Porcine parvovirus antibody detection kit based on indirect immunofluorescence method and its application

Abstract

The present invention relates to the technical field of in vitro detection reagents, and specifically to a porcine circovirus antibody detection kit based on an indirect immunofluorescence method and its application. The porcine circovirus Cap antigen provided by the present invention can simultaneously bind to serum antibodies induced by two genotypes, TTSuV1 and TTSuV2, and has high specificity. Based on the Cap antigen and the IFA method, the present invention establishes a serological detection kit and detection method for TTSuV antibodies, which can simultaneously detect serum antibodies induced by two genotype viruses, TTSuV1 and TTSuV2, and has good specificity, stability and repeatability, effectively avoiding the problems encountered in the establishment process of the ELISA method, and can better meet the needs of TTSuV serological clinical detection, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro detection reagents, in particular to a porcine parvovirus antibody detection kit based on an indirect immunofluorescence method and an application thereof. Background Art

[0002] Torque teno sus virus (TTSuV) is a small, non-enveloped, single-stranded circular DNA virus belonging to the genus Torquevirus in the family Toriviridae. Its genome is approximately 2.8 kb long and contains three to four overlapping open reading frames (ORFs) as well as a non-coding region with high GC content. Torque teno sus virus (TTSuV) was first discovered in 1997 when a non-enveloped, single-stranded circular virus was detected in the serum of a Japanese patient with acute hepatitis after a blood transfusion. It was subsequently identified in domestic animals such as pigs, chickens, cattle, sheep, cats, and dogs.

[0003] TTSuV is divided into two genotypes, TTSuV1 and TTSuV2, whose genomes share only approximately 50% similarity. In recent years, TTSuV infection has been reported in pigs in various countries. TTSuV is primarily transmitted through the fecal-oral route, but can also be transmitted vertically, such as through the placenta and uterus. While TTSuV infection alone does not produce specific clinical symptoms, TTSuV often co-infects with other swine viruses, such as porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), creating unknown synergistic effects that can exacerbate the disease.

[0004] Currently, existing TTSuV detection methods are limited to PCR for viral pathogen detection or ELISA for serological testing. There are no commercially available kits specifically for detecting TTSuV antibodies in clinical practice. Therefore, the development of a rapid, efficient, and sensitive TTSuV antibody detection method is crucial. Furthermore, research on the pathogenicity of TTSuV is limited. Sequence analysis of TTSuV and the development of antibody detection methods can provide a deeper understanding of the virus's infection status in different pig populations, providing a theoretical basis and technical support for the early detection and prevention of the virus.

[0005] Huang Yaowei et al. reported on the Western blot and ELISA antibody detection methods for TTSuV2 (DOI: 10.1016 / j.virusres.2011.03.013, and patent application CN201080047299). Yang Xiaoxiao et al. reported on the establishment of an indirect ELISA antibody detection method for TTSuV1 (Yang Xiaoxiao et al., Journal of Beijing Agricultural College 2024, 39(1): 43-46). First, although the ELISA method has been widely used in the serological detection of many epidemic diseases, many kits still have many problems in terms of specificity and stability. An important reason for the above problems is that the ELISA coated plates used in the method have poor stability and large non-specific reactions. In addition, the antibody detection methods reported above are all targeted at one of TTSuV2 and TTSuV1, and there is no kit that can detect antibodies to both TTSuV1 and TTSuV2. Summary of the Invention

[0006] The present invention provides a porcine parvovirus antibody detection kit based on an indirect immunofluorescence assay (IFA) and an application thereof.

[0007] The present invention analyzes and detects the prevalence of TTSuVs strains in large-scale pig farms in recent years, screens the prevalent strains of TTSuV1 and TTSuV2, combines the genome sequence analysis of TTSuV1 and TTSuV2, selects relatively conserved sequences of the Cap genes of TTSuV1 and TTSuV2, and obtains a fusion protein that simultaneously expresses part of the Cap genes of TTSuV1 and TTSuV2 through optimized combination. The fusion protein is used as an antigen molecule to develop a TTSuV antibody detection kit based on the indirect immunofluorescence method.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides a porcine circovirus antibody detection kit based on an indirect immunofluorescence method, wherein the kit comprises cells expressing porcine circovirus Cap antigen;

[0010] The amino acid sequence of the Cap antigen is shown in SEQ ID NO.1.

