CN111424070B - Total bilirubin detection kit containing bacillus subtilis laccase - Google Patents
Total bilirubin detection kit containing bacillus subtilis laccase Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
Abstract
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 comprises: buffer solution, surfactant, mannitol, sodium nitrite, sodium azide, sodium ethylene diamine tetracetate and the balance of water; the reagent R2 comprises: 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.
Description
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 ferriporphyrin compounds in erythrocytes, bilirubin has a structure formed by connecting four pyrrole rings through a methyl bridge in a molecule. Heme released after the 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 part, prevent the part from producing oxygen free radical, regulate myocardial cell bilirubin antioxidant activity, 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 with 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, capillary hepatitis 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: 100mmol/L buffer solution, 1-10g/L surfactant, 10-250mmol/L mannitol, 0.05-0.2g/L sodium nitrite, 0.2-2g/L sodium azide, 0.001-5g/L sodium ethylene diamine tetracetate and the balance of water;
the reagent R2 comprises: buffer solution 100mmol/L, KCl 100mmol/L, polyethylene glycol-600-500 g/L, sodium cholate 20mmol/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 =7.0-9.5, glycine-sodium hydroxide buffer at pH =7.0-9.5, disodium hydrogenphosphate-sodium dihydrogenphosphate buffer at pH =7.0-9.5, or boric acid-potassium chloride-sodium hydroxide buffer at pH = 7.0-9.5.
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 =7.0-9.5, glycine-sodium hydroxide buffer at pH =7.0-9.5, disodium hydrogenphosphate-sodium dihydrogenphosphate buffer at pH =7.0-9.5, or boric acid-potassium chloride-sodium hydroxide buffer at pH = 7.0-9.5.
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 amplification 2 Transferring the strain into an escherichia coli expression host bacterium BL21 (DE 3) by a transformation method to obtain a recombinant bacterium;
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 bacillus subtilis laccase has short fermentation period and low production cost, and can realize rapid mass production.
(2) The total bilirubin detection kit prepared by using 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 known 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.
Example 1
The bacillus subtilis laccase is obtained by the following method:
(1) Constructing escherichia coli containing a bacillus subtilis laccase gene: designing an upstream primer (shown in SEQ ID No. 2) and a downstream primer (shown in SEQ ID No. 3) of a Bacillus subtilis laccase gene (shown in SEQ ID No.1 in nucleotide sequence), taking a Bacillus subtilis (CICC 20613) genome as a template, performing PCR amplification by using the upstream primer and the downstream primer, integrating an amplification product into an escherichia coli plasmid pET-28a (sold in markets) in an enzyme digestion-connection mode to form a recombinant plasmid pET-28a-laccase, selecting escherichia coli DH5 alpha (DE 3) as a host cell for plasmid amplification, and performing CaCl amplification on the amplified plasmid 2 Transferring the strain into an escherichia coli expression host bacterium BL21 (DE 3) by a transformation method to obtain a recombinant bacterium;
(2) Fermenting and purifying the recombinant bacteria obtained in the step (1) to prepare the bacillus subtilis laccase, which comprises the following specific steps:
inoculating the recombinant bacteria obtained in the step (1) to an LB liquid culture medium, and culturing in an incubator at 37 ℃ for 12h to obtain a seed solution; inoculating the seed solution into new LB liquid culture medium at a ratio of 1%, culturing at 37 deg.C and 220rpm until OD600 is 0.8, adding IPTG and CuCl at addition amount of 1mg/L and 1mmol/L respectively 2 Then fermenting for 20 hours at 16 ℃ and 220 rpm; and after fermentation, 10000rmp are centrifuged to obtain thalli, a high-pressure cell crusher is used for crushing cells, and purified by an Ni-NTA affinity chromatography column to obtain the purified bacillus subtilis laccase.
The formula of the LB liquid culture medium: 5g/L yeast powder, 10g/L, naCl g/L peptone, 50mg/L kanamycin and the balance of water.
GenBank number of the bacillus subtilis laccase gene (SEQ ID No. 1) is: JN043511.1;
an upstream primer 5' -CGCGGATCCATGACACTTGAAAAATTTGTGG (SEQ ID No. 2);
the downstream primer 5' -CGGCTCGAGCTATTTATGGGGATCAGTTATATCC (SEQ ID No. 3).
