Artificial porcine circovirus, preparation method and application thereof

Abstract

The invention relates to an artificial porcine circovirus, a preparation method and an application thereof. An Avi sequence as shown in SEQ ID NO.2 is inserted into a porcine circovirus full genome sequence, and a specific insertion position is the C-terminus of a PCV Cap protein. The porcine circovirus is one of PCV2, PCV3 and PCV4. An Avi tag is added to the C-terminus of the PCV Cap protein, and quantum dots are labeled on the PCV modified with the Avi tag by using a biotin-avidin system. The obtained labeled PCV can be used for vaccine development, establishment of a virus infection detection method, and research on virus infection and pathogenic mechanism, and further tracer analysis of PCV infection in cells is performed.

Description

Technical Field

[0001] The present invention belongs to the field of virus molecular biology technology and bioengineering, and specifically relates to an artificial porcine circovirus (PCV) and a preparation method. The artificially prepared labeled PCV can be used for the study of porcine circovirus infection and pathogenic mechanism. Background Art

[0002] Porcine circovirus (PCV) is a single-stranded DNA virus without an envelope structure. So far, four subtypes of porcine circovirus have been discovered, namely PCV1, PCV2, PCV3 and PCV4. The genome size is about 1700-2000nt, and it is a single-stranded circular DNA molecule structure. The circular genome mainly contains two open reading frames (ORF) in opposite directions. ORF1 encodes the replication-related Rep protein, and ORF2 encodes the Cap protein. The Cap protein is the main antigenic protein of the virus, which contains the main antigenic determinant cluster of the virus. It has stronger immunogenicity than the Rep protein and plays an important role in the process of virus transmission and infection.

[0003] PCV1 is generally considered non-pathogenic, while PCV2 is the main source of porcine circovirus disease characterized by multisystemic wasting syndrome in weaned piglets, porcine respiratory disease complex, dermatitis and nephropathy syndrome, etc., and can cause immunosuppression in infected pigs. A large number of literature reports that PCV3 and PCV4 have certain pathogenicity and may also be related to respiratory diseases, diarrhea, dermatitis and nephropathy syndrome in pigs. Their infection rates in pig herds are gradually increasing, and they have become porcine circovirus subtypes worthy of attention. It is worth noting that the infection and pathogenic mechanisms of porcine circovirus are still not very clear.

[0004] In order to further observe the entire life cycle of the virus after infection and understand the virus infection and pathogenic mechanism, dynamic tracing analysis of the virus is a common method. However, traditional fluorescent labeling methods, such as fluorescent dyes and fluorescent protein labeling, have problems such as low fluorescence intensity, easy quenching, and difficulty in long-term tracking. Quantum dots are fluorescent nanomaterials with significant photostability and brightness, and are widely used as fluorescent labeling probes. The excellent luminescence properties of quantum dots make them very suitable for labeling small-sized viruses, thereby achieving long-term single-particle tracing. Avi-Tag refers to adding a label consisting of 15 amino acid residues to the N-terminus or C-terminus of the target protein. The label can be specifically recognized by the biotin ligase BirA and covalently binds biotin to the lysine residue of the target protein Avi-Tag label, thereby achieving biotinylation of the target protein. Subsequently, the quantum dot-labeled streptavidin binds to the biotinylated virus, and the quantum dots can be labeled on the virus for virus tracing detection and functional research.

[0005] At present, there is no artificially prepared porcine circovirus with Avi-Tag used in vaccine development, establishment of virus infection detection methods, and research on virus infection and pathogenic mechanisms. Summary of the invention

[0006] The present invention provides an artificial porcine circovirus. An Avi tag is added to the C-terminus of the PCV Cap protein, and quantum dots are labeled on the Avi-tag modified PCV (PCV-Avi) by using a biotin-avidin system. The obtained labeled PCV can be used for vaccine development, establishment of a virus infection detection method, research on virus infection and pathogenic mechanism, and further trace analysis of PCV infection in cells.

[0007] The present invention provides an artificial DNA fragment, which is an Avi sequence as shown in SEQ ID NO.2 inserted into the whole genome sequence of porcine circovirus, and the specific insertion position is the C-terminus of PCV Cap protein; the porcine circovirus is one of PCV2, PCV3 and PCV4.

