Recombinant duck plague virus vaccine strain as well as construction method and application thereof

A technology for duck plague virus and duck viral enteritis, which is applied to vaccines, viruses, antiviral agents, etc., and can solve problems such as unclear classification, low efficiency, and heavy workload

Active Publication Date: 2020-03-24
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

However, for viruses such as DEV, which lack basic research, unclear classification, and unknown non-essential genes, using the first or second method to construct recombinant viruses has a large workload and low efficiency
The difficulty of the third method lies in the establishment of polymysmid infectious clones. If this platform is successfully constructed, it will be able to quickly and effectively construct recombinant viruses.

Method used

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  • Recombinant duck plague virus vaccine strain as well as construction method and application thereof
  • Recombinant duck plague virus vaccine strain as well as construction method and application thereof
  • Recombinant duck plague virus vaccine strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1. Construction of Genome Fosmid Library of DEV Vaccine Strain

[0077] The Fosmid library of the DEV genome was constructed according to the instructions of the "CopyControl Fosmid Library Production Kit" kit from EPICENTRE.

[0078] The method is as follows: the DNA of the DEV vaccine strain virus (CVCC AV1222) (GeneBank EU082088) (China Veterinary Microbiological Culture Collection Management Center, catalog number AV1222; purchased from the China Shanghai Zhiyu Medical Instrument Co., Ltd.) was pumped several times to cut the DNA fragments with T4 DNA polymerase (T4DNA Polymerase, purchased from New England Biolabs) and alkaline phosphatase (Alkaline Phosphatase, purchased from New England Biolabs). End smoothing and dephosphorylation treatment, pulse electrophoresis (with Bio-Rad CHEF The XA Pulsed Field system performs pulse electrophoresis. The conditions of pulse electrophoresis are: the electrophoresis buffer is 0.5xTBE, the agarose gel concentration...

Embodiment 2

[0079] Example 2. Selection for rescue of DEV cosmids

[0080] After the library was successfully constructed, 286 clones were picked to extract the cosmids, and the alkaline lysis method was used to [5] Extract the cosmid and send it to Dalian Bao Biological Co., Ltd. to sequence the end of the DEV DNA fragment inserted into pCC1 Fos. The sequences of the sequencing primers are as follows:

[0081] Primer 1: 5'-TAATACGACTCACTATAGGG-3'

[0082] Primer 2: 5'-GCCAAGCTATTTAGGTGAGA-3'

[0083] After terminal sequencing analysis, a total of 250 clones with complete Fse I-Sbf I-Pme I adapters connected to both ends of the insert fragment were obtained. Multiple sets of 5-cosmid combinations for rescue of DEV were selected from these 250 clones. Both ends of the DEV DNA fragments cloned in each group contain Fse I-Sbf I-Pme I joints, which can overlap each other and can be spliced ​​to cover the entire DEV genome.

Embodiment 3

[0084] Example 3. Virus rescue

[0085] The DNA of the selected cosmids was extracted with Qiagen's medium extraction kit. The selected cosmids were linearized with FseI, SbfI or PmeI endonuclease (both purchased from New England Biolabs), and the reaction conditions were as follows: 20 U of SbfI endonuclease (FseI or PmeI endonuclease could also be used) Dicer), cosmid 10 μg, reacted at 37°C for 1 hour, extracted with phenol / chloroform, and precipitated with ethanol to prepare DEV DNA for transfection.

[0086]Refer to the calcium phosphate method of Reddy SM (2002) to co-transfect the second generation chicken embryo fibroblasts (CEF) with 5 segments of DEV DNA [28] After multiple repetitions, 3 groups of 5-cosmid combinations showed typical lesions of DEV virus after 4-6 days of transfection. A group of 5-cosmid combinations with good repeatability was selected for subsequent experiments. Harvest the rescued virus co-transfected with the group of 5 cosmids, named dDEV, an...

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Abstract

The invention provides a recombinant duck plague virus vaccine strain as well as a construction method and application thereof. The recombinant duck plague virus vaccine strain provided by the invention comprises one or more antigen coding sequences inserted in a spacer region between US8 and US1 genes of a duck enteritis virus (DEV) genome. The invention also relates to the method for constructing the recombinant duck plague virus vaccine strain, and the application of the recombinant duck plague virus vaccine strain for preparing a vaccine for preventing diseases caused by duck virus and / orbacterial infection. The recombinant duck plague virus vaccine strain provided by the invention does not affect the immune effect of the DEV, and provides immune protection against diseases caused byother duck virus and / or bacterial infection.

Description

technical field [0001] The invention belongs to the field of recombinant virus vaccines, more specifically to the field of recombinant duck viral enteritis virus vaccines. The invention provides a recombinant duck viral enteritis virus bivalent vaccine strain CCTCC V201840 expressing avian influenza virus hemagglutinin (HA) gene, named rDEV-HA H5 / H7, its construction method and application. Background technique [0002] Duck enteritis virus (DEV), also known as duck viral enteritis virus. It can cause acute, febrile and septic acute infectious diseases in ducks, geese and other birds of Anseriformes. However, there are relatively few studies on DEV compared with other herpes viruses. Therefore, the eighth report of the International Commission on Classification of Virology classified it as a herpes virus [1] , without being able to classify it further. Until recently, its full sequence was not fully tested [2] . Since 2007, our laboratory has started the sequencing of ...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K39/295A61K39/145A61K39/25A61P31/16A61P31/22
CPCC12N7/00A61K39/12A61P31/16A61P31/22C12N2760/16122C12N2760/16134C12N2710/16721C12N2710/16734A61K2039/70A61K2039/5256A61K2039/552Y02A50/30
Inventor 陈化兰柳金雄陈普成姜永萍邓国华施建忠
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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