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

A feline and attenuated vaccine technology, applied in biochemical equipment and methods, virus/bacteriophage, recombinant DNA technology, etc., can solve problems such as weak innovation, lack of international competitiveness, and backward prevention and control technology

Inactive Publication Date: 2012-12-05
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] The construction of the recombinant rabies virus eukaryotic expression vector of embodiment 1 expression exogenous gene

[0031] 1 Materials and methods

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

[0033] Plasmid pCI ( Figure 11 ), pCDNA3.1(+) ( Figure 12 ), rabies virus SRV9 strain, and BSR cells were purchased from the Military Veterinary Research Institute of the Academy of Military Medical Sciences of the 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 future use.

[0034]Phusion DNA polymerase, T4 DNA ligase and restriction endonuclease were all purchased from NEB Company, competent cells were purchased from Takata Company, gel recovery kit and plasmid extraction kit were purchased from Axygen Company, TRIzol and mouse source were used for RNA extraction Reverse transcriptase and liposomes were purchased from Invi...

Embodiment 2

[0068] The vector construction and identification of the recombinant rabies virus of embodiment 2 expressing eGFP

[0069] 1 Materials and methods

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

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

[0072] 1.2 Construction of expression eGFP vector

[0073] According to the sequence of eGFP, eGFP fragments with restriction sites at both ends were obtained by PCR (see Table 4 for PCR primer sequences), and eGFP was inserted into pCI-SRV9-PMIn, pCI-SRV9- From sPMIn, pCI-SRV9-PM-eGFP and pCI-SRV9-sPMIn-eGFP were obtained. After being digested with NheI+XhoI, they were ligated into the pCDNA3.1(+) vector and named pD-SRV9-PM-eGFP and pD-SRV9-sPM-eGFP respectively.

[0074] Table 4 Primer sequences ...

Embodiment 3

[0090] Example 3 Construction and Identification of the Vector of Recombinant Rabies Virus Expressing Canine Parvovirus VP2 Gene

[0091] 1 Materials and methods

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

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

[0094] 1.2 DNA extraction and PCR

[0095] Parvoviral DNA was extracted according to the instructions of the kit.

[0096] 1.3 Construction of recombinant virus expressing parvovirus VP2

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

[0098] Table 5 Primer sequences

[0099]

[0100] Note: Bold are ...

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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 invention relates to a method for preparing canine and / or feline disease recombinant live vector vaccines based on RNA virus rescue technology. Background technique [0002] With the improvement of people's living standards and changes in lifestyles in our country, the number and types of canids raised have increased sharply. According to incomplete statistics, there are currently about 150 million domestic dogs in my country. Although the breeding of dogs, cats and other animals has been included in government management (including epidemic prevention) in some large cities, the rapid spread and spread of animal infectious diseases has become a huge obstacle restricting 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; on the other hand, pet-derived rabies, influenza, toxopla...

Claims

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Application Information

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Patent Type & Authority Applications(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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