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Feline calicivirus infectious clone and construction method and application thereof

An infectious cloning, feline calicivirus technology, applied in the field of positive-sense RNA virus expression vector and its construction, can solve the problems of low titer and low infectivity, and achieve high titer, stable passage, and good infectivity

Inactive Publication Date: 2012-07-11
广东省农业科学院兽医研究所
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  • Abstract
  • Description
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Problems solved by technology

[0006] However, the existing feline calicivirus infectious clones are generally not highly infective and have low titers

Method used

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  • Feline calicivirus infectious clone and construction method and application thereof
  • Feline calicivirus infectious clone and construction method and application thereof
  • Feline calicivirus infectious clone and construction method and application thereof

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Embodiment Construction

[0038] full-length amplification

[0039] The full sequence of PCV is shown in GenBank ID: GU214989.1.

[0040] Primer used: FCVNORZ:5′-GCA GAGCTC TCTGGCTAACTGTAAAAGAAATTTGAGAC-3' (SEQ ID NO: 5) (the underlined part is the Sac I restriction site). The first half is the pCDNA sequence, and the second half is the FCV sequence. FCVhouB: 5′-TTTTCTAGAG CGGCCGC TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 6). (The underline is the Not I restriction site). Using a plasmid containing full-length DCV as a template, use PrimeSTAR TM HSDNA Polymerase amplification, reaction system: PrimeSTAR 0.5μL, dNTP (2.5mM) 4μL, 5×PS Buffer 10μL, FCVNORZ (10mM) 1μL, FCVhouB (10mM) 1μL, pFCV0.5μL, ddH 2 O up to 50 μL. PCR program: 95°C for 5 minutes; 98°C for 10s, 59°C for 15s, 72°C for 4min, 30 cycles; 72°C for 10min.

[0041] Identification of PCR products by agarose gel electrophoresis

[0042] Prepare 1% agarose gel with 1×TAE buffer solution, add 5 μL of PCR product to each sa...

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Abstract

The invention discloses feline calicivirus infectious clone, which is obtained by connecting a sequence with an expression vector after connecting restriction enzyme cutting sites with two ends of a full-length complementary deoxyribonucleic acid (cDNA) of feline calicivirus (FCV). Experimental results show that the FCV obtained through the clone production has similar characteristics with parentvirus, has good infectivity, can enable cells to be denatured obviously, and is high in titer and stable in passage.

Description

technical field [0001] The invention relates to a positive-sense RNA virus expression vector and its construction method and application, in particular to a feline calicivirus infectious clone and its construction method and application. Background technique [0002] The base sequence of the single-stranded positive-strand RNA virus is exactly the same as that of the mRNA, which can directly function as the viral mRNA. Therefore, this type of virus is called a positive-sense RNA virus, and its naked RNA is infectious. The genome replication of this type of virus is characterized by first synthesizing (-) RNA and then using it as a template for synthesizing (+) RNA. Racaniello and Baltimore successfully constructed the infectious clone of animal RNA virus for the first time in 1981. They cloned the full-length cDNA of the poliovirus (Pliovirus, PV) genome into the pBR322 vector, and transfected mammalian cells with the recombinant plasmid Finally, infectious virus particles...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12R1/93
Inventor 向华徐敏黄元陈晶
Owner 广东省农业科学院兽医研究所
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