A bivalent nanobody against a consensus epitope of genotype 1 and 2 porcine reproductive and respiratory syndrome virus and preparation method and application thereof

Abstract

The application discloses a bivalent nanobody against common epitopes of genotype 1 and 2 porcine reproductive and respiratory syndrome viruses and a preparation method and application thereof, and belongs to the technical field of animal epidemic disease detection. The application provides a bivalent nanobody against common epitopes of genotype 1 and 2 PRRSV and a preparation method and application thereof, and an expression preparation method of a fusion protein of the bivalent nanobody and HRP, and simultaneously evaluates the application of the fusion protein of the bivalent nanobody and HRP in detecting anti-genotype 1 and 2 PRRSV antibodies in pig serum.

Description

Technical Field

[0001] The present invention relates to the technical field of animal disease detection, and in particular to a bivalent nanobody against a common epitope of porcine reproductive and respiratory syndrome virus type 1 and type 2, and a preparation method and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an acute, highly contagious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). It primarily causes reproductive disorders in sows, including abortion, premature birth, and weak piglets, as well as respiratory disorders and growth retardation in piglets and finishing pigs. It is a highly contagious disease that severely harms the swine industry. The latest classification standard has divided the two original genotypes of PRRS into two distinct species: Betaarterivirus suid 1 (European type / PRRSV-1) and Betaarterivirus suid 2 (North American type / PRRSV-2). In my country, PRRSV-2 is the predominant strain. However, in recent years, PRRSV-1 infections have been reported in various farms across China. Epidemiological surveys have shown that PRRSV-1 infections in swine farms are often co-infected with PRRSV-2. Furthermore, monitoring anti-PRRSV antibodies is a primary method for evaluating vaccine immunity and monitoring PRRSV infection in pig farms. However, PRRS antibody detection kits currently on the domestic market are primarily imported, and domestically produced products are unstable. Therefore, it is necessary to develop an ELISA kit with high sensitivity, good stability, and the ability to simultaneously detect antibodies against genotype 1 and 2 PRRSV to meet the clinical needs of PRRS antibody testing.

[0003] Nanobodies, as a new type of third-generation genetically engineered antibody, are the smallest known antibodies with antigen-binding ability. Compared with traditional monoclonal antibodies, their molecular weight is only about 15kDa, making them easy to express, prepare, and genetically engineer in vitro. For example, nanobodies can be coupled with reporter genes (green fluorescent protein, horseradish peroxidase, etc.) to prepare fusion proteins, which can be widely used in the development of animal disease detection technologies. Currently, there are many papers reporting the application of nanobodies in the development of animal disease detection methods, such as the establishment of competitive ELISA methods based on nanoantibodies for African swine fever and avian influenza antibodies. This type of method uses a fusion protein of nanoantibodies and horseradish peroxidase (HRP) as a competitive probe, eliminating the need for in vitro labeling and the use of enzyme-labeled secondary antibodies, avoiding batch instability of labeled antibodies and reducing the complexity of the production process. At the same time, nanoantibodies and HRP fusion proteins can be mass-produced using yeast or suspension cell culture, reducing production costs and having good market application prospects.

[0004] There have been many research reports on methods for detecting PRRSV antibodies. For example, Sorensen et al. developed a double-blocking ELISA method that can distinguish between genotype 1 and 2 PRRSV antibodies, but whether this method is suitable for detecting currently prevalent strains remains to be verified. Brown et al. developed a dual ELISA based on the PRRSV Nsp7 protein that can distinguish between genotype 1 and 2 PRRSV antibodies, but this has not yet been applied in the Chinese market. Methods and kits for detecting anti-genotype 1 and 2 PRRSV antibodies based on nanoantibody technology have not yet been reported. Currently, the main clinical test kit for detecting anti-genotype 1 and 2 PRRSV antibodies in my country uses the IDEXX PRRS X3 Ab kit as the gold standard. This kit is developed based on the indirect ELISA method and is expensive. Summary of the Invention

[0005] The purpose of the present invention is to provide a bivalent nanobody against the common epitope of porcine reproductive and respiratory syndrome virus type 1 and type 2, and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a fusion protein of a bivalent nanobody against gene type 1 and 2 PRRSV N protein and horseradish peroxidase, the amino acid sequence of which is shown in SEQ ID NO.13.

[0008] The second technical solution of the present invention is a DNA molecule encoding the fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.14.

[0009] The fusion protein is applied to the preparation of a product for detecting anti-gen 1 and 2 type PRRSV antibodies.

[0010] The fourth technical scheme of the present application is a product for detecting anti-gen 1 and 2 type PRRSV antibodies, which comprises the fusion protein.

