Polyclonal antibody based on pedv orf3 recombinant protein and established indirect elisa detection method

Abstract

The application discloses a PEDV ORF3 recombinant protein, which is encoded by a gene base sequence of a sequence table SEQ.ID.No.1 or has an amino acid sequence of SEQ.ID.No.2. The designed ORF3 recombinant gene can normally encode a protein, and the coded protein does not have a transmembrane domain. Researches show that the recombinant PEDV ORF3 recombinant protein has good antigenicity and immunogenicity, the antibody prepared based on the protein has high antibody titer and good specificity. Accordingly, the inventors also establish a corresponding indirect ELISA detection method, the method has the characteristics of strong specificity, good repeatability, high sensitivity and the like, can accurately identify PEDV, has the advantages of simplicity, rapidness, economy and the like, and the result is accurate, and the like, provides a new selection and reliable method for diagnosis of PED, and provides technical support for monitoring PEDV infection on a pig population. The clinical application prospect is good.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal antibodies, and particularly relates to a polyclonal antibody prepared based on a PEDV ORF3 recombinant protein and an established indirect ELISA detection method. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a pig intestinal disease that can affect pigs of all ages. The disease is characterized by acute, highly contagious and contact. Pigs infected with the disease will have clinical symptoms such as vomiting, watery diarrhea, dehydration and loss of appetite. The morbidity is close to 100%, and the mortality is more than 50%, which has caused great harm to the pig breeding industry in China and brought serious economic losses.

[0003] In clinical practice, when detecting PEDV serum samples, enzyme-linked immunosorbent assay (ELISA) is one of the commonly used diagnostic methods. This technology uses the specificity of antigen-antibody and enzyme to quickly detect a large number of samples, and is simple to operate and suitable for detection in primary and laboratory. At present, ELISA detection methods based on PEDV S, M and N proteins have been reported and are relatively mature, but ELISA detection methods based on ORF3 protein are relatively few.

[0004] The ORF3 gene is located between the S and E genes of the PEDV genome, about 675 bp, encoding 225 aa. The encoded ORF3 protein contains four transmembrane domains and usually exists in the form of a tetramer, forming an ion channel to facilitate the release of the virus to the outside of the cell. The presence of multiple transmembrane domains also makes it difficult to express the ORF3 protein in prokaryotes, and it needs to be modified to reduce the transmembrane domain, and finally realize the prokaryotic expression of the ORF3 protein. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a polyclonal antibody prepared based on a PEDV ORF3 recombinant protein and an established indirect ELISA detection method. The modified recombinant protein does not contain a transmembrane domain, and the antibody prepared accordingly has a high antibody titer and good specificity. The constructed indirect ELISA detection method has strong specificity, good repeatability and high sensitivity.

[0006] To solve the above technical problems, the present application adopts the following technical solutions:

[0007] PEDV ORF3 recombinant protein, which is encoded by the gene base sequence of SEQ.ID.No. 1 or has the amino acid sequence of SEQ.ID.No. 2.

[0008] The preparation method of the PEDV ORF3 recombinant protein is performed according to the following steps:

[0009] <1> Amplification of the modified gene and construction of the recombinant expression vector

[0010] The modified ORF3 recombinant gene fragment is amplified by two pairs of specific primers with 17GXCZ-1ORF3d as a template and is connected to pMD18T; the obtained target fragment and the prokaryotic expression vector pET32a are double-digested, purified and then constructed by T4 ligase to obtain the recombinant expression vector pET32a-ORF3; the obtained expression vector is transformed into DH-5α competent cells, a single colony is selected for plasmid extraction, and the recombinant plasmid is double-digested to verify whether the recombination is successful;

[0011] <2> Induced expression of the recombinant plasmid and verification

[0012] The verified recombinant plasmid pET32a-ORF3 is transformed into BL21 competent cells, a single colony is selected for culture in LB(Amp+), and the pET32a empty plasmid is transformed as a control; the bacterial cells are collected for ultrasonic disruption, and the expression of the ORF3 recombinant protein is detected by SDS-PAGE.

[0013] The preparation method of the PEDV ORF3 recombinant protein is performed according to the following steps:

[0014] Virus cDNA is obtained: the total reverse transcription system is 25μL, including oligo dT 1μL, 5×Buffer 5μL, dNTPmix 2μL, reverse transcriptase 0.5μL, RNasin 0.5μL, RNA 16μL; the reverse transcription program is 42℃ for 1h;

[0015] Amplification of the target fragment A: the total PCR reaction system is 50μL, including 2×Taq Master Mix 25μL, ORF3-1F and ORF3-2R each 1μL, ddH2O 17μL, cDNA 6μL, the program is 95℃ for 2min; 95℃ for 15s, 55℃ for 30s, 72℃ for 30s, a total of 35 cycles; 72℃ for 10min, 4℃ for preservation;

[0016] Amplification of the target fragment B: the total PCR reaction system was 50 μL, including 2×Taq Master Mix 25 μL, ORF3-1R and ORF3-2F each 1 μL, ddH2O 17 μL, cDNA 6 μL; the program was the same as that for amplifying the fragment A;

[0017] Amplification of the modified gene: the gel recovery products A and B were obtained by respectively recovering the target fragments A and B, and the mixed gel recovery products A and B were amplified by using primers ORF3-1F and ORF3-1R to obtain the modified gene fragment; the total PCR reaction system was 50 μL, including 2×Taq Master Mix 25 μL, ORF3-1F and ORF3-1R each 1 μL, ddH2O 17 μL, gel recovery product A 3 μL, gel recovery product B 3 μL; the program was the same as the above program to obtain the modified gene; wherein, the specific primers used have the base sequences of SEQ.ID.No.3 to 6 in the sequence listing;

[0018] The total system of T4 ligase was 10 μL, including T4 DNA ligase 1 μL, double enzyme digestion recombinant plasmid pMD18T-ORF3 reaction liquid recovery product 7 μL, double enzyme digestion empty plasmid pET32a reaction liquid recovery product 1 μL, T4 DNA ligase Buffer 1 μL, and the connection was performed in a 16℃ metal bath for 16 h;

[0019] Inducing expression of the recombinant plasmid: 2 mL of the cultured bacterial liquid was added into 200 mL of LB (Amp+), and amplification was performed on a horizontal shaker at 37℃; when the OD600 value was 0.6, 240 μL of IPTG was added to induce for 16 h on a horizontal shaker at 16℃. 562nm

[0020] The rabbit polyclonal antibody of the PEDV ORF3 recombinant protein described above.

