3AB-based ELISA (enzyme-linked immunosorbent assay) detection method and kit thereof

Abstract

The invention provides an ELISA (enzyme-linked immuno sorbent assay) detection method based on 3AB, which can be used for distinguishing porcine sapelovirus (PSV) infection and immunization, and is beneficial to prevention and control of PSV. The invention also provides a related kit.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, to a 3AB-based ELISA detection method and a kit thereof. Background Art

[0002] Porcine sapelovirus (PSV) is a non-enveloped single-stranded positive-sense RNA virus belonging to the Picornaviridae family and the genus Sapelovirus. In the 1960s, British scientists first isolated it from the intestines of diarrheal pigs, so it is also called porcine enterovirus 8 (PEV8). After its first discovery in the UK, traces of porcine Sapelovirus were also found in other countries. Porcine Sapelovirus is mainly transmitted through the fecal-oral route, causing severe diarrhea, pneumonia, neurological disorders, and fertility disorders in pigs, but there are also subclinical symptoms and asymptomatic infections without obvious symptoms.

[0003] The PSV genome is about 7.5kb in size and contains 4 structural proteins (3AB, VP2, VP3, VP1) and 7 non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C, 3D). The 3AB gene is 366bp in size, encoding 122 amino acids, and the protein size is about 14kDa. In order to control the disease caused by porcine Sapelo virus, the injection of inactivated immune vaccine can play a preventive role. In the prevention and treatment of PSV, the differential diagnosis of immune animals and infected animals is very important.

[0004] However, there are currently no differential diagnostic methods and related kits for PSV infection and immunity in the art. Summary of the invention

[0005] The object of the present invention is to provide an ELISA detection method based on 3AB, which can be used to distinguish porcine sapelovirus (PSV) infection and immunity, and is beneficial to PSV prevention and control. The present invention also provides a related kit.

[0006] In a first aspect of the present invention, there is provided the use of porcine Sapelo virus (PSV) recombinant protein 3AB in the preparation of a reagent / kit for distinguishing between PSV infection and immunity, or distinguishing whether PSV is infected; the amino acid sequence of the PSV recombinant protein 3AB is shown in SEQ ID NO:4.

[0007] In one or more embodiments, the reagent / kit includes: a PSV recombinant protein 3AB antigen coated on a solid phase carrier; and by detecting the PSV recombinant protein 3AB antigen, PSV infection and immunity, or whether PSV infection occurs, can be distinguished.

[0008] In one or more embodiments, the detection method comprises:

[0009] (a) adding a sample to be tested to a solid phase carrier coated with PSV recombinant protein 3AB antigen; the amino acid sequence of the PSV recombinant protein 3AB antigen is shown in SEQ ID NO: 4;

[0010] (b) Detecting whether the PSV recombinant protein 3AB antigen-antibody complex exists in the system of (a); if the complex exists, it indicates that PSV exists in the sample to be tested or the sample to be tested is a PSV-infected sample; otherwise, it indicates that PSV does not exist in the sample to be tested or the sample to be tested is a PSV-immunized sample.

[0011] In one or more embodiments, the detection method further includes one or more (eg, all) of the following steps: washing the solid phase carrier, blocking, adding a sample for incubation, adding an enzyme-labeled antibody for incubation, color development, terminating the reaction, and reading the OD value.

[0012] In one or more embodiments, the solid phase carrier includes, but is not limited to: a well plate (such as an ELISA plate), microbeads (such as magnetic beads), a dish, a glass slide, a test strip, and the like.

[0013] In one or more embodiments, the reagent / kit is an ELISA detection reagent / kit; preferably an indirect ELISA detection reagent / kit.

[0014] In one or more embodiments, the reagent / kit further includes: blocking solution, washing solution, positive control, negative control, enzyme-labeled antibody, color development solution, and stop solution.

[0015] In one or more embodiments, the blocking solution is 5% to 15% skim milk powder, preferably 10±1% skim milk powder.

[0016] In one or more embodiments, the washing solution is phosphate-buffered saline (PBS-Tween), preferably PBST.

[0017] In one or more embodiments, the positive control comprises or is a PSV-positive serum sample.

[0018] In one or more embodiments, the negative control includes PSV negative serum samples, PSV immune serum samples, and other virus serum samples.

[0019] In one or more embodiments, the other viruses include CSFV, BVDV, PEDV, PRV, FMDV, PRRSV.

[0020] In one or more embodiments, the enzyme-labeled antibody is HRP goat anti-pig IgG.

[0021] In one or more embodiments, the color developing solution is a TMB substrate color developing solution.

[0022] In one or more embodiments, the stop solution is H2SO4.

[0023] In one or more embodiments, the detection method includes one or more of the following features:

[0024] (A) The coating concentration of the PSV recombinant protein 3AB is 0.25-2 μg / ml, preferably 0.5-2 μg / ml, and more preferably 0.5±0.1 μg / ml;

[0025] (B) The dilution factor of the sample is 1:400 to 1:3200, preferably 1:1600;

[0026] (C) The dilution multiple of the enzyme-labeled antibody is 1:10000 to 1:25000, preferably 1:20000;

[0027] (D) The incubation condition of the enzyme-labeled antibody is 37±1°C for 15 to 45 minutes;

[0028] (E) The reaction conditions for the color development are 5 to 30 minutes at 37±1°C, preferably 5 to 15 minutes at 37±1°C;

[0029] (F) The OD value is the OD value at a wavelength of 450 nm.

[0030] In one or more embodiments, the preparation method of the PSV recombinant protein 3AB includes: amplifying the PSV recombinant protein 3AB gene sequence; transferring it into a vector to construct a recombinant plasmid; inducing expression of the recombinant plasmid; and separating and purifying to obtain the PSV recombinant protein 3AB.

[0031] In one or more embodiments, the PSV recombinant protein 3AB gene sequence is amplified using primers such as SEQ ID NO:1 and SEQ ID NO:2.

[0032] In one or more embodiments, the vector is a pET-28a vector.

[0033] In one or more embodiments, the induced expression is induced expression by adding IPTG.

[0034] In a second aspect of the present invention, a method for detecting porcine Sapelo virus (PSV) for non-diagnostic purposes is provided, comprising:

[0035] (a) adding a sample to be tested to a solid phase carrier coated with PSV recombinant protein 3AB antigen; the amino acid sequence of the PSV recombinant protein 3AB antigen is shown in SEQ ID NO: 4;

[0036] (b) Detecting whether the PSV recombinant protein 3AB antigen-antibody complex exists in the system of (a); if the complex exists, it indicates that PSV exists in the sample to be tested or the sample to be tested is a PSV-infected sample; otherwise, it indicates that PSV does not exist in the sample to be tested or the sample to be tested is a PSV-immunized sample.

[0037] In one or more embodiments, the method further comprises one or more (eg, all) of the following steps: washing the solid phase carrier, blocking, adding a sample for incubation, adding an enzyme-labeled antibody for incubation, color development, terminating the reaction, and reading the OD value.

[0038] In one or more embodiments, the detection method includes one or more of the following features:

[0039] (A) The coating concentration of the PSV recombinant protein 3AB is 0.25-2 μg / ml, preferably 0.5±0.1 μg / ml;

[0040] (B) the dilution factor of the sample is 1:400 to 1:3200, preferably 1:1600;

[0041] (C) The dilution multiple of the enzyme-labeled antibody is 1:10000 to 1:25000, preferably 1:20000;

[0042] (D) The incubation condition of the enzyme-labeled antibody is 37±1°C for 15 to 45 minutes;

[0043] (E) The reaction conditions for the color development are 5 to 30 minutes at 37±1°C, preferably 5 to 15 minutes at 37±1°C;

[0044] (F) The OD value is the OD value at a wavelength of 450 nm;

[0045] (G) The blocking solution is 5% to 15% skim milk powder, preferably 10±1% skim milk powder;

[0046] (H) The sample is a serum sample; more preferably, the serum sample includes a PSV-positive serum sample, a PSV-negative serum sample, a PSV-immune serum sample, or other virus serum samples; more preferably, the other viruses include CSFV, BVDV, PEDV, PRV, FMDV, and PRRSV;

[0047] (I) the enzyme-labeled antibody is HRP goat anti-pig IgG;

[0048] (J) the color developing solution is a TMB substrate color developing solution;

[0049] (K) The stop solution is H2SO4.

