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Carrier pigeon Newcastle disease virus genetic engineering modified attenuated strain as well as preparation method and application thereof

A technology of Newcastle disease virus and genetic engineering, which is applied in the field of genetically engineered attenuated strains of homing pigeon Newcastle disease virus and its preparation, and can solve problems such as unclear species and optimized preparation process.

Pending Publication Date: 2022-07-22
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation process is not optimized for the pigeons to be used, the specific species of pigeons to be used are not clear, and the route of intramuscular injection in the chest is not suitable for homing pigeons (the inactivated vaccination of homing pigeons can only be inoculated subcutaneously in the neck)

Method used

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  • Carrier pigeon Newcastle disease virus genetic engineering modified attenuated strain as well as preparation method and application thereof
  • Carrier pigeon Newcastle disease virus genetic engineering modified attenuated strain as well as preparation method and application thereof
  • Carrier pigeon Newcastle disease virus genetic engineering modified attenuated strain as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Construction of PNDV-aYQ

[0052] 1 material

[0053] 1.1 Viruses and cells

[0054] 1.1.1 The PNDV-YQ strain was isolated, identified and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry in 2012 in a loft of a sick homing pigeon. It is preserved in the General Microbiology Center of China Microorganism Culture Collection Management Committee, and the preservation number is CGMCC No: 24459.

[0055] 1.1.2 BSR-T7 cells expressing T7 polymerase were provided by Zhonghai Biotechnology Co., Ltd.

[0056] 1.2 Plasmids and strains

[0057] 1.2.1 pOK12 plasmid The cloning vector used in this experiment was provided by China Agricultural University.

[0058] 1.2.2 pCI-neo eukaryotic cell expression vector, provided by Zhejiang University.

[0059] 1.2.3 DH5α Escherichia coli competent cells, provided by Southeast University.

[0060] 1.3 SPF chicken embryos 10-day-old SPF chicken embryos were purchased fro...

Embodiment 2

[0122] Preparation of pigeon Newcastle disease inactivated vaccine (PNDV-aYQ strain)

[0123] 1 material

[0124] 1.1 Genetic engineering of the virus species An attenuated strain of pigeon Newcastle disease virus (PNDV-aYQ strain) was constructed, which was passaged and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry.

[0125] 1.2 SPF chicken embryos 10-day-old SPF chicken embryos were purchased from Beijing Boehringer Ingelheim Viton Biotechnology Co., Ltd.

[0126] 1.3 Adjuvant Montanide TM ISA 763A VG, produced by SEPPIC.

[0127] 1.4 Ultrafiltration concentration membrane package 300KD ultrafiltration membrane package (Pall); 100KD ultrafiltration membrane package (Sartorius Vivaflow200), purchased from Beijing Jinhao Tiancheng Biotechnology Co., Ltd.

[0128] 1.5 Formaldehyde (C.P 40% formaldehyde solution) was purchased from Beijing Yili Fine Chemicals Co., Ltd.

[0129] 1.6 Shearing machine produc...

Embodiment 3

[0169] Immune Efficacy Test of Laboratory Products of Inactivated Newcastle Disease Vaccine (PNDV-aYQ) in Pigeons

[0170] 1 material

[0171] 1.1 Three batches of pigeon Newcastle disease inactivated vaccine (PNDV-aYQ) with vaccine batch numbers of 201701, 201702 and 201703 were developed by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry.

[0172] 1.2 The 20 test animals homing pigeons (about 30 days old) were provided by Beijing Shunyi Mulin Breeding Farm.

[0173] 1.3 Efficacy test virus strain PNDV-YQ strain F3 generation ELD 50 =10 -8.3 / 0.1ml was isolated, identified and preserved by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences.

[0174] 1.4 Pigeon Newcastle disease virus HI antigen and negative and positive serum batch number is 201701, all prepared by the Institute of Animal Husbandry and Veterinary Sciences, Beijing Academy of Agriculture and F...

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Abstract

The invention discloses a carrier pigeon Newcastle disease virus genetic engineering modified attenuated strain as well as a preparation method and application of the carrier pigeon Newcastle disease virus genetic engineering modified attenuated strain. The method specifically comprises the following steps: carrying out site-directed mutagenesis on amino acids of three cleavage sites of F protein of a carrier pigeon Newcastle disease virus strain (PNDV-YQ strain) by using a reverse genetic manipulation technology, constructing and obtaining a full-length cDNA plasmid pOK-YQ of a low-toxicity gene in a segmented cloning manner, respectively cloning ORF genes of NP, P and L of the PNDV-YQ strain to an eukaryotic expression vector pCIneo, and carrying out expression by using a recombinant vector pCIneo, according to the invention, helper plasmids pCI-NP, pCI-P and pCI-L are constructed and obtained. After a BSR-T7 cell is co-transfected by the full-length plasmid and a helper plasmid, a recombinant attenuated virus, namely the pigeon Newcastle disease virus (PNDV-aYQ strain), is rescued. The strain of virus is a low-pathogenicity or non-pathogenicity strain, and has good proliferation characteristics and stable hereditary characteristics. Compared with the existing newcastle disease vaccine, the inactivated vaccine prepared from the virus strain has the advantages of low side reaction, early immune production period and high protection rate.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the technical field of avian virus genetic engineering research and vaccine preparation, in particular to a carrier pigeon Newcastle disease virus genetically modified attenuated strain and a preparation method and application thereof. Background technique [0002] Pigeon Newcastle disease (PND), also known as pigeon plague, is the number one disease that threatens the healthy development of the carrier pigeon industry. In the early 1980s, it was first popular in Africa, then spread to Europe and the United States and other countries, and spread to my country around 1986. The disease spreads rapidly, pigeons of different breeds and ages are susceptible, and the incidence rate is as high as 50% to 80%. In the early stage of the disease, the sick pigeons are depressed, disheveled, closed eyes and necks; with the development of the disease course, the sick pigeons appear legs Neurological...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/45C12N15/85A61K39/17A61P31/14C12R1/93
CPCC12N7/00C12N15/85C07K14/005A61K39/12A61P31/14C12N2760/18121C12N2760/18122C12N2760/18134C12N2800/106A61K2039/5252A61K2039/5254A61K2039/552A61K2039/545A61K2039/54
Inventor 杨志远孙晔林健程水生刘月焕高洁段会娟潘雨王楠赵际成黄程刘立新程慧敏潘洁
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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