Synthetic peptide antigen of cap protein of porcine circovirus type 2, polyclonal antibody and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly discloses a synthetic peptide antigen of porcine circovirus type 2 Cap protein, a polyclonal antibody and application thereof. In order to solve the technical defects of low specificity and inaccurate detection of the amino acid sequence of the synthetic peptide antigen in the prior art, the application provides a synthetic peptide antigen of PCV2 Cap protein, a polyclonal antibody and application thereof. The application obtains an amino acid sequence of an antigen peptide by performing antigen peptide prediction on PCV2 Cap protein, and the applicant synthesizes the antigen peptide by using an Fmoc solid phase method, and immunizes a New Zealand white rabbit with the obtained antigen peptide to prepare a polyclonal antibody of Cap protein, the antibody can effectively react with a transiently expressed Cap protein and a Cap protein in PCV2b-infected PK15 cells, and provides a powerful tool for detection and biological function research of the PCV2 Cap protein.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a synthetic peptide antigen of PCV2 Cap protein, a polyclonal antibody, and their applications. Background Technology

[0002] Porcine circovirus (PCV) belongs to the family Circoviridae (Polyoviridae). Circoviridae ) Circovirus genus ( Circovirus Porcine circovirus (PCV) is a non-enveloped virus and the smallest known single-stranded circular DNA virus. Four genotypes have been identified: PCV1, PCV2, PCV3, and PCV4. PCV2 is globally recognized as a porcine pathogen with significant economic impact. PCV2 infection can lead to immunosuppression in pigs, resulting in secondary infections, decreased vaccine response, and other problems. Acute infection mortality in piglets can reach 10%. PCV2 affects pigs of all ages and can cause a range of porcine circovirus-associated diseases (PCVADs), including weaned piglet multisystemic wasting syndrome, porcine dermatitis-nephropathy syndrome, porcine respiratory disease syndrome, and reproductive disorders in sows. PCVAD has become one of the most important diseases in the global pig industry, causing severe economic losses to pig production in various countries.

[0003] Currently, eight genotypes of PCV2 have been reported, ranging from PCV2a to PCV2h, with PCV2a, PCV2b, and PCV2d being the predominant genotypes in my country. The PCV2 genome is only about 1.7 kb in size and contains 11 open reading frames (ORFs), including ORF1, ORF2, and ORF3. ORF2, located on the negative strand, is relatively conserved and encodes the approximately 27.8 kDa capsid protein, Cap. Cap protein is the only structural protein of PCV2, participating in viral particle assembly and serving as a major immunogenic protein with strong immunogenicity. Furthermore, Cap protein enhances PCV2's replication adaptability within cells, playing a crucial role in viral transmission within the host population and being a key factor determining PCV2 pathogenicity and virulence. Therefore, Cap protein is frequently used as a key target protein in research on PCV2 pathogenic mechanisms and vaccine development.

[0004] However, current experimental tools available for PCV2 Cap protein research remain relatively scarce. Therefore, developing rapid and efficient technical tools for detecting Cap protein is of significant theoretical and practical value for a deeper understanding of the PCV2 pathogenesis mechanism and for advancing related vaccine development.

[0005] Chinese invention patent 200810118570.5 discloses a synthetic peptide antigen of porcine circovirus type 2, an ELISA kit and an immunomicrosphere detection reagent developed using the synthetic peptide antigen, and uses it to specifically detect porcine circovirus type 2 antibodies. However, this ELISA kit is susceptible to contamination by impurities and interference from non-specific proteins, and the selected amino acid sequence has low specificity, resulting in a high risk of false positives when detecting porcine circovirus type 2 antibodies.

[0006] The Chinese literature "Identification of B-cell epitopes of porcine circovirus type 2 Cap protein and preparation of synthetic peptide polyclonal antibodies" screens dominant B-cell epitopes of Cap protein (such as 65–87 aa, 165–180 aa), synthesizes peptides conjugated with KLH to immunize rabbits, which can be used to prepare highly specific polyclonal antibodies for Western blot detection and IFA.