[0050] In a third aspect of the present invention, a use of porcine Sapelo virus (PSV) recombinant protein 3AB in identifying porcine Sapelo virus (PSV) for non-diagnostic purposes is provided, wherein the amino acid sequence of the PSV recombinant protein 3AB is shown in SEQ ID NO:4.

[0051] In one or more embodiments, the application includes:

[0052] (a) adding a sample to be tested to a solid phase carrier coated with PSV recombinant protein 3AB antigen; the amino acid sequence of the PSV recombinant protein 3AB antigen is shown in SEQ ID NO: 4;

[0053] (b) Detecting whether the PSV recombinant protein 3AB antigen-antibody complex exists in the system of (a); if the complex exists, it indicates that PSV exists in the sample to be tested or the sample to be tested is a PSV-infected sample; otherwise, it indicates that PSV does not exist in the sample to be tested or the sample to be tested is a PSV-immunized sample.

[0054] In one or more embodiments, the method for determining whether the 3AB antigen-antibody complex exists includes: if the OD value is greater than a threshold value, determining that the sample to be tested is a PSV-infected sample; otherwise, determining that the sample to be tested is a PSV-immune sample.

[0055] In one or more embodiments, the threshold value is 0.417.

[0056] In one or more embodiments, the detection method is as described in any embodiment of the present invention.

[0057] In a fourth aspect of the present invention, a method for distinguishing whether a sample to be tested is infected with PSV is provided, comprising:

[0058] (a) using the reagent / kit described in any embodiment of the present invention to detect the recombinant protein 3AB in the sample to be tested, and reading the OD value;

[0059] (b) judging whether the sample is a PSV-infected sample or a PSV-uninfected sample according to the OD value obtained in (a); if the OD value is greater than a threshold value, judging that the sample to be tested is a PSV-infected sample; otherwise, judging that the sample to be tested is a PSV-uninfected sample.

[0060] In one or more embodiments, the threshold value is 0.417.

[0061] A fifth aspect of the present invention provides a method for distinguishing PSV infection or immunity in a sample to be tested, comprising:

[0062] (a) using the reagent / kit described in any embodiment of the present invention to detect the recombinant protein 3AB in the sample to be tested, and reading the OD value;

[0063] (b) judging whether the sample is a PSV-infected sample or a PSV-immune sample according to the OD value obtained in (a); if the OD value is greater than a threshold value, judging that the sample to be tested is a PSV-infected sample; otherwise, judging that the sample to be tested is a PSV-immune sample.

[0064] In one or more embodiments, the threshold value is 0.417.

[0065] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 , 3 PCR amplification results of AB gene. M: DNA molecular mass standard, 1: negative control, 2: 3AB gene.

[0067] Figure 2 , double enzyme digestion identification results of recombinant plasmid pET-28a-3AB. M: DNA molecular mass standard, 1: recombinant plasmid pET-28a-3AB.

[0068] Figure 3 , SDS-PAGE analysis results of the induced expression product of the recombinant plasmid pET-28a-3AB protein. M: protein molecular weight standard; 1: before pET-28a-3AB induction; 2: product after pET-28a-3AB induction; 3: supernatant after pET-28a-3AB cleavage; 4: precipitate after pET-28a-3AB cleavage.

[0069] Figure 4 , SDS-PAGE electrophoresis results of recombinant 3AB protein purification. M: protein molecular weight standard, 1: flow-through, 2: 20mmol / L imidazole 10th wash, 3-6: 200mmol / L imidazole elution.

[0070] Figure 5 , 3 The results of SDS-PAGE electrophoresis after desalting and concentration of AB protein. M: protein molecular weight standard, 1: purified and concentrated 3AB protein.

[0071] Figure 6 , 3AB protein immunoblotting results. M: protein molecular weight standard, 1: purified recombinant 3AB protein.

[0072] Figure 7 , ROC curve of ELISA test of immune pig serum samples. AUC=0.9954, P<0.0001.

[0073] Figure 8 , dot plot of ELISA test of immune pig serum samples. Critical value = > 0.417, sensitivity = 97.92%, specificity = 100.0%, 95% confidence interval = 99.78% ~ 100.0%.

[0074] Fig. 9 , sensitivity test results.

[0075] Fig.10 , specificity test results.

[0076] Fig.11 , 3AB gene sequence comparison results from Coxsackievirus, Echovirus, Poliovirus, Enterovirus, Rhinovirus, Sapelovirus, Cardiovirus, Hepatovirus and Foot-and-mouth (FMDV). DETAILED DESCRIPTION

[0077] After in-depth research, the inventors constructed a pET-28a-3AB recombinant plasmid using a prokaryotic expression system to express the recombinant protein 3AB, and used the 3AB protein as an antigen to establish an ELISA method (preferably an indirect ELISA method) based on the recombinant protein 3AB, which can be used to distinguish PSV-infected and vaccinated animals, thereby facilitating PSV prevention and control. The present invention also provides a related kit.

[0078] In a specific embodiment, the present invention uses PSV recombinant protein 3AB to coat the ELISA plate, uses the matrix titration method to determine the optimal working conditions of ELISA (preferably the indirect ELISA method), and analyzes the specificity and sensitivity of the method. In the ELISA method, the optimal coating concentration of PSV recombinant protein 3AB is 0.5 μg / ml, the optimal dilution of the sample is 1:1600; the optimal blocking solution is 10% skim milk powder-PBST; the optimal dilution of HRP-labeled goat anti-pig IgG is 1:20000, the optimal incubation time is 30 minutes; the optimal color development time is 10 minutes, and the OD 450nm The threshold value is 0.417. The repeatability of this method is good, and the coefficient of variation of repeated tests within the sample batch is 0.22%-4.61%, and the coefficient of variation between batches is 1.03%-7.38%, both of which are less than 10%. This method can still detect positive results when the serum is diluted to 1:12800 times, and has high sensitivity. Although proteins such as 3AB or 3ABC have also been identified and studied in other viruses such as FMDV, there are extremely significant differences between different viruses. Fig.11 The results of the comparison of 3AB gene sequences from Coxsackievirus, Echovirus, Poliovirus, Enterovirus, Rhinovirus, Sapelovirus (PSV), Cardiovirus, Hepatovirus and Foot-and-mouth (FMDV) are shown. The results show that the sequence of PSV 3AB is different from the 3AB sequences of other viral sources. In addition, the method has strong specificity and does not cross-react with other pathogens (such as CSFV, PEDV, PRV, PRRSV, FMDV, BVDV positive serum).

[0079] The present invention successfully established a porcine PSV recombinant protein 3AB ELISA detection method, which can be applied to the analysis of PSV recombinant protein 3AB in porcine serum, to distinguish whether the test sample is infected with PSV or to distinguish between porcine PSV infection and immunity, which is beneficial to the prevention and control of PSV.

