Porcine reproductive and respiratory syndrome virus NADC30 strain and its application

Abstract

The present invention discloses a porcine reproductive and respiratory syndrome virus NADC30 strain and its application. Specifically disclosed is a porcine reproductive and respiratory syndrome virus PRRSV NADC30 strain, whose deposit number at the General Microbiology Center of the China Culture Collection Administration of Microorganisms is CGMCC No.26423. The virus strain of the present invention can proliferate well on Marc145 cells, has a high culture titer, has high immunogenicity and good genetic stability, and the inactivated vaccine prepared using it has high immunogenicity, is safe and reliable, more efficient and has long-lasting immunity, can induce the body to produce high levels of antibodies, achieve 100% protection against strong toxic attacks, and the duration of immunity can reach 7 months. The virus strain of the present invention can also be used to detect and evaluate viral antibodies, prepare infection models, develop new diagnostic strategies and / or treatment strategies, etc., and has a wide range of clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products, and specifically relates to a porcine reproductive and respiratory syndrome virus NADC30 strain and an application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome virus NADC30 (PRRSV NADC30) is a single-stranded, positive-sense RNA virus with spherical virions measuring 55 to 60 nanometers in diameter. There are two serotypes, and the strain isolated in my country is the American strain. In recent years, the NADC30 strain circulating in Chinese pig farms is genetically closely related to the NADC30 strain reported in the United States. This strain has reassorted with other subgroups of PRRSV circulating in China (classical PRRSV, HP-PRRSV, or vaccine strains), resulting in a new class of viruses that Chinese researchers collectively refer to as PRRSV NADC30 strains. This strain has become the predominantly circulating PRRSV strain. Infection in sows can cause abortion, while infection in piglets can lead to severe respiratory symptoms, causing significant economic losses to the global swine industry.

[0003] The most effective means of preventing PRRSV NADC30 infection is vaccination. Currently, research on PRRSV NADC30 in my country is in its infancy, and no such vaccines or diagnostic products are available on the market. There is an urgent need to develop a vaccine against porcine reproductive and respiratory syndrome virus NADC30. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to effectively prevent porcine reproductive and respiratory syndrome virus NADC30 or diseases caused by porcine reproductive and respiratory syndrome virus NADC30 infection. The technical problem to be solved is not limited to the technical subject matter described herein, and those skilled in the art will clearly understand other technical subjects not described herein through the following description.

[0005] In order to solve the above technical problems, the present invention first provides a porcine reproductive and respiratory syndrome virus NADC30 virus strain, which is the PRRSV NADC30 strain, and its preservation number in the General Microbiology Center of the China Culture Collection Administration of Microorganisms is CGMCC No.26423.

[0006] The present invention also provides a vaccine for preventing porcine reproductive and respiratory syndrome virus NADC30 infection or diseases caused by porcine reproductive and respiratory syndrome virus NADC30 infection, wherein the vaccine comprises the virus strain PRRSV NADC30.

[0007] Furthermore, the virus strain PRRSV NADC30 is inactivated.

[0008] Furthermore, the vaccine may further comprise an adjuvant.

[0009] The vaccine may be an inactivated vaccine.

[0010] The adjuvant may include biological adjuvants, plant adjuvants, inorganic compound adjuvants, synthetic adjuvants, organic adjuvants, Freund's adjuvants, liposome adjuvants, nano adjuvants and oil-free adjuvants, but is not limited thereto.

[0011] Furthermore, the adjuvant may be 605 adjuvant. The composition of the 605 adjuvant described herein may be: 3.5 g disodium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 7 g sodium chloride, 7 g trehalose, 7 g astragalus polysaccharide, 3 g sodium glutamate, 13 g gelatin, 7 g polyethylene glycol 3000, 3 g dextran, and 1000 ml water for injection.

[0012] Furthermore, the vaccine may further comprise a pharmaceutically acceptable carrier.

[0013] The pharmaceutically acceptable carrier is selected from diluents, excipients, fillers, binders, wetting agents, disintegrants, preservatives, stabilizers, absorption promoters, adsorption carriers, surfactants, lubricants, aerosolizers, suspending agents, plasticizers and dispersants.

[0014] Furthermore, the vaccine may further comprise a protective agent.

[0015] The protective agent may include dimethyl sulfoxide (DMSO), complexes (such as skim milk, gelatin, protein, peptone, dextrin, serum and methylcellulose), sugars (such as sucrose, lactose, maltose, glucose and fructose), salts (such as calcium lactate, sodium glutamate, sodium chloride, potassium chloride, ammonium acetate and sodium thiosulfate), alcohols (such as sorbitol, glycerol, mannitol, inositol and xylitol), acids (such as citric acid, tartaric acid and amino acids), and polymers (such as dextran, polyethylene glycol and polyvinyl pyrrolidone (PVP)), but are not limited thereto.

[0016] The vaccine may be in the form of nasal drops or injections (such as injection solutions or powder for injection).

[0017] Furthermore, the active ingredient of the vaccine may be an inactivated PRRSV NADC30 strain.

[0018] Furthermore, the virus in the vaccine (PRRSV NADC30 strain) is greater than 10 6.0 TCID 50 / ml, 10 6.5 TCID50 / ml, 10 7.0 TCID 50 / ml, 10 7.5 TCID 50 / ml, 10 8.0 TCID 50 / ml, 10 8.5 TCID 50 / ml or 10 9.0 TCID 50 / ml of the virus titer exists.