[0007] Based on this, the present invention is proposed. Summary of the Invention

[0008] To address the shortcomings of existing technologies in synthesizing peptide antigens, such as low specificity of amino acid sequences and inaccurate detection, this invention provides a synthetic peptide antigen of PCV2 Cap protein, a polyclonal antibody, and their applications. This invention obtains the amino acid sequence of an antigenic peptide through antigenic peptide prediction of the PCV2 Cap protein. The applicant synthesizes the antigenic peptide using the Fmoc solid-phase method and immunizes New Zealand white rabbits with the obtained antigenic peptide to prepare a polyclonal antibody for the Cap protein. This antibody can effectively react with transiently expressed Cap protein and Cap protein in PK15 cells infected with PCV2b, providing a powerful tool for the detection and biological function research of PCV2 Cap protein.

[0009] In a first aspect, the present invention provides a synthetic peptide antigen of PCV2 Cap protein, comprising a carrier-coupled protein and a synthetic peptide, wherein the amino acid sequence of the synthetic peptide is SEQ ID NO. 1. The carrier-coupled protein is preferably a KLH carrier protein.

[0010] Secondly, the present invention provides a polyclonal antibody against PCV2 Cap protein prepared using the above-mentioned synthetic peptide antigen. This antibody is prepared by immunizing animals with the synthetic peptide antigen formed by C-terminal modification of the PCV2 Cap synthetic peptide shown in SEQ ID NO. 1 and coupling it with a KLH carrier protein. The preferred animal is the New Zealand white rabbit.

[0011] Thirdly, the present invention provides a method for preparing the above-mentioned polyclonal antibody against PCV2 Cap protein, which includes the following steps: (a) Preparation of synthetic peptide antigen: The antigen peptide shown in SEQ ID NO. 1 was obtained by artificial synthesis and purified by coupling with KLH carrier protein to obtain synthetic peptide antigen; (b) Immunization of New Zealand white rabbits: The synthetic antigenic peptide (500 μg / rabbit) was emulsified with Freund's adjuvant at a ratio of 1:1 and then injected subcutaneously to immunize New Zealand white rabbits, and serum containing polyclonal antibodies was obtained.

[0012] Preferably, the immunization of New Zealand white rabbits is performed via subcutaneous or intramuscular injection.

[0013] The preparation method described above also includes polyclonal antibody purification and titer detection. Polyclonal antibody purification includes purifying serum using Protein G affinity chromatography to obtain polyclonal antibodies. The titer detection step includes detecting the titer of the anti-PCV2 Cap polyclonal antibody using an indirect ELISA method.

[0014] Fourthly, the present invention provides the application of the aforementioned Cap protein polyclonal antibody, specifically the application of the anti-Cap protein polyclonal antibody in the transient expression of Cap protein and the detection of PCV2. One application is the use of the aforementioned anti-PCV2 Cap protein polyclonal antibody in the detection of the transient expression level of PCV2d Cap protein in cells using Western blotting.

[0015] Preferably, the cell is HEK-293T cell, and the PCV2d Cap protein is one or more of HA-PCV2a Cap, HA-PCV2b Cap, and HA-PCV2d Cap protein.

[0016] The present invention also provides another application of the above-mentioned anti-PCV2 Cap protein polyclonal antibody, namely, the application of the above-mentioned anti-PCV2 Cap protein polyclonal antibody in the detection of PCV2b infection. Preferably, the PCV2 detection is to detect the Cap protein in cells infected with PCV2b by PK15.

[0017] This invention predicts the antigenic peptide of PCV2 Cap protein and prepares a synthetic peptide polyclonal antibody. This antibody can be effectively used for the detection of the dominant PCV2 genotype Cap protein, providing a powerful tool for future PCV2 research. Compared with existing technologies, this invention has the following advantages: (1) The PCV2 Cap protein polyclonal antibody of the present invention can be used to detect Cap protein of different dominant gene subtypes. The high sensitivity and specificity of the antibody were demonstrated by ELISA and Western blot detection, providing an effective tool for the study of the pathogenesis mechanism of PCV2 and vaccine prevention and control.

