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A method for constructing canine distemper virus reverse genetics system

A reverse genetics, plasmid technology, applied in the field of pathogenic microorganism genomics research, can solve the problems of low efficiency, error, time-consuming and so on

Active Publication Date: 2016-03-09
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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  • A method for constructing canine distemper virus reverse genetics system
  • A method for constructing canine distemper virus reverse genetics system
  • A method for constructing canine distemper virus reverse genetics system

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Experimental program
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Embodiment Construction

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

[0037] 1. Amplification of the whole CDV3 gene

[0038] 1. Primer design

[0039] According to the CDV3 (GenBank: EU726268.1) sequence in GeneBank, 9 pairs of primers were designed using Primer5.0 (Table 1), and some of the adjacent fragment PCR amplification primers The PCRCloningSystem 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’-CGAGCTCCGCTTCTAGGAATCTCACTT-3’

SEQ ID NO: 7

F4r

5’-ATCATATCACCTCCAGAGTATC-3’

SEQ ID NO: 8

F5f

5’-AGTCCTGTGGTGCCTCGGAAT-3’

SEQ ID...

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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 pathogenic microbial genomics research, and specifically relates to a method for constructing a reverse genetic system of canine distemper virus. Background technique [0002] Canine distemper is caused by canine distemper virus (Canine Distemper Virrus, CDV) infection, which is highly contact and fatal acute sepsis. In the Paramyxovirus family, CDV belongs to the measles virus genus with measles virus and rinderpest virus. It is a serologically closely related genus in the Paramyxovirus family, but its host specificity is different. Canine distemper virus infection in dogs can lead to recessive infection, intestinal symptoms, or respiratory symptoms, sometimes accompanied by central nervous system symptoms. Neurological symptoms may also be the late manifestations of canine distemper infection. [0003] Canine distemper virus genome is a non-segmented RNA virus with a total length of 15690 bp. There is a high...

Claims

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Application Information

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Patent Type & Authority Patents(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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