Recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of avian leukosis virus subgroup J and construction method and application of recombined chicken Marek's disease virus vaccine strain

A technology of avian leukosis virus and chicken Marek's disease, applied in the field of recombinant chicken Marek's disease virus vaccine strain and its construction

Active Publication Date: 2016-06-22
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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Problems solved by technology

[0007] The present invention constructs a recombinant live vector vaccine expressing ALV-J protective antigen through the infe

Method used

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  • Recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of avian leukosis virus subgroup J and construction method and application of recombined chicken Marek's disease virus vaccine strain
  • Recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of avian leukosis virus subgroup J and construction method and application of recombined chicken Marek's disease virus vaccine strain
  • Recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of avian leukosis virus subgroup J and construction method and application of recombined chicken Marek's disease virus vaccine strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] The construction of embodiment 1 MDV vaccine 814 strain genome Fosmid libraries

[0094] Marek's disease virus vaccine strain 814 (Zhang, F., Liu, C.J., Zhang, Y.P., et al. Comparative full-length sequence analysis of Marek's disease virus vaccine 814. Arch Virol. 2012, 157 (1): 177-183. Institute preserved, provided. The GenBank accession number of the whole genome sequence of the 814-strain attenuated Marek's disease virus vaccine is JF742597.

[0095] The MDV genome Fosmid library was constructed according to the instructions of the Epicentre CopyControlTMFosmidLibraryProductionKit kit. Methods as below:

[0096] Genomic DNA of 814 strains of MDV vaccine was aspirated repeatedly 50-100 times with a 200 μ L pipette tip by physical means, and passed through pulsed field electrophoresis (CHEF of BioRad Company). XAPulsedField ElectrophoresisSystem system, the conditions are: electrophoresis buffer 0.5 × TBE, gel concentration 1%, program 5-220kb) analysis, until its...

Embodiment 2

[0098] Embodiment 2 virus rescue

[0099] According to the sequencing analysis of recombinant cosmid ends, 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. The selected cosmid DNA was extracted with the extraction kit from QIAGEN Company. The extracted cosmids were linearized with NotI (NEB Company): NotI endonuclease 100U, cosmids 10 μg, 37°C for 2h. The digested product was extracted with phenol-chloroform-isoamyl alcohol, and precipitated with ethanol to prepare MDV genomic DNA for transfection.

[0100] The five MDV genomic DNA fragments were co-transfected into the secondary CEF cells by calcium phosphate transfection method. The preparation method of CEF cells is as follows: take 9-10 day-old SPF chicken embryos, aseptically take out the chicken embryos, place them in a plate filled with...

Embodiment 3

[0104] Example 3 Construction of a recombinant mutant cosmid inserting the CAG-ALVGE expression framework (SEQ ID No.1) inside the US2 gene of the MDV genome

[0105] Based on the MDV multi-segment cosmid rescue system established above, within the US2 gene of the MDV genome in the selected 5-cosmid group member p814-5, specifically, the US2 gene has a total of 813bp, and in the present invention, the US2 gene is deleted. The 15th to 630th nucleotides are replaced by inserting the CAG-ALVGE expression frame (the nucleotide sequence of the CAG-ALVGE expression frame is SEQ ID NO.1, and its structure is CMV enhancer-chicken β-actin promoter-Gag gene -IRES2 sequence-Env gene-sv40PolyA), construct a recombinant mutant cosmid p814-5US2ALVGE.

[0106] The construction process of the recombinant mutant cosmid p814-5US2ALVGE is briefly described as follows:

[0107] 3.1 Construction of pKSKanccdB plasmid

[0108] Three pairs of primers shown in Table 1 were used for multiplex PCR am...

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Abstract

The invention discloses a recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of an avian leukosis virus subgroup J (ALV-J) and a construction method and application of the recombined chicken Marek's disease virus vaccine strain, and belongs to the technical field of medicine or veterinary medicine. By means of the recombination and clone technology, a gene segment CAG-ALVGE containing the ALV-J Gag and Env genes and a CAG promoter sequence is inserted in a US2 gene of the strain 814 of the chicken Marek's disease virus, a recombined cosmid with a CAG-ALVGE expression frame inserted in a US2 gene is constructed, and the recombined chicken Marek's disease virus vaccine strain for expressing the Gag and Env genes is obtained through salvation of the recombined cosmid. Research shows that the obtained vaccine strain has the same in-vitro replication ability as a parent virulent vaccine strain 814 and good hereditary stability, and can resist attacks of a very virulent MDV strain and a very virulent ALV-J strain at the same time. It can be seen that the obtained recombined MDV vaccine strain can be used for preparing medicine for preventing or treating avian leukosis and the chicken Marek's disease.

Description

technical field [0001] The present invention relates to a recombinant chicken Marek's disease virus vaccine strain and its construction method and application, in particular to a recombinant chicken Marek's disease virus vaccine strain expressing J subgroup avian leukosis virus Gag and Env genes and its construction method and application , The invention belongs to the technical field of medicine or veterinary medicine. Background technique [0002] Avian leukemia is a neoplastic disease caused by the excessive proliferation of certain cellular components in hematopoietic tissues of poultry caused by avian leukemia virus (ALV). According to the difference of pathogenicity and host, ALV is divided into A-J10 subgroups, among which A-D are exogenous viruses, which mainly infect laying hens, and A and B subgroups mainly induce lymphocytic leukemia in chickens. J subtype avian leukemia virus (ALV-J) is a new enveloped subgroup isolated from broiler chickens by Payne of England ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85A61K39/295A61K39/21A61K39/245A61P31/14C12R1/93
CPCA61K39/12A61K2039/70C07K14/005C12N7/00C12N15/85C12N2710/16321C12N2710/16334C12N2710/16351C12N2740/11022C12N2740/11034C12N2800/107
Inventor 李凯高玉龙王笑梅刘长军张艳萍崔红玉高立祁小乐王永强
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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