Recombinant duck enteritis virus (DEV) vaccine strain for expressing avian influenza virus haemagglutinin (HA) gene and constructing method and application thereof

A technology of duck viral enteritis and avian influenza virus, which is applied in the field of recombinant duck viral enteritis virus vaccine and recombinant virus vaccine, can solve the problems of heavy workload, unclear classification and low efficiency

Active Publication Date: 2012-03-14
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 enteritis virus (DEV) vaccine strain for expressing avian influenza virus haemagglutinin (HA) gene and constructing method and application thereof
  • Recombinant duck enteritis virus (DEV) vaccine strain for expressing avian influenza virus haemagglutinin (HA) gene and constructing method and application thereof
  • Recombinant duck enteritis virus (DEV) vaccine strain for expressing avian influenza virus haemagglutinin (HA) gene and constructing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Construction of Genome Fosmid Library of DEV Vaccine Strain

[0056] The Fosmid library of the DEV genome was constructed according to the instructions of the "CopyControl Fosmid Library Production Kit" kit from EPICENTRE.

[0057] 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 (T4 DNA 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

[0058] Example 2. Selection for rescue of DEV cosmids

[0059] 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:

[0060] Primer 1: 5'-TAATACGACTCACTATAGGG-3'

[0061] Primer 2: 5'-GCCAAGCTATTTAGGTGAGA-3'

[0062] 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-SbfI-Pme I joints, which can overlap each other and can be spliced ​​to cover the entire DEV genome.

Embodiment 3

[0063] Example 3. Virus rescue

[0064] The DNA of the selected cosmids was extracted with Qiagen's medium extraction kit. The selected cosmids were linearized with Fse I, Sbf I or Pme I endonuclease (both purchased from New England Biolabs), and the reaction conditions were as follows: 20 U of SbfI endonuclease (Fse I or Pme I 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.

[0065] 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 dD...

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Abstract

The invention provides a recombinant duck enteritis virus (DEV) vaccine strain CCTCC NO:V201125 named rDEVus78Ha-Re6 for expressing an avian influenza virus haemagglutinin (HA) gene, and a constructing method and application thereof. The vaccine strain is prepared by the following steps of: inserting a genetic fragment SV40-HA comprising the avian influenza virus HA gene and an SV40 promoter sequence into a spacer between a US7 gene and a US8 gene of a DEV by adopting a recombinant cloning technology; constructing a cosmid for inserting an SV40-HA expression frame between the US7 gene and theUS8 gene; and saving to obtain the recombinant DEV vaccine strain CCTCC NO:V201125 for expressing the avian influenza virus HA gene. The invention further relates to a method for constructing the recombinant DEV vaccine strain and application of the recombinant DEV vaccine strain to preparation of a vaccine for preventing duck viral enteritis and avian influenza.

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 NO: V201125 expressing avian influenza virus hemagglutinin (HA) gene, named rDEVus78Ha-Re6, 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85A61K39/295A61K39/145A61K39/245A61P31/16A61P31/22C12R1/93
Inventor 陈化兰柳金雄步志高邓国华
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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