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Recombinant influenza virus carrying Helicobacter pylori, host cell and preparation method and application thereof

A technology of Helicobacter pylori and influenza virus, applied in botany equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve problems such as preventing Helicobacter pylori infection

Active Publication Date: 2021-09-24
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this therapy can only remove Helicobacter pylori in the stomach and cannot prevent the infection of Helicobacter pylori in humans, and in many countries in the world, Helicobacter pylori is resistant to antibiotics such as clarithromycin and metronidazole. Medicinal properties

Method used

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  • Recombinant influenza virus carrying Helicobacter pylori, host cell and preparation method and application thereof
  • Recombinant influenza virus carrying Helicobacter pylori, host cell and preparation method and application thereof
  • Recombinant influenza virus carrying Helicobacter pylori, host cell and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Construction of recombinant NS fragment

[0047] 1. Use molecular biology methods to carry out synonymous point mutations at the RNA splicing site in the NS fragment, and 525-CCAGGA-530 is mutated to 525-CCCGGG-530.

[0048] 2. The Helicobacter pylori gene is synthesized by gene synthesis means.

[0049] 3. Ligate the mutated NS fragment with the exogenous Helicobacter pylori gene through the self-splicing polypeptide according to the open reading frames of NS1 and NS2 to obtain a recombinant NS fragment, such as Figure 4 shown. 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

[0050] Example 2: Rescue of recombinant influenza virus

[0051] Co-transfect influenza virus wild-type PB2, PB1, PA, HA, NP, NA, M and recombinant plasmids into 293T or COS cells, or the co-culture cell line of 293T or COS and MDCK, and replace it with TPCK after 6 hours Trypsin in DMEM medium. The final concentration of TPCK trypsin was 1ug / ml. at 37°C, 5% CO 2 environment, cultivated for 48 hours, and collected the supernatant. The collected supernatant was clarified to infect MDCK cells. After 48-72 hours, the supernatant was collected for plaque purification, and after three rounds of plaque purification, the virus was amplified in MDCK to obtain an influenza virus vaccine strain carrying the Helicobacter pylori gene.

Embodiment 3

[0052] Example 3: Plaque Purification of Influenza Virus

[0053] Before virus adsorption, digest MDCK cells, spread them into 6-well plate, the number of cells per well is 10 6 . After MDCK adhered to the wall and grew into a single layer of cells, the culture medium was aspirated and washed twice with PBS. The collected virus-containing supernatant was diluted 10-fold with PBS solution containing 0.3% BSA and added to a six-well plate in an amount of 400ul per well, and an appropriate amount of auxiliary wells should be set for each gradient. The adsorption time was 1 h, and the residual supernatant was washed with PBS after the adsorption was completed. Mix 2×DMEM with melted low melting point agarose 1:1 and add TPCK trypsin, the final concentration is 1ug / ml. Add 2ml of the mixture into each well, and put it into a 37°C incubator for 3 days after cooling and solidifying. After 3 days, the plaque growth was observed.

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Abstract

The invention discloses a recombinant influenza virus carrying Helicobacter pylori, a host cell, a preparation method and application thereof. The present invention is a recombinant influenza virus composed of Helicobacter pylori antigen or antigen dominant epitope integrated into NS segment of influenza virus genome. The recombinant influenza virus carrying Helicobacter pylori of the present invention can be stably passaged in host cells or chicken embryos, and can be used for the development of Helicobacter pylori vaccines, the development of related drugs, and the production of Helicobacter pylori proteins using chicken embryos or cells as bioreactors .

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant influenza virus carrying Helicobacter pylori, a host cell and a preparation method and application thereof. Background technique [0002] Type A influenza virus (influenzaAvirus), belonging to Orthomyxoviridae, its genome is composed of 8 negative polarity RNA segments (vRNA). In influenza virus, eight RNA segments of its genome combine with three polymerase proteins (PB2, PB1, PA) and nucleoprotein (NP) to form active ribonucleoprotein polymers (RNPs) (Eisfeld A J, Neumann G , Kawaoka Y. At the centre: influenza A virus ribonucleoproteins [J]. Nature Reviews Microbiology, 2015, 13(1): 28-41.). When influenza A virus infects host cells, hemagglutinin (HA) mediates the binding of virus particles to sialic acid receptors on host cells. After the influenza virus enters the cell by membrane fusion, the virus will release RNPs, and the RNPs enter the nucleus to start the r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/31C12N5/10A61K39/02A61K39/145A61K39/295A61P31/04A61P31/16
CPCA61K39/0208A61K39/0225A61K39/12A61K39/295A61K2039/70A61P31/04A61P31/16C07K14/20C12N7/00C12N2760/16121C12N2760/16134
Inventor 朱应聂龙宇佘应龙刘实
Owner WUHAN UNIV
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