[0008] When the porcine circovirus described in the present invention is PCV2, the above-mentioned DNA fragment is as shown in SEQ ID NO.5, which is obtained by inserting the Avi sequence ggcctgaacg acatcttcga ggcccagaag atcgagtggcacgag shown in SEQ ID NO.2 between the 1028th and 1029th bases of the PCV2 full genome sequence (GenBank: JQ955679.1) shown in SEQ ID NO.4.

[0009] When the porcine circovirus described in the present invention is PCV3, the above-mentioned DNA fragment is as shown in SEQ ID NO.3, which is obtained by inserting the Avi sequence shown in SEQ ID NO.2 between the 1345th and 1346th bases of the PCV3 full genome sequence (GenBank: MF318451.1) shown in SEQ ID NO.1.

[0010] When the porcine circovirus described in the present invention is PCV4, the above DNA fragment is as shown in SEQ ID NO.7, which is obtained by inserting the Avi sequence shown in SEQ ID NO.2 between bases 1049-1050 of the PCV4 full genome sequence (GenBank: MT311854.1) shown in SEQ ID NO.6.

[0011] The Avi sequence shown in SEQ ID NO.2 is inserted between bases 1028-1029 of the PCV2 full genome sequence shown in SEQ ID NO.4, thereby obtaining the DNA fragment shown in SEQ ID NO.5; the Avi sequence shown in SEQ ID NO.2 is inserted between bases 1345-1346 of the PCV3 full genome sequence shown in SEQ ID NO.1, thereby obtaining the DNA fragment shown in SEQ ID NO.3; the Avi sequence shown in SEQ ID NO.2 is inserted between bases 1049-1050 of the PCV4 full genome sequence shown in SEQ ID NO.6, thereby obtaining the DNA fragment shown in SEQ ID NO.7.

[0012] The present invention also provides a recombinant vector, which comprises the above DNA fragment, specifically, the above DNA fragment replaces the sequence between Kpn I and Sac I sites on the pBluescript SK (+) plasmid.

[0013] The present invention also provides a method for artificially preparing porcine circovirus, specifically, the above-mentioned recombinant vector is transfected into a receptor cell to obtain the artificial porcine circovirus. The receptor cell is a PK-15 cell.

[0014] The present invention also provides an artificial porcine circovirus prepared by the method.

[0015] The present invention also provides an artificial porcine circovirus cell model, specifically a recombinant cell obtained by infecting pig cells with the artificial porcine circovirus.

[0016] The present invention also provides an application of an artificial porcine circovirus or an artificial porcine circovirus cell model in porcine circovirus and vaccine research, or in porcine circovirus drug screening, or in a porcine circovirus infection detection method. The above application includes mixing the artificial porcine circovirus and a biotinylation reagent, incubating them, and then adding a quantum dot reagent to incubate to obtain a quantum dot-labeled artificial porcine circovirus.

[0017] The present invention inserts an Avi sequence on the basis of the existing PCV full genome, thereby realizing the artificial preparation of PCV2-Avi virus, PCV3-Avi virus and PCV4-Avi virus with an Avi tag.

[0018] The constructed infectious clone pSK-PCV2-Avi is formed by inserting the artificially synthesized PCV2-Avi gene sequence DNA shown in SEQ ID NO.5 into the pBluescript SK(+) plasmid, and the Avi sequence shown in SEQ ID NO.2 is located at a specific position upstream of the PCV2 Cap gene sequence.

[0019] The constructed infectious clone pSK-PCV3-Avi is formed by inserting the artificially synthesized PCV3-Avi gene sequence DNA shown in SEQ ID NO.3 on the basis of the pBluescript SK(+) plasmid, and the Avi sequence shown in SEQ ID NO.2 is located at a specific position upstream of the PCV3 Cap gene sequence.

[0020] The constructed infectious clone pSK-PCV4-Avi is formed by inserting the artificially synthesized PCV4-Avi gene sequence DNA shown in SEQ ID NO.7 into the pBluescript SK(+) plasmid, and the Avi sequence shown in SEQ ID NO.2 is located at a specific position upstream of the PCV4 Cap gene sequence.

[0021] The infectious clone pSK-PCV-Avi (including pSK-PCV2-Avi, pSK-PCV3-Avi and pSK-PCV4-Avi) can replicate and express in PK-15 cells. The artificially synthesized PCV-Avi full genome on the infectious clone pSK-PCV-Avi can use the regulatory sequence DNA at both ends and its own conserved sequence to specifically connect into a single-stranded circular PCV-Avi DNA during the replication process, and assemble into the PCV-Avi virus with the expressed viral protein.

