
tcagtcacta ccgtctgttt cgagacagca tgtcacccag acctcgtgtt gggcagggct 420
tgcgtccctg aggctccaga gatgggaatc ggagattact tgccaccaga ggttcctagg 480
ttgagaaggg agcctcccat cgtgacaccc gagaggtgga gcccacatct gatcgttctg 540
agggctactc caaacgatac cggactgtac actctccacg acgcttcagg tcctagagcc 600
gtgttcttcg tggccgtggg cgacaggcca ccagctccag ccgacccagt tggcccagca 660
aggcacgagc ccaggtttca cgccctcggt ttccactctc agttgttcag cccaggcgac 720
accttcgatc tgatgcccag ggttgttagc gacatgggag actcacgcga gaatttcacc 780
gctactctgg actggtacta cgctagggca ccaccacgct gcttgttgta ctacgtttac 840
gagccctgta tctaccaccc aagggcaccc gaatgcctca ggcctgttga cccagcatgt 900
agcttcacct ctccagctag agctaggctc gtcgcacgca gagcatacgc ttcatgcagc 960
cctctgctgg gtgataggtg gctgaccgcc tgtccctttg acgcattcgg cgaagaggtg 1020
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aaggagctga ccgctcctgc acgcgctcct ggcactcctt ggggacccgg aggaggtgac 1200
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ggaaagggct ctgagtggat tgagtcaatc aattctagcg ctaaactgta tcagtggcca 180
ggactgaagt ccagagtgac tcctagcgtt gacacatctg attaccagtt cagctggaag 240
ctctctgtga ctgctggcga cactgcccag tactgtgcct cacagagcct cactattgtt 300
tacccttggt ctactgacag aaggcagtac caactgaccc tggtgactcc atctagggcc 360
tcaaagggac cctccgtgtt cccactggca ccttccagca aatccacatc tggaggcacc 420
gcagccctcg gctgtctcgt caaggactac ttccctgaac ctgtcaccgt ctcatggaac 480
tcaggcgcac tgacttcagg agtgcacacc ttccctgccg ttctgcaaag ctctggtctg 540
tattctctct cttccgtggt cacagtccca tcatctagct tgggtactca aacttacatt 600
tgcaacgtta atcacaagcc atctaatacc aaagttgaca agaaggtcga acctaagtca 660
tgtgacaaga cccacacctg ccctccttgc ccagcacccg aactgttggg aggtccatct 720
gtgttcctct tcccacccaa gccaaaggac actctgatga tttctcgcac accagaagtc 780
acttgcgtgg tggtggacgt tagccatgaa gatcctgaag ttaagttcaa ctggtatgtc 840
gatggcgtcg aagtccataa tgccaagacc aagcccagag aagagcagta caattcaact 900
taccgcgtgg tgtccgtctt gaccgttctg caccaggatt ggctcaacgg taaggagtac 960
aaatgcaagg tgtctaacaa ggcattgcca gcacctatcg agaagactat ctccaaggct 1020
aaaggccagc caagggagcc tcaggtgtat actctccctc cttctcgcga tgagctgaca 1080
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gagtgggaga gcaacggcca acctgagaat aactataaga ccactccacc cgttctggat 1200
agcgacggct cattcttctt gtactctaaa ctgactgtgg acaagtctag gtggcagcaa 1260
ggcaacgtgt tctcctgctc agtcatgcac gaagccctgc acaatcacta cacacagaag 1320
agcctgtctc tctctcctgg caag 1344
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caatcatatc tcacacaacc agagtcagtt ccatacgcta caggacagcg ctggactatc 60
agctgcactt ggcagtcaga gaactacggc tggccattcc agcgctggta tcctcagctc 120
tggtacggta ctgctcctaa gagcttgatc tacgtcaact tctgggtgcc tgattctgtt 180
ccctggaggt tctccggatc ttactcagga acatctgcac agctgattcc cgctggtttg 240
caggcattca gggagactga ctactggtgt caatctggca cagctaaggg tctgcgctac 300
gtcgtgttcg gcagaggagg caggctgaca agactgtatc agcccaccgc tgctccaagc 360
gtgaccctgt ttcctccctc ctccgaagaa ctccaggcta acaaggcaac cctcgtgtgt 420
ttgatctcag acttctaccc tggcgctgtg accgtggcct ggaaagctga ttcctctccc 480
gtcaaagccg gtgttgaaac caccactcca tcaaagcaaa gcaacaacaa gtacgctgca 540
agcagctacc tctccctgac tcctgaacag tggaagtccc atcgctccta ctcttgccaa 600
gtcacacatg aaggatctac cgtggagaag actgtcgctc caaccgaatg ctca 654
<210>6
<211>435
<212>PRT
<213> Pseudorabies Virus (Pseudorabies virus)
<400>6
Met Arg Pro Phe Leu Leu Arg Ala Ala Gln Leu Leu Ala Leu Leu Ala
1 5 10 15
Leu Ala Leu Ser Thr Glu Ala Pro Ser Leu Ser Ala Glu Thr Thr Pro
20 25 30
Gly Pro Val Thr Glu Val Pro Ser Pro Ser Ala Glu Val Trp Asp Leu
35 40 45
Ser Thr Glu Ala Asp Asp Asp Asp Leu Asn Gly Asp Leu Asp Gly Asp
50 55 60
Asp Arg Arg Ala Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu Ala
65 70 75 80
