Mixed preparation for preventing and treating porcine reproductive and respiratory syndrome virus infection as well as preparation method and application thereof

Abstract

The invention discloses a mixed preparation for preventing and treating porcine reproductive and respiratory syndrome virus (PRRSV) infection and a preparation method and application thereof, and belongs to the technical field of biological and animal epidemic disease prevention and treatment. The invention provides a mixed preparation for preventing and treating PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) infection as well as a preparation method and application of the mixed preparation as well as sequences of two nano antibodies and porcine IgG1Fc contained in the mixed preparation, and meanwhile, the activity of the mixed preparation for resisting PRRSV infection in vivo is evaluated. The mixed preparation is applied to prevention and treatment of PRRSV infected pigs, a new strategy and preparation are provided for development of drugs for treating the PRRSV, and the mixed preparation has a good market application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biological and animal disease prevention and treatment, and in particular to a mixed preparation for preventing and treating porcine reproductive and respiratory syndrome virus (PRRSV) infection, and a preparation method and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease with a high degree of contact characteristics. Its pathogen is porcine reproductive and respiratory syndrome virus (PRRS virus, PRRSV). The disease was first reported in the United States in 1987, and then the disease was also reported in Europe. PRRSV infection can cause respiratory disorders in pigs of different ages. Infection of pregnant sows can cause severe reproductive disorders such as premature birth, fetal death, and mummification. In addition, PRRSV infection in pigs can cause immunosuppression, secondary infection with other viruses and bacteria, and lead to increased mortality in pigs. PRRS seriously endangers the healthy development of the pig industry worldwide and is one of the important epidemic diseases that causes serious economic losses to the pig industry. In 2006, highly pathogenic PRRSV (HP-PRRSV) infection broke out in my country, causing severe high fever (41℃-42℃), high morbidity (50%-100%) and high mortality (20%-100%) in pigs. The outbreak has resulted in the culling of more than 20 million pigs in my country, causing severe economic losses to my country's pig industry. In recent years, the prevalence of HP-PRRSV strains in my country's pig herds has gradually shifted to NADC-30 and NADC-34 strains. The prevalence of different lineages of PRRSV strains in my country's pig herds has made the prevention and control of the disease increasingly complex.

[0003] At present, the most effective prevention and control measure for the disease is vaccine immunization, and the vaccines available on the market to prevent the disease are mainly attenuated vaccines. However, due to the error-prone nature of the RNA polymerase replication process of PRRSV and the frequent recombination between different PRRSV strains, the virus has evolved rapidly, resulting in up to 20% differences in nucleotide sequences between different strains, thus forming multiple lineages. The existing commercial vaccines have poor protection against different strains, especially heterologous strains. Therefore, in order to effectively prevent and control PRRS, new prevention and control strategies still need to be developed. In recent years, the development of anti-PRRSV infection drugs has gradually become one of the hot spots in the research of new strategies for the prevention and control of the disease. For example, many Chinese herbal medicines (epigallocatechin gallate, tanshinone ⅡA, etc.) have been proven to effectively inhibit the replication of PRRSV in host cells in vitro; small interfering RNA targeting viral genes can also inhibit the replication of viruses in cells. However, due to problems such as production costs or routes of administration, the use of anti-PRRSV drugs such as Chinese herbal medicines or nucleic acid inhibitors in clinical practice is still relatively rare. In addition, antibody drugs have also been a research hotspot. A variety of monoclonal antibodies against PRRSV GP3, N and M proteins have been prepared and have been shown to have a certain effect of inhibiting PRRSV proliferation in vitro or in vivo. However, monoclonal antibodies have disadvantages such as high production cost and poor stability, and have not yet been used in clinical practice.

