Bufo melanostictus schneider cystatin as well gene and application thereof

A technology of toad cystatin and cystatin, applied in the field of biomedicine, can solve the problem of less research on molecular mechanism and so on

Inactive Publication Date: 2013-06-26
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Black-orbited toad, Anura, Bufoidae, Bufo genus, is one of the main sources of traditional Chinese medicine "dried toad", generally caught in summer and autumn; tastes pungent, cool, poisonous, and returns to liver, spleen, lung meridian, It has the effects of detumescence, detoxification, pain relief, and diuresis. It is often used in the treatment of chronic bronchitis, carbuncle furuncle, sore throat, edema, and dysuria. There are also folk prescriptions for cancer (especially breast cancer), but At present, there are few studies on the molecular mechanism of its efficacy

Method used

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  • Bufo melanostictus schneider cystatin as well gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Cloning of Cystatin Gene from Bufo melanogaster

[0026] 1. Extraction of total RNA from toad skin

[0027] A, the live black-orbited toad was cleaned with water, put into liquid nitrogen and quick-frozen for 4 hours, got skin tissue, weighed, got 300mg skin tissue, added 10ml total RNA extraction buffer (Trizol solution, U.S. Invitrogen company product), in Homogenize in a 20ml glass homogenizer for 10 minutes;

[0028] B. Add an equal volume of phenol / chloroform solution, oscillate and mix well, place at room temperature for 10 minutes, centrifuge at 12000rpm at 4°C for 10 minutes, and absorb the upper aqueous phase liquid;

[0029] C. Add 1 / 2 volume of isopropanol to the supernatant, place it at room temperature for 10 minutes, centrifuge at 7500g for 10 minutes at 4°C, wash the precipitate once with 75% ethanol, dry it, and the precipitate at the bottom of the tube is Total RNA from toad skin.

[0030] 2. Synthesis of the skin cDNA library of Toad me...

Embodiment 2

[0094] Example 2: Prokaryotic expression and purification of melanocarpus cystatin

[0095] 1. Construction of prokaryotic expression vector for cystatin from Bufo melanocarpi

[0096] Primers were designed according to the gene encoding cystatin in Buad melanogaster, with two forward nested primer sequences, adding a Kpn I restriction endonuclease site and an enterokinase site, and the F1 sequence was 5'-GGTACCGACGACGACGACAAGATGGA-3 '(SEQ ID NO:5); F2 sequence is 5'-CGACAAGATGGATCAAAAAGG-3'(SEQ ID NO:6); reverse primer R sequence is 5'-AAGCTTCTAAAAATAACAGATGGG-3'(SEQ ID NO:7), add Hind The III restriction site is subjected to double enzyme digestion after PCR and gel recovery; the operation steps of PCR and gel recovery are the same as Step 3 in Example 1 above.

[0097] Prepare the following digestion systems in PCR tubes:

[0098] PCR product / circular vector 50 ul

[0099] wxya 2 O 30ul

[0100] 10×M Buffer 10ul

[0101] Kpn I 5ul(15U)

...

Embodiment 3

[0129] Embodiment 3: the application of melanocarpus cystatin

[0130] Detection of cathepsin B inhibitory activity of melanocarpus cystatin.

[0131] With the chromogenic substrate Z-Arg-Arg-pNA (Benzyloxycarbonyl-L-arginyl-L-arginine-p-nitroani-lide) as the reaction substrate, bovine cathepsin B (0.2ng / reaction) and reaction buffer ( 0.1 M sodium acetate, pH 5.5, containing 1mM DTT, 2mM EDTA and 4mM cysteine), different concentrations of Bufabufa cystatin (final concentration 2–8μM), mixed and left at room temperature for 5min, added Z-Arg -Arg-pNA to a final concentration of 400uM, after incubation at 37°C for 30min, detect the absorbance at 410nm (recorded as Abo I), set the recombinant cystatin added as a control (denoted as Abo C), calculate the inhibition rate, calculate The method is (Abo C-Abo I) / Abo C × 100%.

[0132] The result is as figure 1 It shows that cystatin from Bufo melanogaster has strong cathepsin B inhibitory activity, and can inhibit 75% of cathep...

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Abstract

The invention discloses a bufo melanostictus schneider cystatin and a gene of the bufo melanostictus schneider cystatin. The amino acid sequence of the bufo melanostictus schneider cystatin is shown as SEQ ID NO: 2, and the nucleotide sequence of the gene for coding the bufo melanostictus schneider cystatin is shown as SEQ ID NO: 1. The bufo melanostictus schneider cystatin obtained by means of prokaryotic expression has strong cathepsin-B inhibiting activity, is convenient to produce in vitro and can be used as a cathepsin-B inhibitor.

Description

technical field [0001] The invention provides a cystatin gene and application thereof, belonging to the technical field of biomedicine. Background technique [0002] Black-orbited toad, Anura, Bufoidae, Bufo genus, is one of the main sources of traditional Chinese medicine "dried toad", generally caught in summer and autumn; tastes pungent, cool, poisonous, and returns to liver, spleen, lung meridian, It has the effects of detumescence, detoxification, pain relief, and diuresis. It is often used in the treatment of chronic bronchitis, carbuncle furuncle, sore throat, edema, and dysuria. There are also folk prescriptions for cancer (especially breast cancer), but At present, there are few studies on the molecular mechanism of its efficacy. [0003] In the process of tumor development, cathepsin B plays an important role. Cathepsin B can directly degrade or activate plasminogen activator and matrix metalloproteinase, etc., and indirectly degrade many extracellular matrix comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C12N15/15
Inventor 宋玉竹刘娃张阿梅韩芹芹纪森林
Owner KUNMING UNIV OF SCI & TECH
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