Construction method of porcine circovirus type 4 circular DNA fragment, and preparation method and application of porcine circovirus type 4
By self-cyclizing the construction of pig circovirus type 4 circular DNA fragments and transfecting cells, the problem of PCV4 infectious cloning in the prior art was solved, the virus rescue and detection was achieved, and effective tools were provided for molecular virology research and vaccine development.
Patent Information
- Application Number
- CN202510623137.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-08
AI Technical Summary
The existing technology is difficult to successfully rescue infectious clones of pig circovirus type 4 (PCV4), and lacks effective construction and rescue methods, which affects molecular virology research and vaccine development.
The 4-cyclic DNA fragment of pig circovirus type 4 was constructed by self-cyclization. The recombinant plasmid pB-PCV4 was used for single-enzyme cleavage and then self-ligated cyclization. The PK-15 cells were transfected for cell-virgin passage, and the virus was detected in combination with Western-blotting, indirect immunofluorescence and fluorescence quantitative PCR.
The successful rescue of the pig circovirus type 4 with infectious activity provides tools for molecular virology research and vaccine development, and achieves effective detection and passage of the virus.
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Figure CN120442658A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the fields of genetic engineering and biotechnology, and particularly relates to a method for constructing a porcine circovirus type 4 circular DNA fragment, a method for preparing porcine circovirus type 4 and an application thereof. Background Art
[0002] PCV4 was first discovered in 2019 in a herd of pigs with porcine respiratory disease, diarrhea, and dermatitis and nephropathy syndrome in Hunan Province, my country. Its genome is 1,770 bp in size and contains two major open reading frames (ORFs). ORF1, located on the positive strand, is 891 bp long and encodes the 296-amino acid Rep protein. ORF2, located on the negative strand, is 687 bp long and encodes the 228-amino acid Cap protein.
[0003] PCV4 can infect pigs of all ages, including weaned pigs, finishing pigs, and multiparous sows. PCV4 infection is not significantly correlated with sex, age, or health status. Clinically, it may be associated with reproductive failure, abortion, stillbirth, mummified fetuses, postpartum nonspecific necrosis (PND), diarrhea, and respiratory and neurological symptoms. PCV4 has been detected in samples of the brain, liver, spleen, lung, kidney, small intestine, and lymph nodes, demonstrating its widespread tissue distribution and tropism. PCV4 is excreted through nasal fluid, feces, and saliva of infected pigs, contaminating feed, drinking water, and the surrounding environment. It can also be transmitted horizontally through the digestive and respiratory tracts. Furthermore, it can be transmitted vertically from sows to piglets. PCV4 poses a potential threat to the swine industry. Currently, no PCV4 has been successfully isolated. There are two main methods for constructing infectious clones of PCV4. One method involves constructing a recombinant plasmid containing the viral gene and transfecting cells to obtain the virus. Another method involves cloning the viral gene onto a plasmid, recovering the full-length viral genome fragment, and then ligating it overnight with T4 DNA ligase for in vitro self-circularization. The virus is then transfected into cells to obtain the virus. Currently, there are few reports on infectious clones of PCV4. Successful rescue of PCV4 from infectious clones has been achieved by constructing a recombinant plasmid containing the viral gene and then transfecting cells to obtain the virus. There are no reports of PCV4 rescue using other methods. Summary of the Invention
[0004] In response to the deficiencies in the prior art, the present invention provides a method for constructing a circular DNA fragment of porcine circovirus type 4, a method for preparing porcine circovirus type 4, and applications thereof. The present invention successfully rescues the virus by transfecting cells using self-circularized PCV4 for the first time, and can detect PCV4 by Western blotting, indirect immunofluorescence, and fluorescent quantitative PCR, providing an effective tool for molecular virology research and vaccine development.
[0005] The technical solution provided by the present invention is as follows:
[0006] The invention provides a method for constructing a porcine circovirus type 4 circular DNA fragment. The circular DNA fragment is obtained by self-ligation and circularization after single enzyme digestion of a recombinant plasmid pB-PCV4, wherein the nucleotide sequence of the recombinant plasmid pB-PCV4 is shown in SEQ ID NO: 1; the recombinant plasmid pB-PCV4 comprises a modified PCV4 full gene sequence and a pBluescript II SK(+) vector, wherein EcoRI restriction sites are introduced into the 5' end and the 3' end of the PCV4 full gene sequence, respectively.
[0007] Furthermore, the modification of the PCV4 full gene sequence includes: A base sequence before the EcoRI restriction site of the porcine circovirus type 4 gene published by NCBI was moved to the end of the gene, and a second EcoRI restriction site was introduced after the moved sequence to form a full PCV4 gene sequence with EcoRI sites at both ends.
