A monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus and its application.

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus (PEDV) and its applications. Its heavy chain variable region CDR sequences are SEQ ID NO. 1-3, and its light chain variable region CDR sequences are SEQ ID NO. 4-6. This antibody specifically recognizes the PEDV / S1 protein, and its binding can be competitively blocked by PEDV-specific neutralizing antibodies in serum, making it suitable for establishing a blocking ELISA method. Detection tools based on this antibody exhibit high specificity and sensitivity, with blocking rates exceeding 50% against PEDV-positive serum. This invention can be used to prepare detection kits or evaluate vaccine immunization efficacy, providing an efficient and accurate technical means for PEDV infection diagnosis and immune monitoring.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus antibodies and application thereof. BACKGROUND

[0002] Porcine epidemic diarrhea is a digestive tract disease caused by porcine epidemic diarrhea virus (PEDV), which mainly causes vomiting, diarrhea and even death in piglets. PEDV can be transmitted through fecal-oral, breast milk and respiratory tract, and can infect pigs of all breeds and ages. The infection of PEDV is still in a high prevalence situation in China, and is listed as a class II animal disease. Moreover, the genetic evolution of PEDV is complex and variable, which has a serious impact on the development of China's pig industry.

[0003] PEDV belongs to the Coronaviridae family and is a single-stranded, enveloped RNA virus. Its genome encodes 16 non-structural proteins and 4 structural proteins: surface spike protein (S), membrane protein (M), envelope protein (E), and internal nucleocapsid protein (N). The S protein is composed of S1 and S2 subunits, with S1 responsible for binding to host receptors. The protein contains the main neutralizing epitopes of the virus and can stimulate the production of neutralizing antibodies, often used as a target antigen for epidemiological studies and serological diagnosis of PED.

[0004] Currently, there is no specific drug for treating PEDV infection, and vaccination is still an effective way to control PEDV. However, due to the large genetic variation of the virus, the effectiveness of the current vaccine is greatly challenged. Therefore, establishing and applying a fast and effective detection method for routine PEDV screening is the key to controlling porcine epidemic diarrhea. Molecular diagnostic methods such as qRT-PCR detect viral nucleic acids to determine whether pigs carry the virus. The operation requires special equipment, and the reagents are expensive, with the risk of nucleic acid contamination. Immunological methods such as ELISA detect antibodies such as IgG and IgA in serum, milk or saliva, which can be used for infection diagnosis or evaluation of post-immune antibody levels. In addition, there are neutralization tests for evaluating the protective effect of antibodies. However, these methods still have problems such as poor detection effect or complicated operation. Therefore, more accurate detection methods are still needed to achieve accurate diagnosis and effective prevention and control of PEDV. Studies have shown that specific antibodies can be detected in serum, milk, saliva and other types of samples after PEDV infection. The IgG level curve of S protein-specific antibodies can be detected as early as 7 days after infection, peaks at 14 days, and can last for up to 6 months. Therefore, the detection of S protein antibodies can effectively improve the accuracy of PEDV infection diagnosis and antibody level evaluation.

[0005] Blocking ELISA is a commonly used immunological detection technique, widely used in the fields of infectious disease diagnosis, vaccine immune effect evaluation, etc. There are commercial kits for antibody detection of African swine fever (ASF), classical swine fever (CSF) and the like applied in clinics. The monoclonal antibody required in the blocking method is usually a specific antibody against a key protein of the virus, thus greatly reducing cross-reactions, and the anti-matrix interference ability is stronger than that of the indirect method and other antibody detection methods, and the detection result is more accurate. In the blocking ELISA, the high biological activity of the coating antigen and the specific blocking monoclonal antibody are the keys to ensure the accuracy of the detection method, and the correct folding and modification of the antigen are extremely important. PEDV / S protein is a transmembrane glycoprotein, and the prokaryotic expression system cannot perform post-translational modification on it, and cannot accurately display the conformation of the antigen, so the antigenicity is poor, while the eukaryotic expression system can produce proteins with a conformation close to the natural conformation, so that the detection is more accurate, and it is more suitable for the evaluation of vaccine immune effect and the serological diagnosis of PEDV infection. SUMMARY

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides a monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus antibody and its application, which solves the deficiencies of the prior detection method in specificity, accuracy or operation convenience, and realizes the efficient and accurate evaluation of PEDV antibody level in a pig population.

[0007] In order to achieve the above-mentioned purpose, the main technical solutions adopted by the present application include:

[0008] In a first aspect, the present application provides a monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus antibody, wherein the heavy chain variable region of the monoclonal antibody 1D3 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 1-SEQ ID NO. 3.

[0009] The light chain variable region of the monoclonal antibody 1D3 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 4-SEQ ID NO. 6.

[0010] The binding of the monoclonal antibody 1D3 to PEDV / S1 protein can be competed by PEDV specific antibodies (such as antibodies produced after infection or immunization) in animal serum samples that can bind to S1 protein.

[0011] A monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus antibody, further, a monoclonal antibody 1D3 that can be used for blocking ELISA to detect porcine epidemic diarrhea virus antibody, or a monoclonal antibody 1D3 targeting porcine epidemic diarrhea virus.

[0012] In a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1D3 is shown as SEQ ID NO. 7.

[0013] In a further embodiment, the amino acid sequence of the light chain variable region of monoclonal antibody 1D3 is shown as SEQ ID NO. 8.

[0014] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1D3 is shown as SEQ ID NO. 9.

[0015] In a further embodiment, the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1D3 is shown as SEQ ID NO. 10.

[0016] In a second aspect, the present application provides a use of the above-mentioned monoclonal antibody 1D3 in the preparation of a medicament for preventing or treating porcine epidemic diarrhea.

