A porcine circovirus type 3 virus strain and its application

Abstract

The present invention discloses a porcine circovirus type 3 strain and its application. Specifically disclosed is a PCV3 XY strain, whose collection number in the General Microbiology Center of China National Microbiological Culture Collection Committee is CGMCC No.26424. The virus strain of the present invention can proliferate well on PK15 cells, has high immunogenicity and good genetic stability, and the inactivated vaccine prepared by using the virus strain has high immunogenicity, is safe and reliable, more efficient and has long-lasting immunity, has a good immune protection effect on PCV3 virus infection, and the duration of immunization of piglets can reach 4 months. The virus strain of the present invention can also be used to detect PCV3 antibodies, and to prepare a virus infection model, and then used in the study of the pathogenesis of PCV3 infection, antiviral drug screening, development of new diagnostic strategies and / or treatment strategies, and evaluation of vaccine immune protection effects, 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 in particular relates to a porcine circovirus type 3 strain and an application thereof. Background Art

[0002] Porcine circovirus type 3 (PCV3) is a member of the Circovirus genus of the Circoviridae family. It is a non-enveloped, single-stranded, positive-chain circular DNA virus and the main pathogen causing a multi-systemic disease in pigs. Clinical infection is mainly manifested by dysfunction of the respiratory, urinary, intestinal, lymphatic, cardiovascular, nervous, reproductive systems and skin. It can also lead to immunosuppression, leading to secondary infection or mixed infection, causing huge economic losses to the global pig industry every year. The diameter of porcine circovirus type 3 virus is 13 to 25 nm. The genome is a single-stranded positive-chain circular DNA virus with a length of about 2,000 bp and a homology of less than 40% with PCV1 / 2.

[0003] The most effective means of preventing PCV3 infection is vaccination. Currently, there are no vaccines or diagnostic products for this disease on the market in my country. It is difficult to isolate and culture porcine circovirus type 3 (PCV3), and it is difficult to obtain strains with high toxicity and good immunogenicity. In view of this, the development of a highly immunogenic, safe, reliable, more efficient, and long-lasting vaccine against porcine circovirus type 3 can effectively prevent and control PCV3 infection and reduce economic losses in the pig industry, which has important economic value and social benefits. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to effectively prevent PCV3 infection or diseases caused by PCV3 infection. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0005] In order to solve the above technical problems, the present invention first provides a porcine circovirus type 3 virus strain, which is PCV3 XY strain, and its preservation number in the General Microbiological Center of China National Microbiological Culture Collection is CGMCC No.26424.

[0006] The present invention also provides a vaccine for preventing porcine circovirus type 3 infection or diseases caused by porcine circovirus type 3 infection, wherein the vaccine comprises the virus strain PCV3 XY strain.

[0007] Furthermore, the virus strain PCV3 XY strain 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 are 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 also contain 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 injection preparations (such as injection solutions or powder injections).

[0017] Furthermore, the active ingredient of the vaccine may be an inactivated PCV3 XY strain.

[0018] Furthermore, the virus (PCV3 XY strain) in the vaccine is present in a concentration greater than 10 5.0 TCID 50 / ml, 10 5.5 TCID 50 / ml, 10 6.0 TCID 50 / ml, 10 6.5 TCID 50 / ml, 107.0 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 virus titer exists.

[0019] Furthermore, the virus content (PCV3 XY strain content) in the vaccine may be 10 5.0～7.0 TCID 50 / ml, 10 5.0～7.5 TCID 50 / ml, 10 5.0～8.0 TCID 50 / ml, 10 5.0～8.5 TCID 50 / ml, 10 5.0～9.0 TCID 50 / ml, 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 circovirus type 3 infection or a disease caused by porcine circovirus type 3 infection, the method may comprise the following steps:

[0021] A1) culturing the virus strain PCV3 XY strain 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 fluid with an adjuvant to obtain the vaccine.

