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Total bilirubin detection kit containing bacillus subtilis laccase

A Bacillus subtilis and detection kit technology, applied in the field of biomedicine, can solve the problems of expensive bilirubin reagents, high production costs, and short storage time, and achieve rapid mass production, low production costs, and short fermentation cycles Effect

Active Publication Date: 2020-07-17
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the short storage time and high production cost of general bilirubin oxidase, the enzymatic detection of bilirubin reagents is expensive, and it is at a disadvantage in clinical application.

Method used

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  • Total bilirubin detection kit containing bacillus subtilis laccase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Bacillus subtilis laccase is obtained by the following method:

[0025] (1) Construction of Escherichia coli containing the Bacillus subtilis laccase gene: design the upstream primer (SEQ ID No. 2) and downstream of the Bacillus subtilis laccase gene (the nucleotide sequence is shown in SEQ ID No. 1) Primer (shown in SEQ ID No. 3), using Bacillus subtilis (CICC20613) genome as a template, using upstream primers and downstream primers for PCR amplification, and integrating the amplified products into by enzyme digestion-ligation In the Escherichia coli plasmid pET-28a (commercially available), a recombinant plasmid pET-28a-laccase is formed. Escherichia coli DH5α (DE3) is selected as the host cell for plasmid amplification, and the amplified plasmid is passed through CaCl 2 Transformation method into E. coli expression host strain BL21 (DE3) to obtain recombinant strain;

[0026] (2) Ferment and purify the recombinant bacteria obtained in step (1) to prepare Bacillus subtilis...

Embodiment 2

[0034] A test kit for preparing total bilirubin by using Bacillus subtilis laccase, which is composed of reagent R1, reagent R2, reagent R3, and reagent R4:

[0035] Reagent R1 includes: pH=8.0 Tris-HCl buffer solution 100mmol / L, sodium lauryl sulfate 3g / L, mannitol 100mmol / L, sodium nitrite 0.1g / L, sodium azide 0.2g / L, Sodium ethylenediaminetetraacetate 0.5g / L, the balance is water;

[0036] Reagent R2 includes: pH=8.0 Tris-HCl buffer solution 100mmol / L, KCl 100mmol / L, polyethylene glycol-600 50g / L, sodium cholate 20mmol / L, the balance is water;

[0037] Reagent R3: Bacillus subtilis laccase (prepared in Example 1).

[0038] Reagent R4: total bilirubin standard.

[0039] Test the total bilirubin with the kit:

[0040] Take reagent R3 Bacillus subtilis laccase and put it into reagent R2 to make a concentration of 10000U / L, mix well, and let it stand for 5 minutes to obtain enzyme-containing reagent;

[0041] Take 0.04μmol of total bilirubin standard product and add double distilled water...

Embodiment 3

[0054] A test kit for preparing total bilirubin by using Bacillus subtilis laccase, which is composed of reagent R1, reagent R2, reagent R3, and reagent R4:

[0055] Reagent R1 includes: pH=7.0 Tris-HCl buffer 100mmol / L, TritonX-100 0.5g / L, Triton-405 0.5g / L, mannitol 10mmol / L, sodium nitrite 0.05g / L, stack Sodium nitride 0.2g / L, sodium edetate 0.001g / L, the balance is water;

[0056] Reagent R2 includes: pH=7.0 Tris-HCl buffer 100mmol / L, KCl 100mmol / L, polyethylene glycol-600 5g / L, sodium cholate 20mmol / L, and the balance is water;

[0057] Reagent R3: Bacillus subtilis laccase (prepared in Example 1);

[0058] Reagent R4: total bilirubin standard.

[0059] Test the total bilirubin with the kit:

[0060] Take reagent R3 Bacillus subtilis laccase and put it into reagent R2 to make a concentration of 5000 U / L, mix well, and let stand for 5 minutes to obtain enzyme-containing reagent;

[0061] Take 0.04μmol of total bilirubin standard product and add double distilled water to 2mL, and diss...

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Abstract

The invention discloses a total bilirubin detection kit containing bacillus subtilis laccase. The kit is composed of a reagent R1, a reagent R2, a reagent R3 and a reagent R4; the reagent R1 includesa buffer solution, a surfactant, mannitol, sodium nitrite, sodium azide, sodium ethylene diamine tetracetate, and the balance of water; the reagent R2 includes a buffer solution, KCl, polyethylene glycol-600, sodium cholate, and the balance of water; the reagent R3 is bacillus subtilis laccase; and the reagent R4 is a total bilirubin standard product. The bacillus subtilis laccase has a short fermentation cycle and low production costs, and can realize rapid mass production; the total bilirubin detection kit prepared by using the bacillus subtilis laccase can overcome the problems of poor stability and high costs of original bilirubin oxidase; and the kit provided by the invention has the advantages of good stability and low costs.

Description

Technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a detection reagent, in particular to a total bilirubin detection kit containing Bacillus subtilis laccase. Background technique [0002] Bilirubin is an important indicator of liver function, and the determination of total bilirubin has very important clinical significance. As the main metabolite of iron porphyrin compounds in red blood cells, the structure of bilirubin is formed by four pyrrole rings connected by methyl bridges in the molecule. The heme released from the destruction of red blood cells in the blood produces biliverdin under the action of heme oxidase, and produces bilirubin under the action of biliverdin reductase. Bilirubin is an effective antioxidant, has the ability to capture oxygen free radicals, and can protect lipids and lipoproteins from oxidation. It has a synergistic effect with other antioxidant defense systems in the human body. It exists in the muscl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26C12N9/02C12N15/53C12N1/21C12N15/70C12R1/19
CPCC12Q1/26C12N9/0061C12N15/70C12Y110/03002
Inventor 齐崴尤生萍张成玉苏荣欣
Owner TIANJIN UNIV
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