Application of AIV (avian influenza virus) H7N9 NA (neuraminidase) inhibitor

A bird flu virus and neuraminidase technology, applied in antiviral agents, medical preparations containing active ingredients, organic active ingredients, etc., can solve problems such as high price, low oral availability, and small volume of distribution

Inactive Publication Date: 2015-06-03
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the clinical use of zanamivir and oseltamivir has certain limitations, such as the low oral availability of zanamivir, small volume of distribution in the body, and rapid renal clearance, so it can only be used as a local drug
Oseltamivir (trade name: Tamiflu), as

Method used

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  • Application of AIV (avian influenza virus) H7N9 NA (neuraminidase) inhibitor
  • Application of AIV (avian influenza virus) H7N9 NA (neuraminidase) inhibitor
  • Application of AIV (avian influenza virus) H7N9 NA (neuraminidase) inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Expression of influenza virus A / Anhui / 1 / 2013 (H7N9) neuraminidase

[0017] Obtain the gene sequence of influenza virus A / Anhui / 1 / 2013 (H7N9) neuraminidase from the database GISAID (Global Initiative on Sharing Avian Influenza Data) (serial number: EPI439509), and optimize its codons to make it suitable Highly expressed in CHO-K1 cells. The gene sequence is artificially synthesized, named N9, digested by Hind III and Xho I, the digested product is ligated into pcDNA 3.1(+) plasmid digested with the same digestion, and the ligated product is transformed into TOP10 competent cells and coated Amp icillin resistant plate was cultured for 14-18 hours, and a single colony was picked. After the colony was amplified by shaking flask culture, the plasmid was extracted and identified by restriction enzyme digestion, and the cloned neuraminidase gene was sequenced. The recombinant plasmid was identified by double enzyme digestion and expressed in CHO-K1 cells, and a stable cell line ...

Embodiment 2

[0023] Expression of mutant influenza virus A / Anhui / 1 / 2013 (H7N9) neuraminidase

[0024] In the influenza virus A / Anhui / 1 / 2013 (H7N9) neuraminidase gene sequence, arginine 294 was mutated to lysine, and the gene was named N9-R294K. The recombinant expression plasmid pCDNA3.1(+)-N9-R294K was constructed, and a stable cell line was constructed to prepare recombinant neuraminidase N9-R294K. The method is the same as above.

[0025] The results are attached Figure 4 , 5, 6, Figure 4 It showed that the recombinant plasmid pCDNA3.1(+)-N9-R294K was extracted and detected by 1% agarose gel electrophoresis, and a plasmid with a size of 4.1 kb was obtained. The plasmid pCDNA3.1(+)-N9-R294K was double digested with restriction endonucleases Hind III and Xho I, and a 1395bp gene fragment was obtained, which was sequenced. The sequencing result was completely consistent with the sequence of gene N9-R294K , Indicating that the recombinant plasmid pCDNA3.1(+)-N9-R294K was successfully constru...

Embodiment 3

[0027] Inhibition of baicalin on N9 neuraminidase activity

[0028] A 96-well blackboard was used in the experiment, and baicalin was dissolved in PBS and diluted to 500, 250, 125, and 62.5 μM. Add 35μl of MES buffer to each well, add 30μl of recombinantly expressed influenza virus A / Anhui / 1 / 2013(H7N9) neuraminidase solution, and then add 10μl of baicalin of different concentrations, and incubate at 37°C for 5 min. For light operation, add 25μl of neuraminidase specific fluorescent substrate MUNANA to each well, incubate at 37°C for 20min, and finally add 100μl of stop solution 150μl (14mmol / 1 NaOH solution, containing 83% ethanol) to each well. Fluorescence microplate reader measures fluorescence at excitation wavelength 360nm and emission wavelength 440nm. Calculate the inhibition rate of baicalin on neuraminidase activity. The experiment also set up a blank control group without drugs. Inhibition rate of neuraminidase enzyme activity = (fluorescence value of blank control g...

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Abstract

The invention belongs to the technical field of antiviral drugs in natural active matters, and particularly relates to an application of baicalin which has the activity of inhibiting AIV (avian influenza virus) H7N9 NA (neuraminidase). The baicalin has the activity of inhibiting the AIV H7N9 NA. Researches show that the baicalin has the obvious activity of inhibiting wild type and oseltamivir-resistant AIV H7N9 NA and has the application of preparation of drugs for resisting AIVs. The AIV NA is the wild type and oseltamivir-resistant AIV A/Anhui/1/2013 (H7N9) NA recombined and expressed in CHO-K1 cells. Research results show that the baicalin has the application of preparation of the drugs for resisting the AIV H7N9.

Description

Technical field: [0001] The invention belongs to the technical field of antiviral drugs in biochemistry, and specifically relates to a use of baicalin for inhibiting avian influenza virus H7N9 neuraminidase. Background technique: [0002] The H7N9 subtype avian influenza virus is one of the influenza A viruses, which can cause a severe infectious disease of poultry, belonging to the Orthomyxoviridae family, RNA viruses. Studies have revealed that the H7N9 virus may have come from genetic recombination between avian influenza viruses carried by wild birds migrating to Southeast Asia from Eurasia and ducks and chickens in Shanghai, Zhejiang, Jiangsu and other places in China. Neuraminidase (NA) of influenza virus is a surface glycoprotein with enzymatic activity, which is highly conserved in all influenza A and B viruses. [0003] Commonly used influenza virus neuraminidase inhibitors (zanamivir, oseltamivir, etc.) can simultaneously inhibit influenza A and B viruses, and play an im...

Claims

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Application Information

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IPC IPC(8): A61K31/7048A61P31/16
Inventor 周长林窦洁王婷婷郁洁王慧靳京
Owner CHINA PHARM UNIV
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