[0021] The preparation method of the rabbit polyclonal antibody described above, in which the PEDV ORF3 recombinant protein is emulsified with Freund's adjuvant to immunize rabbits to obtain polyclonal antibody serum.

[0022] The preparation method of the rabbit polyclonal antibody described above, which is performed according to the following steps: a New Zealand white rabbit weighing about 2 kg is selected, 1 mg of PEDV ORF3 recombinant protein is emulsified with an equal volume of Freund's complete adjuvant for the first immunization, and the rabbit is boosted at the 14th day, the 21st day, the 28th day and the 35th day after the first immunization, and the emulsification is performed by using 0.5 mg of PEDV ORF3 recombinant protein and an equal volume of Freund's incomplete adjuvant; the heart blood of the rabbit is collected on the seventh day after the completion of the immunization, and the serum is separated for use.

[0023] ​The PEDV ORF3 recombinant protein-based indirect ELISA antibody detection kit comprises a coated enzyme label plate, and the PEDV ORF3 recombinant protein is used as a coating antigen.

[0024] The above-mentioned indirect ELISA antibody detection kit further comprises positive serum, negative serum, HRP-rabbit anti-pig IgG, coating solution, washing solution, blocking solution, coloring solution and termination solution.

[0025] The PEDV ORF3 recombinant protein-based indirect ELISA detection method for non-diagnostic purposes is characterized in that the following steps are performed:

[0026] Coating: the PEDV ORF3 recombinant protein is used as an antigen to coat an enzyme label plate, and after the coating is completed, the unbound protein and impurities are cleaned with a washing solution;

[0027] Blocking: a blocking solution is added, the blocking solution is discarded after the blocking is completed, and the impurities are cleaned with a washing solution;

[0028] Sample detection: a sample to be detected is added, the solution is discarded after incubation is completed, and a washing solution is used for cleaning;

[0029] Enzyme label antibody addition: HRP-rabbit anti-pig IgG is added, the solution is discarded after incubation is completed, and a washing solution is used for cleaning;

[0030] Termination: a coloring agent is added, the termination solution is added after color development in the dark, and a D 450nm value is measured by using an enzyme label instrument.

[0031] In the above-mentioned indirect ELISA detection method, the amount of the PEDV ORF3 recombinant protein added in each well in the coating is 100 ng, the coating condition is 37°C for 1 h; the blocking solution is 1% BSA, 100 μL is added in each well, and the blocking time is 2 h; the dilution degree of the enzyme label antibody in the enzyme label antibody addition is 1:1 000, 100 μL is added in each well, the incubation condition is 37°C for 40 min; the incubation time in the sample detection and the enzyme label antibody addition is 40 min; and the color development time is 15 min.

[0032] Since the PEDV ORF3 protein is a transmembrane ion channel protein, it is difficult to be expressed in prokaryotes in vitro. The inventors obtain a PEDV ORF3 recombinant protein by analyzing and modifying the protein, which is encoded by the gene base sequence of SEQ. ID. No. 1 or has the amino acid sequence of SEQ. ID. No. 2. The designed ORF3 recombinant gene can normally encode protein, and the encoded protein has no transmembrane domain. Studies show that the recombinant PEDV ORF3 recombinant protein has good antigenicity and immunogenicity, and the antibody prepared based on the protein has high antibody titer and good specificity. Accordingly, the inventors also establish a corresponding indirect ELISA detection method, which has the characteristics of strong specificity, good repeatability, high sensitivity, etc., can accurately identify PEDV, and has the advantages of simplicity, rapidness, economy, accuracy, etc., provides a new choice and reliable method for the diagnosis of PED, and provides technical support for monitoring the infection of PEDV to pig population. The clinical application prospect is good. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is an ORF3 protein nucleotide sequence comparison chart.

[0034] Figure 2 is an ORF3 protein transmembrane domain prediction chart, in which: A: 17GXCZ-1 ORF3c ORF3 protein transmembrane domain; B: 17GXCZ-1 ORF3d ORF3 protein transmembrane domain; C: recombinant pET32a-ORF3 protein transmembrane domain.

[0035] Figure 3 is an ORF3 recombinant gene amplification PCR identification result chart, in which: M. DNA standard DL 2 000; A. Recombinant ORF3 gene front section; B. Recombinant ORF3 gene rear section; C. Recombinant ORF3 gene full length.

[0036] Figure 4 is an ORF3 recombinant gene enzyme digestion identification result chart, in which: M1. DNA standard DL 5 000; M2. DNA standard DL 2 000; 1. Recombinant ORF3 gene (288bp); 2. pET32a empty plasmid (5 900bp).

[0037] Figure 5 is an ORF3 protein solubility analysis chart, in which: M. Protein Mark (14-100kDa); 1. Empty control; 2. ORF3 recombinant protein induction supernatant sample; 3. ORF3 recombinant protein induction precipitate sample.

[0038] Figure 6is the ORF3 protein purification result figure, in which: M. protein Mark (14-100 kDa); 1-5. Purified ORF3 recombinant protein samples; 6. Empty control.

[0039] Figure 7 is the Western-Blot verification result figure, in which: M1. Protein Mark (10-180 kDa); 1. ORF3 recombinant protein WB verification.

[0040] Figure 8 is the WB verification result figure of polyclonal antibody, in which: M2. Protein Mark (14-100 kDa); 1. Rabbit polyclonal antibody WB verification.

[0041] Figure 9 is the IFA verification result figure of polyclonal antibody.

[0042] Figure 10 is the determination result figure of polyclonal antibody titer.

[0043] Figure 11 is the specificity test result figure of indirect ELISA method.

