Nanometer antibody or antigen binding fragment thereof, coding gene, recombinant expression vector and application of nanometer antibody or antigen binding fragment thereof as reagent or kit

Abstract

The invention discloses a nano antibody or an antigen binding fragment, a coding gene and a recombinant expression vector of a targeted porcine circovirus type 2 Rep protein, and application of the nano antibody or the antigen binding fragment, the coding gene and the recombinant expression vector as a reagent or a kit. The nanobody or the antigen binding fragment thereof comprises at least one complementary determinant selected from the group consisting of CDR1, CDR2 and CDR3. The high-affinity nano antibody molecule aiming at the porcine circovirus type 2 (PCV2) Rep protein, which is constructed by the invention, is combined with a competitive enzyme-linked immunosorbent assay system, so that high-sensitivity, high-specificity and high-stability quantitative detection on the anti-PCV2 antibody in porcine serum can be effectively realized. According to the technical scheme, multiple limitations of a traditional antibody detection method in the aspects of stability, production consistency, detection flux and cross reactivity are broken through, and a set of standardized diagnosis platform suitable for grass-roots breeding institutions, large-scale epidemiological screening and vaccine immune effect evaluation is constructed.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to nanobodies or antigen-binding fragments thereof targeting the Rep protein of porcine circovirus type 2, encoding genes, recombinant expression vectors, and their applications as reagents or kits. Background Technology

[0002] Porcine Circovirus Type 2 (PCV2) infection has become one of the most serious viral diseases facing the global swine industry. This virus belongs to the genus *Circovirus* of the family Circoviridae. It is a single-stranded circular DNA virus with a genome length of approximately 1.76 kb. It primarily causes porcine multisystemic wasting syndrome (PMWS), post-weaning multisystemic wasting, and subclinical infection. Clinical manifestations include growth retardation, respiratory distress, diarrhea, and immunosuppression. Mortality rates in infected weaned pigs can exceed 20%. Epidemiological data show that the natural infection rate of PCV2 in swine herds is as high as 30% to 70%. Its high infectivity, persistent infection, and synergistic pathogenicity with other pathogens cause huge economic losses to farms, the slaughter chain, and international trade. It is estimated that the global swine industry loses more than US$1 billion annually due to PCV2-related diseases. There is an urgent need to develop efficient, accurate, and portable molecular detection technologies to support early diagnosis, vaccine efficacy evaluation, and virus surveillance.

[0003] However, current PCV2-based detection systems still face multiple technical obstacles: traditional polyclonal antibodies rely on animal immunization, which leads to batch variations, cross-reactivity, and ethical issues; while monoclonal antibodies improve specificity, hybridoma screening is time-consuming, has low stability, and high production costs, making it difficult to meet the screening needs of large-scale pig farms; existing commercial ELISA or PCR reagents mostly target the Cap protein, ignoring Rep expression in the early replication stage, resulting in insufficient sensitivity for low viral load samples and a high risk of false negatives; antigenic variations caused by viral genotype diversity (ai type) further weaken the broad-spectrum recognition ability of existing antibodies; at the same time, detection methods rely on complex equipment or species-specific reagents, limiting their application in grassroots veterinary settings or field environments.

[0004] Current technologies have not yet achieved a breakthrough in balancing performance optimization and cost control at the level of antibody engineering and detection integration. Traditional antibody molecules are large in size and have poor thermal stability, making them prone to denaturation and inactivation at high temperatures or in complex sample matrices, affecting the shelf life of reagent kits and on-site reliability. Their prokaryotic or eukaryotic expression systems are inefficient and complex to purify, failing to guarantee consistency in industrial production. Antibody modification flexibility is limited, making it difficult to construct multimodal detection platforms. Although nanobody technology has shown advantages in other viral fields such as high affinity, small molecular weight, and ease of engineering, specific nanobodies against the PCV2 Rep protein have not yet been systematically screened and optimized, and their integration with immunochromatography or fluorescence detection technologies lacks a mature pathway. Current diagnostic systems have significant shortcomings in terms of sensitivity, specificity, variant coverage, ease of operation, and cost-effectiveness.