Experiments prove that: the bacillus subtilis laccase obtained by the method has the advantages of short fermentation period (fermentation can be completed after the inducer is added for 20 hours), low production cost (fermentation cost per liter is about 1.52 yuan), and quick and large-scale production.
Example 2
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: 100mmol/L of Tris-HCl buffer solution with the pH =8.0, 3g/L of sodium dodecyl sulfate, 100mmol/L of mannitol, 0.1g/L of sodium nitrite, 0.2g/L of sodium azide, 0.5g/L of sodium ethylene diamine tetracetate and the balance of water;
the reagent R2 comprises: 100mmol/L, KCl mmol/L Tris-HCl buffer solution with pH =8.0, 600 50g/L polyethylene glycol, 20mmol/L sodium cholate and the balance of water;
a reagent R3: bacillus subtilis laccase (prepared in example 1).
Reagent R4: total bilirubin standard.
And (3) detecting total bilirubin by using the kit:
adding a reagent R3, namely the bacillus subtilis laccase into a reagent R2 to prepare a mixture with the concentration of 10000U/L, uniformly mixing, and standing for 5min to obtain an enzyme-containing reagent;
adding 0.04 mu mol of total bilirubin standard substance into 2mL of double distilled water, and dissolving to obtain total bilirubin standard substance solution (referred to as standard substance solution for short);
a sample to be tested: fresh, hemolysis free serum.
Detection wavelength: 450nm.
The method comprises the following steps:
1) Adding 270 mu L of reagent R1 into the sample tube and the standard tube respectively;
2) Adding 10 mu L of sample to be detected into the sample tube, adding 10 mu L of standard substance solution into the standard substance tube, placing the sample tube and the standard substance tube into a spectrophotometer, measuring absorbance at 450nm, and respectively recording as A1 sample and A1 standard substance;
3) Respectively adding 20 mu L of enzyme-containing reagent into the sample tube and the standard tube, uniformly mixing, incubating at 25 ℃ for 5min, and measuring absorbance at 450nm to be respectively marked as an A2 sample and an A2 standard;
calculating the concentration of the sample to be detected:
wherein Δ a sample = A1 sample-A2 sample; Δ a standard = A1 standard-A2 standard
The concentration of total bilirubin in the sample to be tested is 45.7 mu mol/L.
Besides the spectrophotometer, the applicable instrument can also be selected from various semi-automatic and full-automatic biochemical analyzers.
Example 3
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: 100mmol/L, tritonX-100.5g/L Tris-HCl buffer solution with the pH =7.0, 0.5g/L triton-405, 10mmol/L mannitol, 0.05g/L sodium nitrite, 0.2g/L sodium azide, 0.001g/L sodium ethylene diamine tetracetate and the balance of water;
the reagent R2 comprises: 100mmol/L, KCl mmol/L Tris-HCl buffer solution with pH =7.0, 5g/L polyethylene glycol-600, 20mmol/L sodium cholate and the balance of water;
a reagent R3: bacillus subtilis laccase (prepared in example 1);
a reagent R4: total bilirubin standard.
Detection of total bilirubin with the kit:
adding a reagent R3, namely the bacillus subtilis laccase, into a reagent R2 to prepare a mixture with the concentration of 5000U/L, uniformly mixing, and standing for 5min to obtain an enzyme-containing reagent;
adding 0.04 mu mol of total bilirubin standard substance into 2mL of double distilled water, and dissolving to obtain total bilirubin standard substance solution (referred to as standard substance solution for short);
a sample to be tested: heparin anticoagulant plasma.
Detection wavelength: 450nm.
The method comprises the following steps: the procedure was as in example 2.
The concentration of total bilirubin in the sample to be tested is 45.2 mu mol/L.
Example 4
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: 100mmol/L of Tris-HCl buffer solution with pH =9.5, 5g/L of betaine, 5g/L of polysorbate, 250mmol/L of mannitol, 0.2g/L of sodium nitrite, 2g/L of sodium azide, 5g/L of sodium ethylene diamine tetracetate and the balance of water;
the reagent R2 comprises: 100mmol/L, KCl 100mmol/L Tris-HCl buffer solution with pH =9.5, polyethylene glycol-600 g/L, 20mmol/L sodium cholate, and the balance of water;
a reagent R3: bacillus subtilis laccase (prepared in example 1)
Reagent R4: total bilirubin standard.
