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Serine protease inhibitors of bufo melanocarpi and its genes and applications

A protease inhibitor, bufaserine technology, applied in the field of black-orbited toad research, to achieve the effect of strong inhibition of trypsin activity, convenient in vitro production, and strong inhibition of trypsin activity

Inactive Publication Date: 2019-02-12
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serine protease inhibitors of Bufo melanocarpius have not been reported yet

Method used

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  • Serine protease inhibitors of bufo melanocarpi and its genes and applications
  • Serine protease inhibitors of bufo melanocarpi and its genes and applications
  • Serine protease inhibitors of bufo melanocarpi and its genes and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Cloning of the Serine Protease Inhibitor Gene of Bufo melanogaster

[0058] 1. Extraction of total RNA from toad skin

[0059] 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;

[0060] 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;

[0061] 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.

[0062] 2. Synthesis of the skin cDNA l...

Embodiment 2

[0119] Example 2: Prokaryotic expression and purification of bufo melanocarpi serine protease inhibitor

[0120] 1. Construction of a prokaryotic expression vector for a serine protease inhibitor from Bufo melanogaster

[0121] Primers were designed according to the gene encoding the serine protease inhibitor of Bufia melanogaster, with two forward nested primer sequences, adding a Kpn I restriction endonuclease site and an enterokinase site, and the F1 sequence was 5'-GGTACCGACGACGACGACAAGATGC- 3' (SEQ ID NO: 5); F2 sequence is 5'-ACAAGATGCATCCTTGTTCTGGTTG-3' (SEQ ID NO: 6); reverse primer R sequence is 5'-AAGCTTCTATTTCACCTTTGGACATTC-3' (SEQ ID NO: 7), adding After the Hind III restriction site was recovered by PCR and gel, double enzyme digestion was performed; the operation steps of PCR and gel recovery were the same as Step 3 in Example 1 above.

[0122] Prepare the following digestion systems in PCR tubes:

[0123]

[0124] Digest at 37°C for 10 hours, add 20 μl of 6...

Embodiment 3

[0145] Example 3: Application of Serine Protease Inhibitors in Bufo melanogaster

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PUM

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Abstract

The invention discloses a duttaphrynus melanostictus serine protease inhibitor and a gene thereof. The amino acid sequence of the duttaphrynus melanostictus serine protease inhibitor is as shown in SEQ ID NO:2. The amino acid sequence of the gene encoding the duttaphrynus melanostictus serine protease inhibitor is as shown in SEQ ID NO:1. The duttaphrynus melanostictus serine protease inhibitor obtained through the prokaryotic expression means has high trypsin inhibition activity, has the advantages of being convenient to produce in vitro and high in trypsin inhibition activity, and can be used as a trypsin inhibitor.

Description

technical field [0001] The invention relates to the research field of toads melanocarpus, in particular to a serine protease inhibitor of toads melanocarbitus and its gene and application. 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 proven prescriptions for cancer (especially breast cancer) among the people, but At present, there are few studies on the molecular mechanism of its efficacy. [0003] Serine protease (serine protease) is a kind of proteolytic enzyme with serine as the active center. It belongs to proteolytic enzymes and exists in various or...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C12N15/15C12N15/10A61K38/57
CPCA61K38/00C07K14/8121C12N15/102C12Q2549/119
Inventor 宋玉竹卢文成刘锐张阿梅韩芹芹刘娃
Owner KUNMING UNIV OF SCI & TECH
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