A mixed preparation for preventing and treating porcine reproductive and respiratory syndrome virus infection and its preparation method and application

Abstract

The application discloses a mixed preparation for preventing and treating porcine reproductive and respiratory syndrome virus (PRRSV) infection, a preparation method and application thereof, and belongs to the technical field of biological and animal epidemic disease prevention and treatment. The application provides a mixed preparation for preventing and treating PRRSV infection, a preparation method and application thereof, and sequences of two strains of nanobodies and pig IgG1 Fc contained in the mixed preparation, and in-vivo anti-PRRSV infection activity of the mixed preparation is evaluated. The mixed preparation is applied to prevention and treatment of PRRSV infected pigs, and provides a new strategy and preparation for development of a therapeutic drug for the disease, and 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 control, 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, highly contagious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). The disease was first reported in the United States in 1987 and subsequently in Europe. PRRSV infection can cause respiratory problems in pigs of all ages. Infection in pregnant sows can lead to severe reproductive impairments, including premature birth, fetal death, and mummified fetuses. Furthermore, PRRSV infection in pigs can cause immunosuppression, leading to secondary viral and bacterial infections and increased mortality. PRRS poses a serious threat to the healthy development of the global swine industry and is a major disease causing significant economic losses. In 2006, an outbreak of highly pathogenic PRRSV (HP-PRRSV) infection in my country caused severe hyperthermia (41-42°C), high morbidity (50%-100%), and high mortality (20%-100%) in pigs. This outbreak has led to the culling of over 20 million pigs in my country, causing severe economic losses to the pig industry. In recent years, the prevalence of HP-PRRSV strains in Chinese pig populations has gradually shifted to NADC-30 and NADC-34-like strains. The prevalence of different lineages of PRRSV strains in Chinese pig populations has made disease prevention and control increasingly complex.

[0003] Currently, the most effective prevention and control measure for PRRS is vaccination, and the vaccines available on the market are primarily attenuated. However, the error-prone nature of PRRSV RNA polymerase replication and the frequent recombination between different PRRSV strains drive rapid viral evolution, resulting in up to 20% nucleotide sequence diversity between strains, leading to the formation of multiple lineages. Existing commercial vaccines offer limited protection against different strains, particularly heterologous strains. Therefore, effective prevention and control of PRRS requires the development of new prevention and control strategies. In recent years, the development of anti-PRRSV drugs has become a hot topic in research on new prevention and control strategies. For example, many Chinese herbal medicines (epigallocatechin gallate, tanshinone IIA, etc.) have been shown to effectively inhibit PRRSV replication in host cells in vitro; small interfering RNAs targeting viral genes can also inhibit viral replication in cells. However, due to production costs and administration routes, the clinical use of anti-PRRSV drugs such as Chinese herbal medicines or nucleic acid inhibitors remains limited. Furthermore, antibody drugs have also been a research hotspot. Several monoclonal antibodies targeting PRRSV GP3, N, and M proteins have been developed and demonstrated to inhibit PRRSV proliferation in vitro and in vivo. However, monoclonal antibodies have drawbacks such as high production costs and poor stability, and have not yet been used clinically.

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

[0005] PRRSV infects pigs with strict monocyte macrophage tropism, which mainly infects porcine alveolar macrophages (PAM) in vivo. Therefore, the development of anti-PRRSV infection drugs mainly focuses on whether it can be efficiently delivered into PAM cells. It is known that porcine IgG1 Fc receptor (FcγRs) is the main protein on the surface of PAM cells, which can bind to the Fc fragment of porcine IgG1 and endocytose into porcine alveolar macrophages (PAM cells). There is no report on the use of nanobody technology for the prevention and treatment of PRRSV. SUMMARY

[0006] The purpose of the present application 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 prior art. The present application provides the sequences of two nanobodies Nb5 and Nb1-14 capable of neutralizing PRRSV infection, and the amino acid sequences of their fusion with the Fc fragment of porcine IgG1. The expression preparation method of the above-mentioned fusion protein and the preparation method of the mixed preparation thereof are also disclosed. The role of the mixed preparation in preventing and treating PRRSV infection is evaluated. The present application provides a new material for the prevention and control of PRRSV.

[0007] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0008] One of the technical solutions of the present application is a nanobody Nb1-14 for neutralizing PRRSV, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0009] The second technical solution of the present application is a DNA molecule encoding the nanobody Nb1-14, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0010] The third technical solution of the present application is a nanobody Nb5 for neutralizing PRRSV, the amino acid sequence of which is shown in SEQ ID NO. 3.

