Anti-gene type 2 PRRSV (porcine reproductive and respiratory syndrome virus) bivalent nano antibody and horseradish peroxidase fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a fusion protein of an anti-gene type 2 PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) bivalent nano antibody and horse radish peroxidase (HRP) as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention also evaluates the application of the fusion protein in detecting the anti-gene type 2 PRRSV antibody in porcine serum. The anti-gene type 2 PRRSV bivalent nano antibody and the HRP fusion protein are used as competitive reagents to be applied to competitive ELISA for detecting the anti-gene type 2 PRRSV antibody in porcine serum, the detection method is simple to operate, short in sample detection time, free of use of an enzyme-labeled secondary antibody and low in production cost, and the coincidence rate with an existing IDEXX PRRSV antibody detection commercialized kit is as high as 96.92%. The sensitivity is higher than that of ELISA (Enzyme-Linked Immuno Sorbent Assay) taking a monovalent nano antibody as a competitive reagent, and the competitive rate with positive serum can be increased by about 20%. Therefore, the subsequent development of the anti-PRRSV antibody ELISA detection kit by using the divalent nano antibody has a good market application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-genotype 2 PRRSV bivalent nano antibody and horseradish peroxidase (HRP) fusion protein, and a preparation method and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an acute, highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). It mainly causes reproductive disorders such as abortion, premature birth, and weak piglets in sows, as well as respiratory disorders and growth retardation in piglets and fattening pigs. It is a highly contagious disease that seriously endangers the pig farming industry. The latest classification standard has divided its original two genotypes into two different species, namely Betaarterivirus suid 1 (European type / PRRSV-1) and Betaarterivirus suid 2 (North American type / PRRSV-2). Rapid diagnosis of PRRSV in the early stage of infection can effectively control its spread and prevalence. At present, PRRSV-2 is the main prevalent strain in my country, and PRRSV-1 is only prevalent in certain areas.

[0003] ELISA refers to a qualitative or quantitative detection method that uses antigen-antibody specific binding to adsorb antigens or antibodies onto a polystyrene solid phase carrier to perform an immune response, including indirect ELISA, competitive ELISA, double antibody sandwich ELISA, etc. The ELISA method has the advantages of only a small amount of antigen for coating, fast diagnosis, high specificity, and low cost, and is widely used in detecting anti-PRRSV antibodies. In 1992, French scholar Albina used the virus prepared by inoculating porcine alveolar macrophages with the virus as the coating antigen to establish an indirect ELISA for anti-PRRSV antibodies in porcine serum. The biggest drawback of this method is that it is prone to non-specific reactions. In order to overcome this problem, some scholars established a competitive ELISA, using PRRSV polyantibodies to detect antigens, and the specificity was greatly improved. PRRSV GP5 protein has good antigenicity and can induce the body to produce neutralizing antibodies. Therefore, GP5 protein is often used as a coating antigen for diagnostic kits. Companies on the market that use GP5 protein as a coating antigen to establish detection kits include French LIS Company, etc. Chinese scholars Fang Liurong and others fused GP5 protein with GST protein to establish an indirect ELISA, which has a good detection effect on anti-PRRSV antibodies. However, due to the high variability of GP5 protein of different PRRSV strains, this method is prone to false negatives. PRRSV-N protein is highly conserved in different PRRSV strains and can induce a strong immune response in the host. Therefore, PRRSV-N protein is also often used as a target antigen to establish anti-PRRSV antibodies. For example, Li Subing et al. used the prokaryotic expressed N protein as a coating antigen to establish an indirect ELISA for detecting anti-PRRSV antibodies, and the result of the commercial kit of IDEXX Company in the United States was consistent with 84%. Duan Hong et al. developed a competitive ELISA for detecting type 2 PRRSV antibodies based on nanobody-HRP fusion protein as a sensitive probe. The method has a high consistency rate with the IDEXX ELISA kit. However, when this method is applied to clinical serum detection, a high false negative occurs. Therefore, on the basis of this ELISA, the present invention continues to optimize the competitive reagent and coating antigen to establish a highly sensitive ELISA for the detection of anti-PRRSV antibodies in clinical pig serum. In addition, the most commonly used PRRSV antibody detection kit in clinical practice in my country is still the PRRS X3 Ab kit from IDEXX, which is developed based on indirect ELISA. However, there is no development of kits based on competitive ELISA. Summary of the invention

[0004] The purpose of the present invention is to provide a bivalent nano antibody and horseradish peroxidase fusion protein against genotype 2 PRRSV and its preparation method and application to solve the problems existing in the above-mentioned prior art. Based on competitive ELISA, the present invention further optimizes the competitive reagent and the coating antigen to establish a highly sensitive ELISA, which is applied to the detection of anti-PRRSV antibodies in clinical pig serum.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a bivalent nanobody against genotype 2 PRRSV, whose amino acid sequence is shown in SEQ ID NO.1.