[0005] Therefore, providing a novel antibody-detection synergistic technology that integrates molecular stability, production scalability, multifunctionality of detection, and target adaptability is a problem that this invention urgently needs to solve. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to solve the problems existing in the current field of porcine circovirus type 2 (PCV2) serological diagnosis, such as poor stability of antibody reagents, long preparation cycle, high cost, significant batch-to-batch variability, insufficient detection sensitivity and specificity, and difficulty in adapting to high-throughput screening requirements. Therefore, this invention provides a nanobody or its antigen-binding fragment, encoding gene, recombinant expression vector, and application as a reagent or kit that targets the Rep protein of porcine circovirus type 2.

[0007] To achieve the above objectives, the present invention provides a nanobody or antigen-binding fragment thereof targeting the Rep protein of porcine circovirus type 2, wherein the nanobody or antigen-binding fragment comprises at least one complementary determinant cluster selected from CDR1, CDR2, and CDR3; wherein... CDR1 is an amino acid sequence as shown in SEQ ID No:1 (specifically, its sequence is: GSWYTYNTFALN), or a mutant sequence that has at least 90% (not limited to 90%, the sequence identity here only needs to be no less than 90%, for example, it can also be 95%, 98%, etc.) sequence identity with the amino acid sequence shown in SEQ ID No:1 and is capable of specifically binding to the porcine circovirus type 2 Rep protein; CDR2 is an amino acid sequence as shown in SEQ ID No:2 (the specific sequence of which is: SMVGNLTFWAD), or a mutant sequence that has at least 90% (not limited to 90%, the sequence identity here only needs to be not less than 90%, for example, it can also be 95%, 98%, etc.) sequence identity with the amino acid sequence shown in SEQ ID No:2 and is capable of specifically binding to the porcine circovirus type 2 Rep protein; CDR3 is an amino acid sequence as shown in SEQ ID No:3 (the specific sequence of which is: YFVRVSPSFNYRK), or a mutant sequence that has at least 90% (not limited to 90%, the sequence identity here only needs to be no less than 90%, for example, it can also be 95%, 98%, etc.) sequence identity with the amino acid sequence shown in SEQ ID No:3 and is capable of specifically binding to the porcine circovirus type 2 Rep protein.

[0008] That is, the nanobody or its antigen-binding fragment provided by the present invention can specifically bind to the porcine circovirus type 2 Rep protein. Its complementary determinant clusters (CDRs) include one or more of CDR1, CDR2, and CDR3.

[0009] Preferably, the nanobody or its antigen-binding fragment described in this invention includes CDR1, CDR2, and CDR3. More preferably, the amino acid sequence of CDR1 is shown in SEQ ID No:1, the amino acid sequence of CDR2 is shown in SEQ ID No:2, and the amino acid sequence of CDR3 is shown in SEQ ID No:3.

[0010] The present invention also provides a coding gene for encoding the aforementioned nanobody or its antigen-binding fragment. Preferably, based on the nanobody or its antigen-binding fragment including CDR1, CDR2, and CDR3, the nucleotide sequence of the coding gene is selected from: a) the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; b) a nucleic acid sequence that can hybridize with the complementary strand of the nucleotide sequence shown in SEQ ID NO:4 under strict hybridization conditions and encodes the aforementioned nanobody or its antigen-binding fragment.

[0011] More preferably, the encoding gene includes the nucleotide sequences shown in SEQ ID No:4, SEQ ID No:5, and SEQ ID No:6. Specifically, the nucleotide sequence of SEQ ID No:4 (specifically: GGGAGCTGGTACACCTATAATACATTCGCACTAAAT) encodes the amino acid sequence shown in SEQ ID No:1; the nucleotide sequence of SEQ ID No:5 (specifically: TCGATTGGTCGGCAATCTAACCTTCTGGGCTGAC) encodes the amino acid sequence shown in SEQ ID No:2; and the nucleotide sequence of SEQ ID No:6 (specifically: TATTTTGTAAGGGTTTCGCCCTCGTTTAACTATCGAAAA) encodes the amino acid sequence shown in SEQ ID No:3.

[0012] The present invention also provides a recombinant expression vector containing the coding gene as described above.

[0013] The present invention also provides a recombinant host cell containing the coding gene as described above, or containing the recombinant expression vector as described above.

[0014] Furthermore, the preparation of the nanobody or its antigen-binding fragment of the present invention can be achieved by culturing the recombinant host cells described above, and then isolating and purifying the nanobody or its antigen-binding fragment from the culture.