[0019] Furthermore, the virus content in the vaccine (the content of PRRSV NADC30 strain) can be 10 6.0～7.0 TCID 50 / ml, 10 6.0～7.5 TCID 50 / ml, 10 6.0～8.0 TCID 50 / ml, 10 6.0～8.5 TCID 50 / ml, 10 6.0～9.0 TCID 50 / ml, 10 7.0～ 8.0 TCID 50 / ml, 10 7.0～8.5 TCID 50 / ml, 10 7.0～9.0 TCID 50 / ml, 10 8.0～8.5 TCID 50 / ml or 10 8.0～9.0 TCID 50 / ml.

[0020] The present invention also provides a method for preparing a vaccine for preventing porcine reproductive and respiratory syndrome virus NADC30 infection or a disease caused by porcine reproductive and respiratory syndrome virus NADC30 infection, the method comprising the following steps:

[0021] A1) culturing the virus strain PRRSV NADC30 to obtain a virus culture fluid;

[0022] A2) inactivating the virus culture fluid to obtain an inactivated virus culture fluid;

[0023] A3) Mixing the inactivated virus culture solution with an adjuvant to obtain the vaccine.

[0024] In the above method, the culturing method in A1) comprises: inoculating the virus strain PRRSV NADC30 into cells, chicken embryos, tissues or animals for culturing.

[0025] Furthermore, the culturing method described in A1) may include: inoculating the PRRSV NADC30 strain into cells for culturing.

[0026] Furthermore, the cells are animal cells that can be used for virus propagation, including passage cells, primary cells and diploid cells.

[0027] Furthermore, the cells can be selected from PK-15 (PK15) cells, IBRS-2 cells, Vero cells, Marc-145 (Marc145) cells, CHO cells, COS cells, MDCK cells, MDBK cells, MDOK cells, CRFK cells, Hela cells, 293 cells, BSC-1 cells, LLC-MK cells, CV-1 cells, WI-38 cells, TCMK cells, MRC-5 cells, BHK cells and chicken embryo cells.

[0028] Furthermore, the inactivation method described in A2) can be carried out using physical or chemical methods well known to those skilled in the art, for example, common inactivators such as formaldehyde solution, alkylating agents (such as acetylethyleneimine (AEI), diethyleneimine (BEI) and glycidaldehyde (GDA)) and β-propionyl lactone can be used for inactivation.

[0029] Furthermore, the inactivation method described in A2) may include: mixing binary ethyleneimine (BEI) with the virus culture solution described in A1) to inactivate the virus, and finally adding sodium thiosulfate to the mixture to terminate the inactivation.

[0030] Furthermore, the virus titer of the PRRSV NADC30 strain in the virus culture fluid is greater than or equal to 10 7.0 TCID 50 / ml, 10 7.5 TCID 50 / ml, 10 8.0 TCID 50 / ml, 10 8.5 TCID 50 / ml or 10 9.0 TCID 50 / ml.

[0031] Furthermore, the virus titer of the PRRSV NADC30 strain in the virus culture fluid is greater than or equal to 10 8.0 TCID 50 / ml.

[0032] In the above method, the adjuvant can be a biological adjuvant, a plant adjuvant, an inorganic compound adjuvant, a synthetic adjuvant, an organic adjuvant, Freund's adjuvant, a liposome adjuvant, a nano adjuvant or an oil-free adjuvant.

[0033] In the above method, the adjuvant may be 605 adjuvant, and the composition of the 605 adjuvant may be: 3.5 g disodium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 7 g sodium chloride, 7 g trehalose, 7 g astragalus polysaccharide, 3 g sodium glutamate, 13 g gelatin, 7 g polyethylene glycol 3000, 3 g dextran, and 1000 ml water for injection.

[0034] Furthermore, the volume ratio of the inactivated virus culture solution and the adjuvant in A3) can be 2:1.

[0035] Furthermore, the culturing method described in A1) may include inoculating the PRRSV NADC30 strain into Marc145 cells for culturing. The culturing medium may be DMEM cell maintenance medium, which may be DMEM medium containing 200 units / ml of penicillin, 200 μg / ml of streptomycin, and 20 ml / L of fetal bovine serum.

[0036] In one embodiment of the present invention, the method for preparing the virus culture fluid comprises: diluting the PRRSV NADC30 strain with DMEM cell maintenance medium at 1:10, then inoculating the diluted virus fluid into Marc145 suspension cells at an MOI of 0.1, culturing in a cell shaker incubator or bioreactor at 37°C containing 5% CO2 for 2 to 3 days, harvesting the cell culture, and measuring the virus content in the cell culture to 10 7.0~8.0 TCID 50 / ml, can be used as antigens for preparing vaccines.

[0037] In one embodiment of the present invention, the cell culture is inactivated using binary ethyleneimine (BEI), that is, 2% (volume percentage) of 0.1 mol / L BEI solution is added, stirred while adding to fully mix, and stirred at 37°C for 20 to 24 hours to inactivate, and then a filter-sterilized 1 mol / L sodium thiosulfate solution is added to the inactivated virus solution so that the final volume of sodium thiosulfate is 30% of the volume of the added BEI solution. The solution is fully mixed and inactivated to obtain a virus inactivated solution of porcine reproductive and respiratory syndrome virus PRRSV NADC30 strain.

[0038] The present invention also provides a kit, which contains the virus strain PRRSV NADC30 strain or its antigenic substance.

