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A kind of cicadae protease capable of inhibiting HIV-1 reverse transcriptase and its preparation method

A technology of protease and protease activity, which is applied in the field of cicadae protease and its preparation, and can solve problems such as outstanding drug resistance and toxic side effects

Inactive Publication Date: 2011-12-07
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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  • A kind of cicadae protease capable of inhibiting HIV-1 reverse transcriptase and its preparation method
  • A kind of cicadae protease capable of inhibiting HIV-1 reverse transcriptase and its preparation method
  • A kind of cicadae protease capable of inhibiting HIV-1 reverse transcriptase and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Separation, purification and identification of Cordyceps sobolifera protease

[0034] 1. Extraction and coarse separation

[0035] Suspend Cordyceps sobolifera (Liu, Z.-Y., Liang, ZQ, Whalley, AJS, Liu, A.-Y. & Yao*, Y.-JA new species of Beauveria) with 0.15M NaCl solution The anamorph of Cordyceps sobolifera.Fungal Diversity, 2001, 7: 61-70. The public can obtain the fruit body from Beijing Academy of Agriculture and Forestry Sciences). The fruit body is broken with a tissue masher until the fruit body is pasty. Place it at 4℃. Hours later, centrifuge at 8000rpm (12000g) for 15min, collect the supernatant solution to obtain the crude extract; add (NH 4 ) 2 SO 4 To 80% saturation, stand at 4°C for 4 hours, centrifuge at 8000 rpm (12000 g) for 15 minutes, collect the precipitate to obtain the crude protein extract, dissolve the protein crude extract precipitate in distilled water and dialyze in distilled water for 12-16 hours.

[0036] 2. Ion exchange column chromat...

Embodiment 2

[0057] Example 2. Characterization of protease properties of cicada flower

[0058] Take the pure cicada flower protease obtained in Example 1, according to the protease activity detection method of Example 1, to study the optimum temperature, thermal stability, optimum pH value, the effect of ions on the cicada flower protease activity, protease Enzymatic properties such as the effect of inhibitors on cicada protease.

[0059] 1. Optimal temperature

[0060] Take the pure cicada flower protease obtained in Example 1, and use 0.1M pH7.2 phosphate buffer to prepare an enzyme solution with a concentration of 10mM. According to the protease activity detection method in Example 1, first prepare the same reaction system and mix well. , And then react for 15 minutes at 20°C, 30°C, 40°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, 85°C, and measure the protease activity, and measure it at 37°C for 15 minutes The enzyme activity is set at 100%, and the relative enzyme activity is plotted against t...

Embodiment 3

[0076] Example 3. Functional detection of Cordyceps sobolifera protease inhibiting HIV-1 reverse transcriptase activity

[0077] 1. Accurately weigh the pure cicada flower protease obtained in Example 1, and configure them into different dosage solutions (see Table 4) to detect the activity of inhibiting HIV-1 reverse transcriptase;

[0078] 2. Use the Non-radioactive reverse transcriptase ELISA kit (Roche, 11468120910), follow the kit instructions, and 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 Positive control -A405 sample ) / (A405 Positive control -A405 Negative control )×100%

[0080] A405 Positive control : The reaction system do...

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

Claims

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Application Information

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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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