Construction of recombinant MDV (Marek's Disease Virus) and vIL-8 (Viral Interleukin 8) double-gene deleted strain and application thereof

A technology of Marek's disease and recombinant virus, applied in the field of animal virology, to achieve the effect of good immune protection

Active Publication Date: 2017-05-31
北京邦卓生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Knocking out the Meq gene can make MDV lose its virulent tumorigenicity, but the Meq deletion strain can still cause slight immunosuppression

Method used

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  • Construction of recombinant MDV (Marek's Disease Virus) and vIL-8 (Viral Interleukin 8) double-gene deleted strain and application thereof
  • Construction of recombinant MDV (Marek's Disease Virus) and vIL-8 (Viral Interleukin 8) double-gene deleted strain and application thereof
  • Construction of recombinant MDV (Marek's Disease Virus) and vIL-8 (Viral Interleukin 8) double-gene deleted strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] ——Construction of recombinant MDV BZ-1 strain

[0038] 1. Replace the meq gene in the MDV BJ07 strain virus with the marker gene rpsl-neo

[0039] According to references (Schumacher D, Tischer BK, Fuchs W, Osterrieder N: Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA cloned as abacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant. Journal of virology 2000,74(23):11088-11098.),构建MDVBJ07株(CGMCC No.13598)强毒的BAC感染性克隆毒rBJ07。 According to the sequences shown in Table 1, a pair of primers MDV-rpsl-neo-F / R (SEQ ID NO: 3 and SEQ ID NO: 4) were synthesized. The uppercase letters in the primers indicate the 50 bp sequences of the MDV genome located upstream and downstream of the meq gene in rBJ07, respectively, and the lowercase letters indicate the 5′ and 3′ sequences of rpsL-neo of the marker gene Counter-Selection BACModification Kit kit. Using rpsL-neo template DNA as a template for PCR reaction, a 1419bp PCR ta...

Embodiment 2

[0058] - Validation of recombinant virus MDV BZ-1 strain

[0059] BZ-1 was seeded on 6-well cell culture plates grown into monolayers of CEFs at a dose of 100 PFU / well. At the same time, a BJ07 virus control was set up and cultured for 3-5 days. After MDV-specific plaques appeared, the cell culture medium was poured out, fixed with cold acetone:ethanol (3:2) fixative solution at room temperature for 8 minutes, and washed once with PBS buffer. , add 500ul (working concentration) antibody (anti-meq gene serum) as the primary antibody, put it in a 37°C incubator for 45 minutes, wash it three times with PBS, and dry at room temperature, add 500ul (working concentration) FITC-labeled fluorescent antibody as a The secondary antibody was reacted in an incubator at 37°C for 45 min, washed three times with PBS, dried by drying, and covered with 50% glycerol (glycerol:PBS=1:1) to prevent water volatilization, and observed under an inverted fluorescence microscope. The results showed th...

Embodiment 3

[0061] ——The pathogenicity of BZ-1 strain virus to chickens

[0062] BZ-1 infected 25 1-day-old SPF chickens with a dose of 5000 PFU. After 13 weeks of infection, there was no MD-specific death, no lesions, and no tumor formation. The body weight and the ratio of the central immune organs thymus and bursa to body weight were all the same as those of BZ-1. There was no significant difference between the blank control chickens, which did not cause immunosuppression in the flock.

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Abstract

The invention provides a recombinant serum I type MDV (Marek's Disease Virus) (a BZ-1 strain). The recombinant serum I type MDV is actually characterized in that pathogenic associated genes (meq and vIL-8 (Viral Interleukin 8) genes) of an isolated MDV virulent strain BJ07 are knocked out at the same time, and a constructed MDV gene deleted strain (the BZ-1 strain) has no pathogenicity, cannot induce tumor of chicken and has no immunosuppressive action on chicken flocks; the BZ-1 strain is used as a vaccine prepared by a strain produced by MD (Marek's Disease) live vaccine, chicken MD induced by a very virulent MDV can be prevented, and the protective immune effect is superior to that of a CVI988/Rispens strain vaccine which is most widely applied in domestic and foreign markets at present.

Description

technical field [0001] The invention relates to the construction and application of a recombinant Marek's disease virus meq and vIL-8 double gene deletion strain, and belongs to the field of animal virology. Background technique [0002] Marek's disease (MD) is one of the most common lymphoproliferative diseases in chickens caused by Marek's disease virus (MDV). Lymphoid cell infiltration, proliferation and tumor formation are characterized. [0003] The disease was first discovered by the Hungarian veterinary pathologist Marek in 1907 and named Marek's disease in 1961. At present, it is prevalent in all major chicken-raising countries and regions in the world, and it is also one of the most important poultry diseases in the economic sense in my country, causing serious threats and huge economic losses to my country's chicken production. [0004] The Meq gene of the Marek's disease virus genome contains a base leucine zipper (bZIP) domain at the N-terminus similar to the j...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K39/245A61P31/22C12R1/93
CPCA61K39/12A61K2039/552C07K14/005C12N7/00C12N2710/16321C12N2710/16334C12N2710/16343
Inventor 毕建敏
Owner 北京邦卓生物科技有限公司
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