[0039] The kit can be used to detect porcine reproductive and respiratory syndrome virus NADC30-specific antibodies in the serum of a subject (or patient), and further used for diagnosis or auxiliary diagnosis of porcine reproductive and respiratory syndrome virus NADC30 infection or diseases caused by porcine reproductive and respiratory syndrome virus NADC30 infection. For example, porcine reproductive and respiratory syndrome virus NADC30 infection can be detected or diagnosed based on immunological technology principles of specific antigen-antibody binding.

[0040] The antigenic substance may include components of the PRRSV NADC30 strain, such as protein or polysaccharide components of the pathogen obtained through processing and extraction; the antigenic substance may also include a culture (such as a cell culture) containing the PRRSV NADC30 strain. Those skilled in the art are familiar with how to prepare antigenic substances from the PRRSV NADC30 strain pathogen described herein. For example, the antigenic substance may be obtained through dilution, concentration, or extraction, or by constructing a recombinant vector or recombinant cell containing the DNA molecule shown in SEQ ID No. 1.

[0041] The kit may be a detection kit, and further, the kit may be a chemiluminescence immunoassay kit, an enzyme-linked immunosorbent assay kit, an immunoblotting assay kit, an immunochromatography assay kit, a flow cytometry assay kit, an immunohistochemistry assay kit, a colloidal gold immunoassay kit or a fluorescent immunoassay kit, but is not limited thereto.

[0042] The test sample of the kit can be a blood sample (such as whole blood, plasma, serum), a tissue sample or a cell sample, but is not limited thereto.

[0043] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0044] The components of the kit can be provided in the form of solutions, such as aqueous solutions. When provided in aqueous solutions, the concentrations or amounts of these components can be readily determined by those skilled in the art based on individual needs. For example, for storage purposes, the components can be provided in a higher concentration. When in working condition or use, the higher concentration solution can be diluted to reduce the concentration to the working concentration.

[0045] The present invention also provides the use of the virus strain PRRSV NADC30 in any of the following:

[0046] B1) Use in the preparation of vaccines for preventing porcine reproductive and respiratory syndrome virus NADC30 infection or diseases caused by porcine reproductive and respiratory syndrome virus NADC30 infection;

[0047] B2) Use in the preparation of a product for inducing a specific immune response against porcine reproductive and respiratory syndrome virus NADC30 virus in a subject;

[0048] B3) Application in screening and / or preparing antibodies against porcine reproductive and respiratory syndrome virus NADC30;

[0049] B4) Use in the detection and / or evaluation of antibodies against porcine reproductive and respiratory syndrome virus NADC30;

[0050] B5) Application in the preparation of porcine reproductive and respiratory syndrome virus NADC30 antibody detection reagent;

[0051] B6) Application in the preparation of porcine reproductive and respiratory syndrome virus NADC30 infection cell model or animal model.

[0052] The vaccine described herein may be an inactivated vaccine.

[0053] Furthermore, the method for screening and / or preparing antibodies against porcine reproductive and respiratory syndrome virus NADC30 in B3) may be to screen and / or prepare antibodies using the virus strain PRRSV NADC30 as an immunogen.

[0054] Furthermore, the method for preparing the antibody may include: immunizing an animal with the virus strain PRRSV NADC30, and obtaining the antibody from the immunized animal.

[0055] The antibody may include a monoclonal antibody, a polyclonal antibody, a single-chain antibody, or a single-domain antibody.

[0056] Furthermore, the animals may include mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, chickens, cats, sheep, cows, horses, monkeys, alpacas, etc., but are not limited thereto.

[0057] Furthermore, the method for screening antibodies may include: contacting a candidate antibody drug with the virus strain PRRSV NADC30 or its antigenic substance, and measuring the activity or toxicity of the virus strain.

[0058] Furthermore, the method for detecting and / or evaluating porcine reproductive and respiratory syndrome virus NADC30 antibodies described in B4) may include: using the PRRSV NADC30 strain described in the present invention or any kit described in the present invention to detect or quantitatively detect the sample to be tested, thereby detecting whether porcine reproductive and respiratory syndrome virus NADC30 antibodies are present in the sample to be tested, and / or evaluating the titer, activity, affinity, etc. of porcine reproductive and respiratory syndrome virus NADC30 antibodies.

[0059] Antigen-antibody reactions are the core and foundation of immunological testing technology. As is well known to those skilled in the art, based on the principle of specific antigen-antibody binding, using known antigens to detect and analyze specific antibodies can assist in clinical diagnosis, efficacy observation, prognosis assessment, and observation of vaccination effectiveness. This is also of particular importance in epidemiological investigations of infectious diseases. Methods for detecting antibodies using known antigens are well known to those skilled in the art, including precipitation reactions, agglutination tests, fluorescent immunoassays, radioimmunoassays, enzyme immunoassays, chemiluminescent immunoassays, and immunoassay techniques related to point-of-care testing (POCT), such as colloidal gold immunoassays and fluorescent immunochromatography.

[0060] The cell model or animal model described in B6) can be used to study the pathogenic mechanism of porcine reproductive and respiratory syndrome virus NADC30 infection, screen antiviral drugs, develop new diagnostic strategies and / or therapeutic strategies, and evaluate the immune protection effect of vaccines.

[0061] The product mentioned in B2) may be a reagent, a preparation, a drug, a pharmaceutical composition or a kit.

[0062] The nucleotide sequence of the gene encoding the structural protein (GP5 protein) of the virus strain PRRSV NADC30 described herein (GP5 gene) can be shown as SEQ ID No. 1. The amino acid sequence of the GP5 protein can be shown as SEQ ID No. 2.