Detection of total bilirubin with the kit:
adding a reagent R3 bacillus subtilis laccase into a reagent R2 to prepare a mixture with the concentration of 20000U/L, uniformly mixing, and standing for 5min to obtain an enzyme-containing reagent;
adding 0.04 mu mol of total bilirubin standard substance into 2mL of double distilled water, and dissolving to obtain total bilirubin standard substance solution (referred to as standard substance solution for short);
a sample to be tested: EDTA anticoagulated plasma.
Detection wavelength: 450nm.
The method comprises the following steps: the same procedure as in example 2 was used.
The concentration of total bilirubin in the sample to be tested is 45.6 mu mol/L.
The storage conditions and the validity period are as follows: the original reagent is preserved at the temperature of 2-8 ℃ and the validity period is 12 months.
Examples 2, 3 and 4 compare with commercial kits:
the "high-value total bilirubin fresh non-hemolytic serum" samples were each assayed using the kits listed in Table 1, and the concentration of total bilirubin in the samples was determined to be 325. Mu. Mol/L by the laboratory. The measurement and results are shown in Table 1.
Table 1:
| reagent kit | Number of measurements | Mean value (μmol/L) | Relative deviation% |
| Commercial (improved) diazo method total bilirubin determination kit | 10 | 328.7 | 4.7 |
| Commercial enzyme method total bilirubin determination kit | 10 | 321.5 | 3.8 |
| Example 2 | 10 | 324.3 | 2.4 |
| Example 3 | 10 | 323.9 | 2.9 |
| Example 4 | 10 | 324.1 | 2.6 |
The results show that: compared with the detection results of the kit in the market, the kit has no obvious difference; but the data show that the kit has stronger specificity, higher precision and accurate and reliable result.
Experiments prove that the total bilirubin detection kit prepared by using 100mmol/L of glycine-sodium hydroxide buffer solution with pH = 8.0/100 mmol/L, pH =8.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with pH = 8.0/L, pH =8.0 is used for replacing 100mmol/L of Tris-HCl buffer solution with pH =8.0 in example 2, and the detection effect is similar to that in example 2.
Experiments prove that the detection effect of the total bilirubin detection kit prepared by using 100mmol/L of 100mmol/L, pH =7.0 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution 100mmol/L, pH =7.0 boric acid-potassium chloride-sodium hydroxide buffer solution 100mmol/L instead of 100mmol/L of Tris-HCl buffer solution with pH =7.0 in example 3 is similar to that of example 3.
Experiments prove that the total bilirubin detection kit prepared by using 100mmol/L of 100mmol/L, pH =9.5 glycine-sodium hydroxide buffer solution/100 mmol/L, pH =9.5 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution/100 mmol/L of L, pH =9.5 boric acid-potassium chloride-sodium hydroxide buffer solution respectively replaces 100mmol/L of 9.5 Tris-HCl buffer solution in example 4, and the detection effect is similar to that of example 4.
