Recombinant Newcastle disease virus (NDV) for expressing VP3 gene of new duck parvovirus and application of recombinant NDV

A technology of Newcastle disease virus and duck parvovirus, applied in the field of reverse genetics application, can solve the problem of lack of preparation method of NDPV-VP3 protein

Inactive Publication Date: 2018-05-15
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides a recombinant Newcastle disease virus expressing the new duck parvovirus VP3 gene and its application, so as to solve the technical problem of lacking a preparation method of NDPV-VP3 protein in the prior art

Method used

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  • Recombinant Newcastle disease virus (NDV) for expressing VP3 gene of new duck parvovirus and application of recombinant NDV
  • Recombinant Newcastle disease virus (NDV) for expressing VP3 gene of new duck parvovirus and application of recombinant NDV
  • Recombinant Newcastle disease virus (NDV) for expressing VP3 gene of new duck parvovirus and application of recombinant NDV

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Embodiment 1

[0031] 1. Construction of recombinant Newcastle disease virus expressing NDPV-VP3

[0032] The NDPV-VP3 gene was amplified by RT-PCR using the Shandong isolate of the new duck parvovirus. Design primers VP3-F and VP3-R with reference to the nucleotide sequence of the new duck parvovirus VP3 gene (GenBank No. KT343253) included in GenBank, and use Trizol reagent to extract NDPV virus RNA, and the fragment size of the amplified product NDPV-VP3 is 1605bp ; The nucleotide sequence of the amplified product NDPV-VP3 is shown in SEQ ID NO:1. The recovered PCR product was connected to pMD18-T-Vector, and it was confirmed by sequencing that NDPV-VP3 was correctly inserted into the vector. The primers are designed as follows:

[0033]

[0034] The NDPV-VP3 gene was connected with the pLMV-RFP linearized vector, and the connected product was transformed into Stbl2 competent cells, and positive clones were screened out to obtain a recombinant NDV virus plasmid expressing NDPV-VP3. ...

Embodiment 2

[0053] A recombinant Newcastle disease virus expressing the novel duck parvovirus VP3 gene, the recombinant Newcastle disease virus is constructed by the following method:

[0054] 1. Construction of recombinant Newcastle disease virus expressing NDPV-VP3

[0055] (1a) The NDPV-VP3 gene was obtained by amplifying the novel duck parvovirus Shandong isolate by RT-PCR method. The recovered PCR product was connected to pMD18-T-Vector, and it was confirmed by sequencing that NDPV-VP3 was correctly inserted into the vector.

[0056] (1b) Ligate the NDPV-VP3 gene with the pLMV-RFP linearized vector, transform the ligated product into Stbl2 competent cells, screen out positive clones, and obtain a recombinant NDV virus plasmid expressing NDPV-VP3.

[0057] More preferably, in the step (1a),

[0058] The primers of the NDPV-VP3 gene PCR amplification are:

[0059] Upstream primer: 5'-ATGGCAGAGGGAGGAGGCGGAGC-3',

[0060] Downstream primer: 5'-TTACAGATTTTGAGTTAGATATCTG-3';

[0061] ...

Embodiment 3

[0079] A recombinant Newcastle disease virus expressing the novel duck parvovirus VP3 gene, the recombinant Newcastle disease virus is constructed by the following method:

[0080] 1) Take duck parvovirus, use RT-PCR method to amplify the NDPV-VP3 gene, recover the PCR product, and connect it to pMD18-T-Vector;

[0081] 2) connecting the NDPV-VP3 gene to the pLMV-RFP linearized vector, and transforming the ligated product into Stbl2 competent cells, screening out positive clones, and obtaining the recombinant NDV virus plasmid;

[0082] 3) Inoculate the recombinant poxvirus vTF7-3 expressing T7 polymerase in a 24-well plate of monolayer BHK-21 cells at a titer of MOI=3, and after incubation for 1 h, pLMV-VP3, pcDNA-NP, pcDNA- P and pcDNA-L plasmids were co-transfected into BHK-21 cells. After 72 hours of transfection, the harvested and rescued recombinant virus was inoculated into 10-day-old SPF chicken embryos, and the chicken embryo allantoic fluid with positive HA and HI te...

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Abstract

The invention provides a recombinant Newcastle disease virus (NDV) for expressing a VP3 gene of a new duck parvovirus and application of the recombinant NDV. The ORF of the VP3 gene of the new duck parvovirus is inserted into a full-length transcription vector pLMV-RFP of the NDV by utilizing a reverse genetic manipulation platform of an established NDV PHY.LMV42 strain, so as to construct a recombinant plasmid pLMV-NDPV-VP3, and the recombinant plasmid and three auxiliary plasmids are together transfected with BHK-21 cells to obtain a recombinant vaccine strain rLMV-NDPV-VP3 which is higher in reproductive performance and capable of expressing NDPV-VP3 protein. The NDPV-VP3 gene is located in a non-coding region between a P gene and an M gene of the NDV, and a recombinant virus rLMV-NDPV-VP3 is obtained through reverse genetic technique. The recombinant NDV for expressing the VP3 gene of the new duck parvovirus can be used for preventing duck new parvovirus disease and duck Newcastledisease.

Description

technical field [0001] The invention relates to the application field of reverse genetics, in particular to a recombinant Newcastle disease virus expressing a novel duck parvovirus VP3 gene and an application thereof. Background technique [0002] Since 2014, a large area of ​​ducks in Shandong, Anhui, Jiangsu and other places has broken out a disease characterized by growth retardation, duck (upper and lower) beak shortening, drooping tongue, lameness, paralysis, diarrhea, and easy breakage of wings and legs. An infectious disease with main clinical features, commonly known as "duck short beak-dwarf syndrome" or "duck tongue disease". The pathogen of the disease was identified as a novel duck parvovirus (NDPV), which was closely related to gosling plague virus, but they were not in the same evolutionary branch. Duck short beak-dwarf syndrome mainly occurs in meat ducks aged 10-25 days. The affected ducks often become emaciated due to difficulty in eating and drinking water...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85C12N15/35A61K39/295A61K39/17A61K39/23A61P31/14A61P31/20C12R1/93
CPCA61K39/12A61K2039/5256A61K2039/70C07K14/005C12N7/00C12N15/85C12N2750/14322C12N2750/14334C12N2760/18121C12N2760/18134C12N2760/18152
Inventor 郑文卿李亚杰杨保收付旭彬梁武
Owner TIANJIN RINGPU BIO TECH
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