Gene 1 and 2 type porcine reproductive and respiratory syndrome virus shared epitope bivalent nano antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-gene 1 and 2 type porcine reproductive and respiratory syndrome virus common epitope bivalent nano antibody as well as a preparation method and application thereof, and belongs to the technical field of animal epidemic disease detection. The invention provides a bivalent nano antibody sharing epitopes for resisting genes 1 and 2 PRRSV (Porcine Reproductive and Respiratory Syndrome Virus), a preparation method and application of the bivalent nano antibody, and an expression preparation method of the bivalent nano antibody and HRP (Horse Radish Peroxidase) fusion protein, and also evaluates the application of the bivalent nano antibody and the HRP fusion protein in detecting the antibodies for resisting the genes 1 and 2 PRRSV in porcine serum.

Description

Technical Field

[0001] The present invention relates to the technical field of animal disease detection, and in particular to a bivalent nano antibody 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 (PRRS 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 suid2 (North American type / PRRSV-2). PRRSV-2 is the main prevalent strain in my country. However, in recent years, PRRSV-1 infection has also been reported in different farms across the country. Epidemiological surveys have shown that if PRRSV-1 infection occurs in pig farms, it is often co-infected with PRRSV-2. In addition, the monitoring of anti-PRRSV antibodies is also the main way for pig farms to evaluate vaccine immunity and monitor PRRSV infection. However, the PRRS antibody detection kits in the domestic market are mainly imported, and the stability of domestic related products is not good. Therefore, it is necessary to develop an ELISA kit with high sensitivity, good stability, and the ability to simultaneously detect anti-gene type 1 and 2 PRRSV antibodies to meet the clinical PRRS antibody detection needs.

[0003] As a new type of third-generation genetically engineered antibody, nanobodies are the smallest antibodies known to have antigen binding ability. Compared with traditional monoclonal antibodies, their molecular weight is only about 15kDa, which is easy to express and prepare in vitro and genetically engineered. For example, nanobodies can be coupled with reporter genes (green fluorescent protein, horseradish peroxidase, etc.) to prepare fusion proteins, and fusion proteins can be widely used in the development of animal disease detection technology. At present, there are many literature reports on 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 egg of nanoantibodies and horseradish peroxidase (HRP) as a competitive probe, without 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 suspended cell culture, reducing production costs and having good market application prospects.

[0004] There have been many research reports on the detection methods of PRRSV antibodies. For example, Sorensen et al. established a double-blocking ELISA method that can distinguish between genotype 1 and 2 PRRSV antibodies, but whether this method is suitable for the detection of currently prevalent strains remains to be verified. Brown et al. established a dual ELISA based on the PRRSV Nsp7 protein, which can distinguish between genotype 1 and 2 PRRSV antibodies, but it has not yet been applied in the Chinese market. Methods and kits for detecting anti-genotype 1 and 2 PRRSV antibodies based on nano-antibody technology have not been reported. At present, the clinical detection kit for anti-genotype 1 and 2 PRRSV antibodies in my country mainly uses the IDEXX company's PRRS X3 Ab kit as the gold standard for detection. The 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 epitopes of porcine reproductive and respiratory syndrome virus type 1 and type 2, and a preparation method and application thereof, 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, and its amino acid sequence is shown in SEQ ID NO.13.

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

[0009] The third technical solution of the present invention is the use of the fusion protein in the preparation of products for detecting antibodies against gene types 1 and 2 PRRSV.

[0010] A fourth technical solution of the present invention is a product for detecting antibodies against gene types 1 and 2 PRRSV, comprising the fusion protein.

[0011] A fifth technical solution of the present invention is a method for detecting antibodies against PRRSV gene types 1 and 2 for non-disease diagnosis or treatment purposes, comprising 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 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-gene type 1 and 2 PRRSV antibodies.

[0013] Based on the above technical solution, the present invention has the following technical effects:

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

[0015] 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.

[0016] Figure 1 The gene sequences encoding the monovalent nanoantibodies PRRSV-N-1a3 and PRRSV-N-Nb3 were amplified by PCR respectively; wherein, M is Maker; 1 is the fusion of the amplified monovalent nanoantibody PRRSV-N-1a3 gene and the rigid Linker; 2 is the fusion of the amplified monovalent PRRSV-N-Nb3 gene and the rigid Linker.

[0017] Figure 2 It is a gene sequence encoding a tandem bivalent nanobody PRRSV-N-1a3-Nb3 amplified by overlapping PCR; wherein M is Maker; 1 is a gene sequence encoding a bivalent nanobody PRRSV-N-1a3-Nb3 amplified by overlapping PCR.

