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H9N2 subtype avian influenza virus ns1 gene deletion attenuated live vaccine candidate strain and its construction method and application

A technology of avian influenza virus and live attenuated vaccine, applied in the field of AIV strains of BJ-94-like and Y280-like lineages, can solve problems such as the inability to protect SPF chickens well, and achieve a real immune protection effect, Good security and good cross-protection effect

Active Publication Date: 2018-01-09
YANGZHOU UNIV
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  • Claims
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AI Technical Summary

Problems solved by technology

Under laboratory conditions, after SPF chickens were vaccinated with inactivated vaccines prepared by early strains, they were challenged with the current popular H9N2 subtype AIV by nasal drops, and high levels of shedding still occurred (Sun Y, Pu J, JiangZ , et al.Genotypic evolution and antigenic drift of H9N2influenza viruses inChina from1994to 2008.Vet Microbiol,2010,146:215-225.); and the inactivated vaccine prepared by using some epidemic strains can not protect SPF chickens well against H9N2 Virus attack (Liu Dong, Gong Xiao, Liu Xiaodong, et al. 2013. Analysis of H9N2 subtype avian influenza epidemic and screening of vaccine strains. China Animal Quarantine. 2013.30. (6): 45-50; Wan Xiaopeng, Zeng Xian Ying, Tian Guobin, et al. Screening of inactivated vaccine seed viruses of H9N2 subtype avian influenza epidemic strains. Chinese Journal of Preventive Veterinary Medicine. 2010.32(4):277-280)

Method used

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  • H9N2 subtype avian influenza virus ns1 gene deletion attenuated live vaccine candidate strain and its construction method and application
  • H9N2 subtype avian influenza virus ns1 gene deletion attenuated live vaccine candidate strain and its construction method and application
  • H9N2 subtype avian influenza virus ns1 gene deletion attenuated live vaccine candidate strain and its construction method and application

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Embodiment Construction

[0016] Concrete test scheme among the present invention:

[0017] 1. Gene deletion and rescue of vaccine strains

[0018] 1.1 Primer design

[0019]According to the NS gene sequence of A / chicken / Taixing / 10 / 2010 (TX) strain virus (Genbank sequence number: JN653684, A / chicken / Taixing / 10 / 2010 (TX) strain (Zhu Y, Yang Y, Liu W, et al .Comparison of biological characteristics of H9N2avian influenza viruses isolated from different hosts, Archives of virology, 2015, 160:917-927) Design NS1 gene deletion primers (Table 1).

[0020] Table 1 Primer design

[0021]

[0022] 1.2 Overlapping-PCR to delete and modify the NS1 gene

[0023] Using the whole viral nucleic acid of the TX strain as a template, full-length primers (NS-1 and NS-2) reverse transcription PCR (RT-PCR) to amplify the NS gene, using a 25 μL system: 10×Buffer 2.5 μL, Mg 2+ 1.5 μL, 0.5 μL of dNTP, 0.5 μL of each primer NS-1 and NS-2 (both at a concentration of 25 μmol / μL), 0.5 μL of Taq DNA polymerase (1 U / μL), 2.5...

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Abstract

The invention relates to an NS1 gene deleted and live-attenuated vaccine candidate strain rTX-NS1-128 of H9N2 subtype avian influenza virus and its establishing method and application. The NS1 gene deleted and live-attenuated vaccine candidate strain rTX-NS1-128 is characterized in that an A / chicken / Taixing / 10 / 2010TX strain is used as a female parent to delete the length of an NS1 gene, and only 128 amino acids indicated in SEQ ID NO.15 are reserved in an NS1 protein. The NS1 gene deleted and live-attenuated vaccine candidate strain can be replicated only in a trachea, no contact transmission occurs, and safety is good. The NS1 gene deleted and live-attenuated vaccine candidate strain is available for independent use, as an effective tool to control the H9 subtype avian influenza virus, is also useful with an inactivated vaccine to allow truer immunity protection, and is able to play an important role in controlling the H9 subtype avian influenza.

Description

technical field [0001] The invention relates to a method for modifying the length of H9N2 subtype avian influenza virus (AIV) NS1 protein to construct a broad-spectrum H9N2 subtype AIV attenuated live vaccine by reverse genetic technology, and the AIV mainly involved is BJ-94-like and Y280-like lineage strains. Background technique [0002] The H9N2 subtype avian influenza virus is prevalent worldwide. Since its first isolation in mainland China in 1994, it has spread widely and gradually formed an endemic disease in my country, causing huge economic losses to the aquaculture industry in our country. In addition to infecting poultry, H9N2 subtype AIV can also infect mammals (pigs and humans, etc.). Although H9N2 subtype AIV infects humans only causes subclinical or mild clinical symptoms, H9N2 subtype AIV can recombine with other subtypes of avian influenza viruses to produce new lethal human influenza viruses, such as H5 subtype avian influenza virus and H7N9 Subtype of i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/04C12N15/63C12N15/44A61K39/145A61P31/16C12R1/93
Inventor 彭大新陈素娟朱寅彪杨达施少华刘秀梵
Owner YANGZHOU UNIV
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