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Cordyceps sobolifera protease capable of inhibiting human immunodeficiency virus-1 (HIV-1) reverse transcriptase and preparation method for Cordyceps sobolifera protease

A technology of protease and protease activity, applied in the field of cicada protease and preparation, can solve problems such as outstanding drug resistance, toxic and side effects, etc.

Inactive Publication Date: 2012-11-21
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mitsuya et al. first discovered that deoxythymidine AZT has a significant effect of inhibiting HIV, and then found that deoxynucleosides, acyclic nucleosides, and nucleoside analogs can effectively inhibit the replication of HIV, but the resistance of these inhibitors Drug problem is becoming more and more prominent, and has serious toxic side effects

Method used

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  • Cordyceps sobolifera protease capable of inhibiting human immunodeficiency virus-1 (HIV-1) reverse transcriptase and preparation method for Cordyceps sobolifera protease
  • Cordyceps sobolifera protease capable of inhibiting human immunodeficiency virus-1 (HIV-1) reverse transcriptase and preparation method for Cordyceps sobolifera protease
  • Cordyceps sobolifera protease capable of inhibiting human immunodeficiency virus-1 (HIV-1) reverse transcriptase and preparation method for Cordyceps sobolifera protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, separation, purification and identification of cicada flower (Cordyceps sobolifera) protease

[0034] 1. Extraction and coarse separation

[0035] Suspending Cicada (Cordyceps sobolifera) with 0.15M NaCl solution (Liu, Z.-Y., Liang, Z.Q., Whalley, A.J.S., Liu, A.-Y. & Yao*, Y.-J.A new species of Beauveria, The anamorph of Cordyceps sobolifera. Fungal Diversity, 2001, 7: 61-70. The public can obtain from the Beijing Academy of Agriculture and Forestry Sciences) fruiting body, use a tissue breaker to break the fruiting body into a paste, and place it at 4°C for 12 After 1 hour, 8000rpm (12000g) centrifugal 15min, collect supernatant solution, obtain crude extract; Add (NH 4 ) 2 SO 4 To 80% saturation, stand at 4°C for 4 hours, centrifuge at 8000rpm (12000g) for 15min, collect the precipitate to obtain a crude protein extract, dissolve the crude protein precipitate in distilled water and dialyze in distilled water for 12-16 hours.

[0036] 2. Separation a...

Embodiment 2

[0057] Embodiment 2, the characterization of cicadae protease properties

[0058] Get the cicadae protease pure product that embodiment 1 obtains, according to the protease activity detection method of embodiment 1, research the optimum temperature of cicadae protease, thermostability, optimum pH value, ion on the impact of cicadae protease activity, protease The enzymatic properties such as the influence of inhibitor on cicadae protease.

[0059] 1. Optimum temperature

[0060] Get the cicadae protease pure product that embodiment 1 obtains, be mixed with the enzyme liquid that concentration is 10mM with 0.1M pH7.2 phosphate buffer solution, press the protease activity detection method of embodiment 1, first be made into identical reaction system after mixing , and then reacted at 20°C, 30°C, 40°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, and 85°C for 15 minutes to measure the protease activity, which was measured at 37°C for 15 minutes The enzyme activity was determined as 100%...

Embodiment 3

[0076] Example 3, Functional Detection of Cordyceps sobolifera Protease Inhibiting HIV-1 Reverse Transcriptase Activity

[0077] 1. Accurately take the pure product of cicadae protease that embodiment 1 obtains, be configured into different dosage solutions (see table 4), to detect the activity that suppresses HIV-1 reverse transcriptase;

[0078] 2. Use the Non-radioactive reverse transcriptase ELISA kit (Roche Company, 11468120910), and follow the instructions of the kit. For specific methods, refer to the literature Shuang Zhao & Yongchang Zhao & Shuhong Li & Jingkun Zhao & Guoqing Zhang & Hexiang Wang & Tzi Bun Ng. A novel lectin with highly potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from the edible wild mushroom Russula delica. Glycoconj J (2010) 27: 259-265, the results are shown in Table 4 and Figure 8 .

[0079] Inhibition rate (%) = (A405 正对照 -A405 样品 ) / (A405 正对照 -A405 负对照 )×100%

[0080] A405 正对照 : The reaction system does ...

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Abstract

The invention discloses a Cordyceps sobolifera protease capable of inhibiting human immunodeficiency virus-1 (HIV-1) reverse transcriptase and a preparation method for the Cordyceps sobolifera protease. The preparation method comprises the following steps of: 1) breaking Cordyceps sobolifera cells; 2) collecting a crude protein extract; and 3) separating and purifying protease from the crude protein extract collected in the step 2) to obtain a Cordyceps sobolifera extract. The protease has the molecular weight of 31KD, has the N-terminal amino acid sequence shown as a sequence 1 in a sequencetable, belongs to serine protease, and has the highest activity under the following conditions: the pH value is 12-12.5 and the temperature is 55-65DEG C. The Cordyceps sobolifera extract prepared bythe method has obvious inhibition effect on the HIV-1 reverse transcriptase, and has important value in the field of new anti-AIDS (acquired immune deficiency syndrome) medicine research.

Description

technical field [0001] The invention relates to a cicadae protease capable of inhibiting HIV-1 reverse transcriptase and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] Protease is an enzyme that catalyzes the hydrolysis reaction of proteins. It can act on the peptide bonds in protein molecules to form shorter peptide chains or amino acids. Proteases widely exist in organisms, regulate protein metabolism, and perform various functions at various levels such as cells, organs, and organisms. There are many kinds of proteases, and the application fields are also very wide, mainly involving detergents, leather, textiles, food, medicine, environmental protection and resources, etc. In recent years, with the development of biological research technology, the development of protease resources and the search for new high-quality proteases have always been the focus of researchers from all over the world. [0003] Cicada flower (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/58A61K38/48A61P31/18C12R1/79
Inventor 王守现刘宇王贺祥张国庆赵爽许峰耿小丽王兰青孟莉莉
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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