[0011] The specific sequence of SEQ ID NO.1 is shown below.

[0012] MPGDRFHSGIQDPSKVQNTVLNPWDYDCDGIVRKDTLKRLLELPTETEEEEPSRTLCADTPTEASQSAFLRGDEKEETSEGEETATSSSITSAESTTEGDGSSDDDETRK.

[0013] The above-mentioned Cap antigen is a fusion protein that contains partial capsid protein Cap fragments of both genotypes TTSuV1 and TTSuV2. It can simultaneously bind to serum antibodies against TTSuV1 and TTSuV2, has high specificity, can improve the specificity and sensitivity of the test kit, and can achieve simultaneous and efficient detection of serum antibodies against TTSuV1 and TTSuV2.

[0014] The cells mentioned above carry the gene encoding the Cap antigen.

[0015] Based on the amino acid sequence and codon coding rules of the above-mentioned Cap antigen, those skilled in the art can obtain the sequence of the gene encoding the Cap antigen. Due to the degeneracy of the codons, the sequence of the gene encoding the Cap antigen is not unique, and all genes that can encode the Cap antigen are within the scope of protection of the present invention.

[0016] Preferably, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2.

[0017] The specific sequence of SEQ ID NO.2 is shown below.

[0018] GCCACCATGCCAGGAGACAGATTCCACAGCGGCATCCAGGACCCCAGCAAGGTGCAGAACACAGTGCTGAATCCTTGGGATTACGATTGCGACGGCATCGTGAGAAAGGATACACTGAAGAGACTGCTGGAGCTGCCCACAGAGACAGAGGAGGAGGAGCCCAGCAGGAC CCGTGTGCCGATACACCTACAGAGGCCAGCCAGTCCGCCTTTCGAGAGGCGACGAGAAGGAGGAGACCAGCGAGGGCGAGGAGACCGCCACATCCAGCTCCATCACATCCGCCGAGAGCAGCACCGAGGGCGACGGAAGCTCCGATGACGACGAGACCAGAAAGTGA.

[0019] The sequence shown in SEQ ID NO.2 comprises the expression-promoting sequence GCCACC and the coding sequence of the Cap antigen from 5' to 3'. The coding sequence of the Cap antigen is obtained by codon optimization. The coding gene sequence can significantly increase the expression level of the Cap antigen in host cells.

[0020] The cells mentioned above are mammalian cell lines used for protein expression.

[0021] Preferably, the cells are HEK293T cells.

[0022] Preferably, the gene encoding the Cap antigen is located on a mammalian cell expression plasmid.

[0023] Further preferably, the mammalian cell expression plasmid is pcDNA3.1(+).

[0024] In some specific embodiments of the present invention, the pcDNA3.1(+) plasmid carrying the gene encoding the Cap antigen is introduced into HEK293T cells to obtain cells expressing the porcine parvovirus Cap antigen.

[0025] The cells expressing porcine circovirus Cap antigen described above are fixed in the wells of a perforated glass slide.

[0026] Preferably, the fixation can be performed using acetone.

[0027] In some specific embodiments of the present invention, the method for preparing the above-mentioned perforated glass slide fixed with cells expressing porcine parvovirus Cap antigen comprises the following steps: introducing a DNA molecule containing the gene encoding the Cap antigen into the expression vector pcDNA3.1 (+), constructing a recombinant vector pcDNA-TTSuV-1, and transfecting it into HEK293T cells, then fixing the positively transfected cells on the glass slide with acetone, sealing with alkaline glycerol, and placing it at -20°C for use.