[0080] Methods for distinguishing whether a test sample is infected with PSV / distinguishing between PSV infection and immunity

[0081] Based on the above research, the present invention provides a method for distinguishing whether a test sample is infected with PSV, or a method for distinguishing between PSV infection and immunity, comprising using the ELISA described in the present invention, preferably the indirect ELISA detection method, to detect the PSV recombinant protein 3AB, thereby determining whether the test sample is infected with PSV, or distinguishing between PSV infection and immunity.

[0082] As used herein, the "PSV infection" refers to the process in which the PSV virus invades the body through oral administration or injection and colonizes in the body and continuously replicates.

[0083] As used herein, the "PSV immunization" refers to the oral or injection administration of an inactivated PSV vaccine into healthy animals (eg, pigs) to activate the in vivo immune response and produce an antiviral effect.

[0084] It can also be said that the present invention provides the use of PSV recombinant protein 3AB in the preparation of a reagent for distinguishing PSV infection and immunity; the nucleotide sequence of the PSV recombinant protein 3AB is shown in SEQ ID NO:3.

[0085] In one or more embodiments, the reagent is an indirect ELISA detection reagent.

[0086] In one or more embodiments, the method comprises:

[0087] (a) applying a sample to be tested to a solid phase carrier (such as an ELISA plate) coated with a 3AB antigen; the nucleotide sequence of the 3AB antigen is shown in SEQ ID NO: 3;

[0088] (b) Detecting whether a 3AB antigen-antibody complex exists in the system of (a); if the complex exists, it indicates that porcine Sapelo virus (PSV) exists in the sample to be tested or the sample to be tested is a sample infected with porcine Sapelo virus (PSV); otherwise, it indicates that porcine Sapelo virus (PSV) does not exist in the sample to be tested or the sample to be tested is a sample immune to porcine Sapelo virus (PSV).

[0089] It should be understood that when the detection method of the present invention is used to distinguish whether the test sample is infected with PSV, or to distinguish between PSV infection and immunity, when OD 450 nm When the reading of OD is greater than the threshold, the test sample is judged to be a sample infected with PSV, or the sample is infected with PSV; otherwise, when OD 450 nm When the reading is less than the threshold, the detected sample is judged to be a sample not infected with PSV, or the sample is not infected with PSV. In one or more specific embodiments, the threshold is 0.417.

[0090] In one or more embodiments, the method for distinguishing whether a sample is infected with PSV comprises: (a) using the detection method of the present invention to detect the recombinant protein 3AB in the sample, reading the OD 450 nm (b) OD obtained in (a) 450 nm The value determines whether the sample is infected with PSV; if OD 450 nm When the reading of is greater than the threshold, the sample is judged to be a sample infected with PSV; otherwise, the sample is judged to be a sample not infected with PSV. In one or more specific embodiments, the threshold is 0.417.

[0091] In one or more embodiments, the method for distinguishing PSV infection or immunity comprises: (a) detecting the recombinant protein 3AB in the sample using the detection method of the present invention, reading the OD 450 nm (b) OD obtained in (a) 450nm The sample is judged to be a PSV-infected sample or a PSV-immune sample by OD 450 nm When the reading of is greater than the threshold, the sample is judged to be a PSV-infected sample; otherwise, the sample is judged to be a PSV-immune sample. In one or more specific embodiments, the threshold is 0.417.

[0092] Reagent test kit

[0093] The present invention also provides a porcine Sapelo virus (PSV) (e.g., indirect ELISA) kit, the kit comprising: a PSV recombinant protein 3AB antigen coated on a solid phase carrier (e.g., an ELISA plate); the amino acid sequence of the PSV recombinant protein 3AB antigen is shown in SEQ ID NO:4.

[0094] In one or more embodiments, the nucleotide sequence of the PSV recombinant protein 3AB antigen is as shown in SEQ ID NO:3.

[0095] In one or more embodiments, the kit may further include: blocking solution, washing solution, positive control, negative control, enzyme-labeled antibody, color development solution, and stop solution.

[0096] The present invention also provides the use of the kit in identifying porcine sapero virus (PSV) for non-diagnostic purposes. The kit is used to detect the sample by ELISA (preferably indirect ELISA) detection method to determine whether the sample contains porcine sapero virus (PSV).

[0097] Detection Methods

[0098] The present invention provides a detection method based on PSV recombinant protein 3AB (e.g., indirect ELISA), comprising:

[0099] (a) adding a sample to be tested to a solid phase carrier coated with PSV recombinant protein 3AB antigen; the amino acid sequence of the PSV recombinant protein 3AB antigen is shown in SEQ ID NO: 4;

[0100] (b) Detecting whether the PSV recombinant protein 3AB antigen-antibody complex exists in the system of (a); if the complex exists, it indicates that PSV exists in the sample to be tested or the sample to be tested is a PSV-infected sample; otherwise, it indicates that PSV does not exist in the sample to be tested or the sample to be tested is a PSV-immunized sample.

[0101] In one or more embodiments, the detection method includes: (1) coating a solid phase carrier (such as an ELISA plate) with the PSV recombinant protein 3AB as an antigen; (2) washing the solid phase carrier; (3) blocking; (4) adding a sample for incubation; (5) adding an enzyme-labeled antibody for incubation; (6) color development; (7) terminating the reaction; and (8) reading the OD value.

[0102] In one or more embodiments, the detection method based on PSV recombinant protein 3AB is a method for non-disease diagnosis and treatment purposes.

[0103] In one or more embodiments, in step (1), the coating concentration of the PSV recombinant protein 3AB as an antigen is 0.25 μg / ml to 2 μg / ml, for example, 0.5 μg / ml to 2 μg / ml or 0.5 μg / ml to 1 μg / ml. In one or more preferred embodiments, the coating concentration of the recombinant protein 3AB as an antigen is 0.5±0.1 μg / ml.

[0104] In one or more embodiments, in step (1), the coating is to dilute the recombinant protein 3AB with a coating buffer and coat at 37±1° C. for 1 to 3 hours. In one or more specific embodiments, the coating buffer is a phosphate buffer.

[0105] In one or more embodiments, in step (2), the solid phase carrier (such as an ELISA plate) is washed by washing with phosphate-tween buffer (such as PBST).

[0106] In one or more embodiments, in step (3), the blocking is: adding a blocking solution for blocking. In one or more embodiments, the blocking solution is skim milk powder or BSA. In one or more embodiments, the blocking solution includes but is not limited to: 5% to 15% skim milk powder, 1% to 3% BSA buffer; preferably, the blocking solution is 10±1% skim milk powder.

[0107] In one or more embodiments, in step (4), the sample includes serum and plasma. In one or more preferred embodiments, the sample is serum. In one or more specific embodiments, the serum includes: positive sample serum and negative sample serum. As described in the present invention, the positive sample includes or is: serum of challenged pigs infected with PSV; the negative sample includes but is not limited to: serum of healthy pigs, serum of immune pigs, serum of pigs infected with other viruses (for example but not limited to: CSFV, BVDV, PEDV, PRV, FMDV, PRRSV). Therefore, the ELISA detection method described in the present invention can distinguish samples infected with PSV from samples not infected with PSV.

[0108] In one or more embodiments, in step (4), the sample (eg, serum) may be diluted before incubation, and the dilution factor may be 1:400 to 1:3200, for example, 1:1600.

[0109] In one or more embodiments, in step (5), the enzyme-labeled antibody is goat anti-pig IgG labeled with HRP.

[0110] In one or more embodiments, in step (5), the enzyme-labeled antibody may be diluted before incubation, and the dilution multiple may be 1:10000 to 1:25000, for example, 1:20000.

[0111] In one or more embodiments, in step (5), the incubation condition of the enzyme-labeled antibody can be 37±1° C. for 15 to 45 min.