Pro Pro Ala His Leu Val Asn Val Ser Glu Gly Ala Asn Phe Thr Leu
85 90 95
Asp Ala Arg Gly Asp Gly Ala Val Leu Ala Gly Ile Trp Thr Phe Leu
100 105 110
Pro Val Arg Gly Cys Asp Ala Val Ser Val Thr Thr Val Cys Phe Glu
115 120 125
Thr Ala Cys His Pro Asp Leu Val Leu Gly Arg Ala Cys Val Pro Glu
130 135 140
Ala Pro Glu Met Gly Ile Gly Asp Tyr Leu Pro Pro Glu Val Pro Arg
145 150 155 160
Leu Arg Arg Glu Pro Pro Ile Val Thr Pro Glu Arg Trp Ser Pro His
165 170 175
Leu Ile Val Leu Arg Ala Thr Pro Asn Asp Thr Gly Leu Tyr Thr Leu
180 185 190
His Asp Ala Ser Gly Pro Arg Ala Val Phe Phe Val Ala Val Gly Asp
195 200 205
Arg Pro Pro Ala Pro Ala Asp Pro Val Gly Pro Ala Arg His Glu Pro
210 215 220
Arg Phe His Ala Leu Gly Phe His Ser Gln Leu Phe Ser Pro Gly Asp
225 230 235 240
Thr Phe Asp Leu Met Pro Arg Val Val Ser Asp Met Gly Asp Ser Arg
245 250 255
Glu Asn Phe Thr Ala Thr Leu Asp Trp Tyr Tyr Ala Arg Ala Pro Pro
260 265 270
Arg Cys Leu Leu Tyr Tyr Val Tyr Glu Pro Cys Ile Tyr His Pro Arg
275 280 285
Ala Pro Glu Cys Leu Arg Pro Val Asp Pro Ala Cys Ser Phe Thr Ser
290 295 300
Pro Ala Arg Ala Arg Leu Val Ala Arg Arg Ala Tyr Ala Ser Cys Ser
305 310 315 320
Pro Leu Leu Gly Asp Arg Trp Leu Thr Ala Cys Pro Phe Asp Ala Phe
325 330 335
Gly Glu Glu Val His Thr Asn Ala Thr Ala Asp Glu Ser Gly Leu Tyr
340 345 350
Val Leu Val Met Thr His Lys Gly His Val Ala Thr Trp Asp Tyr Thr
355 360 365
Leu Val Ala Thr Ala Ala Glu Tyr Val Thr Val Ile Lys Glu Leu Thr
370 375 380
Ala Pro Ala Arg Ala Pro Gly Thr Pro Trp Gly Pro Gly Gly Gly Asp
385 390 395 400
Gly Ala Ile Tyr Val Asp Gly Val Thr Thr Pro Ala Pro Pro Ala Arg
405 410 415
Pro Trp Asn Pro Tyr Gly Arg Thr Thr Pro Gly Gly Leu His His His
420 425 430
His His His
435

Claims (6)

1. A detection kit for porcine pseudorabies virus gE blocking ELISA antibody comprises an enzyme-linked reaction plate and an enzyme-labeled antibody, wherein the enzyme-linked reaction plate is coated with porcine pseudorabies virus gE protein, and the enzyme-labeled antibody is a monoclonal antibody which is labeled by horse radish peroxidase and is used for resisting the porcine pseudorabies virus gE protein; the enzyme-linked reaction plate is a detachable 96-hole enzyme label plate; the porcine pseudorabies virus gE protein is obtained by utilizing a baculovirus expression system and an insect cell suspension culture process for expression; the sequence of the porcine pseudorabies virus gE protein is a sequence 6 in a sequence table; the monoclonal antibody of the porcine pseudorabies virus gE protein contains a heavy chain variable region and a light chain variable region; the heavy chain variable region and the light chain variable region are both composed of a determinant complementary region and a framework region;
the determinant complementary regions of the heavy chain variable region and the light chain variable region are each comprised of CDR1, CDR2, and CDR 3;
the amino acid sequence of the CDR1 of the heavy chain variable region is shown as the amino acids from 25 th to 34 th of SEQ ID No. 1;
the amino acid sequence of the CDR2 of the heavy chain variable region is shown as amino acids 52-59 of SEQ ID No. 1;
the amino acid sequence of the CDR3 of the heavy chain variable region is shown as amino acids 94-108 of SEQ ID No. 1;
the amino acid sequence of the CDR1 of the light chain variable region is shown as the amino acids at the 24 th to 35 th positions of SEQ ID No. 2;
the amino acid sequence of the CDR2 of the light chain variable region is shown as amino acids 50-59 of SEQ ID No. 2;
the amino acid sequence of the CDR3 of the light chain variable region is shown as amino acids 94-100 of SEQ ID No. 2.