[0018] (2) The antibody preparation method described in this invention can be used to prepare antibodies of various synthetic peptides and has a wide range of applications. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 : HA and polyclonal antibody detection of transiently expressed Cap protein. a: HA antibody detection; b: 218-232aa synthetic peptide polyclonal antibody detection; Figure 2 Western blot analysis of Cap protein expression in PCV2-infected PK15 cells using a synthetic peptide polyclonal antibody (218-232aa). Detailed Implementation

[0020] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0021] Unless otherwise stated, the culture media, reagents and solutions used in the following examples are commercially available products or can be prepared by methods known in the art.

[0022] Example 1: Preparation of PCV2 Cap protein synthetic peptide antigen and polyclonal antibody A relatively conserved antigenic peptide (218aa-232aa: YVQFREFNLKDPPLN) from the PCV2 Cap protein sequence was selected as the antigenic peptide for preparing polyclonal antibodies. This peptide was conjugated to a KLH carrier protein and purified. The synthesized peptide was then diluted with physiological saline and emulsified with Freund's adjuvant at a 1:1 ratio before immunization in New Zealand white rabbits. A negative control was obtained by intravenous blood collection before the initial immunization. Immunization was performed via subcutaneous or intramuscular injection at a dose of 500 µg per rabbit. Booster immunizations were administered every 14 days. On day 7 after six immunizations, polyclonal antibody serum was collected via intravenous or cardiac blood collection. The polyclonal antibodies were purified using Protein G affinity chromatography and stored in PBS buffer containing 20% ​​glycerol.

[0023] Example 2: Determination of antibody titer using indirect ELISA. The synthetic peptide and KLH-coupled protein were diluted with PBS to 1.25 µg / mL, and coated at 100 µL / well overnight at 4°C. The next day, the liquid in the ELISA plate was discarded, and the plate was blocked with 2% BSA at room temperature (25°C) for 2 h. Anti-Cap protein polyclonal antibody was diluted to different concentrations: 1:6250, 1:12500, 1:25000, 1:50000, 1:100000, and 1:200000. Rabbit serum taken before the first immunization was diluted 1:1000 as a negative control, and incubated at 37°C for 1 h. After washing three times with PBS, horseradish peroxidase-labeled goat anti-rabbit secondary antibody was added and incubated at 37°C for 1 h. TMB was added for color development, and the absorbance was measured at 450 nm after the color development was terminated. The results of the Cap polyclonal antibody titer test are shown in Table 1: After dilution by 200,000 times, the ELISA titers of the polyclonal antibodies obtained by immunizing the Cap synthetic peptide in three stages were still positive. The ELISA titers of the Cap polyclonal antibodies were all greater than 1:200,000, which can ensure that the polyclonal antibodies can be used for the detection of Cap protein. Note: OD 450 >0.2, indicating a positive antibody result; OD 450 <0.2, is considered negative.

[0024] Example 3 Specificity analysis of polyclonal antibodies To verify whether the prepared Cap protein synthetic peptide polyclonal antibody could be used for subsequent experiments, the overexpression of HA-PCV2a Cap, HA-PCV2b Cap, and HA-PCV2d Cap plasmids in HEK-293T cells was detected using the obtained Cap protein polyclonal antibody. HEK-293T cells were passaged into 6-well plates. When the cells reached 70%-80% confluency, the recombinant plasmids HA-PCV2a Cap, HA-PCV2b Cap, and HA-PCV2d Cap were transfected into HEK-293T cells using PEI transfection reagent. After 24 h of Cap protein overexpression, protein samples were collected and detected using mouse anti-HA antibody and the prepared rabbit anti-Cap polyclonal antibody as primary antibodies. Figure 1 As shown, both the HA antibody and the prepared rabbit-derived anti-Cap polyclonal antibody exhibited an overexpressed Cap protein band of approximately 27.8 kDa, consistent with the expected size. These results indicate that the anti-Cap polyclonal antibody prepared by immunizing the 218-232aa synthetic peptide can specifically recognize eukaryotically expressed Cap protein.

