A recombinant vaccine strain of genotype vii Newcastle disease virus with hn protein mutation

A technology of Newcastle disease virus and recombinant vaccine, which is applied in the field of veterinary biological products, can solve the problems of immune failure, unable to completely prevent the spread and transmission of Newcastle disease virus, and the loss of breeding industry

Active Publication Date: 2022-05-20
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Vaccine immunization is an important means of preventing Newcastle disease. Currently, the widely used Newcastle disease vaccines mainly include II series vaccine B1, IV series vaccine LaSota, clone30, V4 live vaccine, A-VII oil emulsion inactivated vaccine and LaSota oil emulsion inactivated vaccine etc. Although these vaccines can provide certain immune protection, they cannot completely prevent the spread and spread of Newcastle disease virus, especially the prevalence of genotype VII Newcastle disease virus in my country has caused huge losses to the breeding industry.
The genotype VII Newcastle disease vaccine currently immunized in farms is developed for the genotype VII.1.1 subtype, which has the risk of immune failure against the current genotype VII.2 subtype strain

Method used

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  • A recombinant vaccine strain of genotype vii Newcastle disease virus with hn protein mutation
  • A recombinant vaccine strain of genotype vii Newcastle disease virus with hn protein mutation
  • A recombinant vaccine strain of genotype vii Newcastle disease virus with hn protein mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Rescue of LaSota strain

[0039] 1. Virus purification

[0040] The purification of the virus is to obtain a single virus clone. The LaSota strain was purified by the limiting dilution method. The detailed steps are as follows: Dilute the virus liquid by 10 times, and inoculate the virus liquid of each titer for 9 to 11 days after dilution SPF chicken embryos (100 μL / piece) were inoculated with 5 pieces per dilution. After 4 days, the chicken embryo allantoic fluid was harvested to measure the activity of HA. The allantoic fluid with the highest dilution factor with HA activity was selected as the next-generation purified virus fluid. The same method was used for doubling dilution and chicken embryo inoculation, and the virus was continuously purified for 5 generations, and the 5th generation virus liquid was stored in aliquots, which was used as the original seed virus for the next test.

[0041] 2. Virus sequence determination

[0042] Using the purifie...

Embodiment 2

[0061] Example 2: Rescue of recombinant Newcastle disease vaccine candidate seeds

[0062] 1. Virus screening and purification

[0063] The chicken-derived Newcastle disease virus of subtype VII.2 isolated and preserved in the laboratory was selected for reproduction and rejuvenation, and then 5 strains were purified by limited dilution method. The hemagglutination titers of different generations of each strain were See Table 5. Finally, the YN1106 / 2017 strain after 5 generations of purification was selected as the original seed virus for the construction of full-length cDNA clones.

[0064] Table 5: Hemagglutination titer table of different strains of genotype VII Newcastle disease virus at each generation

[0065]

[0066] 2. Determination and analysis of the full-length genome sequence of YN1106 / 2017 strain

[0067] Using the purified YN1106 / 2017 virus reverse transcription genome cDNA as a template, using the designed 12 pairs of primers (Table 6) to amplify the full...

Embodiment 3

[0087] Example 3 Safety test and immune effect test of rLa-VII-YN17 vaccine strain on SPF chickens

[0088] 1. Preparation of inactivated vaccine

[0089] The rLa-VII-YN17 vaccine strain was diluted 10 000 times with sterilized normal saline, inoculated with 9-11-day-old SPF chicken embryos, 0.1 mL per embryo, and incubated at 37°C. The dead embryos within 24h after inoculation were discarded, and the dead embryos were kept at 4°C in time for 24h~120h, and the mixed samples were collected. 8 , the virus titer is 10 9.65 EID 50 / 0.1mL. The Newcastle disease virus solution with the determined titer was introduced into the inactivation tank, and the final concentration of 0.1% formaldehyde solution was added to make it fully mixed, inactivated at 4°C for 48 hours, and shaken every 2 hours during the period. Oil adjuvant inactivated vaccine is prepared from the inactivated virus stock solution according to conventional methods.

[0090] 2. Safety test of overdose vaccination ...

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Abstract

The present invention provides a gene VII Newcastle disease virus recombinant vaccine strain with HN protein mutation, wherein the gene fragment used in the process of constructing the gene VII Newcastle disease virus recombinant vaccine strain contains the toxic attenuated gene VII.2 subunit Envelope glycoprotein F protein gene and HN protein gene of Newcastle disease virus type. The recombinant vaccine strain of gene VII Newcastle disease virus provided by the invention has the biological characteristics of high growth titer and low pathogenicity in chicken embryos, is genetically stable, has good immune protection effect against Newcastle virus, and can effectively inhibit shedding , can be used to prevent and control the current popular genotype VII Newcastle disease virus, and has broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, in particular to a recombinant vaccine strain of Newcastle disease virus type VII with HN protein mutation and a construction method and application thereof. Background technique [0002] Newcastle disease (ND) is an acute, highly contagious infectious disease caused by Newcastle disease virus (NDV) virulent strains infecting poultry. The virulent strains of NDV can cause typical features such as dyspnea, diarrhea, neurological disorders, and mucosal and serosa hemorrhage and necrosis in infected birds. The fatality rate can reach 100%, causing serious economic losses to the poultry industry. The World Organization for Animal Health (OIE) lists Newcastle disease as a legally notified animal disease, and the Ministry of Agriculture and Rural Affairs of my country lists it as a first-class animal disease. It is listed as a major animal disease with priority control. [0003]...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/45C12N7/04C12N15/85A61K39/17A61P31/14C12R1/93
CPCC07K14/005C12N7/00C12N15/85A61K39/12A61P31/14C12N2760/18121C12N2760/18122C12N2760/18134C12N2760/18162A61K2039/5252A61K2039/5254A61K2039/552Y02A50/30
Inventor 于晓慧王静静刘华雷李峥舒波李阳蒋文明
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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