[0112] In one or more embodiments, in step (6), the color development is: adding a color developing solution for color development. In one or more embodiments, the color developing solution is a TMB substrate solution. In one or more embodiments, the reaction conditions of the color development can be: color development at 37±1°C for 5 to 30 minutes, for example, color development at 37±1°C for 5 to 15 minutes.

[0113] In one or more embodiments, in step (7), the termination reaction is: adding a stop solution to stop the color development. In one or more embodiments, the stop solution is H2SO4.

[0114] It should be understood that the conditions not specifically described in the method are known to those skilled in the art. For example, before adding the next reaction solution (e.g., blocking solution, sample, enzyme-labeled antibody, color development solution or stop solution) to the solid phase carrier (e.g., ELISA plate), the previous reaction solution is removed (e.g., blotted, dried or patted dry) by a washing step. Usually, the number of washings is greater than 1, for example, 2, 3 or more times.

[0115] It should be understood that in step (8), the OD value reading is related to the label of the enzyme-labeled antibody and the color developing solution. When the label is HRP and the color developing solution is TMB substrate, the OD value reading is the OD value read at a wavelength of 450nm (for example, using an ELISA reader).

[0116] In one or more embodiments, in step (8), the OD value reading includes: the sample reading infected with PSV (positive sample reading, also known as P value) and the sample reading not infected with PSV (negative sample reading, also known as N value). In one or more embodiments, the P / N value is the ratio of the P value to the N value.

[0117] Preparation of PSV recombinant protein 3AB

[0118] The amino acid sequence of the “PSV recombinant protein 3AB” of the present invention is shown in SEQ ID NO:4.

[0119] In the detection method or kit of the present invention, PSV recombinant protein 3AB is used as the coating antigen. The preparation method of the coating antigen comprises the following steps:

[0120] (1) PSV 3AB gene amplification: PCR amplification of the PSV 3AB gene to obtain the PSV 3AB gene;

[0121] (2) Construction of recombinant plasmid pET-28a-3AB: pET-28a plasmid was digested with restriction endonuclease, PSV 3AB gene was connected to pET-28a plasmid after digestion with ligase, and transformed into Escherichia coli BL21 (DE3) competent cells for culture, and then PCR was performed to identify positive clones to obtain PCR-positive bacterial liquid, and the plasmid of PCR-positive bacterial liquid was extracted, and the positive recombinant plasmid pET-28a-3AB was obtained by enzyme digestion and identification;

[0122] (3) Induced expression of recombinant protein pET-28a-3AB: The positive recombinant plasmid pET-28a-3AB was transformed into Escherichia coli BL21 (DE3) expression bacteria, and IPTG was added to induce expression to obtain PSV 3AB recombinant protein;

[0123] (4) Obtaining PSV recombinant protein 3AB: Purifying the induced expressed PSV 3AB recombinant protein to obtain PSV recombinant protein 3AB.

[0124] In one or more embodiments, the PSV 3AB gene is amplified using primers of SEQ ID NOs: 1-2.

[0125] In one or more embodiments, the nucleotide sequence of the PSV 3AB gene is as shown in SEQ ID NO:3.

[0126] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0127] Materials and Methods

[0128] 1. Virus strains, bacterial strains and vectors

[0129] PSV strain SHCM2019 (Genbank accession number: MN685785), pET-28a(+) plasmid and expression host bacteria BL21(DE3).

[0130] 2. Reagents and equipment

[0131] Reagents: BCA Protein Assay Kit, reverse transcription kit, FastPfu, FastPfu buffer, nucleic acid marker, T4DNA ligase, 10K buffer, and restriction enzymes BamHⅠ and Hind IIⅠ were purchased from Bao Biotechnology (Dalian) Co., Ltd. Agarose gel DNA recovery kit (DP2019-03), plasmid extraction kit (DP103-03) and protein markers (MP205, MP206) were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; IPTG was purchased from Biovision, USA; horseradish peroxidase (HRP) labeled goat anti-swine IgG was purchased from Abcam; TMB substrate colorimetric solution was purchased from Wuhan Keqian Substrate Biological Products Co., Ltd.; porcine Sapelo virus challenge positive serum, healthy pig serum and immune pig serum samples were prepared and stored according to conventional methods.

[0132] Equipment: 4℃ centrifuge (SIGMA 3K30 / Z and 3K15), microplate reader (SpectraMax ABS full wavelength microplate reader), shaker (HYL-X modular shaker), magnetic stirrer (85-2A), electronic balance (DENVER INSTRUMENT SI-203), ultrasonic crusher (SONICS VCX750), ultra-high sensitivity rapid imaging system (Ameisham Imager 680), membrane transfer instrument (eBlot L1 rapid wet transfer instrument), 96-well microplate (labselect 31111B), plate washer (ExCellBio), PCR instrument (BIO-RAD), protein electrophoresis tank (BIO-RAD), gel imaging system (BIO-RAD), micropipette (Eppendorf), NC membrane (GenScript), room temperature high-speed centrifuge (Thermo Scentific Pico 21), dry thermostat (refrigeration type) JX-20 Tuohe constant temperature metal bath, vortex mixer (KMC-1300V), ice machine (Panasonic IMS-30 fully automatic snowflake ice machine),

[0133] 3. Serum samples

[0134] Healthy pig sera: 46 samples of healthy pig sera that were not challenged with PSV or immunized with vaccines.

[0135] Immunized pig sera: Healthy anti-PSV antibody-negative pigs were immunized with PSV inactivated vaccine, and pig sera were collected 21 to 60 days after the second immunization, for a total of 75 samples.

[0136] Both healthy pig serum and immune pig serum samples were negative serum samples.

[0137] Virus-challenged pig sera: Pig sera were collected 5, 7, and 14 days after healthy pigs were artificially infected with 50-100 TCID50 PSV, and used as positive sera, with a total of 62 samples.

[0138] Other serum samples: CSFV-positive serum was purchased from Beijing Jinbaiao Biotechnology Co., Ltd., and bovine viral diarrhea virus (Bovine vial diarrhoea virus, BVDV)-positive serum was purchased from Ingenasa, Spain; PEDV, PRV, FMDV, and PRRSV-positive serum were prepared and stored according to conventional methods.

[0139] 4. Primer design and synthesis

[0140] Referring to the PSV SHCM2019 strain sequence, a pair of specific primers were designed for the 3AB gene and combined with the pET-28a restriction site. The upstream primer F: 5'- GGATCC GGACCAGTGCAAGTGCCAGA-3' (SEQ ID NO: 1, the underlined part is the BamH I restriction site), downstream primer R: 5'- AAGCTT CTGCACGACTGCTTTCCTGAG-3' (SEQ ID NO: 2, the underlined part is the Hind III restriction site), synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the amplified fragment length is 366 bp.

[0141] 5. Construction and identification of recombinant plasmid pET-28a-3AB

[0142] Use an RNA extraction kit to extract RNA from PSV strain SHCM2019, use a reverse transcription kit to reverse transcribe the RNA into cDNA, and use cDNA as a template for RT-PCR amplification.

[0143] RT-PCR reaction system: FastPfu polymerase 1μl, dNTP 5μl, FastPfu buffer 10μl, viral cDNA 2μl, upstream and downstream primers 1μl each, add ddH2O to make up to 50μl.

[0144] RT-PCR reaction conditions: 94℃ pre-denaturation for 4min; 95℃ denaturation for 30s, 64℃ annealing for 30s, 72℃ extension for 35s, 32 cycles; 72℃ extension for 8min. After detecting the PCR product by 1% agarose gel electrophoresis, the gel was cut to recover the 3AB fragment, and then double-digested with BamH I and Hind III and connected with the pET-28a vector with the same double enzyme digestion at 16℃ for 12h. After connection, it was transformed into Escherichia coli BL21 (DE3) competent cells, coated on a solid LB plate containing 50μg / ml kanamycin and inverted in a 37℃ incubator for 12h to 16h. After picking a single colony, shake the bacteria overnight. After the recombinant plasmid was identified by bacterial liquid PCR, it was sent to Boshang Biotechnology (Shanghai) Co., Ltd. for sequencing. At the same time, the plasmid was extracted for enzyme digestion verification, and the positive plasmid was named pET-28a-3AB.