[0017] In a third aspect, the present application provides a use of the above-mentioned monoclonal antibody 1D3 in the preparation of a tool for detecting PEDV antibody.

[0018] In a further embodiment, the tool comprises a reagent, a kit or a test strip.

[0019] In a further embodiment, the kit comprises a blocking ELISA kit, which detects PEDV antibody by a blocking ELISA method.

[0020] In a fourth aspect, the present application provides a use of the above-mentioned monoclonal antibody 1D3 in the evaluation of the immune effect of PEDV vaccine.

[0021] Beneficial effects:

[0022] The monoclonal antibody 1D3 provided by the present application for detecting PEDV antibody has high specificity and binding activity, and can specifically recognize the key epitope of PEDV / S1 protein. Based on its unique CDRs (SEQ ID NO. 1-6), the antibody can effectively bind to PEDV / S1 protein, and the binding can be competitively blocked by PEDV-specific antibodies in animal serum, so it is particularly suitable for establishing a blocking ELISA detection method. Experimental results show that the blocking ELISA kit established based on monoclonal antibody 1D3 has a high blocking rate (> 50%) for PEDV antibody positive serum, and no cross reaction (blocking rate < 10%) for negative serum and other positive serum of swine fever, blue ear disease, porcine transmissible gastroenteritis and other pig diseases, and has excellent specificity, sensitivity and repeatability, which can accurately detect the PEDV antibody level in serum samples, and provides an efficient and reliable tool for vaccine immune effect evaluation and serological diagnosis of PEDV infection. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Result image for blue-white spot screening;

[0024] Figure 2 Result image for morphological observation of Sf9 cells after transfection;

[0025] Figure 3 Result image for denaturing PAGE electrophoresis of PEDV / S1 recombinant protein (12%);

[0026] Figure 4 Result image for non-denaturing PAGE electrophoresis of PEDV / S1 recombinant protein (8%);

[0027] Figure 5 Result image for ELISA identification of PEDV / S1 recombinant protein;

[0028] Figure 6 Result image for identification of binding activity of different monoclonal antibodies with PEDV / S1 and PEDV / S recombinant proteins;

[0029] Figure 7 Result image for identification of binding activity of monoclonal antibody 1D3.

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor. DETAILED DESCRIPTION

[0031] The embodiments of the present application will be further described in detail below with reference to the drawings and examples. The detailed description and drawings of the following examples are used to exemplarily illustrate the principles of the present application, but cannot be used to limit the scope of the present application, and the present application can be implemented in many different forms, and is not limited to the specific embodiments disclosed in the text, but includes all technical solutions falling within the scope of the claims.

[0032] The present application provides these embodiments in order to make the present application thorough and complete, and fully express the scope of the present application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of components and steps, the composition of materials, numerical expressions and values set forth in these embodiments should be interpreted as merely exemplary, and not as a limitation.

[0033] The application selects the gene sequence of the S1 subunit of PEDV / S protein, uses an insect-baculovirus expression system to obtain an expression vector capable of efficiently secreting and expressing S1 protein, a recombinant baculovirus, and purifies the trimeric S1 protein with high purity and good activity. At the same time, specific monoclonal antibodies are screened by using hybridoma technology, and a blocking ELISA method for detecting PEDV / S1 protein antibodies is established based on the monoclonal antibodies, which can specifically detect PEDV positive serum, and has no cross with classical swine fever virus antibody positive serum, blue ear virus antibody positive serum and porcine transmissible gastroenteritis virus antibody positive serum, and has important reference value for the development of diagnostic reagents for PEDV antibody detection.

[0034] The application is used for detecting porcine epidemic diarrhea virus antibody, specifically for blocking ELISA detection of porcine epidemic diarrhea virus antibody.

[0035] The application is used for in vitro detection of the level of PEDV specific antibody in animal serum by blocking ELISA method, wherein the antibody level is evaluated by calculating the blocking rate of monoclonal antibody 1D3 and PEDV / S1 protein binding, and the method does not involve the diagnosis or treatment process of the disease.

[0036] The application of monoclonal antibody 1D3 in evaluating the immune effect of porcine epidemic diarrhea virus vaccine, the evaluation includes detecting the antibody level or blocking rate in animal serum.

[0037] The PEDV / S1 protein in the application includes S1 protein naturally existing in porcine epidemic diarrhea virus and S1 recombinant protein expressed by gene recombination technology.

[0038] The application provides a blocking ELISA kit for detecting porcine epidemic diarrhea virus antibody, the kit comprising: a solid phase carrier, the surface of which is coated with PEDV / S1 recombinant protein as an antigen; enzyme-labeled monoclonal antibody 1D3 as a detection antibody for binding with PEDV / S1 recombinant protein; positive control and negative control for providing PEDV antibody positive and negative detection standards respectively; washing solution for washing unbound components; color developing solution and termination solution for generating and terminating color developing reaction; wherein the binding of monoclonal antibody 1D3 and PEDV / S1 recombinant protein can be blocked by serum containing PEDV antibody, so as to realize the detection of PEDV antibody in the sample.

[0039] Example 1:

[0040] 1. Source of PEDV / S recombinant protein

[0041] The S1 protein of PEDV-CH-SBC / 2013 strain, i.e. G2-a type, is selected, which is a recombinant protein containing S1 region expressed by using an insect-baculovirus expression system, contains a His tag, has a trimer structure, is identified to be capable of reacting with a positive monoclonal antibody, and has good biological activity.