[0024] In the above method, the culturing method in A1) comprises: inoculating the virus strain PCV3 XY strain into cells, chicken embryos, tissues or animals for culturing.

[0025] Furthermore, the culturing method described in A1) may include: inoculating the PCV3 XY 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] Further, the cells can be selected from PK-15 (PK15) cells, IBRS-2 cells, Vero cells, Marc-145 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 diethyleneimine (BEI) with the virus culture solution described in A1) to inactivate the virus, and finally adding sodium thiosulfate to the mixed solution to terminate the inactivation.

[0030] Furthermore, the virus titer of the PCV3 XY strain in the virus culture fluid is greater than or equal to 10 5.0 TCID 50 / ml, 10 5.5 TCID 50 / ml, 10 6.0 TCID 50 / ml, 10 6.5 TCID 50 / ml, 10 7.0 TCID 50 / ml, 10 7.5 TCID 50 / ml, 10 8.0 TCID 50 / ml, 108.5 TCID 50 / ml or 10 9.0 TCID 50 / ml.

[0031] Furthermore, the virus titer of the PCV3 XY strain in the virus culture fluid is greater than or equal to 10 7.0 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 culture method in A1) may include: inoculating the PCV3 XY strain into PK15 cells for culture. The culture medium may be DMEM cell maintenance medium, and the DMEM cell maintenance medium may be a DMEM culture 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 PCV3 XY strain with DMEM cell maintenance fluid at a ratio of 1:10, then inoculating the diluted virus fluid into PK15 suspension cells at an MOI of 0.1, and placing the culture fluid at 37°C with 5% CO 2 The cells were cultured in a cell shaker incubator or bioreactor for 4 to 5 days, and the cell culture was harvested. The virus content in the cell culture was determined to be 10 7.0~8.5 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 diethyleneimine (BEI), that is, 2% (volume percentage) of 0.1 mol / L BEI solution is added, stirred while adding to fully mix, and inactivated at 37°C for 20 to 24 hours, and then a filtered and 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, and the mixture is fully mixed for inactivation blocking to obtain a porcine circovirus type 3 virus inactivated solution.

[0038] The present invention also provides a kit, which contains the virus strain PCV3 XY strain or its antigenic substance.

[0039] The kit can be used to detect PCV3 virus-specific antibodies in the serum of a subject (or patient), and then used to diagnose or assist in the diagnosis of PCV3 virus infection or a disease caused by PCV3 virus infection. For example, the PCV3 virus infection can be detected or diagnosed based on the immunological technology principle of antigen-antibody specific binding.

[0040] The antigenic substance may include components of the PCV3 XY strain, such as protein or polysaccharide components of the pathogen obtained by processing and extraction; the antigenic substance may also include a culture (such as a cell culture) containing the PCV3 XY strain. Those skilled in the art are familiar with how to prepare the antigenic substance from the pathogen PCV3 XY strain of the present invention, for example, the antigenic substance may be obtained by dilution, concentration or extraction steps, 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 may be provided in the form of a solution, such as an aqueous solution. In the case of being in the form of an aqueous solution, the concentration or content of these components can be easily determined by those skilled in the art according to different requirements. For example, for storage purposes, the concentration of the components may be present in a higher form, and when in working state or use, the concentration may be reduced to the working concentration by diluting the above-mentioned higher concentration solution.

[0045] The present invention also provides the use of the virus strain PCV3 XY strain in any of the following:

[0046] B1) Use in the preparation of vaccines for preventing porcine circovirus type 3 infection or diseases caused by porcine circovirus type 3 infection;

[0047] B2) Use in the preparation of a product for inducing a specific immune response against porcine circovirus type 3 virus in a subject;

[0048] B3) Application in screening and / or preparing antibodies against porcine circovirus type 3;

[0049] B4) Application in the detection and / or evaluation of antibodies against porcine circovirus type 3;

[0050] B5) Application in the preparation of porcine circovirus type 3 antibody detection reagent;

[0051] B6) Application in the preparation of porcine circovirus type 3 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 porcine circovirus type 3 antibodies in B3) may be to screen and / or prepare antibodies using the PCV3 XY virus strain as an immunogen.