[0015] The present invention also provides a reagent or kit for in vitro detection, diagnosis or monitoring of porcine circovirus type 2 infection, wherein the reagent or kit uses the nanobody or its antigen-binding fragment as described above as a capture reagent or detection reagent.

[0016] Specifically, the reagents or reagents in this invention, along with the nanobodies or antigen-binding fragments provided by this invention, specifically target and bind to PCV2 Rep to achieve specific recognition and detection. Furthermore, the specific detection method can employ conventional methods that are understandable and usable by those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography, or immunofluorescence. Further details will not be elaborated upon here.

[0017] The beneficial effects of this invention are as follows: The nanobody targeting the PCV2 Rep protein and its biodetection technology provided by this invention fundamentally solve the technical defects of traditional PCV2 antibody detection methods, such as large batch-to-batch antibody variability, high production costs, and insufficient detection sensitivity. This nanobody has a small molecular weight and stable structure, maintaining activity under room temperature transportation and long-term storage conditions, significantly reducing reagent cold chain costs by more than 30%. Its prokaryotic expression system ensures high consistency in antibody production. The special conformation of complementarity-determining region 3 (CDR3) enables it to recognize occult epitopes that traditional antibodies cannot reach, significantly improving detection specificity and showing no cross-reactivity with three common porcine viruses. The direct detection principle avoids the use of species-specific secondary antibodies, allowing the system to be directly applied to serum detection of different pig breeds without adjusting reagent formulations. The detection sensitivity reaches 1:20,000, and the quantitative range covers the titer range of common clinical antibodies. This technical solution demonstrates its high reliability and practicality, providing standardized, low-cost, and high-throughput technical support for the precise prevention and control of PCV2. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a predicted 3D structure diagram of the nanobody protein screened in Example 1; Figure 2 This is an SDS-PAGE analysis result of the expression of PCV2 REP Nb in Escherichia coli in Example 2; Figure 3 This is a graph showing the SDS-PAGE analysis results of the purified PCV2 REP Nb from Example 3; Figure 4 This is a graph showing the results of cross-reactivity detection of the nanobody PCV2 REP Nb with CSFV, PRRSV, and PEDV in Example 4. Figure 5 This is a graph showing the results of the PCV2 REP Nb specificity assay using the blocking ELISA method in Example 5. Detailed Implementation

[0019] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0020] In this invention, the screening and identification of the phage library is carried out by directly using a commercial alpaca nanobody library to obtain the target nanobody and its related gene information. The commercial alpaca nanobody library used was purchased from Pujian Biotechnology (Wuhan) Technology Co., Ltd.

[0021] The oligo-dT primers were commercially available products purchased from Genewiz Biotechnology Co., Ltd.; pCANTAB 5E was a commercially available product purchased from Shanghai Boson Biotechnology Co., Ltd.; E. coli competent cells were commercially available products purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the E. coli expression vector pET28a was a commercially available product purchased from Sangon Biotech Co., Ltd.; and the HRP-conjugated anti-E-Tag antibody was a commercially available product purchased from Abcam (catalog number ab193349).

[0022] CSFV, PRRSV, and PEDV strains are attenuated vaccines of the corresponding strains. Among them, the CSFV attenuated vaccine (swine fever live vaccine, strain C) was purchased from Wuhan Keqian Biotechnology Co., Ltd.; the PRRSV attenuated vaccine (swine reproductive and respiratory syndrome live vaccine) was purchased from Huawit (Jiangsu) Biopharmaceutical Co., Ltd.; and the PEDV attenuated vaccine (swine epidemic diarrhea virus attenuated vaccine) was purchased from Shanghai Haili Biotechnology Co., Ltd.