[0063] The present invention screened and obtained a porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 strain, and provided a method for preparing an inactivated vaccine using the virus strain. The virus strain of the present invention can be well propagated on Marc145 cells, has a high culture titer, has high immunogenicity and good genetic stability, and the inactivated vaccine prepared using the virus strain has high immunogenicity, is safe and reliable, more efficient, and has long-lasting immunity. It has a good immune protection effect against porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 infection, can induce the body to produce high levels of antibodies, and achieve 100% protection against strong toxic attacks. The immune protection period is long, and the duration of immunity can reach 7 months. In addition, the virus strain of the present invention can also be used to screen, prepare, detect and / or evaluate porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 antibodies, as well as to prepare porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 infected cell models or animal models, and further used in the study of the pathogenic mechanism of porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 infection, antiviral drug screening, development of new diagnostic strategies and / or treatment strategies, and evaluation of vaccine immune protection effects, and has broad clinical application value.

[0064] Definition of terms

[0065] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0066] The term "adjuvant" generally refers to a nonspecific immunopotentiating substance that is injected into the body before or simultaneously with an antigen to enhance or modify the immune response to the antigen. These adjuvants can be used as vaccine components to boost antibody levels in humans and animals, and they can also serve as nonspecific immunopotentiators for adjunctive anti-tumor and anti-infective therapies. The adjuvants are well known to those skilled in the art, and include, but are not limited to: biological adjuvants such as Bacillus Calmette-Guérin (BCG), Corynebacterium pumilus (CP), lipopolysaccharide (LPS), cytokines (such as GM-CSF), cholera toxin (CTX), Escherichia coli heat-labile enterotoxin (LT), and plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides (such as pachymannan), glycolipids, and lectins); inorganic compound adjuvants such as aluminum adjuvants (such as aluminum hydroxide and aluminum phosphate) and calcium adjuvants (such as calcium carbonate); synthetic adjuvants such as CpG oligodeoxynucleotides (CpG ODN), polyinosinic:cytidylic acid (poly I:C), and levamisole; organic adjuvants such as mineral oil; liposomes such as immunostimulating complexes (ISCOM); and Freund's adjuvants such as complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA). CFA contains inactivated Mycobacterium tuberculosis (BCG) and mineral oil, and can enhance both humoral and cellular immune responses. IFA, containing only mineral oil, can enhance the body's antibody response. CFA and IFA are the most commonly used adjuvants in animal research. These adjuvants can be pharmaceutically acceptable.

[0067] The term "virulence" is also called virulence or titer, which is intended to indicate the infectivity of the virus. For example, the titer can be expressed as the TCID50 infectious dose (TCID50) of the tissue cell culture medium. 50 )express.

[0068] The term "inactivation" generally refers to destroying the biological activity, reproductive capacity, and pathogenicity of microorganisms while minimizing the impact on their immunogenicity. Inactivated microorganisms are primarily used in the production of inactivated vaccines. The chemical agents or drugs used to inactivate microorganisms are called inactivators.

[0069] The term "inactivated vaccine" usually refers to a biological preparation made by inoculating highly immunogenic pathogenic microorganisms or their metabolites into animals, chicken embryos, tissues or cells for growth and reproduction, and then inactivating them to make them lose their pathogenicity but still retain their immunogenicity.

[0070] Preservation Instructions

[0071] Reference biological material (strain): PRRSV NADC30

[0072] Proposed taxonomic name: Porcine reproductive and respiratory syndrome virus

[0073] Depository: General Microbiology Center of China Culture Collection Administration

[0074] Abbreviation of depository unit: CGMCC

[0075] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0076] Deposit date: June 5, 2023

[0077] CGMCC registration number: CGMCC No.26423 BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The porcine reproductive and respiratory syndrome virus PRRSV NADC30 strain CGMCC No.26423 of the present invention was inoculated into Marc145 cells to observe the cytopathic effect (CPE); wherein, Figure 1 Middle A is the infected Marc145 cells; Figure 1 Middle B is a normal Marc145 cell.

[0079] Figure 2 These are the PCR detection results of the porcine reproductive and respiratory syndrome virus PRRSV NADC30 strain CGMCC No.26423 strain of the present invention; wherein, the order of gel electrophoresis loading is as follows: lanes 1 to 5: PCR identification results of F1, F3, F5, F7, and F10 generations of porcine reproductive and respiratory syndrome virus PRRSV NADC30 strain CGMCC No.26423 strain, respectively; lane 6: positive control for porcine reproductive and respiratory syndrome virus PRRSV NADC30 strain; lane 7: negative control (water), M represents Marker, and from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, and 250 bp. DETAILED DESCRIPTION

[0080] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0081] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0082] The reagents and materials used in the following examples are as follows:

[0083] The gel imaging system (model: Tanon 1600R) was purchased from Beijing Yuanpinghao Biotechnology Co., Ltd.;

[0084] PCR instrument (model: Mastercycler) was purchased from Eppendorf;

[0085] Nucleic acid and protein electrophoresis instrument (model: DYY-6C) was purchased from Beijing Liuyi Instrument Factory;

[0086] DL 2000 DNA Marker (Cat. No. 3427A) and DL 10000 DNA Marker (Cat. No. 3584A) were purchased from Dalian Takara Bioengineering Co., Ltd.;

[0087] EasyPure Viral DNA / RNA Kit Viral DNA / RNA purification kit (catalog number: ER201-01) and RT-PCR kit (catalog number AE411-02) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0088] Marc145 (Monkey Kidney cell) cells were purchased from the China Veterinary Drug Administration, China Veterinary Culture Collection Center (CVCC), CVCC number CL32. The cells were domesticated to pure suspension cells by our company. DMEM medium (catalog number: 12100046) and 0.25% Trypsin-EDTA (1×) trypsin (catalog number: 25200072) were purchased from Gibco.