[0007] The second technical solution of the present invention is a DNA molecule encoding the bivalent nanobody, whose nucleotide sequence is shown in SEQ ID NO.2.

[0008] The third technical solution of the present invention is a fusion protein, comprising the bivalent nanobody and horseradish peroxidase, and its amino acid sequence is shown in SEQ ID NO.3.

[0009] The fourth technical solution of the present invention is a DNA molecule encoding the fusion protein, whose nucleotide sequence is shown in SEQ ID NO.4.

[0010] A fifth technical solution of the present invention is the use of the fusion protein in the preparation of a product for detecting antibodies against genotype 2 PRRSV.

[0011] The sixth technical solution of the present invention is a product for detecting antibodies against genotype 2 PRRSV, comprising the fusion protein.

[0012] The seventh technical solution of the present invention is a method for detecting anti-genotype 2 PRRSV antibodies for non-disease diagnosis or treatment purposes, comprising the following steps:

[0013] The ELISA plate is coated with PRRSV as an antigen, the fusion protein is used as a competitive reagent, the sample to be tested is mixed with the fusion protein and 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-genotype 2 PRRSV antibodies.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The present invention uses anti-genotype 2 PRRSV bivalent nano antibody and HRP fusion protein as competitive reagents in competitive ELISA for detecting anti-genotype 2 PRRSV antibodies in pig serum. The detection method 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 96.92% with the existing IDEXX PRRSV antibody detection commercial kit. It is also more sensitive than ELISA using monovalent nano antibodies as competitive reagents, and its competitive rate with positive serum can be increased by about 20%. Therefore, the subsequent development of anti-PRRSV antibody ELISA detection kits using the bivalent nano antibodies has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 It is the PCR amplification encoding the PRRSV2-N-Nb19 protein gene; M is Maker; 1 is the fusion of the monovalent PRRSV2-N-Nb19 gene downstream and the rigid Linker; 2 is the fusion of the monovalent PRRSV2-N-Nb19 gene upstream and the rigid Linker.

[0018] Figure 2 PCR amplification encodes the PRRSV2-N-Nb19-Nb19 gene; wherein M is Maker; 1 overlapping PCR connects the downstream of the amplified monovalent PRRSV2-N-Nb19 gene with the upstream of the amplified monovalent PRRSV2-N-Nb19 gene.

[0019] Figure 3 1 is the result of double enzyme digestion of PRRSV2-N-Nb19-Nb19 gene and pCMV-N1-HRP empty vector, where M is Maker, 1 is the result of double enzyme digestion of pCMV-N1-HRP empty vector, and 2 is the result of double enzyme digestion of PRRSV2-N-Nb19-Nb19.

[0020] Figure 4 The pCMV-N1-HRP-PRRSV2-N-Nb19-Nb19 recombinant positive vector was identified by bacterial liquid PCR, where M is Maker, 1-3 are positive clones, and 4 is a negative control.

[0021] Figure 5After transfecting HEK-293T cells with the constructed bivalent nanobody and HRP fusion protein recombinant expression plasmid, IFA was used to identify the expression of PRRSV2-N-Nb19-Nb19-HRP fusion protein in HEK-293T cells.

[0022] Figure 6 ELISA was used to analyze the secretion and expression of PRRSV2-N-Nb19-Nb19-HRP fusion protein into the culture supernatant of HEK-293T cells, as well as the titer of the fusion protein in the supernatant.

[0023] Figure 7 This is Western blot analysis of purified PRRSV particles, where M stands for Maker.

[0024] Figure 8 The competition rates of the two coating antigens were compared by competitive ELISA analysis using prokaryotically expressed purified PRRSV2-N protein and purified PRRSV particles as coating antigens and fusion protein as competition probe.

[0025] Fig. 9 In order to use purified PRRSV particles as coating antigen, monovalent nanobody and HRP fusion protein PRRSV2-N-Nb19-HRP and divalent nanobody and HRP fusion protein PRRSV2-N-Nb19-Nb19-HRP were used as competition probes, and competition ELISA analysis was used to compare the competition rates of the two competition probes.

[0026] Fig.10 In order to use purified PRRSV particles as coating antigen and PRRSV2-N-Nb19-Nb19-HRP as competition reagent, five anti-PRRSV antibody-positive pig sera were diluted into the fusion protein at dilution ratios of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:1280, and mixed to analyze the sensitivity of the competitive ELISA assay.

[0027] Fig.11 In order to use purified PRRSV particles as coating antigens and PRRSV-N-Nb19-Nb19-HRP as competition reagents, PEDV-positive serum, ASFV-positive serum, and PCV2-positive serum were diluted into the fusion protein at a ratio of 1:10 and mixed. At the same time, the confirmed PRRSV-positive pig serum was used as a positive control and diluted into the fusion protein at a ratio of 1:10 and mixed to identify the specificity of the competitive ELISA detection method. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0034] An embodiment of the present invention provides a bivalent nanobody against genotype 2 PRRSV, and its amino acid sequence is shown in SEQ ID NO.1.