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

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

[0013] The sixth technical solution of the present application is a fusion protein Nb5-pFc, the amino acid sequence of which 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 formulation for preventing and treating PRRSV infection, its preparation method, and its use, as well as the sequences of two nanobodies and porcine IgG1 Fc contained in the mixed formulation. The mixed formulation's in vivo anti-PRRSV activity was also evaluated. The mixed formulation's application in preventing and treating PRRSV infection in pigs provides a new strategy and formulation for the development of therapeutic drugs for the disease, with promising market prospects. 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0018] Figure 1 The figure shows 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 to analyze the neutralizing activity of the eukaryotically 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 being challenged with the virus.

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

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

[0026] Figure 9 HE staining of lung tissue from 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

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

[0032] Unless otherwise specified, the technical solutions described in the present invention are conventional in the art. The reagents and raw materials used were purchased from commercial sources or publicly available sources unless otherwise specified. The reactions were carried out under the conditions described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or under 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, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0035] The present invention also provides a nanobody 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, whose nucleotide sequence is shown in SEQ ID NO.4.

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

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

[0039] An 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 porcine IgG1 Fc fragments with nanobodies for expression, using them as delivery molecules to deliver the nanobodies into PAM cells, thereby combating PRRSV infection. Furthermore, porcine IgG1 Fc fragments readily form dimers when expressed in vitro, and when fused with nanobodies, they can also form a traditional antibody Y-shaped structure, further enhancing the neutralizing activity of the nanobodies.

[0042] Based on the properties of nanobodies and porcine IgG1 Fc fragments, the present invention fused porcine IgG1 Fc fragments with two anti-PRRSV neutralizing nanobodies to produce two fusion proteins. These two fusion proteins were then mixed to create a mixed formulation. Animal experiments have demonstrated that this mixed formulation has good anti-PRRSV infection activity. This mixed formulation is expected to become a new biological agent for the treatment of PRRS.

[0043] The present invention utilizes two nano-antibodies Nb1-14 and Nb5 that can neutralize PRRSV infection to be linked and cloned with pig IgG1Fc fragments respectively, and uses HEK 293F suspension cells for secretory expression, thereby preparing fusion proteins Nb1-14-pFc and Nb5-pFc. The two fusion proteins are then mixed in a 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 the pigs, reduce the level of virus replication in the pigs, 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 Nanobody Nb1-14 used in the present invention is (SEQ ID NO. 1): ESGGGSVQA GGSLRLSCTASGYTYLMTWFRQAPGKEREGVATIYTAGGTTFYVNSVKGRFTISQDK 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 for 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 encoding the gene was amplified by two rounds of nested PCR. After two rounds of restriction digestion, the gene was ligated into a PMECS vector 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 nanoantibodies 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 GENEWIZ, and the recombinant cloning plasmid pcDNA3.1 was constructed.

[0055] The recombinant plasmid was transformed into Trans(5α) competent cells and cultured 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 to 1000 mL). After shaking and culturing at 37°C for 12 h, the plasmids were 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 constructed plasmids pcDNA3.1-Nb1-14-pFc and pcDNA3.1-Nb5-pFc were mixed in Union 293 medium and then efficiently transfected into HEK 293F cells using PEI (linearized polyisocyanate). For specific procedures, refer to the instructions of the purchased transfection kit.

[0058] (1) One day before transfection, 2.0×10 6 cells / mL, and 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, can be used directly; if the cell density is lower than 4.0×10 6 cells / mL, the cells were collected by centrifugation (800 rpm, 3-5 min) and the cells were plated 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 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 solution A; 80 μg of PEI was mixed with 600 μL of culture medium to form solution B; then, solution A was added to solution B and gently pipetted to mix, and the mixture was allowed to stand at room temperature for 15 minutes.

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

[0063] (5) For cultures longer than 6 days, add ≤5% feed (and optionally <5g / 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 to 3-6g / 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, 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 onto the chromatography column at a flow rate of approximately 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 approximately 2 mL / min until the A280 absorbance of the effluent approaches 0 and stabilizes.

[0072] (7) Elute the target protein. Gently add 5-10 volumes of 0.1 M glycine (pH = 2.8) to avoid resin suspension. Maintain a flow rate of approximately 1 mL / min. Collect the effluent and immediately add 1 / 10 volume of 1 M Tris-base (pH = 8.0) to neutralize the effluent to pH 7.5. 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 0.01M 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 and place the concentrator tube in a low-temperature horizontal rotor refrigerated centrifuge. 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 plated at a density of 1 x 10 6 / mL in a 24-well cell culture plate, 500 μL per well, and incubated at 37°C in a 5% CO2 incubator for 3 hours until the cells were completely adherent.