[0008] Furthermore, the self-ligation circularization after single enzyme digestion of the recombinant plasmid pB-PCV4 comprises the following steps: The modified PCV4 full gene sequence was cloned into the EcoRI site of the pBluescript II SK(+) vector to obtain the recombinant plasmid pB-PCV4; The recombinant plasmid pB-PCV4 was digested with EcoRI to isolate and recover the complete PCV4 gene fragment; The recovered PCV4 full gene fragment was self-ligated and circularized by T4 ligase to obtain a circular DNA fragment.
[0009] The present invention also provides a method for preparing porcine circovirus type 4, wherein the circular DNA fragment constructed by the above method is transfected into PK-15 cells, and the cells are passaged with the virus to obtain porcine circovirus type 4 with infectious activity.
[0010] The present invention also provides porcine circovirus type 4 prepared by the preparation method described above.
[0011] The present invention also provides a porcine circovirus type 4 cell model, which is obtained by infecting pig cells with the porcine circovirus type 4 described above, and the pig cells are PK-15 cells.
[0012] The present invention also provides the use of the porcine circovirus type 4 or the porcine circovirus type 4 cell model described above in preparing a porcine circovirus type 4 vaccine, or in screening porcine circovirus type 4 drugs.
[0013] The present invention also provides a pharmaceutical composition or vaccine, comprising the porcine circovirus type 4 or the porcine circovirus type 4 cell model described above, and further comprising a pharmaceutically acceptable carrier and / or excipient.
[0014] Beneficial effects
[0015] The present invention successfully rescues the virus by transfecting cells using self-circularized PCV4 for the first time, synthesizing the modified PCV4 full gene into a pBluescript II SK(+) vector, separating the modified PCV4 full gene from the pBluescript II SK(+) vector by a single enzyme digestion method, and self-ligating the fragments using T4 DNA ligase to obtain circular DNA fragments. The circular DNA fragments are then transfected into PK-15 cells for virus-carrying cell passage, thereby obtaining porcine circovirus type 4 with infectious activity. PCV4 can be detected by Western blotting, indirect immunofluorescence, and fluorescent quantitative PCR, providing an effective tool for molecular virology research and vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is the plasmid map of pSK(+)-PCV4.
[0017] Figure 2 Construction strategy for PCV4.
[0018] Figure 3 The pBluescript II SK(+) vector and the modified PCV4 full gene were separated by EcoRI single enzyme digestion.
[0019] Figure 4 This is Western-blotting identification of WT-PCV4.
[0020] Figure 5 Indirect immunofluorescence identification of WT-PCV4.
[0021] Figure 6 Fluorescence quantitative PCR identification of WT-PCV4. DETAILED DESCRIPTION
[0022] The experimental methods in this example are conventional methods unless otherwise specified, and all raw materials are commercially available products.
[0023] Example 1 Construction of recombinant plasmid pB-PCV4
[0024] The PCV4 full gene sequence (GenBank: MZ593773.1) was obtained from NCBI. The base sequence preceding the EcoRI restriction site was moved to the end of the PCV4 gene, with the EcoRI restriction site as the boundary. Another EcoRI restriction site was introduced after the moved base sequence. The final synthesized PCV4 full gene contained two EcoRI restriction sites. Starting from the EcoRI restriction site of the pBluescriptII SK(+) vector, the modified PCV4 full gene was synthesized into the pBluescript II SK(+) vector to obtain the recombinant plasmid pB-PCV4 ( Figure 1 and Figure 2 ).
[0025] Example 2 Large-scale preparation of recombinant plasmid pB-PCV4
[0026] 2.1 Plasmid transformation
[0027] (1) Thaw the competent cells on ice, take 50 mL of the thawed competent cells, add 1 mL of plasmid to the competent cells, mix gently, and let it stand on ice for 30 min.
[0028] (2) Place the mixture of plasmid and competent cells in a 1.5 mL EP tube, heat shock the mixture in a 42°C water bath for 90 seconds, quickly transfer to ice, and let it stand for 2 minutes.
[0029] (3) Add 1 mL of antibiotic-free LB culture medium to the EP tube and culture on a shaker at 37°C for 1 h.
[0030] (4) After centrifuging the EP tube at 5,000 rpm for 5 min, discard 900 mL of the supernatant and use the remaining liquid to mix the precipitate. Spread it on a plate containing ampicillin and culture it upside down in a 37°C incubator overnight for 14 h.