[0035] The embodiment of the present invention also provides a DNA molecule encoding the bivalent nanobody, and its nucleotide sequence is shown in SEQ ID NO.2.

[0036] The embodiment of the present invention also provides a fusion protein, comprising the bivalent nanobody and horseradish peroxidase, whose amino acid sequence is shown in SEQ ID NO.3, and is named PRRSV2-N-Nb19-Nb19-HRP.

[0037] The embodiment of the present invention also provides a DNA molecule encoding the fusion protein, and its nucleotide sequence is shown in SEQ ID NO.4.

[0038] The embodiment of the present invention also provides the use of the fusion protein in preparing a product for detecting anti-genotype 2 PRRSV antibodies.

[0039] The embodiment of the present invention also provides a product for detecting antibodies against genotype 2 PRRSV, comprising the fusion protein.

[0040] The embodiment of the present invention also provides a method for detecting anti-genotype 2 PRRSV antibodies for non-disease diagnosis or treatment purposes, comprising the following steps:

[0041] The ELISA plate is coated with PRRSV as an antigen, the fusion protein is used as a competitive reagent, the sample to be tested is mixed with the fusion protein and 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-genotype 2 PRRSV antibodies.

[0042] In some specific embodiments, the formula for calculating the competition rate is:

[0043] Competition rate = (1-OD of the pig serum sample to be tested 450nm Value / negative pig serum sample OD 450nm value)×100%.

[0044] In some specific embodiments, the method for determining whether the sample to be tested contains anti-genotype 2 PRRSV antibodies is: if the competition rate of the pig serum to be tested is greater than 19.98%, it is determined to be positive, otherwise it is negative.

[0045] The present invention also provides a method for preparing the anti-PRRSV-2 bivalent nanobody and HRP fusion protein, comprising the following steps:

[0046] (1) Construction of recombinant eukaryotic expression vector: By designing primers and amplifying the target fragment, the nucleotide sequences of two monovalent anti-PRRSV2-N-Nb19 nanobodies were concatenated to form a bivalent nanobody PRRSV2-N-Nb19-Nb19.

[0047] Preferably, two monovalent Nanobodies are connected by a rigid Linker.

[0048] (2) The above-mentioned bivalent nanoantibody PRRSV2-N-Nb19-Nb19 was connected to the pCMV-N1-HRP vector by double enzyme digestion to obtain a positive plasmid.

[0049] Preferably, the double digestion enzymes are Pst I and Not I.

[0050] (3) After the eukaryotic expression vector constructed above is transfected into cells using PEI transfection reagent, the supernatant obtained is the anti-PRRSV-2 bivalent nanobody and HRP fusion protein.

[0051] Preferably, the host cell is a HEK-293T cell.

[0052] Preferably, the transfection expression time is 48h.

[0053] The present invention also provides an application of the fusion protein in detecting anti-PRRSV-2 antibodies in pig serum, comprising the following steps:

[0054] (1) Purified PRRSV particles were coated on ELISA plates;

[0055] (2) mixing the bivalent nanobody and the HRP fusion protein with the pig serum to be tested to obtain a mixture;

[0056] (3) adding the mixture of step (2) to the ELISA plate coated in step (1) and incubating, and after incubation, adding a color developing solution and reacting in the dark;

[0057] (4) adding a stop solution in step (3) to terminate the reaction;

[0058] (5) OD determination by automatic microplate reader 450nm The competition rate of the test serum was calculated.

[0059] (6) The method for determining whether the pig serum contains anti-PRRSV-2 antibodies is as follows: if the competition rate of the pig serum to be tested is greater than 19.98%, it is determined to be positive, otherwise it is negative.

[0060] Preferably, PRRSV propagated in MARC-145 cells is purified by sucrose gradient centrifugation to obtain antigen-coated PRRSV particles.

[0061] Preferably, the incubation temperature is 37° C. and the incubation time is 1 h.

[0062] Preferably, the color developing solution is TMB color developing solution.

[0063] Preferably, the stop solution is 3M H2SO4.

[0064] The present invention provides a bivalent nano antibody and HRP fusion protein against PRRSV-2-N protein and a preparation method thereof, and simultaneously evaluates the application of the bivalent nano antibody and HRP fusion protein in detecting anti-PRRSV-2 antibodies in pig serum.

[0065] Example 1

[0066] Preparation of anti-PRRSV-2 bivalent nanobody and horseradish peroxidase fusion protein

[0067] 1.1 Construction of fusion protein eukaryotic expression vector

[0068] (1) Based on the monovalent nanobody gene sequence (SEQ ID NO. 5), specific primers were designed. The primer sequences were synthesized by Xi'an Qingke Biotechnology Co., Ltd. and their sequences are shown in Table 1.

