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H5 and H7 subtype avian influenza virus genetic engineering subunit vaccine, and preparation method and application thereof

A technology of avian influenza virus and subunit vaccines, which is applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of expensive chicken embryo processing costs and high labor costs, and is conducive to large-scale production, The effect of short time consumption and simple preparation method

Inactive Publication Date: 2021-04-20
乾元浩生物股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, domestic H5 and H7 subtype avian influenza vaccines are mainly produced with chicken embryos. Due to the particularity of chicken embryos, they are easily affected by the incubation cycle, and the live virus is handled during the production process, resulting in high labor costs, and the chicken embryos after production need to be expensive processing fees

Method used

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  • H5 and H7 subtype avian influenza virus genetic engineering subunit vaccine, and preparation method and application thereof
  • H5 and H7 subtype avian influenza virus genetic engineering subunit vaccine, and preparation method and application thereof
  • H5 and H7 subtype avian influenza virus genetic engineering subunit vaccine, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Design and Construction of Shuttle Carrier

[0033] According to the H5 Clade2.3.4.4d (GenBank: QOJ99767.1), Clade2.3.2.1d (GenBank: QBF65380.1) published by the NCBI database, the HA protein sequence of the H7 highly pathogenic branch epidemic strain (GenBank: ASB31508.1 ), intercept the 1-400 amino acid residues of each HA protein, design the coding gene sequence, add the start codon ATG to the N-terminus, add 6×His tag CATCATCATCATCACCAC and the stop codon TAA sequence to the end, and carry out insect cell codon Optimization (the coding gene sequence of the optimized HA protein truncated body is respectively shown in SEQ ID NO: 4-6), sent to the biological company for synthesis, and connected to the pFastBac1 vector (purchased from Invitrogen Corporation) by means of homologous recombination ), pFastBac1-54HA, pFastBac1-52HA and pFastBac1-7HA were obtained respectively.

Embodiment 2

[0034] Example 2 Screening of Positive Recombinant Bacmid and Obtaining of Recombinant Baculovirus

[0035]1. Take 5 μg each of the shuttle vectors pFastBac1-54HA, pFastBac1-52HA, and pFastBac1-7HA, and transform them into DH10Bac competent cells, incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, incubate on ice for 5 minutes, add anti-antibody-free LB medium, and keep at 37°C Incubate on a shaker at 190rpm for 4 hours, take 10 μl of the bacterial solution and coat the three-antibody plate, and incubate in a constant temperature incubator at 37°C for 48 hours. After the blue and white spots are obvious, pick the white spots and add the third antibody (containing kanamycin, gentamicin and tetracycline) LB medium for overnight culture.

[0036] 2. Collect the bacterial liquid, collect the bacterial cells by centrifugation at 12000 rpm, extract the recombinant Bacmid by isopropanol precipitation, and detect the concentration of Bacmid with a spectrophotometer. Th...

Embodiment 3

[0038] Embodiment 3 Propagation of recombinant baculovirus and expression of protein

[0039] 1. Take sf9 cells from serum-free suspension culture until the cell density is 2.5×10 6 cells / mL, inoculate P0 generation recombinant virus according to 0.1% volume ratio, place at 27°C, 120rpm for suspension culture, after 72-96h, when the cells expand and the refractive index decreases, harvest the supernatant as P1 generation recombinant virus, and continue in this way Passed to 5-6 generations, the P5 or P6 generation recombinant virus was used as the seed virus for protein expression.

[0040] 2. Acquire hi5 cells from serum-free suspension culture until the cell density is 2.5×10 6 cells / mL, inoculate P5 or P6 generation virus at 0.1% volume ratio, place at 27°C, 120rpm for suspension culture, wait for 72-96h after the cells expand and the refractive index decreases, harvest the supernatant, and perform Western Blot detection with anti-His tag antibody Protein expression, the ...

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Abstract

The invention provides an H5 and H7 subtype avian influenza virus genetic engineering subunit vaccine, and a preparation method and application thereof. The extracellular region of H5 and H7 subtype avian influenza virus HA protein is captured to construct a shuttle vector containing an HA extracellular region gene, an sf9 insect cell is transfected, a recombinant virus is saved, through an hi5 cell, secretory expression is carried out, purification is carried out through affinity chromatography to obtain an HA protein antigen, and an adjuvant is added and emulsified to prepare the H5 and H7 subtype avian influenza virus genetic engineering subunit vaccine. The preparation method disclosed by the invention is simple, overcomes the defects generated by producing vaccines by chick embryos, is short in time consumption, is high in an expression quantity, can realize cell suspension culture in a reactor, and is favorable for large-scale production. The prepared genetic engineering subunit vaccine has a good immune effect, can effectively prevent infection of H5 and H7 subtype avian influenza viruses and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a genetically engineered subunit vaccine of H5 and H7 subtype avian influenza virus and its preparation method and application. Background technique [0002] Avian influenza is an acute, febrile and highly contagious disease caused by avian influenza virus, which has caused huge losses to the breeding industry in our country. Hemagglutinin (HA) and neuraminidase (NA) on its surface are the two main viral envelope glycoproteins. According to the different antigenic properties, type A influenza virus can be divided into 18 HA subtypes and 11 NA subtypes. Subtypes, of which H1-H16 and N1-N9 are present in wild waterfowl. Since the first report of the H5N1 subtype avian influenza in 1996, the H5 subtype avian influenza virus has spread to the whole world. So far, the H7N9 subtype avian influenza has also caused multiple waves of infection. [0003] Influenz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11C12N15/44C12N15/866C12N5/10
Inventor 李海鹰丁向东王增福岳建新李吉轩张秀美袁野孙灵睿史张艳赵成全李京京
Owner 乾元浩生物股份有限公司
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