[0023] The nanobody targeting the PCV2 Rep protein provided in this invention is obtained through the following steps: Healthy adult alpacas are selected as the immunization host; recombinantly expressed and purified full-length PCV2 Rep protein is used as the immunogen; an alternating immunization regimen of Freund's complete adjuvant and Freund's incomplete adjuvant is employed for 6 weeks, with subcutaneous injections at multiple sites once a week, each immunization dose being 200 micrograms; peripheral venous blood is collected on day 7 after the last immunization, lymphocytes are isolated, total ribonucleic acid is extracted, and complementary deoxyribonucleic acid is synthesized by reverse transcription; the variable region gene fragment of the heavy chain antibody is amplified using nested polymerase chain reaction, with the upstream primer sequence containing the leader peptide coding region and the downstream primer sequence containing the initiation portion of the constant region; the amplified product is purified by agarose gel electrophoresis, ligated into a phage vector digested with restriction endonucleases, and transformed into competent Escherichia coli TG1 strain to construct... A primary phage display library was constructed. Recombinant Rep protein was used as the target antigen, coated onto the wells of an ELISA plate, and subjected to three rounds of biopanning. After each round, bound phages were recovered and amplified, with the Tween 20 concentration in the washing buffer gradually increased to enhance the screening pressure. After panning, single colonies were picked, plasmids were extracted and sequenced, and candidate clones with highly diverse complementarity-determining regions and conserved framework regions were screened. The nanobody genes of the candidate clones were subcloned into the prokaryotic expression vector pET-28a, transformed into *E. coli* BL21 competent cells, and induced to express by isopropyl thiogalactoside. The expression product was purified by nickel column affinity chromatography to obtain soluble nanobody proteins with a purity greater than 95%. This method can replace other camelid animals, immune adjuvants, or expression vectors to achieve similar high-affinity antibody acquisition and purification.

[0024] Furthermore, the obtained nanobody's complementarity-determining region (CDR) contains three hypervariable loop regions. CDR3, in particular, has a 13-residue amino acid sequence and its spatial conformation allows it to penetrate deep into the recessed neutralizing epitope region on the Rep protein surface, exhibiting high recognition ability, especially for conformational antigenic epitopes at the junction of the hypervariable regions of the Rep protein. This nanobody maintains complete binding activity within a pH range of 3.0 to 10.0, retains over 90% of its antigen-binding capacity after heating at 80°C for 30 minutes, and shows no loss of activity after incubation in 0.5% sodium dodecyl sulfate solution for 1 hour. Its equilibrium dissociation constant with the Rep protein (i.e., the PCV2Rep recombinant protein) is less than 10. -9 The molar per liter indicates that it has nanomolar affinity. This characteristic ensures that nanobodies function stably under various environmental conditions and can replace traditional antibodies or other single-domain antibodies for the detection of complex samples.

[0025] The nanobodies of this invention, after being labeled with horseradish peroxidase, are used to construct a competitive enzyme-linked immunosorbent assay (ELISA) system. The labeling method employs a modified sodium periodate oxidation method, specifically: the purified nanobodies are diluted to 1 mg / mL with sodium carbonate buffer, and a sodium periodate solution with a final concentration of 10 mmol / L is added. The reaction is carried out at 4°C in the dark for 30 minutes. After the reaction, the nanobodies are dialyzed overnight at 4°C with sodium acetate buffer to remove unreacted oxidants. An equal volume of horseradish peroxidase solution is added, and the reaction is carried out at room temperature in the dark for 2 hours. The reaction is terminated by adding sodium borohydride solution with a final concentration of 50 mmol / L, and the mixture is allowed to stand at 4°C for 2 hours. Finally, the nanobodies are purified by dialyzing with phosphate buffer to obtain the enzyme-labeled nanobodies working solution, which has a specific activity of 3.5 enzyme units bound per microgram of nanobodies and a labeling efficiency greater than 95%. This labeling process can be replaced by other enzymes (such as alkaline phosphatase), labels, or chemical reactions (such as click chemistry) to achieve similar signal amplification and detection functions.

[0026] The method for constructing the competitive enzyme-linked immunosorbent assay (ELISA) system further employed in this invention includes: diluting recombinant Rep protein to 5 μg / mL with carbonate buffer, adding 100 μL per well to a 96-well ELISA plate, and coating overnight at 4°C; discarding the coating solution, washing three times with phosphate buffer containing 0.05% Tween 20 for 3 minutes each time; adding 200 μL of phosphate buffer containing 5% skim milk powder to each well, and blocking at 37°C for 2 hours; discarding the blocking solution, drying, sealing, and storing at 4°C for later use; and adding 50 μL of phosphate buffer to each well for detection. Add 50 μL of enzyme-labeled nanobody working solution to the swine serum sample to be tested, and incubate at 37°C for 1 hour; discard the reaction solution and wash 5 times; add 100 μL of tetramethylbenzidine chromogenic substrate and incubate at room temperature in the dark for 15 minutes; stop the reaction by adding 50 μL of 2 mol / L sulfuric acid; measure the absorbance at 450 nm using an ELISA reader; calculate the relative content of anti-Rep antibody in the sample according to the standard curve, which is established by serial dilution of known concentrations of positive swine serum, ranging from 1:100 to 1:6400. This system can be extended to other solid-phase carriers (such as magnetic beads), chromogenic systems, or automated equipment to achieve high-throughput and portable detection.

