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Recombined duck virus enteritis viral vaccine strain CCTCC for expressing bird flu virus hemagglutinin (HA) gene (rDEVus78Ha) as well as establishing method and application thereof

A technology of duck viral enteritis and avian influenza virus, applied in application, antiviral agents, genetic engineering, etc., can solve problems such as unclear classification, heavy workload, and low efficiency

Active Publication Date: 2012-07-11
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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  • Recombined duck virus enteritis viral vaccine strain CCTCC for expressing bird flu virus hemagglutinin (HA) gene (rDEVus78Ha) as well as establishing method and application thereof
  • Recombined duck virus enteritis viral vaccine strain CCTCC for expressing bird flu virus hemagglutinin (HA) gene (rDEVus78Ha) as well as establishing method and application thereof
  • Recombined duck virus enteritis viral vaccine strain CCTCC for expressing bird flu virus hemagglutinin (HA) gene (rDEVus78Ha) as well as establishing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Construction of Genome Fosmid Library of DEV Vaccine Strain

[0050] The Fosmid library of the DEV genome was constructed according to the "CopyControl Fosmid Library Production Kit" kit instructions of EPICENTRE company.

[0051] 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 for cutting, and T4DNA Polymerase (T4DNA Polymerase, purchased from New England Biolabs) and alkaline phosphatase (Alkaline Phosphatase, purchased from New England Biolabs) were used to smooth the ends of the DNA fragments. Phosphorylation and dephosphorylation treatment, pulse electrophoresis (using Bio-Rad CHEF The XA Pulsed Field system performs pulse electrophoresis. The conditions of pulse electrophoresis are: the electrophoresis buffer is ...

Embodiment 2

[0052] Example 2. Selection for rescue of DEV cosmids

[0053] 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 pCC1Fos. The sequences of the sequencing primers are as follows:

[0054] Primer 1: 5'-TAATACGACTCACTATAGGG-3'

[0055] Primer 2: 5'-GCCAAGCTATTTAGGTGAGA-3'

[0056] After terminal sequencing analysis, a total of 250 clones with complete FseI-SbfI-PmeI 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 FseI-SbfI-PmeI joints, which can overlap each other and can be spliced ​​to cover the entire DEV genome.

Embodiment 3

[0057] Example 3. Virus rescue

[0058] 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 can also be used) Enzyme), cosmid 10 μg, 1 hour at 37°C, phenol / chloroform extraction, ethanol precipitation to prepare DEV DNA for transfection.

[0059] 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, and use the dDEV and the par...

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Abstract

The invention provides a recombined duck virus enteritis viral vaccine strain CCTCC for expressing bird flu virus hemagglutinin HA gene with V201025, which is named as rDEVus78Ha, as well as an establishing method and application thereof. Particularly, by utilizing the recombination clone technology, the gene segment SV40-HA including the bird flu virus hemagglutinin HA gene and SV40 promoter sequence is inserted in the spacer between the US7 and US8 genes of the duck virus enteritis virus, so as to establish and obtain the cosmid inserted into the SV40-HA expression cassette between the US7 and US8 genes, as a result, the recombined duck virus enteritis viral vaccine strain CCTCC for expressing bird flu virus hemagglutinin HA gene with V201025 is saved and obtained. The invention also relates to a method for establishing the recombined duck virus enteritis viral vaccine strain and the application of the recombined duck virus enteritis viral vaccine strain to preparing the vaccine for preventing duck virus enteritis and bird flu.

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 vaccine strain CCTCC V201025 expressing avian influenza virus hemagglutinin (HA) gene, named rDEVus78Ha, 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 the genome o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/44A61K39/215A61P31/12
Inventor 陈化兰柳金雄步志高姜永萍
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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