[0044] Figure 12 is the sensitivity test result figure of indirect ELISA method. DETAILED DESCRIPTION

[0045] The experimental materials used in the following examples are as follows:

[0046] <1> Strains, plasmids and serum samples

[0047] PEDV ORF3 gene complete strain 17GXCZ-1 ORF3c (NCBI Accession No: MT547180.1) and PEDV ORF3 gene truncated strain 17GXCZ-1 ORF3d (NCBI Accession No: MT547179.1) are both preserved in the laboratory of the applicant, and have been published in the article Genetic Diversity of Porcine Epidemic Diarrhea Virus With a Naturally Occurring Truncated ORF3 Gene Found in Guangxi, China.

[0048] The pET32a empty plasmid was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0049] The 1129 serum samples to be detected were from 24 large-scale pig farms in different regions of Guangxi from 2019 to 2021.

[0050] Positive serum samples of Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine delta corona virus (PDCoV), Porcine rotavirus (PoRV), Pseudorabies virus (PRV), Procine enterovirus G (EVG), Classic swine fever virus (CSFV), Foot-and-mouth disease virus (FMDV), porcine circovirus 2 (PCV-2), and African swine fever virus (ASFV) were stored by the applicant's laboratory.

[0051] <2> Main experimental apparatus

[0052] SCIENTZ-IIE series touch ultrasonic cell pulverizer was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; FLoid TM Cell imaging workstation was purchased from Thermo Fisher Scientific (China) Co., Ltd.; microplate reader was purchased from Bomeilabo Biotech Co., Ltd.

[0053] <3> Experimental reagents

[0054] Endonuclease BamH I and Xho I were purchased from Novagen Biotech Co., Ltd.; His-tag antibody (mouse monoclonal antibody) and horseradish peroxidase-labeled goat anti-mouse IgG (H+L) were purchased from Biyun Tian Biotechnology Co., Ltd.; enzyme-labeled plate was purchased from Leiyin Biotechnology Co., Ltd.; goat anti-rabbit IgG-FITC, goat anti-rabbit IgG-HRP, goat anti-pig IgG-HRP, 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI), coating solution, and termination solution were purchased from Beijing Solerbio Technology Co., Ltd.; PEDV antibody detection kit was purchased from Biovet Inc. in Canada.

[0055] Example 1 Preparation of recombinant PEDV ORF3 recombinant protein of the application

[0056] I. Experimental methods

[0057] 1.1 Construction of recombinant plasmid pET32a-ORF3

[0058] 1.1.1 Sequence analysis and modification

[0059] PEDV ORF3 protein is a transmembrane protein, making its expression in prokaryotes difficult. Therefore, online analysis software was used to express it. http: / / www.detaibio.com / tools / transmembrane.html The amino acid sequences of the ORF3 protein of PEDV Guangxi strains 17GXCZ-1ORF3c and 17GXCZ-1ORF3d were analyzed for transmembrane domains. Based on the analysis results, the nucleotide sequence of the ORF3 protein of 17GXCZ-1ORF3d was modified so that the designed recombinant ORF3 protein nucleotide sequence can encode the protein normally, and the encoded protein does not have a transmembrane domain.

[0060] 1.1.2 Primer Synthesis

[0061] Based on the nucleotide sequence of 17GXCZ-1ORF3d, primers were designed for the two identical parts of the sequence as follows, corresponding to the base sequences of SEQ.ID.No.3 to SEQ.ID.No.6 in the sequence listing.

[0062] ORF3-1F, 5'- CGCGGATCC ATGTTTCTTGGACTTTTTCAATACA-3' (The underlined part is the restriction site of the endonuclease BamHI);

[0063] ORF3-2R, 5′-GCTCAACAGTAAAGAAGTAAATAAAAACACTGGTGAAAA-3′;

[0064] ORF3-2F, 5'-ACTTCTTTACTGTTGAGCTTCTTGATGGCAAGA-3';

[0065] ORF3-1R, 5'- CCGCTCGAG TTCACTAATTGTAGCATACTCGTCT-3' (The underlined part is the restriction site of the endonuclease Xho I).

[0066] 1.1.3 Amplification of the target fragment

[0067] The total viral RNA of 17GXCZ-1 ORF3c was extracted according to the method of Axy Prep Body Fluid Viral DNA Miniprep Mix, and the RNA was reversely transcribed into cDNA. The obtained cDNA was amplified by primers ORF3-1F and ORF3-2R and primers ORF3-2F and ORF3-1R, respectively, to obtain amplification product fragments A and B. The PCR reaction system was as follows: 2×Taq Master Mix 25 μL, ORF3-1F and ORF3-2R each 1 μL, ddH2O 17 μL, cDNA 6 μL, total system 50 μL. The reaction program was as follows: 95℃ 2min; 95℃ 15s, 55℃ 30s, 72℃ 30s, a total of 35 cycles; 72℃ 10min, 4℃ preservation.

[0068] The gel recovery products A and B were obtained by respectively recovering the gel of fragments A and B. The mixed gel recovery products A and B were amplified by primers ORF3-1F and ORF3-1R to obtain the target fragment. The PCR reaction system was as follows: 2×Taq Master Mix 25 μL, ORF3-1F and ORF3-1R each 1 μL, ddH2O 17 μL, gel recovery product A 3 μL and gel recovery product B 3 μL, total system 50 μL, the program was the same as above.

[0069] 1.1.4 Construction of recombinant plasmid

[0070] The obtained target fragment was gel recovered and cloned into pMD-18T vector. The plasmid was extracted and sent to Guangzhou Huada Gene Technology Co., Ltd. for sequencing. The empty plasmid pET32a and the correctly sequenced recombinant plasmid pMD18T-ORF3 were double-enzyme cut by endonuclease BamH I and Xho I in a 37℃ constant temperature water bath for 16h. The double-enzyme cutting system was as follows: recombinant (empty) plasmid 33 μL, endonuclease BamH I 7 μL, endonuclease Xho I 7 μL, 10×Buffer 10 μL, ddH2O 43 μL, total system 100 μL.