[0018] Figure 3It is the result of double enzyme digestion of the gene sequence of the tandem bivalent nano antibody PRRSV-N-1a3-Nb3 and the pCMV-N1-HRP empty vector, wherein M is Maker; 1 and 2 are the results of double enzyme digestion of PRRSV-N-1a3-Nb3; 3 is the result of double enzyme digestion of the pCMV-N1-HRP empty vector.

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

[0020] Figure 5 After transfecting HEK-293T cells with the constructed bivalent nanobody and HRP fusion protein recombinant expression plasmid, IFA was used to identify the expression of PRRSV-N-1a3-Nb3-HRP fusion protein in HEK-293T cells.

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

[0022] Figure 7 This is Western blot analysis of purified PRRSV particles, where M stands for Maker.

[0023] Figure 8 The prokaryotically expressed purified PRRSV-N protein and purified PRRSV particles were used as coating antigens, and three fusion proteins PRRSV-N-1a3-HRP, PRRSV-N-Nb3-HRP and PRRSV-N-1a3-Nb3-HRP were used as competition probes, respectively. Competition ELISA analysis was used to compare the competition rates of the two coating antigens and the three competition probes.

[0024] Fig. 9 In order to use purified PRRSV particles as coating antigen and PRRSV-N-1a3-Nb3-HRP fusion protein as competition probe, PRRSV antibody positive and negative pig sera were diluted and mixed with the fusion protein at dilution ratios of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280 to analyze the sensitivity of the competitive ELISA assay.

[0025] Fig.10In order to identify the specificity of the competitive ELISA detection method, purified PRRSV particles were used as coating antigens and PRRSV-N-1a3-Nb3-HRP was used as a competitive reagent. PEDV-positive serum, TGEV-positive serum, PCV2-positive serum, PRV-positive serum, and PPV-positive serum were diluted into the fusion protein at a ratio of 1:10 and mixed. At the same time, the determined PRRSV-positive pig serum was used as a positive control. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] 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.

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

[0033] 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.14.

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

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

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

[0037] 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-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 anti-gene type 1 and 2 PRRSV antibodies 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 also 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 nano antibody with a common epitope against gene type 1 and 2 PRRSV and a preparation method thereof, as well as a preparation method of the bivalent nano antibody and a horseradish peroxidase (HRP) fusion protein and an application of the fusion protein in detecting antibodies against gene type 1 and 2 PRRSV in pig serum. Two nano antibodies with different common epitopes against nucleocapsid (N) proteins of gene type 1 and 2 PRRSV are connected in series using a rigid linker, and then the series-connected bivalent nano antibodies are fused and expressed with HRP to prepare a bivalent nano antibody 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 gene type 1 and 2 PRRSV in pig serum. The method can simultaneously detect antibodies against gene type 1 and 2 PRRSV 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 great market application prospects.

[0046] Example 1

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

[0048] 1.1 Construction of fusion protein eukaryotic expression vector

[0049] (1) Based on the gene sequences of two monovalent nanobodies (1a3 and Nb3) against the common epitopes of PRRSV N protein of gene type 1 and 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 PRRSV-N protein of gene type 1 and 2 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] GAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTACTACATGGGCTGGTTCCGAAAGCCTCCAGGGAAGGAGCGCGAGGGGGTCGCAGCTTTATACTCCTAGTGGTAGCACATACACTGCCAACTCCGTGAAA AGCCGATTCACCATCAAAGACGCCGCCAAGAACACGGTGCCCCAAATGAACAGCCTACAACCTGAGGACACTGCCATGTACTACAATGCGGCAGATCGGAATCCGGGCGGGGTACTATCCTCCCGCGGGTATCACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

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

[0055] ESGGGSVQAGGSLTLSCAASGFPVNNYYMGWFRQAPGRELEGVASIAGDDGTV YTNSVKGRFTIFRDNVNNTLYLRINSLKPEDTAIYYCAAESGRVRAQWLIANAFKYW GQGTQVTVSS.

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

[0057] GAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGACACTCTCCTGTGCAGCCTCTGGATTTCCCGTCAATAATTACTACATGGGCTGGTTTCGCCAGGCTCCAGGGAGGGAGCTCGAGGGGGTCGCGTCTATTGCTGGTGATGATGGCACAGTCTACACAAACTCCGTGAAGGGT CGATTCACCATCTTTCGCGACAACGTCAACAACACGCTGTATCTGCGAATAAACAGCCTAAAACCTGAGGACACTGCCATTTACTACTGTGCGGCAGAAAGCGGGCGGGTTCGCGCACAGTGGTTAATAGCGAATGCTTTTAAATACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

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

[0059]

[0060] First, primers F1-240509-PstⅠ and R1-240509-rigid linker were used to amplify the gene sequence of the monovalent nanoantibody PRRSV-N-1a3 against the common epitopes of PRRSV types 1 and 2. The reaction system is shown in Table 2.

