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Fructose detection method based on fructokinase reaction system

A fructokinase and detection method technology, applied in the detection field, can solve the problems of limited detection range and low specificity of fructose detection means, and achieve the effect of wide detection range, good application prospect and high specificity detection

Pending Publication Date: 2022-05-13
南京盛德瑞尔医药科技有限公司
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Problems solved by technology

[0004] However, the specificity of existing fructose detection methods is not high, and the detection range is limited. Therefore, a fructose detection method based on the fructokinase reaction system is proposed.

Method used

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  • Fructose detection method based on fructokinase reaction system
  • Fructose detection method based on fructokinase reaction system
  • Fructose detection method based on fructokinase reaction system

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Experimental program
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Embodiment

[0059] 1. Prokaryotic expression of FK protein

[0060] 1. Construction of prokaryotic expression system

[0061] The target gene was synthesized according to the standard procedure, using Escherichia coli expression vector plasmid pET-28a, inserting the FK protein gene at the restriction site XhoI / NcoI to construct the recombinant expression vector pET-28a-FK, screening for kanamycin resistance gene, HIS Tag protein purification, such as figure 2 shown.

[0062] 2. Expression and purification of prokaryotic recombinant proteins

[0063] 2.1 Protein expression

[0064] (1) Transformation into expression bacteria by conventional methods

[0065] 1. Add 2 µL of pET-28a-FK plasmid to 50 µL of BL21 (DE 3) competent medium on a clean bench and mix on ice for 30 min.

[0066] 2. Heat shock: Put the mixture into a water bath, heat shock in a water bath at 42°C for 90 seconds, then take it out quickly.

[0067] 3. After cooling on ice for 3 minutes, add 900 µL of antibiotic-fre...

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Abstract

The invention discloses a fructose detection method based on a fructokinase reaction system, and belongs to the technical field of detection, the detection method is based on the FK activity of fructokinase and the principle of determination through a three-step enzyme kinetics method, and the method specifically comprises the following steps: step 1, plasmid construction of fructokinase genes and expression and purification of fructokinase proteins; step 2, enzymatic detection: constructing a fructose detection method on the basis of FK enzyme kinetic detection; and step 3, taking substitutes of blood, urine and beverages as examples to detect fructose of a standard substance, and further detecting the fructose content of a sample, so that high-specificity detection of fructose is realized, the detection range is relatively wide, and therefore, the method has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a fructose detection method based on a fructokinase reaction system. Background technique [0002] Fructose is the most common ketohexose widely found in nature. Containing 6 carbon atoms in fructose, it is also a monosaccharide, an isomer of glucose, and a component of sucrose. [0003] When the human body ingests fructose, it is absorbed through the intestinal tract and directly enters the intracellular metabolism without relying on insulin, which is one of the important factors that cause fat deposition in human organs. When there is too much fructose in the diet, it is potentially dangerous to the liver, arteries and heart. Diabetics also have increased levels of fructose in the blood circulation, congenital fructosuria caused by fructokinase loss-of-function mutations. After eating foods rich in fructose, the fructose content in the blood increases and is excreted in the...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C12Q1/32C12N9/12C12N15/54C12N15/70G01N21/31
CPCC12Q1/485C12Q1/32C12N9/1205C12N15/70G01N21/31C12Y207/01004G01N2333/904G01N2333/91215C12N2800/101
Inventor 李长红朱秋莎周雪
Owner 南京盛德瑞尔医药科技有限公司
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