[0071] The double-digestion products were recovered using the OMEGA Cycle-Pure Kit (100). Ligation was performed using T4 DNA ligase in a 16°C metal bath for 16 hours. The ligation system consisted of: 1 μL T4 DNA ligase, 7 μL recovered product from the double-digestion of recombinant plasmid pMD18T-ORF3, 1 μL recovered product from the double-digestion of empty vector plasmid pET32a, and 1 μL T4 DNA ligase buffer, for a total volume of 10 μL. The ligation products were transformed into DH-5α competent cells, plated on LA plates, and cultured overnight. Single colonies were then picked and cultured overnight in LB broth, and plasmids were extracted. Double digestion identification was performed using the following system: 5 μL recombinant pET32a-ORF3 plasmid, 0.3 μL BamH I restriction enzyme, 0.3 μL Xho I restriction enzyme, 1 μL 10× Buffer, and 3.4 μL ddH2O, for a total volume of 10 μL. Finally, the protein was sent to BGI Genomics Co., Ltd. in Guangzhou for sequencing. The amino acid sequence of the recombinant ORF3 protein was analyzed for transmembrane domains using online analysis software.

[0072] 1.2 Prokaryotic expression and purification of pET32a-ORF3 protein

[0073] 1.2.1 Prokaryotic expression and identification of expression form of pET32a-ORF3 recombinant plasmid

[0074] The recombinant plasmid pET32a-ORF3, with correct results in both enzyme digestion and sequencing, was transformed into BL21 competent cells and plated for spot culture. A portion of the cultured bacterial solution was added to 200 mL of LB broth and amplified in a horizontal shaker at 37°C. The bacterial solution to be amplified was D... 562nm When the concentration was 0.6, 240 μL of IPTG was added and induction was continued at 16°C in a horizontal shaker for 16 h. After induction, the cells were centrifuged at 8,000 rpm for 15 min at 4°C, the supernatant was discarded, and the bacterial pellet was collected. The pellet was washed three times with PBS, and then resuspended in 10 mL of PBS. The pellet was then placed on ice for ultrasonic lysis. The ultrasonic power was 400 W, with a 3-second working time followed by a 5-second rest time, for a total of 2 min. Three cycles were performed, followed by an ice bath for 5 min. This process was repeated until the bacterial cells were clear and transparent. The completely lysed sample was centrifuged at 8,000 rpm for 15 min at 4°C. The supernatant was poured into a 10 mL centrifuge tube, and the pellet was resuspended in 10 mL of PBS. 40 μL of the supernatant and pellet sample were transferred to a centrifuge tube, 10 μL of 2×SDS loading buffer was added, and the mixture was boiled at 100°C for 10 min. SDS-PAGE electrophoresis was then performed for identification.

[0075] 1.2.2 Purification and Western blotting verification of pET32a-ORF3 recombinant protein

[0076] The above precipitated samples were purified using a His-Tagged Protein Purification Kit, and then subjected to SDS-PAGE electrophoresis. The purified protein was subjected to SDS-PAGE electrophoresis. After electrophoresis, the protein was verified by Western blotting (WB). The specific operation steps are as follows: PVDF membrane was immersed in methanol solution for 5 min. Filter paper, PVDF membrane and gel were immersed in membrane transfer buffer for 10 min and placed in a transfer apparatus. The transfer apparatus current was set to 5V, 30mA constant current, and the transfer time was 1 h. The membrane was washed three times with washing buffer for 5 min each time, and blocked with 5% skim milk powder on a horizontal shaker at 37℃ for 2 h. The membrane was washed three times with washing buffer for 5 min each time, and incubated with primary antibody (His-tag mouse monoclonal antibody) at 4℃ for 16 h. The membrane was washed three times with washing buffer for 5 min each time, and incubated with secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG) at 37℃ for 1 h. The membrane was washed three times with washing buffer for 5 min each time, and then incubated with chromogenic solution in the dark for 30 s. The results were observed in a protein imaging analyzer.

[0077] II. Experimental Results

[0078] 2.1 Analysis of transmembrane domains of the modified ORF3 recombinant protein

[0079] The nucleotide sequences of strains 17GXCZ-1ORF3c, 17GXCZ-1ORF3d, and the modified ORF3 protein were compared, and the results are as follows: Figure 1 As shown, transmembrane domain prediction analysis was performed on its amino acid sequence, and the results are as follows. Figure 2 As shown, strain 17GXCZ-1ORF3c ( Figure 2 The ORF3 protein of type -A contains 5 transmembrane domains, and the 17GXCZ-1ORF3d strain ( Figure 2 The -B) ORF3 protein contains a transmembrane domain, which affects its normal prokaryotic expression. The modified recombinant ORF3 protein ( Figure 2 -C) It lacks a transmembrane domain but can still encode and translate normally, laying the foundation for the successful expression of recombinant proteins.

[0080] 2.2 Amplification results of the target fragment

[0081] The first segment of the ORF3 recombinant gene was amplified using primers ORF3-1F and ORF3-2R. Figure 3 Fragment A), 190 bp in size; the latter part of the ORF3 recombinant gene was amplified using primers ORF3-2F and ORF3-1R. Figure 3Fragment B), 134 bp in size; gel recovery was performed on fragments A and B, and mixed gel recovery products A and B were amplified using primers ORF3-1F and ORF3-1R to obtain the ORF3 recombinant gene Figure 3 Fragment C), 288 bp in size. The results showed that the three fragments all had clear bands near the target size.

[0082] 2.3 Identification of recombinant plasmid pET32a-ORF3

[0083] The recombinant plasmid pET-32a-ORF3 was identified using endonuclease BamH I and Xho I Figure 4 ), and the target fragment was 288 bp in size, and there was a clear band near the target fragment.

[0084] 2.4 Identification of expression form of pET32a-ORF3 recombinant protein

[0085] After ultrasonic disruption of the obtained protein sample, SDS-PAGE electrophoresis was performed, and the results showed Figure 5 ) that the empty control had a clear band at 20.4 kDa, the ORF3 recombinant protein induced supernatant sample had no specific band, and the ORF3 recombinant protein induced precipitate sample had a clear band near 29.5 kDa, proving that the recombinant ORF3 protein was expressed in the form of inclusion bodies.

[0086] 2.5 Identification of expression form of pET32a-ORF3 recombinant protein

[0087] The pET32a-ORF3 recombinant protein expressed in the form of inclusion bodies was purified using His-Tagged Protein Purification Kit. The purified sample was identified using SDS-PAGE electrophoresis, and the results showed Figure 6 ) that the empty control had a clear band at 20.4 kDa, proving that the control was correct; the sample had a clear band at 29.5 kDa, the band was single, clear and thick, and there was no obvious impurity band.

