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Bdellin-hm, a kazal-type trypsin inhibitor and its coding gene and application

A technology of trypsin inhibition and phenanthrene, which is applied in the field of biomedicine, can solve the problems that have not yet been reported on the study of phenanthrene Kazal-type serine protease inhibitors

Active Publication Date: 2019-03-01
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are no relevant research reports on the Kazal-type serine protease inhibitors

Method used

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  • Bdellin-hm, a kazal-type trypsin inhibitor and its coding gene and application
  • Bdellin-hm, a kazal-type trypsin inhibitor and its coding gene and application

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Embodiment Construction

[0021] The substantive content of the present invention will be described in further detail below by way of examples.

[0022] Separation and purification of phenanthrene trypsin inhibitor Bdellin-HM:

[0023] Ⅰ. DEAE Sephadex A-50 anion exchange column:

[0024] The crude extract of the head of 3 g of lyophilized Hirudo phenanthae was dissolved in 20 ml of 50 mM Tris hydrochloric acid buffer (pH 8.9), centrifuged at 12000 rpm for 10 minutes, and the supernatant was taken to be loaded on an equilibrated DEAE Sephadex A-50 anion-exchange column ( 5 × 60 cm), with gradient elution of Tris hydrochloric acid buffer containing different concentrations of sodium chloride, and eluted 200 tubes (collected with an automatic part collector, 15 ml per tube, flow rate 1.5 ml / min). The specific sodium chloride concentration is: 0-0.2M NaCl (0, 0.2M NaCl each 1L), 0.2-0.4M NaCl (0.2, 0.4M NaCl each 1L), 0.4-0.6M NaCl (0.4, 0.6M NaCl each 1L ), 1M NaCl. The concentration of protein or pol...

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Abstract

The invention relates to a Hirudinaria manillensis Kazal trypsin inhibitor Bdellin-HM, and an encoded gene and application thereof, belonging to the technical field of biomedicine. The molecular weight of the Hirudinaria manillensis Kazal trypsin inhibitor Bdellin-HM is 17432.8 Dalton, the isoelectric point is 4.84, the complete sequence is disclosed as SEQ ID NO:1, and the 1st and the 5th cysteines, the 2nd and the 4th cysteines, and the 3rd and the 6th cysteines form 3 pairs of intramolecular disulfide bonds. The encoded gene disclosed as SEQ ID NO:2 is composed of 504 nucleotides, and the 54th-501st of the encoded mature peptide part is nucleotide. Separation and purification are carried out to obtain the Hirudinaria manillensis Kazal trypsin inhibitor Bdellin-HM, and cloning is carried out to obtain the cDNA (complementary deoxyribonucleic acid) sequence. The inhibitor has obvious trypsinase inhibition activity (Ki-8.45nM), but the inhibiting actions on elastase, chymotrypsin, kallikrein, thrombin, fibrinolysin, FXIIa, FXIa and FXa are not observed; and thus, the inhibitor can be used for preparing the trypsin inhibitor.

Description

Technical field: [0001] The invention provides a Kazal-type trypsin inhibitor Bdellin-HM of Hirudinaria manillensis and its coding gene and application, belonging to the technical field of biomedicine. Background technique: [0002] Leeches are a kind of traditional Chinese medicine in my country. The anticoagulant effect of leeches is recorded in "Erya" in the 2nd century BC, "Compendium of Materia Medica" and "Shen Nong's Materia Medica" by Li Shizhen in the Ming Dynasty. "Compendium of Materia Medica" said: "Salty to remove blood, bitterness is better than blood. The salty and bitterness of leeches is used to remove blood accumulation, and it is the liver meridian blood to divide the medicine, so it can pass the liver meridian and gather blood." Chinese medicine believes that leeches are a traditional broken medicine. Blood medicine has the effects of dispelling blood stasis, dredging meridians, and benefiting water channels. It is mainly used to treat blood stasis amenor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C12N15/15
CPCC07K14/8135
Inventor 赖仞龙承波赖艳梅李博文
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI