Reverse genetic operating system for Marek's disease virus attenuated vaccine strain 814 and application thereof in virus rescue

A technology for Marek's disease and attenuated vaccine, applied in the field of reverse genetic operating system, which can solve the problems of high copy number and low library stability

Inactive Publication Date: 2015-08-12
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, Cosmid is a traditional cosmid, which uses the same replicon as the plasmid, and its high copy number makes the library built with low stability and certain bias

Method used

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  • Reverse genetic operating system for Marek's disease virus attenuated vaccine strain 814 and application thereof in virus rescue
  • Reverse genetic operating system for Marek's disease virus attenuated vaccine strain 814 and application thereof in virus rescue
  • Reverse genetic operating system for Marek's disease virus attenuated vaccine strain 814 and application thereof in virus rescue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of Genome Fosmid Library of MDV Vaccine 814 Strains

[0046] 1. Materials and Methods

[0047] 1.1 Strains

[0048] The attenuated vaccine strain 814 of Marek's disease virus has been recorded in the following literature: Sequence determination and analysis of genome repeat region of Marek's disease virus "814" vaccine strain, Zhang Feng et al., Chinese Journal of Preventive Veterinary Medicine, 32(4), 2010. It is now preserved and provided by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The GeneBank accession number of the whole genome sequence of the 814-strain attenuated Marek's disease virus vaccine is JF742597.

[0049] 1.2 Main reagents and instruments

[0050] Micrococcal nuclease, low-range PFG Marker, and β-agarase I were purchased from NEB Company; CopyControlTM Fosmid Library Production Kit was purchased from Epicentre Company; Calcium Phosphate Transfection Kit was purchased from Invitro...

Embodiment 2

[0068] Embodiment 2: virus rescue and identification

[0069] 1. Method

[0070] 1.1 Virus rescue

[0071] 1) Selection of cosmids for virus rescue: According to the cosmid end sequencing analysis, 6 groups of 5 cosmid combinations were selected. The 5 cosmids in each combination were cloned with 814 genomic DNA fragments of MDV vaccine, which contained overlapping regions and could be spliced ​​to cover the complete MDV genome.

[0072] 2) Extraction and linearization of recombinant cosmids: the selected cosmid DNA was extracted with a medium extraction kit from QIAGEN Company. The extracted cosmids were linearized with NotI (purchased from NEB Company). The reaction conditions were: 100 U of NotI endonuclease, 10 μg of cosmids, and 37° C. for 2 hours. The digested product was extracted twice with equal volume of phenol-chloroform-isoamyl alcohol and once with chloroform, centrifuged at 8000rpm for 5min each time, and the supernatant was taken; 1 / 10 volume of 3M sodium ace...

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Abstract

The invention discloses a reverse genetic operating system for a Marek's disease virus attenuated vaccine strain 814 and an application thereof in virus rescue. A genome of the Marek's disease virus attenuated vaccine strain 814 is cut and then subjected to segmented cloning into a Fosmid cosmid vector pCC1Fos, and a Fosmid library of the genome of the vaccine strain 814 is constructed. On the basis of genome sequencing on the library, 5 recombinant cosmids which are cloned with the vaccine strain 814 genome segment, mutually contain overlapping regions and can be spliced to cover the whole genome of the strain 814 are selected. The 5 cosmids are used for co-transfecting chicken embryo fibroblasts (CEF), and viruses having same biological characteristics with an original MDV vaccine strain are successfully rescued. With the application of the established reverse genetic operating system, firstly, the attenuated vaccine strain can be rescued and used as a vaccine for MDV prevention; and more importantly, with the use of the reverse genetic operating system, virus transformation can be implemented in bacteria, so as to be used in development of MDV live vector vaccines.

Description

technical field [0001] The present invention relates to the field of virus genetic manipulation, more specifically, the present invention relates to a reverse genetic operating system of Marek's disease virus attenuated vaccine 814 strain and its application in virus rescue. Background technique [0002] Marek's disease (MD) is a contagious neoplastic disease of chickens caused by Marek's disease virus (MDV), characterized by lymphoid tissue hyperplasia and tumor formation, peripheral nerves, gonads, iris, various organs, muscles and The skin was infiltrated with mononuclear cells. MDV spreads through the air and is highly contagious. Infected chickens produce tumors and die, causing huge economic losses. Marek's disease is one of the main diseases of chickens, and it is also the most important chicken disease at home and abroad since the 1950s. [0003] MDV is a cell-associated herpes virus whose genome is linear double-stranded DNA. MDV includes three serotypes, serotyp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N7/01C12R1/93
Inventor 李凯刘长军王笑梅张艳萍高玉龙崔红玉高立
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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