Nucleic acid molecule for coding aryl sulfatase and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to a nucleic acid molecule for coding aryl sulfatase and application of the nucleic acid molecule. The wild aryl sulfatase is derived from klebsiella pneumoniae, nucleic acid molecules suitable for encoding the aryl sulfatase are obtained through a codon optimization method, the nucleic acid molecules can be generated through escherichia coli expression and can be efficiently expressed in a culture medium, the yield is greatly increased and is 24 times that of naturally extracted aryl sulfatase, and the method is suitable for industrial production. The enzymolysis effect of the prepared aryl sulfatase is equivalent to that of natural aryl sulfatase, and production application can be met.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a nucleic acid molecule encoding aromatic sulfatase and an application thereof. Background Art

[0002] Genetic recombination technology can modify and recombine specific genes. This operation is carried out in a laboratory environment with high accuracy. Through precise gene cutting, connection and introduction steps, it can ensure that the target gene is accurately inserted into the genome of the host cell, thereby avoiding unnecessary gene mutations and erroneous expressions. Genetic recombination technology has broad application prospects in many fields. In the agricultural field, it can be used to cultivate new varieties of high-yield, high-quality and stress-resistant crops; in the medical field, it can be used in gene therapy, biopharmaceuticals and vaccine research and development; in the industrial field, it can be used in the production of bioenzyme preparations and biofuels. In addition, genetic recombination technology also plays an important role in environmental protection, food safety and biosafety.

[0003] As an important class of enzymes, sulfatases have a wide range of applications. Sulfatases can be used in the food industry to improve the taste and quality of food by hydrolyzing sulfate compounds in food. For example, sulfates in some foods may affect their flavor or stability, and removing them with sulfatases can optimize these properties. Sulfatases can gently degrade the sulfate groups on agar, thereby increasing the gel strength of agar and improving its performance. Sulfatases also have potential applications in drug development. For example, some drug precursors need to be converted into active pharmaceutical ingredients through the action of sulfatases. Therefore, the study of sulfatases helps the development and optimization of new drugs.

[0004] However, the currently common natural sulfatases have low yields and unstable enzyme activities, which cannot meet the needs of production applications. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a nucleic acid molecule suitable for encoding an aromatic sulfatase derived from Klebsiella and its application.

[0006] The present invention provides a nucleic acid molecule encoding an aromatic sulfatase having:

[0007] (1) the nucleotide sequence shown in SEQ ID NO: 1; or

[0008] (2) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (1) but is different from the nucleotide sequence shown in (1) due to the degeneracy of the genetic code; or

[0009] (3) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (1) or (2), and having the same or similar functions as the nucleotide sequence shown in (1) or (2); or

[0010] (4) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (1), (2) or (3).

[0011] The invention provides a transcription unit of aromatic sulfatase, comprising: a promoter nucleic acid molecule and a terminator.

[0012] The present invention provides a recombinant vector, characterized in that it comprises a backbone vector and the nucleic acid molecule.

[0013] In some embodiments, the backbone vector comprises at least one of the pET series vectors.

[0014] In some specific embodiments, the backbone vector is pET22b.

[0015] In some embodiments, a purification tag is further included, wherein the purification tag includes a His tag.

[0016] In some specific embodiments, the insertion sites of the nucleic acid molecule in the backbone vector are Nde I and Xho I.

[0017] The invention provides a host cell transformed or transfected with the recombinant vector.

[0018] In some embodiments, the host cell is a prokaryotic cell.

[0019] In some specific embodiments, the host cell is Escherichia coli BL21 (DE3).

[0020] The present invention provides the use of the nucleic acid molecule, the transcription unit, the recombinant vector and / or the host cell in preparing at least one of the following (5) to (7):

[0021] (5) Reagents or kits for treating sulfate groups in samples;

[0022] (6) Food additives;

[0023] (7) Antibacterial, antiviral or anti-tumor drugs.

[0024] The present invention provides a reagent or a kit, comprising the nucleic acid molecule, the transcription unit, the recombinant vector and / or the host cell, and an acceptable auxiliary agent.

[0025] The present invention provides a food additive, comprising the nucleic acid molecule, the transcription unit, the recombinant vector and / or the host cell, and an auxiliary material acceptable to food.

[0026] The present invention provides a medicine, comprising the nucleic acid molecule, the transcription unit, the recombinant vector and / or the host cell, and pharmaceutically acceptable excipients.

[0027] The present invention provides a nucleic acid molecule encoding an aromatic sulfatase and an application thereof. The wild-type aromatic sulfatase used in the present invention is derived from Klebsiella pneumoniae. A nucleic acid molecule suitable for encoding the aromatic sulfatase is obtained by a codon optimization method. The aromatic sulfatase can be expressed and produced by Escherichia coli, and is efficiently expressed in a culture medium. The yield is greatly improved, which is 24 times that of the naturally extracted aromatic sulfatase. The prepared aromatic sulfatase has an enzymatic hydrolysis effect comparable to that of the natural aromatic sulfatase, and can meet production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure shows the purification result of the codon-optimized aromatic sulfatase protein in Example 3. DETAILED DESCRIPTION

[0029] The present invention provides nucleic acid molecules encoding aromatic sulfatase and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0030] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0031] Example 1 Construction of an Optimization Scheme for Aromatic Sulfatase Sequences

[0032] The aromatic sulfatase gene of Klebsiella pneumoniae (Uniprot number: A0A0H3H1F1) was synthesized by Suzhou Jinweizhi Company after codon optimization and ligated into the pET22b vector (purchased from Novagen) using double restriction enzymes Nde I and Xho I, and named AS01-pET22b.

