Marek's disease virus infectious recombinant cloning system and its construction method and application

A Marek's disease and infectious technology, applied in the field of Marek's disease virus infectious recombinant cloning system and its construction, virus infectious recombinant cloning system, can solve problems such as lag and achieve good safety effects

Active Publication Date: 2017-12-12
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

In addition, compared with other herpesviruses, the research on MDV is relatively lagging behind, and there are few reports on the insertion sites of foreign genes in its genome

Method used

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  • Marek's disease virus infectious recombinant cloning system and its construction method and application
  • Marek's disease virus infectious recombinant cloning system and its construction method and application
  • Marek's disease virus infectious recombinant cloning system and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of Genome Fosmid Library of MDV Vaccine 814 Strains

[0048] 1.1 Strains

[0049] 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 strain 814. Arch Virol. 2012, 157(1): 177-183 .) Preserved and provided by Harbin Veterinary Research Institute, 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.

[0050] 1.2 MDV culture and virus genome extraction

[0051] The attenuated Marek's disease virus vaccine strain 814 was inoculated into primary chicken embryo fibroblast (CEF) cells, and when the virus grew to about 90% of the cells, the cells were repeatedly frozen and thawed three times, and the cells and supernatant were collected. Centrifuge the cells and supernatant at 32000rpm for 45min, discard the supernatant; wash th...

Embodiment 2

[0063] Example 2 Virus rescue

[0064] 2.1 Virus rescue

[0065] 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.

[0066] 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 (NEB Company): NotI endonuclease 100U, cosmids 10 μg, 37°C for 2h. 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 acetate and 2 times volume of absolute ethanol were added, Precipitate at -20°C for 2 hours; centrifuge at 1...

Embodiment 3

[0078] Example 3 Mutation of recombinant cosmid

[0079] Based on the above established 5-cosmid infectious cloning platform, the UL41 gene, between the UL45 and UL46 genes of the MDV genome in p814-3, between the UL55 and LORF10 genes of the MDV genome in p814-4, and the MDV genome in p814-5 The eGFP expression framework was inserted into the US10 gene and US2 gene of the genome to construct 5 recombinant mutant cosmids p814-3UL41eGFP, p814-3UL45 / 46eGFP, p814-4UL55 / 10eGFP, p814-5US10eGFP, p814-5US2eGFP (the construction mode can be found in Figure 6 H-L). The process is briefly described as follows:

[0080] 3.1 Construction of pKS KanccdB

[0081] Three pairs of primers shown in Table 1 were used to perform multiplex PCR amplification on pDEST22 plasmid (Invitrogen Company) and pMOD6 plasmid (Epicentre Company) respectively.

[0082] Table 1 PCR primers for cloning Kan-ccdB expression framework

[0083] Primer name

Sequence (5'-3')

R1F

GCG TCTA...

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Abstract

The invention discloses a Marek's disease virus (MDV) infectivity recombinant cloning system, and a construction method and application thereof and belongs to the field of biotechnologies. The MDV infectivity recombinant cloning system comprises a plurality of cosmids, wherein an MDV vaccine strain gene segment is cloned in each cosmid; the MDV vaccine strain gene segment contains mutually superposed regions and can be spliced to cover an integral MDV vaccine strain genome; an exogenous gene is inserted into a non-essential region for replication of at least one of the cosmids. The MDV infectivity recombinant cloning system can quickly and efficiently clone an exogenous gene expression cassette into the MDV genome to further realize rescue of the recombinant MDV. The recombinant cloning system can be used for very conveniently inserting different foreign genes into the corresponding regions so as to construct an MDV living-vector multi-vaccine.

Description

technical field [0001] The invention relates to a virus infectious recombination cloning system, in particular to a Marek's disease virus infectious recombination cloning system and its construction method and application, belonging to the field of biotechnology. 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. 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. MDV belongs to the genus Marek's disease-like virus of the α-herpesvirus subfamily, and is a cell-associated herpes virus. Its genome is a linear double-stranded DNA, about 180kb, and its composition mode is TR L -U L -IR L -IR S -U S -TR S . MDV encodes at least 103 proteins, most of which remain unidentified. MDV includes three serotypes, serotype 1 virus is tumorige...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/38C12N7/01A61K39/245A61P31/22C12R1/93
Inventor 李凯王笑梅刘长军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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