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HN protein mutant gene VII type Newcastle disease virus recombinant vaccine strain

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: 2022-01-07
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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  • HN protein mutant gene VII type Newcastle disease virus recombinant vaccine strain
  • HN protein mutant gene VII type Newcastle disease virus recombinant vaccine strain
  • HN protein mutant gene VII type Newcastle disease virus recombinant vaccine strain

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: the virus liquid is diluted 10 times, and after dilution, the virus liquid of each titer is inoculated for 9 to 11 days. Age SPF chicken embryos (100 μL / piece), inoculate 5 pieces for each dilution. After 4 days, the allantoic fluid of chicken embryos was harvested to measure the activity of HA. The allantoic fluid with the highest dilution factor of HA activity was selected as the next-generation purified virus fluid. Doubling dilution and chicken embryo inoculation were carried out in the same way, the virus was continuously purified for 5 generations, and the 5th generation virus solution was subpackaged and preserved, and used as the original seed virus for the next test.

[0041] 2. Determination of virus sequence

[0042] Usi...

Embodiment 2

[0061] Embodiment 2: Rescue of recombinant Newcastle disease vaccine candidate virus

[0062] 1. Virus screening and purification

[0063] Select the currently popular Chinese Newcastle disease virus of gene VII.2 subtype, which is isolated and preserved in the laboratory, for reproduction and rejuvenation, and then use the limiting dilution method to purify 5 strains. The hemagglutination titers of different generations of each strain See Table 5. Finally, the YN1106 / 2017 strain purified for 5 generations was selected as the original seed virus for the construction of full-length cDNA clones.

[0064] Table 5: Table of hemagglutination titers of different strains of genotype VII Newcastle disease virus in each generation

[0065]

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

[0067] Using the purified YN1106 / 2017 viral reverse-transcribed genomic cDNA as a template, the YN1106 / 2017 genome was amplified by PCR using 12 p...

Embodiment 3

[0087] Example 3 rLa-VII-YN17 vaccine strain to SPF chicken safety test and immune effect test

[0088] 1. Preparation of inactivated vaccine

[0089] The rLa-VII-YN17 vaccine strain was diluted 10 000 times with sterilized saline, inoculated with 9-11-day-old SPF chicken embryos, 0.1 mL per embryo, and incubated at 37°C. Discard the dead embryos within 24 hours after inoculation, put the dead embryos at 4°C in time between 24h and 120h, collect the mixed samples, and measure the HA for seedling toxicity to be 2 8 , the virus titer was 10 9.65 EID 50 / 0.1mL. Introduce the Newcastle disease virus solution with a determined titer into the inactivation tank, add formaldehyde solution with a final concentration of 0.1%, make it fully mixed, inactivate at 4°C for 48 hours, and shake it every 2 hours during this period. The oil adjuvant inactivated vaccine was prepared from the inactivated virus stock solution by a conventional method.

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

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Abstract

The invention provides an HN protein mutant gene VII type Newcastle disease virus recombinant vaccine strain. A gene segment used in the process of constructing the gene VII type Newcastle disease virus recombinant vaccine strain contains a envelope glycoprotein F protein gene and an HN protein gene of a gene VII.2 subtype Newcastle disease virus with weakened toxicity. The gene VII type Newcastle disease virus recombinant vaccine strain provided by the invention has the biological characteristics of high growth titer and low pathogenicity in a chicken embryo, is stable in heredity, has a good immune protection effect on the Newcastle disease virus, can effectively inhibit toxin expelling, can be used for preventing and controlling the currently popular gene VII type Newcastle disease virus, and has a wide application prospect.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(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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