[0042] 1.1, Baculovirus expression vector construction of PEDV / S1 protein:

[0043] Based on the baculovirus expression vector pFastBac-l, the GP67 secretory signal peptide sequence is added at the N-terminal of the pFastBac-l vector to promote the secretory expression of the target protein, so that the protein can be secreted into the cell culture supernatant for easy purification. In addition, since the natural S protein of PEDV virus is a trimer structure, in order to make S1 also form a trimer conformation close to nature, a flexible linker (GSS) and a Foldon sequence derived from the C-terminal domain of T4 bacteriophage fibritin are added at the C-terminal of the vector. The truncated mutant (containing 27 aa, and the conventional one contains 33 aa) is used to enhance expression and improve stability. At the same time, a 6×His tag is also added to facilitate efficient purification of the target protein in the later stage. The sequence is as follows:

[0044] Nucleotide sequence of GP67 secretory signal peptide (shown in SEQ ID NO. 11): ATGCTACTAGTAAATCAGTCACACCAAGGCTTCAATAAGGAACACACAAGCAAGATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGCGGATCCCGGG.

[0045] Amino acid sequence (shown in SEQ ID NO. 12): MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPG.

[0046] The S1 protein gene sequence of PEDV gene reference PEDV-CH-SBC / 2013 strain (Accession: KC787542) is (shown in SEQ ID NO. 13):

[0047]

[0048] The amino acid sequence is (as shown in SEQ ID NO. 14):

[0049] LPQDVTRCSANTNFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFVSHIRGGHGFEIGISQEPFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIPAHMSEHSVVGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCTANCIGYAANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLTIPKIYGLGQFFSFNQTIDGVCNGAAVQRAPEALRFNINDTSVILAEGSIVLHTALGTNFSFVCSNSSDPHLATFAIPLGAIQVPYYCFLKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTAIQRILYCDDPVSQLKCSQVAFDLDDGFYPISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSNSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGF.

[0050] Foldon sequence:

[0051] The nucleotide sequence (as shown in SEQ ID NO. 15) is GGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTG.

[0052] Amino acid sequence (shown in SEQ ID NO. 16): GYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0053] Linker:

[0054] Nucleotide sequence (shown in SEQ ID NO. 17): GGCAGCAGC.

[0055] Amino acid sequence (shown in SEQ ID NO. 18): GSS.

[0056] The sequence of SEQ ID NO. 17 contains less than 10 specifically defined nucleotides, and the non-intentionally skipped sequence must contain at least 10 specifically defined nucleotides, therefore, the sequence table is marked as intentionally skipped sequence, and the sequence table is recorded as 000; the sequence of SEQ ID NO. 18 contains less than 4 specifically defined amino acids, and the non-intentionally skipped sequence must contain at least 4 specifically defined amino acids, therefore, the sequence table is marked as intentionally skipped sequence, and the sequence table is recorded as 000.

[0057] His tag:

[0058] Nucleotide sequence (shown in SEQ ID NO. 19): CATCATCATCATCATCATTAA.

[0059] Amino acid sequence (shown in SEQ ID NO. 20): HHHHHH*, the last TAA in the nucleotide is a stop codon, which does not encode amino acids, and the stop is represented by *, and the sequence table does not show the "*" number.

[0060] Complete sequence (shown in SEQ ID NO. 21):

[0061]

[0062] The constructed recombinant plasmid pFastBac-PEDV / S1 was sequenced to determine the correct insertion of the fragment, and then the downstream test was performed.

[0063] 1.2, obtain recombinant bacmid

[0064] The recombinant plasmid pFastBac-PEDV / S1 was transposed into DH10Bac competent cells, and blue-white spot screening was performed to obtain recombinant bacmid Bacmid-PEDV / S1. Specifically, 1 ng of recombinant plasmid pFastBac-PEDV / S1 was added to 50 μl of DH10Bac competent cells, mixed gently, and placed on ice for 30 min. Then it was quickly transferred to a 42°C water bath for 60 s, immediately placed on ice for 2 min, and then 900 μl of LB medium without antibiotics was added. The mixture was incubated at 37°C on a shaker at 200 rpm for 4 h. The transformed competent cells were diluted 10-fold with antibiotic-free LB medium and evenly spread on solid LB plates containing three antibiotics (kanamycin 50 ug / ml, gentamicin 7 ug / ml, tetracycline 10 ug / ml, X-Gal 100 ug / ml, IPTG 40 ug / ml). The plates were incubated in a 37°C incubator under inverted light for two days, and then the colonies were observed. Independent and individual white colonies were picked for identification. See Figure 1 , after the pFastBac-PEDV / S1 was transformed into DH10Bac cells, the cells were spread on solid LB plates containing three antibiotics and incubated to produce multiple white colonies. The white colonies indicate that the transposition has successfully occurred, i.e., the target gene has been integrated from the pFastBac plasmid into the Bacmid. These white colonies contain the recombinant baculovirus genome, which will be picked for identification in the following steps.

[0065] 1.3, identification of recombinant bacmid Bacmid-PEDV / S1

[0066] The white independent colonies were inoculated into liquid LB medium containing three antibiotics and incubated at 37°C, 200 rpm overnight. The bacterial suspension was used as a template for amplification and sequencing identification using universal primer M13F and S1 gene-specific primer.

[0067] The primer sequence is: M13-F (as shown in SEQ ID NO. 22): GTTTTCCCAGTCACGAC; S1 gene specific reverse primer (as shown in SEQ ID NO. 23): GCGCGGCACCAGCTGGAAACCAGGCAACTCCCTAGTA; The PCR amplification reaction program is: 98℃ 5min; 98℃ 20s, 55℃ 30s, 72℃ 70s, 30 cycles; 72℃ 5min. The PCR amplification product is sent for sequencing, and after identification, the bacteria are extracted from the bacmid.