[0031] 2.2 Plasmid extraction
[0032] (1) Column equilibration: Prepare two CP4 adsorption columns and two collection tubes, place the CP4 adsorption column in the collection tube, add 500 mL of equilibration solution BL to the CP4 adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
[0033] (2) Place 45 mL of bacterial culture in a 50 mL EP tube and centrifuge at 7,000 rpm for 5 min. Discard the supernatant and retain the precipitate.
[0034] (3) Add 2 mL of P1 solution to a 50 mL EP tube and vortex to mix the bacterial pellet.
[0035] (4) Add 2 mL of P2 solution to the 50 mL EP tube and gently invert to mix. The bacterial solution will become clear and viscous.
[0036] (5) Add 2.8 mL of P3 solution to a 50 mL EP tube and quickly and gently invert to mix. A white flocculent precipitate will be visible in the tube.
[0037] (6) Take four 2 mL EP tubes and add 1.7 mL of the above mixture to each tube. Centrifuge at 12,000 rpm for 10 min. A white precipitate will be visible at the bottom of the tube.
[0038] (7) Add the supernatant collected in the previous step to the CP4 adsorption column after column equilibration in 4 times, adding 800 μL at a time, and try not to aspirate the precipitate. Centrifuge at 12,000 rpm for 1 min each time, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
[0039] (8) Add 600 μL of PW rinse solution to each adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Repeat this step once.
[0040] (9) Place the adsorption column back into the collection tube, spin at 12,000 rpm for 2 min, let it stand for 5 min, and remove the rinse solution.
[0041] (10) Place the two adsorption columns in two clean 1.5 mL centrifuge tubes. Add 100 μL of EB eluent to the CP4 adsorption column, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes. To improve the recovery efficiency of the plasmid, add the liquid in the centrifuge tube back to the adsorption column and centrifuge at 12,000 rpm for 2 minutes. Measure the concentration using a microspectrophotometer and record the results.
[0042] Example 3 Construction of circular DNA fragments
[0043] 3.1 Identification of recombinant plasmid by single enzyme digestion
[0044] The constructed recombinant plasmid pB-PCV4 was digested with EcoRI endonuclease. The digestion system (100 mL) consisted of 5 mL EcoRI, 35 mL recombinant plasmid pB-PCV4, 50 mL ddH2O, and 10 mL buffer. After mixing all reagents evenly, the digestion was incubated at 37°C for 3 h. The digestion product was cleaned and recovered for subsequent experiments.
[0045] 3.2 Gel separation of PCV4 complete gene and pBluescript II SK(+) vector
[0046] The cleaned and recovered products were subjected to 1% agarose gel electrophoresis at 120V for 50 min to separate the gene and vector ( Figure 3 The full PCV4 gene is 1770 bp in size, and the vector is 2961 bp. Excise the 1770 bp band from the agarose gel and place it in a clean 2 mL polypropylene (EP) tube. Label the tube. Add an appropriate amount of PE solution to the tube to completely cover the gel. Incubate the gel at 55°C for 5 min, inverting the tube to ensure complete gel dissolution.
[0047] Add the gel-dissolved mixture to a CA5 adsorption column. Place the CA5 adsorption column in a collection tube. Let it sit at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid from the collection tube and return the CA5 adsorption column to the collection tube. Add 600 mL of PW rinse buffer to the CA5 adsorption column. Centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid from the collection tube and return the CA5 adsorption column to the collection tube. Repeat this step once. Evacuate the CA5 adsorption column at 12,000 rpm for 2 minutes. Place the CA5 adsorption column in a 1.5 mL EP tube and let it sit at room temperature for 2 minutes. Add 40 mL of TB eluent to the adsorption column and let it sit at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes. Label the EP tube used to collect the DNA solution.
[0048] 3.3 PCV4 full gene self-circularization
[0049] The recovered PCV4 full gene was self-ligated using T4 ligase. The ligation system (10 mL) included 8 mL of the target gene, 1 mL of T4 DNA ligase, and 1 mL of buffer. All reagents were pipetted and mixed thoroughly. The ligation was then incubated at 16°C overnight to obtain rearranged circularized DNA fragments.
[0050] Example 4 Rescue of WT-PCV4
[0051] 4.1 Cell transfection
[0052] (1) Take a 6-well plate and plate the subcultured PK-15 cells in the 6-well plate. Add 2 mL of cell suspension to each well and adjust the cell density. It is best to allow the cells to grow to about 80% within 24 hours.
[0053] (2) When the cells grow to about 80%, two wells in the 6-well plate are transfected with circularized DNA fragments, and the other two wells are transfected with pBluescript II SK(+) empty vector as a control.
