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Porcine circovirus I type infectious clone and virus rescued thereby and application thereof

An infectious cloning, porcine circovirus technology, applied in application, virus/phage, botanical equipment and methods, etc., can solve problems such as unclear pathogenicity, and achieve the effect of easy operation

Inactive Publication Date: 2009-05-06
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCV1 exists as a PK-15 cell-derived contaminating virus, and little is known about the origin and evolution of the virus, the mechanism of molecular genetic variation, etc. How pathogenic in co-infection conditions is unclear

Method used

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  • Porcine circovirus I type infectious clone and virus rescued thereby and application thereof
  • Porcine circovirus I type infectious clone and virus rescued thereby and application thereof
  • Porcine circovirus I type infectious clone and virus rescued thereby and application thereof

Examples

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Embodiment 1

[0030] Example 1 Construction of Infectious Clones, Rescue, Subculture and Determination of Recombinant Viruses

[0031] 1 Materials and methods

[0032] 1.1 Viruses, cells, antibodies, strains and vectors

[0033] The PCV1 virus genome was obtained from the contaminated pig kidney passage cell line (PK-15); the Escherichia coli strain (Top10), vector (pUC19) and restriction endonucleases used for gene cloning were products of Bao Bioengineering (Dalian) Co., Ltd. ; Transfection reagent Lipofectamine 2000 and RPMI1640 culture medium are Gibco products; PCV1-free pig kidney passage cell line (PK-15) was imported from abroad. Rabbit anti-PCV1 / Cap and PCV2 / Cap recombinant protein single-factor serum was self-made.

[0034] 1.2 Primer design and PCR amplification

[0035] Primers were designed according to the PCV1 sequence NC001792 provided by GenBank:

[0036] PCV1-F907: 5'- GTC GAC GGAAGTACCCGAAGGCCGATTTGA-3' (SEQ ID NO.1), the corresponding sequence is 907-936;

[0037]...

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Abstract

The invention discloses infective clone for porcine circovirus type 1 and rescued virus and application thereof. Two genomes of the porcine circovirus type 1 are connected in cis and inserted into a vector to construct and obtain an infective molecular clone. A Sal I enzyme cutting site as a molecular target is inserted in the infective molecular clone. A rescued recombinant virus (PCV1 / G strains) carries a molecular marker, and the preservation number of the rescued recombinant virus is CGMCC NO.2658. An antigen of the recombinant virus is only reacted with a univalent specific antibody of PCV1 / Cap protein, but has no cross with an antibody against PCV2 / Cap protein. The recombinant virus and a parental virus are identified by combination of PCR and RFLP. The virus strain cultured in vitro has stable propagation property and high virus titer, and the rescued virus strain can be used for detecting the antibody of the virus. The invention lays a foundation for research such as genesis and evolution, genetic variation rule, molecular differential diagnosis, and the like for porcine circovirus in future.

Description

technical field [0001] The present invention relates to an infectious clone of a virus, in particular to an infectious clone of porcine circovirus type 1 and a recombinant porcine circovirus type 1 virus rescued from the infectious clone, and also relates to the construction of the infectious clone The method and the application of the virus rescued by the infectious clone in diagnostic technology belong to the field of virus molecular biology and veterinary immunology. Background technique [0002] According to the pathogenicity, antigenicity and nucleotide sequence of porcine circovirus (Porcine circovirus, PCV), it can be divided into two genotypes, namely PCV1 and PCV2. PCV1 was first isolated from a contaminated pig kidney cell line (PK-15) by German scholar Tischer et al. (1974). It was confirmed by artificially infecting pigs that the virus did not cause significant clinical symptoms and pathological changes (Tischer I, Gelderblom H, Vetteermann W, et al.A very small...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/63C12N7/01G01N33/569
Inventor 刘长明危艳武陆月华张朝霞袁婧黄立平
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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