[0088] 2.6 Western-blotting verification of pET32a-ORF3 recombinant protein

[0089] WB verification was performed on the purified protein, using His-tag antibody (mouse monoclonal antibody) as the primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG (H+L) as the secondary antibody, and the results showed Figure 7 ) that the target fragment was 29.5 kDa in size, and there was a clear band at the target fragment, proving that the purified pET32a-ORF3 recombinant protein could specifically bind to the His tag.

[0090] Preparation and application of rabbit polyclonal antibody against the modified PEDV ORF3 recombinant protein of the application

[0091] I. Experimental methods

[0092] 1.1 Preparation of polyclonal antibody against the modified ORF3 recombinant protein

[0093] 2 mL of blood was collected from the marginal ear vein of a New Zealand white rabbit weighing 2.1 kg, and the serum was separated as a negative control. The recombinant protein 1 mg was emulsified with an equal volume of Freund's complete adjuvant for the first immunization. The recombinant protein 0.5 mg was emulsified with an equal volume of Freund's incomplete adjuvant for the booster immunization on the 14th day, 21st day, 28th day and 35th day after the first immunization. The serum was separated from the heart blood collected on the seventh day after the completion of the immunization, and stored in a refrigerator at -20°C.

[0094] 1.2 WB verification of polyclonal antibody

[0095] SDS-PAGE electrophoresis was performed on the recombinant ORF3 recombinant protein, and a PVDF membrane was soaked in a methanol solution for 5 min. The filter paper, PVDF membrane and gel were soaked in a membrane transfer buffer for 10 min, and placed in a membrane transfer instrument for 1 h. The gel was washed with washing solution three times, each for 5 min, and blocked with 5% skimmed milk powder at 37°C on a horizontal shaker for 2 h. The gel was washed with washing solution three times, each for 5 min, and incubated with primary antibody (prepared rabbit polyclonal antibody) at 4°C for 16 h. The gel was washed with washing solution three times, each for 5 min, and incubated with secondary antibody (goat anti-rabbit IgG-HRP) at 37°C for 1 h. The gel was washed with washing solution three times, each for 5 min, and developed with color developing solution for 30 s in the dark. The results were observed in a protein imaging analyzer.

[0096] 1.3 Indirect immunofluorescence test of rabbit anti-ORF3 recombinant protein polyclonal antibody

[0097] The prepared rabbit anti-ORF3 recombinant protein polyclonal antibody was subjected to indirect immunofluorescence test (Immunofluorescence assay, IFA) to verify whether the antibody could bind to the antigen. The specific operation steps were as follows: Vero cells were inoculated in a 96-well cell culture plate and cultured in a 37°C incubator; PEDV 17GXCZ-1ORF3c and 17GXCZ-1ORF3d virus liquid were diluted 10 times, respectively, to 10 -6; When the cells reached about 80%, the cells were infected, and the last two rows were added with culture medium as negative control, and were placed in a 37°C incubator for 36h; after the cells showed obvious cytopathic effect, the cells were washed with 100μL DPBS-T for 3 times, 5min each time, and the cells were fixed with 100μL pre-cooled ice methanol at-20°C for 30min; the ice methanol was discarded, and the cells were washed with 100μL DPBS-T for 3 times, 5min each time, 100μL 1% BSA was added to each well, and the cells were blocked at room temperature for 1h; the 1% BSA was discarded, and the cells were washed with 100μL DPBS-T for 3 times, 5min each time, 100μL 1:100 diluted primary antibody (rabbit anti-ORF3 protein polyclonal antibody) was added to each well, and the cells were incubated at 4°C for 16h; the primary antibody was discarded, and the cells were washed with 100μL DPBS-T for 3 times, 5min each time, 100μL 1:100 diluted secondary antibody (FITC goat anti-rabbit IgG) was added to each well, and the cells were incubated at 37°C for 1h in the dark; the secondary antibody was discarded, and the cells were washed with 100μL DPBS-T for 3 times, 5min each time, 100μL DAPI was added to each well, and the cells were incubated at 37°C in the dark for 15min; the DAPI was discarded, and the cells were washed with 100μL DPBS-T for 3 times, 5min each time, 50μL DPBS-T was added to each well, and the cells were observed under a fluorescence microscope in the dark.

[0098] 1.4 Detection of the titer of polyclonal antibody by indirect ELISA method

[0099] The prepared rabbit anti-ORF3 polyclonal antibody was determined by using the optimized indirect ELISA method. The specific operation steps are as follows: the recombinant protein pET32a-ORF3 of the determination concentration was diluted by using the protein coating solution, and the ELISA enzyme-labeled plate was coated with the protein at a concentration of 200 ng / well at 4°C for 16 h. The coating solution was discarded, and the plate was washed with PBST for 3 times, 5 min each time, 100 μL of 1% bovine serum albumin (BSA) was added to each well, and the plate was incubated at 37°C for 2 h. The blocking solution was discarded, and the plate was washed with PBST for 3 times, 5 min each time, 100 μL of rabbit anti-ORF3 polyclonal antibody with different dilution ratios (1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, 1:1024000, 1:2048000) was added to each well, and the same dilution ratio of rabbit negative serum was set as a control, and the plate was incubated at 37°C for 1 h. The primary antibody was discarded, and the plate was washed with PBST for 3 times, 5 min each time, 100 μL of secondary antibody (goat anti-rabbit HRP labeled antibody) diluted at 1:5000 was added to each well, and the plate was incubated at 37°C for 1 h. The secondary antibody was discarded, and the plate was washed with PBST for 3 times, 5 min each time, 100 μL of TMB substrate solution was added to each well for reaction, and the plate was placed at 37°C for 10 min. 100 μL of stop solution was added to terminate the reaction, and the wavelength of 450 nm of the enzyme-labeled instrument was set to read the D 450nm values of each dilution ratio, and the data was saved.

