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Recombinant pseudo-rabies virus expressing swine parvovirus VP2 gene and vacine and its preparation method

A technique for porcine pseudorabies virus and pseudorabies virus, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, antiviral agents, etc., and can solve problems such as unfavorable clinical application, sterility of breeding pigs, and improvement

Inactive Publication Date: 2004-02-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(4) cause sterility in breeding pigs
In order to ensure the amount of antigens for each pathogen, the multivalent vaccine developed in this way will inevitably lead to an increase in the vaccination dose, which is not conducive to clinical application.

Method used

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  • Recombinant pseudo-rabies virus expressing swine parvovirus VP2 gene and vacine and its preparation method
  • Recombinant pseudo-rabies virus expressing swine parvovirus VP2 gene and vacine and its preparation method
  • Recombinant pseudo-rabies virus expressing swine parvovirus VP2 gene and vacine and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Cloning and sequencing of the VP2 gene of embodiment 1:

[0035] Propagate PPV on the passaged pig kidney cells (IBRS-2), adopt the method of synchronous inoculation, that is, inoculate the PPV seed virus according to 1 / 10 of the culture medium while dispersing the cells, stop the culture 30-36 hours after inoculation, and collect the infection by centrifugation cells, and then extract porcine parvovirus RF-DNA (quoted from Moliter T W, Joo H S, Coliett M S. Virol, 1984, 137: 241-254). In order to obtain the full gene of PPV VP2, a pair of PCR primer oligonucleotides was designed according to the PPV genome sequence reported by Bergeron et al. The sequence is as follows:

[0036] Upstream primer: 5'-TTAGGATCCCAATGAGTGAAAATGTGGAAC-3'

[0037] Downstream primers: 5'-TACAGGATCCGTAAACACATGAGAGCTTG-3'The upstream and downstream primers respectively contain a BamH I restriction endonuclease site, and the BamH I site in the upstream primer ...

Embodiment 2p

[0046] Example 2 Construction of pUSK-VP2 transfer vector:

[0047] Vectors were constructed by conventional methods. Digest the pUSK vector with BamH I, and after agarose gel electrophoresis confirms that the digestion is complete, precipitate with absolute ethanol, wash with 75% ethanol, dissolve with water after the ethanol is completely evaporated, dephosphorylate with CIAP, and electrophoresis with 1% agarose gel Recycle later. The PCR product of the VP2 gene was digested by BamH I, and mixed with the digested and dephosphorylated pUSK vector after recovery, at T 4 Ligating under the action of DNA ligase, and then converting the ligated product into DH 5 αEscherichia coli, a small amount of plasmid was prepared, identified by enzyme digestion and its connection direction was determined, so as to obtain the pUSK-VP2 transfer vector. Such as figure 2 shown.

Embodiment 3

[0048] Embodiment 3 recombinant pseudorabies virus TK - / gG - / VP 2 + The build:

[0049] Propagation of PrV TK on IBRS-2 cells - / gG - / LacZ + Genomic DNA was extracted from the parental strain, because there is only one EcoR I restriction site in the genomic DNA of PrV, and it is located in the gG gene. PrVTK with EcoR I - / gG - / LacZ + The genomic DNase was cut into two segments, and liposome-mediated transfection technology was used to combine them with pUSK-VP 2 The transfer vector was co-transfected into IBRS-2 cells, and the entire VP2 gene was transferred into the genomic DNA of the PrV TK and gG double gene deletion strain through homologous recombination, and the LacZ gene was replaced at the same time, and the virus was collected after the cytopathic disease occurred. The virus liquid was inoculated on PK-15 cells, covered with DMEM containing 1% low-melting point agarose (such as the product of American GIBCO Company), and ke...

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Abstract

The present invention relates to main structure gene VP2 of artificial constructed pseudorabies virus, PrV and porcine parvovirus, PPV. In the pseudorabies virus genome in which the main toxicity gene (TK) and virus generation nonessential gene (gG) are deleted the VP2 gene of porcine parvovirus can be site-specifically inserted to make it be positioned in strong late promoter downstream of pseudorabies virus, and the inserted exogenous gene coded protein has good immunogenicity, and can stimulate swine to produce protective immune reaction for resisting two virulent challenges of porcine parvovirus and pseudorabies virus. Said invention also includes recombinant pseudorabies virus, Hzau AVL-PRppvV-VP2, vaccine prepared by using it and its preparation method.

Description

technical field [0001] The invention belongs to the technical field of virology, in particular to an artificially constructed pseudorabies virus and porcine parvovirus genetically engineered bivalent vaccine strain (PrV TK - / gG - / VP 2 + ), there are two gene mutations in its genome, which lead to the inactivation of TK and gG genes, and using DNA recombination method, a foreign gene is inserted downstream of the gG promoter, which is the main structural gene of porcine parvovirus VP 2 . [0002] The invention also relates to the recombinant genetic engineering vaccine and its preparation method. Background technique [0003] Pseudorabies is an acute infectious disease caused by pseudorabies virus (Pseudorabies virus, PrV) infecting pigs, cattle, sheep, dogs, cats, rabbits, mice, minks, foxes and other domestic and wild animals. store and disseminator. (Mettenleiter et al., Comp Immu Microbiol Infect Dis.1991, 14:151-163) other animals except pigs are sporadic, usual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/125A61P31/12C12N7/01C12N15/35
Inventor 陈焕春吕建强赵俊龙金梅林何启盖吴斌方六荣
Owner HUAZHONG AGRI UNIV
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