[0061] Table 2 PCR amplification reaction system

[0062]

[0063] 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.

[0064] 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, and the gene sequence encoding the monovalent nanobody PRRSV-N-1a3 was obtained.

[0065] Similarly, primers F2-240509-rigid linker and R2-240509-NotⅠ were used to PCR amplify the monovalent nanoantibody PRRSV-N-Nb3 gene sequence against the common epitope of PRRSV type 1 and 2. The upstream of the gene sequence was connected to the rigid linker, and the PCR reaction system is shown in Table 3.

[0066] Table 3 PCR amplification reaction system

[0067]

[0068] 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.

[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 using the EasyPure Quick Gel Extraction Kit to obtain the gene sequence encoding the monovalent nanobody PRRSV-N-Nb3.

[0070] Using the PCR products recovered from the two gels as templates, primers F3-240509-PstⅠ and R3-240509-NotⅠ were used for overlapping PCR amplification, and the gene of the monovalent nanoantibody PRRSV-N-1a3 was connected to the gene of PRRSV-N-Nb3 using a rigid linker. The reaction system is shown in Table 4.

[0071] Table 4 Overlapping PCR amplification reaction system

[0072]

[0073]

[0074] 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 1 min; and extension at 72°C for 7 min.

[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 using the EasyPure Quick Gel Extraction Kit.

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

[0077] ESGGGSVQAGGSLRLSCAASGSTYYMGWFRKPPGKEREGVAALYTPSGSTYTANSVKSRFTIKDAAKNTVPQMNSLQPEDTAMYYNAADRNPGGVLSSRGYHYWGQGTQVTVSSEAAAKEAAAKE AAAKESGGGSVQAGGSLTLSCAASGFPVNNYYMGWFRQAPGRELEGVASIAGDDGTVYTNSVKGRFTIFRDNVNNTLYLRINSLKPEDTAIYYCAAESGRVRAQWLIANAFKYWGQGTQVTVSS.

[0078] The nucleotide sequence encoding the above-mentioned 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 epitopes of PRRSV genotypes 1 and 2

[0081] The above-recovered PCR product 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).

[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 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 ).

[0087] 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.

[0088] Table 7 Reagent-digested overlapping PCR products and vector pCMV-N1-HRP connection system

[0089]

[0090] The amino acid sequence of the fused protein obtained by connection is (SEQ ID NO.13): ESGGGSVQAGGSLRL SCAASGSTYYMGWFRKPPGKEREGVAALYTPSGSTYTANSVKSRFTIKDAAKNTVPQMNSLQPEDTAMYYNAADRNPGGVLSSRGYHYWGQGTQVTVSSEAAAKEAAAKEAAAKESGGGSVQAGGSLTLSCAASGFPVNNYYMGWFRQAPGRELEGVASIAGDDGTVYTNSVKGRFTIFRDNVNNTLYLRINSLKPEDTAIYYCAAESGRVRAQWLIANAFKYWGQGTQVTVSSAAASSSGSGMQLTPTFYDNSCPNVSNIVRDIIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFSVIDRMKAAVESACPGTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKSQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPDATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRRNCRVVNSNSDLHHHHHHH。

[0091]

[0092] 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-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 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.

[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 culture supernatant of the transfected cells collected above 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 detecting antibodies against PRRSV genotypes 1 and 2 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] ①Take 10 μL of recombinant expression E. coli pET28a-N bacterial solution (expressing PRRSV-N protein) and add 5 mL of kanamycin resistance (K + ) in LB medium and cultured overnight at 37°C 200 rpm. The construction method of recombinant expression Escherichia 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] 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.

[0107] ③ After collecting the bacterial solution and ultrasonically disrupting it, SDS-PAGE was used to analyze the expression of the recombinant PRRSV-N protein.

[0108] 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.

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

[0110] c) Staining After SDS-PAGE electrophoresis, the protein gel was stained for 2 hours.

[0111] d) Decolorization: Wash the protein gel with deionized water and add decolorizing solution. The decolorization time depends on the clarity of the protein bands.

[0112] (2) Purification of PRRSVN recombinant protein

[0113] ① The bacterial solution with large-scale induced expression was centrifuged at 12,000 g for 2 min at 4°C to collect the bacteria.

[0114] 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).

[0115] 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.

[0116] ④ Take a nickel column (Ni Resin) and balance the column with 5 column volumes of Buffer A. 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.