[0068] 1.4, Extraction of recombinant bacmid

[0069] Take 40ul of bacteria detection positive bacteria liquid and add it to 4ml LB medium (containing 7ug / ml gentamicin, 100ug / ml kanamycin, 10ug / ml tetracycline) and culture at 37℃ 200rpm overnight. Take 4mL of overnight bacteria, centrifuge at 12000rpm for 2min to collect the bacterial body, and discard the supernatant. Add 1ml solution I (25 mM Tris-Cl, 10 mM EDTA, 50 mM glucose, RNaseA 100ug / ml, pH 8.0) to the centrifuge tube, blow the bacterial pellet evenly and transfer the suspension to a new 4mL centrifuge tube; add 1ml solution II (0.2M NaOH, 10g / L SDS) to the centrifuge tube, gently roll for 6-10 times, and the bacterial body is fully lysed, and the liquid becomes clear and thick. Add 1ml solution III (5M potassium acetate 60mL, 11.5mL glacial acetic acid, 28.5mL deionized water) to the centrifuge tube, gently roll the EP tube for 6-10 times, at which time white flocculent precipitate can be observed in the tube, centrifuge at 12000rpm for 10min at room temperature. After centrifugation, take the clear supernatant to a 4mL centrifuge tube, slowly add an equal volume of pre-cooled isopropanol, mix well after inverting, and place on ice for 10min, centrifuge at 12000rpm for 15min, discard the supernatant. Add 2mL of pre-cooled 70% ethanol to wash the precipitate, centrifuge at 12000rpm for 10min and discard the supernatant. Repeat the washing. Dry the precipitate in a biological safety cabinet, dissolve with 80ul of enzyme-free water, and store at -20℃.

[0070] 1.5, Obtain recombinant baculovirus (Baculovirus)

[0071] The correctly identified recombinant bacmid Bacmid-PEDV / S1 is transfected into Sf9 cells (Spodoptera frugiperda ovary cells) to obtain recombinant baculovirus. The Sf9 cells in good condition are cultured in advance at 0.5*10 6The total amount of holes is inoculated in 6-hole cell culture plates. 1 ug of sterile recombinant bacmid Bacmid-PEDV / S1 is added to 100 ul of insect cell serum-free medium (Yiqiao God, MSF1) and mixed gently, and 6-8 ul of transfection reagent Cellfectin (Thermo, 10362100) is added to 100 ul of insect cell serum-free medium and mixed, and the recombinant bacmid and transfection reagent are mixed well, and incubated at room temperature for 30 min. The cells in the hole plate are washed with serum-free medium, and 800 ul of serum-free medium is added to the recombinant bacmid-transfection reagent mixture, mixed well, and added to the 6-hole plate, and a control with only transfection reagent and a normal cell control are set up. Incubate at 27°C for 5h, discard the culture medium, add 2ml of serum-free medium to each hole, and incubate in a 27°C incubator in the dark. Observe the changes in the cells. When the transfection holes appear to be large, round and fused, etc. (about 5-7 days), while the transfection reagent control and cell control holes are normal, indicating successful infection, the cell culture supernatant is harvested, which is the P1 generation of recombinant virus liquid. See Figure 2 , the left graph is the morphology of the diseased sf9 cells, and the right graph is the morphology of the control normal sf9 cells. In the left graph, the cells appear to be significantly larger and round, and some cells begin to fuse to form larger cell clumps, which is a typical characteristic of cell disease. In the right graph, the cell morphology is uniform, spindle-shaped or oval, and grows tightly attached to the wall, with no signs of disease. The comparison of the two graphs clearly shows the effect of recombinant bacmid transfection on Sf9 cells, confirming the successful acquisition and expression of recombinant baculovirus.

[0072] The cell culture supernatant of the 6-well plate was centrifuged, and the culture supernatant of the transfection well and the cell control well was concentrated with a 30kD ultrafiltration concentration tube, then the concentrated supernatant was diluted 100 times, and coated a 96-well enzyme-labeled plate (coating buffer: sodium carbonate 1.59g, sodium bicarbonate 2.93g, constant volume to 1L of pure water, pH 9.6) at an amount of 50ul / well at 4°C overnight, and the coating solution was discarded the next day, and 3% sucrose+2% BSA was blocked at 37°C for 2h, then the plate was washed once with PBST (0.05% Tween-20 in PBS), and then dried. After blocking, 10ug / ml, 1ug / ml, 100ng / ml gradient diluted PEDV / S protein monoclonal antibody (Guangzhou Qianxun Biological) was added at 50ul / well, and PBS was added as a control. Incubate at 37°C for 30 minutes, wash the plate thoroughly, then add 50ul / well of HRP-labeled goat anti-mouse IgG (5000 times, diluted with PBS), incubate at 37°C for 30 minutes, wash the plate thoroughly, then add TMB developing solution (Beijing Meikewande), 50ul / well, develop at room temperature for 10 minutes, stop the color development reaction with a stop solution, and read the OD450nm detection value with an enzyme-labeled instrument.

[0073] As shown in Table 1, after the transfection well concentrated supernatant was diluted 100 times and coated on the enzyme-labeled plate, there was a positive reaction with the commercialized monoclonal antibody, and the antibody did not react with the control well concentrated supernatant coated on the enzyme-labeled plate, indicating that the target protein was secreted and expressed in the transfection cell culture supernatant, and the harvested supernatant could be used as P1 generation venom for the next step of infection and passage.