[0022] The preparation method of the present invention is suitable for artificially preparing and labeling PCV-Avi virus. The obtained PCV-Avi virus can be used for vaccine development, establishment of virus infection detection methods, and research on virus infection and pathogenic mechanisms. The related research can provide a theoretical basis for the epidemic caused by PCV infection and an effective tool for preventing and controlling the epidemic. It has broad application prospects in clinical and laboratory research.

[0023] The present invention uses a method for labeling PCV with an Avi tag combined with quantum dots to obtain labeled PCV-Avi virus. It has been verified that the method for labeling PCV provided by the present invention can achieve the expected effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the construction of the pSK-PCV3-Avi recombinant plasmid of an embodiment of the present invention;

[0025] Figure 2 It is the electron microscopy negative staining result of the embodiment of the present invention;

[0026] Figure 3 This is a schematic diagram of quantum dot labeling of PCV3-Avi virus according to an embodiment of the present invention;

[0027] Figure 4 This is a diagram showing the immunofluorescence analysis results of quantum dot-labeled viruses according to an embodiment of the present invention. DETAILED DESCRIPTION

[0028] In the present invention, unless otherwise specified, all parts and percentages are weight units, and all equipment and raw materials can be purchased from the market or are commonly used in the industry. The following embodiments are introduced by taking the method of marking PCV3 and its application as an example, and other PCVs (including PCV2 and PCV4, etc.) can be marked in this way. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0029] The method for labeling PCV3 provided by the present invention is completed on the basis of artificially preparing PCV3-Avi virus, wherein the PCV3-Avi virus is first biotinylated and then reacted with quantum dots to complete the preparation of quantum dot-labeled PCV3-Avi virus. The specific preparation method of quantum dot-labeled PCV3-Avi virus particles is as follows:

[0030] S1. PCV3-Avi virus and biotinylation reagent were mixed and incubated at 4°C for 16 h to obtain biotinylated PCV3-Avi virus;

[0031] S2, after centrifugation at 5000 rpm for 3 min, the quantum dot reagent was incubated with biotinylated PCV3-Avi virus particles at room temperature for 3 h, or at 4°C for 30 min at a final concentration of 10 ng / μl;

[0032] S3. Then, add no more than 4 ml of the above mixed solution to an AmiconR Ultra 10K ultrafiltration centrifuge tube and centrifuge at 7,500×g for 40 min.

[0033] S4. Add the initial amount of PBS reagent and use a pipette to absorb the recovered sample to obtain the quantum dot-labeled PCV3-Avi virus.

[0034] The preparation method of the PCV3-Avi virus provided by the present invention is as follows:

[0035] S1. Inserting the Avi sequence shown in SEQ ID NO.2 between bases 1345 and 1346 of the PCV3 full genome sequence shown in SEQ ID NO.1 to obtain an artificially synthesized PCV3-Avi gene sequence shown in SEQ ID NO.3;

[0036] S2, replacing the sequence between the Kpn I and Sac I sites on the pBluescript SK(+) plasmid with the artificially synthesized PCV3-Avi gene sequence to construct a recombinant plasmid pSK-PCV3-Avi, i.e., the infectious clone pSK-PCV3-Avi;

[0037] S3. After the infectious clone pSK-PCV3-Avi is transfected into PK-15 cells, the cells are cultured, and the supernatant is collected and centrifuged to obtain the artificially prepared PCV3-Avi virus stock solution.

[0038] Example 1: Preparation and labeling of PCV3-Avi virus

[0039] (I) Construction of PCV3 infectious clone

[0040] The artificially prepared PCV3-Avi virus in this embodiment has a gene sequence of the artificially synthesized PCV3-Avi gene sequence as shown in SEQ ID NO. 3. The artificially synthesized PCV3-Avi gene sequence is obtained by inserting the Avi sequence shown in SEQ ID NO. 2 into a specific upstream position of the PCV3 full genome sequence shown in SEQ ID NO. 1. The details are as follows:

[0041] 1) inserting the Avi sequence shown in SEQ ID NO.2 between bases 1345 and 1346 of the PCV3 full genome sequence shown in SEQ ID NO.1 (GenBank: MF318451.1) to obtain an artificially synthesized PCV3-Avi gene sequence shown in SEQ ID NO.3;

[0042] 2) The artificially synthesized PCV3-Avi gene sequence is used to replace the sequence between the Kpn I and Sac I sites on the pBluescript SK(+) plasmid to construct a recombinant plasmid pSK-PCV3-Avi, thereby obtaining the PCV3 infectious clone pSK-PCV3-Avi with an Avi tag sequence. Figure 1 shown.