[0054] Furthermore, the method for preparing antibodies may include: immunizing an animal with the PCV3 XY virus strain, and obtaining the antibodies 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 PCV3 XY virus strain or an antigenic substance thereof, and determining the activity or toxicity of the virus strain.

[0058] Furthermore, the method for detecting and / or evaluating porcine circovirus type 3 antibodies described in B4) may include: using the PCV3 XY 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 circovirus type 3 antibodies exist in the sample to be tested, and / or evaluating the titer, activity, affinity, etc. of porcine circovirus type 3 antibodies.

[0059] Antigen-antibody reaction is the core and foundation of immunological testing technology. It is well known to those skilled in the art that based on the principle of specific binding between antigen and antibody, the use of known antigen detection and analysis of specific antibodies can be used to assist clinical diagnosis, efficacy observation, prognosis judgment and observation of vaccination effects. It also has special and important significance in epidemiological surveys of infectious diseases. Methods for detecting antibodies using known antigens are well known to those skilled in the art, such as precipitation reaction, agglutination test, fluorescent immunoassay, radioimmunoassay, enzyme immunoassay, chemiluminescent immunoassay, and POCT (point-of-care testing) related immunoassay techniques (such as colloidal gold immunoassay, fluorescent immunochromatography), etc.

[0060] The cell model or animal model described in B6) can be used to study the pathogenic mechanism of PCV3 infection, screen antiviral drugs, develop new diagnostic strategies and / or treatment 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 ORF2 structural protein (CAP protein) of the PCV3 XY strain described herein (CAP gene) may be shown as SEQ ID No. 1. The amino acid sequence of the CAP protein may be shown as SEQ ID No. 2.

[0063] The present invention screened and obtained a porcine circovirus type 3 virus strain PCV3 XY 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 PK15 cells, 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, has a good immune protection effect on porcine circovirus type 3 virus infection, and the duration of immunization of piglets can reach 4 months. In addition, the virus strain of the present invention can also be used for screening, preparing, detecting and / or evaluating porcine circovirus type 3 antibodies, and for preparing porcine circovirus type 3 infected cell models or animal models, and then for the study of the pathogenic mechanism of PCV3 infection, antiviral drug screening, development of new diagnostic strategies and / or treatment strategies, and vaccine immune protection effect evaluation, etc., and has a wide range of 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. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0066] The term "adjuvant" generally refers to a non-specific immune-enhancing substance that is injected into the body in advance or simultaneously with an antigen to enhance the body's immune response to the antigen or change the type of immune response. The adjuvant can be used as a vaccine component to enhance the level of antibodies in humans and animals, and can also be used as a non-specific immune enhancer for adjuvant treatment of anti-tumor and anti-infection. The adjuvant is well known to those skilled in the art, including but not limited to: biological adjuvants such as 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 lectin); 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 acid:cytidylic acid (poly I: C) and levamisole. Organic adjuvants such as mineral oil; liposomes such as immunostimulating complexes (ISCOM); Freund's adjuvants such as Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA). CFA contains inactivated Mycobacterium tuberculosis (BCG) and mineral oil, which can enhance the body's humoral and cellular immune responses. IFA contains only mineral oil ingredients, which can enhance the body's antibody response. CFA and IFA are the most commonly used adjuvants in animal experiments. The adjuvant can be a pharmaceutically acceptable adjuvant.

[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 tissue cell culture median infectious dose (TCID 50 )express.

[0068] The term "inactivation" generally refers to destroying the biological activity, reproductive capacity and pathogenicity of microorganisms, but without affecting their immunogenicity as much as possible. Inactivated microorganisms are mainly used to produce inactivated vaccines. Chemical agents or drugs used to inactivate microorganisms are called inactivators.

