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Homocysteine detection using fusion enzymes

A homocysteine, fusion enzyme technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as difficult control, restricting catalytic efficiency, etc., to improve accuracy and stability, and improve detection. horizontal effect

Inactive Publication Date: 2014-08-13
王学忠 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In a continuous enzyme-catalyzed process like the homocysteine ​​enzyme cycle, the product (intermediate product) of the previous enzyme is the substrate for the next enzyme-catalyzed reaction, and these enzymes are often in a free state, especially in liquid reagents. In , the distance between enzyme molecules is free and evacuated, and it is difficult to carry out any control, which restricts its catalytic efficiency.

Method used

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  • Homocysteine detection using fusion enzymes
  • Homocysteine detection using fusion enzymes
  • Homocysteine detection using fusion enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Expression and purification of embodiment 1 fusion enzyme protein

[0026]The fusion enzyme protein uses a flexible linker peptide (GGGGS) 2 , applied to the fusion of homocysteine ​​S-methyltransferase (EC2.1.1.10) and adenosylhomocysteinase (EC3.3.1.1), and adenosylhomocysteinase (EC3.3.1.1) and methionine adenosyltransferase fusion (EC2.5.1.6), the construction method of the fusion gene is consistent:

[0027] Design the upstream primer with the EcoR I restriction site for the first enzyme gene, and the downstream primer with GGGGS and the 5' end partial sequence of the second enzyme gene; design the 3' end partial sequence with GGGGS and the first enzyme gene An upstream primer, and a downstream primer with a Xhol I restriction site for the second enzyme gene fragment. Using the American ABI gradient PCR instrument (Veriti TM 96), the amplification system and reaction conditions of the two are the same (total volume 100ul, 10ul of 10*PCR buffer, 10ul of 100umo / L ...

Embodiment 2

[0039] Embodiment 2 Utilizes the comparison of the detection accuracy of the reagent prepared by fusion enzyme and common enzyme cycle reagent

[0040] Reagents prepared using fusion enzymes contain the following components:

[0041] Reagent components

Dosage per liter

Phosphate buffer, pH7.6, 37°C

100mM

TCEP

26.5mM

Sodium azide

7.7mM

ATP

2mM

adenosine deaminase

3ku

H-G-Aase

10ku

A-E-Mase

10ku

bovine serum albumin

0.5％

alpha-ketoglutarate

7.5mM

Mannitol

60mM

Methionine

0.5mM

Glycerin

100g

reduced coenzyme NADH

0.29mM

glutamate dehydrogenase

2ku

[0042] The contrast reagent is a commercially available general enzyme cycle reagent, which is calibrated with a prepared homocysteine ​​standard solution with a concentration of 15.1 μM. The detection sample is a prepared linear sample, and the concentrations of h...

Embodiment 3

[0054] Embodiment 3 utilizes the comparison of the thermal stability of the reagent prepared by fusion enzyme and common enzyme cycle reagent

[0055] The liquid dual-reagent reagent prepared by fusion enzyme contains the following components:

[0056] Reagent 1:

Dosage per liter

Tris buffer, pH9.1, 20.0°C

100mM

TCEP

26.5mM

Sodium azide

15.0mM

Methionine

0.5mM

alpha-ketoglutarate

7.5mM

Triton X-100

2.0g

bovine serum albumin

2.0g

reduced coenzyme NADH

0.29mM

adenosine deaminase

7ku

glutamate dehydrogenase

3ku

[0057] Reagent 2:

Dosage per liter

HEPES buffer, pH7.4, 20.0°C

50mM

Mannitol

60mM

ATP

10mM

[0058] Sodium azide

15.0mM

PEG2000

0.5g

Tween-20

3.0g

bovine serum albumin

1.0g

H-E-Aase

50ku

A-G-Mase

70ku

[0059] The co...

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Abstract

The invention discloses a method for detecting homocysteine concentration, and the method is characterized in that to-be-detected substrate homocysteine is catalyzed by gene engineering fusion enzymes to obtain product adenosine, the adenosine production rate is in direct proportion to homocysteine content in a sample, and the homocysteine content in the sample can be detected by detecting the adenosine. The used gene engineering fusion enzymes are a fusion enzyme of homocysteine methyltransferase (EC2.1.1.10) and adenosine homocysteine enzyme (EC3.3.1.1), and a fusion enzyme of the adenosine homocysteine enzyme (EC3.3.1.1) and methionine adenosyltransferase (EC2.5.1.6). According to the method, an efficient fusion enzyme protein can be established for detecting the homocysteine, by use of efficient catalytic activity of the fusion enzymes, the sensitivity of ordinary enzymatic reaction and the stability of a detection reagent can be greatly improved, and a new methodological improvement area is proposed for an enzyme cycle detection method.

Description

technical field [0001] The invention belongs to the technical field of medical examination and determination, and in particular relates to a homocysteine ​​determination reagent. Background technique [0002] Homocysteine ​​(Homocysteine) is a sulfur-containing amino acid that is produced in cells by demethylation of methionine. Recent studies have shown that homocysteine ​​produces superoxide and peroxide damages vascular endothelial cells, changes the function of coagulation factors, increases the tendency of thrombosis, promotes atherosclerosis and thrombosis, and increases the incidence of cardiovascular diseases and Mortality increases, so measuring the concentration of homocysteine ​​in blood is of great clinical significance. When homocysteine ​​accumulates in cells and enters the blood circulation, most of it exists in the oxidized form and binds to proteins, while the reduced form of homocysteine ​​accounts for only 1% in the blood. To determine the content of tot...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C12Q1/34C12Q1/527C12Q1/32
Inventor 王学忠
Owner 王学忠
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