[0043] (II) Rescue and electron microscopic identification of PCV3-Avi virus

[0044] 1 Plasmid: PCV3 infectious clone pSK-PCV3-Avi with Avi tag sequence.

[0045] 2 Cells: porcine kidney epithelial cells (PK-15).

[0046] 3 The experimental process is as follows:

[0047] 1) Cell transfection

[0048] Referring to the instruction manual of Lipofectamine 3000, the infectious clone pSK-PCV3-Avi was transfected into PK-15 cells, cultured, and the supernatant was collected and centrifuged to obtain the artificially prepared PCV3-Avi virus stock solution.

[0049] The procedure is as follows: seed cells in a 6-well plate until 70-90% of the cells are transfected. Add 5 μl of Lipofectamine TM Add 3000 reagent to 125 μl of Opti-MEMTM medium and mix well for later use. Add 5 μg DNA (0.5-5 μg / μl) of pSK-PCV3-Avi plasmid solution and 10 μl of P3000 TM Reagent (2 μl / μg DNA) was added to 125 μl of Opti-MEM TM Culture medium. Mix the two tubes evenly and incubate at room temperature for 10-15 minutes. Gently aspirate the mixture with a pipette and slowly and evenly drip it onto the cultured PK-15 cells. Gently shake the mixture to allow it to fully contact the cells. Steadily transfer the cells to a 37°C, 5% CO2 incubator for culture.

[0050] 2) Blind transmission of the virus

[0051] Blind virus culture refers to the process of inoculating new cells for culture based on the presence of the virus without being sure whether the virus exists.

[0052] In the present invention, the transfected cells are recorded as the 0th generation, the cells after transfection are cultured for 72 hours, the cells are lysed by repeated freezing and thawing to collect the recombinant virus, and the virus after freezing and thawing is directly inoculated into new cells as the first generation of blind transmission, and then blind transmission is performed every 72 hours as one generation, and so on. The lysate obtained by repeated freezing and thawing of each generation is the PCV3-Avi virus stock solution.

[0053] 3) Electron microscopic identification of PCV3-Avi virus

[0054] After blind cell passage, cells were lysed by repeated freeze-thaw method, and virus samples were collected after centrifugation. The shape of virus particles was observed by negative staining under electron microscope. Viruses that were blindly passaged to the fourth generation were collected and observed by electron microscope after negative staining. Figure 3 As shown, typical porcine circovirus particles can be clearly observed in the test samples, and the diameter of the spherical virus particles is about 20nm (as shown in Figure 2 ), demonstrating that the PCV3-Avi virus was successfully rescued.

[0055] (III) Biotinylation (BirA method) and quantum dot labeling of PCV3-Avi virus

[0056] 1 Virus: PCV3-Avi virus with Avi tag obtained in (ii) above.

[0057] 2 The experimental process is as follows:

[0058] 1) Prepare a reaction system according to Table 1, gently pipette to mix, and incubate at 4° C. for 16 h. The rescued virus with the Avi tag can be biotin-labeled to obtain the biotinylated PCV3-Avi virus.

[0059] Table 1

[0060]

[0061] 2) Quantum dot labeling of biotinylated PCV3-Avi virus

[0062] Quantum dot-labeled streptavidin purchased from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd. was used for labeling. The labeling steps are as follows: After the quantum dot reagent is centrifuged at 5000rpm for 3 minutes, it is incubated with 500μl of biotinylated PCV3-Avi virus particles at room temperature for 3 hours, or at 4°C for 30 minutes at a final concentration of 10ng / μl. Then, no more than 4ml of the above mixture is added to the AmiconRUltra 10K ultrafiltration centrifuge tube and centrifuged at 7,500×g for 40 minutes. Subsequently, the starting amount of PBS reagent is added, and the recovered sample is aspirated with a pipette to obtain the quantum dot-labeled PCV3-Avi virus, such as Figure 3 shown.

[0063] (IV) Immunofluorescence analysis of labeled viruses

[0064] 1 Virus: PCV3-Avi virus labeled with quantum dots prepared in (iii) above.