[0004] Nanobodies are gradually being widely used in the fields of biology, medicine, energy, catalysis and environment due to their small molecular force, low in vitro production cost and easy genetic engineering. In 2019, the U.S. Food and Drug Administration approved the marketing of a nanoantibody drug called CabliviTM for the treatment of thrombotic thrombocytopenic purpura, which is the first nanoantibody drug approved for marketing. Nanoantibodies are also widely used in the research and development of therapeutic preparations for human infectious diseases. For example, the development of antiviral nanoantibodies for human immunodeficiency virus-1 (HIV-1), influenza virus (Influenzaviruses), hepatitis C virus (HCV), respiratory syncytial virus (RSV) and enterovirus have been reported.

[0005] PRRSV infects pigs with strict mononuclear macrophage tropism, and mainly infects porcine alveolar macrophages (PAM) in vivo. Therefore, the research and development of anti-PRRSV infection drugs mainly focuses on whether they can be efficiently delivered to PAM cells. It is well known that porcine IgG1 Fc receptors (Fcγreceptors, FcγRs) are the main proteins on the surface of PAM cells, which can bind to porcine IgG1 Fc fragments and internalize them into porcine alveolar macrophages (PAM cells). At present, there are no reports on the use of nano-antibody technology for the prevention and treatment of PRRSV. Summary of the invention

[0006] The purpose of the present invention is to provide a mixed preparation for preventing and treating porcine reproductive and respiratory syndrome virus (PRRSV) infection, and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The present invention provides the sequences of two nano antibodies Nb5 and Nb1-14 that can neutralize PRRSV infection, and their amino acid sequences after being fused with the Fc fragment of pig IgG1 respectively. At the same time, the expression preparation method of the above-mentioned fusion protein and the preparation method of its mixed preparation are disclosed. The role of the mixed preparation in preventing and treating PRRSV infection is evaluated. The present invention provides a new material for the prevention and control of PRRSV.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a nano antibody Nb1-14 that neutralizes PRRSV, and its amino acid sequence is shown in SEQ ID NO.1.

[0009] The second technical solution of the present invention is a DNA molecule encoding the nanobody Nb1-14, whose nucleotide sequence is shown in SEQID NO.2.

[0010] The third technical solution of the present invention is a nano antibody Nb5 that neutralizes PRRSV, and its amino acid sequence is shown in SEQ ID NO.3.

[0011] The fourth technical solution of the present invention is a DNA molecule encoding the nanobody Nb5, whose nucleotide sequence is shown in SEQ ID NO.4.

[0012] The fifth technical solution of the present invention is a fusion protein Nb1-14-pFc, whose amino acid sequence is shown in SEQ ID NO.5.

[0013] The sixth technical solution of the present invention is a fusion protein Nb5-pFc, whose amino acid sequence is shown in SEQ ID NO.6.

[0014] The seventh technical solution of the present invention is a mixed preparation for preventing and treating PRRSV infection, comprising the fusion protein Nb1-14-pFc and the fusion protein Nb5-pFc.

[0015] Based on the above technical solution, the present invention has the following technical effects:

[0016] The present invention provides a mixed preparation for preventing and treating PRRSV infection, a preparation method and application thereof, as well as the sequences of two nanoantibodies and pig IgG1 Fc contained in the mixed preparation, and the activity of the mixed preparation against PRRSV infection in vivo is evaluated. The mixed preparation is applied to the prevention and treatment of PRRSV infection in pigs, providing a new strategy and preparation for the development of therapeutic drugs for the disease, and has a good market application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 The SDS-PAGE analysis of the recombinant proteins Nb5-pFc and Nb1-14-pFc after eukaryotic expression and purification, where 1 is Nb5-pFc and 2 is Nb1-14-pFc.

[0019] Figure 2 Immunofluorescence assay (IFA) was used to analyze the neutralizing activity of the eukaryotic expressed and purified recombinant fusion proteins Nb5-pFc and Nb1-14-pFc against PRRSV. The inoculated control group was only infected with PRRSV, the Nb53-pFc group was an irrelevant fusion protein control group, and the Nb1-14-pFc group and Nb5-pFc group were two fusion protein test groups, respectively.

[0020] Figure 3 Western blot analysis was performed on the neutralizing activity of the eukaryotic expressed and purified recombinant proteins Nb5-pFc and Nb1-14-pFc against PRRSV. The inoculation control group was only infected with PRRSV, the Nb53-pFc group was an irrelevant fusion protein control group, and the Nb1-14-pFc group and the Nb5-pFc group were two fusion protein test groups, respectively.

