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Sequence for regulating proliferation of PCV2 virus and application thereof

A type 2 virus, sequence technology, applied in the field of molecular biology and biomedicine, can solve the problem of lack of

Active Publication Date: 2019-07-26
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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  • Sequence for regulating proliferation of PCV2 virus and application thereof
  • Sequence for regulating proliferation of PCV2 virus and application thereof
  • Sequence for regulating proliferation of PCV2 virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Design of gRNAs targeting the PCV2 genome

[0025] 1 Design of gRNAs targeting PCV2 genome

[0026] According to the PCV2SD strain gene sequence (Genbank Accession Number JQ653449) on GenBank in NCBI and the whole genome structure of PCV2, design 8 gRNA targeting fragments related to PCV2 replication: gRNA-Oc8, gRNA-H3, gRNA-O13, gRNA-O134 , gRNA-O26, gRNA-NPS, gRNA-NQT and gRNA-NAT, respectively target the octamer structure of the PCV2 replication origin, the hexamer of the side arm of the stem-loop structure, the overlapping region of ORF1 and ORF3, ORF1, ORF3 and The overlapping region of ORF4, the overlapping region of ORF2 and ORF6, the fragment of ORF1 encoding 23-25aa glycosylation site NPS, the fragment of ORF1 encoding 256-258aa glycosylation site NQT and the fragment of ORF1 encoding 286-288aa glycosylation site Fragments of NAT, such as figure 1 shown.

[0027]Use the CRISPR / Cas9 gRNA design website (http: / / crispr.mit.edu / ) provided by Zhang Feng...

Embodiment 2

[0050] Embodiment 2 is suitable for the establishment of the CRISPR / Cas9 system of editing PCV2 genome

[0051] 1PCV2 Cas9 / gRNAs recombinant plasmid construction

[0052] 1.1 Synthesis of gRNAs dimers

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

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

[0055] name Sense chain 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 GCGGACCCCAAACCACATAAA TTTATGTGGTTGGGGTCCGC gRNA-NQT AGAATACTGCGGGCCAAAAA TTTTTGGCCCGCAGTATTCT gRNA-NAT CAGCTGTAGAAGCTCTCTAT ATAGAGAGCTTCTACAGCTG

[0056] Dilute t...

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Abstract

The invention provides a gRNAs sequence for regulating the proliferation of a PCV2 virus. 8 gRNA targeting fragments of gRNA-Oc8, gRNA-O13, gRNA-O134, gRNA-O26, gRNA-NPS, gRNA-NQT and gRNA-NAT associated with PCV2 replication are designed, respectively targeting the octamer structure of a PCV2 replication origin, the overlapping region of ORF1 and ORF3, the overlapping region of ORF1, ORF3 and ORF4, the overlapping region of ORF2 and ORF6, the 23-25aa glycosylation locus of ORF1, the 256-258aa glycosylation locus of ORF1 and the 286-288aa glycosylation locus of ORF1. A recombinant plasmid PCV2Cas9 / gRNAs is obtained with pX458 as an expression vector, and the positive Cas9 / gRNAs cloning is subjected to sequencing and identification. At the same time, a PK-15 cell line transfected with therecombinant plasmid PCV2 Cas9 / gRNAs is successfully obtained, and the recombinant plasmid PCV2 Cas9 / gRNAs can effectively edit the PCV2 genome and inhibit the proliferation and replication of PCV2 inPK-15 cells.

Description

technical field [0001] The invention belongs to the fields of molecular biology and biomedicine, and in particular relates to a sequence for regulating the proliferation of PCV2 virus. Background technique [0002] The CRISPR / Cas9 system uses gRNA (tracrRNA+crRNA) to specifically recognize the PAM sequence of the target DNA, triggering Cas9 nuclease to cleave complementary double-stranded DNA at a specific point. It has the advantages of simple preparation, high editing site specificity and easy simultaneous editing of multiple It has been widely used in 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 successively, and CRISPR / Cas9 technology has been successfully applied to effectively edit double-stranded DNA viruses and single-stranded RNA viruses. So far, there has been no report on the ...

Claims

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Application Information

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Patent Type & Authority Applications(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