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

[0082] (3) Neutralization test: The plated cell plate was placed in advance at 4°C to maintain the cell temperature at 4°C (to prevent the occurrence of cell internalization), and the above-mentioned mixed solution of fusion protein and virus incubated at 4°C was inoculated into the cells, 500 μL per well, and placed at 4°C for 2 hours of virus adsorption, washed twice with PBS (K+) to remove residual unabsorbed virus, and the medium was replaced with DMEM medium containing 3% FBS, 500 μL per well, and incubated at 37°C in a 5% CO2 incubator for 24 hours. After observation under a fluorescence microscope, the results showed that the number of green fluorescence after mixing the fusion protein Nb5-pFc and Nb1-14-pFc with the virus was significantly lower than that of the non-virus group and the irrelevant control group (Nb53-pFc), indicating that the fusion proteins Nb5-pFc and Nb1-14-pFc had neutralizing activity against PRRSV Figure 2 ).

[0083] (4) 24 hours after inoculation, the cell culture supernatant was discarded, and the cells were washed three times with preheated PBS solution. 100 μL of NP40 lysis buffer containing 1% PMSF was added, and the cells were lysed on ice for 10 minutes. The insoluble matter was discarded after centrifugation. The cell protein after lysis was boiled and subjected to SDS-PAGE, and the protein was transferred to a PVDF membrane. The membrane was blocked with PBS'T containing 5% skim milk powder for 1 hour, washed, and anti-PRRSV N protein specific monoclonal antibody 6D10 (1 μg / mL) was added and incubated at room temperature for 1 hour. After washing, HRP-labeled goat anti-mouse IgG was added and incubated for 1 hour. Finally, ECL luminescent solution was used for color development, and the results were photographed to evaluate the virus neutralization effect of Nb5-pFc and Nb1-14-pFc. The results showed that Nb5-pFc and Nb1-14-pFc significantly inhibited the replication of PRRSV ( Figure 3 ).

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

[0085] Strain: The strain used for the challenge was PRRSV-JXA1.

[0086] Experimental animals: 20 healthy weaned piglets at 28 days of age, which were negative for PRRSV antibodies and antigens.

[0087] Experimental design: 20 non-immune negative pigs were randomly divided into 4 groups, 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 unrelated mixed preparation (Nb53-pFc) treatment control group; the challenge method was neck intramuscular injection (10 5 TCID 50 The mixed preparation was injected intramuscularly into the neck. All animals were euthanized and autopsied on day 14.

[0088] Table 1 Animal test protocol for the mixed preparation to inhibit PRRSV replication 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 unrelated mixed preparation treatment control group was higher than 40.5℃ on the 3rd and 4th day after the challenge, respectively, and continued 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 continued 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-challenged 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 the nanoantibody 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 the 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-challenged control group were injected with the nanoantibody 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, feces and morbidity of each group of pigs were observed daily, and the survival rate of the piglets was recorded. The pigs in the challenge control group and the unrelated mixed preparation treatment control group 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 unchallenged 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-challenged control group and the mixed preparation treatment group 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 challenge, and the viral load in the nasal swabs was tested by fluorescent quantitative RT-PCR. Serum was collected from pigs on days 0, 1, 3, 5, 7, and 14 after challenge, and the viral load was tested by fluorescent quantitative RT-PCR. These results indicate that treatment with the combined formulation significantly reduced the viral load in the pigs' serum and the amount of toxins excreted into 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 pigs in the challenge control group and the unrelated mixed preparation treatment control group. 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 challenge, 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 challenge control group and the irrelevant mixed preparation treatment control group were thickened, and there was red blood cell and inflammatory cell infiltration in the alveoli. The alveolar walls of the pigs in the non-challenge control group and the mixed preparation treatment group were normal, and no red blood cell and inflammatory cell infiltration was seen ( Figure 9 ), all pigs were dissected 14 days after infection, and lungs were collected and fixed in 4% paraformaldehyde. HE staining was used to observe lung histopathological changes. These results indicate that the mixed preparation treatment significantly reduced lung damage in pigs after PRRSV infection.

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

[0095] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection 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 according to claim 1, 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 according to claim 3, 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 comprises 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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