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

A technology of duck viral enteritis and duck Tembusu virus, applied in the field of recombinant duck viral enteritis virus vaccine and recombinant virus vaccine, can solve the problems of unclear classification, low efficiency, heavy workload, 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 E protein
  • Construction and application of recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus E protein
  • Construction and application of recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus E protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Construction of Genome Fosmid Library of DEV Vaccine Strain

[0051] The Fosmid library of the DEV genome was constructed according to the instructions of the "CopyControl Fosmid Library Production Kit" kit from EPICENTRE.

[0052] 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). Carry out terminal smoothing and dephosphorylation treatment, pulse electrophoresis (use Bio-Rad company CHEF The XA Pulsed Field system performs pulse electrophoresis. The conditions of pulse electrophoresis are: the electrophoresis buffer is 0.5xTBE, the aga...

Embodiment 2

[0053] Example 2. Selection for rescue of DEV cosmids

[0054] 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:

[0055] Primer 1: 5'-TAATACGACTCACTATAGGG-3'

[0056] Primer 2: 5'-GCCAAGCTATTTAGGTGAGA-3'

[0057] 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-Sbf I-Pme I joints, which can overlap each other and can be spliced ​​to cover the entire DEV genome.

Embodiment 3

[0058] Example 3. Virus rescue

[0059]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 Sbf I endonuclease (Fse I or Pme I could also be used Endonuclease), cosmid 10 μg, acted at 37°C for 1 hour, extracted with phenol / chloroform, and precipitated with ethanol to prepare DEV DNA for transfection.

[0060] 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 u...

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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 CCTCC V201214 expressing secreted duck Tembusu virus E protein, named rDEV-TE-tPAS, and 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. 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 started the sequencing o...

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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