[0011] The fifth technical scheme of the present application is a method for detecting anti-gen 1 and 2 type PRRSV antibodies for non-disease diagnosis or treatment purposes, which comprises the following steps:

[0012] The ELISA plate is coated with PRRSV as an antigen, the fusion protein is used as a competitive reagent, the sample to be detected is mixed with the fusion protein and then added to the coated ELISA plate, and after incubation, the competition rate is calculated according to the OD value to determine whether the sample to be detected contains anti-gen 1 and 2 type PRRSV antibodies.

[0013] Based on the above technical scheme, the present application has the following technical effects:

[0014] The present application provides a bivalent nanobody against common epitopes of gen 1 and 2 type PRRSV, a preparation method and application thereof, and an expression and preparation method of the bivalent nanobody and HRP fusion protein, and simultaneously evaluates the application of the bivalent nanobody and HRP fusion protein in detecting anti-gen 1 and 2 type PRRSV antibodies in pig serum. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical schemes in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0016] Figure 1 PCR is used to amplify the gene sequences encoding the monovalent nanobodies PRRSV-N-1a3 and PRRSV-N-Nb3, respectively; wherein M is Maker; 1 is the amplification of the fusion of the monovalent nanobody PRRSV-N-1a3 gene and the rigid Linker; and 2 is the amplification of the fusion of the monovalent PRRSV-N-Nb3 gene and the rigid Linker.

[0017] Figure 2 PCR is used to amplify the gene sequences encoding the monovalent nanobodies PRRSV-N-1a3 and PRRSV-N-Nb3, respectively; wherein M is Maker; 1 is the amplification of the fusion of the monovalent nanobody PRRSV-N-1a3 gene and the rigid Linker; and 2 is the amplification of the fusion of the monovalent PRRSV-N-Nb3 gene and the rigid Linker.

[0018] Figure 3The gene sequence of the bivalent nanobody PRRSV-N-1a3-Nb3 in series and the double enzyme digestion results of the pCMV-N1-HRP empty vector, wherein M is Maker; 1 and 2 are the double enzyme digestion results of PRRSV-N-1a3-Nb3; 3 is the double enzyme digestion results of the pCMV-N1-HRP empty vector.

[0019] Figure 4 The recombinant positive plasmid pCMV-N1-HRP-PRRSV-N-1a3-Nb3 constructed by PCR identification of the bacterial liquid, wherein M is Maker, 2-4 are positive clones, and 1 is a negative control.

[0020] Figure 5 IFA identification of the expression of PRRSV-N-1a3-Nb3-HRP fusion protein in HEK-293T cells after the constructed bivalent nanobody and HRP fusion protein recombinant expression plasmid were transfected into HEK-293T cells.

[0021] Figure 6 ELISA analysis of the secretion expression of PRRSV-N-1a3-Nb3-HRP fusion protein into the culture supernatant of HEK-293T cells, and the titer of the fusion protein in the supernatant.

[0022] Figure 7 Western blot analysis of purified PRRSV particles, wherein M is Maker.

[0023] Figure 8 Competitive ELISA analysis and comparison of the competition rates of two coating antigens and the competition rates of three competitive probes by using prokaryotic expression and purified PRRSV-N protein and purified PRRSV particles as coating antigens, respectively, and using three fusion proteins PRRSV-N-1a3-HRP, PRRSV-N-Nb3-HRP and PRRSV-N-1a3-Nb3-HRP as competitive probes, respectively.

[0024] Figure 9 Using purified PRRSV particles as coating antigens and PRRSV-N-1a3-Nb3-HRP fusion protein as competitive probes, PRRSV antibody positive and negative pig serum was diluted at dilution ratios of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280, mixed into the fusion protein, and the sensitivity of the competitive ELISA detection method was analyzed.

[0025] Figure 10In order to take the purified PRRSV particles as the coated antigen and the PRRSV-N-1a3-Nb3-HRP as the competitive reagent, the PEDV positive serum, the TGEV positive serum, the PCV2 positive serum, the PRV positive serum and the PPV positive serum are mixed in the fusion protein at a dilution ratio of 1:10, and the determined PRRSV positive pig serum is used as the positive control to identify the specificity of the competitive ELISA detection method. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not restrictive of the application. It will be appreciated that those of ordinary skill in the art will be able to devise numerous alternative arrangements without departing from the scope of the application.

[0027] It should be understood that the terms used in the specification of the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated by reference, the content of the specification prevails.

[0029] Various modifications and changes can be made to the specific embodiments of the present application described in this specification without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples are illustrative only.

[0030] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".

[0031] The technical solutions described in the present application are conventional solutions in the art unless otherwise specified, and the reagents or raw materials used are purchased from commercial channels or are already disclosed.