[0069] The nucleotide sequence encoding the anti-PRRSV-2 monovalent Nanobody is (SEQ ID NO.5): GAGTCTGG GGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTACTACATGGGCTGGTTCCGAAAGCCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGCTCTTTATACTCCTAGTGGTAGCACATACACTGCCAACTCCGTGAAAAGCC GATTCACCATCAAAGACGCCGCCAAGAACACGGTGCCCCAAATGAACAGCCTACAACCTGAGGACACTGCCATGTACTACAATGCGGCAGATCGGAATCCGGGCGGGGTACTATCCTCCCGCGGGTATCACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

[0070] Table 1 Specific primer sequences for PCR amplification of bivalent nanobody genes

[0071]

[0072] First, primers F1-240509-PstⅠ and R1-240509-rigid linker were used to amplify the gene sequence of the monovalent PRRSV2-N-Nb19 nanoantibody, and the reaction system is shown in Table 2.

[0073] Table 2 PCR amplification reaction system

[0074]

[0075]

[0076] The reaction program was pre-denaturation at 94°C for 5 min; 34 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 35 s; and extension at 72°C for 7 min.

[0077] The PCR product was identified by 1% agarose gel electrophoresis, and the results showed a band at the 400 bp position ( Figure 1 ). The PCR product of 400 bp was recovered using the EasyPure Quick Gel Extraction Kit according to the instructions, thereby obtaining the gene encoding the monovalent nanobody PRRSV2-N-Nb19.

[0078] Similarly, primers F2-240509-rigid linker and R2-240509-NotⅠ were used to PCR amplify the monovalent PRRSV2-N-Nb19 gene. The gene sequence was connected to the downstream of the monovalent nanoantibody sequence amplified above. The two monovalent sequences were connected with a rigid linker. The PCR reaction system is shown in Table 3.

[0079] Table 3 PCR amplification reaction system

[0080]

[0081] 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, and 72°C for 35 s; and extension at 72°C for 7 min.

[0082] 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 using the EasyPure Quick Gel Extraction Kit.

[0083] Using the PCR products recovered from the two gels as templates, primers F3-240509-PstⅠ and R3-240509-NotⅠ were used to perform overlapping PCR amplification to connect the two monovalent PRRSV2-N-Nb19 gene sequences. The reaction system is shown in Table 4.

[0084] Table 4 Fusion PCR amplification reaction system

[0085]

[0086] 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, and 72°C for 1 min; and extension at 72°C for 7 min.

[0087] 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 using the EasyPure Quick Gel Extraction Kit.

[0088] The amino acid sequence of the anti-PRRSV-2 bivalent nanobody is (SEQ ID NO. 1): ESGGGSVQAGGS LRLSCAASGSTYYMGWFRKPPGKEREGVAALYTPSGSTYTANSVKSRFTIKDAAKNTVPQMNSLQPEDTAMYYNAADRNPGGVLSSRGYHYWGQGTQVTVSSEAAAKEAAAKEAAAKESGGGSVQAGGSLRLSCAASGSTYYMGWFRKPPGKEREGVAALYTPSGSTYTANSVKSRFTIKDAAKNTVPQMNSLQPEDTAMYYNAADRNPGGVLSSRGYHYWGQGTQVTVSS;

[0089] The nucleotide sequence encoding the anti-PRRSV-2 bivalent nanobody is (SEQ ID NO.2): GAGTCTGG GGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTACTACATGGGCTGGTTCCGAAAGCCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGCTCTTTATACTCCTAGTGGTAGCACATACACTGCCAACTCCGTGAAAAGCCGATTCACCATCAAAGACGCCGCCAAGAACACGGTGCCCCAAATGAACAGCCTACAACCTGAGGACACTGCCATGTAC TACAATGCGGCAGATCGGAATCCGGGCGGGGTACTATCCTCCCGCGGGTATCACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGAAGCCGCAGCAAAGGAAGCCGCAGCAAAGGAAGCCGCAGCAAAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTACTACATGGGCTGGTTCCGAAAGCCTCCAGGGAAGGAGCG CGAGGGGGTCGCAGCTCTTTATACTCCTAGTGGTAGCACATACACTGCCAACTCCGTGAAAAGCCGATTCACCATCAAAGACGCCGCCAAGAACACGGTGCCCCAAATGAACAGCCTACAACCTGAGGACACTGCCATGTACTACAATGCGGCAGATCGGAATCCGGGCGGGGTACTATCCTCCCGCGGGTATCACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

[0090] (2) Construction of eukaryotic expression vector of bivalent nanobody and HRP fusion protein

[0091] The recovered PCR product (750 bp) and pCMV-N1-HRP vector were double-digested with Pst I and Not I at the same time. The specific enzyme digestion system is shown in the following table (Tables 5 and 6).