[0145] 6. Inducible expression of recombinant protein pET-28a-3AB

[0146] The positive plasmid pET-28a-3AB was transformed into competent E. coli BL21 (DE3) cells, and the positive clones were picked and inoculated into LB medium containing 50 μg / ml kanamycin, and cultured at 37°C overnight. The next day, the positive bacterial solution cultured overnight was added to LB medium (800 ml) containing 50 μg / ml kanamycin at an inoculum of 1:100 and continued to be cultured. 600 When the pH reached 0.6, IPTG was added to a final concentration of 1 mM, and the cells were collected by centrifugation after continued cultivation for 8 h. The cells were resuspended in pre-cooled PBS and then ultrasonically disrupted at 4 °C, 12000 r / min, and centrifuged for 10 min. The supernatant and precipitate were collected for SDS-PAGE analysis.

[0147] 7. Purification and identification of recombinant protein pET-28a-3AB

[0148] After collecting the induced expression bacteria (LB medium containing 50 μg / ml kanamycin in a total volume of 800 ml), resuspend the bacteria with pre-cooled PBS, wash three times, add washing buffer (8 M urea, 50 mM Tris-HCl, 0.5 M NaCl, pH 8.0) and resuspend, then place on ice for ultrasonic treatment for 10 min (ultrasonication 4 s, stop 6 s), centrifuge at 4 ° C, 12000 r / min for 10 min after the end of ultrasonication, and collect the supernatant. The supernatant was added to the balanced His tag purification resin (mixed with the wash buffer and the His tag purification resin for 4 hours, and centrifuged at 4°C, 1000r / min for 1 minute, the wash buffer was discarded, and the previous operation was repeated twice to reach equilibrium), and after binding on a 4°C shaker for 3 hours, it was added to the purification column, and the filler was washed with 3 column volumes of wash buffer and wash buffer (8M urea, 20mM imidazole, 50mM Tris-HCl, 0.5M NaCl, pH8.0), and the protein was eluted with 2 column volumes of elution buffer (8M urea, 200mM imidazole, 50mM Tris-HCl, 0.5M NaCl, pH 8.0). The target protein was desalted and concentrated by ultrafiltration centrifuge tube, and finally the concentration was determined by BCA protein quantification kit, and the aliquot was stored at -80°C. Take 40 μl of purified 3AB protein and add 1 / 4 volume of 5×SDS-PAGE Loading Buffer, mix thoroughly, boil for 10 minutes, and perform SDS-PAGE electrophoresis. After the electrophoresis, take out the gel and stain it with Coomassie Brilliant Blue staining solution for 30 minutes. Then put the protein gel on a shaker at room temperature overnight to decolorize, take a picture in the gel imaging system and save it. At the same time, perform Western blot identification on the purified 3AB protein, the steps are as follows:

[0149] (1) Add 1 / 4 volume of 5× SDS-PAGE Loading Buffer to the purified 3AB protein sample, mix thoroughly, boil for 10 min, and load 10 μl per well for SDS-PAGE electrophoresis;

[0150] (2) After electrophoresis, add 5 ml of equilibration solution to the WB incubation box, place the cut NC membrane into the WB incubation box, and soak both sides for about 1 min;

[0151] (3) Place the filter paper, NC membrane, gel, and filter paper in the transfer apparatus in order from bottom to top, and remove the bubbles. Turn on the power, start the eBlot L1 fast wet transfer apparatus, and transfer the membrane.

[0152] (4) Gently remove the NC membrane after transfer and wash the NC with TBST buffer three times, each time for 15 minutes;

[0153] (5) Block with rapid blocking solution at room temperature for 30 min;

[0154] (6) After blocking, wash three times with TBST, 15 min each time, dilute mouse 3AB polyclonal antibody (primary antibody) at 1:1000 with TBST, add to the NC membrane, and incubate at 37°C, 180 rpm, on a shaker for 2 h;

[0155] (7) After the primary antibody incubation, the membrane was washed three times with TBST for 15 min each time, and HRP-labeled goat anti-mouse IgG (H+L) (secondary antibody) was diluted 1:2000 with TBST and added to the NC membrane, and incubated at 37°C on a shaker at 180 rpm for 1 h;

[0156] (8) After the secondary antibody incubation, wash three times with TBST, each time for 15 min, then add an appropriate amount of chemiluminescent reagent to the NC membrane for color development, expose it for observation, and take pictures.

[0157] 8. Establishment and optimization of indirect ELISA serum antibody differential diagnosis method

[0158] 8.1. Selection of optimal antigen coating concentration and serum dilution

[0159] Using the square array titration method, phosphate buffer was used as the coating solution to dilute the 3AB protein to 2μg / ml, 1μg / ml, 0.5μg / ml, and 0.25μg / ml. Each concentration was coated in two columns, 50μl was coated in each well, and the coating was carried out at 37℃ for 2h. Wash with PBST (0.05% Tween-20 in 0.01mol / L PBS, pH7.4) washing solution for 5 times, 3min each time, and pat dry after washing. Add blocking solution, 200μl / well, block at 37℃ for 1h, wash as above, and pat dry after washing. Dilute the negative and positive samples with PBS at a gradient of 1:400, 1:800, 1:1600, and 1:3200, respectively, add them to the ELISA reaction plate, 100μl / well, act at 37℃ for 1h, and wash as above. After patting dry, add 1:20000 diluted HRP-labeled goat anti-swine IgG enzyme-labeled secondary antibody, 100μl / well, react at 37℃ for 30min, wash and pat dry as above. Add TMB substrate solution, 100μl / well, react at 37℃ in the dark for 10min. Add 2mol / L H2SO4, 100μl / well, terminate the reaction, read the sample at 450nm wavelength on the microplate reader, the reading of the positive sample is the P value, the reading of the negative sample is the N value, and the P / N value is the ratio of the P value to the N value. The antigen concentration and serum dilution of the hole with the maximum P / N value are taken as the optimal antigen coating concentration and serum dilution.

[0160] 8.2. Optimal blocking solution concentration

[0161] Coat the ELISA plate with the optimal coating concentration of the antigen determined in 8.1, dilute skim milk powder with PBST to a final concentration of 5%, 10%, and 15%, and dilute BSA to a final concentration of 1% and 3%, add 100 μl of blocking solution to each well, and block at 37°C for 1 hour. The other conditions are the same as above, and compare the P / N values ​​of each group of negative and positive standards to select the optimal blocking solution concentration.

[0162] 8.3. Optimal enzyme-labeled antibody action time and optimal enzyme-labeled antibody dilution

[0163] After adding HRP-labeled goat anti-swine IgG enzyme-labeled antibody to the ELISA plate at the recommended dilution multiple (1:20000), the plate was incubated at 37°C for 30 min, 45 min, and 60 min, respectively. The other steps were the same as above. The P / N values ​​of each group of negative and positive samples were compared to select the appropriate enzyme-labeled antibody action time.

[0164] 8.4 Determination of critical value

[0165] 149 samples of pig sera (48 samples of infected pig sera, 60 samples of vaccinated pig sera, and 41 samples of healthy negative pig sera) were collected and tested for OD under optimized conditions. 450nm Graph Pad Prism 8.0.2 software was used to perform receiver operating characteristic (ROC) curve analysis and calculate the optimal critical value.