[0021] Figure 4 This is the body temperature change curve of pigs in animal experiments.

[0022] Figure 5 Survival rate of pigs for animal experiments.

[0023] Figure 6 This is an animal experiment to detect the amount of toxins excreted by pigs after virus attack.

[0024] Figure 7 Serum viral load in animal experiments pigs.

[0025] Figure 8 The purpose is to observe clinical lesions in the lungs of pigs for animal experiments.

[0026] Fig. 9 HE staining of lung tissue of pigs used in animal experiments. DETAILED DESCRIPTION

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The technical schemes described in the present invention, unless otherwise specified, are all conventional schemes in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions provided by the manufacturer.

[0033] An embodiment of the present invention provides a nanobody Nb1-14 that neutralizes PRRSV, and its amino acid sequence is shown in SEQ ID NO.1.

[0034] The embodiment of the present invention also provides a DNA molecule encoding the nanobody Nb1-14, and its nucleotide sequence is shown in SEQ ID NO.2.

[0035] The embodiment of the present invention also provides a nano antibody Nb5 that neutralizes PRRSV, and its amino acid sequence is shown in SEQ ID NO.3.

[0036] The embodiment of the present invention also provides a DNA molecule encoding the nanobody Nb5, and its nucleotide sequence is shown in SEQ ID NO.4.

[0037] The embodiment of the present invention further provides a fusion protein Nb1-14-pFc, whose amino acid sequence is shown in SEQ ID NO.5.

[0038] The embodiment of the present invention further provides a fusion protein Nb5-pFc, whose amino acid sequence is shown in SEQ ID NO.6.

[0039] The embodiment of the present invention further provides a mixed preparation for preventing and treating PRRSV infection, comprising the fusion protein Nb1-14-pFc and the fusion protein Nb5-pFc.

[0040] In some specific embodiments, the mass ratio of the fusion protein Nb1-14-pFc to the fusion protein Nb5-pFc is 1:1.

[0041] The present invention fuses the porcine IgG1 Fc fragment with the nano antibody and uses it as a delivery molecule to bring the nano antibody into PAM cells to achieve the purpose of resisting PRRSV infection. In addition, the porcine IgG1 Fc fragment is easy to form a dimer when expressed in vitro, and after being fused with the nano antibody, it can also form a traditional antibody Y-shaped structure, further improving the neutralizing activity of the nano antibody.

[0042] Based on the characteristics of nano antibodies and porcine IgG1 Fc fragments, the present invention fuses porcine IgG1 Fc fragments with two strains of anti-PRRSV neutralizing nano antibodies to express, prepares two fusion proteins, and then mixes the two fusion proteins to prepare a mixed preparation. Animal experiments have shown that the mixed preparation has good anti-PRRSV infection activity. The mixed preparation is expected to become a new type of biological preparation for the treatment of PRRS.

[0043] The present invention utilizes two nano antibodies Nb1-14 and Nb5 that can neutralize PRRSV infection to connect and clone with pig IgG1Fc fragments respectively, and uses HEK 293F suspension cells for secretory expression, that is, fusion proteins Nb1-14-pFc and Nb5-pFc are prepared. Then the two fusion proteins are mixed according to 1:1 (mass ratio) to prepare a mixed preparation. When pigs are infected with PRRSV, the mixed preparation is injected into the neck muscle, which can significantly improve the survival rate of pigs, reduce the replication level of the virus in the pig body, and alleviate the clinical manifestations of PRRSV infection in pigs. The mixed preparation of the present invention has the characteristics of simple production process, stable properties, low production cost, high safety, and high efficiency in preventing and treating PRRSV infection in pigs. Therefore, the mixed preparation has a good market application prospect as a biological preparation for preventing and treating PRRSV infection.