2. The detection kit for the porcine pseudorabies virus gE blocking ELISA antibody according to claim 1, characterized in that: the amino acid sequence of the heavy chain variable region is shown as 1 st to 123 th sites of SEQ ID No.1 in a sequence table; the amino acid sequence of the light chain variable region is shown as 1 st to 117 th sites of SEQ ID No.2 in the sequence table.
3. The detection kit for the porcine pseudorabies virus gE blocking ELISA antibody according to claim 2, characterized in that: the method for obtaining the enzyme-linked reaction plate comprises the steps of dissolving the porcine pseudorabies virus gE protein in 100 mu l of carbonate solution with the pH value of 9.6, adding the solution into a 96-hole polystyrene enzyme-linked reaction plate, placing 0.1 mu g-1 mu g of the porcine pseudorabies virus gE protein in each hole for 8-12 hours at the temperature of 2-8 ℃ to ensure that the coating antigen is fully combined with the enzyme-linked reaction plate, then adding PBS buffer solution containing 10mg/ml of bovine serum albumin with the pH value of 7.4 according to 300 mu l/hole, carrying out sealing treatment at the temperature of 37 ℃ for 2-3 hours, drying the enzyme-linked reaction plate, and carrying out sealing storage at the temperature of 4 ℃.
4. The detection kit for the porcine pseudorabies virus gE blocking ELISA antibody according to claim 2, characterized in that: the kit also comprises a substrate solution A, a substrate solution B and a stop solution; the substrate solution A is a citrate phosphate buffer solution containing 0.6mg/ml of urea hydrogen peroxide, the substrate solution B is a tetramethylbenzidine solution containing 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when in use; the stop solution is a 2mol/L sulfuric acid solution.
5. The detection kit for the porcine pseudorabies virus gE blocking ELISA antibody according to claim 2, characterized in that: the kit also comprises a sample diluent and a 20-time concentrated washing solution; the sample diluent was phosphate buffer containing 5mg/ml casein with a value of 0.01M, pH of 7.4; the concentrated washing solution is 0.01M phosphate buffer solution with the pH value of 7.4 and contains 0.8-1.2% of Tween-20; the kit also comprises positive control serum and negative control serum, wherein the positive control serum is porcine serum collected after artificial infection of porcine pseudorabies virus; the negative control serum is porcine serum without porcine pseudorabies virus pathogen infection and vaccine inoculation.
6. Use of the porcine pseudorabies virus gE blocking ELISA antibody detection kit of any one of claims 1-5 for the preparation of a reagent for detecting a sample to be detected for porcine pseudorabies virus infection or vaccination; the sample to be tested is a swine serum infected with a wild strain or applied with a non-gE gene deletion virus vaccine.
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CN113009139B (en) * 2021-02-26 2022-07-29 中牧实业股份有限公司 Enzyme linked immunosorbent assay kit for detecting porcine pseudorabies virus antigen and application thereof
CN114740201B (en) * 2022-05-16 2023-03-21 北京亿森宝生物科技有限公司 Chemiluminescent detection kit for antibodies gE and gI of porcine pseudorabies virus and application of chemiluminescent detection kit
CN115819562B (en) * 2022-11-15 2024-12-10 北京纳百生物科技有限公司 Heavy chain and light chain variable regions of anti-pseudorabies virus gB monoclonal antibody and their application
CN115819563B (en) * 2022-12-05 2024-11-19 北京纳百生物科技有限公司 Monoclonal antibody against pseudorabies virus gE protein and its application

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