Recombinant rabies virus for expressing canine distemper virus structural protein and application of recombinant rabies virus

A technology of rabies virus and canine distemper virus, which is applied in the field of immunology, veterinary biopharmaceuticals, and genetic engineering, can solve problems such as incomplete control, achieve high levels of neutralizing antibodies, easy mass production, and high expression levels of foreign proteins Effect

Pending Publication Date: 2020-04-03
CHANGCHUN SR BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many countries have fully immunized dogs and wild animals, the disease has not yet been fully controlled

Method used

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  • Recombinant rabies virus for expressing canine distemper virus structural protein and application of recombinant rabies virus
  • Recombinant rabies virus for expressing canine distemper virus structural protein and application of recombinant rabies virus
  • Recombinant rabies virus for expressing canine distemper virus structural protein and application of recombinant rabies virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions. The construction of the recombinant rabies virus of embodiment 1 expression canine distemper virus fusion protein (F) and canine distemper virus hemagglutinin protein (H)

[0047] 1. Materials

[0048] 1.1 Plasmids, cells and antibodies

[0049] The full-length infectious clone plasmid of rabies virus and BSR cells were preserved by Changchun Sino Biotechnology Co., Ltd. FITC-labeled mouse anti-RABV N protein monoclonal antibody was purchased from FDI FUJIREBIO; goat anti-CDV polyclonal was purchased from VMRD; FITC-lab...

Embodiment 2

[0104] Example 2 BHK cell suspension culture recombinant virus

[0105] 1. Materials

[0106] 1.1 Virus: Recombinant rabies virus SRV9-CDVH, SRV9-CDVF, constructed from Example 1.

[0107] 1.2 Cells: BHK suspension cells and NA cells were identified and preserved by Changchun Sino Biotechnology Co., Ltd.

[0108] 1.3 Medium: DMEM medium was purchased from Corning Company, and CD BHK-21 medium was purchased from Gbico Company.

[0109] 2. Method

[0110] 2.1 Growth curve of recombinant virus in BHK cells in vitro

[0111] Infect BHK suspension cells with recombinant viruses SRV9-CDVH and SRV9-CDVF according to MOI=0.5 and MOI=1, respectively, at 37°C, 5% CO 2 , 120rpm conditions, during the culture period, virus supernatant samples were collected at 24 hours, 48 ​​hours, 72 hours, 96 hours, and 120 hours to detect virus titers and draw virus growth curves.

[0112] Infect BHK suspension cells with the recombinant virus SRV9-CDVH and SRV9-CDVF according to MOI=1, at 37°C, 5...

Embodiment 3

[0119] The genetic stability research of embodiment 3 recombinant virus SRV9-CDVH, SRV9-CDVF exogenous gene

[0120] 1. Materials

[0121] 1.1 Virus: Recombinant rabies virus SRV9-CDVH, SRV9-CDVF, constructed from Example 1.

[0122] 1.2 Cells: BHK suspension cells, identified and preserved by Changchun Sino Biotechnology Co., Ltd.

[0123] 1.3 Medium: CD BHK-21 medium was purchased from Gbico.

[0124] 2. Method

[0125] According to the sequence information of the inserted CDV-H and CDV-F-TMCD genes in the recombinant virus genome, a pair of specific primers were designed for RT-PCR identification using PrimerPremier software. The primer sequences are shown in Table 4. Take the SRV9-CDVH, SRV9-CDVF recombinant rabies virus of the F2 generation, F4 generation, F6 generation, F8 generation, F10 generation, F12 generation, F14 generation, F16 generation, F20 generation, F24 generation and F28 generation, and carry out RNA extraction. After reverse transcription, use cDNA to...

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Abstract

The invention provides a recombinant rabies virus for expressing canine distemper virus structural protein and application of the recombinant rabies virus. The recombinant rabies virus takes a rabiesvirus SRV9 strain as a skeleton, a canine distemper virus H gene and a canine distemper virus F gene are inserted between a P gene and an M gene of the full length of rabies virus SRV9cDNA, or betweenan N gene and the P gene, or between the M gene and a G gene, or between the G gene and an L gene respectively, and finally, the recombinant rabies virus strains SRV9-CDVH and SRV9-CDVF are obtainedrespectively through rescue by a reverse genetic manipulation technology. After the prepared recombinant virus is inactivated, an adjuvant is added to prepare a recombinant rabies virus inactivated vaccine for expressing the canine distemper virus structural protein, and after an animal is immunized, corresponding rabies and canine distemper antibody titer can be generated through induction. The prepared vaccine has the advantages of ideal immunogenicity and high safety.

Description

technical field [0001] The invention relates to the fields of genetic engineering, immunology and veterinary biopharmaceuticals, in particular to a recombinant rabies virus expressing canine distemper virus structural protein and application thereof. Background technique [0002] Canine distemper (canine distemper, CD) is an acute, highly contagious infectious disease caused by canine distemper virus (Canine Distemper Virus, CDV) infection. One of the most dangerous diseases. The disease can infect not only canids, but also various animals such as Mustelidae, Procyonidae, and Felines. The incidence rate is extremely high, almost reaching 100%, and the clinical symptoms are diverse. Secondary infection, the mortality rate can be as high as 80%. Canine distemper virus belongs to Paramyxoviridae (Paramyxoviridae), Paramyxovirinae (Paramyxovirinae), and Morbillivirus (Morbillivirus). The genome is 15690bp long, encoding N, P, M, F, H, L and C proteins. Among them, fusion pro...

Claims

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Application Information

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IPC IPC(8): C12N7/01C07K14/13A61K39/295A61K39/175A61K39/205A61P31/14
CPCC07K14/005C12N7/00A61K39/12A61P31/14C12N2760/18422C12N2760/20121C12N2760/18434C12N2760/20134A61K2039/70A61K2039/552A61K2039/5252
Inventor 夏振强金宏丽石晶刘迪殷玉和焦翠翠赵健卢继龙刘彦超
Owner CHANGCHUN SR BIOLOGICAL TECH
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