[0089] Fetal bovine serum (catalog number: BS-1101) was purchased from Inner Mongolia Jinyuankang Bioengineering Co., Ltd.

[0090] The cell maintenance medium in the following examples is DMEM cell maintenance medium, that is, DMEM culture medium containing 200 units / ml of penicillin, 200 μg / ml of streptomycin and 20 ml / l of fetal bovine serum.

[0091] Example 1: Isolation and Identification of Porcine Reproductive and Respiratory Syndrome Virus NADC30

[0092] 1.1 Isolation of porcine reproductive and respiratory syndrome virus

[0093] 1.1.1. Treatment of diseased materials

[0094] Lung tissue collected from pigs suspected of being infected with porcine reproductive and respiratory syndrome virus (NADC30) was placed in transport medium (DMEM medium containing 1000 units / ml penicillin, 1000 μg / ml streptomycin, and 20% fetal bovine serum) and stored overnight at 4°C. The tissue was then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was sterilized by filtration through a 0.22 μm microporous filter. The resulting filtrate was used to inoculate Marc145 cells.

[0095] 1.1.2 Virus Isolation and Passaging

[0096] One ml of the treated filtrate was inoculated into a confluent monolayer of Marc145 cells in the logarithmic growth phase. Adsorb at 37°C for 2 hours, gently shaking every 30 minutes to ensure adequate contact between the filtrate and the cells. After adsorption, the cells were decanted and gently washed twice with PBS. DMEM maintenance medium (containing 200 units / ml penicillin, 200 μg / ml streptomycin, and 20 ml / l fetal bovine serum) was then added. The cells were cultured in a 37°C, 5% CO2 incubator for 48–72 hours and then harvested. This method was repeated for five passages. The harvested cell cultures were frozen and thawed twice at -70°C and centrifuged at 4000 rpm for 15 minutes. The supernatant was stored at -70°C until further use and sampled for viral content. The virus solution was inoculated into Marc-145 cells for virus content detection. After the cells grew into a monolayer, the monolayered Marc-145 cells were digested with 0.1% trypsin and the cell suspension was inoculated into a 96-well plate at 100 μl / well. After the cells grew into a monolayer, the culture medium in the well was discarded; 100 μl of virus solution diluted 10-fold with cell maintenance medium (10 -1 ~10 -8 ), each dilution was repeated 4 times, and normal Marc-145 cells were used as control. The cells were cultured in a 37°C, 5% CO2 incubator for 72 h. The cytopathic effect was observed under an inverted biological microscope, and the number of wells with and without cytopathic effect was recorded. The virus TCID was calculated using the Reed-Muench method. 50 The results are as follows Figure 1 As shown, specific cytopathic effect (CPE) was observed in Marc145 cells inoculated with the virus, indicating that Marc145 cells were infected with the virus and the virus could proliferate on Marc145 cells.

[0097] 1.2 PCR identification of porcine reproductive and respiratory syndrome virus NADC30

[0098] 1.2.1 Viral PCR Testing

[0099] Based on the gene sequence of PRRSV GP5 protein published in Genbank, DNAStar was used to align the nucleotide sequences of all PRRSV GP5 protein genes. Two primers were designed in the conserved region using Oligo 6.0. The expected amplification product was 599 bp. The primer sequences are shown in Table 1.

[0100] Table 1. PRRSV PCR primer sequences and amplification target fragment sizes

[0101]

[0102] Extract viral RNA: Take 200 μl of viral cell culture and use the EasyPure Viral DNA / RNA Kit (product of Quanshijin Company, product number: ER201-01) to extract viral RNA, and place it in a -70℃ refrigerator until use.

[0103] RT-PCR reactions were performed in a 20 μL system: 10 μL of 2× one-step reaction mix, 0.4 μL of ES one-step mix, 1 μL of upstream and downstream primers (both at a concentration of 10 pmol), 2 μL of RNA, and 5.6 μL of RNase-free water, for a total of 20 μL system.

[0104] Reaction conditions: 98°C for 30 s; 30 cycles of 98°C for 10 s, 58°C for 10 s, and 72°C for 30 s; and 72°C for 2 min. After completion of the reaction, the PCR product was identified by electrophoresis on a 1% agarose gel. The PCR product was stored at 4°C. A 5 μl aliquot of the reaction product was added with an appropriate amount of loading buffer, mixed thoroughly, and then electrophoresed on a 1% agarose gel (Golden View, 5 μl / 100 mL) at 120 V for 30 min. After electrophoresis, the gel was removed and analyzed using a gel imager. A specific band of approximately 599 bp was amplified. The PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing analysis, which confirmed that the specific band was a DNA molecule with the nucleotide sequence shown in SEQ ID No. 1. The DNA molecule is the GP5 gene sequence of porcine reproductive and respiratory syndrome virus NADC30, encoding the GP5 protein of porcine reproductive and respiratory syndrome virus NADC30. The amino acid sequence of the GP5 protein is SEQ ID No. 2. A gene alignment of the nucleotide sequence of the DNA molecule with the sequence published by NCBI showed a homology of 99.3% with the existing gene encoding the GP5 protein of porcine reproductive and respiratory syndrome virus NADC30. The results showed that the virus in the above-mentioned viral cell culture is porcine reproductive and respiratory syndrome virus NADC30 (PRRSV NADC30).

