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Method for quickly constructing IBV (Avian Infectious Bronchitis Virus) reverse genetic strain

A bronchitis and reverse genetic technology, applied in the field of coronavirus reverse genetics, can solve the problems of transcript heterogeneity, coronavirus cannot be stably preserved, and the efficiency of enzymatic ligation is low, achieving high positive clone rate and improving rescue Efficiency and simple operation

Active Publication Date: 2017-09-22
ZHEJIANG UNIV
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Problems solved by technology

[0007] The present invention aims to establish a method for quickly constructing a reverse genetic strain of avian infectious bronchitis virus, which overcomes the problem that the cDNA clone of the coronavirus partial replicase gene in the prior art cannot be stably preserved in bacteria, and overcomes the problem that the traditional enzyme The problem of low cutting and ligation efficiency overcomes the problem of transcript heterogeneity caused by in vitro transcription

Method used

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  • Method for quickly constructing IBV (Avian Infectious Bronchitis Virus) reverse genetic strain
  • Method for quickly constructing IBV (Avian Infectious Bronchitis Virus) reverse genetic strain
  • Method for quickly constructing IBV (Avian Infectious Bronchitis Virus) reverse genetic strain

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Embodiment 1

[0056] The chicken infectious bronchitis virus vaccine strain H120 used in the present invention is preserved by the virus seed bank of this laboratory; the passaged cell BHK-21 used is preserved by the cell bank of this laboratory.

[0057] The cloning vector pBeloBAC11 and the Escherichia coli DH10B strain were donated by Professor Huang Yaowei, School of Animal Science, Zhejiang University.

[0058] RNA extraction reagents, PCR high-fidelity enzymes, homologous recombination kits, and site-directed mutagenesis kits were purchased from Vazyme Company, reverse transcription kits were purchased from Thermo Company, restriction enzymes BamHI and XhoI were purchased from Takara Company, and transfection Reagent jetPRIME was purchased from Dakwei Biotechnology Co., Ltd.

[0059] The primers were synthesized by Sangon Bioengineering Co., Ltd., and the sequence determination was completed by Boshang Biotechnology Co., Ltd.

[0060] 1. Construction strategy of full-length cDNA of I...

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Abstract

The invention discloses a method for quickly constructing an IBV (Avian Infectious Bronchitis Virus) reverse genetic strain, and belongs to the technical field of coronavirus reverse genetics. The constructing method comprises the following steps: quickly completing construction containing IBV genomic full-strength cDNA (Complementary Deoxyribose Nucleic Acid) clone by taking a BAC (Bacterial Artificial Chromosome) vector as a framework and applying an in-vitro homologous recombination technology, directly transfecting cells by a constructed recombinant plasmid, and transcribing in the cells, thus obtaining a transcript having infectivity; completing virus packaging; inoculating SPF (Specific Pathogen Free) chick embryo to a mixed solution of the cells and a culture medium and passing from generation to generation, thus obtaining the IBV reverse genetic strain. The constructing method disclosed by the invention has the advantages of simple operation and high positive cloning efficiency, the obtained IBV reverse genetic strain has passage stability, and an effective tool is provided for researching pathogenesis of the virus in vitro, developing a novel vaccine and the like; according to the method disclosed by the invention, transcription is carried out in the cells by utilizing a CMV (Cytomegalovirus) promoter added on a 5' terminal, and the rescue efficiency of the virus is greatly increased by utilizing an HDVR (Hepatitis Delta Virus Ribozyme) sequence added on a 3' terminal.

Description

technical field [0001] The invention relates to the technical field of coronavirus reverse genetics, in particular to a method for quickly constructing a reverse genetic strain of avian infectious bronchitis virus. Background technique [0002] Avian infectious bronchitis (IB) is one of the major infectious diseases that endanger the poultry industry. It is a highly contagious disease caused by avian infectious bronchitis virus (IBV). , mainly characterized by respiratory symptoms, decreased egg production of laying hens, decreased egg quality, kidney lesions and gastrointestinal lesions. IBV is prevalent in the world, and due to the great differences in pathogenicity and tissue tropism of different IBV strains, the serotypes are extremely complex, the lack of effective cross-protection between different serotypes, and the continuous emergence of new mutant strains, resulting in Prevention and control of IBV is very difficult. [0003] The causative agent of IB, IBV, is a ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N7/00C12R1/93
CPCC12N7/00C12N15/85C12N2770/20021C12N2770/20052C12N2800/204
Inventor 廖敏周继勇石婷婷曹尚尚颜焰
Owner ZHEJIANG UNIV
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