阅读说明：本技术 含有枯草芽孢杆菌漆酶的总胆红素检测试剂盒 (Total bilirubin detection kit containing bacillus subtilis laccase ) 是由 齐崴 尤生萍 张成玉 苏荣欣 于 2020-03-03 设计创作，主要内容包括：本发明公开了一种含有枯草芽孢杆菌漆酶的总胆红素检测试剂盒,由试剂R1、试剂R2、试剂R3和试剂R4组成：试剂R1包括：缓冲液、表面活性剂、甘露醇、亚硝酸钠、叠氮化钠、乙二胺四乙酸钠、余量为水；试剂R2包括：缓冲液、KCl、聚乙二醇-600、胆酸钠、余量为水；试剂R3为枯草芽孢杆菌漆酶；试剂R4为总胆红素标准品。枯草芽孢杆菌漆酶发酵周期短、生产成本低、可实现快速大批量生产。利用枯草芽孢杆菌漆酶制备的总胆红素检测试剂盒,可以克服原胆红素氧化酶稳定性差、成本高等问题,本发明的试剂盒稳定性好,成本低。(The invention discloses a total bilirubin detection kit containing bacillus subtilis laccase, which consists of a reagent R1, a reagent R2, a reagent R3 and a reagent R4: the reagent R1 includes: buffer solution, surfactant, mannitol, sodium nitrite, sodium azide, sodium ethylene diamine tetracetate and the balance of water; the reagent R2 includes: buffer solution, KCl, polyethylene glycol-600, sodium cholate and the balance of water; reagent R3 is Bacillus subtilis laccase; reagent R4 is total bilirubin standard. The fermentation period of the bacillus subtilis laccase is short, the production cost is low, and quick mass production can be realized. The total bilirubin detection kit prepared by utilizing the bacillus subtilis laccase can overcome the problems of poor stability, high cost and the like of the original bilirubin oxidase, and the kit disclosed by the invention is good in stability and low in cost.)

1. A detection kit for preparing total bilirubin by using bacillus subtilis laccase is characterized by comprising a reagent R1, a reagent R2, a reagent R3 and a reagent R4:

the reagent R1 comprises buffer solution 100 mmol/L, surfactant 1-10 g/L, mannitol 10-250 mmol/L, sodium nitrite 0.05-0.2 g/L, sodium azide 0.2-2 g/L, sodium ethylene diamine tetracetate 0.001-5 g/L and the balance of water;

the reagent R2 comprises buffer solution 100 mmol/L, KCl 100 mmol/L, polyethylene glycol-6005-500 g/L, sodium cholate 20 mmol/L and the balance of water;

the reagent R3: a bacillus subtilis laccase;

the reagent R4: total bilirubin standard.

2. The detection kit according to claim 1, wherein the buffer in the reagent R1 is Tris-HCl buffer at pH 7.0 to 9.5, glycine-sodium hydroxide buffer at pH 7.0 to 9.5, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.0 to 9.5, or boric acid-potassium chloride-sodium hydroxide buffer at pH 7.0 to 9.5.

3. The detection kit according to claim 1, wherein the surfactant in the reagent R1 is at least one of sodium dodecyl sulfate, TritonX-100, Triton-405, betaine and polysorbate.

4. The detection kit according to claim 1, wherein the buffer in the reagent R2 is Tris-HCl buffer at pH 7.0 to 9.5, glycine-sodium hydroxide buffer at pH 7.0 to 9.5, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.0 to 9.5, or boric acid-potassium chloride-sodium hydroxide buffer at pH 7.0 to 9.5.

5. The detection kit of claim 1, wherein the bacillus subtilis laccase is obtained by the following method:

(1) constructing escherichia coli containing a bacillus subtilis laccase gene: designing an upstream primer of a bacillus subtilis laccase gene and a downstream primer of the bacillus subtilis laccase gene, carrying out PCR amplification by using a bacillus subtilis genome as a template and utilizing the upstream primer and the downstream primer, integrating an amplification product into an escherichia coli plasmid pET-28a in an enzyme digestion-connection mode to form a recombinant plasmid pET-28a-laccase, and carrying out CaCl-mediated amplification₂Transferring into Escherichia coli expression host bacteria B L21 (DE3) by transformation method to obtain recombinant bacteria;

the nucleotide sequence of the bacillus subtilis laccase gene is shown as SEQ ID No. 1; the nucleotide sequence of the upstream primer of the bacillus subtilis laccase gene is shown as SEQ ID No. 2; the nucleotide sequence of the downstream primer of the bacillus subtilis laccase gene is shown as SEQ ID No. 3;

(2) and (2) fermenting and purifying the recombinant bacteria obtained in the step (1) to prepare the bacillus subtilis laccase.

Technical Field

The invention belongs to the technical field of biological medicines, relates to a detection reagent, and particularly relates to a total bilirubin detection kit containing bacillus subtilis laccase.