[0092] Table 5 pCMV-N1-HRP vector double enzyme digestion system

[0093]

[0094] Table 6 PCR product double enzyme digestion system

[0095]

[0096] The digestion conditions were 37°C for 16 h. After digestion, the digestion products were recovered using the commercial kit EasyPure Quick GelExtraction Kit. The digestion results were analyzed by agarose gel electrophoresis, and the digestion fragments of about 5000 bp and 750 bp were obtained respectively ( Figure 3 ).

[0097] The above-recovered PCR digestion product was ligated into the digested vector pCMV-N1-HRP using T4 DNA ligase, and the ligation was carried out at 16° C. for 16 h. The ligation system is shown in Table 7.

[0098] Table 7 Reagent-cut overlapping PCR products and vector pCMV-N1-HRP connection system

[0099]

[0100]

[0101] The amino acid sequence of the fused protein obtained by connection is (SEQ ID NO.3): ESGGGSVQAGGSLRLS CAASGSTYYMGWFRKPPGKEREGVAALYTPSGSTYTANSVKSRFTIKDAAKNTVPQMNSLQPEDTAMYYNAADRNPGGVLSSRGYHYWGQGTQVTVSSEAAAKEAAAKEAAAKESGGGSVQAGGSLRLSCAASGSTYYMGWFRKPPGKEREGVAALYTPSGSTYTANSVKSRFTIKDAAKNTVPQMNSLQPEDTAMYYNAADRNPGGVLSSRGYHYWGQGTQVTVSSAAASSSGSGMQLTPTFYDNSCPNVSNIVRDIIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFSVIDRMKAAVESACPGTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKSQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPDATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRRNCRVVNSNSDLHHHHHHH;

[0102] The DNA nucleotide sequence of the fused protein obtained by connection is (SEQ ID NO.4): GAGTCTGGGGG AGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTACTACATGGGCTGGTTCCGAAAGCCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGCTCTTTATACTCCTAGTGGTAGCACATACACTGCCAACTCCGTGAAAAGCCGATTCACCATCAAAGACGCCGCCAAGAACACGGTGCCCCAAATGAACAGCCTACAACCTGAGGACACTGCCATGTACTACAATGCGGCAGATCGGAATCCGGGCGGGGTACTATCCTCCCGCGGGTATCACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAAGCCGCAGCAAAGGAAGCCGCAGCAAAGGAAGCCGCAGCAAAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTACTACATGGGCTGGTTCCGAAAGCCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGCTCTTTATACTCCTAGTGGTAGCACATACACTGCCAACTCCGTGAAAAGCCGATTCACCATCAAAGACGCCGCCAAGAACACGGTGCCCCAAATGAACAGCCTACAACCTGAGGACACTGCCATGTACTACAATGCGGCAGATCGGAATCCGGGCGGGGTACTATCCTCCCGCGGGTATCACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCCAGTAGTAGTGGCAGTGGTATGCAGCTGACCCCCACATTCTACGATAACTCTTGTCCCAACGTGTCCAACATCGTTCGTGACATCATCGTGAATGAGCTGAGGAGCGACCCTAGGATCGCCGCCAGCATTCTGAGGCTGCACTTCCACGACTGCTTCGTGAATGGCTGCGACGCCTCCATTTTACTGGATAACACCACCAGCTTTCGTACCGAAAAGGACGCTTTCGGCAACGCCAACAGCGCTCGTGGCTTCAGCGTGATCGATCGTATGAAAGCCGCCGTGGAAAGCGCTTGTCCCGGCACAGTGTCTTGTGCCGATTTATTAACCATTGCCGCCCAGCAGTCCGTGACACTGGCCGGCGGACCTAGCTGGAGAGTGCCTCTGGGTCGTAGGGATTCTTTACAAGCCTTTTTAGATTTAGCCAACGCCAATTTACCCGCCCCTTTCTTCACTTTACCTCAGCTCAAGGACAGCTTCAGAAACGTGGGTTTAAATAGGAGCAGCGATTTAGTGGCTTTATCCGGCGGCCACACATTTGGCAAGAGCCAGTGTCGTTTCATTATGGATCGTCTGTACAATTTCAGCAACACCGGTTTACCCGACCCCACACTGAACACCACCTATCTCCAGACTTTAAGAGGTTTATGCCCTTTAAACGGCAATCTGTCCGCTTTAGTGGATTTCGACTTACGTACCCCCACAATCTTCGACAACAAATACTACGTGAATTTAGAGGAGCAGAAGGGTTTAATCCAGAGCGACCAAGAACTGTTTTCCAGCCCCGATGCCACCGACACCATCCCTCTGGTGAGGAGCTTCGCCAACTCCACCCAGACCTTCTTCAACGCCTTTGTGGAGGCCATGGATCGTATGGGCAACATTACCCCTCTGACCGGCACCCAAGGTCAGATTCGTAGGAACTGTAGGGTGGTGAATAGCAACAGCGATTTACATCATCACCACCATCACCAC。

[0103] The ligation product was transformed into Trans(5α) competent cells and cultured in a 37°C incubator for 12 h. A single colony was picked and inoculated into 10 mL of LB liquid medium. After shaking culture at 37°C for 12 h, PCR identification of the bacterial solution showed that 10 were positive ( Figure 4 The positive bacterial solution was sent for sequencing, and the positive plasmid was named pCMV-N1-HRP-PRRSV2-N-Nb19-Nb19.