[0074] Table 1: Identification of protein expressed in transfection well culture supernatant

[0075]

[0076] P1 generation venom 200ul was added to the surface of T25 culture flask Sf9 cells (70% density), and incubated at 27°C until 90% of the cells showed pathological changes, and the supernatant was collected as P2 generation recombinant baculovirus. Continue to pass to obtain high titer P3 generation recombinant baculovirus, at this time the venom virus titer is high and relatively stable. Take P3 generation baculovirus venom and add it to the suspension culture of Sf9 cells at a ratio of 1:500, the cell density is about 1*10 5 / ml, and incubate in a 27°C 120rpm shaker, until more than 90% of the cells show pathological changes, then centrifuge to collect the supernatant for protein purification.

[0077] 2. Purification of recombinant protein

[0078] The recombinant protein expressed with a histidine tag was purified using a TED nickel ion affinity chromatography column (Solarbio). The A buffer was 50 mM PB, 300 mM NaCl, pH 8.0, and the B buffer was 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The chromatography column was equilibrated with the A buffer, and then the collected culture supernatant was centrifuged at 12000 rpm for 10 min. The supernatant was filtered with a 0.45 um filter, diluted 1:1 with the A buffer, and slowly loaded onto the chromatography column. After loading, the chromatography column was washed with the A buffer, and finally eluted with the B buffer gradient. The elution peak of the target protein was collected and dialyzed overnight at 4°C with the A buffer. The purified protein was subjected to 12% SDS-polyacrylamide gel electrophoresis and 8% non-denaturing polyacrylamide gel electrophoresis, and the size and purity of the target protein were observed after Coomassie blue staining. The electrophoresis results of the purified protein are shown in Figure 3 、 Figure 4 The protein concentration was determined using a ultramicro spectrophotometer and stored at -20°C. Figure 3 M: protein Marker, R is the PEDV / S1 protein in 12% denaturing gel electrophoresis results, Figure 4 M: protein Marker, NR is the PEDV / S1 recombinant protein in 8% non-denaturing gel electrophoresis results.

[0079] As shown in Figure 3 , under denaturing conditions, the PEDV / S1 protein can be seen near 95kDa with obvious main band and high purity, slightly larger than the expected size of 88kDa. Because the PEDV / S1 protein has glycosylation modification, the band may be slightly larger than expected during electrophoresis. Under non-denaturing conditions, the protein is not damaged by reducing agents such as SDS and mercaptoethanol, and can maintain its natural conformation, as shown in Figure 4 , near 270kDa there is an obvious main band, consistent with the expected size of the trimeric protein, indicating that the expressed protein has a trimeric conformation.

[0080] 3. Indirect ELISA to identify the activity of PEDV / S1 recombinant protein

[0081] The purified PEDV / S1 recombinant protein was coated on the enzyme-labeled plate, and the indirect ELISA method was used to identify the reaction with the positive monoclonal antibody. The positive monoclonal antibody was a commercially available PEDV S monoclonal antibody (Guangzhou Qianxun Biological). The recombinant protein was coated in the microplate (coating buffer: carbonate buffer, sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, constant volume to 1 L of pure water, pH 9.6), the coating concentration was 1 μg / mL, 50 μL / well, 4°C overnight. The next day, the coating solution was discarded, and 3% sucrose + 2% BSA was used for blocking, 150 μL per well, 37°C for 2 hours, then washed with PBST (0.05% Tween-20 in PBS) once, and dried. The monoclonal antibody was diluted with PBS at a gradient of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL, and 50 μL was added to the coated antigen microplate, and PBS was used as a negative control, 37°C for 30 min. The liquid in the well was shaken out, the plate was washed with PBST 4 times, and dried. Then 50 μL of HRP-labeled goat anti-mouse IgG (5000 times, diluted with PBS) was added to each well, 37°C for 30 min, and the plate was washed 4 times, and dried. Then 50 μL of TMB color developing liquid (Beijing Meikewande) was added to each well, and the color was developed at room temperature for 10 min. Finally, 50 μL of TMB stop solution was added to terminate the reaction, and the OD450nm value was measured by an enzyme-labeled instrument. The results are shown in Table 1. Figure 5 . Figure 5 The results show that the purified PEDV / S1 recombinant protein can specifically react with the positive monoclonal antibody, indicating that the recombinant protein has good biological activity and can be used as a coating or immunizing antigen for monoclonal antibody screening.

[0082] 4. Screening of PEDV / S protein monoclonal antibody

[0083] Mouse immunization.

[0084] The purified PEDV / S1 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected into 6-week-old female BALB / c mice in multiple points, with a dose of 30 μg per mouse. At weeks 2 and 4, the mice were given booster immunization by subcutaneously injecting an equal volume of Freund's incomplete adjuvant at the same dose. At week 6, the mice were immunized by directly injecting the spleen with an insulin needle at a dose of 5 ug per mouse. Seven days after the last immunization, the mouse serum was collected for antibody titer detection. The mouse with higher titer was selected for intraperitoneal boost with 20 μg of recombinant PEDV / S1 protein, and the mouse spleen was collected 3 days later for preparation of hybridoma cells.

[0085] 5. Screening of hybridoma cells

[0086] After all the spleen cells of the immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase, the fusion cells were placed in HAT medium for screening culture. When the fusion cells grew to 1 / 2 of the bottom of the well, positive clones that reacted with both PEDV / S1 and PEDV / S recombinant proteins (Jiangshan, DRA252, PEDV-CV777 strain) were obtained by indirect ELISA. Since the immunogen contains a His tag, the background components need to be screened to select specific cell strains targeting the PEDV / S1 recombinant protein. The positive cells were cloned into a monoclonal state by limiting dilution, and the cell strains were expanded and frozen.