[0032] An embodiment of the present invention provides a fusion protein of a bivalent nanobody against genotype 1 and 2 PRRSV N protein and horseradish peroxidase, the amino acid sequence of which is shown in SEQ ID NO.13.

[0033] The embodiment of the present invention also provides a DNA molecule encoding the fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.14.

[0034] The embodiment of the present invention also provides the use of the fusion protein in preparing a product for detecting antibodies against genotype 1 and 2 PRRSV.

[0035] An embodiment of the present invention also provides a product for detecting antibodies against genotype 1 and 2 PRRSV, comprising the fusion protein.

[0036] The present invention also provides a method for detecting antibodies against PRRSV genotypes 1 and 2 for purposes other than disease diagnosis or treatment, comprising the following steps:

[0037] An ELISA plate is coated with PRRSV as an antigen and the fusion protein as a competitive reagent. The sample to be tested is mixed with the fusion protein and then added to the coated ELISA plate. After incubation, the competition rate is calculated based on the OD value to determine whether the sample to be tested contains anti-gene type 1 and 2 PRRSV antibodies.

[0038] In some specific embodiments, the formula for calculating the competition rate based on the OD value is:

[0039] Competition rate = (1-OD of the pig serum sample to be tested 450nm Value / negative pig serum sample OD 450nm value)×100%.

[0040] In some specific embodiments, the method for determining whether the sample to be tested contains antibodies against genotype 1 and 2 PRRSV is:

[0041] If the competition rate of the pig serum to be tested is greater than 25.67%, it is judged as positive, otherwise it is negative.

[0042] In some specific embodiments, after the incubation reaction, the step of adding a color developing solution and a strong acid to terminate the reaction is further included;

[0043] The color developing solution is TMB color developing solution; the strong acid is 3M H2SO4.

[0044] In some specific embodiments, the incubation reaction conditions include: an incubation temperature of 37° C. and an incubation time of 1 h.

[0045] The present invention provides a bivalent nanobody against a common epitope of PRRSV genotypes 1 and 2, a preparation method thereof, a preparation method of a fusion protein of the bivalent nanobody and horseradish peroxidase (HRP), and the use of the fusion protein in detecting antibodies against PRRSV genotypes 1 and 2 in pig serum. Two nanobodies against different common epitopes of the nucleocapsid (N) protein of PRRSV genotypes 1 and 2 are connected in series using a rigid linker, and then the series-connected bivalent nanobodies are fused with HRP for expression to prepare a bivalent nanobody and HRP fusion protein. Purified PRRSV particles are used as coating antigens and the prepared fusion protein is used as a detection probe to establish a method for detecting antibodies against PRRSV genotypes 1 and 2 in pig serum. This method can simultaneously detect antibodies against PRRSV genotypes 1 and 2 in pig serum, is simple to operate, takes a short time to detect samples, does not require the use of enzyme-labeled secondary antibodies, has low production costs, and has a compliance rate of up to 95.90% with the existing IDEXX PRRSV antibody detection commercial kit. This tandem bivalent nanoantibody is a key material for the subsequent development of commercial kits for detecting antibodies against gene types 1 and 2 PRRSV, and has good market application prospects.

[0046] Example 1

[0047] Preparation of bivalent nanobody against the common epitope of PRRSV genotypes 1 and 2 and horseradish peroxidase fusion protein

[0048] 1.1 Construction of fusion protein eukaryotic expression vector

[0049] (1) Based on the gene sequences of two monovalent nanoantibodies (1a3 and Nb3) against the common epitopes of PRRSV N protein type 1 and type 2, specific primers were designed respectively. The primer sequences were synthesized by Xi'an Qingke Biotechnology Co., Ltd. and their sequences are shown in Table 1.

[0050] The amino acid sequence of the monovalent Nanobody 1a3 against the common epitope of genotype 1 and 2 PRRSV-N protein is (SEQ ID NO.1):

[0051] ESGGGSVQAGGSLRLSCAASGSTYYMGWFRKPPGKEREGVAALYTPSGSTYTA NSVKSRFTIKDAAKNTVPQMNSLQPEDTAMYYNAADRNPGGVLSSRGYHYWGQGT QVTVSS.

