A h9n2 subtype avian influenza virus with interchanged ha and ns1 deletion gene packaging signals and its construction method and application

A technology of avian influenza virus and construction method, which is applied in the field of H9N2 subtype avian influenza virus and its construction, and can solve problems such as the virulence return of live attenuated vaccines and the like

Active Publication Date: 2021-09-03
YANGZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, live attenuated vaccines also have certain defects, such as the exchange of internal gene segments between the vaccine strain and the wild-type AIV in the environment, resulting in the emergence of new epidemic strains and the return of the virulence of the attenuated live vaccine.

Method used

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  • A h9n2 subtype avian influenza virus with interchanged ha and ns1 deletion gene packaging signals and its construction method and application
  • A h9n2 subtype avian influenza virus with interchanged ha and ns1 deletion gene packaging signals and its construction method and application
  • A h9n2 subtype avian influenza virus with interchanged ha and ns1 deletion gene packaging signals and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) Vector construction

[0048]The 8 fragments of the H9N2 TX strain used in the present invention were cloned into the pHW2000 plasmid, and the NS1 gene deletion plasmid was constructed simultaneously (see Chen S, ZhuY, Yang D, YangY, Shi S, Qin T, et al.Efficacy of Live-Attenuated H9N2 Influenza Vaccine Candidates Containing NS1 Truncationsagainst H9N2 Avian Influenza Viruses[J].Front Microbiol.2017;8:1086.) were named as pHW291-PB2, pHW292-PB1, pHW293-PA, pHW294-HA, pHW295-NP, pHW296-NA, pHW297-M, pHW298-NS, pHW298-NS1-128. The nucleotide sequences of the NS-HAmut-NS and HA-NS1-128mut-HA genes that exchange HA and NS1 deletion gene packaging signals are shown in sequence as SEQ ID NO.1 and SEQ ID NO.2. The two recombinant genes were artificially synthesized and connected to the pHW2000 plasmid vector. The site-directed mutagenesis and primers are shown in Table 1, the head-to-tail synonymous mutation primers of HA and NS1-128 ORF box are shown in Table 2, and the ...

Embodiment 2

[0075] The hemagglutination titer of the virus, the half-infection dose of SPF chicken embryos and the growth curve on MDCK cells

[0076] Take 25 μl chicken embryo allantoic fluid and add it to 25 μl PBS to make 2 1 -2 11 Add 25 μl of 1% chicken red blood cell suspension after doubling dilution, and make a negative control well at the same time, place it in a 37°C incubator for 10 minutes, and read the hemagglutination titer. Compared with the parental virus and the NS1 gene deletion virus, the HA titer decreased by 3 and 1 titers, respectively.

[0077] Table 8 Virus HA titer

[0078]

[0079] The stock solution of viral allantoic fluid was made into PBS containing antibiotics for 10 5 ~10 10 Gradual dilution, inoculate five 9-day-old SPF chicken embryos, 0.1ml / each, and incubate in a 37°C incubator for 72h. The allantoic fluid was collected, the HA titer of the allantoic fluid was measured, and the half infection dose was calculated. The results are shown in Table 9...

Embodiment 3

[0086] HA and NS fragment recombination assay

[0087] Will 1×10 6 The MDCK cells were cultured in a 6-well plate, and the rTX wild strain and NS1-128 (mut) packaging signal exchanged NS truncated attenuated virus were simultaneously inoculated at an infectious dose of MOI=10, and after adsorption for 1 hour, washed with PBS for 3 hours. Then add 1ml anti-blood-free DMEM containing TPCK trypsin with a final concentration of 2μg / ml for cell culture. After 12 hours after infection, the supernatant was collected by freezing and thawing three times, and centrifuged for later use.

[0088] The MDCK cells with a monolayer were cultured in a 12-well plate, and the plate was washed three times with PBS after discarding the medium. The virus was diluted to 10 with anti-blood-free MEM medium gradient -5, add 200 μl virus diluent along the well wall, shake the plate to mix. The 12-well plate was incubated at 37°C for 1 h, and the plate was shaken every 15 minutes to mix once. Then r...

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Abstract

The invention provides an H9N2 subtype avian influenza virus with interchangeable HA and NS1 deletion gene packaging signals, its construction method and application, and belongs to the technical field of vaccines. An H9N2 subtype avian influenza virus with exchanged HA and NS1 deletion gene packaging signals, including recombinant NS‑HAmut‑NS and HA‑NS1‑128mut‑HA genes. The nucleotide sequences of the recombinant NS‑HAmut‑NS and HA‑NS1‑128mut‑HA genes are shown in sequence as SEQ ID NO.1 and SEQ ID NO.2. The recombinant H9N2 subtype avian influenza virus constructed by using the gene of the present invention can effectively avoid reassortment of HA and NS fragments with wild strains, and has attenuation properties and good challenge protection effect.

Description

technical field [0001] The invention belongs to the technical field of vaccines, and in particular relates to an H9N2 subtype avian influenza virus with interchangeable HA and NS1 deletion gene packaging signals and its construction method and application. Background technique [0002] H9N2 subtype avian influenza virus (AIV) can infect a variety of poultry and wild birds. Although H9N2 subtype AIV strains are less pathogenic to poultry, they cause huge economic losses to the poultry industry, mainly causing respiratory symptoms, immunosuppression and decreased egg production. H9N2 AIV has the property of binding to mammalian receptors, and studies have confirmed that it can be transmitted between ferrets through aerosols. In addition, H9N2 AIV can provide some or all of its internal genes to recombine with H5N1, H7N9, H10N8, and H5N6 subtypes of AIV to produce reassortant viruses. The H5N6 subtype AIV of the internal gene cassette; in 2013, six internal gene fragments wer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/44C12N15/88C12N7/04A61K39/145A61P31/16C12R1/93
CPCA61K39/12A61K2039/5254A61K2039/552A61P31/16C07K14/005C12N7/00C12N15/88C12N2760/16121C12N2760/16122C12N2760/16134C12N2760/16152C12N2760/16162
Inventor 陈素娟王辉彭大新秦涛杜元钊楚电峰
Owner YANGZHOU UNIV
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