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Gene VII-type newcastle disease virus strain, vaccine composition thereof and preparing method and application of vaccine composition

A technology for Newcastle disease virus and vaccine composition, applied in the field of attenuated vaccine strains, can solve the problems of difficult separation of genotype VII Newcastle disease virus strains, obvious differences in chicken embryo growth characteristics, and inability to provide ideal immune protection efficacy and the like

Pending Publication Date: 2016-10-05
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Judging from the epidemic characteristics of Newcastle disease in the past ten years, most of the clinically isolated Newcastle disease virus strains belong to genotype VII except the traditional types I and II, while the genotypes of conventional Newcastle disease vaccine strains Mainly types I and II (such as V4, LaSota, etc.), the new isolates of Newcastle disease are far from the conventional Newcastle disease vaccine strains in terms of genetic distance. Ideal immune protection potency
[0003] However, the virulence of different Newcastle disease virus isolates is very different, and the growth characteristics on chicken embryos are significantly different, and the isolation of suitable gene VII Newcastle disease virus strains is extremely difficult.

Method used

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  • Gene VII-type newcastle disease virus strain, vaccine composition thereof and preparing method and application of vaccine composition
  • Gene VII-type newcastle disease virus strain, vaccine composition thereof and preparing method and application of vaccine composition
  • Gene VII-type newcastle disease virus strain, vaccine composition thereof and preparing method and application of vaccine composition

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Experimental program
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Effect test

Embodiment 1

[0071] Isolation and identification of embodiment 1 gene VII type Newcastle disease virus strain

[0072] 1.1 Sample collection and processing

[0073] The brain, heart, liver, spleen, kidney, air sac and other tissues of dead chickens in Shandong, Guangdong, Sichuan, Jiangsu, Henan and other places were collected, and physiological saline or phosphate buffered saline (PBS, PH7. 4) Grind into an emulsion, add penicillin (final concentration 1000U / ml) and streptomycin (final concentration 1mg / ml) into the solution, act at 37°C for 1 hour or overnight at 4°C, centrifuge at 1000rpm for 10 minutes, and take the supernatant for later use.

[0074] 1.2 Virus isolation and identification

[0075] Inoculate 9-11-day-old SPF chicken embryos with the supernatant obtained in step 1.1, discard dead embryos within 24 hours after inoculation, and irradiate embryos every day 24-120 hours after inoculation, place dead chicken embryos at 4°C, The chicken embryos were harvested 120 hours after ...

Embodiment 2

[0107] Example 2 HN1101 Newcastle Disease Virus Main Immunogenicity Gene Determination

[0108] 2.1 Extraction of HN1101 Newcastle Disease Virus RNA

[0109] Utilize the Trizol method to extract the viral genome RNA in the allantoic fluid of chicken embryos, the detailed steps are as follows:

[0110] (1) Take 250 μl of chicken embryo allantoic fluid, add 750 μl Trizol (invitrogen, USA), shake and mix well, and place at room temperature for 5 minutes;

[0111] (2) Add 200 μl of chloroform to each tube, cap the centrifuge tube tightly, shake the centrifuge tube vigorously for 15 seconds, place at room temperature for 10 minutes, and centrifuge at 12000 rpm for 15 minutes;

[0112] (3) Take the upper aqueous phase and place it in a new centrifuge tube, add 700 μl of isopropanol, place at 4°C for 10 minutes, and centrifuge at 12,000 rpm for 10 minutes;

[0113] (4) Discard the supernatant, add 75% ethanol at a ratio of at least 1ml per milliliter of Trizol solution, mix well, a...

Embodiment 3

[0139] The attenuation of embodiment 3HN1101 strain Newcastle disease virus strain

[0140] Referring to Hu Shunlin's doctoral dissertation of Yangzhou University, the establishment and application of the reverse genetic technology platform of goose-derived Newcastle disease virus and the reverse genetic operating system of Newcastle disease La Sota vaccine strain and its application (patent publication number: CN 1772909A) using segmental cloning Methods The whole genome cDNA clone (transcription vector) was constructed, and the clones NP, P and L were respectively amplified into the eukaryotic expression vector pCI-neo as the helper plasmid for NDV rescue. The rescue process of NDV is to construct a whole genome cDNA clone and helper plasmids containing genes NP, P and L to co-transfect mammalian cells expressing T7 RNA polymerase. The positive-sense single-stranded RNA, the polymerase protein expressed by the auxiliary plasmid wraps the positive-sense single-stranded RNA of...

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Abstract

The invention discloses a gene VII-type newcastle disease virus strain with good immunogenicity and a low virulent strain subjected to passage attenuation through the newcastle disease virus strain. A newcastle disease virus strain F gene comprises a nucleotide sequence of a protein sequence shown by substantially-coded SEQ ID NO.2, and a virus strain HN gene comprises a nucleotide sequence of a protein sequence shown by substantially-coded SEQ ID NO.6. The newcastle disease virus strain is high in toxicity, and the growth rate on a chicken embryo is high; compared with a conventional newcastle disease virus strain, the gene VII-type newcastle disease virus strain has the advantages of being good in safety, high in immune protection capacity and immune efficacy and the like.

Description

technical field [0001] The invention relates to a virulent strain of gene VII type Newcastle disease virus strain, an attenuated vaccine strain weakened by reverse genetic technology, and a vaccine composition prepared by the attenuated strain and the preparation of the vaccine composition The method and application belong to the field of biomedicine. Background technique [0002] Newcastle disease is an important infectious disease caused by Newcastle disease virus that causes a high degree of mortality in a variety of poultry. Newcastle disease virus has existed in China for decades. Due to the widespread use of vaccines in recent years, the morbidity and mortality of Newcastle disease have been well controlled. However, since the early 1990s, the outbreak of atypical Newcastle disease At the same time, clinically, the phenomenon that Newcastle disease virus and other respiratory pathogens cause disease together is relatively common. Therefore, Newcastle disease is still ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C07K14/125C12N7/00A61K39/17A61P31/14
Inventor 张许科孙进忠王同燕田克恭
Owner PU LIKE BIO ENG
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