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Recombinant gene VII type Newcastle disease virus strain and vaccine composition, preparation method and application thereof

A technology for Newcastle disease virus and vaccine composition, applied in the field of recombinant gene type VII Newcastle disease virus strains, can solve problems such as low production efficiency, and achieve the effects of reducing production costs, improving economic benefits, and broad-spectrum immunogenicity

Active Publication Date: 2020-01-21
LUOYANG HUIZHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it was found that the production efficiency of the rescued strain was low during actual large-scale production, and further improvement was needed in terms of virus content and hemagglutination activity.

Method used

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  • Recombinant gene VII type Newcastle disease virus strain and vaccine composition, preparation method and application thereof
  • Recombinant gene VII type Newcastle disease virus strain and vaccine composition, preparation method and application thereof
  • Recombinant gene VII type Newcastle disease virus strain and vaccine composition, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Isolation and identification of gene VII type Newcastle disease virus

[0052] 1.1 Isolation of Newcastle disease virus

[0053] The respiratory secretions and air sac tissues of infected chickens with typical symptoms of Newcastle disease were collected from farms in Henan, Hunan, etc., added to the PBS buffer containing penicillin-streptomycin double antibody, and the grinding solution was centrifuged at 1000 rpm After 10 minutes, take the supernatant and inoculate 9-11 day-old chicken embryos, 0.1ml / embryo, seal with paraffin and place in a 37°C incubator for 120 hours. Observe the death of chicken embryos every day, discard the dead chicken embryos within 24 hours, and harvest the allantoic fluid after 24-120 hours of dead chicken embryos are placed in a refrigerator at 2-8°C for 1 hour; The allantoic fluid of undead chicken embryos harvested for 120 hours was blindly passed for 2 generations according to the above method. If no dead chicken embryos appea...

Embodiment 2

[0065] Construction and identification of embodiment 2 recombinant gene VII type Newcastle disease virus strain

[0066] 2.1 Construction

[0067] Primer design: Design primer pairs 1, 2, and 3, using the constructed N7a strain of attenuated gene VII Newcastle disease strain as the backbone, and replace the P gene of the N7a strain with the P gene of a different strain.

[0068] 2.1.1 Amplification of the P protein gene of 5 strains of VII strains isolated in Example 1.3

[0069] Genomic RNA of each NDV strain was reverse-transcribed, and cDNA clones of five gene VII clinical strains HN-07, HuN-01, HuN-03, FJ-02, and GD-05 were constructed. The sequences of primer pairs 1, 2, and 3 are as follows:

[0070] Primer pair 1:

[0071] F1: 5-GCgtcgacAACCCGCCCAGAGCCCAAG-3; SaI

[0072] R1: 5-TCGCACAACTGCAACCAATCCAGCT-3;

[0073] Primer pair 2:

[0074] F2: 5-TCGCACAACTGCAACCAATCCAGCT-3;

[0075] R2: 5-GAGTgccggcTTGAATGATGACTTT-3Nae I

[0076] Primer pair 3:

[0077] F3: 5-GC...

Embodiment 3

[0106] The preparation of embodiment 3 vaccine composition

[0107] 3.1 Preparation of virus solution

[0108] Dilute the NDV N7a strain and the purified N7a-HuN-01 strain and N7a-FJ-02 strain in Example 2.3 to 10 with sterile PBS buffer solution according to the dilution factor in Table 3. 5.5 EID 50 / 0.1ml, inoculate 20 10-day-old SPF chicken embryos via the allantoic cavity route, 0.1ml per embryo, and continue to incubate at 37°C. The embryos that died between 24 and 120 hours after inoculation were placed at 2 to 8°C in time, and the mixed samples were collected at 120 hours, and the HA titer and virus content of each virus solution were measured by sampling. The results are shown in Table 3.

[0109] Table 3 HA antibody titer and virus content determination results of each strain after culture

[0110]

[0111] According to the virus content, each strain was diluted to 10 8.0 EID 50 / 0.1ml, 10 9.0 EID 50 / 0.1ml (because the virus content of N7a-HuN-01 strain is...

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Abstract

The invention discloses a recombinant gene VII type Newcastle disease virus attenuated strain rN7a strain, and further discloses a vaccine composition containing the rN7a strain or a culture-inactivated antigen of the rN7a strain. The rN7a strain is the attenuated strain obtained by replacing a P protein gene sequence of a Newcastle disease virus N7a strain with the preservation number of CCTCC NO:V201545 with a P protein gene sequence as shown in SEQ ID No.1. The rN7a strain has high virus titer and high HA titer after culture. The vaccine composition can provide complete protection to a variety of strains.

Description

technical field [0001] The invention relates to a recombinant gene VII type Newcastle disease virus strain, its vaccine composition, preparation method and application, and belongs to the field of biotechnology. Background technique [0002] Newcastle disease (ND), also known as Asian chicken plague and pseudofowl plague, is a disease caused by serotype I avian paramyxovirus (ie Newcastle disease virus, NDV) among the nine serotypes of avian paramyxoviruses in chickens and turkeys. It is an acute and highly contagious infectious disease of poultry, often showing symptoms of sepsis. Newcastle disease is a serious, highly contagious, viral disease that is widely distributed in many countries around the world and has caused major economic losses. At the same time, clinically, the phenomenon of synergistic pathogenicity of Newcastle disease virus and other respiratory pathogens is also relatively common. is a major threat to the poultry industry. [0003] Judging from the epid...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/86C12N15/66A61K39/17A61P31/14C07K14/125
CPCC12N7/00C07K14/005C12N15/86C12N15/66A61K39/12A61P31/14C12N2760/18121C12N2760/18143C12N2760/18122C12N2760/18134A61K2039/5252A61K2039/552Y02A50/30
Inventor 田克恭韩水仲孙进忠张许科
Owner LUOYANG HUIZHONG BIOTECH
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