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Preparation method of recombination live vector vaccines for diseases of canid and/or feline

A live vector vaccine, feline technology, applied in the field of preparation of recombinant live vector vaccines for canine and/or feline epidemic diseases, can solve the problems of lack of international competitiveness, few new vaccine varieties, backward prevention and control technology, etc.

Inactive Publication Date: 2014-04-23
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, my country's dog, cat and other animal disease prevention and control technology is relatively backward, mainly manifested in the lack of new vaccine varieties, backward technology, weak innovation, and lack of international competitiveness. As a result, my country's animal vaccine market mainly relies on the import of foreign products

Method used

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  • Preparation method of recombination live vector vaccines for diseases of canid and/or feline
  • Preparation method of recombination live vector vaccines for diseases of canid and/or feline
  • Preparation method of recombination live vector vaccines for diseases of canid and/or feline

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of recombinant rabies virus eukaryotic expression vector expressing foreign genes

[0031] 1 Materials and methods

[0032] 1.1 Plasmids, cell lines, strains and reagents

[0033] Plasmid pCI ( Figure 11 ), pCDNA3.1(+) ( Figure 12 ), rabies virus SRV9 strain, and BSR cells were purchased from the Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Chinese People's Liberation Army. BSR cells were cultured in DMEM containing 5% fetal bovine serum, rabies virus SRV9 strain was amplified on BSR cells, and frozen at -70°C for use.

[0034]Phusion DNA polymerase, T4 DNA ligase, and restriction endonuclease were purchased from NEB Company, competent cells were purchased from Takata Company, gel recovery kit and plasmid extraction kit were purchased from Axygen Company, TRIzol, mouse-derived RNA was extracted Reverse transcriptase and liposomes were purchased from Invitrogen Company, and fetal bovine serum was purchased ...

Embodiment 2

[0068] Example 2 Vector construction and identification of recombinant rabies virus expressing eGFP

[0069] 1 Materials and methods

[0070] 1.1 Plasmids, cell lines, strains and reagents

[0071] Plasmids pD-SRV9-PMIn and pD-SRV9-sPMIn were constructed in Example 1, and the plasmid pCI-eGFP containing eGFP ( Figure 13 ) is preserved by the Military Veterinary Research Institute of the Academy of Military Medical Sciences of the Chinese People's Liberation Army.

[0072] 1.2 Construction of expression eGFP vector

[0073] According to the sequence of eGFP, the eGFP fragment with restriction sites at both ends was obtained by PCR method (see Table 4 for PCR primer sequences), and eGFP was inserted into pCI-SRV9-PMin and pCI-SRV9- Among sPMin, pCI-SRV9-PM-eGFP and pCI-SRV9-sPMin-eGFP were obtained. After double digestion with NheI+XhoI, it was ligated into pCDNA3.1(+) vector and named as pD-SRV9-PM-eGFP and pD-SRV9-sPM-eGFP respectively.

[0074] Table 4 Primer sequences ...

Embodiment 3

[0090] Example 3 Construction and identification of recombinant rabies virus vector expressing canine parvovirus VP2 gene

[0091] 1 Materials and methods

[0092] 1.1 Plasmids, cell lines, strains and reagents

[0093] Plasmid pD-SRV9-PM-eGFP was constructed in Example 2, and canine parvovirus CPV (strain CR86106) was purchased from Institute of Military Veterinary Medicine, Academy of Military Medical Sciences.

[0094] 1.2 DNA extraction and PCR

[0095] Extract parvovirus DNA according to the kit instructions.

[0096] 1.3 Construction of recombinant virus expressing parvovirus VP2

[0097] According to the sequence of parvovirus VP2, a VP2 fragment with restriction sites at both ends was obtained by PCR method (see Table 5 for PCR primer sequences), and VP2 was inserted into pD-SRV9-PM-eGFP by BsiWI+PmeI double restriction digestion. pD-SRV9-VP2 was obtained.

[0098] Table 5 Primer sequences

[0099]

[0100] Note: Bold is protective base

[0101] 1.4 Virus res...

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Abstract

The invention provides a method for preparing recombination live vector vaccines for diseases of canid and / or feline based on an RNA (ribose nucleic acid) virus rescuing technique, which is characterized in that a recombination virus expression vector capable of expressing main protective antigens of diseases of the canid and / or the feline based on a reverse genetic manipulation system is established. A rabies virus is conjunctly transfected by the expression vector and assistant plasmids to copy a permissive host cell to rescue the recombination virus, so that multivalent live vector vaccines are prepared. The growth curve of the virus presents that the rescued maternal virus has no obvious difference from the growth kinetic of the recombination virus which expresses the protective antigen genes of main disease pathogens of the canid and / or the feline, and a foundation is established for successfully preparing gene recombination live vector vaccines of main diseases of the canid and / or the feline.

Description

technical field [0001] The present invention relates to a method for preparing canine and / or feline animal disease recombinant live vector vaccine based on RNA virus rescue technology. Background technique [0002] With the improvement of people's living standards and the change of lifestyle, the number and species of canine animals have risen sharply. According to incomplete statistics, there are about 150 million domestic dogs in my country. Although the breeding of dogs, cats and other animals in some large cities has been brought into government management (including epidemic prevention), the rapid spread and spread of animal infectious diseases has become a huge obstacle to the development of my country's breeding and pet industries. On the one hand, infectious diseases such as canine distemper, canine infectious hepatitis, and canine coronavirus enteritis seriously threaten the health of pets; Zoonotic diseases such as leptospirosis and brucellosis seriously affect h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/79C12N7/01A61K48/00A61P31/14
Inventor 杨松涛王化磊金宏丽冯娜赵永坤郑学星高玉伟王承宇王铁成黄耕夏咸柱
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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