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Method for constructing canine distemper virus (CDV) reverse genetics system

A technology of canine distemper virus and reverse genetics, which is applied in the field of construction of canine distemper virus reverse genetics system, can solve problems such as low efficiency, errors, and time-consuming

Active Publication Date: 2014-08-13
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

, it is very easy to make mistakes during the construction process, and it takes a long time to lead to low efficiency

Method used

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  • Method for constructing canine distemper virus (CDV) reverse genetics system
  • Method for constructing canine distemper virus (CDV) reverse genetics system
  • Method for constructing canine distemper virus (CDV) reverse genetics system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0036] The method of the present invention will be described in detail below in conjunction with the examples.

[0037] 1. Amplification of the whole CDV3 gene

[0038] 1. Primer design

[0039] According to CDV3 (GenBank: EU726268.1) sequence in Gene Bank, use Primer5.0 to design 9 pairs of primers (Table 1), wherein part of the adjacent fragment PCR amplification primers according to The PCR Cloning System specification requires design.

[0040] Table 1: CDV gene amplification primer sequence information

[0041] Primer name

Primer sequence

Sequence listing order

F1f

5'-ACCAGACAAAGTTGGCTAT-3'

SEQ ID NO:1

F1r

5'-AGTAAGCATCCTCATCTTGG-3'

SEQ ID NO:2

F2f

5'-ATGAGGATGCTTACTAAGATGC-3'

SEQ ID NO:3

F2r

5'-TGGATCTATTACTCTGACTTGG-3'

SEQ ID NO:4

F3f

5'-AGAGTAATAGATCCAGGACTCG-3'

SEQ ID NO:5

F3r

5'-AGGCACCACAGGACTAAC-3'

SEQ ID NO:6

F4f

5'-CGAGCTCCGCTTCTTAGGAATCTCACTT-3' ...

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Abstract

The invention aims to provide a method for constructing a canine distemper virus (CDV) reverse genetics system. According to the method provided by the invention, in design of a primer, a full consideration is taken to ensure the integrity of the open reading frame of each gene segment when plasmid FA, FB and FC are constructed, particularly plasmid FB contains the complete H gene and F gene, the H gene and F gene are a research content of CDV main evolution and have an important impact on virulence and immunogenicity; the construction of FB plasmid provides convenience for the genetic manipulation of the CDV main research content in the future application of the method disclosed by the invention, in addition, selections of each primer and enzyme cutting sites are also based on the consideration and the isogenic cloning operation can be replaced by various major gene fragments complete enzyme cutting. In conclusion, compared with the existing method, the method has the advantages that the times of gene cloning can be greatly reduced by constructing the plasmids FA, FB and FC and convenient conditions are provided for future application with different purposes.

Description

technical field [0001] The invention belongs to the technical field of genomics research of pathogenic microorganisms, and in particular relates to a method for constructing a canine distemper virus reverse genetic system. Background technique [0002] Canine distemper is caused by canine distemper virus (Canine Distemper Virus, CDV) infection, has highly contact, fatal acute sepsis. In the paramyxovirus family, CDV belongs to the genus Morbillivirus with measles virus and rinderpest virus. It is a serologically closely related genus in the Paramyxoviridae family, but the host specificity is different. Canine distemper virus infection in dogs can result in occult infection, with intestinal symptoms, or with respiratory symptoms, sometimes with central nervous system symptoms. Neurological symptoms may also be a late manifestation of distemper infection. [0003] The canine distemper virus genome is a non-segmented RNA virus with a full length of 15690bp, which is highly co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/85C12N15/66C12R1/93
Inventor 单虎蒋文明杜翔黄娟杨瑞梅张传美秦志华
Owner QINGDAO AGRI UNIV
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