[0028] To facilitate indirect immunofluorescence detection, the kit may further comprise one or more selected from a washing solution, a fluorescein-labeled secondary antibody, a positive standard control, a negative control, and a sealing reagent.

[0029] The fluorescein-labeled secondary antibody is used to bind to serum antibodies to detect antigen-antibody complexes. Preferably, the fluorescein-labeled secondary antibody is FITC-labeled goat anti-swine IgG.

[0030] The washing solution is used to wash the slides after each step of the reaction. Preferably, the washing solution is PBS buffer.

[0031] The sealing agent is used to seal the slide. Preferably, the sealing agent is alkaline glycerol.

[0032] The above-mentioned kit comprises the following steps when used to detect porcine parvovirus antibodies:

[0033] Add the test sample to the well of the perforated glass slide fixed with cells expressing porcine circovirus Cap antigen and incubate at 35-37°C for 0.8-1.2h;

[0034] After washing with washing buffer, add fluorescein-labeled secondary antibody and incubate at 35-37℃ in the dark for 25-35 minutes;

[0035] After washing with washing solution, add sealing reagent for sealing and fluorescence observation;

[0036] The fluorescence signal is used to determine whether the sample to be tested contains porcine parvovirus antibodies.

[0037] In the present invention, the porcine parvovirus antibody is a serum antibody.

[0038] Preferably, the sample to be tested is pig serum.

[0039] In a second aspect, the present invention provides any of the following uses of the above-described kit:

[0040] (1) Application in the preparation of products for detecting porcine parvovirus antibodies;

[0041] (2) Use in the preparation of products for diagnosing porcine parvovirus infection;

[0042] (3) Application in the detection of porcine parvovirus antibodies for non-disease diagnosis purposes.

[0043] In the above (1) and (3), the antibody is a pig serum antibody.

[0044] In a third aspect, the present invention provides an indirect immunofluorescence detection method for porcine parvovirus antibodies for non-disease diagnosis purposes, the method comprising: using the above-mentioned kit to detect a sample to be tested.

[0045] Preferably, the sample to be tested is pig serum.

[0046] Preferably, the above detection method comprises:

[0047] Add the test sample to the well of the perforated glass slide fixed with cells expressing porcine circovirus Cap antigen and incubate at 35-37°C for 0.8-1.2h;

[0048] After washing with washing buffer, add fluorescein-labeled secondary antibody and incubate at 35-37℃ in the dark for 25-35 minutes;

[0049] After washing with washing solution, add sealing reagent for sealing and fluorescence observation;

[0050] The fluorescence signal is used to determine whether the sample to be tested contains porcine parvovirus antibodies.

[0051] In a fourth aspect, the present invention provides a porcine circovirus Cap antigen, the amino acid sequence of the Cap antigen is shown in SEQ ID NO.1.

[0052] Those skilled in the art will understand that the Cap antigen obtained by adding a protein tag, signal peptide, or the like to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1, which does not affect the function of the amino acid sequence shown in SEQ ID NO.1, is an equivalent variation of the Cap antigen shown in SEQ ID NO.1, and is therefore also within the scope of protection of the present invention.

[0053] In a fifth aspect, the present invention provides a nucleic acid molecule encoding the porcine circovirus Cap antigen described above.

[0054] Based on the amino acid sequence and codon encoding rules of the above-mentioned Cap antigen, those skilled in the art can obtain the sequence of the nucleic acid molecule encoding the Cap antigen. Due to the degeneracy of the codons, the sequence of the nucleic acid molecule is not unique, and all nucleic acid molecules that can encode the Cap antigen are within the scope of protection of the present invention.

[0055] In some embodiments of the present invention, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0056] In a sixth aspect, the present invention provides a biomaterial, which comprises the nucleic acid molecule described above; the biomaterial is an expression cassette, a vector or a host cell.

[0057] The expression cassette mentioned above is obtained by connecting the nucleic acid molecule with a promoter and one or more selected from a terminator, an enhancer, a translation promoting sequence and the like.