[0065] 2 The experimental process is as follows:

[0066] 1) When the PK-15 cells grew to 50-70%, PCV3-Avi virus without quantum dot labeling and PCV3-Avi virus labeled with quantum dots were inoculated onto the cells and incubated at 37°C for 1 hour. The cells were washed with PBS twice to remove the unadsorbed PCV3-Avi.

[0067] 2) Add 4 nM streptavidin, incubate at 4°C for 1 h, and wash twice with PBS;

[0068] 3) Add 80% pre-cooled acetone and fix overnight at -20°C;

[0069] 4) Wash three times with PBS at room temperature on a shaker for 5 min each time, add 1% BSA to PBST to make a blocking solution, and block on a shaker at room temperature for 1 h;

[0070] 5) Wash three times with PBST for 5 min each time, and incubate with rabbit anti-PCV3 Cap protein polyclonal antibody at 37°C on a shaker for 3 h;

[0071] 6) Wash three times with PBST for 5 min each time, add FITC-labeled goat anti-rabbit secondary antibody and incubate at 37°C in the dark for 45 min;

[0072] 7) Wash three times with PBST in the dark for 5 min each time, add DAPI (diluted 1:500 with PBST) and incubate at 37°C in the dark for 7 min;

[0073] 8) Wash three times with PBST in the dark for 5 min each time, add ddH2O and store at 4℃.

[0074] The confocal microscopy system was used to detect the excitation light of quantum dots, viral Cap proteins, and cell nuclei. The results showed that the unlabeled virus only had immunofluorescence (green fluorescence) and no quantum dot fluorescence (red fluorescence). The quantum dot-labeled virus could show two different fluorescence signals at the same time, and the two fluorescence signals overlapped with each other, indicating that the quantum dots and Cap proteins were co-localized. Figure 4 As shown, quantum dot-labeled viruses can successfully trace viral infection.

[0075] The results show that the PCV3-Avi virus rescued by the present invention has the ability to infect PK-15 cells and proliferate and propagate in the cells, and the virus can be labeled with quantum dots after biotinylation.

[0076] The PCV3 infectious clone pSK-PCV3-Avi with an Avi tag prepared by the present invention can successfully rescue the PCV3-Avi virus, and can be used as an active ingredient in related vaccine development and pathogenic mechanism research.

[0077] Therefore, the present invention inserts the Avi sequence on the basis of the existing PCV3 full genome, and realizes the artificial preparation of PCV3-Avi virus with Avi tag. The constructed infectious clone pSK-PCV3-Avi is formed by inserting the artificially synthesized PCV3-Avi gene sequence DNA shown in SEQ ID NO.3 on the basis of the pBluescript SK(+) plasmid, and the Avi sequence shown in SEQ ID NO.2 is located at a specific position upstream of the PCV3 Cap gene sequence.

[0078] The infectious clone pSK-PCV3-Avi disclosed in the present invention can be replicated and expressed in PK-15 cells, wherein the artificially synthesized PCV3-Avi full genome on the infectious clone pSK-PCV3-Avi can utilize the regulatory sequence DNA at both ends and its own conserved sequence to specifically connect into a single-stranded circular PCV3-Avi DNA with a length of 2045 nt during the replication process, and assemble with the expressed viral protein into the PCV3-Avi virus.

[0079] The preparation method disclosed in the present invention is suitable for artificially preparing and labeling PCV3-Avi virus. The obtained PCV3-Avi virus can be used for vaccine development, establishment of virus infection detection methods, and research on virus infection and pathogenic mechanisms. The related research can provide a theoretical basis for epidemic diseases caused by PCV infection and an effective tool for preventing and controlling the epidemic diseases. It has broad application prospects in clinical and laboratory research.

[0080] In other embodiments of the present invention, PCV2 is labeled by the same method, thereby achieving artificial preparation of PCV2-Avi virus with Avi tag, which is different from Example 1 in that the artificially synthesized Avi tag sequence shown in SEQ ID NO.2 is inserted between bases 1028-1029 of PCV2 shown in SEQ ID NO.4 (taking PCV2 sequence as an example, GenBank: JQ955679.1), to obtain an artificially synthesized fragment shown in SEQ ID NO.5. The fragment is used to replace the sequence between the Kpn I and Sac I sites on the pBluescript SK(+) plasmid to construct a recombinant plasmid pSK-PCV2-Avi, thereby obtaining a PCV2 infectious clone pSK-PCV2-Avi with an Avi tag sequence. The infectious clone can be used to artificially prepare PCV2-Avi virus with Avi tag.