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

[0070] Collection Instructions

[0071] Biological material (strain): PCV3 XY

[0072] Proposed taxonomic name: Porcine circovirus type 3

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

[0074] Abbreviation of depository unit: CGMCC

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

[0076] Date of deposit: June 5, 2023

[0077] CGMCC Registration Number: CGMCC No.26424 BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is the indirect immunofluorescence observation result of PK15 cells inoculated with the porcine circovirus type 3 virus CGMCC No.26424 strain of the present invention. Among them, Figure 1 A in the middle is the infected PK15 cell. Figure 1 Middle B is a normal PK15 cell.

[0079] Figure 2 It is the PCR detection result of the porcine circovirus type 3 virus CGMCC No.26424 strain of the present invention; wherein, the order of loading samples for gel electrophoresis is as follows: lane 1: negative control (water); lanes 3-7: PCR identification results of F3, F4, F5, F6, and F7 generations of porcine circovirus type 3 virus CGMCC No.26424 strain, respectively; lane 2: porcine circovirus type 3 virus positive control; M represents Marker, and from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp. DETAILED DESCRIPTION

[0080] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0081] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

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

[0083] CO 2 The incubator (model: 3111) was purchased from Thermo Company;

[0084] A high-speed desktop centrifuge (model: TGL-16C) was purchased from Shanghai Anting Scientific Instrument Factory;

[0085] The gel imaging system (model: Tanon 1600R) was purchased from Beijing Yuanpinghao Biotechnology Co., Ltd.;

[0086] PCR instrument (model: Mastercycler) was purchased from Eppendorf;

[0087] Nucleic acid and protein electrophoresis instrument (model: DYY-6C) was purchased from Beijing Liuyi Instrument Factory;

[0088] DL 2000 DNA Marker (Cat. No. 3427A) and DL 10000 DNA Marker (Cat. No. 3584A) were purchased from Dalian Bao Bioengineering Co., Ltd.;

[0089] EasyPure Viral DNA / RNA Kit Viral DNA / RNA Purification Kit (Cat. No.: ER201-01), a product of Beijing Quanshijin Biotechnology Co., Ltd., Porcine Circovirus Type 3 Fluorescence PCR Kit (Model: AP97291), a product of Shanghai Fusheng Industrial Co., Ltd.;

[0090] PK15 (Madin-Darby Bobine Kidney cell) cells were purchased from the Chinese Veterinary Microbial Culture Collection Center (CVCC) of the Chinese Institute of Veterinary Drug Administration. The cell line number is CVCC CL24. The cells were domesticated into 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.

[0091] Fetal bovine serum (catalog number: BS-1101) was purchased from Inner Mongolia Jinyuankang Bioengineering Co., Ltd.

[0092] Example 1: Isolation and identification of porcine circovirus type 3

[0093] 1.1. Isolation of porcine circovirus type 3

[0094] 1.1.1. Treatment of diseased materials

[0095] The collected inguinal lymph nodes of the suspected porcine circovirus type 3 infected pigs were 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. Then centrifuged at 10000 r / min for 10 min, the supernatant was filtered and sterilized with a 0.22 μm microporous filter membrane, and the obtained diseased material filtrate was used to inoculate PK15 cells.

