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Recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes and construction method thereof

A technology of Newcastle disease virus and goose parvovirus, applied in the field of recombinant Newcastle disease virus expressing goose parvovirus VP3 gene and its construction, can solve the problem of virulence reversion, insufficient control of gosling plague pandemic, potential infection and detoxification And other issues

Active Publication Date: 2014-12-10
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Gosling plague is a serious hazard to the goose breeding industry. Currently, the commonly used vaccines for preventing gosling plague infection are divided into two types: goose breeding vaccines and gosling vaccines. Goose breeding vaccines can be divided into attenuated vaccines adapted to duck embryos and goose embryo vaccines. Strongly virulent vaccines, vaccines for goslings are also divided into duck embryo-adapted attenuated vaccines and goose embryo virulent vaccines. Strongly virulent vaccines can produce better maternal antibodies and pass them to goslings through the yolk, but may cause the spread of virulent viruses , the attenuated vaccine has no pathogenicity, but has the disadvantage of weak protection, which is not enough to control the pandemic of gosling plague
In addition, the attenuated vaccine is unstable and prone to virulence reversion, which may be selected as a risk of potential infection and detoxification. The oil emulsion vaccine produces immunity slowly and has a series of problems such as large dosage, complicated preparation process, and high cost. Difficult to meet the needs of intensification

Method used

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  • Recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes and construction method thereof
  • Recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes and construction method thereof
  • Recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 Construction of Newcastle disease virus reverse genetics operating system

[0034] 1 Materials and methods

[0035] 1.1 Materials

[0036] Newcastle disease virus NA-1 strain (GenBank: DQ659677.1); BHK-21 cells (ATCC No.CCL-10), the culture medium is DMEM containing 10% fetal bovine serum; plasmids pCI (Promega) and pBluescript II KS+ ( Clontech); Phusion DNA polymerase, T4 DNA ligase and other restriction endonucleases were purchased from NEB Company; competent cells were purchased from TaKaRa Company; gel recovery kit and plasmid extraction kit were purchased from OMEGA Company; RNA extraction TRIzol, mouse-derived reverse transcriptase MLV, calcium phosphate transfection kit, RPMI1640 culture medium were purchased from Invitrogen; chicken anti-NDV whole virus hyperimmune serum; fluorescein (FITC)-labeled goat anti-chicken secondary antibody was purchased from Sigma; The fluorescent microscope was BX51FL; Olympus was purchased from Olympus Company, cell...

Embodiment 2

[0059] Example 2 Mutation of F cleavage site and construction of full-length cDNA clone expressing goose parvovirus (GPV) VP3 gene

[0060] 1 Materials and methods

[0061] 1.1 Materials

[0062] With embodiment 1.

[0063] 1.2 Construction of full-length plasmid expressing VP3 gene

[0064] On the basis of the above-mentioned NDV genome full-length cDNA clone pCI-NA-1, design mutation primers as follows:

[0065]

[0066] Note: The italics in the primer Fmut-R are the cleavage site sequence of F protein, and the mutation site is underlined.

[0067]The PCR method is used to mutate the 4883-4900nt of the full-length cDNA clone, so that the encoded amino acid is mutated from the highly toxic characteristic sequence RRQKRF to the weakly toxic characteristic sequence GRQGRL. The open reading frame sequence of GPV GD-01 strain VP3 (GenBank Accession No.DQ665790) was obtained from GenBank, and primers were designed, and the upstream primer was introduced into the PmeI restri...

Embodiment 3

[0086] Embodiment 3 Chicken embryo growth characteristics comparison

[0087] 1 Materials and methods

[0088] 1.1 Materials

[0089] SPF chicken embryos were purchased from Beijing Merial, 96-well hemagglutination plate, fresh chicken blood, phosphate buffered saline (PBS), incubator, etc.

[0090] 1.2 Method

[0091] In order to determine the growth characteristics of rescued recombinant virus rNA-1, rNA-VP3 and maternal wild-type NA-1 chick embryo, the recombinant virus rNA-1 and rNA-VP3 F3 generation allantoic fluid and wild-type rNA-1 allantoic fluid Diluted in PBS to 1×10 4 EID 50 Inoculate the allantoic cavity of chicken embryos with SPF. Samples were taken at 24 hours, 48 ​​hours, 72 hours and 96 hours after inoculation, 6 embryos were randomly selected at each time point, the allantoic fluid was collected, and the EID was measured after mixing 50 .

[0092] 2 results

[0093] In order to identify the growth characteristics of the rescue mutant strain and the w...

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Abstract

The invention discloses a recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes and a construction method thereof, belonging to the field of recombinant virus vaccines. The recombinant Newcastle disease virus is a goose isolate, the cleavage site amino acid sequence of F protein of the recombinant Newcastle disease virus is GRQGRL, and the P3 gene is positioned in a noncoding region between the P gene and M gene of Newcastle disease virus. The transcription plasmid pCI-NA-VP3(SEQ ID NO.1) and the transcription helper plasmid pcDNA-N, pcDNA-P and pcDNA-L(SEQ ID NO. 2-4) cotransfect a host cell licensed by application of the Newcastle disease virus to culture a transfected host cell, and the recombinant Newcastle disease virus can be saved from a cell suspension of the transfected host cell. The recombinant Newcastle disease virus for expressing goose parvovirus VP3 genes can be used as a bivalent living-vector vaccine for preventing Newcastle disease virus and goose parvovirus.

Description

technical field [0001] The invention relates to the field of recombinant virus vaccines, more specifically, the invention relates to a recombinant Newcastle disease virus expressing goose parvovirus VP3 gene and a construction method thereof. Background technique [0002] Newcastle disease (ND) is an acute, febrile, septic and highly contagious infectious disease caused by Newcastle disease virus (NDV). It is characterized by high fever, dyspnea, diarrhea, nervous disturbance, and mucosal and serosal hemorrhage. The disease first broke out in Batavia, Indonesia in 1926. Since the fourth pandemic in the 1990s, the main epidemic strain in many countries in the world, including China, is a member of the gene VII virus in class II. NDV can infect almost all birds and can spread widely with the migration of migratory birds. Among all poultry species, chickens and turkeys are the most susceptible, often causing more than 90% mortality in the entire flock. Waterfowl such as duck...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85A61K39/295A61K39/17A61K39/12A61P31/14A61P31/20C12R1/93
Inventor 丁壮王建忠丛彦龙李芹李想
Owner JILIN UNIV
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