[0044] The amino acid sequence of the Nanobody Nb1-14 used in the present invention is (SEQ ID NO.1): ESGGGSVQAGGSLRLSCTASGYTYLMTWFRQAPGKEREGVATIYTAGGTTFYVNSVKGRFTISQDK TKKTVYLEMNSLKPEDTAMYYCAAADQGDSPRSWLAPNRYTYWGQGTQVTVSS;

[0045] The nucleotide sequence of Nanobody Nb1-14 used in the present invention is (SEQ ID NO.2): GAGTCTGG GGGAGGGTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATACACTTACTTGATGACCTGGTTCCGCCAGGCTCCAGGGAAGGCGCGAGGGGGTCGCGACTATTTATACTGCGGGTGGTACCACATTCTATGTCAACTCCGTGAAGGGCCGATTCACC ATCTCCCAAGACAAGACAAAGAAAACGGTGTATCTGGAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCGGCAGATCAGGGGGACTCGCCTCGTTCTTGGCTGGCCCCAAATAGGTATACGTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA;

[0046] The amino acid sequence of the Nanobody Nb5 used in the present invention is (SEQ ID NO.3): ESGGDSVQAG GSLRLSCAVSDLQFGSYCMGWFRQAPGKVREGVASIDVGGRTKYADSVKGRFTVTR DNANNTLYLQMNSLKPEDTFTYVCARGYGAGCPRNPSDYAYWGQGTQVTVSS;

[0047] The nucleotide sequence of Nanobody Nb5 used in the present invention is (SEQ ID NO.4): GAGTCTGGGG GAGACTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGTCTCCGACCTCCAATTCGGTAGCTACTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGTGCGCGAGGGGGTCGCGAGTATTGATGTTGGAGGCAGGACGAAGTACGCAGACTCCGTGAAGGGCCG ATTCACCGTCACCCGAGACAACGCCAACAATACTCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACCTTCACGTACGTCTGTGCCCGGGGTTATGGTGCGGGCTGTCCTAGGAACCCAAGTGACTATGCGTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA;

[0048] The amino acid sequence of the fusion protein Nb1-14-pFc used in the present invention is (SEQ ID NO.5): MHSSAL LCCLVLLTGVRAQVQLQESGGGSVQAGGSLRLSCTASGYTYLMTWFRQAPGKEREGVATIYTAGGTTFYVNSVKGRFTISQDKTKKTVYLEMNSLKPEDTAMYYCAAADQGDSPRSWLAPNRYTYWGQGTQVTVSSTKTKPPCPICPGCEVAGPSVFIFPPKPKDTLMISQTPEVTCVVVDVS KEHAEVQFSWYVDGVEVHTAETRPKEEQFNSTYRVVSVLPIQHQDWLKGKEFKCKVNNVDLPAPITRTISKAIGQSREPQVYTLPPPAEELSRSKVTVTCLVIGFYPPDIHVEWKSNGQPEPEGNYRTTPPQQDVDGTFFLYSKLAVDKARWDHGETFECAVMHEALHNHYTQKSISKTQGK;

[0049] The amino acid sequence of the fusion protein Nb5-pFc used in the present invention is (SEQ ID NO.6): MHSSALLC CLVLLTGVRAQVQLQESGGDSVQAGGSLRLSCAVSDLQFGSYCMGWFRQAPGKVREGVASIDVGGRTKYADSVKGRFTVTRDNANNTLYLQMNSLKPEDTFTYVCARGYGAGCPRNPSDYAYWGQGTQVTVSSTKTKPPCPICPGCEVAGPSVFIFPPKPKDTLMISQTPEVTCVVVDVSK EHAEVQFSWYVDGVEVHTAETRPKEEQFNSTYRVVSVLPIQHQDWLKGKEFKCKVNNVDLPAPITRTISKAIGQSREPQVYTLPPPAEELSRSKVTVTCLVIGFYPPDIHVEWKSNGQPEPEGNYRTTPPQQDVDGTFFLYSKLAVDKARWDHGETFECAVMHEALHNHYTQKSISKTQGK.