[0096] 1.1.2 Virus isolation and passage

[0097] 1 ml of the filtrate of the above-treated diseased material was inoculated into the PK15 cells that had grown a monolayer and were in the logarithmic growth phase. The cells were adsorbed at 37°C for 2 h and gently shaken once every 30 min to allow the filtrate of the diseased material to fully contact the cells. After adsorption, the liquid was poured off and the cells were gently washed twice with PBS. Then, DMEM maintenance solution (DMEM medium containing 200 units / ml of penicillin, 200 μg / ml of streptomycin and 20 ml / L of fetal bovine serum) was added and incubated at 37°C and 5% CO. 2 Culture in an incubator for 72-96 hours and harvest. Follow this method for 5 consecutive blind generations, freeze and thaw the harvested cell culture at -70℃ for 2 times, centrifuge at 4000 r / min for 15 min, take the supernatant and store at -70℃ for later use, use porcine circovirus type 3 polyclonal antibody as primary antibody, FITC-labeled goat anti-mouse IgG (product of Shanghai Huzhen Industrial Co., Ltd., item number: HZ1115) as secondary antibody, and perform indirect immunofluorescence assay (IFA) to detect PCV3 virus. The results are as follows Figure 1 As shown, specific immunofluorescence was observed in the virus-inoculated PK15 cells, while no fluorescence was observed in the normal PK15 cells that were not inoculated with the virus, indicating that the PK15 cells were infected with PCV3 and the virus could proliferate on PK15.

[0098] 1.2. PCR Identification of Porcine Circovirus Type 3

[0099] 1.2.1. Virus PCR detection

[0100] According to the gene sequence of PCV3 CAP protein published in Genbank, DNAStar compared the nucleotide sequences of all PCV3 CAP protein genes, and designed two primers in the conserved region using Oligo 6.0. The amplified product was expected to be 695 bp. The primer sequences are shown in Table 1 below.

[0101] Table 1. PCV3 PCR primer sequences and amplification target fragment sizes

[0102]

[0103] Extract viral DNA: Take 200 μl of viral cell culture and use EasyPure Viral DNA / RNA Kit (product of Quanshijin Company, product number: ER201-01) to extract viral DNA, and place it in a -70℃ refrigerator for use.

[0104] The PCR reaction was carried out in a 20 μL system: 2×PCR Mix 10 μL, upstream and downstream primers (both at a concentration of 10 pmol) 1.0 μL each, template DNA 2.0 μL, ddH 2 O 6.0 μl.

[0105] PCR reaction conditions: 94℃ 5 min; 94℃ 30 s, 55℃ 45 s, 72℃ 60 s, 30 cycles; 72℃ 10min. PCR products were stored at 4℃. Take 5 μl of the PCR reaction product, add an appropriate amount of loading buffer, mix well, and electrophoresed at 120V for 30 min in 1% agarose gel (golden view concentration is 5 μl / 100 mL). After the electrophoresis, take out the gel and observe the results in a gel imager. The results showed that a specific band of about 695 bp was amplified, and the PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing analysis. The sequencing results showed that the obtained specific band was a DNA molecule with a nucleotide sequence of SEQ ID No.1. The DNA molecule is the CAP gene sequence of porcine circovirus type 3, encoding the CAP protein of porcine circovirus type 3, and the amino acid sequence of the CAP protein is SEQ ID No.2. The nucleotide sequence of the DNA molecule was compared with the sequence published by NCBI, and the homology with the existing porcine circovirus type 3 CAP protein encoding gene was as high as 99%. The results showed that the virus in the above-mentioned viral cell culture was porcine circovirus type 3 (Porcine Circovirus Type 3, PCV3).

[0106] Example 2: Virus subculture and determination of virus titer and toxicity

[0107] 2.1 Virus proliferation culture

[0108] The isolate was inoculated into PK15 monolayer cells at an inoculation rate of 5:100 and cultured for 72 to 96 hours, then harvested as the F1 generation. The virus fluid after harvest 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 qualified isolates were frozen for seed preservation. The study eventually screened out a PCV3 virus strain, named PCV3 XY strain. The PCV3 XY strain was continuously passaged in cells to allow the virus to better adapt to cell proliferation and increase the virus content (toxin titer). The seed virus (PCV3 XY strain) was continuously passaged to the F7 generation, with the virus harvest of at least 100 ml in each generation, and the harvested virus was stored at -70°C. At the same time, the viruses after continuous passage 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 .