[0105] Example 2: Virus subculture and determination of titer and virulence

[0106] 2.1 Virus proliferation and culture

[0107] The isolate was inoculated with Marc145 monolayer cells at an MOI of 0.1 and cultured for 48 to 72 hours, then harvested as the F1 generation. The harvested virus solution was subjected to a series of tests including sterility test, exogenous virus test, specificity, and virus titer determination according to the methods in the appendix of Part III of the Chinese Pharmacopoeia of Veterinary Medicine, and the isolates with high titer and purity were selected for cryopreservation. The study eventually screened out a PRRSV NADC30 virus strain, named PRRSV NADC30 strain, as a backup seed virus, and then the PRRSV NADC30 strain was used for continuous cell propagation to allow the virus to better adapt to cell proliferation and increase the virus content (toxin titer). The seed virus was continuously propagated to the F10 generation, and the virus harvest volume of each generation was at least 100 ml. The harvested virus was stored at -70°C. At the same time, the viruses after continuous propagation were subjected to PCR detection, and all of them could be amplified to specific target bands. The results are shown in the table. Figure 2 .

[0108] The porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 strain (passage 5) obtained by screening in the present invention was deposited with the General Microbiology Center of the China Culture Collection Administration on June 5, 2023, with the deposit number CGMCC No. 26423. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. This strain is also referred to herein as the porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 strain CGMCC No. 26423, or simply the PRRSV NADC30 strain.

[0109] 2.2. Toxicity determination

[0110] The acclimated virus was labeled as F1 generation and passaged for 10 generations to detect the virus content. The virus solution was inoculated into Marc-145 cells for virus content detection. After the cells grew into a monolayer, the monolayered Marc-145 cells were digested with 0.1% trypsin and the cell suspension was inoculated into a 96-well plate at 100 μl / well. After the cells grew into a monolayer, the culture medium in the well was discarded; 100 μl of virus solution diluted 10-fold with cell maintenance medium (10) was added to each well. -1 ~10 -8 ), each dilution was repeated 4 times, and normal Marc-145 cells were used as control. The cells were cultured in a 37°C, 5% CO2 incubator for 72 h. The cytopathic effect was observed under an inverted biological microscope, and the number of wells with and without cytopathic effect was recorded. The virus TCID was calculated using the Reed-Muench method. 50 The highest virus titer before acclimation is 10 6.33 TCID 50 / ml, using Marc145 suspension cell culture up to 10 8.0 TCID 50 / ml. Its toxicity can be increased by more than 10 times compared with the toxicity before virus acclimation.

[0111] 2.3 Toxicity assay

[0112] Virulence was determined for the third generation virus culture. Virulence was expressed as the minimum infectious dose (MID), which was determined by diluting the virus 10-fold to 10 -6 , take 10 -4 , 10 -5 , 10 -6Piglets aged 49 to 56 days, negative for PRRSV antibodies and antigens (purchased from Chengxin Farm in Mancheng District, Baoding City, Hebei Province), were inoculated intranasally with 5 ml of the virus at three dilutions. Each pig was observed for 14 consecutive days after challenge. Blood samples were collected for viremia on days 7, 10, and 14 after challenge. Viral PCR was performed according to the method described in 1.2 of Example 1, and the minimum infectious dose (MID) of PRRSV was calculated. The results showed that the MID of PRRSV was 5×10 6.0 TCID 50 / head.

[0113] Example 3. Preparation and testing of inactivated porcine reproductive and respiratory syndrome virus NADC30 vaccine

[0114] 3.1 Virus Reproduction

[0115] The third generation virus seed of porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 strain CGMCC No. 26423 was diluted 1:10 with DMEM cell maintenance medium (DMEM medium containing 200 units / ml of penicillin, 200 μg / ml of streptomycin and 20 ml / l of fetal bovine serum). The diluted virus solution was then inoculated into well-grown Marc145 suspension cells at an MOI of 0.1 and cultured in a cell shaker incubator at 37°C with 5% CO2 for 2-3 days. The cell culture was harvested and the virus content was determined to be 10 7.0~ 8.0 TCID 50 / ml, can be used as antigens for preparing vaccines.

[0116] 3.2. Inactivation of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) NADC30 strain

[0117] The cell culture was inactivated using binary ethyleneimine (BEI) by adding 2% of a 0.1 mol / L BEI solution, stirring while adding to thoroughly mix, and stirring at 37°C for 20-24 hours to inactivate the virus. A sterile, filtered 1 mol / L sodium thiosulfate solution was then added to the inactivated virus solution, so that the final volume of sodium thiosulfate was 30% of the volume of the added BEI solution. The solution was thoroughly mixed to inactivate the virus, resulting in an inactivated virus solution of porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 strain, which was then stored at 2-8°C. The inactivated virus solution was sampled for sterility and inactivation testing, and qualified for use in vaccine formulation. The sterility test is carried out in accordance with the method in the appendix of Part III of the Chinese Pharmacopoeia of Veterinary Medicine; the inactivation test method is to inoculate two bottles of Marc145 monolayer cells with the inactivated virus solution of porcine reproductive and respiratory syndrome virus PRRSV NADC30 strain, discard the virus solution after adsorption for 1 hour, add 10 times the volume of maintenance solution of the virus inactivation solution, culture at 37°C for 3 days, harvest the cell culture medium and blindly propagate it for one generation according to the same method, and inoculate Marc145 cells for detection without the occurrence of specific cytopathic effect (CPE) to be judged as qualified.