[0052] The nucleotide sequence encoding the above-mentioned common epitope monovalent Nanobody 1a3 is (SEQ ID NO.2):

[0053] GAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTACTACATGGGCTGGTTCCGAAAGCCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGCTCTTTATACTCCTAGTGGTAGCACATACACTGCCAACTCCGTGAAAAGCCGATTCACCATCAAAGACGCCGCCAAGAACACGGTGCCCCAAATGAACAGCCTACAACCTGAGGACACTGCCATGTACTACAATGCGGCAGATCGGAATCCGGGCGGGGTACTATCCTCCCGCGGGTATCACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0054] The amino acid sequence of the monovalent Nanobody® Nb3 against the common epitope of the type 1 and 2 PRRSV-N proteins is (SEQ ID NO. 3):

[0055] ESGGGSVQAGGSLTLSCAASGFPVNNYYMGWFRQAPGRELEGVASIAGDDGTV YTNSVKGRFTIFRDNVNNTLYLRINSLKPEDTAIYYCAAESGRVRAQWLIANAFKYW GQGTQVTVSS.

[0056] The nucleotide sequence encoding the above-mentioned monovalent Nanobody® Nb3 against the common epitope is (SEQ ID NO. 4):

[0057] GAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGACACTCTCCTGTGCAGCCTCTGGATTTCCCGTCAATAATTACTACATGGGCTGGTTTCGCCAGGCTCCAGGGAGGGAGCTCGAGGGGGTCGCGTCTATTGCTGGTGATGATGGCACAGTCTACACAAACTCCGTGAAGGGTCGATTCACCATCTTTCGCGACAACGTCAACAACACGCTGTATCTGCGAATAAACAGCCTAAAACCTGAGGACACTGCCATTTACTACTGTGCGGCAGAAAGCGGGCGGGTTCGCGCACAGTGGTTAATAGCGAATGCTTTTAAATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0058] Table 1 Primer sequences for PCR amplification of bivalent nanobody gene sequences

[0059]

[0060] The gene sequence of the monovalent nanobody PRRSV-N-la3 against the common epitope of genotype 1 and 2 PRRSV was amplified using primers F1-240509-Pst I and R1-240509-rigid linker, and the reaction system is shown in Table 2.

[0061] Table 2 PCR amplification reaction system

[0062]

[0063] The reaction procedure was pre-denaturation at 94°C for 5 min, 34 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 35 s, and extension at 72°C for 7 min.

[0064] The PCR product was identified by 1% agarose gel electrophoresis, and the result showed that there was a band at the position of 400 bp. Figure 1 The 400 bp PCR product was recovered using the gel recovery kit EasyPure Quick Gel Extraction Kit according to the instructions, and the gene sequence encoding the monovalent nanobody PRRSV-N-la3 was obtained.

[0065] The monovalent nanobody PRRSV-N-Nb3 gene sequence which was amplified by PCR using primers F2-240509-rigid linker and R2-240509-Not I and which was connected with a rigid linker at the upstream, and which was against the common epitope of genotype 1 and 2 PRRSV, had a reaction system as shown in Table 3.

[0066] Table 3 PCR amplification reaction system

[0067]

[0068] The reaction procedure was 5 min pre-denaturation at 94℃, 30 s at 94℃, 30 s at 58℃, 35 s at 72℃, 34 cycles, and 7 min extension at 72℃.

[0069] The PCR product was subjected to 1% agarose gel electrophoresis, and the results showed that a 400 bp target band was obtained. Figure 1 Then, the PCR product was recovered by using a kit EasyPure Quick Gel Extraction Kit, and the gene sequence encoding the monovalent nanobody PRRSV-N-Nb3 was obtained.

[0070] The PCR product recovered twice by using a kit was used as a template, and the primers F3-240509-Pst I and R3-240509-Not I were used to perform overlapping PCR amplification, and the gene of the monovalent nanobody PRRSV-N-1a3 was connected with the gene of PRRSV-N-Nb3 by using a rigid linker, and the reaction system was as shown in Table 4.

[0071] Table 4 Overlapping PCR amplification reaction system

[0072]

[0073]

[0074] The reaction procedure was 5 min pre-denaturation at 94℃, 30 s at 94℃, 30 s at 58℃, 1 min at 72℃, 34 cycles, and 7 min extension at 72℃.

[0075] The PCR product was subjected to 1% agarose gel electrophoresis, and the results showed that a 750 bp target band was obtained. Figure 2 Then, the PCR product was recovered by using a kit EasyPure Quick Gel Extraction Kit.

[0076] The amino acid sequence of the bivalent nanobody against the common epitope of PRRSV-N protein of genotype 1 and 2 PRRSV was (SEQ ID NO. 11):

[0077] ESGGGSVQAGGSLRLSCAASGSTYYMGWFRKPPGKEREGVAALYTPSGSTYTANSVKSRFTIKDAAKNTVPQMNSLQPEDTAMYYNAADRNPGGVLSSRGYHYWGQGTQVTVSSEAAAKEAAAKEAAAKESGGGSVQAGGSLTLSCAASGFPVNNYYMGWFRQAPGRELEGVASIAGDDGTVYTNSVKGRFTIFRDNVNNTLYLRINSLKPEDTAIYYCAAESGRVRAQWLIANAFKYWGQGTQVTVSS.