[0081] In other embodiments of the present invention, PCV4 is labeled by the same method, thereby achieving artificial preparation of PCV4-Avi virus with Avi tag, which is different from Example 1 in that the artificially synthesized Avi tag sequence shown in SEQ ID NO.2 is inserted into the 1049-1050 bases of PCV4 shown in SEQ ID NO.6 (taking PCV4 sequence as an example, GenBank: MT311854.1), and finally the artificially synthesized fragment shown in SEQ ID NO.7 is obtained. The fragment is used to replace the sequence between Kpn I and Sac I sites on the pBluescript SK (+) plasmid to construct a recombinant plasmid pSK-PCV4-Avi, and the PCV4 infectious clone pSK-PCV4-Avi with Avi tag sequence is obtained. The infectious clone can be used to artificially prepare PCV4-Avi virus with Avi tag.

[0082] Through the above method, PCV2-Avi virus and PCV4-Avi virus with Avi label can be obtained. At the same time, PCV2 and PCV4 with Avi label can also be effectively labeled with quantum dots, and finally PCV2 and PCV4 labeled with quantum dots can be obtained.

[0083] Therefore, the labeling method in Example 1 can also be used to label PCV2 and PCV4.

[0084] In Comparative Example 1 of the present invention, the difference from Example 1 is that the artificially synthesized Avi tag sequence as shown in SEQ ID NO.2 is inserted into other positions of PCV3 (other positions except the specific position between the 1345th and 1346th bases), as a result, the PCV3-Avi virus cannot be successfully rescued and the subsequent virus labeling cannot be completed.

[0085] In Comparative Example 2 of the present invention, the difference from Example 1 is that the Avi tag sequence shown in SEQ ID NO.2 is replaced with the GFP sequence, and the new sequence obtained is used to replace the sequence between the Kpn I and Sac I sites on the pBluescript SK(+) plasmid, resulting in the failure to successfully prepare quantum dot-labeled PCV3.

[0086] 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 preferred embodiments, it should be understood that the above description is only a specific implementation mode of the present invention and is not used to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A DNA fragment characterized by: The DNA fragment is an Avi sequence as shown in SEQ ID NO.2 inserted into the whole genome sequence of porcine circovirus, and the specific insertion position is the C-terminus of PCV Cap protein; the porcine circovirus is one of PCV2, PCV3 and PCV4; The Avi sequence shown in SEQ ID NO.2 is inserted between bases 1028-1029 of the PCV2 full genome sequence shown in SEQ ID NO.4, thereby obtaining the DNA fragment shown in SEQ ID NO.5; the Avi sequence shown in SEQ ID NO.2 is inserted between bases 1345-1346 of the PCV3 full genome sequence shown in SEQ ID NO.1, thereby obtaining the DNA fragment shown in SEQ ID NO.3; the Avi sequence shown in SEQ ID NO.2 is inserted between bases 1049-1050 of the PCV4 full genome sequence shown in SEQ ID NO.6, thereby obtaining the DNA fragment shown in SEQ ID NO.

7.

2. A recombinant vector comprising the DNA fragment as claimed in claim 1.

3. A recombinant vector according to claim 2, characterized in that: The DNA fragment as claimed in claim 1 was replaced on the pBluescript SK(+) plasmid Kpn I and Sac The sequence between the I sites.

4. A method for preparing an artificial porcine circovirus, characterized in that: The recombinant vector as claimed in claim 3 is transfected into a recipient cell to obtain an artificial porcine circovirus; the recipient cell is a PK-15 cell.

5. An artificial porcine circovirus prepared by the preparation method as claimed in claim 4.

6. An artificial porcine circovirus cell model, characterized in that: Specifically, the artificial porcine circovirus as claimed in claim 5 is used to infect pig cells to obtain recombinant cells, and the pig cells are PK-15 cells.

7. Use of the artificial porcine circovirus according to claim 5 or the artificial porcine circovirus cell model according to claim 6 in the preparation of porcine circovirus vaccines, or in the screening of porcine circovirus drugs.

8. The use as claimed in claim 7 comprises mixing the artificial porcine circovirus and the biotinylation reagent and then incubating them, and then adding the quantum dot reagent and incubating them to obtain the quantum dot-labeled artificial porcine circovirus.

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