[0104] 1.2 Expression and preparation of bivalent nanobody and HRP fusion protein

[0105] The successfully constructed positive plasmid pCMV-N1-HRP-PRRSV2-N-Nb19-Nb19 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.

[0106] 48 h after transfection, some of the 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.

[0107] The results showed that the PRRSV2-N-Nb19-Nb19-HRP fusion protein was correctly expressed, and the cells emitted green fluorescence ( Figure 5 Then, the collected cell supernatant was used to detect whether the recombinant PRRSV2-N-Nb19-Nb19-HRP fusion protein was secreted into the HEK-293T cell supernatant using direct ELISA. The results showed that the expressed nanobody and HRP fusion protein were successfully secreted into the cell supernatant ( Figure 6 ). The above results indicate that the culture supernatant of the above transfected cells collected contains PRRSV2-N-Nb19-Nb19-HRP fusion protein.

[0108] Example 2

[0109] Application of anti-PRRSV-2 bivalent nanobody and HRP fusion protein in detecting anti-PRRSV-2 antibodies in pig serum

[0110] 2.1 Screening of the best coating antigen

[0111] 2.1.1 Expression and purification of PRRSV-N protein

[0112] The nucleocapsid N protein encoded by PRRSV ORF7 is a protein with a molecular weight of 15kDa. The N protein content of PRRSV particles is the highest, accounting for about 40% of the total viral protein. The N protein has good antigenicity and immunogenicity, and anti-PRRSV N protein antibodies can be detected 7 days after infection. Therefore, the N protein is an ideal target protein for the development of PRRSV antibody diagnostic kits.

[0113] (1) Expression of PRRSV-N recombinant protein

[0114] ①Take 10 μL of recombinant expression E. coli pET28a-N bacterial solution (expressing PRRSV2-N protein) and add 5 mL of kanamycin-resistant (K+) LB medium, and culture overnight at 37°C 200 rpm. The construction method of recombinant expression E. coli pET28a-N bacteria can be found 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).

[0115] ②Take the activated bacterial solution and inoculate it into K + The cells were cultured in LB medium at 37°C and 200 rpm for 2-3 h until the logarithmic phase was reached, i.e., OD 600nm To 0.6-0.8. Add 0.1 mM IPTG and continue to induce expression for 6-8 hours.

[0116] ③ After collecting the bacterial solution and ultrasonically disrupting it, SDS-PAGE was used to analyze the expression of the recombinant PRRSV2-N protein.

[0117] a) Sample treatment: 20 μL of protein sample was added to 5 μL of 5× SDS-PAGE loading buffer, boiled at 100°C for 10 min, and centrifuged at 12,000 g for 1 min.

[0118] b) SDS-PAGE electrophoresis: Install the electrophoresis tank, add electrophoresis buffer, remove the comb, apply samples, and run electrophoresis at a constant temperature of 200 V for 40 min.

[0119] c) Staining After the SDS-PAGE electrophoresis is completed, the protein gel is placed in a box containing staining solution and stained for 2 hours.

[0120] d) Decolorization: Wash the protein gel with deionized water and add decolorizing solution. The decolorization time depends on the clarity of the protein bands.

[0121] (2) Purification of PRRSVN recombinant protein

[0122] ① Centrifuge the bacterial solution with large-scale induced expression at 12,000 g at 4°C for 2 min to collect the bacteria.

[0123] ② Resuspend the bacteria in Buffer A (0 mM imidazole) at a ratio of 1:20, and then perform ultrasonic disruption of the bacteria (power 30 W, working time 6 s, rest 6 s, ultrasonic 30 min).

[0124] ③After ultrasonication, centrifuge at 12,000g for 10 min at 4°C, collect the supernatant, and filter with a 0.45μm filter to remove impurities.

[0125] ④ Take Ni Resin and 5 column volumes of Buffer A to balance the column. Pass the supernatant collected by centrifugation through the column at a flow rate of 0.5 mL / min, and collect the effluent as A. Then use Buffer B (20 mM imidazole) to elute the impurities, and collect the effluent as B. Finally, use Buffer C (250 mM imidazole) to elute the target protein, and collect the target protein as C.