[0058] The vectors mentioned above may be plasmid vectors, viral vectors, etc. Among them, plasmid vectors include replicative vectors, non-replicative vectors, etc.

[0059] The host cells mentioned above include microbial cells, animal cells, or animal cell lines. The microbial cells include, but are not limited to, Escherichia coli, yeast, etc. The animal cells can be any mammalian cell line used for protein expression, including but not limited to HEK293T cells.

[0060] In some embodiments of the present invention, a cell expressing porcine parvovirus Cap antigen is provided, wherein the cell is preferably a microbial cell or an animal cell (the animal cell does not have the potential to reproduce into an animal individual).

[0061] In a seventh aspect, the present invention provides any of the following uses of the porcine circovirus Cap antigen, the nucleic acid molecule, or the biological material described above:

[0062] (1) Application in the preparation of products for detecting porcine parvovirus antibodies;

[0063] (2) Use in the preparation of products for diagnosing porcine parvovirus infection;

[0064] (3) Application in the detection of porcine parvovirus antibodies for non-disease diagnosis purposes.

[0065] In an eighth aspect, the present invention provides a porcine circovirus detection reagent, comprising the porcine circovirus Cap antigen described above, or comprising cells expressing the porcine circovirus Cap antigen described above. The cells are preferably microbial cells or animal cells (the animal cells do not have the potential to reproduce into individual animals).

[0066] The beneficial effects of the present invention include at least: the porcine circovirus Cap antigen provided by the present invention can simultaneously bind to serum antibodies induced by both genotypes TTSuV1 and TTSuV2, with high specificity. Based on this Cap antigen and the IFA method, the present invention has established a serological detection kit and detection method for TTSuV antibodies. This kit and detection method can simultaneously detect serum antibodies induced by both genotypes of viruses, TTSuV1 and TTSuV2, with good specificity, stability, and repeatability, effectively avoiding the problems encountered during the establishment of the ELISA method, and can better meet the needs of TTSuV serological clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0068] Figure 1 These are the expression test results of the porcine circovirus Cap antigen encoding gene sequence before and after optimization in Example 1 of the present invention.

[0069] Figure 2 Schematic diagram of a glass slide with holes in Example 2 of the present invention.

[0070] Figure 3 This is the result of TTSuV serum antibody detection using the IFA method in Example 3 of the present invention. DETAILED DESCRIPTION

[0071] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0072] Example 1: Design and expression optimization of porcine circovirus Cap antigen

[0073] 1. Design of porcine circovirus Cap antigen

[0074] By analyzing and detecting the prevalence of TTSuVs strains in large-scale pig farms in recent years, the prevalent strains of TTSuV1 and TTSuV2 were screened. Combined with the genomic sequence analysis of TTSuV1 and TTSuV2, relatively conserved sequences of the Cap genes of TTSuV1 and TTSuV2 were selected, and a fusion protein expressing part of the Cap genes of TTSuV1 and TTSuV2 was obtained through optimized combination. The amino acid sequence of the fusion protein is shown in SEQ ID NO.1, and the full-length coding sequence of the fusion protein is 333 bp (nucleotide sequence is shown in SEQ ID NO.3), in which positions 1-3 are the added start codon sequence ATG, positions 4-153 are derived from positions 1-150 of the Cap gene of TTSuV2 GD strain (JF937659), positions 154-330 are derived from positions 151-327 of the Cap gene of TTSuV1 HeN1-A9 strain (MW117137), and positions 331-333 are the added stop codon sequence TGA.

[0075] The sequence shown in SEQ ID NO.3 is specifically shown below.

