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Application of recombinant virus vaccine in galliformes poultry by adopting duck virus enteritis virus vaccine strain as vector

A technology for duck viral enteritis and virus vaccine, which is applied in the directions of application, antiviral agent, virus antigen component, 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

So far, there has been no report that it can be used as a live virus vector to express the protective antigen of the source of infectious diseases of galliformes such as chickens, and can be used to prevent infectious diseases of galliformes

Method used

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  • Application of recombinant virus vaccine in galliformes poultry by adopting duck virus enteritis virus vaccine strain as vector
  • Application of recombinant virus vaccine in galliformes poultry by adopting duck virus enteritis virus vaccine strain as vector
  • Application of recombinant virus vaccine in galliformes poultry by adopting duck virus enteritis virus vaccine strain as vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Construction of Genome Fosmid Library of DEV Vaccine Strain

[0047] The Fosmid library of the DEV genome was constructed according to the instructions of the "CopyControl Fosmid Library Production Kit" kit from EPICENTRE.

[0048] 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

[0049] Example 2. Selection for rescue of DEV cosmids

[0050] 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:

[0051] Primer 1: 5'-TAATACGACTCACTATAGGG-3'

[0052] Primer 2: 5'-GCCAAGCTATTTAGGTGAGA-3'

[0053] 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

[0054] Example 3. Virus rescue

[0055] 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.

[0056] 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 [22] 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 ...

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Abstract

The invention provides an application of living virus vector duck virus enteritis virus (DEV) attenuated vaccine vector in galliformes poultry. The DEV attenuated vaccine vector can be used for expressing relevant genes of the galliformes poultry pathogeny and can carry exogenous genes to be expressed in the galliformes poultry, and the protection for the pathogeny can be well induced in an immune animal body. Through the application of the recombinant duck virus enteritis virus vaccine strain (preservation serial number is CCTCC V201125, named as rDEVus78Ha-Re6) for expressing the avian influenza virus haemagglutinin (HA) genes and the recombinant duck virus enteritis virus vaccine strain (preservation serial number is CCTCC V201220, and named as rDEV F) for expressing newcastle disease virus gene F in chicken, the characteristics of the DEV attenuated vaccine vector can be proved.

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 live virus vector that can be used in chickens of the order Galliformes, that is, a duck viral enteritis virus (DEV) attenuated vaccine strain vector, which can express genes related to the pathogens of chickens of the order Galliformes, and can carry the foreign gene Expressed in galliformes, induces good protection against this pathogen in immunized animals. In this study, the recombinant duck viral enteritis virus vaccine strain expressing the hemagglutinin (HA) gene of avian influenza virus (its deposit number is CCTCC V201125, named rDEVus78Ha-Re6) and the recombinant duck viral enteritis virus expressing the Newcastle disease virus F gene The enteritis virus vaccine strain (its storage number is CCTCC V201220, named rDEV F) is a model virus, which proves the above characterist...

Claims

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Application Information

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IPC IPC(8): C12N15/869A61K48/00A61K39/145A61K39/17A61K39/12A61K39/215A61P31/16A61P31/14C12R1/93
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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