[0087] 6. Screening of positive clones by indirect ELISA

[0088] PEDV / S1 recombinant protein, PEDV / S recombinant protein, and CMV / gB recombinant protein (Jiangshan, DRA263, containing a His tag) were coated in a microplate, respectively (the coating buffer was carbonate buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, constant volume to 1 L of pure water, pH 9.6), and the coating concentration was 1 μg / mL, 4°C overnight; the next day, the coating solution was discarded, and 3% sucrose + 2% BSA was used for blocking, 150 μL per well, 37°C for 2 hours, then washed once with PBST (0.05% Tween-20 in PBS, pH 7.4), and dried. Add 50 μL of cell culture supernatant, 37°C for 30 min. Shake off the liquid in the well, wash the plate 4 times with PBST, dry, then add 50 μL of HRP-labeled goat anti-mouse IgG (Solebo, diluted 5000 times with PBS) per well, 37°C for 30 min, wash the plate 4 more times, dry, then add 50 μL of TMB color developing liquid per well, color develop at room temperature for 10 min, finally add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA), stop the reaction, and measure the OD 450 nm value. The results are shown in Table 2. Positive cell strains that react with PEDV / S1 and PEDV / S recombinant proteins but not with control recombinant proteins were selected for subsequent experiments.

[0089] Table 2: Screening results of monoclonal antibodies

[0090]

[0091] The recombinantly expressed PEDV / S1 protein is of PEDV G2 genotype, while the PEDV / S recombinant protein of Jiangshan organism is of GI genotype, and there is a certain degree of amino acid similarity between the two. The use of these two recombinant proteins as coating antigens has screened 20 cross-reactive monoclonal antibodies that react with both, laying a foundation for the subsequent screening of blocking monoclonal antibodies.

[0092] After the selected hybridoma cell line was expanded, 0.2 ml (containing 2.5 x 10 6 cells) was injected into the abdominal cavity of female BALB / c mice, and about 10 days later, when the abdominal cavity of the mice was significantly enlarged, the ascites was collected using a sterile syringe needle.

[0093] 7. Purification of monoclonal antibodies

[0094] The ascites was centrifuged at 12000 r / min for 10 minutes, 1 ml of the supernatant was diluted 10 times with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), filtered through a 0.22 um filter, and the filtered sample was pumped into a Protein L purification column equilibrated with binding buffer at low speed using a peristaltic pump. The column was connected to a protein purification instrument, and 5-10 column volumes of binding buffer were used for washing until the ultraviolet absorption peak was washed flat, and then eluted with elution buffer (0.1 M glycine, pH 2.7). The elution peak was collected, and the collected sample was neutralized with 1 M Tris-HCl, pH 9, and placed in a dialysis bag (MW: 8000-14000) and dialyzed in a 20 mM PBS pH 7.4 solution at 2-8°C for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 r / min for 5 minutes, and the supernatant was the purified monoclonal antibody.

[0095] The purified monoclonal antibody was diluted to 1 ug / ml with PBS, and further detected for its binding activity to PEDV / S1, PEDV / S recombinant proteins and irrelevant antigen CMV / gB by indirect ELISA. The results are shown in Figure 6 As can be seen from Figure 6 , the purified monoclonal antibody has obvious specific binding to PEDV / S1 recombinant protein and PEDV / S recombinant protein (OD 450 nm value is high), and the binding rate to irrelevant control antigen CMV / gB is very low (OD value is close to background).

[0096] 8. Screening of blocking monoclonal antibodies

[0097] In order to improve the efficiency of screening of blocking monoclonal antibodies, 15 strains of monoclonal antibodies with high binding titer were selected for downstream experiments.

[0098] HRP-labeled monoclonal antibody

[0099] Dilute the selected antibody to be labeled to a final concentration of 2 mg / mL with carbonate coupling buffer (sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, make up to 1 L of pure water, pH 9.6), dissolve 2 mg of HRP in 0.5 mL of ultrapure water, and mix well with 0.5 mL of 0.06 M sodium periodate solution, then add the diluted 1 mg (0.5 mL) antibody solution to the HRP-containing matched tube, mix well by blowing, and incubate at room temperature for 1 h, mixing well at regular intervals during incubation. Add 50 μL of 5 mg / mL sodium borohydride and mix well for 15 min to terminate the labeling reaction, and finally dialyze the labeled antibody in 0.01 M PBS, pH 7.4 buffer overnight, add glycerol at a volume ratio of 1:1, and store at -20°C.

[0100] To test the binding activity of the HRP-labeled mAb, direct ELISA was used for detection, and the enzyme-labeled mAb with strong reaction with PEDV / S1 protein was selected for subsequent blocking mAb testing. PEDV / S1 recombinant protein was coated in a microplate at 1 ug / mL, 50 ul / well, the HRP-labeled antibody was diluted 1000-fold with PBS and directly added to the coated plate, incubated at 37°C for 30 min, the liquid was removed, the plate was washed 4 times, and then 50 μL of TMB color developing solution was added per well for color development at room temperature for 10 min, finally 50 μL of TMB stop solution was added to terminate the reaction, and the OD 450 nm values, and the results are shown in Table 3. Ten strains of HRP-labeled antibodies with a reading value exceeding 1.0 were selected for subsequent blocking tests.