[0076] ATGCCCGGGGACCGCTTCCACAGCGGGATACAAGACCCCTCCAAGGTACAAAACACCGTCCTCAACCCCTGGGACTATGACTGTGATGGGATTGTTAGAAAAGATACTCTCAAAAGACTTCTCGAACTCCCCACAGAGACAGAGGAGGAGGAGCCGTCCAGAACCCT CTGTGCAGATACACCCACAGAAGCATCGCAAAGTGCATTTCTCAGAGGGGACGAAAAAGAGGAAACCTCGGAAGGAGAGGAAACCGCGACATCGTCCAGTATCACGAGTGCCGAAAGCTCTACTGAAGGAGATGGATCGTCTGATGATGATGAAACGAGAAAATGA.

[0077] 2. Expression optimization of porcine circovirus Cap antigen

[0078] The coding sequence of the porcine circovirus Cap fusion protein (i.e., Cap antigen) in the above 1 (the nucleotide sequence is shown in SEQ ID NO.3) was codon-optimized and an expression-promoting sequence was added to increase the expression level of the porcine circovirus Cap antigen. After screening and verification, a Cap antigen encoding gene with codon optimization and addition of an expression-promoting sequence was obtained, and its nucleotide sequence is shown in SEQ ID NO.2.

[0079] HEK293T cells were transfected with the empty pcDNA3.1(+) vector, the pcDNA-TTSuV recombinant vector carrying the sequence shown in SEQ ID NO. 3, and the pcDNA-TTSuV-1 recombinant vector carrying the sequence shown in SEQ ID NO. 2 (codon-optimized and with an expression-enhancing sequence added) to obtain recombinant cells. The recombinant cells were cultured to obtain a cell suspension.

[0080] The expression level of porcine circovirus Cap antigen was detected, and the specific method is as follows.

[0081] (1) Take 20 μL of HEK293T cell suspension transfected with pcDNA3.1(+) empty vector, pcDNA-TTSuV recombinant vector and pcDNA-TTSuV-1 recombinant vector, add an equal volume of 2×SDS loading buffer, boil in boiling water bath for 8-10 min, take 20 μL of the treated sample and add it to the sample tank for SDS-PAGE electrophoresis.

[0082] (2) After electrophoresis, transfer the gel to a NC membrane for Western Blot analysis. Place the NC membrane in β-actin monoclonal antibody, TTSuV antibody positive and negative serum, incubate at 37°C for 1 hour, wash with PBS buffer, add infrared fluorescent dye-labeled goat anti-mouse / pig-IgG secondary antibody, incubate at 37°C in the dark for 1 hour, wash with PBS buffer, scan the NC membrane with the infrared scanning system Odyssey, and calculate the grayscale value of the bands.

[0083] (3) The results are as follows Figure 1 As shown, β-actin monoclonal antibodies reacted specifically with HEK293T cells transfected with pcDNA3.1(+) empty vector, pcDNA-TTSuV recombinant vector and pcDNA-TTSuV-1 recombinant vector, and there was no significant difference in the grayscale values ​​of the bands; TTSuV1- and TTSuV2-positive pig sera reacted specifically with HEK293T cells transfected with pcDNA-TTSuV recombinant vector and pcDNA-TTSuV-1 recombinant vector, and the grayscale value of the bands of HEK293T cells transfected with pcDNA-TTSuV-1 recombinant vector was 1.6 times that of HEK293T cells transfected with pcDNA-TTSuV recombinant vector. TTSuV1- and TTSuV2-positive pig sera did not react with HEK293T cells transfected with pcDNA3.1(+) empty vector. Furthermore, TTSuV1- and TTSuV2-negative pig sera did not react with HEK293T cells transfected with the pcDNA3.1(+) empty vector, the pcDNA-TTSuV recombinant vector, or the pcDNA-TTSuV-1 recombinant vector. These results indicate that codon optimization and the addition of an expression-enhancing sequence significantly increased Cap gene expression (Cap antigen expression increased 1.6-fold compared to pre-optimization levels) and that the gene specifically reacted with antibodies from TTSuV1- and TTSuV2-positive sera.

