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Circovirus 3 type double-copy full-length gene infectious clone plasmid as well as construction method and application thereof

An infectious cloning and double-copy technology, applied in the fields of virology and biology, can solve the problems that the real mechanism has not been elucidated, the virus whole gene sequencing and amplification are difficult, and there is no pathogenicity research.

Active Publication Date: 2021-11-23
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The 3 ORFs encode 3 proteins of the virus, namely capsid protein VP1, scaffolding protein VP2 and apoptosis protein VP3, which are translated from a single polycistronic mRNA by alternately using start codons, but the codons alternate The actual mechanism used has not been elucidated
The biggest difficulty in the construction of GyV3 infectious clones is the amplification of the whole genome, because the 5′ end of the UTR region of the virus contains a region with high GC content, which makes it very difficult to sequence and amplify the whole virus gene. Only the first member of chicken infectious anemia virus (CIAV) has completed the construction of infectious clones and achieved virus rescue in vivo and in vitro, while the other 12 viruses have not constructed infectious clones and have no clear pathogenicity studies

Method used

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  • Circovirus 3 type double-copy full-length gene infectious clone plasmid as well as construction method and application thereof
  • Circovirus 3 type double-copy full-length gene infectious clone plasmid as well as construction method and application thereof
  • Circovirus 3 type double-copy full-length gene infectious clone plasmid as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The PCR amplification of the whole genome of embodiment 1 GyV3

[0041] 1. Primer design and synthesis

[0042] According to the construction strategy (attached figure 1 ), according to the GyV3 SDAU-1 nucleic acid sequence in the NCBI GenBank database, use the SnapGene Viewer software to query the enzyme cutting site of GyV3, and artificially design two pairs of GyV3 full-length gene amplification primers containing the enzyme cutting site. The underlined part is the enzyme cleavage site, and the 5' end is the protective base.

[0043] GyV3 Whole Gene Amplification Primers

[0044]

[0045]

[0046] 2. Preparation of GyV3 template DNA

[0047] The GyV3 template DNA was prepared using the GyV3-SDAU-2 strain's second regression SPF chicken tissue.

[0048] Use cell / tissue / blood DNA extraction kit (Tiangen, Beijing) to extract GyV3 infected chicken kidney DNA;

[0049] 3. PCR amplification and purification of GyV3 whole genome

[0050] In order to accurately a...

Embodiment 2

[0056] Example 2 Construction and identification of single-copy full-length gene recombinant plasmid pcDNA3.1-GyV3

[0057] 1. pcDNA3.1(+) and HindⅢ-GyV3-NotⅠ double digestion and ligation

[0058] Use QuickCut HindⅢ and QuickCut NotⅠ (Takara) to carry out double enzyme digestion on the empty vector pcDNA3.1(+) and HindⅢ-GyV3-NotⅠ, respectively, and add samples to the double enzyme digestion system in the following table (the amount of the empty vector and HindⅢ-GyV3-NotⅠ varies from More than 1 μg), react in a metal bath at 37°C for 10 min, then purify the digested product by agarose gel electrophoresis and gel recovery for use. Ligation of pcDNA3.1(+) and HindⅢ-GyV3-NotI was carried out on the same day as the double digestion, and the ligation system in the table below was added and reacted at 16°C for 30 minutes.

[0059] Double Enzyme Digestion System

[0060]

[0061]

[0062] connection system

[0063]

[0064] 2. Transformation of pcDNA3.1(+) and HindⅢ-GyV3-...

Embodiment 3

[0074] Example 3 Construction of double-copy full-length gene recombinant plasmid pcDNA3.1-2GyV3

[0075] 1. pcDNA3.1-GyV3 and BamHI-GyV3-BamHI digestion and ligation

[0076] Use QuickCut BamHI to digest the GyV3 single-copy cloning plasmids pcDNA3.1-GyV3 and BamHI-GyV3-BamHI respectively, and add samples according to the enzyme digestion system in the following table (the amount of pcDNA3.1-GyV3 and BamHI-GyV3-BamHI should not exceed 1 μg ), react in a metal bath at 37°C for 10 min, and then purify the digested product by agarose gel electrophoresis and gel recovery for use. The ligation of pcDNA3.1-GyV3 and BamHI-GyV3-BamHI was carried out on the same day as the enzyme digestion, and the ligation system in the following table was added, and reacted at 16°C for 30min. The ligation product was stored at -20°C for future use.

[0077] enzyme digestion system

[0078]

[0079] connection system

[0080]

[0081] 2. Transformation of pcDNA3.1-GyV3 and BamHI-GyV3-BamHI ...

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Abstract

The invention relates to the technical field of virology and biology, and relates to a circovirus 3 type (GyV3) double-copy full-length gene infectious clone using a reverse genetics technology as well as a construction method and application thereof. Specifically, the structural characteristics of a cyclic genome of GyV3 and enzyme cutting sites of the GyV3 are utilized, a GyV3 double-copy full-length infectious clone pcDNA3.1-2GyV3 is constructed by amplifying to two sections of virus whole genomes with different starting and ending points by using a high-fidelity enzyme and connecting to eukaryotic expression pcDNA3.1 (+) through two times of connection transformation, and virus rescue and pathogenicity research in an SPF (Specific Pathogen Free) chicken body are realized; and finally, a simple and rapid circle virus reverse genetics operating system is provided, a platform is provided for GyV3 pathogenicity, gene structure and function research and the like, and a foundation is laid for research of genetic engineering vaccines for GyV3.

Description

technical field [0001] The invention relates to the fields of virology and biotechnology, and relates to a circovirus type 3 double-copy full-length gene infectious clone utilizing reverse genetics technology and a construction method and application thereof. Background technique [0002] Gyrovirus type 3 (Gyrovirus 3, GyV3) is the third member of the circovirus genus after chicken infectious anemia virus (Chicken infectious anemia virus, CAV or CIAV), and is classified in the single-stranded circular DNA virus phylum encoding replication-related proteins Anelloviridae (Circular replication-associated protein-encoding single-stranded DNA viruses, CRESS DNA viruses). Since GyV3 was identified and named by macroviromics in Chilean children with unknown etiology of acute gastroenteritis diarrhea in 2012, it has been found in human diarrhea feces of different ages, ferret diarrhea feces and market chicken. Due to the breadth and complexity of its discovery samples, the relevanc...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/85C12N15/34C12R1/92
CPCC12N7/00C12N15/85C07K14/005C12N2750/00021C12N2750/00022C12N2800/107C12N15/70C12N2800/101C12Q1/6851C12Q1/701G01N33/56983G01N2333/01G01N2469/20C12Q2531/113C12Q2563/107C12Q2545/114Y02A50/30
Inventor 成子强张贤文
Owner SHANDONG AGRICULTURAL UNIVERSITY
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