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Canine distemper virus CDV-3 strain infectious cDNA cloning and constructing method and application thereof

A canine distemper virus, CDV-3 technology, applied in recombinant DNA technology, applications, viruses, etc., can solve problems such as threats to human public health safety, economic loss of fur animals, etc., and achieve stable and efficient rescue and rescue efficiency.

Active Publication Date: 2020-04-03
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This not only caused huge economic losses to pet dog and cat breeding and fur animal breeding, but also seriously threatened the safety of human public health

Method used

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  • Canine distemper virus CDV-3 strain infectious cDNA cloning and constructing method and application thereof
  • Canine distemper virus CDV-3 strain infectious cDNA cloning and constructing method and application thereof
  • Canine distemper virus CDV-3 strain infectious cDNA cloning and constructing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of canine distemper virus CDV-3 strain infectious cDNA clone and virus rescue

[0033] 1 Materials and methods

[0034] 1.1 Materials

[0035] 1.1.1 Virus strains and cells

[0036] The CDV-3 strain of canine distemper virus is preserved by the Animal Disease Research Office of the Specialty Products Research Institute of the Chinese Academy of Agricultural Sciences; the 293T and Vero cells are preserved by the disease prevention and control team of the Specialty Products Research Institute of the Chinese Academy of Agricultural Sciences;

[0037] 1.1.2 Main reagents

[0038] Culture medium DMEM, penicillin streptomycin and calf serum FBS were all purchased from Gibco; eukaryotic expression vector pcDNA3.1 was purchased from Invitrogen; cloning vector pEASY-Blunt or pEASY-Simple-Blunt, high-fidelity enzymes Trans StartFastPfu DNA Polymerase and FITC-labeled goat anti-mouse secondary antibody was purchased from Beijing Quanshijin; various restri...

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PUM

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Abstract

The invention discloses a canine distemper virus CDV-3 strain infectious cDNA cloning and constructing method and application thereof. A canine distemper virus CDV-3 strain is used as a template, theoverall length of the CDV-3 is divided into 5 fragments, and RT-PCR amplification is performed. Through enzyme-cutting splicing, the 5 fragments are sequentially inserted into a eukaryotic vector, besides, a hammerhead ribozyme sequence and a hepatitis D ribozyme sequence are respectively added to an F1 head end and an F5 tail end, and canine distemper virus CDV-3 strain infectious cDNA clone is obtained; then three auxiliary plasmids expressing CDV-3N, P and L proteins are constructed; the canine distemper virus CDV-3 infectious cDNA clone and the three auxiliary plasmids are used for performing cotransfection on 293T cells so that saved recombinant CDV-3 viruses (rCDV-3) are obtained. Through research, the highest titer of the obtained rCDV-3 virus can reach 107.667TCID50 / mL, which is 10times higher than that of wtCDV-3. After Vero cells are infected with the rCDV-3, within 36h after infection, the highest virus titer can be achieved; but after the Vero cells are infected with the wtCDV-3, within 72h after infection, the virus content can reach the highest value. A base is established for developing a canine distemper virus novel vaccine and studying the infective mechanism through the canine distemper virus CDV-3 strain infectious cDNA cloning and constructing method disclosed by the invention.

Description

technical field [0001] The invention relates to an infectious cDNA clone and its construction method and application, in particular to an infectious cDNA clone of canine distemper virus CDV-3 strain and its construction method and application. The invention belongs to the field of biotechnology. Background technique [0002] Canine distemper (Canie Distemper, CD) is a highly contagious infectious disease caused by canine distemper virus (Canie Distemper Virus, CDV) infecting dogs, mink and other carnivorous animals. The disease is highly contagious, has a high incidence rate, and has various clinical symptoms. It is easy to form secondary and mixed infections in the late stage of infection. In recent years, the host of natural infection of canine distemper virus has been expanding continuously. Outbreaks of canine distemper have been reported in wild animals such as tigers, lions, leopards and seals, and even giant panda populations. In January 2015, five giant pandas rais...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/45C12N7/01
CPCC07K14/005C12N7/00C12N15/85C12N2760/18421C12N2760/18422
Inventor 薛向红闫喜军卜研陈杰赵建军赵传芳胡博
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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