[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] The MARC-145 cell culture layer was cultured into a monolayer. The medium containing 10% FBSDMEM was discarded, and the medium containing 2% FBSDMEM was replaced for culture. The virus supernatant was added and cultured in a 37°C 5% CO2 incubator. CPE was observed under a low-power microscope. Lesions began to appear in 24-48 hours, and CPE was about 80% in 72-96 hours. The pathological phenomena were: cell vacuolation, rounding, first focal shedding, then large shedding, and finally the whole cell lysis and rupture. The virus liquid was collected and frozen and thawed three times, centrifuged at 3000r / min for 10min, and the supernatant was taken. The cell culture was centrifuged at 8000r / min at 4°C for 30min, the supernatant was taken, and then centrifuged at 45000r / min at 4°C for 2.5h, the supernatant was discarded, and the precipitate was resuspended with 0.01mol / LPBS (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 from low to high density into the centrifuge tube. Centrifuge at 4°C and 43,000 r / min for 2 h. Clear stratification will appear in the centrifuge tube. Collect different stratified solutions for Western blot analysis of purified PRRSV particles, such as Figure 7As shown, the band between 35% and 45% was collected. 5 to 10 times the volume of PBS was added to the centrifuge tube to dilute the virus, and the centrifuge was performed at 45,000 rpm, 4°C, for 2 hours to collect the precipitate, which was dissolved with PBS and stored at -80°C.

[0123] 2.1.3 Selection of the best 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'T, add TMB colorimetric solution (100 μL / well) and color for 15 min in the dark;

[0128] (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.

[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 competition 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'T, 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 doubly diluted PRRSV-positive pig serum, add them to the above-mentioned coated ELISA plate, and incubate at room temperature for 1 h;

[0134] (4) Wash the plate 4 times with PBS'T, add TMB colorimetric solution (100 μL / well) and color for 15 min in the dark;

[0135] (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.

[0136] 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 ( Figure 8 ), so the bivalent nanobody HRP fusion protein was selected as the competitive probe for competitive ELISA.

[0137] 2.3 Application of bivalent nanoantibody 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 them on ELISA plates at 100 ng / well, 200 ng / well and 400 ng / well respectively, and coat them at 4°C overnight;

[0140] 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;

[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 above-mentioned coated ELISA plate, and incubate at 37°C for 1 hour;

[0142] ④ After washing the plate 4 times with PBS, add TMB substrate colorimetric solution and color for 15 min 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 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).

[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 serum 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] ④After 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 serum dilution ratio was 1:10, the OD of positive serum 450nm (P) compared with negative serum OD 450nm (N) value is the smallest (Table 9). Therefore, 1:10 is 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] ① Coat the ELISA plate with 100 ng / well of purified whole virus particles SX-GD at 4°C overnight;

[0159] ② After washing the plate 4 times with PBS'T, block it with PBS'T containing 2.5% skim milk powder at 37℃ for 1h;

[0160] ③ Use blocking solution to dilute PRRSV-N-1a3-Nb3-HRP fusion protein at the optimal ratio, and dilute the pig serum to be tested in the above fusion protein at a ratio of 1:10. Add 100 μL / well of the mixture to the ELISA plate and incubate at 37°C for 1 hour;

[0161] ④ Add TMB substrate colorimetric solution and color for 15 minutes in the dark, terminate with 3M H2SO4, and read OD 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.

[0162] (4) Determination of critical value for competitive ELISA results

[0163] 240 known anti-PRRSV antibody negative and positive 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 25.67% calculated by the ROC curve. That is, when the serum was tested by the competitive ELISA, its PI value>25.67%, the result was judged as positive for anti-porcine reproductive and respiratory syndrome antibody; otherwise, it was negative.

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

[0165] Three anti-swine 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. 9 ).

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

[0167] 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.10 ).

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

[0169] 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 95.90%.

[0170] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating 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 fusion protein of a bivalent nanobody against gene type 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 as claimed in claim 1 in the preparation of a product for detecting antibodies against PRRSV gene types 1 and 2.

4. A product for detecting antibodies against PRRSV gene type 1 and 2, characterized in that: Comprising the fusion protein of claim 1.

5. A method for detecting antibodies against PRRSV genotypes 1 and 2 for purposes other than disease diagnosis or treatment, 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 1 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-gene type 1 and 2 PRRSV antibodies.

6. The method according to claim 5, characterized in that The formula for calculating the competition rate based on the OD value is: Competition rate = (1-OD of the pig serum sample to be tested 450nm Value / negative pig serum sample OD 450nm value)×100%.

7. The method according to claim 5, characterized in that The method for determining whether the sample to be tested contains anti-gene type 1 and 2 PRRSV antibodies is: 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.

8. 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 3M H2SO4.

9. 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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