[0078] The nucleotide sequence encoding the above bivalent nanobody is (SEQ ID NO. 12):

[0079] .

[0080] (2) Construction of a eukaryotic expression vector for a bivalent nanobody and HRP fusion protein against the common epitope of PRRSV genotypes 1 and 2

[0081] The recovered PCR product and pCMV-N1-HRP vector were simultaneously digested with Pst I and Not I. The specific enzyme digestion system is shown in the following table (Tables 5 and 6).

[0082] Table 5 pCMV-N1-HRP phage display vector double enzyme digestion system

[0083]

[0084] Table 6 PCR product double enzyme digestion system

[0085]

[0086] The enzyme digestion condition was 37°C, 16h. After enzyme digestion, the enzyme digestion product was recovered by using commercial kit EasyPure Quick Gel Extraction Kit, and the enzyme digestion result was analyzed by agarose gel electrophoresis, and enzyme digestion fragments of about 5000bp and 750bp were obtained respectively. Figure 3

[0087] The above recovered PCR enzyme digestion product was connected into the enzyme-digested vector pCMV-N1-HRP by using T4 DNA ligase, 16°C, 16h, and the ligation system is shown in Table 7.

[0088] Table 7 Ligation system of enzyme-digested overlapping PCR product and vector pCMV-N1-HRP

[0089]

[0090] ​The amino acid sequence of the fusion protein obtained by the ligation is (SEQ ID NO. 13): ESGGGSVQAGGSLRLSCAASGSTYYMGWFRKPPGKEREGVAALYTPSGSTYTANSVKSRFTIKDAAKNTVPQMNSLQPEDTAMYYNAADRNPGGVLSSRGYHYWGQGTQVTVSSEAAAKEAAAKEAAAKESGGGSVQAGGSLTLSCAASGFPVNNYYMGWFRQAPGRELEGVASIAGDDGTVYTNSVKGRFTIFRDNVNNTLYLRINSLKPEDTAIYYCAAESGRVRAQWLIANAFKYWGQGTQVTVSSAAASSSGSGMQLTPTFYDNSCPNVSNIVRDIIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFSVIDRMKAAVESACPGTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKSQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPDATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRRNCRVVNSNSDLHHHHHHH.

[0091]

[0092] The ligation product was transformed into Trans(5α) competent cells and cultured in a 37°C incubator for 12 hours. A single colony was picked and inoculated into 10 mL of LB liquid medium. After cultured in a shaker at 37°C for 12 hours, PCR identification of the bacterial solution showed that 10 colonies were positive ( Figure 4 The positive bacterial solution was sent for sequencing, and the positive plasmid was named pCMV-N1-HRP-PRRSV-N-1a3-Nb3.

[0093] 1.2 Expression and preparation of bivalent nanobody and HRP fusion protein

[0094] The successfully constructed positive plasmid pCMV-N1-HRP-PRRSV-N-1a3-Nb3 was After the medium and PEI transfection reagent were mixed evenly, they were added to HEK-293T cells in good condition and cultured in a 37°C, 5% CO2 incubator.

[0095] 48 h after transfection, some transfected HEK-293T cells were selected and anti-His monoclonal antibody (1:500 dilution, 5 μL, Quanshijin Biotechnology Co., Ltd.) was used as the primary antibody to detect whether the recombinant fusion protein was expressed in HEK-293T cells by IFA.

[0096] The results showed that the PRRSV-N-1a3-Nb3-HRP fusion protein was correctly expressed and the cells emitted green fluorescence ( Figure 5 ).

[0097] Then, the collected cell supernatant was used to detect whether the recombinant PRRSV-N-1a3-Nb3-HRP fusion protein was secreted into the HEK-293T cell supernatant using direct ELISA.

[0098] The results showed that the expressed nanobody and HRP fusion protein were successfully secreted into the cell supernatant ( Figure 6 ).

[0099] The above results indicate that the collected culture supernatant of the above transfected cells contains PRRSV-N-1a3-Nb3-HRP fusion protein.

[0100] Example 2

[0101] Application of bivalent nanobody against common epitopes of PRRSV genotypes 1 and 2 and HRP fusion protein in the detection of anti-PRRSV genotypes 1 and 2 antibodies in pig serum

[0102] 2.1 Screening of the best coating antigen

[0103] 2.1.1 Expression and purification of PRRSV-N protein

[0104] (1) Expression of PRRSV-N recombinant protein

[0105] ① Take 10 μL of recombinant E. coli pET28a-N bacterial solution (expressing PRRSV-N protein) and add it to 5 mL of LB medium resistant to kanamycin (K + ). Incubate at 37°C and 200 rpm overnight. The construction method of recombinant E. coli pET28a-N bacteria is described in the literature (Development of a Nanobody-Based Competitive Enzyme-Linked Immunosorbent Assay for Efficiently and Specifically Detecting Antibodies against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses, J Clin Microbiol. 2021 Nov 18; 59(12): e0158021. doi: 10.1128 / JCM.01580-21).

