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A sequence for regulating the proliferation of pcv2 virus and its application

A PCV2-SD, type 2 virus technology, applied in the field of molecular biology and biomedicine, can solve problems such as shortage

Active Publication Date: 2022-05-27
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problem of lacking porcine circovirus type 2 regulatory sequence, the present invention provides a sequence for regulating the proliferation of PCV2 virus, which can effectively regulate the proliferation of PCV2

Method used

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  • A sequence for regulating the proliferation of pcv2 virus and its application
  • A sequence for regulating the proliferation of pcv2 virus and its application
  • A sequence for regulating the proliferation of pcv2 virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Design of gRNAs targeting PCV2 genome

[0025] 1 Design of gRNAs targeting PCV2 genome

[0026] According to the PCV2SD strain gene sequence (Genbank Accession NumberJQ653449) and PCV2 genome structure on GenBank in NCBI, 8 gRNA targeting fragments related to PCV2 replication were designed: gRNA-Oc8, gRNA-H3, gRNA-O13, gRNA-O134, gRNA-O26, gRNA-NPS, gRNA-NQT and gRNA-NAT, targeting the octamer structure of the PCV2 origin of replication, the hexamer structure of the stem-loop structure sidearms, the overlapping region of ORF1 and ORF3, ORF1, ORF3 and ORF4, respectively The overlapping region of ORF2 and ORF6, ORF1 encoding the 23-25aa glycosylation site NPS fragment, ORF1 encoding the 256-258aa glycosylation site NQT fragment and ORF1 encoding the 286-288aa glycosylation site NAT fragments, such as figure 1 shown.

[0027]The gRNAs were designed using the CRISPR / Cas9 gRNA design website (http: / / crispr.mit.edu / ) provided by Feng Zhang et al. The obtained gR...

Embodiment 2

[0050] Example 2 Establishment of CRISPR / Cas9 system suitable for editing PCV2 genome

[0051] 1PCV2 Cas9 / gRNAs recombinant plasmid construction

[0052] 1.1 Synthesis of gRNAs dimers

[0053] Synthesize its reverse complementary primer pair according to the gRNAs designed in Example 1, as shown in Table 4:

[0054] Table 4 Sequence information of complementary primer pairs for CRISPR gRNAs

[0055] name Chain of Justice Sense(5'-3') Antisense strand Anti(5'-3') gRNA-Oc8 TTACCAGCGCACTTCGGCAG CTGCCGAAGTGCGCTGGTAA gRNA-H3 TTGCTGCTGAGGTGCTGCCG CGGCAGCACCTCAGCAGCAA gRNA-O134 TGTCAGAAATTTCCGCGGGC GCCCGCGGAAATTTCTGACA gRNA-O13 GGCAACTTACTGATAGAATG CATTCTATCAGTAAGTTGCC gRNA-O26 GACAGTATAACCGATGGTGC GCACCATCGGTTATACTGTC gRNA-NPS GCGGACCCCAACCACATAAA TTTATGTGGTTGGGGTCCGC gRNA-NQT AGAATACTGCGGGCCAAAAA TTTTTGGCCCGCAGTATTCT gRNA-NAT CAGCTGTAGAAGCTCTCTAT ATAGAGAGCTTCTACAGCTG

[0056] Dilute the prim...

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Abstract

The invention provides a gRNAs sequence for regulating the proliferation of PCV2 virus. The present invention designs 8 gRNA targeting fragments related to PCV2 replication: gRNA‑Oc8, gRNA‑O13, gRNA‑O134, gRNA‑O26, gRNA‑NPS, gRNA‑NQT and gRNA‑NAT, respectively targeting the origin of PCV2 replication The octamer structure of ORF1 and ORF3, the overlapping region of ORF1, ORF3 and ORF4, the overlapping region of ORF2 and ORF6, the 23‑25aa glycosylation site of ORF1, the 256‑258aa glycosylation site of ORF1 and the 286‑288aa glycosylation site of ORF1. Using pX458 as the expression vector, the PCV2 Cas9 / gRNAs recombinant plasmid was obtained, and the positive Cas9 / gRNAs clones were sequenced and identified. At the same time, the PK-15 cell line transfected with the PCV2 Cas9 / gRNAs recombinant plasmid was successfully obtained, and the PCV2 Cas9 / gRNAs recombinant plasmid can effectively edit the PCV2 genome and inhibit the proliferation and replication of PCV2 in PK-15 cells.

Description

technical field [0001] The invention belongs to the field of molecular biology and biomedicine, and particularly relates to a sequence for regulating PCV2 virus proliferation. Background technique [0002] The CRISPR / Cas9 system specifically recognizes the target DNA PAM sequence through gRNA (tracrRNA+crRNA), and triggers the Cas9 nuclease to cut complementary double-stranded DNA. It has been widely used for gene editing in mammalian cells. Currently, CRISPR / Cas9 has been successfully applied to precise genome editing in mice, zebrafish, and human cells and even bacteria. The editing of viral genomes by CRISPR / Cas9 has also been reported, and CRISPR / Cas9 technology has been successfully applied to effectively edit double-stranded DNA viruses and single-stranded RNA viruses. There is no report on the editing of single-stranded DNA viruses using CRISPR / Cas9 technology. [0003] Porcine circovirus (PCV) is the smallest virus known to infect mammals. It belongs to the genus ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90A61P31/20
CPCC12N15/113C12N15/85C12N15/907A61P31/20C12N2310/10C12N2310/20C12N2800/107C12N2810/10
Inventor 李俊时建立彭喆吴晓燕郑书轩徐绍建刘畅韩红王硕孙盼盼王妍
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI