Hydrophobia-canine distemper-canine parvovirus genetic recombination virus strain, construction method and application thereof

A canine parvovirus and gene recombination technology, applied in the field of biological virus vaccine research, can solve the problem of less virus research, and achieve the effect of improving expression and preventing transmission

Inactive Publication Date: 2018-05-04
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on rabies is mostly limited to one virus of rabies, and there are relatively few studies on viruses constructed by recombination of multiple antigen genes

Method used

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  • Hydrophobia-canine distemper-canine parvovirus genetic recombination virus strain, construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 (construct rabies-canine distemper-canine parvovirus)

[0023] Construction of rabies-distemper-canine parvovirus gene recombination plasmid, such as figure 1 Shown:

[0024] Rabies SRV 9 Amplify the N, P, M, G, and L genes of the virus;

[0025] The Gibson Assembly connection method was used for recombinant gene connection, and the SRV 9 The G gene of the virus is transferred from the fourth position of the original genome sequence to the second position of the viral genome, and then the eGFP reporter gene is inserted between the G and P genes through the restriction site to construct pcDSRV 9- N-G-eGFP-PML gene recombination vector;

[0026] Amplify the CDV N gene in the canine distemper pMD18-CDV N recombinant plasmid by PCR method;

[0027] pcDSRV by enzyme digestion 9 -N-G-eGFP-PML recombinant vector's eGFP gene terminal restriction site was digested with Hpa I, and the canine distemper CDV N gene was constructed to pcDSRV using the Gibson Assemb...

Embodiment 2

[0032] Embodiment 2 (the recombinant virus of embodiment 1 is rescued by reverse genetic method)

[0033] The rabies-distemper-canine parvovirus (pcDSRV) constructed by reverse genetics 9 -NG-eGFP+CDV N-PM+VP2-L) recombinant plasmids and pcDNA3.1-N, pcDNA3.1-P, pcDNA3.1-M, ​​pcDNA3.1-G and pcDNA3.1-L helper plasmids through lipid The plastid transfection method was co-transfected into BSR cells at a certain concentration, and the rabies-distemper-canine parvovirus "rSRV" was rescued 9 +CDV N+VP2 Gene Recombinant Virus", and RT-PC, indirect immunofluorescence detection.

[0034] (1) Oral immunization of dogs with recombinant rabies virus

[0035] Nine dogs were randomly divided into groups. The first group was the control group injected with the commercially available dog pentavalent inactivated vaccine, and the injection dose was 1mL / dog; the second group was the compound adjuvant + rSRV 9 +CDV N+VP2 gene recombinant virus oral immunization test group, feeding dose of dog 5...

Embodiment 3

[0042] Embodiment 3 (identification of the biological characteristics of the recombinant virus constructed in embodiment 1)

[0043] The rabies "rSRV" that will be constructed 9 +CDV N+VP2” gene recombinant virus, the morphological characteristics of the recombinant virus were observed through an electron microscope; through immunofluorescence experiments, real-time fluorescent quantitative PCR technology, LD 50 In the experiment, the virus titer and virus virulence of the recombinant virus were detected; the antigenicity of the exogenous protein in the virus liquid was qualitatively and quantitatively analyzed by SDS polyacrylamide gel electrophoresis, BCA protein quantification method and Western-blot experiment; Recombinant virus immunization experimental animals (mice and dogs) were tested against rabies-canine distemper-canine parvovirus "rSRV" through double-antibody sandwich ELISA, T lymphocyte transformation test and autologous animal challenge experiment. 9 +CDV N+VP...

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Abstract

The invention discloses a hydrophobia-canine distemper-canine parvovirus genetic recombination virus strain, a construction method and application thereof. Through a homologous recombination method, agene replacement method and a gene insertion method, a full-length genome of a virus is subjected to gene rearrangement, and a rearranged hydrophobia virus genome plasmid with a canine distemper G gene and a canine parvovirus VP2 gene is constructed and is named as a hydrophobia-canine distemper-canine parvovirus, which is pcDSRV9-NG-eGFP+CDV N-PM+VP2-L genome plasmid; the hydrophobia-canine distemper-canine parvovirus is rescued through a reverse genetic method, and is an rSRV9+CDV N+VP2 gene recombinant virus; a gene nucleotide sequence of a recombination hydrophobia virus is SEQ ID NO:1. The recombination gene virus provided by the invention can be applied to researching a trigeminy oral vaccine for hydrophobia, canine distemper and canine parvovirus of canine and other wild animals soas to prevent three infectious diseases.

Description

technical field [0001] The invention relates to the field of biological virus vaccine research, in particular to a rabies-canine distemper-canine parvovirus gene recombinant virus strain, a construction method and application thereof. Background technique [0002] Rabies virus belongs to Rhabdoviridae, and its genome encodes five structural proteins: RNA-dependent RNA polymerase (L), nucleoprotein (N), phosphoprotein (P), matrix protein (M), outer membrane protein (G ), G protein and N protein are the main antigens of rabies virus, which can stimulate the body to produce corresponding antibodies and cellular immunity. Rabies is mainly transmitted through animal bites or scratches. The virus is transmitted along the peripheral nerves to the nerve center, resulting in cell function damage and the death of infected animals. At present, vaccine immunization is the most effective means to prevent and control the disease, relying on vaccine immunization can reduce the occurrence ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/66A61K39/175A61K39/295A61K39/205A61K39/23A61P31/14A61P31/20C12R1/93
CPCA61K39/12A61K2039/5256A61K2039/70C12N7/00C12N15/66C12N15/85C12N2750/14334C12N2760/18434C12N2760/20121C12N2760/20134
Inventor 简子健翟少华夏婷婷苏晓慧魏玉圆王伟毛丽萍程瑶文兆海
Owner XINJIANG AGRI UNIV
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