[0106] ② Take the activated bacterial solution described above and inoculate it into LB medium resistant to K + at a ratio of 1:100. Incubate at 37°C and 200 rpm for 2-3 h to reach the logarithmic phase, i.e., OD 600nm 0.6-0.8. Add 0.1 mM IPTG and continue to induce expression for 6-8 h.

[0107] ③ After collecting the bacterial solution and sonicating it, analyze the expression of recombinant PRRSV-N protein by SDS-PAGE.

[0108] a) Sample processing: Take 20 μL of protein sample and add 5 μL of 5x SDS-PAGE loading buffer. Boil at 100°C for 10 min and centrifuge at 12,000 g for 1 min.

[0109] b) SDS-PAGE electrophoresis: Install the electrophoresis tank, add the electrophoresis buffer, remove the comb, load the sample, and run the electrophoresis at 200 V for 40 min.

[0110] c) Staining: After the SDS-PAGE electrophoresis is complete, stain the protein gel for 2 h.

[0111] d) Decolorization: Wash the protein gel with deionized water, add the decolorizing solution, and the decolorization time is determined according to the clarity of the protein bands.

[0112] (2) Purification of PRRSV-N recombinant protein

[0113] ① Centrifuge the bacterial solution with large-scale induced expression at 12,000g for 2 minutes at 4℃ to collect the bacteria.

[0114] ② The bacteria were resuspended in Buffer A (0 mM imidazole) at a ratio of 1:20, and then ultrasonically disrupted (power 30 W, working time 6 s, rest 6 s, ultrasonication 30 min).

[0115] ③ After sonication, centrifuge at 12,000 g for 10 min at 4°C, collect the supernatant, and filter with a 0.45 μm filter to remove impurities.

[0116] ④ Prepare a nickel resin column and equilibrate it with 5 column volumes of Buffer A. Pass the supernatant collected from the centrifugation through the column at a flow rate of 0.5 mL / min, collecting the flow-through as A. Elute contaminants with Buffer B (20 mM imidazole), collecting the flow-through as B. Finally, elute the target protein with Buffer C (250 mM imidazole), collecting the target protein as C.

[0117] ⑤SDS-PAGE analysis of the purification of PRRSVN recombinant protein.

[0118] 2.1.2 Concentration and purification of PRRSV particles

[0119] (1) PRRSV particle concentration

[0120] MARC-145 cells were cultured into a monolayer. The medium containing 10% FBSDMEM was discarded and replaced with medium containing 2% FBSDMEM. The virus supernatant was added and the cells were incubated at 37°C in a 5% CO2 incubator. Computed toxin (CPE) was observed under a low-power microscope. Lesions began to appear within 24-48 hours, reaching approximately 80% CPE by 72-96 hours. Symptoms included cellular vacuolation and rounding, followed by focal shedding, then large-scale shedding, and finally, complete cell lysis and rupture. The virus solution was collected and repeatedly frozen and thawed three times, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. The cell culture was centrifuged at 8000 rpm for 30 minutes at 4°C, the supernatant collected, and then centrifuged at 45000 rpm for 2.5 hours at 4°C. The supernatant was discarded and the pellet resuspended in 0.01 mol / L PBS (pH 7.2).

[0121] (2) Purification of PRRSV particles by sucrose density gradient centrifugation

[0122] Prepare 25%, 35%, 45%, and 55% sucrose solutions. Add sucrose solutions in increasing densities to the centrifuge tube. Centrifuge at 4°C and 43,000 rpm for 2 hours until distinct layers appear in the tube. Collect the different layers and perform Western blot analysis on the purified PRRSV particles. Figure 7As shown, the band between 35% and 45% was collected. 5 to 10 volumes of PBS were added to the centrifuge tube to dilute the virus, and the tube was centrifuged at 45,000 rpm and 4°C for 2 h to collect the precipitate, which was dissolved in PBS and stored at -80°C.