Background

Bilirubin is an important index of liver function, and the determination of total bilirubin has very important significance clinically. As a main metabolite of the ferriporphyrin compound in erythrocytes, bilirubin is structurally formed by connecting four pyrrole rings through a methyl bridge in a molecule. Heme released after destruction of red blood cells in blood generates biliverdin under the action of heme oxidase, and bilirubin is generated under the action of biliverdin reductase. Bilirubin is an effective antioxidant, has the ability to capture oxygen free radicals, and protects lipids and lipoproteins from oxidation. It has synergistic effect with other antioxidant defense systems in human body, and can be combined with albumin to be present in ventricular myocyte position to prevent the position from producing oxygen free radical, regulate bilirubin antioxidant activity of myocardial cell, and protect ventricular myocyte from being damaged by oxygen free radical. Bilirubin is divided into total bilirubin and indirect bilirubin, and the sum of the indirect bilirubin and the total bilirubin is the total bilirubin. The increase of the total bilirubin amount and the indirect bilirubin increase show that: hemolytic anemia, blood group incompatibility, blood transfusion, malignant disease, neonatal jaundice, etc.; the increase of both direct and indirect bilirubin indicates: acute icterohepatitis, chronic active hepatitis, cirrhosis, toxic hepatitis; the increase of total bilirubin and direct bilirubin indicates that: intrahepatic and extrahepatic obstructive jaundice, pancreatic cancer, cholangiopathy and other bile stasis syndromes; therefore, the determination of total bilirubin in serum, plasma or urine of a human body and the analysis of components are one of the most common items in clinical medical examination, and have a very important meaning in the treatment of diseases.

The current common clinical methods for measuring total bilirubin include a diazo reagent method, a vanadate oxidation method, an enzyme method and the like. The diazo reagent method is applied earliest and still widely at present, but the reagent has poor stability, low linearity, poor quality control and weak anti-interference capability; the vanadate oxidation method has a relatively good correlation with the diazonium reagent method and is relatively stable in the reagent, and therefore, the development is relatively rapid in recent years. But the anti-interference capability is relatively weak, and negative values often appear in the result. The enzymatic determination of bilirubin has the characteristics of simple operation, strong specificity, high precision, strong anti-interference capability and the like, and is gradually and widely applied in clinical application in recent years. However, the general bilirubin oxidase has short storage time and high production cost, so that the reagent for enzymatic bilirubin detection is expensive, and is disadvantageous in clinical popularization and application. In view of the defects in the prior art, the development of a detection kit for total bilirubin, which has good stability, accurate and reliable result and low production cost, is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a total bilirubin detection kit containing bacillus subtilis laccase.

The technical scheme of the invention is summarized as follows:

a detection kit for preparing total bilirubin by using Bacillus subtilis laccase comprises a reagent R1, a reagent R2, a reagent R3 and a reagent R4:

the reagent R1 comprises buffer solution 100 mmol/L, surfactant 1-10 g/L, mannitol 10-250 mmol/L, sodium nitrite 0.05-0.2 g/L, sodium azide 0.2-2 g/L, sodium ethylene diamine tetracetate 0.001-5 g/L and the balance of water;

the reagent R2 comprises buffer solution 100 mmol/L, KCl 100 mmol/L, polyethylene glycol-6005-500 g/L, sodium cholate 20 mmol/L and the balance of water;

the reagent R3: a bacillus subtilis laccase;

the reagent R4: total bilirubin standard.

The buffer in the reagent R1 is Tris-HCl buffer at pH 7.0 to 9.5, glycine-sodium hydroxide buffer at pH 7.0 to 9.5, disodium hydrogenphosphate-sodium dihydrogenphosphate buffer at pH 7.0 to 9.5, or boric acid-potassium chloride-sodium hydroxide buffer at pH 7.0 to 9.5.

The surfactant in the reagent R1 is at least one of sodium dodecyl sulfate, TritonX-100, Triton-405, betaine and polysorbate.

The buffer in the reagent R2 is Tris-HCl buffer at pH 7.0 to 9.5, glycine-sodium hydroxide buffer at pH 7.0 to 9.5, disodium hydrogenphosphate-sodium dihydrogenphosphate buffer at pH 7.0 to 9.5, or boric acid-potassium chloride-sodium hydroxide buffer at pH 7.0 to 9.5.

The bacillus subtilis laccase is obtained by the following method:

(1) constructing escherichia coli containing a bacillus subtilis laccase gene: designing an upstream primer of a bacillus subtilis laccase gene and a downstream primer of the bacillus subtilis laccase gene, carrying out PCR amplification by using a bacillus subtilis genome as a template and utilizing the upstream primer and the downstream primer, integrating an amplification product into an escherichia coli plasmid pET-28a in an enzyme digestion-connection mode to form a recombinant plasmid pET-28a-laccase, and carrying out CaCl-mediated amplification₂Transferring into Escherichia coli expression host bacteria B L21 (DE3) by transformation method to obtain recombinant bacteria;

the nucleotide sequence of the bacillus subtilis laccase gene is shown as SEQ ID No. 1; the nucleotide sequence of the upstream primer of the bacillus subtilis laccase gene is shown as SEQ ID No. 2; the nucleotide sequence of the downstream primer of the bacillus subtilis laccase gene is shown as SEQ ID No. 3;

(2) and (2) fermenting and purifying the recombinant bacteria obtained in the step (1) to prepare the bacillus subtilis laccase.

The invention has the advantages that:

(1) the fermentation period of the bacillus subtilis laccase is short, the production cost is low, and quick mass production can be realized.

(2) The total bilirubin detection kit prepared by utilizing the bacillus subtilis laccase can overcome the problems of poor stability, high cost and the like of the original bilirubin oxidase, and the kit disclosed by the invention is good in stability and low in cost.

Detailed Description

The technical solution of the present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited thereto. The specific embodiments described herein are merely illustrative and explanatory of the application and are not restrictive of the application. The equivalent replacement of the present disclosure, or the corresponding improvement, still belongs to the protection scope of the present invention.

The Bacillus subtilis is named as Bacillus subtilis and purchased from China center for culture Collection of industrial microorganisms (website http:// www.china-cic.org /), the strain preservation number is CICC20613, and the purchase time is 6 months in 2010.