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Construction of cell strain capable of stably expressing porcine circovirus type 2 ORF3 protein and application thereof

A porcine circovirus, a technology for stable expression, applied in the field of biotechnology detection

Pending Publication Date: 2021-08-17
LONGYAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, ORF3 induces lymphocyte apoptosis and reduces the number of lymphocytes in vivo, which may be an important reason for PCV2 to induce immune suppression in pigs, but how ORF3 induces apoptosis remains to be verified

Method used

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  • Construction of cell strain capable of stably expressing porcine circovirus type 2 ORF3 protein and application thereof
  • Construction of cell strain capable of stably expressing porcine circovirus type 2 ORF3 protein and application thereof
  • Construction of cell strain capable of stably expressing porcine circovirus type 2 ORF3 protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 PCV2 ORF3 Gene Amplification

[0025] 1. PCR amplification of PCV2 ORF3 gene According to the PCV2 ORF3 gene sequence in NCBI (No: AY686763), use the software primer 5 to design the specific primers PCV2 ORF3 gene PCV2 ORF3-F and PCV2 ORF3-R, and add restriction sites NheI and BamH I, for the convenience of subsequent identification, a Flag tag sequence was added to the 3' end of the gene (see the list of SEQ ID3 for the sequence). Use the DNA of PK-15 cells infected with PCV2 as a template for PCR amplification. The PCR amplification system is: template 1 μl, PrimerF (10 μM) 1 μL, Primer R (10 μM) 1 μl, PCR Mix (2×) 25 μl, ddH2O up to 50 μl; PCR reaction program: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, a total of 35 cycles, and a final extension at 72°C for 10 min. After the PCR, 10 μL of the amplified product was electrophoresed on a 1% agarose gel, and the DNA was recovered ...

Embodiment 2

[0026] Example 2 Construction of recombinant expression vector pLVZG-CMV-copGFP-Puro

[0027] Construction of recombinant lentiviral expression plasmid: The PCR product obtained in Example 1 and pLVZG-CMV-copGFP-Puro were digested with NheI and BamHI respectively, and the enzyme digestion system was placed in a water bath at 37°C overnight, electrophoresis, and the correct target band Perform gel recovery. The enzyme digestion system is as follows: ddH2O: 11ul; NheI: 1.5ul; BamHI: 1.5ul; DNA: 1ul (1ug / ul); Enzyme Buffer: 5ul; total system: 20ul, temperature 37°C, reaction time: 30min. React at 16°C for 24 hours to construct a recombinant lentiviral expression plasmid, and construct a recombinant lentiviral expression plasmid pLVZG-CMV-ORF3-copGFP-Puro (see figure 1). The recombinant vector was identified by agarose gel electrophoresis after NheI and BamHI double digestion, and the 8kb vector band and the 305bp target band were observed, while the pLVZG-CMV-copGFP-Puro empty ...

Embodiment 3

[0028] Example 3 Obtaining of recombinant lentivirus CMV-ORF3-copGFP-Puro

[0029] 1. Lentiviral Packaging

[0030] 293T cells were planted in a 10 cm culture dish, placed in an incubator at 37°C and 5% CO2, and the cell confluence reached about 80% after 12 hours of passage. Plasmid cotransfection system: pMDLg / pRRE 6 μg, pRSV-Rev 3 μg, pMD2.G 2 μg, shuttle plasmid 9 μg, Lipo3000 40 μL. 1 h before transfection, the medium containing 10% FBS without double antibody was replaced; 6 h after transfection, the medium containing 10% FBS was replaced with fresh medium. 48 hours after transfection, the cell supernatant was collected, and fresh cell culture medium was appropriately supplemented according to the cell growth status, and the cell supernatant was collected again at 72 hours; the collected cell supernatant (DMEM culture medium) was filtered with a 0.45 μm filter, Store at 4°C for later use;

[0031] 2. Virus Concentration and Purification

[0032] Centrifuge the collec...

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Abstract

The invention discloses construction of a cell strain capable of stably expressing porcine circovirus type 2 ORF3 protein and an application thereof, and belongs to the field of biotechnology detection. The construction method comprises the following steps: (1) inserting a PCV2 ORF3 gene into multiple cloning sites of NheI and BamHI of a pLVZG-CMV-copGFP-Puro vector; (2) co-transfecting a 293T cell by using the constructed pLVZG-CMV-ORF3-copGFP-Puro recombinant plasmid and a virus packaging plasmid, culturing, discharging virus, collecting a virus supernatant, and filtering to obtain a recombinant lentivirus solution; and (3) infecting the packaged recombinant lentivirus with PK-15 cells, and screening and identifying through puromycin to obtain the cell strain for stably expressing the PCV2 ORF3 protein. According to the invention, a recombinant lentivirus system is utilized to prepare a cell strain which is fused with tag protein and can stably express PCV2 ORF3 protein, and the cell strain provides a platform for researching PCV2 pathogenesis and is beneficial to deep-level pathogenic mechanism research of PCV2.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to the construction and application of a cell strain stably expressing porcine circovirus type 2 ORF3 protein. Background technique [0002] Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus disease (PCVD), which causes postweaning multisystemic wasting syndrome (PMWS) in piglets. An important disease that threatens the health of swine herds worldwide. PCV2 belongs to the genus Circovirus of the family Circoviridae. The size of the virion is 17-22 nm. It is a symmetrical icosahedron without an envelope. The genome size is about 1.7 kb and contains 11 open reading frames (ORFs). Currently, five ORF-encoded proteins and their functions have been reported, namely ORF1, ORF2, ORF3, ORF4 and ORF5. The typical histopathological symptoms of PCVD caused by PCV2 infection are lymphocyte injury and histiocyte infiltration. The cells infected wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N15/65C12N15/62C12N5/10C12Q1/6851
CPCC12N15/86C12N15/65C07K14/005C12N5/0686C12Q1/6851C12N2740/15043C12N2800/107C12N2750/10022C12N2750/10051C12N2510/02C07K2319/43C12Q2531/113C12Q2563/107
Inventor 陈洪博陆灵光邱龙新段滇宁邵丹颜路琪
Owner LONGYAN UNIV
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