[0101] Table 3: Results of HRP-labeled antibody direct reaction with antigen

[0102]

[0103] 9. Establishment of blocking ELISA method

[0104] Coat the PEDV / S1 recombinant protein in a microplate, the coating buffer is carbonate buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, dilute to 1 L of pure water, pH 9.6, the coating concentration is 1 μg / mL, 50 ul / well, 4°C overnight; the next day, discard the coating solution, block with 3% sucrose + 2% BSA in PBS (pH 7.4) solution, 150 μL per well, 37°C for 2 hours, then wash the plate with PBST solution (0.05% Tween-20 in PBS, pH 7.4) once, and dry. Dilute the PEDV infection positive pig serum and SPF negative pig serum 5 times with PBS, add to the coated plate, 50 μL / well, 37°C for 30 min, shake out the liquid in the well, wash the plate with PBST solution 4 times, dry, then add HRP-labeled monoclonal antibody (diluted 1000 times with PBS) 50 μL / well, 37°C for 30 min, wash the plate again 4 times, dry, then add TMB developing solution (Beijing Meikewande) 50 μL / well, develop at room temperature for 10 min, finally add 50 μL TMB stop solution (Beijing Meikewande), stop the reaction, and measure the OD450nm value with a microplate reader. The PEDV infection positive pig serum is derived from a PEDV infection recovered pig confirmed by RT-qPCR, with a neutralizing antibody titer of ≥ 1:128; the SPF negative pig serum is derived from a healthy pig in a SPF pig farm, confirmed to be PEDV negative by RT-qPCR and neutralization test.

[0105] Select the HRP-PEDV / S1 monoclonal antibody with the highest percentage of inhibition (PI) for condition optimization. Calculate the percentage of inhibition: PI = (SPF serum OD value - positive serum OD value) / SPF serum OD value x 100%. The results are shown in Table 4.

[0106] Table 4: Results of blocking antibody screening

[0107]

[0108] According to the results in Table 4, the blocking effect of HRP-1D3 is the best, and 1D3 is used for blocking ELISA method optimization.

[0109] 10. Optimization of blocking ELISA method

[0110] Coated with coating buffer (Na2CO3 1.59 g, NaHCO3 2.93 g, constant volume to 1 L of pure water, pH 9.6) at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL (PEDV / S1 recombinant protein), 50 μL / well, coated at 4°C overnight, and the coating solution was discarded the next day. Then, 150 μL of 3% sucrose + 2% BSA per well was added for blocking at 37°C for 2 hours, and the blocking solution was discarded. Subsequently, the plate was washed once with PBST (0.05% Tween-20 in PBS, pH 7.4), and then dried. The PEDV positive pig serum and SPF pig serum were diluted 5 times with PBS and added to the enzyme-labeled plate, 50 μL / well, and incubated at 37°C for 30 min. The plate was washed 4 times with PBST, 50 μL of HRP-1D3 diluted 1000, 2000, and 3000 times with PBS was added, 50 μL / well, and incubated at 37°C for 35 min. The plate was washed 4 times again, dried, 50 μL of TMB color developing solution was added per well, and color development was performed at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) was added to terminate the reaction, and the OD was measured with an enzyme-labeled instrument. 450 nm values. The condition with the largest PI value was selected for the next step of testing. The screening process is shown in Table 5.

[0111] Table 5: Test results of the optimal reaction conditions of the blocking ELISA

[0112]

[0113] According to the data in Table 5, the blocking rate was the highest when the PEDV / S1 recombinant protein coating concentration was 1 ug / ml and the HRP-1D3 was diluted 2000 times.

[0114] 11. Detection of pig serum by blocking ELISA

[0115] According to the selected blocking ELISA reaction conditions, 6 PEDV antibody positive pig sera (confirmed PEDV antibody positive by neutralization test), 3 PEDV antibody negative pig sera (confirmed PEDV antibody negative by neutralization test), 1 porcine pestivirus antibody positive serum (confirmed PEDV antibody negative by neutralization test and positive for porcine pestivirus antibody by commercial kit), 1 blue ear antibody positive serum (confirmed PEDV antibody negative by neutralization test and positive for blue ear antibody by commercial kit), 1 porcine transmissible gastroenteritis virus antibody positive serum (confirmed PEDV antibody negative by neutralization test and positive for porcine transmissible gastroenteritis virus antibody), 1 SPF pig serum, and PBS control were further detected. The sera were diluted 5 times, and the detection results are shown in Table 6.

[0116] Table 6: Detection of pig serum by blocking ELISA

[0117]

[0118] PEDV antibody positive pig serum 6, PEDV antibody negative pig serum 3 are all from different pig serum samples identified by China Agricultural University laboratory, and the difference of blocking effect reflects the real biological variation of different individuals, different infection history or immune state. According to the data in table 6, the blocking rate of the 6 PEDV antibody positive pig serum is higher than 50%, and there is obvious blocking reaction, while the blocking rate of PEDV antibody negative pig serum, piglet antibody positive serum, blue ear virus antibody positive serum and porcine infectious gastroenteritis virus antibody positive serum is less than 10%, and there is no obvious blocking effect, which shows that the blocking ELISA method has good specificity.

[0119] The application can be used for preparing detection kits or evaluating the immune effect of vaccines, and provides an efficient and accurate technical means for PEDV infection diagnosis and immune monitoring.

[0120] 12. Identification of blocking monoclonal antibody binding activity

[0121] According to the foregoing indirect ELISA method, the blocking monoclonal antibody 1D3 is gradient diluted according to 10ug / ml, 1ug / ml, 100ng / ml, 10ng / ml, 1ng / ml, 100pg / ml, and other mouse irrelevant single antibody: PRRSV-N single antibody of Jinuobaitai is used as negative control to determine the binding activity of the antibody and PEDV / S1 recombinant protein. The results are as follows Figure 7 The results show that 1D3 still has positive reaction with PEDV / S1 at 1ng / ml dilution, and has high antibody activity.