[0123] 2.1.3 Selection of the optimal coating antigen

[0124] (1) Purified PRRSV N protein and purified PRRSV particles (100 ng / well) were coated on ELISA plates as coating antigens;

[0125] (2) Wash the plate 4 times with PBS'T and block with 2.5% skim milk powder (200 μL / well) at room temperature for 1 h;

[0126] (3) Wash the plate 4 times with PBS'T, mix PRRSV-N-1a3-Nb3-HRP fusion protein or monovalent nanobody and HRP fusion protein PRRSV-N-1a3-HRP or monovalent nanobody and HRP fusion protein PRRSV-N-Nb3-HRP with doubly diluted PRRSV-positive pig serum, add to the above-mentioned coated ELISA plate, and incubate at room temperature for 1 h;

[0127] (4) Wash the plate 4 times with PBS, add TMB colorimetric solution (100 μL / well) and develop the color for 15 min in the dark;

[0128] (5) After the color development is complete, add 3M sulfuric acid (50 μL / well) to terminate the color development and measure the OD using a microplate reader. 450nm value.

[0129] The results showed that the competition rate between the fusion protein and the positive serum was higher when the purified PRRSV particles were used as the coating antigen ( Figure 8 ), so purified PRRSV particles were selected as coating antigens.

[0130] 2.2 Comparison of the competitive rate between bivalent nanobody and HRP fusion protein and monovalent nanobody and HRP fusion protein

[0131] (1) Purified PRRSV N protein and purified PRRSV particles (100 ng / well) were coated on ELISA plates as coating antigens;

[0132] (2) Wash the plate 4 times with PBS'T and block with 2.5% skim milk powder (200 μl / well) at room temperature for 1 h;

[0133] (3) Wash the plate 4 times with PBS, mix the divalent nanobody HRP fusion protein PRRSV-N-1a3-Nb3-HRP or the monovalent nanobody and HRP fusion protein PRRSV-N-1a3-HRP or the monovalent nanobody and HRP fusion protein PRRSV-N-Nb3-HRP with the serially diluted PRRSV-positive pig serum, add them to the coated ELISA plate, and incubate at room temperature for 1 h.

[0134] (4) Wash the plate 4 times with PBS, add TMB colorimetric solution (100 μL / well) and develop the color for 15 min in the dark;

[0135] (5) After the color development is complete, add 3M sulfuric acid (50 μL / well) to terminate the color development and measure the OD using a microplate reader. 450nm value.

[0136] The results showed that after the bivalent nanobody HRP fusion protein was mixed with positive pig serum, its competitive ELISA detection rate was significantly higher than that of the monovalent nanobody HRP fusion protein ( Figure 8 ), so the bivalent nanobody HRP fusion protein was selected as the competitive probe for competitive ELISA.

[0137] 2.3 Application of the bivalent nanobody fusion protein PRRSV-N-1a3-Nb3-HRP in the detection of anti-PRRSV-N antibodies in pig serum

[0138] (1) Determination of the optimal antigen coating amount and the optimal dilution ratio of the fusion protein

[0139] ① Use purified PRRSV particles as coating antigens and coat ELISA plates at 100 ng / well, 200 ng / well, and 400 ng / well, respectively, at 4°C overnight;

[0140] ② After washing the plate 4 times with PBS, block with 2.5% skim milk powder (200 μL / well) at room temperature for 1 h;

[0141] ③ Dilute the PRRSV-N-1a3-Nb3-HRP fusion protein supernatant at 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600, add it to the coated ELISA plate, and incubate at 37°C for 1 hour;

[0142] ④ After washing the plate 4 times with PBS, add TMB substrate color development solution and develop color for 15 minutes in the dark;

[0143] ⑤Terminate with 3M H2SO4 and measure OD using a microplate reader 450nm value.

[0144] The results showed that when the purified PRRSV particles were used as coating antigens at a coating amount of 100 ng / well and the optimal dilution ratio of the fusion protein was 1:800, the OD value of direct ELISA was 450nm The value of is close to 1.0, which is the optimal antigen coating amount and the optimal fusion protein dilution ratio (Table 8).

[0145] Table 8 Antigen coating amount and antibody dilution ratio

[0146]

[0147] (2) Determination of the optimal dilution ratio of pig serum to be tested

[0148] ① Coat the purified PRRSV particles (100 ng / well) as the coating antigen on the ELISA plate at 4°C overnight;

[0149] ② After washing the plate 4 times with PBS'T, block the plate with PBS'T containing 2.5% skim milk powder at room temperature for 1 hour;

[0150] ③ After diluting the PRRSV-N-1a3-Nb3-HRP fusion protein with diluent, dilute the porcine PRRSV antibody positive and negative sera into the fusion protein at a dilution ratio of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, etc., and then add 100 μL / well of the mixture to the coated ELISA plate, set up multiple well detection, and incubate at 37°C for 1 hour;

[0151] ④ After washing the plate 4 times with PBS, add TMB substrate color development solution and develop color for 15 minutes in the dark;

[0152] ⑤Terminate with 3M H2SO4 and measure OD using a microplate reader 450nm value.

