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Antiviral protein and preparation method and application thereof

An anti-virus and protein technology, applied in the field of anti-viral proteins from fungi and its preparation, can solve the problems of unclear anti-viral components and unclear mechanisms

Inactive Publication Date: 2009-11-11
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many problems in the existing research, such as many antiviral ingredients are not clear, the mechanism is not clear, etc.
The present invention discovers a new antiviral protein RC28 derived from fungi, which has not been reported in the existing literature

Method used

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  • Antiviral protein and preparation method and application thereof
  • Antiviral protein and preparation method and application thereof
  • Antiviral protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Separation and purification of RC28 protein

[0055] (1) Coarse separation: the fruiting bodies of Umberella chinensis were collected from high-altitude mountain forests in Xiaojin County, Sichuan Province. After collection, they were immediately stored in ice packs and brought back.

[0056] Discard the handle of the fruiting body, take only the canopy, wash with distilled water, drain, add 5 times the volume of 4°C pre-cooled pH7.2, 0.02M PBS (containing 0.15M NaCl) solution and homogenate. Stir the homogenate mixture gently at 4°C overnight, centrifuge at 10,000g (20min) to remove the precipitate, and filter the supernatant with filter paper to remove the suspended matter. Use -20°C pre-cooled acetone to add the supernatant filtrate in a ratio of 2:1, mix immediately, After standing at -20°C for more than 2h, centrifuge at 10,000g (4°C) for 30min, discard the supernatant, dissolve the precipitate in 100ml, pH7.2, 0.02M (containing 0.3M NaCl) PBS solution, c...

Embodiment 2

[0060] Embodiment 2 Partial property experiment of antiviral protein RC28

[0061] (1) Stability: Treat the RC28 solution with boiling water at 100°C for 10 minutes, or use 0.25% trypsin to digest overnight at 37°C, then measure the activity against herpes virus type I, compared with the control group without any treatment, all There is more than 60% activity retention, indicating that RC28 has good thermal stability and enzymolysis resistance.

[0062] (2) When the pH value of the solution is less than 4, RC28 precipitates, but after returning to a neutral environment, the activity and solubility are well preserved; at the same time, from the perspective of ion exchange properties, the isoelectric point of RC28 is about 4.

[0063] (3) The exact molecular weight of RC28 was determined to be 28.251kD by MALDI-TOF mass spectrometry.

[0064] (4) After SDS electrophoresis, RC28 was transferred to PVDF membrane, quickly stained with Coomassie brilliant blue R250, the band was ex...

Embodiment 3

[0070] Example 3 Determination of the complete sequence of RC28 protein

[0071] 1. Extract the total RNA of Rozites caperata

[0072] During the growing season of Umberella rugosa, the fresh tender tissues of the canopy were collected in the wild, stored in liquid nitrogen immediately and brought back, and the total RNA was extracted by the Trizol method, and identified by agarose gel electrophoresis, which confirmed that the obtained RNA was of good quality , no significant degradation occurred, and the electrophoresis results were as follows Figure 6 shown;

[0073] 2. Use the 3'-RACE method to amplify the cDNA of RC28

[0074] (1) Based on the obtained RC28 N-terminal sequence and referring to the codon preference of fungi Basidiomycetes, two sets of RACE degenerate primers were designed:

[0075] The outer primer is designed from the amino acid sequence GKLNMYNYA: GGNAAGYTBAACTGGTACAACTACGC

[0076] The inner primer is designed by the amino acid sequence YAVNEGFT: TA...

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Abstract

The invention discloses an antiviral protein RC28, the amino acid thereof is shown in SEQ ID NO.1, and the preparation method thereof comprises the following steps: crude separation is carried out to obtain crude extract of RC28, molecular sieve chromatography and ion exchange chromatography are used to further carry out separation and purification to obtain RC28 protein product. The antiviral protein RC28 of the invention has good antiviral activity, and provides an efficient and safe new way for treating virus infection, in particular to enveloped virus infection diseases.

Description

technical field [0001] The invention relates to a protein and a preparation method thereof, in particular to an antiviral protein from fungi and a preparation method thereof. Background technique [0002] Viral diseases account for about 60% of human and animal infectious diseases and seriously endanger life and health. Therefore, research on antiviral drugs has attracted widespread attention and has become one of the hot spots in international research and development. However, due to the particularity of virus structure, survival and reproduction methods, it is not easy to distinguish it from host cells. Therefore, most antiviral drugs have either weak antiviral effects or severe side effects. It is very difficult to develop ideal antiviral drugs. The clinical efficacy of the existing antiviral drugs is not ideal, and there are problems such as narrow application range, poor penetration, large toxic and side effects, and there is a lack of effective drugs for new viruses s...

Claims

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Application Information

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IPC IPC(8): C07K14/37C07K1/14A61K38/16A61P31/12A61P27/02A61P31/16A61P11/00A61P35/02
Inventor 刘旭阳龚萌程惊秋皮耐诺·弗兰克
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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