A kind of Newcastle disease virus recombinant vaccine strain of pigeon origin and its construction method and application

A technology of Newcastle disease virus and epidemic virus, applied in the direction of viruses, vaccines, applications, etc., can solve the problem of lack of pigeon Newcastle disease vaccine products, etc.

Active Publication Date: 2022-06-21
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to construct and obtain a recombinant vaccine strain of pigeon-derived Newcastle disease virus and its construction method and application, thereby solving the problem of lack of pigeon Newcastle disease vaccine products in the prior art

Method used

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  • A kind of Newcastle disease virus recombinant vaccine strain of pigeon origin and its construction method and application
  • A kind of Newcastle disease virus recombinant vaccine strain of pigeon origin and its construction method and application
  • A kind of Newcastle disease virus recombinant vaccine strain of pigeon origin and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Rescue of LaSota strain

[0044] 1. Virus purification

[0045] 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. 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.

[0046] 2. Virus sequence determination

[0047] Using the purified viral reverse-transcribed genomic cDNA as a template, 13 pairs of primers d...

Embodiment 2

[0068] Example 2 Rescue of vaccine candidate seed virus for recombinant pigeons

[0069] 1. Virus screening and purification

[0070] The pigeon-derived Newcastle disease virus of the VI.2.1.2.2 subtype isolated and preserved in the laboratory was selected for reproduction and rejuvenation, and then 5 strains were purified by the limiting dilution method. The hemagglutination titers are shown in Table 5. Finally, the QH1344 / 2017 strain after 5 generations of purification was selected as the original seed virus for the construction of full-length cDNA clones.

[0071] Table 5: Hemagglutination titer table of different strains of pigeon-derived Newcastle disease virus at each generation

[0072]

[0073] 2. Determination and analysis of the full-length genome sequence of QH1344 / 2017 strain

[0074]Using the purified QH1344 / 2017 virus reverse transcription genome cDNA as a template, using 13 pairs of primers designed to amplify the full-length sequence of type VI NDV (Table...

Embodiment 3

[0093] Example 3 Safety test and immune effect test of rLa-VI-QH17 vaccine strain on non-immunized pigeons

[0094] 1. Preparation of inactivated vaccine

[0095] The rLa-VI-QH17 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. 10.5 , the virus titer is 10 9.48 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. The same dose of LaSota inactivated va...

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Abstract

The invention provides a pigeon-derived Newcastle disease virus recombinant vaccine strain, wherein the gene fragment used in the process of constructing the pigeon-derived Newcastle disease virus recombinant vaccine strain contains the envelope glycoprotein F of the pigeon-derived Newcastle disease virus with attenuated toxicity protein gene and HN protein gene. The preservation number of a specific recombinant attenuated vaccine strain of pigeon-derived Newcastle disease virus is CGMCC No.21900. The pigeon-derived Newcastle disease virus recombinant vaccine strain provided by the invention has the biological characteristics of high growth titer and low pathogenicity in chicken embryos, is genetically stable, has a good immune protection effect on pigeon-derived Newcastle virus, and can effectively inhibit Detoxification can be used to prevent and control the Newcastle disease virus from pigeons that is currently prevalent, filling the gap in my country that lacks special vaccine products for the prevention and control of pigeon Newcastle disease.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, in particular to a pigeon-derived Newcastle disease virus recombinant vaccine strain and a construction method and application thereof. Background technique [0002] Newcastle disease (ND) is an important infectious disease caused by virulent strains of Newcastle disease virus (NDV) infecting poultry. The disease is a highly contagious, acute septicemic infectious disease characterized by respiratory and digestive tract damage. It spreads rapidly and has a high fatality rate, causing huge economic losses to the world poultry industry. The World Organization for Animal Health (OIE) lists Newcastle disease as a statutory reportable animal disease, and the Ministry of Agriculture and Rural Affairs of my country lists it as a first-class animal disease. [0003] Newcastle disease virus NDV is a single-stranded, negative-stranded, non-segmented RNA virus belonging to the family Paramyxov...

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
CPCC07K14/005C12N7/00C12N15/85A61K39/12A61P31/14C12N2760/18121C12N2760/18122C12N2760/18134C12N2760/18162A61K2039/5252A61K2039/5254A61K2039/552
Inventor 于晓慧王静静刘华雷舒波李峥李阳蒋文明
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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