[0153] The results showed that when the serum dilution ratio was 1:10, the OD 450nm (P) compared with negative serum OD 450nm The (N) value was the smallest (Table 9). Therefore, 1:10 was the optimal dilution ratio for the established competitive ELISA test serum.

[0154] Table 9 ELISA test results at different serum dilutions

[0155]

[0156] (3) Competitive ELISA operating procedures

[0157] After optimizing the reaction conditions of competitive ELISA, the operating procedures of this method are as follows:

[0158] ①The purified whole virus particles SX-GD 100 ng / well were coated on the enzyme-labeled plate, and the plate was coated at 4°C overnight;

[0159] ②After washing the plate with PBS'T for 4 times, the plate was blocked with PBS'T containing 2.5% skim milk at 37°C for 1 hour;

[0160] ③The PRRSV-N-1a3-Nb3-HRP fusion protein was diluted with the blocking solution at the optimal ratio, and the pig serum to be detected was diluted at 1:10 in the above fusion protein. 100 μL / well of the mixed solution was added to the enzyme-labeled plate, and incubated at 37°C for 1 hour;

[0161] ④The TMB substrate color developing solution was added to develop color for 15 minutes in the dark, and 3M H2SO4 was added to stop the reaction. The OD value was read. 450nm According to the formula: competition rate (PI) = (1-OD value of the pig serum sample to be detected / OD value of the negative pig serum sample) x 100%, the competition rate (PI value) of the detected serum was calculated. 450nm 450nm

[0162] (4) Determination of the critical value of the competition ELISA result

[0163] 240 portions of known anti-PRRSV antibody negative serum and positive serum were selected to determine the Cut-off value of the competition ELISA. According to the optimal reaction conditions of the competition ELISA determined before, the 240 serum samples were detected. The results showed that, according to the calculation of the ROC curve, the Cut-off value of the competition ELISA was 25.67%. That is, when the PI value of the serum detected by the competition ELISA is greater than 25.67%, the result is determined to be positive for the anti-swine reproductive and respiratory syndrome antibody; otherwise, it is negative.

[0164] (5) Determination of the sensitivity of the competition ELISA

[0165] Three portions of anti-swine PRRSV antibody positive pig serum were selected for dilution by multiples, and then detected by the established competition ELISA. The results showed that when the positive serum was diluted at 1:320, the competition ELISA detection was still positive, indicating that the established competition ELISA had good sensitivity. Figure 9

[0166] (6) Determination of the specificity of the competition ELISA

[0167] The 23 known negative serum samples were detected by the ELISA, and all 23 samples showed negative results. PEDV positive serum, ASFV positive serum, and PCV2 positive serum were detected, and the established PRRSV positive pig serum was used as a positive control. The results were statistically analyzed. The results showed that the method did not have cross-reaction to the above positive control serum.​​​ Figure 10 ).

[0168] (7) Evaluation of the coincidence rate of the developed competitive ELISA with the commercial ELISA kit

[0169] In order to evaluate the clinical application of the competitive ELISA detection method, 240 clinical sera were detected by the competitive ELISA and the commercial ELISA kit, respectively, and the results showed that the coincidence rate was 95.90%.

[0170] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For ordinary skilled users in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required to be exhausted. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the claims of the present application.

Claims

1. A fusion protein of a bivalent nanobody against genotype 1 and 2 PRRSV N protein and horseradish peroxidase, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

13.

2. A DNA molecule encoding the fusion protein according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

14.

3. Use of the fusion protein according to claim 1 in the preparation of a product for detecting antibodies against genotype 1 and 2 PRRSV.

4. A product for detecting antibodies against PRRSV genotypes 1 and 2, characterized in that: Comprising the fusion protein according to claim 1.

5. A method for detecting antibodies against genotype 1 and 2 PRRSV for purposes other than disease diagnosis or treatment, characterized in that: The following steps are involved: An ELISA plate is coated with PRRSV as an antigen and the fusion protein according to claim 1 is used as a competitive reagent. The sample to be tested is mixed with the fusion protein and then added to the coated ELISA plate. After incubation, the competition rate is calculated according to the OD value to determine whether the sample to be tested contains anti-gene type 1 and anti-2 PRRSV antibodies.

6. The method according to claim 5, characterized in that After the incubation reaction, the step of adding a color developing solution and a strong acid to terminate the reaction is also included; The color developing solution is TMB color developing solution; the strong acid is 3 M H2SO4.

7. The method according to claim 5, characterized in that The incubation reaction conditions include: incubation temperature of 37° C. and incubation time of 1 h.

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