[0027] In this invention, the cross-reactivity of common prevalent porcine viruses was verified through a cross-inhibition experiment. Specifically, three common porcine infectious viruses (PRRSV, CSFV, and PEDV) were selected, and their Rep proteins were prepared and coated onto ELISA plates. The same positive porcine serum was used for detection. The results showed that the difference in the inhibition rate of the detection signal among the different strains was less than 10%, indicating that the system has a broad-spectrum recognition ability for the currently prevalent viruses. Simultaneously, the system showed no cross-reactivity with positive sera from classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine reproductive and respiratory syndrome virus, achieving 100% specificity. This verification ensures the robustness of the system in a mutant environment and can be extended to the evaluation of more strains or related viruses.

[0028] The entire construction process of the present invention will be further described in detail below through specific embodiments. Preparation examples, screening and identification of phage libraries

[0029] The PCV2 Rep recombinant protein was prepared according to the method disclosed in "Porcine circovirus type 2 (PCV2) Cap and Rep proteins are involved in the formation of inclusion bodies in cultured porcine epithelial cells" (Fort M et al., Vet Microbiol, 2010). Specifically: (1) the PCV2 Rep gene was cloned into the pET-28a vector and transformed into BL21(DE3) strain; (2) the culture was carried out at 37°C until OD200. 600 =0.6, add 1mM IPTG to induce for 4h; (3) Sonicate the bacterial cells and centrifuge to collect the supernatant; (4) Purify by Ni-NTA affinity chromatography (binding buffer: 20mM Tris-HCl pH8.0, 500mM NaCl; elution buffer: same as above plus 250mM imidazole).

[0030] The purified PCV2 Rep recombinant protein was selected as the immunogen. This recombinant protein was expressed in the E. coli expression system by genetic engineering technology and purified by affinity chromatography. The purity was greater than 95%, the molecular weight was about 23.8 kDa, and the complete spatial conformation epitopes were retained.

[0031] The purified immunogen (i.e., PCV2 Rep recombinant protein) was used to screen a commercial alpaca nanobody library to obtain nanobody clones that can specifically recognize and bind to the target antigen.

[0032] Next, based on the nanobody clones obtained above, a set of specific primers covering the full length of the variable region (VHH) of the alpaca heavy chain antibody were designed and synthesized (the standard alpaca VHH primer sequences, refer to "Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs," Conrath KE et al., J Biol Chem, 2001, whose upstream primer sequence contains the leader peptide coding region and the downstream primer sequence contains the constant region start portion; specifically, the upstream primer CALL001 is as shown in SEQ ID No:9: 5'-GTCCTGGCTGCTCTTCTACAAGG-3'; the downstream primer CALL002 is as shown in SEQ ID No:10: 5'-GGTACGTGCTGTAAACTTTCATCACTG-3'). A VHH gene fragment of approximately 400-450 bp in length was obtained by two rounds of nested PCR amplification. After two rounds of PCR, the amplified products were verified and purified by agarose gel electrophoresis, and then ligated into the linearized phage display vector pCANTAB 5E by restriction enzyme digestion (using Not I and Pst I). The resulting solution was transformed into competent *E. coli* TG1 cells to construct a primary phage display library with a capacity greater than 5 × 10⁻⁶ cells. 6 Independent cloning. Subsequently, using recombinant PCV2 Rep protein (100 ng / well) coated on the bottom of 96-well microplates as a solid-phase target, three rounds of biopanning were performed. Each round of biopanning included: transferring the phage library (approximately 10... 12 PFU and the coating antigen (i.e., PCV2 Rep recombinant protein) were incubated at 37°C for 1 hour. Unbound or non-specifically bound phages were washed away. Specifically bound phages were eluted with glycine-HCl solution (pH 2.2). After neutralization of the elution buffer, logarithmically growing E. coli TG1 was infected, and the phages were amplified and recovered for the next round of panning. Specifically, after each round of panning, the specific bound phages were recovered and amplified, and the Tween 20 concentration in the wash buffer was gradually increased to enhance the screening pressure.