[0166] 8.5 Repeatability test

[0167] Intra-batch repeat test: The purified 3AB protein from the same batch was coated on the ELISA plate, and 12 samples with different serum antibody levels were taken for ELISA test (6 PSV-positive sera and 6 PSV-immune sera). Each sample was repeated 3 wells, and the test results were statistically analyzed and the standard deviation SD and mean value were calculated. and coefficient of variation CV.

[0168] Batch repeat test: 3AB proteins purified from 3 different batches were coated on ELISA plates, and 12 groups of samples with different serum antibody levels were taken for ELISA test (6 PSV-positive sera and 6 PSV-negative sera). Each sample was repeated 3 wells, and the test results were statistically analyzed and the standard deviation SD and mean value were calculated. and coefficient of variation CV.

[0169] 8.6. Sensitivity test

[0170] Three positive samples were taken and diluted into eight gradients of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800, and the test results were subjected to sensitivity analysis.

[0171] 8.7 Specificity Test

[0172] The method established above was used to detect CSFV, PEDV, PRV, PRRSV, FDM, BVDV and PSV positive sera as well as PSV immune sera to evaluate the specificity of the method.

[0173] 8.8. Clinical Sample Testing

[0174] The ELISA method established by the present invention is used to detect samples with known serum antibody levels.

[0175] 9. Sequence

[0176] PSV recombinant protein 3AB nucleotide sequence (SEQ ID NO: 3)

[0177] GGACCAGTGCAAGTGCCAGAATGCGTGAGGGAAGATGAGGTGAAGAAGAAGAAGGTTAATTCAGAGAGAGACATCCCACATGATGTAATGGAGCTTGTTAGATGCACTAAATCCCC

[0178] AATTATAATAGATGAGTTAGAAAA

[0179] GGCTGGTTTTATTATTCCTGTGGAAGCTGAAGTCATACGCCAGACCACTAATGTAAA

[0180] TAATGTAACACAG

[0181] ATTGTTTCAGCAACTTTAGCTAGTTTGGCAGCTATAATTTCTGTAGGGACTGTAGTTTATTTAATGGTTAGGTTGTTTTCCTCGAAGCAAGGAGCATACTCTGGCGCACCTAAACCAGAAACCAGGAAACCTGTACTCAGGAAAGCAGTCGTGCAG

[0182] PSV recombinant protein 3AB amino acid sequence (SEQ ID NO: 4)

[0183] GPVQVPECVREDEVKKKKVNSERDIPHDVMELVRCTKSPIIIDELEKAGFIIPVEAEVIRQ

[0184] TTNVNNVTQIVSATLASLAAIISVGTVVYLMVRLFSSKQGAYSGAPKPETRKPVLRKAVVQ

[0185] Example

[0186] Example 1. Construction and identification of expression vector

[0187] The 3AB gene was amplified using the cDNA of PSV SHCM2019 strain as a template. After agarose gel electrophoresis analysis, a band of approximately 366 bp was obtained, which was consistent with the expected size (see Figure 1 ).

[0188] The 3AB gene was cloned into the pET-28a prokaryotic expression vector to obtain the recombinant plasmid pET-28a-3AB. The recombinant plasmid was identified by double digestion with restriction endonucleases EcoR I and Sal I. The results were as follows (see Figure 2 ) showed a vector band of about 5490 bp and a target band of about 366 bp, the size of which was consistent with the expectation. At the same time, the positive recombinant plasmid was sequenced, and the sequencing results were consistent with the target sequence.

[0189] Example 2: Inducible expression of pET-28a-3AB recombinant protein

[0190] IPTG was used to induce expression in the pET-28a-3AB recombinant bacterial solution. After ultrasonic disruption of the bacterial cells, the supernatant and precipitate were centrifuged and analyzed by SDS-PAGE. The results showed that the size of the expressed protein was about 19 kDa, which was consistent with the predicted results, and the expressed target protein mainly existed in the form of inclusion bodies ( Figure 3 ).

[0191] Example 3: Purification and specificity test of recombinant protein pET-28a-3AB

[0192] The protein was dissolved in 8M urea, and the supernatant was collected after ultrasonic disruption and purified by His affinity chromatography ( Figure 4 ). SDS-PAGE electrophoresis showed that the target protein could be eluted after adding elution buffer containing 200mM imidazole ( Figure 4 The purified protein was then desalted and concentrated using an ultrafiltration centrifuge tube. The protein concentration was measured using a BCA protein concentration assay kit and was approximately 726 μg / ml. SDS-PAGE electrophoresis showed that the purified PSV 3AB protein had a single band ( Figure 5 ). Further Western blot detection using mouse 3AB polyclonal antibody showed that PSV 3AB protein could be specifically recognized by 3AB polyclonal antibody ( Figure 6 ), indicating that PSV 3AB protein has good reactogenicity.

[0193] Example 4. Optimal antigen coating concentration and positive serum dilution

[0194] The results of the array titer are shown in Table 1. When the antigen coating concentration is 0.5 μg / ml, the P / N value is the largest. Therefore, the optimal antigen coating concentration is 0.5 μg / ml, and the optimal positive sample dilution multiple is 1:1600.

[0195] Table 1 Matrix titer (OD 450nm )

[0196]

[0197]

[0198] Example 5, Optimal Concentration of Blocking Solution

[0199] Blocking was performed with different concentrations of skim milk powder and BSA. The results are shown in Table 2. The P / N value was the largest when blocking with 10% skim milk powder, which was determined to be the optimal blocking solution concentration.

[0200] Table 2 Selection of optimal blocking solution concentration

[0201]

[0202] Example 6. Optimal enzyme-labeled antibody dilution and optimal enzyme-labeled antibody action time

[0203] Based on the optimal conditions explored in Examples 1 to 5 (i.e., the optimal coating concentration of PSV recombinant protein 3AB is 0.5 μg / ml, the optimal dilution of the sample is 1:1600; the optimal blocking solution is 10% skim milk powder-PBST), the working conditions of the enzyme-labeled secondary antibody were measured. The results are shown in Table 3. After the enzyme-labeled antibody was added, when the dilution was 1:20000, the P / N value was the highest at 37°C for 30 min.

[0204] Table 3 Selection of optimal enzyme-labeled antibody dilution and enzyme-labeled antibody action time

[0205]

[0206] Example 7, Optimal Substrate Action Time

[0207] Based on the optimal conditions explored in Examples 1 to 6 (i.e., the optimal coating concentration of PSV recombinant protein 3AB is 0.5 μg / ml, the optimal dilution of the sample is 1:1600; the optimal blocking solution is 10% skim milk powder-PBST; the optimal dilution of HRP-labeled goat anti-pig IgG is 1:20000, and the optimal incubation time is 30 min), different substrate reaction times are used for ELISA detection in this example.

[0208] The results showed that the optimal reaction time of the substrate was 10 min.

[0209] Table 4 Selection of the optimal reaction time of substrate

[0210]

[0211] Example 8, determination of critical value

[0212] The ELISA method established by the present invention (i.e., the optimal coating concentration of PSV recombinant protein 3AB is 0.5 μg / ml, the optimal dilution of the sample is 1:1600; the optimal blocking solution is 10% skim milk powder-PBST; the optimal dilution of HRP-labeled goat anti-swine IgG is 1:20000, the optimal incubation time is 30 minutes; the optimal color development time is 10 minutes) is used to detect the serum OD450nm values ​​of healthy negative (41 samples), vaccinated (60 samples) and infected pig sera (48 samples), and the most appropriate critical value is determined by ROC curve analysis. Figure 7 The ROC curve analysis results shown are based on the OD corresponding to the maximum value of the Youden index. 450nm As the optimal critical value, that is, 0.417. The area under the ROC curve is 0.9994, and the 95% confidence interval is 99.78% to 100.0%, indicating that the ELISA method can effectively distinguish between positive (infected pig sera) and negative sample groups (vaccinated and healthy pig sera). Figure 8 ) were further analyzed for diagnostic sensitivity and specificity.