[0122] 13. Variable region gene sequence of single antibody

[0123] Total RNA of hybridoma cells is extracted by using RNeasy Mini Kit (Cat. No. 74104), and cDNA is synthesized by reverse transcription with Random Primers; mouse antibody variable region universal primers are designed, VH and VL genes are amplified by 2 rounds of PCR, and Age1 and Bsiw1 enzyme cutting sites are introduced in the primer of the 3rd round of PCR. The PCR product is cut and purified, then connected to pUC19 vector, transformed into TOP10 strain, and after 37℃ culture for 14h, single colonies are picked for sequencing to obtain the gene sequence of the light and heavy chains of the single antibody.

[0124] The sequence of the monoclonal antibody 1D3 is as follows:

[0125] The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 1D3 is as shown in SEQ ID NO. 10:

[0126] GAGACCACCGTGACCCAGAGCCCCAGCTACCTGGCCGCCAGCCCCGGCGAGACCATCACCATCAACTGCAGGGCCAGCAAGAGCATCGGCAAGTACCTGGCCTGGTTCCAGGAGAAGCCCGGCAAGACCAACAAGCTGCTGATCTACAGCGGCAGGGTGGAGAGCTGGGGCATCCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCATGTACTACTGCAGGGGCCACATCCAGTTCAGCAACCCCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0127] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D3 is shown in SEQ ID NO. 3:

[0128] ETTVTQSPSYLAASPGETITINCRASKSIGKYLAWFQEKPGKTNKLLIYSGRVESWGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCRGHIQFSNPFGGGTKLEIKRTV.

[0129] The light chain CDR regions are annotated as follows:

[0130] The sequence of the complementarity determining region CDR-L1 of the monoclonal antibody 1D3 is shown in SEQ ID NO. 4: RASKSIGKYLA;

[0131] The sequence of the complementarity determining region CDR-L2 of the monoclonal antibody 1D3 is shown in SEQ ID NO. 5: SGRVESW;

[0132] The sequence of the complementarity determining region CDR-L3 of the monoclonal antibody 1D3 is shown in SEQ ID NO. 6: RGHIQFSNP.

[0133] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D3 is shown in SEQ ID NO. 9:

[0134] GAGGTGAAGCTGGAGGAGTCTGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGCTCTGTCACTGGCGACTCCATCACCAGTGATTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGAATACATGGGATACATAAGTTACAGCGGTAGCACTTACTACAATCCATCTCTCAGCAGTCGAATCTCCATCACTCGCGACACATCCAAGAACCAGTTCTACCTGCAGTTGAATTCTGTGACCACTGAGGACACAGCCACATATTACTGTGCAAGATCTCAACAACTCTACTATCATTACGCCGTGAAGGACTTTGACTTCTGGGGCCCAGGCACCACTCTCACAGTCTCCTCA.

[0135] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1D3 is shown as SEQ ID NO. 7:

[0136] EVKLEESGPSLVKPSQTLSLTCSVTGDSITSDYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLSSRISITRDTSKNQFYLQLNSVTTEDTATYYCARSQQLYYHYAVKDFDFWGPGTTLTVSS.

[0137] The heavy chain CDR region is labeled as:

[0138] The sequence of the complementarity determining region CDR-H1 of monoclonal antibody 1D3 is shown as SEQ ID NO. 1: SDYWN;

[0139] The sequence of the complementarity determining region CDR-H2 of monoclonal antibody 1D3 is shown as SEQ ID NO. 2: YISYSGSTYYNPSLSS;

[0140] The sequence of the complementarity determining region CDR-H3 of monoclonal antibody 1D3 is shown as SEQ ID NO. 3: SQQLYYHYAVKDFDF.

[0141] So far, the embodiments of the present application have been described in detail. In order to avoid obscuring the concept of the present application, some details known in the art are not described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein according to the above description.

[0142] Although some specific embodiments of the present application have been described in detail by way of example with reference to the drawings, it is to be understood that the above examples are intended to be illustrative only and are not intended to limit the scope of the present application. It is to be appreciated that variations and modifications to the above embodiments can be made without departing from the scope and spirit of the present application.

Claims

1. A monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus antibody, characterized in that, The heavy chain variable region of the monoclonal antibody 1D3 comprises three complementarity determining regions, the amino acid sequences of which are shown as SEQ ID NO. 1-SEQ ID NO. 3, respectively; The light chain variable region of the monoclonal antibody 1D3 comprises three complementarity determining regions, the amino acid sequences of which are shown as SEQ ID NO. 4-SEQ ID NO. 6, respectively.

2. The monoclonal antibody 1D3 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D3 is shown as SEQ ID NO.

7.

3. The monoclonal antibody 1D3 according to claim 2, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody 1D3 is shown as SEQ ID NO.

8.

4. The monoclonal antibody 1D3 according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D3 is shown as SEQ ID NO.

9.

5. The monoclonal antibody 1D3 according to claim 4, characterized in that, The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D3 is shown as SEQ ID NO.

10.

6. Use of the monoclonal antibody 1D3 according to claim 1 in the preparation of a tool for detecting porcine epidemic diarrhea virus antibodies.

7. Use according to claim 6, characterized in that, The tool comprises a reagent, a kit or a test strip.

8. Use according to claim 7, characterized in that, The kit comprises a blocking ELISA kit for detecting porcine epidemic diarrhea virus antibodies by a blocking ELISA method.

9. Use of the monoclonal antibody 1D3 according to claim 1 for the evaluation of the immunization effect of a porcine epidemic diarrhea vaccine, characterized in that, The use is for non-disease diagnostic purposes.

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