[0118] Among them, 0.1 mol / L BEI solution was prepared by mixing equal volumes of 0.2 mol / L 2-bromoethylamine hydrobromide (BEA, purchased from Shanghai Youen Chemical Co., Ltd.) and 0.4 M sodium hydroxide solution, placing it in a 37°C water bath and shaking it every 10 minutes for 60 minutes to prepare a BEI (diethyleneimine) solution with a final concentration of 0.1 M, which was then sterile filtered before use.

[0119] 3.3 Vaccine Preparation

[0120] The qualified porcine reproductive and respiratory syndrome virus PRRSV NADC30 strain inactivated antigen was diluted to 10 6.0 TCID 50 / ml and 10 7.0 TCID 50 / ml, then mixed with the company's patented adjuvant (605 adjuvant) at a 2:1 ratio (volume ratio) and stirred at 100-300 r / min for 30 minutes to thoroughly mix. The 605 adjuvant (i.e., 605 oil-free adjuvant) is disclosed in the authorized patent ZL201310018498.X. The formula used in this application is: 3.5 g disodium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 7 g sodium chloride, 7 g trehalose, 7 g astragalus polysaccharide, 3 g sodium glutamate, 13 g gelatin, 7 g polyethylene glycol 3000, 3 g dextran, and 1000 ml water for injection.

[0121] Three groups of vaccines were prepared in the experiment, as shown in Table 2.

[0122] Table 2. Vaccine preparation trial grouping table

[0123]

[0124] 3.4 Safety of inactivated porcine reproductive and respiratory syndrome virus vaccine

[0125] Five healthy piglets aged 14 to 21 days (purchased from Chengxin Farm in Mancheng District, Baoding City, Hebei Province) were injected with vaccine No. 1, vaccine No. 2 and the immune control group vaccine through the neck muscle, 4.0 ml per piglet, and observed for 14 consecutive days. No clinical symptoms of porcine reproductive and respiratory syndrome virus or local and systemic adverse reactions caused by vaccination occurred.

[0126] 3.5. Efficacy of inactivated porcine reproductive and respiratory syndrome virus vaccine

[0127] As shown in Table 3, 20 healthy piglets (14 to 21 days old, negative for porcine reproductive and respiratory syndrome virus (PRRSV) antibody antigen) (purchased from Chengxin Farm in Mancheng District, Baoding City, Hebei Province) were used. Five piglets were injected intramuscularly with the inactivated PRRSV NADC30 strain 1 vaccine (1.0 ml / pig), five piglets were injected intramuscularly with the inactivated PRRSV NADC30 strain 2 vaccine (1.0 ml / pig), and five piglets were injected intramuscularly with the control vaccine (2.0 ml / pig). Twenty days after the second vaccination, each piglet received a booster vaccination with the same dose. The control group received a freeze-thawed Marc145 cell suspension. Blood was collected 14 days after the second vaccination, and serum was isolated. Neutralizing antibodies in the serum were measured according to the neutralization test method in the Appendix of the Chinese Pharmacopoeia of Veterinary Medicine. A challenge protection test was also conducted. The 15 immunized piglets and the five control piglets were intranasally infected with 5×10 PRRSV NADC30 strain CGMCC No. 26423 virus. 6.0 TCID 50 The animals were observed for 14 consecutive days after challenge. Blood samples were collected on days 7, 10, and 14 post-challenge for viral serum testing. The detection primers and methods were the same as those described in "1.2. PCR Identification of Porcine Reproductive and Respiratory Syndrome Virus NADC30" in Example 1. The experimental groups are shown in Table 3.

[0128] Table 3. PRRSV NADC30 strain inactivated vaccine efficacy test groups

[0129]

[0130] The serum neutralizing antibody titer is determined by the serum neutralization test (fixed virus dilution serum method), and the specific procedure is as follows:

[0131] (1) Preparation

[0132] a. Serum inactivation: Before testing, all serum samples were inactivated in a 56°C water bath for 30 minutes and stored frozen in aliquots.

[0133] b. Cell Preparation: Marc145 cells were cultured in 96-well cell culture plates until they became confluent monolayers.

[0134] c. Preparation of working antigen: Dilute the virus of known titer to 200 TCID using cell culture medium (Gibco product, catalog number: 12100046). 50 / ml.

[0135] (2) Operation method

[0136] a. Take a 48-well cell culture plate and add 200 μl of cell basal culture medium to each well;

[0137] b. Take 200 μl of the test serum and positive serum respectively, add them to the corresponding wells in the first column, mix thoroughly, then pipette 200 μl and add it to the second well. After mixing, pipette 200 μl and add it to the third well. Repeat this dilution process until the eighth well. After mixing, pipette 200 μl and discard.

[0138] c. Add 200 μl of working antigen to each well, mix thoroughly, and incubate at 37°C for 1 hour.

[0139] d. Take a well-grown monolayer of Marc145 cells cultured in a 96-well plate, discard the cell culture medium, and wash one to two times with 0.01 mol / L PBS.

[0140] e. Add 100 μl of neutralized virus solution to each well. Repeat for four wells at each dilution. Incubate the cells in a 37°C, 5% CO2 incubator for 1 hour. Add 100 μl of cell maintenance medium to each well and continue incubation for another 4 days. Determine the neutralizing antibody titer using indirect immunofluorescence assay (IFA).