[0213] The results showed that the sensitivity was 97.92% and the specificity was 100.0%, indicating that the ELISA method was suitable as a diagnostic test method.

[0214] Example 9, Repeatability Test

[0215] The established ELISA method was used (the optimal coating concentration of PSV recombinant protein 3AB was 0.5 μg / ml, the optimal dilution of the sample was 1:1600; the optimal blocking solution was 10% skim milk powder-PBST; the optimal dilution of HRP-labeled goat anti-swine IgG was 1:20000, the optimal incubation time was 30 min; the optimal color development time was 10 min, and the OD 450nm The optimal critical value was 0.417) and 6 PSV-positive sera and 6 PSV-negative sera were tested intra- and inter-batch.

[0216] The results showed that the coefficient of variation of the intra-batch repeated tests was 0.22%-4.61%, and the coefficient of variation between batches was 1.03%-7.38%, both of which were less than 10%, indicating that the method had good stability and high repeatability.

[0217] Table 6 Repeatability test

[0218]

[0219]

[0220] Example 10, sensitivity test

[0221] Three PSV-positive serum samples were taken and diluted at 8 dilutions, namely 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800, and ELISA detection was performed using the same method as in Example 9.

[0222] The results are as follows Fig. 9 As shown, when the serum was diluted to 1:12800 times, all three positive samples were detected as positive, indicating that the method has high sensitivity.

[0223] Example 11, specificity test

[0224] Although other viruses also have proteins named 3AB, Fig.11 As shown in the figure, the sequence alignment of 3AB genes from Coxsackievirus, Echovirus, Poliovirus, Enterovirus, Rhinovirus, Sapelovirus (PSV), Cardiovirus, Hepatovirus and Foot-and-mouth (FMDV) showed that the sequence of PSV 3AB was significantly different from that of 3AB from other viruses. The sequence identity of PSV3AB and 3AB of Foot-and-mouth (FMDV) was only about 31.9%.

[0225] Further, the same method as in Example 9 was used to detect CSFV, PEDV, PRV, PRRSV, FMDV, BVDV, and PSV positive and negative sera. The detection conditions were: the coating concentration of PSV recombinant protein 3AB was 0.5 μg / ml, the sample dilution was 1:1600; the blocking solution was 10% skim milk powder-PBST; the HRP-labeled goat anti-swine IgG dilution was 1:20000, the incubation time was 30 minutes; the color development time was 10 minutes, and the OD 450nm The critical value was 0.417. The results showed that only PSV serum was positive, indicating that the method has strong specificity and does not cross-react with other pathogens ( Fig.10 ).

[0226] Example 12: Clinical sample testing

[0227] The same method as in Example 9 was used to test known serum samples, including 62 PSV-positive sera, 75 PSV-immune sera (16 sera from 21 days after second immunization, 19 sera from 28 days after second immunization, 37 sera from 35 days after second immunization, and 3 sera from 60 days after second immunization), and 46 negative sera.

[0228] The results showed that 61 out of 62 positive sera were positive and 1 was negative, with a compliance rate of 98.39%; 75 out of 75 immune pig sera were negative, with a compliance rate of 100.0%; 46 out of 46 negative samples were negative, with a compliance rate of 100.0%, indicating that the differential diagnosis ELISA established by the present invention can effectively distinguish PSV-infected and vaccinated pigs.

[0229] Table 8 Clinical sample test results

[0230]

[0231] discuss

[0232] The present invention expresses and prepares the PSV nonstructural protein 3AB and uses it as an antigen to establish an indirect ELISA method that can distinguish PSV vaccine-immunized animals from infected animals. The performance of this ELISA detection method is evaluated using a series of sera collected from uninfected, infected and vaccinated animals.

[0233] PSV was first discovered in the UK in 1958. Currently, there are reports of PSV in countries around the world, but PSV has only one serotype. In recent years, epidemiological surveys on PSV have found that the infection rate of PSV is on the rise. In order to control diseases caused by porcine Sapelo virus, inactivated immune vaccines can be injected to play a preventive role. At the same time, in order to prevent the abuse of vaccines, the differential diagnosis and detection methods of immune animals and infected animals are very important in the prevention and control of PSV. Therefore, according to the research and development of FMD detection methods, an ELISA method was established using PSV 3AB protein for differential diagnosis of PSV infection and vaccine-immunized animals.

[0234] The PSV polyprotein 3AB was expressed using the E. coli prokaryotic expression system, and the 3AB protein was expressed in the form of inclusion bodies. The hydrophilic N-terminus of the recombinant protein is crucial to increase its solubility when expressed in E. coli. Therefore, the structural properties of 3AB were analyzed using the Hopp and Woods prediction method, and it was found that the N-terminus of 3A has weak hydrophilicity. On SDS-PAGE, it was observed that the size of the His-tagged 3AB protein differed by nearly 6 kDa from the calculated size of about 13 kDa. This was due to the size of the His-tagged protein itself in the expression vector, but it had no effect on the target protein. In the FMDV study, 3AB and 3ABC were considered to be the best antigens, and the established methods and the detected data were reliable. 3AB and 3ABC proteins are early products after translation, among which 3C protein has protease hydrolysis activity and is responsible for cleavage during the expression process. In the expression of 3AB protein, the baculovirus expression system was used to express this type of protein in the early stage. However, this method requires cell culture, which usually produces lower protein yields than bacterial systems, so the cost is higher, while the E. coli expression system is the opposite. The PSV 3AB protein has poor solubility. The inventors used 8M urea to dissolve the inclusion bodies and denature them, and then purified them. Finally, they used ultrafiltration tubes to desalt and concentrate the purified 3AB protein to renature it into its natural structure.

[0235] An indirect ELISA method for capturing antibodies in porcine serum was established using 3AB. Compared with competitive ELISA targeting only one or two antigen epitopes, an indirect ELISA format was selected due to the possibility of increased sensitivity caused by the binding of multiple antigen epitopes in serum. The purified 3AB protein showed good reactivity by Western-blotting. The optimization results of the ELISA method conditions showed that the best coating concentration of antigen was 0.5μg / ml, the best serum dilution was 1:1600, the best blocking solution was 5% skim milk powder, and the best enzyme-labeled secondary antibody working concentration was 1:20000 and acted at 37℃ for 30min. A total of 149 positive sera, immune sera and negative sera were used. The optimal critical value was determined to be 0.417 by ROC curve analysis, and its diagnostic sensitivity and specificity were 97.92% and 100.0%, respectively. The method has good repeatability, sensitivity and specificity. This method was used to test 149 known serum samples, and 61 out of 62 positive sera were correctly detected, with a detection compliance rate of 98.39%. Both vaccinated and healthy negative sera were correctly detected, with a detection compliance rate of 100%, indicating that this method is applicable to the differential diagnosis of PSV-infected and vaccine-immunized animals.

[0236] In summary, the present invention uses a prokaryotic expression system to express and purify the PSV 3AB protein, and establishes a differential diagnosis i-ELISA method that can distinguish PSV-infected, healthy and vaccine-immunized animals, which provides a guarantee for the detection of PSV and the control of vaccine abuse.

[0237] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. Use of porcine Sapelo virus (PSV) recombinant protein 3AB in the preparation of a reagent / kit for distinguishing between PSV infection and immunity, or for distinguishing whether PSV is infected; the amino acid sequence of the PSV recombinant protein 3AB is shown in SEQ ID NO:

4.

