Influenza virus H1N1 subtype neuraminidase as well as gene, inhibitor screening model and application thereof

A neuraminidase, H1N1 technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of tedious and time-consuming preparation of neuraminidase, safety issues, etc. Sensitive effect

Inactive Publication Date: 2014-06-25
SHANGHAI INST OF PHARMA IND CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to provide a kind of neuraminidase for the preparation of neuraminidase used in the existing influenza virus H1N1 subtype neuraminidase inhibitor screening model which is cumbersome and time-consuming, and has safety problems. Influenza A virus neuraminidase and its gene and preparation method, and the construction method of the screening model of influenza virus neuraminidase inhibitor

Method used

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  • Influenza virus H1N1 subtype neuraminidase as well as gene, inhibitor screening model and application thereof
  • Influenza virus H1N1 subtype neuraminidase as well as gene, inhibitor screening model and application thereof
  • Influenza virus H1N1 subtype neuraminidase as well as gene, inhibitor screening model and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction of recombinant plasmid pPICZαA-NAS

[0032] 1) Obtained 1629 neuraminidase amino acid sequences of influenza A virus H1N1 subtypes reported from April 2009 to March 2010 from the NCBI website, and selected the most representative virus strains after comparison and analysis by CluxtalX software A / Wisconsin / 629-S0247 / 2009 (H1N1) (GenBank: CY051377) was used as the research object. The full-length nucleic acid of the gene is 1407bp, encoding 469 amino acids. According to the expression habit of Pichia pastoris, the gene is codon-optimized, and the sequence of the codon-optimized NA full-length gene is shown in SEQ ID NO.1 in the sequence table. The optimized gene was synthesized by chemical synthesis, and the synthesized full-length gene was connected to the cloning vector pUC57 to obtain the cloning vector pUC57-NA, and the sequence was verified to be completely correct (entrusted to Shanghai Jingjing Biosynthesis).

[0033] 2) Design primers for P...

Embodiment 2

[0037] Example 2 Construction of recombinant Pichia pastoris strain and induced expression of truncated NA

[0038] 1) Prepare KM71 and SMD1168 competent, the specific steps include:

[0039] a. Single colonies of KM71 (purchased from Invitrogen) and SMD1168 (purchased from Invitrogen) grown on YPD plates at 30° C. were picked and inoculated into 20 mL of YPD liquid medium at 250 rpm at 30° C. for overnight cultivation.

[0040] b. Take 0.1-0.5mL overnight culture, inoculate a 2L shake flask containing 500mL fresh medium, and culture overnight until OD600=1.3-1.5.

[0041] c. Collect the cells by centrifugation at 1500g for 5 minutes at 4°C, and suspend the cells with 500 mL of pre-cooled sterilized water.

[0042] d. Centrifuge as above, and suspend the cells with 250 mL of pre-cooled sterile water.

[0043] e. Centrifuge as above, and suspend the cells with 20 mL of pre-cooled 1M sorbitol.

[0044] f. Centrifuge as above, and suspend the cells with 1 mL of pre-cooled 1M s...

Embodiment 3N

[0054] Embodiment 3NA activity measurement and inhibition experiment

[0055] 0.5% methanol induced recombinant Pichia pastoris to secrete and express truncated NA, and the bacterial solution induced for 96 hours was centrifuged at 12000rpm for 5min, and the supernatant was taken as the crude enzyme solution for NA activity assay, and at the same time, it was concentrated by ultrafiltration in a centrifuge tube. The crude enzyme solution was concentrated about 10 times and used for the inhibition experiment of oseltamivir, zanamivir, amantadine and rimantadine on truncated NA. The experimental equipment was BioTek multifunctional microplate reader and 96-well black microplate plate .

[0056] NA activity assay: take 30 μl fermentation supernatant, add 75 μl reaction substrate (final concentration is 32.5mM MES buffer, pH6.5, 4mM CaCl 2 , 100 μM substrate 4-MUNANA), the kinetic method detects the change of fluorescence intensity under the excitation light of 360nm and the emis...

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Abstract

The invention provides influenza A-type influenza virus neuraminidase (NA), a gene and a preparation method thereof as well as a construction method of a screening model of an influenza virus neuraminidase inhibitor. Particularly, influenza A-type influenza virus H1N1 subtype neuraminidase is secretly expressed by pichia pastoris to obtain the neuraminidase with biological activity, and particularly, the truncated neuraminidases of 82 amino acids at N end of an NA full-length sequence is removed, so that an operation process of the NA prepared by totivirus, which is complicated, consumes time and has a safety problem, is avoided. According to the invention, the A-type influenza virus H1N1 subtype neuraminidase inhibitor screening model is also constructed by utilizing the neuraminidase; and the influenza virus H1N1 subtype neuraminidase has the advantages of definite mechanism of drug action, less material consumption, quick screening speed, high sensitivity, easy realization of high throughput screening and the like.

Description

technical field [0001] The invention belongs to the field of screening anti-influenza virus drugs, and in particular relates to a neuraminidase of influenza virus H1N1 subtype and its gene, and uses the enzyme to construct an inhibitor screening model and application thereof. Background technique [0002] Influenza virus has been a serious threat to human health for a long time, and the successive outbreaks of its H5N1 and H1N1 subtypes in recent years have increased the demand for anti-influenza virus drugs worldwide. There are currently four main drugs approved by the FDA for the treatment of influenza: Amantadine, Rimantadine, Oseltamivir, and Zanamivir. [0003] With the continuous increase of drug-resistant cases, human beings are facing the severe test of a large-scale outbreak of influenza virus, and the development of new anti-influenza virus drugs is imperative, which makes the design and synthesis or screening of new neuraminidase inhibitors from compound libraries...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N15/81C12N1/19C12N9/24C12Q1/34C12R1/93C12R1/84
Inventor 朱宝泉林军胡海峰胡又佳
Owner SHANGHAI INST OF PHARMA IND CO LTD
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