[0050] Example 1

[0051] Construction of eukaryotic expression vector for nanobody and porcine IgG1 Fc fusion protein and its expression and purification

[0052] 1.1 Construction of eukaryotic expression vector of nanobody and porcine IgG1 Fc fusion protein

[0053] Bactrian camels were immunized with PRRSV particles purified by sucrose density gradient centrifugation. After five immunizations, peripheral blood lymphocytes were collected, RNA was extracted, and reverse transcribed into cDNA. The nanobody gene was amplified by two rounds of nested PCR, and then ligated to the PMECS vector after two rounds of restriction digestion and electroporated into TG1 competent cells to construct a nanobody library with a capacity of 1.3×10 8 . Then, purified PRRSV particles were used as coating antigens, and phage display technology was used for three rounds of panning to obtain nanobodies that specifically bind to PRRSV particles. Then, after these nanobodies were expressed and purified in prokaryotes, a virus neutralization test was used to screen nanobodies with neutralizing activity. The results showed that the screened nanobodies Nb1-14 and Nb5 had a good effect in neutralizing PRRSV-infected PAM in vitro.

[0054] The above-screened nanoantibodies Nb1-14 and Nb5 were respectively combined with the nucleotide sequence of the porcine IgG1 Fc fragment (including the hinge region, CH2 and CH3 segments, GenBank ID: U03781), and the gene sequences were synthesized by GENEWISE, and the recombinant cloning plasmid pcDNA3.1 was constructed.

[0055] The recombinant plasmid was transformed into Trans(5α) competent cells. After culturing at 37°C for 12 h, a single colony was picked and inoculated into 10 mL of LB liquid medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and deionized water was supplemented to 1000 mL). After culturing on a shaker at 37°C for 12 h, the plasmid was extracted for subsequent experiments. The plasmids were named pcDNA3.1-Nb1-14-pFc and pcDNA3.1-Nb5-pFc, respectively.

[0056] 1.2 Eukaryotic expression of nanobody and porcine IgG1 Fc fusion protein

[0057] The plasmids pcDNA3.1-Nb1-14-pFc and pcDNA3.1-Nb5-pFc constructed above were mixed with Union 293 culture medium, and PEI (linearized poly(imide)) was used to achieve efficient transfection of HEK 293F cells. For specific operations, refer to the instructions of the purchased transfection kit for experiments.

[0058] (1) One day before transfection, 2.0×10 6 The cell density can reach 4.0×10 cells / mL on the second day of culture. 6 cells / mL;

[0059] (2) After cell counting on the second day of culture, the cell viability is >95% and the viable cell density is ≥4.0×10 6 cells / mL, it can be used directly; if the cell density is lower than 4.0×10 6 cells / mL, the cells can be collected by centrifugation (800 rpm, 3-5 min) and the cells are centrifuged at 4.0×10 6 cells / mL density and resuspended in Union 293 medium;

[0060] Note: If transient expression is performed for ≥6 days without adding feed and glucose, the density during transient expression should be controlled at 2.0-3.0×10 6 cells / mL.

[0061] (3) According to the optimized transient transfection process, PEI was used for transfection in a 20 mL system to prepare a mixture of pcDNA3.1-Nb1-14-pFc and pcDNA3.1-Nb5-pFc and PEI. The specific preparation process is as follows: 20 μg of pcDNA3.1-Nb1-14-pFc or pcDNA3.1-Nb5-pFc was mixed with 600 μL of culture medium to form liquid A; 80 μg of PEI was mixed with 600 μL of culture medium to form liquid B; then liquid A was added to liquid B and gently pipetted to mix, and then allowed to stand at room temperature for 15 min.

[0062] (4) adding the mixed solution to the culture medium for culturing;

[0063] (5) For cultures longer than 6 days, add ≤5% feed (and <5 g / L glucose) to the culture medium after 20 hours to further increase viable cell density and protein expression. Detect residual sugar during the process and control the residual sugar concentration at 3-6 g / L.

[0064] (6) Cultivate until the activity is less than 60%, and then terminate the cultivation.

[0065] 1.3 Purification of Nb1-14-pFc and Nb5-pFc fusion proteins

[0066] (1) Expand the culture system according to step 1.2, express and prepare the fusion protein in large quantities, collect the cell culture supernatant, and purify the fusion protein using Protein G.

[0067] (2) Collect the cell supernatant and then concentrate the cell supernatant with PEG6000 to 1 / 20 of the original volume.

[0068] (3) Filter the supernatant to be purified and other protein purification-related reagents through a 0.45 μm filter membrane.

[0069] (4) Assembly of Protein G. Shake the Protein G filler thoroughly, add 1 mL of 0.01 M PBS to a new chromatography column in advance, then pipette 1 mL of filler into the column, allow the filler to settle naturally, and allow the equilibration buffer to flow out at 1 mL / min.

[0070] (5) Loading. Load the sample into the chromatography column at a flow rate of about 1 mL / min and collect the effluent.

[0071] (6) Wash the impurities. Wash the resin with 10-20 column volumes of 0.01 M PBS at a flow rate of about 2 mL / min until the A280 absorbance of the effluent approaches 0 and becomes stable.

[0072] (7) Elute the target protein. Gently add 5-10 times the volume of 0.1 M glycine (pH = 2.8) to avoid resin suspension, maintain a flow rate of about 1 mL / min, collect the effluent, and immediately add 1 / 10 volume of 1 M Tris-base (pH = 8.0) to neutralize the pH value of the effluent to 7.5, and detect the A280 absorbance until it is close to 0.

[0073] (8) SDS-PAGE was used to detect the purification of the fusion protein. The results showed that there was an obvious target band at 40 kDa ( Figure 1 ).

[0074] (9) Protein dialysis. Add the purified protein to a pre-treated dialysis bag and place it in pre-cooled 0.01 M PBS (containing K + , pH = 7.2-7.4) dialysate, stir and dialyze at 4°C, and replace the dialysate every 4-6 hours.

[0075] (10) Protein concentration. Collect the dialyzed protein and concentrate it using a protein ultrafiltration tube with a pore size of 10 kDa. The specific operation is as follows: fill a new ultrafiltration tube with ultrapure water, soak it on ice for 30 minutes to fully wet the filter membrane, and discard the ultrapure water. Add the protein sample, place the concentration tube in a low-temperature horizontal rotor refrigerated centrifuge, and centrifuge and concentrate at 4°C and 4000 r / min.

[0076] (11) After collecting the concentrated protein, the protein concentration was determined using a protein quantification kit (BCA Protein Assay Kit).

[0077] Example 2

[0078] Identification of Nb5-pFc and Nb1-14-pFc fusion proteins neutralizing PRRSV

[0079] 2.1 Identification of biological activities of Nb5-pFc and Nb1-14-pFc fusion proteins

[0080] (1) PAM cells were cultured at a rate of 1×10 6 / mL density, 500 μL per well of a 24-well cell culture plate, and culture at 37°C, 5% CO2 for 3 h until the cells are completely attached.

[0081] (2) Mixing of fusion protein and virus mixture: Neutralization group: Take a sterilized 1.5mL EP tube, add 150μL of fusion protein (2mg / mL, fusion protein Nb1-14-pFc, Nb5-pFc or irrelevant nanoantibody Nb53-pFc) and 0.1MOI of PRRSV-EGFP recombinant strain (virus carries EGFP green fluorescent protein), then add DMEM basal medium to 1.6mL, mix thoroughly and incubate at 4℃ for 16 hours, with 3 replicate wells for each test group (500μL per well); Inoculation control group: 0.1MOI of PRRSV-EGFP recombinant strain was thoroughly mixed with 1.6mL DMEM basal medium, incubated at 4℃ for 16 hours, with 3 replicate wells for each test group (500μL per well);