[0054] (3) Take three 2 mL EP tubes and add 400 mL of Opti-MEM to each of two tubes. Then add 8 mg of the circularized DNA fragment PCV4 and pBluescript II SK(+) empty load, respectively. Add 800 mL of Opti-MEM to the other tube and then add 32 mL of Lipofectamine 2000 transfection reagent. Gently pipette the mixture of the three tubes to mix thoroughly and let it stand at room temperature for 2 minutes.
[0055] (4) Add 800 mL of Opti-MEM containing 32 μL of Lipofectamine 2000 transfection reagent and equally divide it into two tubes of EP tubes containing circularized DNA fragments and empty pBluescript II SK(+). Gently pipette to mix again and let it stand at room temperature for 15 minutes.
[0056] (5) Add each tube of mixed solution to two wells of a 6-well plate, add 400 mL to each well, and gently place it in a cell culture incubator for culture.
[0057] (6) Change the medium 8 h after transfection.
[0058] 4.2 Passaging cells with virus
[0059] Cells were passaged 24 hours after transfection. Protein was extracted from a portion of the cells 48 hours after plating for Western blotting analysis. Virus suspensions were collected from another portion of the cells 48 hours after plating after three freeze-thaw cycles for virus storage and qPCR analysis. The remaining cells were then passaged. The viral suspension obtained from the first passage of cells after transfection was designated F1. Cells were then passaged every 48 hours, and so on for subsequent generations. (The virus obtained from the circularized DNA fragment was designated WT-PCV4.)
[0060] Example 5 Identification of WT-PCV4
[0061] 5.1 Western blotting of WT-PCV4
[0062] 48 hours after each passage, cells were washed once with PBS. 200 mL of RIPA strong lysis buffer (purchased from Shanghai Biotech Biotechnology Co., Ltd.) was added to each well of a 6-well plate and lysed at room temperature for 5 minutes, shaking the plate during this time to thoroughly lyse the cells. Lysed protein samples were added to 1.5 mL EP tubes and placed on ice. The ultrasonic disruptor was set to 70 W power and sonicated for 5 seconds on, then 5 seconds off, for one minute per sample. After sonication, the samples were centrifuged at 12,000 rpm at 4°C for 20 minutes. The supernatant was the total cell and virus protein sample. 120 mL of supernatant was added to each 1.5 mL EP tube, followed by 40 mL of 4x Loading Buffer. Mix thoroughly and heat at 100°C for 10 minutes. SDS-PAGE electrophoresis, transfer to a membrane, incubation with antibodies, and color development were then performed. The primary antibody used was a rabbit polyclonal antibody stored in the laboratory, diluted at a 1:800 ratio. The secondary antibody was a goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase (HRP) (purchased from KPL, USA), with an antibody dilution ratio of 1:5000. The immunoblotting results showed that the virus was successfully rescued and the expression of PCV4 Cap protein was detected ( Figure 4 ).
[0063] 5.2 Indirect immunofluorescence identification of WT-PCV4
[0064] The harvested F6 passage virus suspension was inoculated at a 30% ratio into a digested PK-15 cell suspension. The suspension was then seeded into a 96-well plate, with 100 mL per well. The plate was incubated in a 37°C, 5% CO2 incubator for 48 h. The culture medium was discarded and the cells were washed twice with PBS for 6 min each. The 96-well plate was tapped dry with absorbent paper. Each well was fixed with 100 mL of anhydrous ethanol for 15 min at room temperature. The cells were then washed three times with PBS for 6 min each and tapped dry with absorbent paper. Each well was blocked with 100 mL of 5% skim milk for 1 h at room temperature. The cells were then washed three times with PBS for 6 min each and tapped dry with absorbent paper. A rabbit polyclonal antibody was used as the primary antibody at a dilution of 1:200. A rabbit negative serum control (60 mL per well) was also used and incubated overnight at 4°C. Discard the primary antibody, wash four times with PBS for 6 minutes each time, tap the 96-well plate dry with absorbent paper, use FITC-labeled goat anti-rabbit antibody as the secondary antibody, the antibody dilution ratio is 1:300, 60 mL per well, incubate at room temperature in the dark for 1 hour. Discard the secondary antibody, wash four times with PBS for 6 minutes each time, tap the 96-well plate dry with absorbent paper. Dilute DAPI 1:1000 with PBS, add 60 mL to each well, incubate at room temperature for 10 minutes, wash twice with PBS for 6 minutes each time, tap the 96-well plate dry with absorbent paper, add 100 mL PBS to each well, observe and photograph under an inverted fluorescence microscope. The results showed that the control had no fluorescence, while the PCV4 Cap protein in the cells infected with the sixth-generation virus solution WT-F6 had a specific fluorescent reaction with the PCV4 Cap rabbit polyclonal antibody, indicating that the virus was successfully rescued ( Figure 5 ).
