Newcastle disease virus heat-resistant attenuated strain adapted to baby hamster kidney passage cell and preparation method and application thereof

A technology of Newcastle disease virus and passaged cells, which is applied in the field of new virus strains, heat-resistant and attenuated strains of Newcastle disease virus and preparation thereof, can solve the problem that D90 strains do not have the ability to adapt to passaged cells and heat resistance, and do not have the ability to adapt to passaged cells and cells. Heat resistance and other problems, to achieve the effect of good protection, long shelf life and broad application prospects

Inactive Publication Date: 2011-12-07
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Existing technical documents about Newcastle disease virus strains mainly contain the following items: as the patent application number CN200710003046.9, the document "Newcastle disease virus D90 strain and its application" discloses Newcastle disease virus D90 strains, and also discloses The application of this strain in anti-tumor, but the D90 strain does not have the characteristics of adapting to passage cells and heat resistance; the patent application number is CN20061000169...

Method used

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  • Newcastle disease virus heat-resistant attenuated strain adapted to baby hamster kidney passage cell and preparation method and application thereof
  • Newcastle disease virus heat-resistant attenuated strain adapted to baby hamster kidney passage cell and preparation method and application thereof
  • Newcastle disease virus heat-resistant attenuated strain adapted to baby hamster kidney passage cell and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0028] Preparation of Newcastle Disease Virus Heat-resistant Attenuated Strain Adapted to Young Hamster Kidney Passage Cells

[0029] Using DMEM nutrient solution (serum-free) containing 0.2 μg / ml TPCK-trypsin as the inoculum and cell maintenance solution, the NDVTS09 chicken embryo-adapted strain was continuously passaged in BHK21 cells for 17 times, except for the 11th, 13th and 15th passages. Except for the purified virus by limiting dilution method (dilution is determined by the highest dilution of CPE), the rest are 10 -2 Dilute the inoculum. The biological characteristics of the passaged viruses were measured, and Table 1 was obtained.

[0030] Table 1. Results of subculture of NDV TS09 chicken embryo-adapted strain on BHK cells

[0031]

[0032] Note: - means no cell disease; + ~ ++++ means cell disease rate (respectively 25% ~ 100%);

[0033] a Indicates that the passage method is to purify the virus by the limiting dilution method;

[0034] b Indicates the limi...

Embodiment 2

[0038] The present invention adapts to the cytopathic effect of NDV heat-resistant attenuated strain of BHK21 cells

[0039] The TS09-C strain virus liquid of the present invention was mixed with 10 -3 After dilution, inoculate BHK21 cells grown into a dense monolayer, observe the cell pathological changes, and obtain figure 1 .

[0040] Depend on figure 1 It can be seen that about 48 hours after the cells were inoculated with the virus, oval-shaped vacuoles of various sizes and cell fusion began to appear; within 72 to 96 hours, a large number of cells appeared to be broken, dead, and suspended in the nutrient solution; normal control cells The growth condition is good. The results showed that: TS09-C strain had cytopathic effect on BHK21 cells.

Embodiment 3

[0042] Determination and Analysis of HN Gene Sequence of NDV Heat-resistant Attenuated Strain Adapted to BHK21 Cells in the Present Invention

[0043] According to the HN gene sequence of Newcastle disease virus HB92 strain (GenBnak accession number: AY225110) published in GenBank, a pair of primers were designed to amplify the full-length HN gene, which was synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd. The specific sequence is as follows: NDV-F2: 5'-CGGTTGTAGATGACCAAAGG-3'; NDV-R2: 5'-AATCTAATCACATTAGCACTAGCTG-3';

[0044] The extraction of viral genome RNA was carried out according to the kit instructions. The extracted RNA was dissolved in 17ul DEPC water, 1μl of reverse transcription primer was added, 75°C for 5min, immediately ice-bathed, and then reverse transcription reaction solution: 5×RT Buffer 5μl, dNTPs (10mmol / L) 1 μl and M-MLV 1 μl. 42°C for 30 minutes, 95°C for 5 minutes, and stored at -20°C for PCR reaction.

[0045] PCR reaction system:...

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Abstract

The invention relates to a newcastle disease virus heat-resistant attenuated strain adapted to baby hamster kidney passage cells and a preparation method and an application thereof, and the microbial preservation number is CCTCC No.V201113; the classification term is newcastle disease virus TS09-C strain; the preservation time is May 9th, 2011; the preservation unit is China center for type culture collection; the preservation address is Wuhan university, Wuhan, China; the strain has the following advantages: 1. the strain is fully adapted to BHK21 cells, and has a proliferation titer of up to 108.0TCID50/ml; 2. the strain has good heat resistance, and the HA titer does not decrease obviously after the strain is treated at 56 degree for 1 hour; 3. the strain has an attenuated characteristic, has chicken embryo average lethal time of more than 90 hours, and an intracerebral pathogenicity index of 0.00; 4. a newcastle disease heat-resistant live vaccine (passage cell source) prepared by the attenuated strain has the advantages of heat resistance, long storage life, good protective efficacy, prevention of chicken embryos for laying from carrying heterogenous virus, and the like.

Description

technical field [0001] The invention belongs to the field of microbial viruses, and relates to a new virus strain, in particular to a heat-resistant attenuated strain of Newcastle disease virus adapted to young hamster kidney passage cells and its preparation method and application. The microorganism preservation number is: CCTCC V201113; classification Name: Newcastle disease virus TS09-C strain; preservation time: May 9, 2011; preservation unit: China Center for Type Culture Collection; preservation address: China.Wuhan.Wuhan University. Background technique [0002] Newcastle disease virus (NDV) is a member of the genus Mumpsvirus in the Paramyxoviridae family and is an enveloped negative-strand RNA virus. Newcastle disease (ND) caused by NDV is a highly contagious and devastating disease with a mortality rate of over 90%. Avian Influenza). The prevention and control of ND is mainly based on immune prevention. There are currently two types of ND vaccines produced and u...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/08A61K39/17A61P31/14C12R1/93
Inventor 温国元邵华斌杨峻商雨王红琳罗青平张蓉蓉郭静艾地云罗玲
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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