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Isolation, identification and purification method of a genotype vii chicken Newcastle disease virus strain and its application

A technology for chicken Newcastle disease virus and Newcastle disease virus, which is applied in the field of animal virology and can solve problems such as inability to suppress detoxification, death, and disease in chickens.

Inactive Publication Date: 2019-05-31
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As NDV continues to mutate, it has been reported that although the vaccine can prevent disease in immunized poultry infected with NDV, it cannot inhibit shedding: the virus can still be excreted into the environment through the larynx / cloaca within days after infection, resulting in viral shedding. Persistent transmission and infection; LaSota-vaccinated flocks have also been reported to suffer from morbidity and mortality following infection with certain virulent genotype VII NDV strains

Method used

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  • Isolation, identification and purification method of a genotype vii chicken Newcastle disease virus strain and its application
  • Isolation, identification and purification method of a genotype vii chicken Newcastle disease virus strain and its application
  • Isolation, identification and purification method of a genotype vii chicken Newcastle disease virus strain and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A chicken-origin Newcastle disease virus strain Chicken / China / SD06 / 2014 of gene type VII, the isolation and identification methods of the virus are as follows:

[0027] 1. Virus Isolation

[0028] Take the internal organs (liver, kidney, spleen, etc.) of the diseased chicken and add sterilized saline to grind into a homogenate, freeze and thaw 3 times, centrifuge at 8000 revolutions per minute (rpm) for 15 minutes, discard the upper layer of fat, and absorb the middle liquid . Add penicillin and streptomycin and treat at 4°C for 1 hour, centrifuge again at 8000 rpm for 15 minutes, take the supernatant and inoculate 11-day-old SPF chicken embryos through the allantoic cavity, 0.2ml / piece, and culture them in an incubator for 72 hours. Observe twice a day, discard the contaminated embryos within 24 hours, and collect the allantoic fluid of the remaining chicken embryos.

[0029] 2. Virus Identification

[0030] Two methods of HA-HI test and PCR were used to identify ND...

Embodiment 2

[0047] Phylogenetic evolution and surface protein (HN and F) analysis of embodiment 2 virus

[0048] 1. Viral RNA extraction and RT-PCR

[0049] Total RNA was extracted from NDV chick embryo allantoic fluid by Trizol method (Invitrogen). The primers used for the amplification of HN and F genes were synthesized with reference to reports (Yang Shaohua, Hu Beixia, Xu Chuantian, etc. Biological characteristics and whole genome sequence analysis of three virulent Newcastle disease virus strains. Acta Virus Sinica, 2012, 28(2) : 143-150.). RT-PCR uses PrimeScript TM One Step RT-PCR Kit from Dalian Bao Biotechnology Co., Ltd.,

[0050] The reaction system is 50ul: 2×step-buffer 25μl, RNase Free dH 2 O 14μl, Enzyme mix 2μl, RNA sample 6μl, upstream and downstream primers 1.5μl each;

[0051] The RT-PCR reaction conditions were: reverse transcription at 50°C for 30min, pre-denaturation at 94°C for 5min, denaturation at 94°C for 50s, annealing at 52°C for 1min, extension at 72°C for...

Embodiment 3

[0071] Embodiment 3 This strain is used for the preparation method and experimental method of the antigen used in HA-HI experiment

[0072] 1. Antigen preparation method: use Chicken / China / SD06 / 2014 as seed virus, inoculate 11-day-old SPF chicken embryos, discard dead chicken embryos within 24 hours, aseptically collect chicken embryo allantoic fluid within 24-120 hours, add 0.2% formaldehyde solution was inactivated at 37°C for 24h. Add 0.02% sodium azide as a preservative and 0.1% bovine serum albumin (BSA) as a stabilizer to the inactivated antigen, and store it in a refrigerator at 4°C for later use.

[0073] 2. Chicken / China / SD06 / 2014 antigen HA titer determination: Take a 96-well hemagglutination plate, add 25ul of 0.01M pH7.4PBS buffer to each well of the first 3 rows, add 25ul of antigen to the first well of each row, and double the Ratio diluted to the 12th well. The 12 wells in the fourth row of the 96-well hemagglutination plate were used as a negative control (viru...

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Abstract

The invention relates to the field of animal virology, provides an isolation, identification and purification method and application of a gene VII type Newcastle disease virus strain, and particularly provides a gene VII type (Class II) Newcastle disease virus strain Chicken / China / SD06 / 2014, an isolation, identification and purification method of the strain and part of biological properties of the strain. The preservation number of the strain is CCTCC NO: V201544. The phyletic evolution and protein variation conditions of the strain are analyzed so as to confirm the feasibility that the strain serves as a candidate Newcastle disease vaccine strain and an HA-HI experiment antibody, and thus data is provided for molecular evolution and epidemiologic study of the Newcastle disease virus.

Description

technical field [0001] The invention relates to the field of animal virology, and the invention provides a method for isolating, identifying and purifying a gene VII type chicken Newcastle disease virus strain and an application thereof. Background technique [0002] Newcastle disease virus (NDV) belongs to type I avian paramyxovirus in the Paramyxoviridae family, and is a group of single-stranded negative-sense RNA viruses. The virus can cause Newcastle disease in poultry and wild birds, causing serious harm to the poultry industry. The full-length NDV genome is 15.2kb, encoding 6 major structural proteins (3'-NP-P-M-F-HN-L-5') and 2 non-structural proteins (V and W). HN and F proteins are located on the surface of the virus and are closely related to the infectivity, pathogenicity and antigenicity of the virus. The F protein is also widely used in the phylogenetic classification of NDV. The current NDV unified genotype classification system divides the virus into two gr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K39/17A61P31/14C12R1/93
CPCA61K39/12A61K2039/5252C12N7/00C12N2760/18121C12N2760/18134
Inventor 黄艳艳许传田杨少华张秀美黄庆华张琳吴家强高丹丹
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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