[0033] After three rounds of enrichment, 96 positive clones were randomly selected for indirect ELISA screening. Binding activity was detected using an anti-E-Tag antibody conjugated with VHH and HRP, containing an E-Tag (sequence shown in SEQ ID No:11: GAPVPYPDPLEPR, inherent to the expression vector). Clones with the highest P / N ratio (positive / negative absorbance ratio) were selected for sequence determination and alignment. After removing duplicate sequences, 19 candidate nanobodies with unique CDR3 sequences were obtained. These nanobodies specifically bound to PCV2 Rep protein without binding to the control protein. The one with the highest affinity was designated Nb15 (KD<10). -9 M). In this preparation example, the optimized design of the library screening involved gradually increasing the washing intensity of the panning rounds (from 5 to 15 times) and decreasing the antigen coating concentration (from 200 ng / well to 50 ng / well) to improve the enrichment efficiency of high-affinity clones. The workflow of this preparation example, from immunogen preparation to phage panning and clone screening, forms a closed loop, ensuring the diversity and specificity of the nanobodies. Example 1: Sequence Analysis of PCV2 REP Nb (i.e., nanobody targeting porcine circovirus type 2 Rep protein)

[0034] For the 19 candidate nanobodies obtained in the preparation example, affinity (KD value) and specificity (no binding to control protein) were determined by indirect ELISA. Three candidate nanobodies with a P / N value > 10 were selected for sequence determination and alignment, grouped based on the CDR3 region. The nanobodies with the best reactivity were obtained after screening, and their amino acid sequences are shown in SEQ ID No:7, which is the PCV2 REP Nb used subsequently. [Specifically, its amino acid sequence is: QVQLQESGGDLVQPGGSLRLSCAAGSWYTYNTFALNWVRQAPGKELERVSSMVGNLTFWADRFTISRDNAKNTLYLQLNSLKTEDTAMYYCAKYFVRVSPSFNYRKQRGQGTQVTVSS (CDR3: YFVRVSPSFNYRK)] The screened nanobodies were compared using Clustal Omega software to identify unique sequences. Figure 1 The image shows the predicted 3D structure of the selected nanobody protein. Example 2: Expression and Identification of PCV2 REP Nb

[0035] The coding gene of the nanobody shown in SEQ ID No:7 was codon optimized (the optimized coding gene is shown in SEQ ID No:7). As shown in No:8, its specific sequence is: CAGGTCCAGCTGCAAGAGTCTGGCGGCGATCTTGTGCAGCCGGGGGGTTCACTGCGCCTGTCCTGTGCCGCCGGTTCATGGTATACGTATAACACGTTCGCGCTGAACTGGGTCCGGCAGGCACCGGGAAAAGAATTGGAGAGGGTATCTAGCATGGTGGGAAACCTGACGTTCTGGGCCGACCGCTTTACAATCTCCCGTGATAACGCCAAGAACACCCTGTACCTCCAGCTGAACTCGTTAAAAACTGAAGATACGGCGATGTACTATTGTGCGAAATACTTCGTTCGGGTGTCGCCGAGCTTTAATTACCGTAAACAGCGTGGTCAGGGTACCCAGGTTACGGTCTCATCT), then subcloned into the E. coli expression vector pET28a [The cloning process specifically includes: (1) using SEQ ID The upstream primer shown in No:12, 5'-CGGAATTCCAGGTGCAGCTGCAGGAG-3' (EcoRI site), and the downstream primer shown in SEQ ID No:13, 5'-CCGCTCGAGTTAGCTGACCTGGTACC-3' (XhoI site), were used to amplify the coding gene of the nanobody by PCR; (2) the amplified fragment was digested with two enzymes (EcoRI / XhoI) and the pET28a vector was added; (3) the fragment was ligated with T4 ligase and transformed into DH5α to screen for positive clones; (4) after sequencing verification, the fragment was transformed into BL21(DE3). That is, the vector inserts the HIS and Avi-Tag coding sequences downstream of the VHH sequence using conventional methods in the art, which facilitates the subsequent expression of the fusion protein, and the recombinant plasmid pET28a-Nb15 is constructed. After transforming BL21(DE3) competent cells with the constructed recombinant plasmid pET28a-Nb15, positive clones were screened to obtain BL21(DE3) expression bacteria containing pET28a-Nb15 plasmid. The BL21(DE3) expression bacteria containing pET28a-Nb15 plasmid were inoculated at 1:50 into 5 mL LB (containing 1:1000 diluted kanamycin resistance) and cultured at 37℃ with shaking for 7 h.The BL21(DE3) expression bacteria containing the pET28a-Nb15 plasmid, cultured as described above, were then inoculated at a ratio of 1:50 into 4 mL LB tubes and 400 mL LB Erlenmeyer flasks (both 4 mL and 400 mL LB flasks contained a 1:1000 dilution of kanamycin resistance). The 4 mL LB tubes were cultured at 37°C with shaking for 7 h as an uninduced control. The 400 mL LB Erlenmeyer flasks were cultured at 37°C with shaking for 3 h, then IPTG (final concentration 1 mmol / L) was added to induce expression, and the culture was continued at 37°C with shaking for another 4 h. After the induction, 500 μL of fresh bacterial culture was taken from each of the control group (inoculated in 4 mL LB flasks) and placed into new 2.0 mL EP tubes. The tubes were centrifuged at 5000 rpm for 60 s. The supernatant was discarded, and the precipitate was resuspended in 40 μL PBS, mixed thoroughly by pipetting, and 10 μL of 5× protein loading solution was added. Buffer; incubate at 100℃ for 10 min, centrifuge at 5000 r / min for 5 min, and finally take 10 μL of supernatant for SDS-PAGE detection.