[0100] II. Experimental results

[0101] 2.1 Western-blotting verification of rabbit anti-ORF3 protein polyclonal antibody

[0102] The obtained rabbit polyclonal antibody was subjected to WB verification, the polyclonal antibody was used as the primary antibody, goat anti-rabbit IgG-HRP was used as the secondary antibody, and the target fragment size was 29.5 kDa. The results showed that there was a clear band at the target fragment, and the verification results showed that the prepared polyclonal antibody could be well combined with the purified pET32a-ORF3 recombinant protein. Figure 8

[0103] 2.2 Indirect immunofluorescence test results of rabbit anti-ORF3 recombinant protein polyclonal antibody

[0104] The prepared polyclonal antibody was incubated in 17GXCZ-1 ORF3c and 17GXCZ-1 ORF3d infected Vero, and IFA was performed. The results showed that Figure 9 ​Specific green fluorescent signals appeared in Vero cells infected with 17GXCZ-1 ORF3c and 17GXCZ-1 ORF3d, and the signals were relatively obvious, while no green fluorescent signals were found in Vero cells incubated with 17GXCZ-1 ORF3c and 17GXCZ-1 ORF3d incubated negative serum, and in the control group of Vero cells directly incubated with polyclonal antibody and negative serum. The verification results show that the rabbit anti-ORF3 recombinant protein polyclonal antibody prepared in the present application can not only recognize the PEDV ORF3 gene truncated strain, but also can recognize the PEDV ORF3 gene complete strain.

[0105] 2.3 Determination of titer of rabbit anti-ORF3 polyclonal antibody

[0106] The prepared polyclonal antibody was detected for antibody titer, and the results showed that the D Figure 10 value of the polyclonal antibody incubated hole was 0. 1 1 1 1, and the D 450nm value of the negative serum incubated hole was 0. 0 5 5 5, and the ratio (P / N value) of the D 450nm values of the two holes was 2. 2 2 2 when the antibody dilution was 1 : 512 000, which was greater than 2. 0. Therefore, the titer of the antibody was 1 : 512 000, indicating that under non-denaturing conditions, the recombinant ORF3 recombinant protein has good immunogenicity and can induce a good immune response in New Zealand white rabbits.

[0107] Example 3 Establishment of ELISA detection method of the present application

[0108] I. Experimental method

[0109] 1.1 Indirect ELISA detection method based on PEDV ORF3 recombinant protein

[0110] The collected pig serum samples were detected by PEDV antibody detection kit, and PEDV positive serum and negative serum were screened. The recombinant protein pET32a-ORF3 was coated at 200 ng / well using ELISA enzyme-labeled plate, 4°C coating for 16 h; the coating solution was discarded, and PBST was used for washing 3 times, 5 min each time, 100 μL 1% skimmed milk powder was added to each well, 37°C blocking for 2 h; the blocking solution was discarded, and PBST was used for washing 3 times, 5 min each time, 100 μL of the screened positive serum and negative serum was added to each well, two repeats, 37°C incubation for 1 h; the primary antibody was discarded, and PBST was used for washing 3 times, 5 min each time, 100 μL of 1:5 000 diluted secondary antibody (goat anti-pig HRP labeled antibody) was added to each well, 37°C incubation for 1 h; the secondary antibody was discarded, and PBST was used for washing 3 times, 5 min each time, 100 μL TMB substrate solution was added to each well for reaction, 37°C for 10 min; 100 μL stop solution was added to terminate the reaction, and the D 450nm value of each dilution was read at 450 nm wavelength of the enzyme-labeled instrument, and the data was saved.

[0111] 1.2 Optimization of each condition of indirect ELISA detection method

[0112] 1.2.1 Optimization of optimal antigen coating concentration and serum dilution

[0113] The antigen coating concentration was set to 200 ng / well, 100 ng / well, 50 ng / well, 25 ng / well, 12.5 ng / well, and 6.25 ng / well, respectively; the serum dilution was set to 1:100, 1:200, 1:400, and 1:800, respectively; other conditions were unchanged, and the D 450nm value was read and the data was saved.

[0114] 1.2.2 Optimization of optimal antigen coating condition and secondary antibody dilution

[0115] The antigen coating condition was set to 37°C for 4 h, 37°C for 2 h, 37°C for 1 h, and 4°C overnight, respectively; the secondary antibody dilution was set to 1:1 000, 1:2 000, 1:4 000, and 1:8 000, respectively; other conditions were unchanged, and the D 450nm value was read and the data was saved.

[0116] 1.2.3 Optimization of optimal serum and secondary antibody incubation time

[0117] The serum incubation time was set to 20 min, 40 min, and 60 min, respectively; the secondary antibody incubation time was set to 20 min, 40 min, and 60 min, respectively; other conditions were unchanged, and the D 450nm value was read and the data was saved.

[0118] 1.2.4 Optimization of the best blocking solution and blocking time

[0119] The blocking solution was set to 1% skim milk, 5% skim milk, 1% BSA, and 5% BSA, respectively; the blocking time was set to 1 h, 1.5 h, 2 h, and 2.5 h, respectively; other conditions were unchanged, and the D 450nm value was read and the data was saved.

[0120] 1.2.5 Optimization of the best color development time

[0121] The color development time was set to 1 min, 5 min, 10 min, 15 min, 20 min, and 25 min, respectively; other conditions were unchanged, and the D 450nm value was read and the data was saved.

[0122] 1.2.6 Determination of the critical value of the indirect ELISA method for detecting PEDV antibodies

[0123] ELISA was performed on 15 PEDV-negative serum samples according to the optimized conditions, and the D 450nm value was read and the data was saved.

[0124] 1.2.7 Reproducibility test of the indirect ELISA method for detecting PEDV antibodies

[0125] Three groups of inter-plate and intra-plate repeats were set for positive and negative sera, respectively, and ELISA was performed according to the optimized conditions, and the D 450nm value was read and the data was saved.

[0126] 1.2.8 Specificity test of the indirect ELISA method for detecting PEDV antibodies

[0127] ELISA was performed on positive serum samples of EVG, PoRV, FMDV, PRRSV, PDCoV, PCV, CSFV, ASFV, PRV, and PEDV stored in the laboratory according to the optimized conditions, and the D 450nm value was read and the data was saved.

[0128] 1.2.9 Sensitivity test of the indirect ELISA method for detecting PEDV antibodies

[0129] Three PEDV-positive serum samples were selected, diluted at dilutions of 1:100, 1:200, 1:400, 1:800, and 1:1600, and ELISA was performed on the diluted sera according to the optimized conditions, and the D 450nm value was read and the data was saved.