[0126] ⑤SDS-PAGE analysis of the purification of PRRSVN recombinant protein.

[0127] 2.1.2 Concentration and purification of PRRSV particles coated with antigen

[0128] (1) PRRSV particle concentration

[0129] The cell culture was centrifuged at 8000 r / min for 30 min at 4°C, the supernatant was taken, and then centrifuged at 45000 r / min for 2.5 h at 4°C, the supernatant was discarded, and the precipitate was resuspended in 0.01 mol / L PBS (pH 7.2).

[0130] (2) Sucrose gradient purification of PRRSV particles

[0131] Prepare 25%, 35%, 45% and 55% sucrose solutions. Add sucrose solutions from low to high density into centrifuge tubes. Centrifuge at 4°C and 43,000 r / min for 2 hours until distinct stratification occurs. Collect the band between 35% and 45%. Add 5 to 10 times the volume of PBS to dilute the virus into the centrifuge tube, centrifuge at 45,000 rpm and 4°C for 2 hours, collect the precipitate, dissolve the precipitate with PBS, and freeze at -80°C ( Figure 7 ).

[0132] 2.1.3 Selection of the best coating antigen

[0133] (1) Purified PRRSV N protein and PRRSV (100 ng / well) were coated on ELISA plates as coating antigens;

[0134] (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;

[0135] (3) Wash the plate 4 times with PBS'T, mix the PRRSV2-N-Nb19-Nb19-HRP fusion protein (1:600) with PRRSV-positive pig serum (1:10), add it to the above-mentioned coated ELISA plate, and incubate at room temperature for 1 h;

[0136] (4) Wash the plate 4 times with PBS'T, add TMB colorimetric solution (100 μL / well) and color for 15 min in the dark;

[0137] (5) After color development, add 3M sulfuric acid (50 μL / well) to terminate the color development and measure the OD using an ELISA reader. 450nm value.

[0138] 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.

[0139] 2.2 Comparison of the competition rate between bivalent nanobody and HRP fusion protein and monovalent nanobody and HRP fusion protein

[0140] (1) The purified PRRSV was coated as a coating antigen in an ELISA plate;

[0141] (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;

[0142] (3) Wash the plate 4 times with PBS'T, mix the divalent nanobody HRP fusion protein PRRSV2-N-Nb19-Nb19-HRP or the monovalent nanobody and HRP fusion protein PRRSV2-N-Nb19-HRP with the doubly diluted PRRSV-positive pig serum, add them to the coated ELISA plate, and incubate at room temperature for 1 h;

[0143] (4) Wash the plate 4 times with PBS'T, add TMB colorimetric solution (100 μL / well) and color for 15 min in the dark;

[0144] (5) After color development, add 3M sulfuric acid (50 μL / well) to terminate the color development and measure the OD using an ELISA reader. 450nmvalue.

[0145] The results showed that after the bivalent nanobody HRP fusion protein was mixed with positive pig serum, its competitive rate in competitive ELISA detection was significantly higher than that of the monovalent nanobody HRP fusion protein ( Fig. 9 ), so the bivalent nanobody HRP fusion protein was selected as the competitive probe for competitive ELISA.

[0146] 2.3 Application of bivalent nanoantibody fusion protein PRRSV2-N-Nb19-Nb19-HRP in the detection of anti-PRRSV-2 antibodies in pig serum

[0147] (1) Determination of the optimal antigen coating amount and the optimal dilution ratio of the fusion protein

[0148] ① Use purified PRRSV particles as coating antigens and coat them on ELISA plates at 100 ng / well, 200 ng / well and 400 ng / well respectively, and coat them at 4°C overnight;

[0149] ② After washing the plate 4 times with PBS'T, block the plate with 2.5% skim milk powder (200 μL / well) at room temperature for 1 h;

[0150] ③ Dilute the PRRSV2-N-Nb19-Nb19-HRP fusion protein supernatant at 1:50, 1:100, 1:200, 1:400, 1:800 and 1:1600, add it to the above-mentioned coated ELISA plate, and incubate at 37°C for 1 hour;

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

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

[0153] 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 of the direct ELISA 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).

[0154] Table 8 Antigen coating amount and antibody dilution ratio

[0155]

[0156]

[0157] (2) Determination of the optimal dilution ratio of pig serum to be tested

[0158] ① Coat the purified PRRSV particles (100 ng / well) as the coating antigen on the ELISA plate at 4°C overnight;

[0159] ② After washing the plate 4 times with PBS'T, block the plate with PBS'T containing 2.5% skimmed milk powder at room temperature for 1 hour;

[0160] ③ After diluting the PRRSV2-N-Nb19-Nb19-HRP fusion protein with diluent, dilute the anti-PRRSV antibody positive and negative pig sera at a dilution ratio of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, etc. and mix with the above-diluted fusion protein, then add 100 μL / well of the mixed solution to the coated ELISA plate, set up multiple well detection, and incubate at 37°C for 1 hour;

[0161] ④After washing the plate 4 times with PBS, add TMB substrate colorimetric solution and color for 15 min in the dark;

[0162] ⑤Terminate with 3M H2SO4 and measure OD using a microplate reader 450nm value.