[0036] After confirming protein expression by the strain, the remaining IPTG-induced bacterial culture was centrifuged, washed twice with PBS, and finally resuspended in 20 mL of PBS. The culture was then sonicated on ice (200 W, 10 s sonication, 5 s pause, 60 min sonication, shaking once every 20 min). After sonication, the culture was centrifuged at 13000 rpm for 20 min, and the supernatant and precipitate were analyzed by SDS-PAGE to determine the protein expression pattern. The SDS-PAGE analysis results of the expression are shown in the figure below. Figure 2 As shown, band 1 is the uninduced supernatant, band 2 is the uninduced precipitate, band 3 is the induced supernatant, and band 4 is the induced precipitate. Example 3: Purification and Identification of PCV2 REP Nb

[0037] Following the identification in Example 2, after confirming that PCV2 REP Nb existed in a soluble form, the supernatant was collected by centrifugation and filtered through a 0.45 μm filter. To ensure complete purification and eliminate interference from E. coli, the recombinant expressed protein (i.e., PCV2 REP Nb) was purified by affinity chromatography. Because the recombinant protein PCV2 REP Nb has a polyhistidine (6×His) tag, Ni... 2+ The recombinant protein PCV2 REP Nb was then purified by affinity chromatography. Ni 2+ The column was first washed with 10 times its volume of ultrapure water, then equilibrated with the same volume of binding buffer. The treated PCV2 REP Nb was then added to the equilibrated Ni. 2+In the column, the protein was treated with Washing Buffer and then eluted with Elution Buffer. SDS-PAGE analysis was used to confirm complete purification. Finally, the solution containing PCV2 REP Nb was collected and stored at -20°C. The SDS-PAGE analysis results of the purified PCV2 REP Nb are shown in the figure below. Figure 3 As shown, the left band is the molecular weight marker, and the right band is the purified PCV2 REP Nb. Example 4: PCV2 REP Nb Activity Analysis

[0038] The biological activity of PCV2 REP Nb was identified using an indirect ELISA method. The indirect ELISA method specifically includes: (1) Antigen coating: PCV2 REP recombinant protein was coated onto an enzyme-labeled plate at 100 ng / well and incubated overnight at 4°C.

[0039] (2) Blocking the microplate: Discard the coating solution, wash the plate 4 times with PBST, add 200 μL / well of 1% BSA, and block in a 37℃ incubator for 1 h.

[0040] (3) Sample addition. Discard the blocking solution, wash the plate 4 times with PBST, and dilute the horseradish peroxidase-labeled PCV2 REP Nb at 1:20, 1:200, 1:2000 and 1:20000 respectively. Add 100 μL / well to the microplate and incubate at 37°C for 2 h.

[0041] (4) Incubate enzyme-labeled antibody: Discard PCV2 REP Nb and wash the plate 4 times with PBST.

[0042] (5) Color development: Discard the enzyme-labeled secondary antibody, wash 4 times with PBST, add 100L / well of freshly prepared TMB chromogenic substrate, and incubate in a 37℃ incubator in the dark for 15min.

