Construction and application of recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus M/E protein

A technology for duck tembusu virus and duck virus enteritis, which can be applied in the directions of virus/phage, application, antiviral agent, etc., and can solve the problems of large workload, low efficiency, unclear classification, etc.

Active Publication Date: 2014-03-26
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 effic

Method used

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  • Construction and application of recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus M/E protein
  • Construction and application of recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus M/E protein
  • Construction and application of recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus M/E protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0051] The method is as follows: DNA of DEV vaccine strain virus (CVCC AV1222) (GeneBank EU082088) (China Veterinary Microbial Culture Collection and Management Center, catalogue number AV1222; purchased from China Veterinary Drug Administration) DNA was prepared by physical method using a 25-gauge needle (purchased from Shanghai Zhiyu Medical Instrument Co., Ltd.) aspirated for several times to cut the DNA fragments. T4 DNA polymerase (T4 DNA Polymerase, purchased from New England Biolabs) and alkaline phosphatase (Alkaline Phosphatase, purchased from New England Biolabs) were used to treat the DNA fragments. End smoothing and dephosphorylation, pulse electrophoresis (with Bio-Rad's CHEF The XA Pulsed Field system performs pulse electrophoresis. T...

Embodiment 2

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

[0053] After the library was successfully constructed, 286 clones were picked to extract the cosmid, and the alkaline lysis method was used to extract the cosmid. [5] The cosmids were extracted and sent to Dalian Bao Bio Co., Ltd. to sequence the ends of the DEV DNA fragments inserted into pCC1 Fos. The sequences of the sequencing primers are as follows:

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

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

[0056] After end-sequencing analysis, a total of 250 clones with complete Fse I-SbfI-Pme I linkers connected to both ends of the insert were obtained. Sets of 5 cosmid combinations for DEV rescue were selected from these 250 clones. Both ends of the cloned DEV DNA fragments in each group contain Fse I-Sbf I-PmeI linkers, which can overlap each other and cover the entire DEV genome.

Embodiment 3

[0057] Example 3. Virus rescue

[0058]The DNA of the selected cosmids was extracted with the Qiagen company's medium volume extraction kit. Selected cosmids were linearized with Fse I, Sbf I or Pme I endonucleases (all purchased from New England Biolabs) under the following reaction conditions: Sbf I endonuclease 20U (Fse I or Pme I can also be used endonuclease), 10 μg of cosmid, treated at 37°C for 1 hour, extracted with phenol / chloroform, and precipitated with ethanol to prepare DEV DNA for transfection.

[0059] According to the calcium phosphate method of Reddy SM (2002), 5 segments of DEV DNA were co-transfected into secondary chicken embryo fibroblasts (CEF) [28] , after many repetitions, 3 groups of 5 cosmid combinations were transfected 4-6 days after the typical lesions of DEV virus were seen in CEF, and a group of 5 cosmid combinations with better repeatability was selected for subsequent experiments. The virus rescued by co-transfection of this set of 5 cosmids ...

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Abstract

The invention provides a recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus M/E protein, and a construction method and application thereof. The collection number is CCTCC NO:V201215, and the name is rDEV-TME-tPAS. By using a recombinant clone technique, a gene segment rDEV-TME-tPAS comprising an SV40 promoter, duck Tembusu virus M and E proteins 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 rDEV-TME-tPAS expression frame is inserted between the US7 and US8 genes, thereby obtaining the recombinant duck viral enteritis virus vaccine CCTCC V201215 for expressing secretory duck Tembusu virus M/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 particularly to the field of recombinant duck virus enteritis virus vaccines. The invention provides a recombinant duck virus enteritis virus vaccine strain CCTCC V201215 expressing secreted duck Tembusu virus M protein and E protein, named rDEV-TME-tPAS, and a construction method and application thereof. Background technique [0002] Duck enteritis virus (DEV), also known as duck enteritis virus. It can cause acute, febrile and septicemic infectious diseases in ducks, geese and other Anseriformes. Compared to other herpesviruses, less research has been done on DEV. It was classified as a herpes virus by the Eighth International Committee on the Taxonomy of Virology [1] , and the specific genus classification has not yet been determined; until recently, the entire genome sequence has not been fully sequenced [2] . Since 2007, our laboratory has started the genome sequencing of ...

Claims

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Application Information

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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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