[0141] f. Simultaneously perform viral regression titration on the working antigen, diluting the working antigen to 100 TCID 50 、10TCID 50 , 1 TCID 50 Each dilution was added to 4 wells, 100 μl per well, and then 100 μl of cell maintenance medium was added to each well. After 1 hour at 37°C, all cells were transferred to a 96-well plate with a well-grown monolayer of Marc145 cells and cultured in a 37°C, 5% CO2 incubator for 4 days. TCID 50 Determination;

[0142] g. Control setup: The titer of the positive serum control group is determined simultaneously with the test serum. The negative serum control is performed in 4 wells, the virus control is performed in 4 wells, and the normal cell control is performed in 8 wells.

[0143] (3) Result determination

[0144] The neutralizing antibody titer of the standard positive serum should be no less than 1:32; CPE should appear in each well of the negative serum and virus control; the virus content should be between 30 and 300 TCID by regression titration of the working antigen. 50 The neutralizing antibody titer of the serum to be tested should be calculated according to the Reed-Muench method in Appendix III of the current Chinese Veterinary Pharmacopoeia.

[0145] Neutralizing antibody testing showed that the vaccine prepared with the porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 strain (CGMCC No. 26423) induced high levels of antibodies. A 1.0 ml vaccination dose of the purified and acclimated PRRSV NADC30 strain (CGMCC No. 26423) achieved the same antibody levels and protection against challenge as a 2.0 ml vaccination dose of the first-generation PRRSV virus, as compared to the first-generation virus. This indicates that the PRRSV NADC30 strain (CGMCC No. 26423) has a higher titer and better immunogenicity. Specific data are shown in Table 4.

[0146] After the challenge, the subjects were observed for 14 consecutive days, with body temperature measured daily, clinical symptoms observed and recorded, and blood was collected on the 7th, 10th, and 14th days after the challenge. Virus PCR testing was performed according to the method described in 1.2 of Example 1. Comprehensive evaluation of body temperature (body temperature increased above 40.5°C and lasted for at least 2 days), clinical symptoms (symptoms such as mental depression and loss of appetite), and pathogen detection (detoxification detection) was performed. The challenge protection rate was 4 / 5 or more, that is, more than 80% protection against virulent challenge was achieved. The results are shown in Table 4. The results show that the porcine reproductive and respiratory syndrome virus PRRSV NADC30 strain CGMCC No. 26423 strain can induce high levels of antibodies and produce 100% protection against virulent challenge.

[0147] Table 4. Results of the study on the efficacy of inactivated porcine reproductive and respiratory syndrome virus vaccine

[0148]

[0149] Note: In columns 3-5, the numerator of the fraction is the number of heads judged to be ill but not protected.

[0150] Example 4: Study on the persistence of antibodies to inactivated porcine reproductive and respiratory syndrome virus vaccine

[0151] Healthy piglets, 14 to 21 days old and negative for porcine reproductive and respiratory syndrome virus (PRRSV) antibodies (purchased from Chengxin Farm in Mancheng District, Baoding City, Hebei Province), were divided into two groups. Group 1 was immunized with the vaccine of the present invention (vaccine No. 2) via intramuscular inoculation of 1.0 ml of the vaccine and a booster 21 days after immunization. Group 2 was a control group immunized with frozen-thawed Marc145 cells. Neutralizing antibody titers were determined by blood sampling at 1, 3, 5, and 7 months after immunization. The results, shown in Table 5, indicate that PRRSV antibody levels reached their peak 3 months after immunization and remained at a high level until 7 months after immunization. The results of antibody persistence are also shown in Table 5.

[0152] Table 5. Study on the duration of antibodies against inactivated porcine reproductive and respiratory syndrome virus vaccine

[0153]

[0154] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A vaccine for preventing porcine reproductive and respiratory syndrome virus NADC30 infection or diseases caused by porcine reproductive and respiratory syndrome virus NADC30 infection, characterized in that: The vaccine contains the porcine reproductive and respiratory syndrome virus NADC30 strain, which is the PRRSV NADC30 strain, and its deposit number in the General Microbiology Center of the China Culture Collection Administration of Microorganisms is CGMCC No. 26423; the virus strain is inactivated; The vaccine also contains an adjuvant, which is 605 adjuvant. The composition of the 605 adjuvant is: 3.5 g disodium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 7 g sodium chloride, 7 g trehalose, 7 g astragalus polysaccharide, 3 g sodium glutamate, 13 g gelatin, 7 g polyethylene glycol 3000, 3 g dextran, and 1000 ml water for injection.

2. A method for preparing a vaccine for preventing porcine reproductive and respiratory syndrome virus NADC30 infection or a disease caused by porcine reproductive and respiratory syndrome virus NADC30 infection, characterized in that: The method comprises the following steps: A1) Cultivate the porcine reproductive and respiratory syndrome virus (PRRSV) NADC30 strain to obtain virus culture fluid; A2) inactivating the virus culture fluid to obtain an inactivated virus culture fluid; A3) mixing the inactivated virus culture fluid with an adjuvant to obtain the vaccine; The virus strain is PRRSV NADC30 strain, and its deposit number at the China General Microbiology Center is CGMCC No. 26423; The adjuvant is 605 adjuvant, and the composition of the 605 adjuvant is: 3.5 g disodium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 7 g sodium chloride, 7 g trehalose, 7 g astragalus polysaccharide, 3 g sodium glutamate, 13 g gelatin, 7 g polyethylene glycol 3000, 3 g dextran, and 1000 ml water for injection.

3. The method according to claim 2, characterized in that The culture method described in A1) comprises: inoculating the virus strain into cells, chicken embryos, tissues or animals for culture.

Images