[0043] (6) Termination: Add 3 mol / L H2SO4 to 50 μL / well to terminate the colorimetric reaction and measure OD450.

[0044] Meanwhile, CSFV, PRRSV, and PEDV were used as comparative examples and were detected using the above method.

[0045] Test results as follows Figure 4 As shown. Example 5: Blocking ELISA-specific detection

[0046] To verify the specificity of PCV2 REP Nb, 30 PCV2-negative porcine sera confirmed by commercial ELISA were used for testing, and the samples were uniformly diluted 200-fold. The 30 PCV2-negative sera were subjected to bELISA under the optimal conditions determined above (i.e., the specific operation method described in the construction method of the competitive enzyme-linked immunosorbent assay system given above), and the blocking rates of the 30 negative sera (denoted as PI values) were calculated. The critical value of bELISA was determined according to the blocking rate: Critical value A = average PI value of negative sera + 2 x standard deviation (s); Critical value B = average PI value of negative sera + 3 x standard deviation (s). The critical values A and B were calculated. If the sample blocking rate PI ≥ critical value B, the sample was determined to be PCV antibody positive; if critical value A < PI < critical value B, it was determined to be PCV antibody suspicious, and the sample needed to be retested; if the retest result was still suspicious, the sample was determined to be PCV antibody negative; if P ≤ critical value A, the sample was determined to be PCV antibody negative. At the same time, the cross-reactivity of positive sera with other viruses (such as PRRSV, CSFV, PEDV) was tested. The results showed that the PI values of PRRSV, CSFV, and PEDV serum samples were between 3.56% and 18.48% (lower than the threshold), and the specificity was 100%, with no positive results. The results are as Figure 5 shown.

[0047] In summary, the high-affinity nanobody molecule targeting the Rep protein of porcine circovirus type 2 (PCV2) constructed in the present invention, combined with the competitive enzyme-linked immunosorbent assay system, can effectively achieve high-sensitivity, high-specificity, and high-stability quantitative detection of anti-PCV2 antibodies in porcine sera. This technical solution breaks through multiple limitations of traditional antibody detection methods in terms of stability, production consistency, detection throughput, and cross-reactivity, and constructs a standardized diagnostic platform suitable for grass-roots breeding institutions, large-scale epidemiological screening, and vaccine immunization effect evaluation.

[0048] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0049] In addition, it should be noted that, in the various specific technical features described in the above specific embodiments, they can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0050] Furthermore, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A nanobody or antigen-binding fragment thereof targeting the Rep protein of porcine circovirus type 2, characterized in that, The nanobody or its antigen-binding fragment includes at least one complementary determinant cluster selected from CDR1, CDR2, and CDR3; wherein... CDR1 is an amino acid sequence as shown in SEQ ID No:1, or a mutant sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No:1 and can specifically bind to the porcine circovirus type 2 Rep protein; CDR2 is an amino acid sequence as shown in SEQ ID No:2, or a mutant sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No:2 and can specifically bind to the porcine circovirus type 2 Rep protein; CDR3 is an amino acid sequence as shown in SEQ ID No:3, or a mutant sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID No:3 and is capable of specifically binding to the porcine circovirus type 2 Rep protein.

2. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The nanobody or its antigen-binding fragment includes CDR1, CDR2 and CDR3.

3. The nanobody or its antigen-binding fragment according to claim 2, characterized in that, CDR1 is the amino acid sequence shown in SEQ ID No:1, CDR2 is the amino acid sequence shown in SEQ ID No:2, and CDR3 is the amino acid sequence shown in SEQ ID No:

3.

4. A coding gene for encoding a nanobody or an antigen-binding fragment thereof as described in any one of claims 1-3.

5. The encoding gene according to claim 4, characterized in that, The encoding gene includes nucleotide sequences as shown in SEQ ID No:4, SEQ ID No:5 and SEQ ID No:

6.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the coding gene as described in claim 4 or 5.

7. A recombinant host cell, characterized in that, The recombinant host cell contains the coding gene as described in claim 4 or 5, or contains the recombinant expression vector as described in claim 6.

8. A reagent or kit for in vitro detection, diagnosis, or monitoring of porcine circovirus type 2 infection, characterized in that, The reagents or kits use nanobodies or their antigen-binding fragments as described in any one of claims 1-3 as capture reagents or detection reagents.

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