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Recombinant turkey herpesvirus candidate vaccine strain for expressing gene VII-type Newcastle disease virus fusion protein and preparation method thereof

A technology for turkey herpes virus and Newcastle disease virus, which is applied in the field of genetic engineering vaccines, can solve the problems such as the inability to effectively prevent the detoxification of infected birds and the like

Inactive Publication Date: 2019-10-15
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although La Sota vaccine provided good clinical protection against genotype VII NDV strains, it was not effective in preventing shedding of infected birds

Method used

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  • Recombinant turkey herpesvirus candidate vaccine strain for expressing gene VII-type Newcastle disease virus fusion protein and preparation method thereof
  • Recombinant turkey herpesvirus candidate vaccine strain for expressing gene VII-type Newcastle disease virus fusion protein and preparation method thereof
  • Recombinant turkey herpesvirus candidate vaccine strain for expressing gene VII-type Newcastle disease virus fusion protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of transfer vectors pFR-F and pFR-ΔF

[0034] 1. Cloning and identification of F gene of Newcastle disease virus strain (DT-2014)

[0035] 1.1 Primer design

[0036] Referring to the sequence of the F gene of the gene VII Newcastle disease virus DT-2014 strain (GenBank accession number: MN125616; Seq No.1), a pair of specific primers were designed with primer5.0 software, wherein the upstream primer contained the KOZAK sequence, and the downstream primer Remove the stop codon. The primer sequences are as follows:

[0037] NDF-F (Seq No.3): 5'-GCCACCATGGGCTCCAAACTTTTCTACC-3'

[0038] NDF-R (Seq No.4): 5'-TGCTCTTGTAGTGGCTCTCATCT-3'

[0039] 1.2 Construction of intermediate plasmid pM3-F

[0040] Take 200 μL of sterile allantois from NDV DT-2014 strain of gene type VII, extract RNA, use NDF-F / NDF-R as primers, and perform RT-PCR. After the reaction, perform 1% agarose gel electrophoresis to recover the F gene The bands, recovered product and ...

Embodiment 2

[0071] Example 2: Rescue and purification of recombinant viruses rHVT-NDV-VII-F and rHVT-NDV-VII-ΔF

[0072] 1. Expanded culture of recombinant virus rHVT-EGFP

[0073] The recombinant virus rHVT-EGFP was preserved and provided by the Key Open Laboratory of Livestock and Poultry Infectious Diseases, Ministry of Agriculture, Yangzhou University (Wu Yantao, et al. A recombinant turkey herpes virus strain expressing the HA gene of H9 subtype avian influenza virus. Patent application number: CN201710517830.5, application publication number: CN 107142280 A). The recombinant virus rHVT-EGFP stored in liquid nitrogen was revived, and the revived cell virus liquid was diluted 10 times with M199 medium containing 5% fetal bovine serum. Prepare the second-generation CEF and spread it on a 6-well cell culture plate. After culturing for 2 hours, inoculate the double-diluted virus solution, 900 μL per well, and culture it in a cell culture incubator. After 24 hours, discard the culture so...

Embodiment 3

[0093] Example 3: Evaluation of immune efficacy of recombinant viruses rHVT-NDV-VII-F and rHVT-NDV-VII-ΔF

[0094] 1. Immune protection test of recombinant virus

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Abstract

The invention belongs to the technical field of genetic engineering vaccines, and particularly relates to a recombinant turkey herpesvirus candidate vaccine strain for expressing gene VII-type Newcastle disease virus fusion protein and a preparation method thereof. The recombinant turkey herpesvirus candidate vaccine strain for expressing the gene VII-type Newcastle disease virus fusion protein isnamed as rHVT-NDV-VII-F, and has a preservation number of CCTCC NO: V201904. The recombinant virus can express a gene VII-type NDV F protein, is completely matched with the genotype of a currently domestically popular NDV wild strain, and can be used as a gene engineering vaccine candidate strain for preventing NDV infection.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering vaccines, and in particular relates to a method for constructing a recombinant turkey herpes virus expressing gene VII type chicken Newcastle disease virus fusion protein and the constructed recombinant virus. Background technique [0002] Newcastle disease (ND) is an acute, severe and highly contagious infectious disease of poultry caused by Newcastle disease virus (NDV). NDV is an enveloped single-stranded, non-segmented, negative-strand RNA virus with a genome of 3'-NP-P-M-F-HN-L-5', which encodes nucleocapsid protein (NP) and phosphoprotein (P) in sequence , matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and large protein (L). In addition, RNA editing can occur during transcription of the P gene, producing the nonstructural proteins V and W. The F protein is located on the envelope of the virus and is the main structural protein of the virus, whi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K39/17A61P31/14C12R1/93
CPCA61K39/12A61K2039/5256A61P31/14C12N7/00C12N2710/16021C12N2760/18134
Inventor 吴艳涛白雪雁张小荣张成成郭梦娇曹永忠
Owner YANGZHOU UNIV
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