Sequence listing
<110> Tianjin university
<120> Total bilirubin detection kit containing bacillus subtilis laccase
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atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccggtacag 60
cagtcaaaag atagcacata ctacgaagta accatggagg aatgctacca tcagcttcac 120
cgcgatctcc ctccaacccg cttgtggggc tataacggtt tattccccgg tcccaccatt 180
aaggccaaaa gaaatgaaaa cgtttatgtg aaatggatga ataaccttcc ttcagagcat 240
tttcttccga ttgatcacac cattcatcac agtgacagcc agcatgccga acccgaggtg 300
aaaaccgtcg ttcatttaca cggcggcgtc actccagatg acagcgacgg ttatcctgag 360
gcctggtttt ctaaagactt tgaacaaaca ggcccttatt ttaaacgaga ggtttaccat 420
tatccaaatc agcagcgcgg agctatttta tggtatcacg atcatgctat ggcgctcacg 480
aggctgaatg tgtatgccgg gctcatcggt gcttatatca tccatgaacc aaaggaaaaa 540
cgtctaaagc tcccatcagg tgaatacgat gtgccgcttt tgatcacgga ccgtacgatt 600
aatgaagatg gctctttatt ttatccgagc ggaccggaaa acccttcacc gtcactgcca 660
aatccgtcaa tcgttccagc cttttgcgga gatacaattc tcgtcaacgg gaaggcatgg 720
ccatacatgg aggtcgaacc gagaaaatac cgtttccgcg tcatcaatgc ctctaatacg 780
agaacatata acctgtcact tgataatggc ggagaattta tccagatcgg ttctgacggc 840
ggacttttgc cgcgctccgt caagctaaac tctttcagta tcgcgccagc tgagcgcttt 900
gatatcctca ttgacttcgc cgcgtttgaa ggacaatcga ttattttagc aaacagcgag 960
ggctgcggcg gcgacgttaa tccggaaaca gacgcaaaca tcatgcaatt cagagtcaca 1020
aaaccgttag cccaaaaaga cgaaagcaga aagccaaaat acctggcatc ttacccttcg 1080
gtacagcatg aaagaataca aaacctccga acattgaagc tggcaggcac tcaagaccaa 1140
tacggcagac ccgtccttct tcttaacaac aaacgctggc acgatcctgt cactgaagca 1200
ccgaaagtcg gttctaccga aatatggtcg attatcaacc cgactcgcgg aacacatccg 1260
atccatcttc atttggtctc cttccgtgta ttggaccggc gcccatttga tacagcccgt 1320
tttgaagagc gcggagaact ggcctacacc ggacccgccg ttccgccgcc accaagtgaa 1380
aaaggctgga aagacacggt tcagtcccac gccggtgaag tcctgagaat cgccgtaaca 1440
ttcgggccat acactgggcg gtacgtatgg cattgccaca ttcttgagca tgaagactat 1500
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cgcggatcca tgacacttga aaaatttgtg gatgc 35
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<213> Bacillus subtilis
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ccgctcgagc tagtggtggt ggtggtggtg tttatgg 37
Claims (5)
1. A total bilirubin detection kit prepared by utilizing bacillus subtilis laccase is characterized by comprising a reagent R1, a reagent R2, a reagent R3 and a reagent R4:
the reagent R1 comprises: 100mmol/L buffer solution, 1-10g/L surfactant, 10-250mmol/L mannitol, 0.05-0.2g/L sodium nitrite, 0.2-2g/L sodium azide, 0.001-5g/L sodium ethylene diamine tetracetate and the balance of water;
the reagent R2 comprises: buffer solution 100mmol/L, KCl 100mmol/L, polyethylene glycol-600-500 g/L, sodium cholate 20mmol/L, and the balance of water;
the reagent R3: a bacillus subtilis laccase;
the reagent R4: total bilirubin standards;
the nucleotide sequence of the bacillus subtilis laccase gene is shown as SEQ ID No. 1.
2. The detection kit according to claim 1, wherein the buffer in the reagent R1 is Tris-HCl buffer at pH =7.0-9.5, glycine-sodium hydroxide buffer at pH =7.0-9.5, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH =7.0-9.5, or boric acid-potassium chloride-sodium hydroxide buffer at pH = 7.0-9.5.
3. The detection kit according to claim 1, wherein the surfactant in the reagent R1 is at least one of sodium dodecyl sulfate, triton x-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 =7.0-9.5, glycine-sodium hydroxide buffer at pH =7.0-9.5, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH =7.0-9.5, or boric acid-potassium chloride-sodium hydroxide buffer at pH = 7.0-9.5.
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 treatment on the recombinant plasmid pET-28a-laccase 2 Transferring the strain into an escherichia coli expression host bacterium BL21 (DE 3) by a transformation method to obtain a recombinant bacterium;
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.
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| IT1203658B (en) * | 1982-07-23 | 1989-02-15 | Amano Pharma Co Ltd | METHOD FOR THE QUANTITATIVE DETERMINATION OF PHYSIOLOGICAL COMPONENTS IN BIOLOGICAL FLUIDS |
| JP2856757B2 (en) * | 1989-03-13 | 1999-02-10 | ユニチカ株式会社 | Method for measuring total bilirubin and reagent for measurement |
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| CN104048929A (en) * | 2013-03-11 | 2014-09-17 | 南京澳林生物科技有限公司 | Detection kit for total bilirubin |
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2020
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