[0084] Example 2: Construction of a porcine parvovirus antibody detection kit based on IFA method

[0085] 1 Materials and Methods

[0086] 1.1 Materials

[0087] 1.1.1 Vectors and cells

[0088] The pcDNA3.1(+) vector and HEK293T cells are maintained by the Henan Provincial Animal Disease Diagnosis and Integrated Prevention Engineering Technology Research Center of Nanyang Normal University and are also commercially available. TTSuV1-positive, TTSuV2-positive, and TTSuV-negative porcine sera were prepared and stored in the inventors' laboratory. Sera positive for common porcine viruses, such as porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and pseudorabies virus (PRV), were also maintained in the inventors' laboratory. Clinical porcine serum samples were collected from large-scale pig farms in Nanyang, Zhumadian, Pingdingshan, and Xinyang in Henan Province, and Xiangyang, Suizhou, and Shiyan in Hubei Province, and are maintained in the inventors' laboratory.

[0089] 1.1.2 Main reagents

[0090] Plasmid extraction kits and gel recovery kits were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; T4 DNA ligase and endonucleases Xhol I and Hind III were purchased from TaKaRa; DMEM cell culture medium, fetal bovine serum, and cell culture flasks were purchased from Thermo Fisher Scientific; Lipofectamine 2000 transfection reagent and β-actin monoclonal antibody were purchased from Thermo Fisher Scientific; Sailboat brand slides (with holes) were purchased from Yancheng Feizhou Glass Plastics Co., Ltd.; and FITC-labeled goat anti-swine IgG was purchased from Solarbio. The optimized TTSuV Cap gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0091] 1.2 Experimental Methods

[0092] 1.2.1 Synthesis of TTSuV Cap antigen gene

[0093] The Hind III restriction site CGTAAGCTT and the expression-promoting sequence GCCACC were added upstream of the codon-optimized TTSuV Cap antigen coding sequence (as shown in positions 7-339 of SEQ ID NO. 2, and the encoded protein sequence is shown in SEQ ID NO. 1), and the Xhol I restriction site CTCGAGTG was added downstream. The product was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0094] 1.2.2 Enzyme digestion and ligation

[0095] The synthesized TTSuV Cap antigen gene sequence and the pcDNA3.1(+) vector were double-digested with the endonucleases Hind III and Xhol I, respectively. A 40 μL reaction system was prepared: 4 μL 10× Fast Digest Buffer, 1.5 μL Hind III, 1.5 μL Xhol I, and 33 μL of gene or vector template. Mix thoroughly and incubate at 37°C in an air bath for 1–2 hours. After digestion, the fragments were analyzed by agarose gel electrophoresis, and the correct bands were excised and purified. The target gene was ligated to the vector using T4 ligase. The reaction system was prepared: 1 μL 10× Buffer, 1 μL T4 DNA ligase, 1 μL pcDNA3.1(+) vector, and 7 μL TTSuV Cap antigen gene template. Mix thoroughly and incubate in an air bath at 22°C overnight.

[0096] 1.2.3 Recombinant vector construction

[0097] The ligation product from 1.2.2 was transformed into competent E. coli Top10 cells using the heat shock method. The cells were then evenly plated onto LB agar plates containing ampicillin and incubated at 37°C for 12–14 hours. A single colony was picked and plated into liquid LB medium containing ampicillin and incubated at 37°C for 6 hours before PCR analysis. The PCR reaction system consisted of 0.5 μL of each upstream and downstream primer, 5 μL of 2× PCR Mix, 1 μL of bacterial suspension, and distilled water to a total volume of 10 μL. PCR amplification was then performed using the following protocol: initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, 35 cycles of 10 minutes at 72°C, and detection by agarose gel electrophoresis. The upstream primer sequence is: CGTAAGCTTGCCACCATGCCCGGGGACC (SEQ ID NO. 4); the downstream primer sequence is: CACTCGAGTCATTTTCTCGTTTCATCATCATC (SEQ ID NO. 5). Bacterial fluid containing the correct target band size detected by PCR was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The recombinant vector with the correct sequencing result was named pcDNA-TTSuV-1 and stored at -80°C until further use.

