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Recombinant influenza A virus carrying hepatitis B virus gene, host cell, preparation method for recombinant influenza A virus and application of recombinant influenza A virus

A technology of influenza A virus and hepatitis B virus, applied in the field of biopharmaceuticals, to avoid injection pain, ensure shearing, and high shearing efficiency

Inactive Publication Date: 2020-07-03
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And using influenza virus as the carrier of HBV vaccine development, there is no report at home and abroad

Method used

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  • Recombinant influenza A virus carrying hepatitis B virus gene, host cell, preparation method for recombinant influenza A virus and application of recombinant influenza A virus
  • Recombinant influenza A virus carrying hepatitis B virus gene, host cell, preparation method for recombinant influenza A virus and application of recombinant influenza A virus
  • Recombinant influenza A virus carrying hepatitis B virus gene, host cell, preparation method for recombinant influenza A virus and application of recombinant influenza A virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Construction of recombinant NS fragment

[0045] 1. Use molecular biology methods to carry out synonymous point mutations at the RNA splicing site in the NS fragment, and mutate 525-CCAGGA-530 into 525-CCCGGG-530.

[0046] 2. The hepatitis B virus gene was obtained by PCR from the plasmid preserved in our laboratory.

[0047] 3. As shown in the figure, link the mutated NS fragment to the exogenous hepatitis B virus gene through the two short peptides Linker1 and Linker2 according to the open reading frames of NS1 and NS2, so as to obtain the recombinant NS fragment. .Using molecular biology methods, the target gene sequence is connected with the bidirectional transcription expression vector pHW2000, and positive clones are selected. The constructed recombinant plasmid was identified by sequencing, and the size of the fragment was exactly the same as expected, and there was no gene mutation.

Embodiment 2

[0048] Example 2: Rescue of Recombinant Influenza A Virus Strain Carrying Hepatitis B Virus

[0049] Co-transfect 293T or cos cells with plasmids carrying influenza virus wild-type PB2, PB1, PA, HA, NP, NA, M fragments and recombinant plasmids carrying NS and target fragments, and can also transfect 293T or COS and MDCK The co-cultured cell line was replaced with DMEM medium containing TPCK trypsin after 6 h. The final concentration of TPCK trypsin was 1ug / ml. Incubate for 48 hours at 37° C. in a 5% CO2 cell culture incubator, and collect the supernatant. The collected supernatant removes cell debris and infects MDCK cells. After 48-72 hours, the supernatant of MDCK cells was collected for plaque purification. After three rounds of plaque purification, the virus was amplified in MDCK, and finally a recombinant influenza A virus strain carrying hepatitis B virus was obtained.

Embodiment 3

[0050] Example 3: Plaque Purification of Recombinant Influenza A Virus Strain Carrying Hepatitis B Virus

[0051] Plating: Digest the MDCK cells in the cell flask with trypsin, spread them into 6-well plates, and the number of cells per well is 10 6 . After the MDCK in the six-well plate adhered to the wall and grew into a single layer of cells, the medium was aspirated and washed twice with PBS.

[0052] Adsorption: The collected virus-containing supernatant was diluted 10-fold with PBS solution containing 0.3% BSA, and added to a six-well plate at a dose of 400ul per well, and 2-3 auxiliary wells were set for each gradient. The adsorption time was 1 h, and the plate was shaken every 15 minutes during the adsorption period to distribute the supernatant evenly and prevent the cells from dehydrating.

[0053] Overlay: After the adsorption is completed, wash off the residual supernatant with PBS. Mix 2×DMEM with melted 0.6% low melting point agarose 1:1 and add TPCK trypsin, ...

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Abstract

The invention discloses a recombinant influenza A virus carrying a hepatitis B virus gene, a host cell, a preparation method for the recombinant influenza A virus and application of the recombinant influenza A virus. According to the recombinant influenza A virus disclosed by the invention, by taking an influenza A virus as a vector, and by utilizing a reverse genetics technique, the hepatitis B virus gene is integrated onto an influenza virus genome, and thus, the recombinant influenza virus can passage steadily in the host cell or a chicken embryo. The recombinant influenza A virus can be used for development of hepatitis B vaccines, development of related drugs, and used for producing hepatitis B associated proteins by utilizing the chicken embryo or the cell as a bioreactor.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and specifically refers to a recombinant influenza A virus carrying a hepatitis B virus gene, a host cell, a preparation method and application thereof. Background technique [0002] Influenza virus belongs to Orthomyxoviridae Orthomyxoviridae segmented single-stranded negative-sense RNA virus, the virus genome is about 13.6kb, divided into 8 independent segments of different sizes, encoding the structural proteins of influenza virus (PB1, PB2, PA, HA, NP, NA, M1, M2) and nonstructural proteins (NS1, NS2). Among them, PB1, PB2 and PA constitute the polymerase of the virus, and the nucleoprotein NP acts as a scaffold structure and binds to vRNA. Influenza viruses encode two surface glycoproteins: hemagglutinin HA and neuraminidase NA. HA can be adsorbed to sialic acid variants on the surface of host cells to promote virus infection of cells; NA promotes the release of virus from infected cells ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/85C07K14/02A61K39/29A61P31/20A61P1/16C12R1/93
CPCA61K39/12A61P1/16A61P31/20C07K14/005C12N7/00C12N15/85C12N2730/10122C12N2730/10134C12N2730/10151C12N2760/16121C12N2760/16152C12N2800/107
Inventor 朱应黄玙佘应龙刘实
Owner WUHAN UNIV
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