Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof

A technology for duck viral enteritis and avian influenza virus, which can be used in applications, antiviral agents, genetic engineering, etc., and can solve problems such as low efficiency, large workload, and unclear classification.

Active Publication Date: 2012-07-04
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 virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof
  • Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof
  • Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as 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 instructions of the "CopyControl Fosmid Library Production Kit" kit from EPICENTRE.

[0051] The method is as follows: the DNA of the DEV vaccine strain virus (CVCC AV 1222) (GeneBank EU082088) (China Veterinary Microbiological Culture Collection Center (CVCC), catalog number AV1222; purchased from the China Veterinary Drug Supervision Institute) DNA was used in physical methods and used No. 25 The needle (purchased from Shanghai Zhiyu Medical Instrument Co., Ltd.) was sucked several times to cut the DNA fragments with T4DNA Polymerase (T4DNA Polymerase, purchased from New England Biolabs) and alkaline phosphatase (AlkalinePhosphatase, purchased from New England Biolabs). Carry out terminal smoothing and dephosphorylation treatment, pulse electrophoresis (use Bio-Rad company CHEF The XA Pulsed Field system performs pulse...

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 cosmids were extracted by the alkaline lysis method [5], and sent to Dalian Bao Biological Company 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 pa...

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Abstract

The invention provides a recombinant duck virus enteritis virus vaccine strain CCTCC No.V201026, named as rDEVul41HA, for expressing avian influenza virus hemagglutinin (HA) genes and a construction method as well as application thereof. Specifically, gene segments SV40-HA containing avian influenza virus hemagglutinin HA genes and a SV40 promoter sequence are inserted into UL41 genes of duck virus enteritis virus to construct cosmids with SV40-HA expression cassette inserted into UL41 genes; and by the cosmids, the recombinant duck virus enteritis virus vaccine strain CCTCC V201026 for expressing the avian influenza virus hemagglutinin (HA) genes is saved and obtained. The invention also relates to a method for constructing the recombinant duck virus enteritis virus vaccine strain and application of the recombinant duck virus enteritis virus vaccine strain to preparing vaccines for preventing duck virus enteritis and avian influenza.

Description

technical field [0001] The invention relates 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 V201026 expressing avian influenza virus hemagglutinin (HA) gene, named rDEVul41HA, 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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