[0065] 5.3 Identification of WT-PCV4 by Fluorescence Quantitative PCR
[0066] During cell passage, viral fluid was collected from each passage and viral genomic DNA was extracted using the Biotech Ezup column-based viral DNA extraction kit for detection of rescued PCV4 virus copy number. SYBR Green I fluorescence quantitative PCR was used to amplify the 102 bp portion of the PCV4 Cap sequence for PCV4 virus copy number determination. The specific primer sequences were: PCV4-Cap102 bp-F: 5'-atagtctccatccagttgtat-3'; PCV4-Cap102 bp-R: 5'-tattaccggatcagaaaggtc-3', synthesized by Suzhou GeneWeiZ Biotechnology Co., Ltd. PCR amplification was performed using the F and R specific primers using the extracted viral genomic DNA as template. The 20 mL qPCR reaction system consisted of 10 mL 2Taq Pro Universal SYBR qPCR Master Mix (Novagen), 0.4 mL each of the upstream and downstream primers, 1 mL of template, and 8.2 mL of ddH2O. A Roche LightCycler® 96 instrument was used with pre-denaturation at 95°C for 30 seconds, followed by cycling at 95°C for 10 seconds and 60°C for 30 seconds for 40 cycles. The instrument's default melting curve acquisition program was used to determine the melting curve and calculate the viral copy number. During the passage of cells carrying the virus to the seventh generation, viral fluid from each generation was collected to extract viral DNA for fluorescence quantitative detection. Fluorescence quantitative analysis revealed the presence of the PCV4 Cap gene in each generation of virus, indicating the successful rescue of the viral genome in the cells. The virus was able to replicate and proliferate in PK-15 cells ( Figure 6 ).
[0067] SEQ ID NO: 1 (nucleotide sequence of recombinant plasmid pB-PCV4):
Claims
1. A method for constructing a porcine circovirus type 4 circular DNA fragment, characterized in that: The circular DNA fragment is obtained by self-ligation and circularization after single enzyme digestion of the recombinant plasmid pB-PCV4, and the nucleotide sequence of the recombinant plasmid pB-PCV4 is shown in SEQ ID NO: 1; the recombinant plasmid pB-PCV4 comprises a modified PCV4 full gene sequence and a pBluescript II SK(+) vector, wherein EcoRI restriction sites are introduced at the 5' end and 3' end of the PCV4 full gene sequence, respectively.
2. The method for constructing a porcine circovirus type 4 circular DNA fragment according to claim 1, wherein The modification of the PCV4 full gene sequence includes: A base sequence before the EcoRI restriction site of the porcine circovirus type 4 gene published by NCBI was moved to the end of the gene, and a second EcoRI restriction site was introduced after the moved sequence to form a full PCV4 gene sequence with EcoRI sites at both ends.
3. The method for constructing a porcine circovirus type 4 circular DNA fragment according to claim 2, wherein The self-ligation circularization after single enzyme digestion of the recombinant plasmid pB-PCV4 comprises the following steps: The modified PCV4 full gene sequence was cloned into the EcoRI site of the pBluescript II SK(+) vector to obtain the recombinant plasmid pB-PCV4; The recombinant plasmid pB-PCV4 was digested with EcoRI to isolate and recover the complete PCV4 gene fragment; The recovered PCV4 full gene fragment was self-ligated and circularized by T4 ligase to obtain a circular DNA fragment.
4. A method for preparing porcine circovirus type 4, characterized in that: The circular DNA fragment constructed by the method according to any one of claims 1 to 3 is transfected into PK-15 cells, and the cells are passaged with the virus to obtain porcine circovirus type 4 with infectious activity.
5. Porcine circovirus type 4 prepared by the preparation method according to claim 4.
6. A porcine circovirus type 4 cell model, characterized in that The porcine circovirus type 4 as claimed in claim 5 is infected with pig cells to obtain the cells, wherein the pig cells are PK-15 cells.
7. Use of the porcine circovirus type 4 according to claim 5 or the porcine circovirus type 4 cell model according to claim 6 in preparing a porcine circovirus type 4 vaccine or in screening porcine circovirus type 4 drugs.
8. A pharmaceutical composition or vaccine, characterized in that The invention comprises the porcine circovirus type 4 cell model according to claim 5 or the porcine circovirus type 4 cell model according to claim 6, and further comprises a pharmaceutically acceptable carrier and / or excipient.