2. The use according to claim 1, characterized in that The reagent / kit comprises: a PSV recombinant protein 3AB antigen coated on a solid phase carrier; by detecting the PSV recombinant protein 3AB antigen, PSV infection and immunity, or whether PSV infection or not can be distinguished; Preferably, the detection method comprises: (a) adding a sample to be tested to a solid phase carrier coated with PSV recombinant protein 3AB antigen; the amino acid sequence of the PSV recombinant protein 3AB antigen is shown in SEQ ID NO: 4; (b) detecting whether the PSV recombinant protein 3AB antigen-antibody complex exists in the system of (a); if the complex exists, it indicates that PSV exists in the sample to be tested or the sample to be tested is a PSV-infected sample; otherwise, it indicates that PSV does not exist in the sample to be tested or the sample to be tested is a PSV-immunized sample; More preferably, the detection method further comprises one or more of the following steps: washing the solid phase carrier, blocking, adding a sample for incubation, adding an enzyme-labeled antibody for incubation, color development, terminating the reaction and reading the OD value.

3. The use according to claim 1 or 2, characterized in that: The reagent / kit is an ELISA detection reagent / kit; preferably an indirect ELISA detection reagent / kit.

4. The use according to any one of claims 1 to 3, characterized in that: The reagents / kit also include: blocking solution, washing solution, positive control, negative control, enzyme-labeled antibody, color development solution, and stop solution; Preferably, the blocking solution is 5% to 15% skim milk powder, more preferably 10±1% skim milk powder; Preferably, the washing solution is phosphate-buffered saline (PBST), more preferably PBST; Preferably, the positive control comprises or is a PSV-positive serum sample; Preferably, the negative control includes PSV negative serum samples, PSV immune serum samples, and other virus serum samples; more preferably, the other viruses include CSFV, BVDV, PEDV, PRV, FMDV, and PRRSV; Preferably, the enzyme-labeled antibody is HRP goat anti-swine IgG; Preferably, the color developing solution is a TMB substrate color developing solution; Preferably, the stop solution is H2SO4.

5. The use according to any one of claims 1 to 4, characterized in that: The detection method includes one or more of the following features: (A) The coating concentration of the PSV recombinant protein 3AB is 0.25-2 μg / ml, preferably 0.5-2 μg / ml, and more preferably 0.5±0.1 μg / ml; (B) The dilution factor of the sample is 1:400 to 1:3200, preferably 1:1600; (C) The dilution multiple of the enzyme-labeled antibody is 1:10000 to 1:25000, preferably 1:20000; (D) The incubation condition of the enzyme-labeled antibody is 37±1°C for 15 to 45 minutes; (E) The reaction conditions for the color development are 5 to 30 minutes at 37±1°C, preferably 5 to 15 minutes at 37±1°C; (F) The OD value is the OD value at a wavelength of 450 nm.

6. The use according to any one of claims 1 to 5, characterized in that: The preparation method of the PSV recombinant protein 3AB comprises: amplifying the gene sequence of the PSV recombinant protein 3AB; transferring it into a vector to construct a recombinant plasmid; inducing the expression of the recombinant plasmid; and separating and purifying to obtain the PSV recombinant protein 3AB; Preferably, the PSV recombinant protein 3AB gene sequence is amplified using primers such as SEQ ID NO: 1 and SEQ ID NO: 2; Preferably, the vector is a pET-28a vector; Preferably, the induced expression is induced expression by adding IPTG.

7. A method for detecting porcine Sapelo virus (PSV) for non-diagnostic purposes, comprising: (a) adding a sample to be tested to a solid phase carrier coated with PSV recombinant protein 3AB antigen; the amino acid sequence of the PSV recombinant protein 3AB antigen is shown in SEQ ID NO: 4; (b) detecting whether the PSV recombinant protein 3AB antigen-antibody complex exists in the system of (a); if the complex exists, it indicates that PSV exists in the sample to be tested or the sample to be tested is a PSV-infected sample; otherwise, it indicates that PSV does not exist in the sample to be tested or the sample to be tested is a PSV-immunized sample; Preferably, the method further comprises one or more of the following steps: washing the solid phase carrier, blocking, adding a sample for incubation, adding an enzyme-labeled antibody for incubation, color development, terminating the reaction, and reading the OD value; More preferably, the detection method includes one or more of the following features: (A) The coating concentration of the PSV recombinant protein 3AB is 0.25-2 μg / ml, preferably 0.5±0.1 μg / ml; (B) the dilution factor of the sample is 1:400 to 1:3200, preferably 1:1600; (C) The dilution factor of the enzyme-labeled antibody is 1:10000 to 1:25000, preferably 1:20000; (D) The incubation condition of the enzyme-labeled antibody is 37±1°C for 15 to 45 minutes; (E) The color development reaction conditions are: color development at 37±1°C for 5 to 30 minutes, preferably color development at 37±1°C for 5 to 15 minutes; (F) The OD value is the OD value at a wavelength of 450 nm; (G) The blocking solution is 5% to 15% skim milk powder, preferably 10±1% skim milk powder; (H) The sample is a serum sample; more preferably, the serum sample includes a PSV-positive serum sample, a PSV-negative serum sample, a PSV-immune serum sample, or other virus serum samples; more preferably, the other viruses include CSFV, BVDV, PEDV, PRV, FMDV, and PRRSV; (I) the enzyme-labeled antibody is HRP goat anti-pig IgG; (J) the color developing solution is a TMB substrate color developing solution; (K) The stop solution is H2SO4.

8. Use of porcine sapelo virus (PSV) recombinant protein 3AB in identifying porcine sapelo virus (PSV) for non-diagnostic purposes, wherein the amino acid sequence of the PSV recombinant protein 3AB is shown in SEQ ID NO: 4; Preferably, the application includes: (a) adding a sample to be tested to a solid phase carrier coated with PSV recombinant protein 3AB antigen; the amino acid sequence of the PSV recombinant protein 3AB antigen is shown in SEQ ID NO: 4; (b) detecting whether the PSV recombinant protein 3AB antigen-antibody complex exists in the system of (a); if the complex exists, it indicates that PSV exists in the sample to be tested or the sample to be tested is a PSV-infected sample; otherwise, it indicates that PSV does not exist in the sample to be tested or the sample to be tested is a PSV-immunized sample; More preferably, the method for determining whether the 3AB antigen-antibody complex exists comprises: if the OD value is greater than a threshold value, determining that the sample to be tested is a PSV-infected sample; Otherwise, the sample to be tested is judged to be a PSV immune sample; more preferably, the threshold value is 0.417; More preferably, the detection method is as defined in any one of claims 1 to 6 or as described in claim 7.

9. A method for distinguishing whether a sample to be tested is infected with PSV, comprising: (a) using the reagent / kit defined in any one of claims 1 to 6 to detect the recombinant protein 3AB in the sample to be tested, and reading the OD value; (b) judging whether the sample is a PSV-infected sample or a PSV-uninfected sample according to the OD value obtained in (a); if the OD value is greater than a threshold value, judging that the sample to be tested is a PSV-infected sample; otherwise, judging that the sample to be tested is a PSV-uninfected sample; Preferably, the threshold is 0.

417.

10. A method for distinguishing PSV infection or immunity in a sample to be tested, comprising: (a) using the reagent / kit defined in any one of claims 1 to 6 to detect the recombinant protein 3AB in the sample to be tested, and reading the OD value; (b) judging whether the sample is a PSV-infected sample or a PSV-immune sample according to the OD value obtained in (a); if the OD value is greater than a threshold value, judging that the sample to be tested is a PSV-infected sample; otherwise, judging that the sample to be tested is a PSV-immune sample; Preferably, the threshold is 0.417.

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