Pseudorabies TK*/gE*/gI* gene dificiency mark live vaccine and preparation method thereof

A gene deletion vaccine, pseudorabies virus technology, applied in the field of animal virology, can solve the problems of unclear side effects, g1/gp63 insertion inactivation sequence, deletion, etc.

Inactive Publication Date: 2004-08-25
HUAZHONG AGRI UNIV
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Problems solved by technology

The main process is to develop the g1 / gp63 / LacZ gene deletion strain first, and then develop TK / g1 / gp63 / LacZ; the defect is that the g1 / gp63 line is insertion inactivation rather than sequence deletion, and the LacZ gene is a bacterial gene, not a pseudo The gene of the rabies virus itself, whether it has side effects on animals is not clear

Method used

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  • Pseudorabies TK*/gE*/gI* gene dificiency mark live vaccine and preparation method thereof
  • Pseudorabies TK*/gE*/gI* gene dificiency mark live vaccine and preparation method thereof
  • Pseudorabies TK*/gE*/gI* gene dificiency mark live vaccine and preparation method thereof

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Embodiment 1

[0077] 1. Construction of pseudorabies virus gE, gI double gene deletion transfer vector pFBBS:

[0078] Plasmid pSKFB was double-digested with BstE II and Stu I, the 1247bp fragment was discarded, and the 5.7kb fragment was recovered, filled in with Klenow enzyme, and ligated with T4DNA ligase to obtain a recombinant plasmid. The plasmid contains part of the gD gene, part of the gI gene, part of the gE gene, all of the 11K gene and part of the 28K gene, and is named pFBBS. Using pFBBS as a template and P7 and P8 as primers for PCR, a fragment with a size of about 500 bp was obtained. The PCR product was recovered, connected into pBluescript IISK(+), and sequenced to identify the missing sequence by the dideoxy termination method. See attached figure 1 , attached figure 2 .

[0079] 2. Pseudorabies virus E A strain TK - / gE - / gI - Construction of mutant strains:

[0080] PrV TK linearized with EcoR I from plasmid pFBBS - / gE - / LacZ + Genomic DNA of mutant strains...

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Abstract

The present invention discloses a construction of three-gene defected recombinant pseudorabies virus (PrV) strain, vaccine prepared by said strain, method for constructing said strain and method for preparing said vaccine. The described recombinant pseudorabies virus strain defects genes of TK.gE ang gI, and contains no exogenous gene. The described vacine belongs to the freeze-dried live vaccine made up by virus liquid containing said invention and gelatin. Said invented live vaccine can be inoculated on the piglet, slaughter pig and sow with farrow, and can obtain obious immune effect. Said vaccine has good biological safety, and can be used for preventing and curing pseudorabies.

Description

technical field [0001] The invention belongs to the technical field of animal virology, in particular to an artificially constructed pseudorabies virus (Pseudorabies virus, PrV) gene deletion mutant (PrV TK - / gE - / gI - ), using genetic engineering methods and DNA recombination methods to delete the main virulence gene TK and glycoprotein gene gI, gE coding regions of PrV, resulting in decreased PrV virulence, and this gene deletion mutant can prevent pseudorabies. [0002] The invention also relates to the recombinant pseudorabies virus genetic engineering vaccine and the preparation method and application of the vaccine. Background technique [0003] Pseudorabies virus (PrV) belongs to Herpesviridae a herpesvirus subfamily, has the characteristics of rapid growth on cell culture, strong neurotropism and latent infectivity (Roizmorn B, Desrosiers R, Flecbenstein B, Lopez C, Minson Ad and studdert MJ. The family Herpesviridae; an update....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/205A61P31/20C12N7/01
Inventor 陈焕春刘正飞吴斌金梅林方六荣何启盖周复春
Owner HUAZHONG AGRI UNIV
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