[0082] (3) Neutralization test: The plated cell plates were placed at 4°C in advance to maintain the cell temperature at 4°C (to prevent the occurrence of cell internalization). The mixture of the fusion protein and virus incubated at 4°C was inoculated into the cells, 500 μL per well, and placed at 4°C for virus adsorption for 2 hours. The cells were gently washed twice with PBS (K+) to remove the residual virus that was not adsorbed. The culture medium was replaced with DMEM culture medium containing 3% FBS, 500 μL / well, 5% CO2, and cultured at 37°C for 24 hours. The fluorescence was observed under a fluorescence microscope. The results showed that after the Nb5-pFc and Nb1-14-pFc fusion proteins were mixed with the virus, the green fluorescence number was significantly lower than that of the non-inoculated group and the unrelated control group (Nb53-pFc), indicating that the fusion proteins Nb5-pFc and Nb1-14-pFc have the activity of neutralizing PRRSV ( Figure 2 ).

[0083] (4) 24 hours after inoculation, discard the cell culture supernatant, wash the cells three times with preheated PBS solution, add 100 μL of NP40 lysis solution containing 1% PMSF, lyse on ice for 10 minutes, centrifuge and discard insoluble matter, boil the lysed cell protein sample and perform SDS-PAGE, and transfer the protein to a PVDF membrane. After blocking the membrane with PBS'T containing 5% skim milk powder for 1 hour, wash the membrane, add anti-PRRSVN protein specific monoclonal antibody 6D10 (1 μg / mL), incubate at room temperature for 1 hour, wash the membrane, add HRP-labeled goat anti-mouse IgG and incubate for 1 hour, finally use ECL luminescent liquid to develop color, take pictures and observe the results, evaluate the virus neutralization effect of Nb5-pFc and Nb1-14-pFc, the results show that Nb5-pFc and Nb1-14-pFc significantly inhibit the replication of PRRSV ( Figure 3 ).

[0084] 2.2 Animal experiments to evaluate the inhibitory effect of the mixed preparation on PRRSV replication in vivo

[0085] Strain: The virus strain used for attack was PRRSV-JXA1.

[0086] Experimental animals: 20 healthy weaned piglets aged 28 days, both PRRSV antibodies and antigens were negative.

[0087] Experimental design: 20 non-immune negative pigs were randomly divided into 4 groups, including 5 pigs in the non-challenge control group, 5 pigs in the mixed preparation treatment group (Nb5-pFc and Nb1-14-pFc were mixed in a mass ratio of 1:1), 5 pigs in the challenge control group, and 5 pigs in the irrelevant mixed preparation (Nb53-pFc) treatment control group; the challenge method was neck intramuscular injection (10 5 TCID 50 / head); mixed preparation treatment injection: neck intramuscular injection. All animals were euthanized and autopsied at 14 days.

[0088] Table 1 Animal test scheme for the inhibition of PRRSV replication by mixed preparations in vivo

[0089]

[0090] The rectal temperature of all pigs was measured with a thermometer every morning after the challenge. The temperature of the pigs in the challenge control group and the control group treated with unrelated mixed preparations was higher than 40.5℃ on the 3rd and 4th days after the challenge, respectively, and lasted until the 12th day after the challenge. The temperature of the pigs in the mixed preparation treatment group was higher than 40.5℃ on the 5th day after the challenge and lasted until the 9th day after the challenge, which was significantly shorter than that of the pigs in the single challenge and unrelated mixed preparation treatment groups. The temperature of the pigs in the non-challenge control group was always below 40.5℃ ( Figure 4), where the pigs in the challenge control group were infected with PRRSV (JX-A1 strain), and the pigs in the mixed preparation treatment group were injected with nano antibody mixed preparation (2 mg / kg) 6 hours, 1 day and 3 days after infection with PRRSV (JX-A1 strain); the pigs in the unrelated mixed preparation treatment control group were injected with unrelated mixed preparation (2 mg / kg) 6 hours, 1 day and 3 days after infection with PRRSV (JX-A1 strain); the pigs in the non-challenge control group were injected with nano antibody mixed preparation (2 mg / kg) 6 hours, 1 day and 3 days after injection of PBS. The results show that the mixed preparation treatment significantly reduced the fever symptoms of pigs after PRRSV infection.

