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Fluorogenic substrate for detecting ADAMTS13 enzymatic activity and detection method

A fluorescent substrate and enzyme activity technology, applied in the biological field, can solve the problems of incompatibility and low sensitivity, and achieve the effects of simple operation, high sensitivity, and easy popularization and use.

Inactive Publication Date: 2012-07-04
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low sensitivity of this method, it is not suitable for clinical detection of ADAMTS13 enzyme activity

Method used

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  • Fluorogenic substrate for detecting ADAMTS13 enzymatic activity and detection method
  • Fluorogenic substrate for detecting ADAMTS13 enzymatic activity and detection method
  • Fluorogenic substrate for detecting ADAMTS13 enzymatic activity and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Obtaining of VWF73 fragments:

[0051]Using the plasmid pSVHVWF1 as a template, the upstream primer V73F having the nucleotide sequence shown in SEQ ID NO: 3 and the downstream primer V73B having the nucleotide sequence shown in SEQ ID NO: 4 were used for PCR amplification. The two primers have restriction sites for BamHI and HindIII respectively.

[0052] The PCR amplification reaction system is:

[0053]

[0054] Add sterilized distilled water to make up the total amount of the system to 25 μL.

[0055] The PCR reaction procedure is:

[0056] Pre-denaturation at 94°C for 5 minutes

[0057]

[0058] Extend at 72°C for 7min

[0059] The PCR amplification product was recovered by gel electrophoresis, digested with Bam HI and Hind III, and the digested fragment was ligated with the pMD19T (simple) vector digested with Bam HI and Hind III, transformed into Escherichia coli TG1, and selected For positive clones, the plasmids were extracted and identifi...

Embodiment 2

[0060] Embodiment 2: Recombination of VWF73 fragment and expression vector pQE30:

[0061] After double digestion with Bam HI and Hind III, the VWF73 target gene fragment was excised from the cloning vector with correct DNA sequencing, connected to the expression vector pQE30 digested with the same enzyme using T4 DNA ligase, and transformed into the host strain TG1. The recombinant was identified and named pQE30-VWF73.

Embodiment 3

[0062] Embodiment 3: Transformation of expression vector pQE30-VWF73:

[0063] 1. Preparation of plasmid pQE30-VWF73(Q1599C) containing Q1599C mutation

[0064] Using the correctly identified pQE30-VWF73 plasmid as a template, PCR amplification was performed with the upstream primer Q1599CF having the nucleotide sequence shown in SEQ ID NO:5 and the downstream primer Q1599CB having the nucleotide sequence shown in SEQ ID NO:6.

[0065] The PCR amplification reaction system is:

[0066]

[0067] Add sterilized distilled water to make up the total amount of the system to 20 μL.

[0068] The PCR reaction procedure is:

[0069] Pre-denaturation at 98°C for 40s

[0070]

[0071] Extend at 72°C for 7min

[0072] After the PCR reaction, add 10U methylase DpnI to digest the template. After elimination, the PCR product was transformed into DH5a bacteria (containing the pREP4 plasmid), the plasmid was extracted, sequenced and identified, thereby obtaining the plasmid pQE30-VW...

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Abstract

The invention belongs to the technical field of biology, in particular to a fluorogenic substrate for detecting ADAMTS13 enzymatic activity and a detection method. The fluorogenic substrate for detecting the ADAMTS13 enzymatic activity comprises an amino acid sequence shown as SEQ ID NO:2, and sulfydryl of cysteine at the 4 position and the 15 position at the N end of the amino acid sequence is combined with fluorescein-5- maleimide. Q1599C of the fluorogenic substrate is closer to N1611C modified site, simultaneously the N-end sequence of the fluorogenic substrate does not contain extra amino acid GS, and enzymolysis can be conducted on the fluorogenic substrate through ADAMTS13 more effectively. Therefore, sensitivity for detecting ADAMTS13 enzymatic activity can be improved. The method for detecting the ADAMTS13 enzymatic activity is convenient to operate, accurate in detection result, high in sensitivity and suitable for quick detection of the ADAMTS13 enzymatic activity in blood plasma in hospitals, health departments and medical research and development institutions at different levels.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a fluorescent substrate and a detection method for detecting ADAMTS13 enzyme activity. Background technique [0002] Thrombotic thrombocytopenic purpura (TTP) is a type of microvascular thrombosis-hemorrhagic syndrome, mainly due to the formation of platelet thrombus in the microcirculation, and the purpura formed by the reduction of the number of platelets due to massive consumption. The typical clinical manifestations of the disease are the five syndromes: fever, microangiopathic hemolytic anemia, thrombocytopenia, central nervous system damage, and renal involvement. Due to embolism of arterioles and microvessels, organ ischemic dysfunction and even infarction are caused, and organs that are highly dependent on microcirculation (brain, kidney, etc.) are also prone to symptoms. Due to the acute onset of TTP, plasma exchange is needed in time once symptoms appear. If th...

Claims

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Application Information

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IPC IPC(8): C12Q1/37C07K14/47C07K1/107
Inventor 苏健白霞阮长耿
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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