[0130] 1.2.10 Coincidence rate test of the indirect ELISA method for detecting PEDV antibodies

[0131] At the same time, 30 samples were preliminarily detected by using the PEDV antibody detection kit and the established method, and the D 450nm values were read, the results were judged, and statistics and analysis were performed.

[0132] 1.2.11 Preliminary application of the PEDV indirect ELISA method

[0133] A total of 1129 serum samples collected from 24 pig farms in the laboratory from 2019 to 2021 were detected by using the established method, and the D 450nm values were read, the results were judged, and statistics and analysis were performed.

[0134] II. Experimental results

[0135] 2.1 Optimization results of optimal antigen coating concentration and serum dilution

[0136] The optimal antigen coating concentration and serum dilution were determined by square array method, and the results are shown in Table 1. When the antigen coating concentration was 100 ng / well and the serum dilution was 1:200, the P / N value was the largest, which was 5.292.

[0137] Table 1 Determination of optimal antigen coating concentration and serum dilution

[0138]

[0139] 2.2 Optimization results of optimal antigen coating conditions and secondary antibody dilution

[0140] The optimal antigen coating conditions and secondary antibody dilution were determined by square array method, and the results are shown in Table 2. When the antigen coating conditions were 37℃ for 1h and the secondary antibody dilution was 1:1 000, the P / N value was the largest, which was 6.198.

[0141] Table 4-2 Determination of optimal antigen coating conditions and secondary antibody dilution

[0142]

[0143]

[0144] 2.3 Optimization results of optimal serum and secondary antibody incubation time

[0145] The optimal serum and secondary antibody incubation time were determined by square array method, and the results are shown in Table 3. When the serum and secondary antibody incubation time were both 40 min, the P / N value was the largest, which was 5.557.

[0146] Table 3 Determination of optimal serum and secondary antibody incubation time

[0147]

[0148] 2.4 Optimization results of the best blocking solution and blocking time

[0149] The best blocking solution and blocking time were determined by square array method, and the results are shown in Table 4. When the blocking solution was 1% BSA and the blocking time was 2 h, the P / N value was the largest, which was 7.234.

[0150] Table 4 Determination of the best blocking solution and blocking time

[0151]

[0152]

[0153] 2.5 Optimization results of the best color development time

[0154] Different color development times were set, and the results are shown in Table 5. When the color development time was 15 min, the P / N value was the largest, which was 6.977.

[0155] Table 5 Determination of the best color development time

[0156]

[0157] 2.6 Determination of the critical value of the PEDV indirect ELISA method

[0158] Fifteen negative sera were taken to determine D values according to the optimized method, and the results are shown in Table 6. The average value of the 15 samples was 450nm SD = 0.067, and thus 450nm When D value < 0.351, the result was negative; when D value > 0.418, the result was positive; when 0.418 > D value > 0.351, the result was suspicious, and if suspicious twice, it was determined as negative. 450nm 450nm

[0159] Table 6 Determination of the critical value of the indirect ELISA method

[0160]

[0161] 2.7 Reproducibility test results of the PEDV indirect ELISA method

[0162] The results of inter-plate and intra-plate reproducibility are shown in Table 7, in which the highest CV value was 0.078, which was less than 0.1, indicating that the established method had good reproducibility.

[0163] Table 7 Reproducibility test

[0164] ​​​​

[0165]

[0166] 2.8 Specificity test results of the indirect ELISA method for detecting PEDV

[0167] The established method was used to detect EVG, PoRV, FMDV, PRRSV, PDCoV, PCV, CSFV, ASFV and PRV positive serum samples, with three replicates in each group. The results showed that the D450nm values of the positive serum samples were lower than the positive critical value, except for the PEDV detection result, indicating that the method had high specificity. Figure 11

[0168] 2.9 Sensitivity test results of the indirect ELISA method for detecting PEDV

[0169] The sensitivity test results showed that the D450nm values of the positive serum samples were lower than the positive critical value, except for the PEDV detection result, indicating that the method had high specificity. Figure 12

[0170] 2.10 Coincidence rate test results of the indirect ELISA method for detecting PEDV

[0171] The established method and the PEDV antibody detection kit were used to detect 30 pig serum samples at the same time, and the results are shown in Table 8. Among them, 28 determinations were consistent, with a coincidence rate of 93.33%, indicating that the established indirect ELISA method can be used for clinical sample detection and application.

[0172] Table 8 Comparison results of the indirect ELISA method and the kit

[0173]

[0174] 2.11 Detection results of clinical samples

[0175] The established indirect ELISA method was used to detect 1,129 clinical samples collected, and the results are shown in Table 9. The positive rates of two pig farms were 100% (pig farms 1 and 9); the positive rates of six pig farms were between 80% and 100% (pig farms 4, 11, 13, 15, 16 and 23); the positive rates of eight pig farms were between 50% and 80% (pig farms 2, 3, 5, 6, 7, 8, 14 and 17); and the positive rates of eight pig farms were less than 50% (pig farms 10, 12, 18, 19, 20, 21, 22 and 24).

[0176] Table 9 Detection of clinical samples ​​

[0177]

[0178] The results show (Table 10) that a total of 20 pig farms are detected positive, and the positive rate of pig farms is 83.33%, a total of 731 positive samples are detected, and the sample positive rate is 67.75%. Among them, a total of 397 samples were obtained from 12 pig farms in 2019, 10 pig farms were detected positive, the positive rate of pig farms was 83.33%, 267 positive samples were detected, and the sample positive rate was 67.25%; 399 samples were obtained from 5 pig farms in 2020, 5 pig farms were detected positive, the positive rate of pig farms was 100%, 305 positive samples were detected, and the sample positive rate was 76.44%; 333 samples were obtained from 7 pig farms in 2021, 5 pig farms were detected positive, the positive rate of pig farms was 71.43%, 159 positive samples were detected, and the sample positive rate was 47.75%.

[0179] Table 10 Detection information statistics

[0180]

[0181] In summary, the recombinant ORF3 recombinant protein provided by the present application has good antigenicity, and the indirect ELISA method established based on the protein can accurately detect PEDV, and has strong specificity, high sensitivity, good repeatability, simple operation, convenient and fast, and good clinical application prospect. SEQUENCE LISTING <110> Guangxi University <120> Polyclonal antibody prepared based on PEDV ORF3 recombinant protein and established indirect ELISA detection method <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 288 <212> DNA <213> Artificial Sequence <400> 1 atgtttcttg gactttttca atacacgatt gacacagtcg tcaaagatgt ctctaagtct 60 gccaacttgt cttcggacgc tgtccaagag ttggagctca atgtagttcc aattagacaa 120 gcttcaaatg tgactggttt tcttttcacc agtgttttta tttacttctt tactgttgag 180 cttcttgatg gcaagaagct ttatgtcttt tcgcaacatc aaattgttgg cattactaat 240 gctgcatttg actcaattca actagacgag tatgctacaa ttagtgaa 288 <210> 2 <211> 96 <212> PRT <213> Artificial Sequence <400> 2 Met Phe Leu Gly Leu Phe Gin Tyr Thr He Asp Thr Val Val Lys Asp 1 5 10 15 Val Ser Lys Ser Ala Asn Leu Ser Ser Asp Ala Val Gin Glu Leu Glu 20 25 30 Leu Asn Val Val Pro He Arg Gin Ala Ser Asn Val Thr Gly Phe Leu 35 40 45 Phe Thr Ser Val Phe He Tyr Phe Phe Thr Val Glu Leu Leu Asp Gly 50 55 60 Lys Lys Leu Tyr Val Phe Ser Gin He Gin He Val Gly He Thr Asn 65 70 75 80 Ala Ala Phe Asp Ser He Gin Leu Asp Glu Tyr Ala Thr He Ser Glu 85 90 95 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <400> 3 cgcggatcca tgtttcttgg actttttcaa taca 34 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <400> 4 gctcaacagt aaagaagtaa ataaaaacac tggtgaaaa 39 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <400> 5 acttctttac tgttgagctt cttgatggca aga 33 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <400> 6 ccgctcgagt tcactaattg tagcatactc gtct 34

Claims

1. A PEDV ORF3 recombinant protein, characterized in that The gene base sequence of the sequence table SEQ. ID. No. 1 encodes or has the amino acid sequence of SEQ. ID. No.

2.

2. The preparation method of the PEDV ORF3 recombinant protein according to claim 1, characterized in that The following steps are taken: <1> Amplification of the modified gene and construction of the recombinant expression vector The modified ORF3 recombinant gene fragment is amplified from the 17GXCZ-1 ORF3d template by two pairs of specific primers and connected to pMD18T; the obtained target fragment and the prokaryotic expression vector pET32a are double-digested respectively, purified and then constructed by T4 ligase to obtain the recombinant expression vector pET32a-ORF3; the obtained expression vector is transformed into DH-5α competent cells, a single colony is selected for plasmid extraction, and the recombinant plasmid is double-digested to verify whether the recombination is successful; <2> Induced expression of the recombinant plasmid and verification The verified recombinant plasmid pET32a-ORF3 is transformed into BL21 competent cells, a single colony is selected for culture in LBAmp+, and the pET32a empty plasmid is transformed as a control, the bacterial cells are collected for ultrasonic disruption, and the expression of the ORF3 recombinant protein is detected by SDS-PAGE.

3. The method for preparing PEDV ORF3 recombinant protein according to claim 2, characterized in that... The following operations are taken: Acquisition of viral cDNA: the total reverse transcription system is 25 μL, including oligo dT 1 μL, 5×Buffer 5 μL, dNTPmix 2 μL, reverse transcriptase 0.5 μL, RNasin 0.5 μL, and RNA 16 μL; the reverse transcription program is 42℃ for 1 h; Amplification of the target fragment A: the total PCR reaction system is 50 μL, including 2×Taq Master Mix 25 μL, ORF3-1F and ORF3-2R each 1 μL, ddH2O 17 μL, cDNA 6 μL, the program is 95℃ for 2 min; 95℃ for 15 s, 55℃ for 30 s, 72℃ for 30 s, a total of 35 cycles; 72℃ for 10 min, 4℃ storage; Amplification of the target fragment B: the total PCR reaction system is 50 μL, including 2×Taq Master Mix 25 μL, ORF3-1R and ORF3-2F each 1 μL, ddH2O 17 μL, cDNA 6 μL; the program is the same as that for amplifying fragment A; Amplification of the modified gene: the target fragments A and B are gel recovered respectively to obtain gel recovery products A and B, and the mixed gel recovery products A and B are amplified by primers ORF3-1F and ORF3-1R to obtain the modified gene fragment; The total PCR reaction system is 50 μL, including 2×Taq Master Mix 25 μL, ORF3-1F and ORF3-1R each 1 μL, ddH2O 17 μL, gel recovery product A 3 μL, and gel recovery product B 3 μL; the program is the same as the above program to obtain the modified gene; wherein the specific primers used have the base sequences of SEQ. ID. No. 3 to 6 in the sequence table; The total system of T4 ligase was 10 μL, including T4 DNA ligase 1 μL, double enzyme cutting recombinant plasmid pMD18T-ORF3 reaction liquid recovery product 7 μL, double enzyme cutting empty plasmid pET32a reaction liquid recovery product 1 μL, T4 DNA ligase Buffer 1 μL, and the connection was carried out in 16℃ metal bath for 16 h. The recombinant plasmid was induced for expression: 2 mL of the cultured bacteria solution was added to 200 mL of LB Amp+, and amplification was performed on a horizontal shaker at 37°C. When the OD600 value was 0.6, 240 μL of IPTG was added, and induction was performed on a horizontal shaker at 16°C for 16 h. The ORF3-1F, ORF3-2R, ORF3-2F, and ORF3-1R were base sequences of SEQ.ID.No.3 to SEQ.ID.No.6, respectively. D 562 nm When the OD600 value was 0.6, 240 μL of IPTG was added, and induction was performed on a horizontal shaker at 16°C for 16 h; the ORF3-1F, ORF3-2R, ORF3-2F, and ORF3-1R were base sequences of SEQ.ID.No.3 to SEQ.ID.No.6, respectively.

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