[0109] The PCV3 XY strain (second generation) obtained by the present invention has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on June 5, 2023, with the deposit number CGMCC No. 26424, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. This strain is also referred to as porcine circovirus type 3 virus CGMCC No. 26424, also referred to as XY strain.

[0110] 2.2. Toxicity determination

[0111] The domesticated virus was marked as F1 generation and subcultured for 5 generations. The virus content was detected by indirect immunofluorescence assay (IFA). -4 , 10 -5 , 10 -6 , 10 -7 Four dilutions were prepared. Each dilution was inoculated into four wells of a 96-well plate containing a well-grown PK15 cell monolayer. 100 μl was added to each well and the plate was incubated at 37°C with 5% CO. 2Continue to culture in the cell culture incubator, observe for 5 consecutive days, and perform IFA detection. Discard the cell culture medium in the cell culture plate, add 100 μl of cold acetone fixative to each well, fix at -20℃ for 30 minutes, discard the fixative, and place in a fume hood (2 hours) or air dry. Add 100 μl of PBS containing 5% skim milk to each well, place on a shaker, and gently shake at 37℃ to block for 45 minutes. Discard the blocking solution of the cell culture plate, wash 3 times with PBS buffer, and spin dry. Add 100 μl of PCV3 positive serum (whole blood collected from PCV3 positive pig farms and centrifuged) diluted 50-800 times with pH 7.4 PBS to the PCV3 infected cell wells and the uninfected cell control wells, respectively, place in a 37°C wet box for 1 hour, wash with PBS 5 times, 3 minutes each time; add 100 μl of goat anti-swine IgG labeled with fluorescein isothiocyanate diluted 100 times with pH 7.4 PBS, and place in a 37°C wet box for 1 hour; wash with PBS 5 times, 3 minutes each time, and finally add 100 μl of PBS to each well, and observe the results under an inverted fluorescence microscope. Test establishment conditions: green fluorescence appears in the positive control wells, and no green fluorescence in the negative control wells. The test sample wells that show green fluorescence are judged as PCV3 antigen positive. Record the fluorescent cell wells and calculate the TCID 50 The highest virus titer before domestication is 10 6.5 TCID 50 / ml, using PK15 suspension cell culture up to 10 8.0 TCID 50 / ml. Its virus titer can be increased by more than 10 times compared with the virus titer before domestication.

[0112] 2.3. Toxicity assay

[0113] Virulence was determined for the third generation virus culture. Virulence was expressed as the minimum infectious dose (MID), which was obtained by diluting the virus 10-fold to 10 in cell culture medium. -6 , take 10 -4 , 10 -5 , 10 -6 Three dilutions were used to inoculate 49-56-day-old piglets with negative PCV3 antibodies and antigens (purchased from Chengxin Farm, Mancheng District, Baoding City, Hebei Province) by intranasal and intramuscular injection. The inoculation dose for each pig was 10 ml (4 ml for intranasal and 6 ml for intramuscular injection). There were 5 pigs in each group. After the challenge, the pigs were observed for 14 consecutive days. The inguinal lymph nodes were obtained by dissection on the 14th day after the challenge. The CT value of the virus nucleic acid was detected by the porcine circovirus type 3 fluorescent PCR kit (product of Shanghai Fusheng Company, model: AP97291). The positive test was judged as the disease, and the minimum infectious dose (MID) of PCV3 virus was calculated. The results showed that the MID of PCV3 was 10×10 6.0 TCID 50 / head.

[0114] Example 3: Preparation and testing of porcine circovirus type 3 inactivated vaccine

[0115] 3.1. Virus reproduction

[0116] The second generation virus seed of PCV3 XY strain CGMCC No.26424 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-growing PK15 suspension cells at an MOI of 0.1 and placed at 37°C with 5% CO. 2 The cell culture was harvested after 4 days of culture in a cell shaker incubator, and the viral content was determined at 10 7.0~8.5 TCID 50 / ml, can be used as antigens for preparing vaccines.