[0091] The spirit, feeding, drinking water, feces and morbidity of each group of pigs were observed daily, and the survival rate of piglets was recorded. The pigs in the control group and the control group treated with irrelevant mixed preparations began to die on the 9th and 10th days after the challenge, respectively, and the survival rate of the two groups of pigs was 25% until the 14th day after the challenge. The pigs in the control group and the mixed preparation treatment group did not die on the 14th day after the challenge, and the survival rate was 100% ( Figure 5 ), the above results indicate that the mixed preparation significantly improved the survival rate of pigs after treating PRRSV-infected pigs.

[0092] Fluorescence quantitative RT-PCR was used to quantitatively detect PRRSV in serum and nasal swabs. The results showed that the serum viral load and toxin excretion of pigs in the challenge control group and the unrelated mixed preparation treatment control group reached the highest level on the 5th day after the challenge, and the serum viral load and toxin excretion of pigs in the non-challenge control group and the mixed preparation treatment group after the challenge were significantly lower than those in the challenge control group and the unrelated mixed preparation treatment control group ( Figure 6 and 7 ), nasal swabs were collected on days 0, 1, 3, 5, 7, 10 and 14 after the challenge, and the viral load of the nasal swabs was detected by fluorescent quantitative RT-PCR. The serum of the pigs was collected on days 0, 1, 3, 5, 7 and 14 after the challenge, and the viral load was detected by fluorescent quantitative RT-PCR. The above results show that the mixed preparation treatment significantly reduced the viral load in the serum of the pigs and the amount of toxins discharged to the external environment through the nose.

[0093] All pigs were autopsied on the 14th day after the challenge. The lung lesions were observed in the autopsy of the lungs. The lungs of the pigs in the challenge control group and the unrelated mixed preparation treatment control group had obvious hemorrhage and edema, while the lungs of the pigs in the non-challenge control group and the mixed preparation treatment group were normal ( Figure 8), all pigs were dissected 14 days after the virus attack, the lungs were collected, and the lung autopsy lesions were observed. The above results show that the mixed preparation treatment significantly reduced the damage of PRRSV to the pigs' lungs. Subsequently, the lung pathological changes of the pigs were observed by HE staining. The results showed that the alveolar walls of the pigs in the virus attack control group and the irrelevant mixed preparation treatment control group were thickened, and there were red blood cells and inflammatory cells infiltration in the alveoli. The alveolar walls of the pigs in the non-virus attack control group and the mixed preparation treatment group were normal, and no red blood cells and inflammatory cells were infiltrated ( Fig. 9 ), all pigs were dissected 14 days after the challenge, lungs were collected and fixed in 4% paraformaldehyde, and HE staining was used to observe the pathological changes of lung tissue. The above results show that the mixed preparation treatment significantly reduces the lung damage of pigs after PRRSV infection.

[0094] In summary, the mixed preparation prepared by the present invention has a significant effect of neutralizing PRRSV in vitro experiments, and the results of in vivo toxicity treatment experiments show that intramuscular injection of the mixed preparation can effectively improve the survival rate of PRRSV-infected pigs, reduce the viral load in the blood and alleviate lung damage. Therefore, the mixed preparation provided by the present invention can effectively treat PRRSV infection, reduce the economic losses caused by PRRSV to the pig farming industry, and has significant economic value and application market.

[0095] 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 nanobody Nb1-14 that neutralizes PRRSV, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A DNA molecule encoding the nanobody Nb1-14, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A nanobody Nb5 that neutralizes PRRSV, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

4. A DNA molecule encoding the nanobody Nb5, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

4.

5. A fusion protein Nb1-14-pFc, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

5.

6. A fusion protein Nb5-pFc, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

6.

7. A mixed preparation for preventing and treating PRRSV infection, characterized in that: It includes the fusion protein Nb1-14-pFc according to claim 5 and the fusion protein Nb5-pFc according to claim 6.

8. The mixed preparation according to claim 7, characterized in that The mass ratio of the fusion protein Nb1-14-pFc to the fusion protein Nb5-pFc is 1:1.

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