[0098] 1.2.4 Cell transfection

[0099] First, pipette 4 μL of Lipofectamine 2000 transfection reagent into 246 μL of DMEM medium, gently pipette to mix, and incubate at room temperature for 2 minutes. Then, pipette 6 μg of pcDNA-TTSuV-1 vector into 250 μL of DMEM medium, gently pipette to mix, and incubate at room temperature for 2 minutes. Mix the two and incubate at room temperature for 15 minutes. Remove HEK293T cells cultured in a 6-well plate for 48 hours, discard the medium, and wash three times with DMEM medium. Then, add the transfection solution and incubate at 37°C for 5 hours. Then, discard the transfection solution, add 2 mL of fresh DMEM medium, and incubate at 37°C for 48 hours to obtain positively transfected cells expressing the TTSuV Cap antigen shown in SEQ ID NO. 1.

[0100] 1.2.5 Cytological smear

[0101] (1) Discard the culture medium of the 6-well plate and add PBS buffer for washing;

[0102] (2) Add trypsin for digestion. After the cells are detached, gently blow to make a single-cell suspension, transfer to a 1.5 mL EP tube, and centrifuge at 5000 rpm for 5 min.

[0103] (3) Discard the supernatant, add 1 mL of PBS buffer to the EP tube, gently pipette the cells until mixed, and centrifuge at 5000 rpm for 5 min;

[0104] (4) Discard the supernatant, add 200 μL PBS buffer to the EP tube, and gently pipette to mix. Figure 2 As shown, 20 μL of cell suspension was added to each well of the slide and allowed to dry at room temperature;

[0105] (5) Then fix the slide with pre-cooled acetone for 10-15 minutes, take it out and dry it, and place it at 4℃ for later use.

[0106] A kit was constructed by combining the above-mentioned glass slide fixed with HEK293T cells expressing TTSuV Cap antigen and reagents required for IFA detection such as PBSA buffer.

[0107] Example 3: Preliminary establishment of IFA antibody detection method

[0108] Take the glass slides prepared in Example 2 with HEK293T cells expressing TTSuV Cap protein fixed thereon, add TTSuV antibody positive and negative serum, incubate at 37°C for 1 hour, wash with PBS buffer, add FITC-labeled goat anti-swine IgG, incubate at 37°C in the dark for 30 minutes, wash with PBS buffer, add alkaline glycerol to seal the slides, and then observe under a fluorescence microscope. The results are as follows. Figure 3As shown, sera from pigs positive for TTSuV1 and TTSuV2 reacted specifically with HEK293T cells transfected with the pcDNA-TTSuV-1 recombinant vector, producing green fluorescence. They did not react with HEK293T cells transfected with the pcDNA3.1(+) empty vector or with normal HEK293T cells. Sera from pigs negative for TTSuV did not react with normal HEK293T cells, HEK293T cells transfected with the pcDNA3.1(+) empty vector, or HEK293T cells transfected with the pcDNA-TTSuV-1 recombinant vector. Furthermore, sera positive for common porcine viruses, such as PCV2, PRRSV, CSFV, and PRV, did not react with HEK293T cells transfected with the pcDNA-TTSuV-1 recombinant vector. The above results showed that the transfected TTSuV Cap gene was transiently expressed in HEK293T cells and could only react specifically with TTSuV (TTSuV1 and TTSuV2) positive sera. A TTSuVIFA antibody detection method was preliminarily established, which can realize the simultaneous detection of TTSuV1 and TTSuV2 serum antibodies.