[0163] The results showed that when the pig serum dilution ratio was 1:10, the OD 450nm (P) compared with negative serum OD 450nm (N) value is the smallest (Table 9). Therefore, the 1:10 dilution ratio was selected as the optimal dilution ratio for the established competitive ELISA test serum.

[0164] Table 9 ELISA test results at different serum dilutions

[0165]

[0166] (3) Competitive ELISA operating procedures

[0167] After optimizing the reaction conditions of competitive ELISA, the operating procedures of this method are as follows:

[0168] ① Coat the purified PRRSV particles (100 ng / well) on the ELISA plate at 4°C overnight;

[0169] ② After washing the plate 4 times with PBS'T, block it with PBS'T containing 2.5% skim milk powder (200 μL / well) at room temperature for 1 h;

[0170] ③ Use PBS'T to dilute the PRRSV2-N-Nb19-Nb19-HRP fusion protein at 1:200, and dilute the pig serum to be tested in the above fusion protein at a final concentration of 1:10 after dilution. Add 100 μL / well of the mixture to the ELISA plate and incubate at 37°C for 1 hour;

[0171] ④ Add TMB substrate colorimetric solution and color for 15 min in the dark, terminate with 3M H2SO4, and measure OD using an enzyme-labeled instrument 450nm The competitive rate (PI) = (1-OD of the pig serum sample to be tested) 450nm Value / negative pig serum sample OD 450nm value)×100% to calculate the competition rate (PI value) of the tested serum.

[0172] (4) Determination of critical value for competitive ELISA results

[0173] 240 known anti-PRRSV-2 antibody negative sera and positive pig sera were selected to determine the cut-off value of the competitive ELISA. According to the optimal reaction conditions of the competitive ELISA determined previously, the 240 sera were tested. The results showed that the cut-off value of the competitive ELISA was 19.98% calculated by the ROC curve. That is, when the serum was tested with the competitive ELISA, its PI value>19.98%, the result was judged as positive for anti-PRRSV antibodies; otherwise, it was negative.

[0174] (5) Determination of the sensitivity of competitive ELISA

[0175] Five anti-PRRSV antibody-positive pig sera were selected for multiple dilution and then tested using the established competitive ELISA. The results showed that when the positive serum was diluted 1:320, the competitive ELISA test was still positive, indicating that the established competitive ELISA has good sensitivity ( Fig.10 ).

[0176] (6) Determination of the specificity of competitive ELISA

[0177] The ELISA was used to test 23 known negative sera, and all 23 were negative. PEDV-positive sera, ASFV-positive sera, and PCV2-positive sera were tested, and the confirmed PRRSV-positive pig sera were used as positive controls, and the results were statistically analyzed. The results showed that this method did not cross-react with the above positive control sera ( Fig.11 ).

[0178] (7) Evaluation of the compliance rate between competitive ELISA and commercial ELISA kits

[0179] In order to evaluate the clinical application of the competitive ELISA detection method, 240 clinical sera were tested using competitive ELISA and commercial ELISA kits, and the results showed that the compliance rate was 96.92%.

[0180] Obviously, the above embodiments of the present invention are only examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A bivalent nanobody against genotype 2 PRRSV, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

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

2.

3. A fusion protein, characterized in that It comprises the bivalent nanobody according to claim 1 and horseradish peroxidase, and its amino acid sequence is shown in SEQ ID NO.

3.

4. A DNA molecule encoding the fusion protein according to claim 3, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

4.

5. Use of the fusion protein as claimed in claim 3 in the preparation of a product for detecting antibodies against genotype 2 PRRSV.

6. A product for detecting antibodies against genotype 2 PRRSV, characterized in that: Comprising the fusion protein of claim 3.

7. A method for detecting anti-genotype 2 PRRSV antibodies for non-disease diagnosis or treatment purposes, characterized in that: The following steps are involved: The ELISA plate is coated with PRRSV as an antigen, and the fusion protein described in claim 3 is used as a competitive reagent. The sample to be tested is mixed with the fusion protein and 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-genotype 2 PRRSV antibodies.

8. The method according to claim 7, characterized in that The formula for calculating the competition rate is: Competition rate = (1-OD of the pig serum sample to be tested 450nm Value / negative pig serum sample OD 450nm value)×100%.

9. The method according to claim 7, characterized in that: The method for determining whether the sample to be tested contains anti-genotype 2 PRRSV antibodies is as follows: if the competition rate of the pig serum to be tested is greater than 19.98%, it is determined to be positive, otherwise it is negative.

Images