[0117] 3.2. Inactivation of porcine circovirus type 3

[0118] The cell culture was inactivated by diethyleneimine (BEI), i.e., 2% of 0.1 mol / L BEI solution was added, stirred while adding, and fully mixed, and inactivated at 37°C for 20-24 hours, and then a 1 mol / L sodium thiosulfate solution that had been filtered and sterilized was added to the inactivated virus solution, so that the final volume of sodium thiosulfate was 30% of the volume of the added BEI solution, and fully mixed for inactivation blocking to obtain porcine circovirus type 3 virus inactivated solution (i.e., porcine circovirus type 3 virus inactivated antigen), which was stored at 2-8°C. The inactivated virus solution was sampled for sterility and inactivation tests, and was used for vaccine preparation after passing the tests. The sterility test is carried out according to 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 PK15 monolayer cells with the inactivated solution of porcine circovirus type 3, discard the virus solution after adsorption for 1 hour, add a maintenance solution 10 times the volume of the virus inactivation solution, culture at 37°C for 3 days, harvest the cell culture solution and blindly propagate it for one generation according to the same method. If no typical fluorescence appears during the IFA test, it is judged to be qualified and completely inactivated.

[0119] 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, shaking it every 10 minutes for 60 minutes, and preparing a BEI (diethyleneimine) solution with a final concentration of 0.1 M, which was then used after sterile filtration.

[0120] 3.3 Vaccine preparation

[0121] The inactivated porcine circovirus type 3 antigen that passed the test was diluted to 10 6.0 TCID 50 / ml and 10 7.0 TCID 50 / ml, and then mixed with the company's homemade patented adjuvant (605 adjuvant) at a ratio of 2:1 (volume ratio), stirred at 100-300 r / min for 30 minutes, and fully mixed. Among them, 605 adjuvant (i.e. 605 oil-free adjuvant) is disclosed in the authorized patent ZL201310018498.X). The composition of the 605 adjuvant used in this implementation 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, 1000 ml water for injection.

[0122] Three groups of vaccines were prepared in the experiment, as shown in Table 2.

[0123] Table 2. Vaccine preparation test grouping table

[0124]

[0125] 3.4 Safety of inactivated porcine circovirus vaccine

[0126] Five healthy piglets aged 14 to 21 days (purchased from Chengxin Breeding Farm, Mancheng District, Baoding City, Hebei Province) were injected with No. 1 vaccine, No. 2 vaccine and the immune control group vaccine through the neck muscle, 4.0 ml per piglet. They were observed for 14 consecutive days and no clinical symptoms of porcine circovirus or local and systemic adverse reactions caused by vaccination were found.

[0127] 3.5. Efficacy of inactivated porcine circovirus vaccine

[0128] As shown in Table 3, 20 healthy piglets with negative porcine circovirus antibody antigen at 14 to 21 days of age (purchased from Chengxin Farm, Mancheng District, Baoding City, Hebei Province) were used. Among them, 5 piglets were injected with porcine circovirus type 3 inactivated vaccine No. 1 vaccine, 1.0 ml / head, 5 piglets were injected with No. 2 vaccine, 1.0 ml / head, and 5 piglets were injected with immune control vaccine, 2.0 ml / head. 21 days after immunization, they were boosted with the same dose, and the control group was injected with PK15 cell freeze-thaw. Blood was collected 14 days after the second immunization, serum was separated, and serum agar antibodies were detected; at the same time, the challenge protection test was carried out, and 15 immunized piglets and 5 control piglets were infected with porcine circovirus type 3 virus CGMCC No. 26424 strain 10 ml virus, of which 4 ml was intranasal and 6 ml was injected into the neck muscle. The animals were observed for 14 days after the challenge, and the inguinal lymph nodes were removed on the 14th day after the challenge. The CT value of the viral nucleic acid was detected using the porcine circovirus type 3 fluorescent PCR kit. The experimental groups are shown in Table 3.