[0109] Example 4: Clinical application of IFA antibody detection method

[0110] 984 blood samples were collected from 20 large-scale pig farms in various regions, including Nanyang, Zhumadian, Pingdingshan, and Xinyang in Henan Province, and Xiangyang, Suizhou, and Shiyan in Hubei Province. The IFA method established in Example 3 was used to detect TTSuV antibodies in these samples. The results showed that 535 blood samples were positive for TTSuV antibodies, with a positive rate of 54.4%. The TTSuV antibody positive rate varied significantly across different pig farms, ranging from 18.0% to 76.1% (Table 1). Blood samples were collected from different groups of pigs at a large-scale pig farm and tested for TTSuV antibodies using the IFA method established in Example 2. The results showed that the TTSuV antibody positive rate was lowest in nursery pigs, at 18.0%, while it was highest in sows, at 78.0%. The positive rates in fattening pigs and gilts were 44.0% and 64.0%, respectively. The TTSuV infection rate gradually increased from nursery pigs to fattening pigs, gilts, and sows (Table 2).

[0111] Table 1 TTSuV antibody test results in sera from different pig farms

[0112]

[0113] Table 2 TTSuV antibody detection results in sera from different pig groups

[0114]

[0115] The above results show that the IFA antibody detection method established in the present invention has good specificity, stability and repeatability, can provide an in-depth understanding of the infection situation of TTSuV in pig herds, and provide a theoretical basis and technical support for the early detection and prevention and control of the virus.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A porcine parvovirus antibody detection kit based on indirect immunofluorescence method, characterized in that: The kit comprises cells expressing porcine parvovirus Cap antigen; The amino acid sequence of the Cap antigen is shown in SEQ ID NO.

1.

2. The kit according to claim 1, wherein The cells carry the gene encoding the Cap antigen.

3. The kit according to claim 2, wherein The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

4. The kit according to any one of claims 1 to 3, characterized in that The cells are mammalian cell lines used for protein expression.

5. The kit according to claim 4, characterized in that The cells are HEK293T cells.

6. The kit according to any one of claims 1 to 3 and 5, characterized in that The coding gene of the Cap antigen is located on a mammalian cell expression plasmid.

7. The kit according to claim 6, characterized in that The mammalian cell expression plasmid is pcDNA3.1(+).

8. The kit according to any one of claims 1 to 3, 5 and 7, characterized in that The cells expressing porcine circovirus Cap antigen are fixed in the wells of a perforated glass slide.

9. The kit according to claim 8, characterized in that The kit further comprises one or more selected from a washing solution, a fluorescein-labeled secondary antibody, a positive standard control, a negative control, and a sealing reagent.

10. The kit according to any one of claims 1 to 3, 5, 7, and 9, characterized in that The kit comprises the following steps when used to detect porcine parvovirus antibodies: Add the test sample to the well of the perforated glass slide fixed with cells expressing porcine circovirus Cap antigen and incubate at 35-37°C for 0.8-1.2h; After washing with washing buffer, add fluorescein-labeled secondary antibody and incubate at 35-37℃ in the dark for 25-35 minutes; After washing with washing solution, add sealing reagent for sealing and fluorescence observation; The fluorescence signal is used to determine whether the sample to be tested contains porcine parvovirus antibodies.

11. The kit according to claim 10, characterized in that The sample to be tested is pig serum.

12. Any of the following uses of the kit according to any one of claims 1 to 11: (1) Application in the preparation of products for detecting porcine parvovirus antibodies; (2) Use in the preparation of products for diagnosing porcine parvovirus infection; (3) Application in the detection of porcine parvovirus antibodies for non-disease diagnosis purposes.

13. A porcine circovirus Cap antigen, characterized in that: The amino acid sequence of the Cap antigen is shown in SEQ ID NO.

1.

14. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the porcine circovirus Cap antigen according to claim 13.

15. The nucleic acid molecule according to claim 14, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

2.

16. An expression cassette, vector or host cell comprising the nucleic acid molecule of claim 14 or 15.

17. Any of the following uses of the porcine circovirus Cap antigen according to claim 13, the nucleic acid molecule according to claim 14 or 15, or the expression cassette, vector or host cell according to claim 16: (1) Application in the preparation of products for detecting porcine parvovirus antibodies; (2) Use in the preparation of products for diagnosing porcine parvovirus infection; (3) Application in the detection of porcine parvovirus antibodies for non-disease diagnosis purposes.

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