Bullfrog slow-excitation peptide, coded nucleic acid thereof and application

A technology of bradykinin and bullfrog, applied in the field of biomedicine, to achieve the effect of promoting the contraction of isolated ileum muscle, convenient artificial synthesis, and simple structure

Inactive Publication Date: 2015-10-21
TIANJIN AGRICULTURE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The application of amphibian drugs has a long history in my country, but the research on its active ingredients and pharmacol...

Method used

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  • Bullfrog slow-excitation peptide, coded nucleic acid thereof and application
  • Bullfrog slow-excitation peptide, coded nucleic acid thereof and application
  • Bullfrog slow-excitation peptide, coded nucleic acid thereof and application

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preparation example Construction

[0020] The preparation process of bullfrog bradykinin of the present invention comprises the following steps:

[0021] 1) Cloning of the bullfrog bradykinin gene

[0022] Extraction of total RNA from bullfrog skin, purification of mRNA, synthesis of the first strand of cDNA and construction of a cDNA library, design of primers and screening of bullfrog serine protease inhibitor genes by PCR. The length of the amplification primer is 20 nucleotides, the upstream primer is 5'-ATGTTCACCWTGAAGAAATC-3', and the downstream primer is CLONTECH company SMART TM The sequence of the 3'PCR Primer in the cDNA Library Construction Kit is 5'-ATTCTAGAGGC CGAGGCGGCC-3'. The obtained positive clones were subjected to nucleotide sequence determination. Gene sequencing results show that the gene encoding the bullfrog serine protease inhibitor consists of 458 nucleotides (SEQ ID NO.1), and the sequence from the 5' end to the 3' is:

[0023]

[0024] Coding bullfrog bradykinin is the 156-183...

Embodiment 1

[0027] Embodiment 1: Bullfrog bradykinin gene cloning

[0028] Ⅰ. Extraction of total RNA from bullfrog skin

[0029] A. The live bullfrog was cleaned with double-distilled water, and the medullary cavity of the bullfrog was destroyed with a sterilized needle. 50-100 mg of bullfrog skin tissue was put into a dry-baked mortar, and 1 mL of TRIZOL Reagent (product of Invitrogen Company) was added to quickly Grind in an ice water bath.

[0030] B. After fully mixing, transfer to a 1.5mL centrifuge tube (DEPC tube) and place at 15-30°C for 5min, add an equal volume of chloroform, vortex mix for 15s, and centrifuge at 12000×g for 15min at 4°C.

[0031] C. Add 500 μL of isopropanol to the supernatant, place at 15-30°C for 5 minutes, vortex for 15 seconds to mix well, and centrifuge at 12,000×g for 10 minutes at 4°C. Discard the supernatant, add 1 mL of 75% ethanol to rinse the precipitate, centrifuge at 7500×g for 5 min, and repeat once. Dry in an ultra-clean workbench for 90 seco...

Embodiment 2

[0069] Embodiment 2: Preparation of bullfrog bradykinin

[0070] Ⅰ. Sample preparation method: The amino acid sequence of bullfrog bradykinin was deduced according to the gene encoding bullfrog bradykinin, and its complete sequence was synthesized with an automatic polypeptide synthesizer APEX396. Desalted and purified by HPLC reverse phase C18 column chromatography.

[0071] II. Molecular weight was determined by electrospray ionization (ESI) with an emission voltage of 4.5Kv.

[0072] Ⅲ, the bullfrog bradykinin of purification is identified its purity (flow velocity 1.0mL / min; mobile phase acetonitrile+water+TFA0.01% with high performance liquid chromatography HPLC method; Gradient elution; Chromatographic column C18, detection wavelength 220nm), molecular weight Fast atom bombardment mass spectrometry was used for the determination, the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an autom...

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Abstract

The invention discloses bullfrog slow-excitation peptide, coded nucleic acid thereof and application. The bullfrog slow-excitation peptide is composed of nine parts of amino acid, the molecular weight is 1043.2 daltons, the isoelectric point is 9.50, and the amino acid sequence is SEQ ID NO. 4: Arg Pro Pro Gly Cys Asn Pro Phe Arg. The sequence of the encoded gene of the bullfrog slow-excitation peptide is the 156-183th nucleotide of the sequence shown by the SEQ ID NO.1. The bullfrog slow-excitation peptide is composed of sixteen parts of amino acid, the molecular weight is 1759.0 daltons, the isoelectric point is 10.35, and the sequence of the amino acid is SEQ ID NO. 6: Arg Pro Pro Gly Cys Asn Pro Phe Arg Ile Ala Pro Ala Ser Tyr Leu. According to the bullfrog slow-excitation peptide, the coded nucleic acid thereof and the application, the amino acid structure of the bullfrog slow-excitation peptide is deduced through the coded gene of the bullfrog slow-excitation peptide, and the synthetic bullfrog slow-excitation peptide RR9 and RL16 have very strong activity for promoting contraction of in-vitro ileum muscle of rats. The bullfrog slow-excitation peptide has the advantages of being simple in structure and convenient to synthesize artificially, so that the bullfrog slow-excitation peptide can serve as cardio-cerebral-vascular drug and application of medicine for promoting intestinal contraction.

Description

technical field [0001] The invention relates to a bullfrog (Rana catesbeiana bradykinin) and its encoding nucleic acid and application, belonging to the technical field of biomedicine. Background technique [0002] Bradykinin is a major kinin substance in the kallikrein-kinin system, which can not only cause pathophysiological reactions, but also play a physiological protective role, and participate in the functional regulation of multiple system organs and pathophysiological processes. Such as the regulation of cardiovascular, renal, and central nervous systems, glucose metabolism, cell proliferation, smooth muscle contraction, inflammation, pain, shock, and tissue damage processes. In recent years, many experiments have conducted research on bradykinin through animal models, human body, and molecular biology, especially in the cardiovascular system. It has been confirmed that angiotensin converting enzyme inhibitor (Angiotensin converting enzyme inhibitor, ACEI) and angio...

Claims

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Application Information

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IPC IPC(8): C07K7/18C12N15/16A61K38/08A61K38/10A61P9/00A61P1/00
Inventor 赵瑞利韩文瑜韩俊友马吉飞冯新朱琳
Owner TIANJIN AGRICULTURE COLLEGE
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