[0129] Table 3. Grouping of the efficacy test of inactivated vaccine of porcine circovirus type 3 virus CGMCC No.26424

[0130]

[0131] The method for determining serum antibody titer is the agar diffusion test. Take the separated serum and porcine circovirus type 3 antigen for agar diffusion test. The specific operation is as follows:

[0132] Use a plum blossom-shaped puncher with a diameter of 4 mm and a distance of 4 mm between the central hole and the peripheral hole to punch holes and seal the bottom. Add porcine circovirus type 3 inactivated antigen to the central hole, and add the serum to be tested in multiple dilutions to the 2nd, 3rd, 5th, and 6th holes on the periphery in turn. The amount of liquid added should not overflow the sample well. After the sample is added, let it stand for 5 to 10 minutes, gently invert the plate, put it in a wet box, and place it in a 37°C incubator for 24 to 48 hours to observe the results. Result judgment: A precipitation line appears between the serum test hole and the antigen hole, which is judged as positive. The highest dilution multiple of the serum with a specific precipitation line is used as the agar titer of the sample to be tested. The antibody test results show that the vaccine prepared with porcine circovirus type 3 virus CGMCC No.26424 strain can induce the production of porcine circovirus type 3 antibodies. The vaccine prepared with the purified and domesticated porcine circovirus type 3 virus CGMCC No.26424, when the vaccination dose was 1.0 ml, had higher antibody levels and challenge protection rates than the same vaccine with a vaccination dose of 2.0 ml before the virus was purified and domesticated (first generation), indicating that the porcine circovirus type 3 virus CGMCC No.26424 has a higher culture titer and better immunogenicity.

[0133] 14 days after infection, the inguinal lymph nodes were removed from the mice and the viral nucleic acid CT value was detected using the porcine circovirus type 3 fluorescent PCR kit. Vaccine No. 1, vaccine No. 2 and the immunization control group vaccine were able to significantly reduce the viral load, among which vaccine No. 2 was more effective. The results are shown in Table 4.

[0134] Table 4. Results of the study on the efficacy of inactivated porcine circovirus vaccine

[0135]

[0136] Example 4: Study on the duration of antibodies against porcine circovirus inactivated vaccine

[0137] Healthy piglets with negative porcine circovirus antibody antigen at 14 to 21 days old (purchased from Chengxin Farm, Mancheng District, Baoding City, Hebei Province) were used and divided into 2 groups. Group 1 was the vaccine immunization group (No. 2 vaccine) of the present invention, and 1.0 ml of the vaccine of the present invention was inoculated into the neck muscle, and a booster immunization was performed once 21 days after immunization. Group 2 was the PK15 cell freeze-thaw immunization control group. Blood was collected at 1 month, 3 months, and 4 months after immunization to determine the serum agar titer. The results are shown in Table 5, indicating that the porcine circovirus antibody level reached the highest level 2 months after immunization, and the antibody can still maintain a high level 4 months after immunization. The results of the antibody duration are shown in Table 5.

[0138] Table 5. Study on the duration of antibodies against porcine circovirus inactivated vaccine

[0139]

[0140] 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 the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A vaccine for preventing porcine circovirus type 3 infection or a disease caused by porcine circovirus type 3 infection, characterized in that: The vaccine contains porcine circovirus type 3 virus strain, the virus strain is PCV3 XY strain, its deposit number in the General Microbiological Center of China National Microbiological Culture Collection Committee is CGMCC No.26424, and the virus strain is inactivated; The vaccine also contains an adjuvant, which is a 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 circovirus type 3 infection or a disease caused by porcine circovirus type 3 infection, characterized in that: The method comprises the following steps: A1) Cultivating porcine circovirus type 3 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 PCV3 XY strain, and its deposit number in the General Microbiological Center of China Microbiological Culture Collection Administration is CGMCC No.26424. 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.

4. A kit, characterized in that The kit contains the vaccine according to claim 1.

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