[0025] Example 4: Application of polyclonal antibodies in detecting Cap protein in PCV2-infected PK15 cells Healthy PK-15 cells were seeded into 6-well plates. When the cell density reached approximately 80%–90%, the cell culture medium was discarded, and PCV2 virus (MOI=1) was diluted with serum-free DMEM medium. The diluted virus solution was added to the cell culture plate, and the plate was incubated at 37°C for 2 hours for adsorption. The virus was then discarded, and DMEM medium containing 2% FBS was added. After culturing for 24 hours, cells were harvested and protein samples were prepared. Uninfected PK-15 cells were collected as a negative control. After SDS-PAGE electrophoresis, the proteins were transferred to NC membranes using a wet transfer method. The membranes were then incubated with a 1:1000 dilution of 218-232aa Cap polyclonal antibody. Results are as follows: Figure 2 The results showed that both polyclonal antibodies could detect the Cap protein in PCV2-infected PK15 cells.

[0026] In summary, this invention successfully prepared two polyclonal antibodies for detecting PCV2 Cap protein. These polyclonal antibodies exhibit high sensitivity and specificity, and can be used to detect different genotypes of PCV2 Cap protein through Western blot experiments. They provide important experimental materials for the study of the pathogenesis mechanism of PCV2 and vaccine development, and have broad application prospects.

[0027] This document uses specific embodiments to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the technical solutions and core ideas of this application. Those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A synthetic peptide antigen of PCV2 Cap protein, characterized in that, It includes a carrier-coupled protein and a synthetic peptide, wherein the amino acid sequence of the synthetic peptide is shown in SEQ ID NO.

1.

2. The synthetic peptide antigen of PCV2 Cap protein according to claim 1, characterized in that, The carrier-coupled protein is a KLH carrier protein.

3. A polyclonal antibody against PCV2 Cap protein prepared using the synthetic peptide antigen according to claim 1 or 2, characterized in that, The synthetic peptide antigen, formed by C-terminal modification of the PCV2 Cap synthetic peptide shown in SEQ ID NO. 1 and coupling it with KLH carrier protein, was used as an immunogen to immunize animals.

4. The polyclonal antibody against PCV2 Cap protein according to claim 3, characterized in that, The animal in question is a New Zealand white rabbit.

5. A method for preparing a polyclonal antibody against PCV2 Cap protein as described in claim 3, characterized in that, Includes the following steps: (a) Preparation of synthetic peptide antigen: The amino acid sequence shown in SEQ ID NO. 1 was obtained by artificial synthesis method, and the synthetic peptide antigen was obtained by coupling and purification using KLH carrier protein; (b) Immunization of New Zealand white rabbits: 500 μg / rabbit of synthetic peptide was mixed with Freund's adjuvant at a ratio of 1:1 and emulsified before being injected into New Zealand white rabbits at least six times, with an interval of 14 days between immunizations, to obtain serum containing polyclonal antibodies.

6. The method for preparing the polyclonal antibody against PCV2 Cap protein according to claim 5, characterized in that, The immunization of New Zealand white rabbits is administered via subcutaneous or intramuscular injection.

7. The method for preparing the polyclonal antibody against PCV2 Cap protein according to claim 5, characterized in that, The preparation method further includes polyclonal antibody purification and titer detection. Polyclonal antibody purification includes purifying serum using Protein G affinity chromatography to obtain polyclonal antibodies. The titer detection step includes detecting the titer of the anti-PCV2 Cap polyclonal antibody using an indirect ELISA method.

8. The use of the anti-PCV2 Cap protein polyclonal antibody according to claim 1 or 2 in detecting the transient expression level of PCV2 d Cap protein in cells using Western Blot.

9. The application of the anti-PCV2 Cap protein polyclonal antibody according to claim 8, characterized in that, The cells are HEK-293T cells, and the PCV2d Cap protein is one or more of HA-PCV2a Cap, HA-PCV2b Cap, and HA-PCV2d Cap proteins.

10. The use of the anti-PCV2 Cap protein polyclonal antibody according to claim 1 or 2 in the detection of PCV2b infection.

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