Construction and Application of Recombinant Duck Viral Enteritis Virus Vaccine Expressing Secreted Duck Tembusu Virus E Protein

A technology for duck Tembusu virus and duck viral enteritis, applied in the fields of application, antiviral agent, virus/bacteriophage, etc., can solve problems such as heavy workload, unclear classification, and low efficiency

Active Publication Date: 2016-04-13
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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  • Construction and Application of Recombinant Duck Viral Enteritis Virus Vaccine Expressing Secreted Duck Tembusu Virus E Protein
  • Construction and Application of Recombinant Duck Viral Enteritis Virus Vaccine Expressing Secreted Duck Tembusu Virus E Protein
  • Construction and Application of Recombinant Duck Viral Enteritis Virus Vaccine Expressing Secreted Duck Tembusu Virus E Protein

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 "CopyControlFosmidLibraryProductionKit" kit of EPICENTRE company.

[0051] The method is as follows: the DEV vaccine strain virus (CVCCAV1222) (GeneBankEU082088) (China Veterinary Microbiological Culture Collection Management Center, catalog number AV1222; purchased from the China Veterinary Drug Supervision Institute) DNA was physically used with a 25-gauge needle (purchased from Shanghai Zhi Yu Medical Devices Co., Ltd.) was pumped several times for cutting, and T4DNA Polymerase (T4DNAPolymerase, purchased from NewEngland Biolabs) and alkaline phosphatase (AlkalinePhosphatase, purchased from NewEngland Biolabs) were used to smooth the ends of the DNA fragments and dephosphorylate them. Pulse electrophoresis (using Bio-Rad's CHEFMapper The XAPulsedField system performs pulse electrophoresis, and the ...

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 DEVDNA fragment inserted into pCC1Fos. The sequences of the sequencing primers are as follows:

[0054] Primer1: 5'-TAATACGACTCACTATAGGG-3'

[0055] Primer2: 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. Use FseI, SbfI or PmeI endonuclease (all purchased from NewEngland Biolabs) to linearize the selected cosmid, the reaction conditions are as follows: SbfI endonuclease 20U (FseI or PmeI endonuclease can also be used), cosmid 10 μg , acted at 37°C for 1 hour, extracted with phenol / chloroform, and precipitated with ethanol to prepare DEVDNA for transfection.

[0059] Refer to the calcium phosphate method of ReddySM (2002) to co-transfect the second generation chicken embryo fibroblasts (CEF) with 5 segments of DEVDNA [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 parent...

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Abstract

The invention provides a recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus E protein, and a construction method and application thereof. The collection number is CCTCC V201214, and the name is rDEV-TE-tPAS. By using a recombinant clone technique, a gene segment SV40-E-tPA comprising an SV40 promoter, a duck Tembusu virus E protein and a tPA (Tissue plasminogen activator) signal peptide sequence is inserted into a spacer between the US7 and US8 genes of the duck viral enteritis virus to construct a cosmid in which the SV40-E-tPA expression frame is inserted between the US7 and US8 genes, thereby obtaining the recombinant duck viral enteritis virus vaccine CCTCC V201214 for expressing secretory duck Tembusu virus E protein. The invention also provides a method for constructing a recombinant duck viral enteritis virus vaccine strain and application of the recombinant duck viral enteritis virus vaccine strain in preparing a vaccine for preventing infectious diseases caused by duck viral enteritis virus and duck Tembusu virus.

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 CCTCCV201214 expressing secreted duck Tembusu virus E protein, which is named rDEV-TE-tPAS, its construction method and application. Background technique [0002] Duck enteritis virus (duckenteritisvirus, DEV), also known as duck virus enteritis virus. It can cause acute, febrile and septic acute infectious diseases in ducks, geese and other birds of Anseriformes. Compared with other herpes viruses, DEV has been less studied. The eighth report of the International Committee on Classification of Virology classified it as a herpes virus [1] , and the specific genus and classification have not yet been determined; until recently, the complete sequence of its genome was completely sequenced [2] . Since 2007, our laboratory has ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/34C12N15/63A61K48/00A61P31/20A61P31/22C12R1/93
Inventor 陈化兰柳金雄陈普成姜永萍
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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