[0033] The nucleotide sequence of the above-mentioned optimized aromatic sulfatase gene is as follows:

[0034]

[0035] AS01-pET22b was sequenced using T7 and T7t universal primers, and the sequencing was completed by Shanghai Sangon Biotechnology Co., Ltd. The sequencing results showed that the inserted gene sequence of AS01-pET22b was the same as the sequence shown in SEQ ID NO:1.

[0036] Example 2 Optimizing the expression of aromatic sulfatase genes

[0037] The AS01-pET22b vector in Example 1 was transferred into BL21 (DE3) competent cells, and kanamycin was added to the LB liquid medium at a ratio of 1:1000 for screening. A single colony was picked from the plate and inoculated into 3mL LB medium, and placed in a desktop constant temperature oscillator at 37°C 230rpm. The OD600 value was cultured to 0.6-1.0, 1mL of the bacterial solution was transferred to a 1.5mL centrifuge tube, 1μL 1M IPTG was added, and the induction culture was carried out for 3-4h. 20μL of the bacterial solution was taken and sent to SDS-PAGE to detect the expression. The bacterial solution that was screened for expression was inoculated into LB medium and induced in a 1L shake flask at 37°C for 6h, and 5g of bacteria were harvested.

[0038] Example 3 Purification and preparation of optimized mutant protein of aromatic sulfatase

[0039] (1) Take out 5 g of bacteria from -20℃, calculate the volume of lysis buffer (50 mM Tris-HCl + 300 Mm NaCl + 20 mM imidazole pH 7.4) required at a ratio of 1:20, and measure it accurately (e.g. 5 g of bacteria requires 100 mL 50 mM Tris-HCl + 300 Mm NaCl + 20 mM imidazole pH 7.4).

[0040] (2) Resuspension of bacterial cells: After the cells are thawed on ice, add cold lysis buffer (50 mM Tris-HCl + 300 Mm NaCl + 20 mM imidazole pH 7.4) in portions and pour into a 250 mL beaker. Rinse the centrifuge tube with the remaining buffer and pour into the same beaker. Stir the sample in an ice bath and stir it thoroughly on a magnetic stirrer for about 30 minutes until no obvious bacterial solids are present.

[0041] (3) Bacteria disruption treatment: Pour the bacterial suspension into the material cup of the high-pressure homogenizer, press the "Run" button to start the operation, and collect the outflowing sample into the beaker. After the outflowing liquid flows out evenly, press the cooling button of the cooling water circulation machine, slowly adjust the pressure valve, and the pressure display reads between 800 and 850 bar. Start homogenization and circulate 4 times (one cycle is based on all samples passing through). During this period, when the pressure display reads less than 800 bar, adjust the pressure valve until the reading returns to between 800 and 850 bar. After the cycle is completed, adjust the pressure valve to read '000'.

[0042] (4) Place the high-pressure homogenized sample into a centrifuge cup, balance it and centrifuge it. Centrifugation conditions: 4°C, 8500 rpm, 30 min. Immediately after centrifugation, pour out the supernatant into a 250 mL beaker.

[0043] (5) Filtration: Filter using a 500 mL filter cup and collect the filtrate into another clean 250 mL beaker.

[0044] (6) Use AKTAprime plus for Ni metal chelate chromatography (add 6*His tag to the C-terminus of the protein to achieve Ni column purification of arylsulfatase). Washing: After loading and rebalancing, run the program to 5% B (B refers to B pump, 5% B refers to 5% imidazole on Ni chromatography B pump) to start elution, observe the trend of UV curve changes, and when the UV absorbance value rises, put the waste liquid tube into a clean 500mL beaker to collect 3% eluate. When the UV value is level, stop collecting and put the waste liquid tube back into the waste liquid bucket.

[0045] (7) Dissociation of target protein: Run the program until 10% B begins elution, observe the trend of the UV curve, and start collecting samples when the UV value rises. Place the waste liquid tube in a 250 mL brown bottle and place the brown bottle on ice.

[0046] (8) The purification results are as follows Figure 1 As shown in the SDS-PAGE electrophoresis diagram, the electrophoretic bands are loading (SY), flow-through (LC), 3% B, 10% B, 20% B, and 50% B. The yield of recombinant aromatic sulfatase is 6 mg / g bacteria.

[0047] Example 4 Determination of aromatic sulfatase activity

[0048] 20μL of purified enzyme solution was added with 80μL of 20mmol / L p-nitrosulfate potassium (p-NP, prepared in 50mmol / L Tris-HCl buffer), incubated at 50℃ for 10 min, and then 25μL of 1mol / L NaOH was added to terminate the reaction. The volume was made up to 1mL with distilled water, and the absorbance was measured at 410nm. The activity (U) of aromatic sulfatase is defined as the amount of enzyme required to catalyze the production of 1μmol p-nitrophenol (p-NP) per minute under the above conditions.

[0049] Table 1 shows the results of enzyme activity determination of naturally extracted aromatic sulfatase supernatant and recombinantly expressed aromatic sulfatase supernatant. It can be seen from the table that the expression level of recombinant aromatic sulfatase of Escherichia coli is 24 times that of aromatic sulfatase naturally extracted from Klebsiella pneumoniae.

[0050] The arylsulfatase sequence provided by the present invention is a wild-type (Uniprot number: A0A0H3H1F1) amino acid sequence with a tag added after codon optimization. Ni column purification of the arylsulfatase is achieved by adding a 6*His tag to the C-terminus of the wild-type sequence. It can be efficiently expressed in the host, and the expression product has high activity. Experiments show that 30 mg of arylsulfatase can be obtained per liter of culture medium after fermentation.

[0051] Table 1

[0052]

[0053] Example 5 Application of arylsulfatase in the treatment of S-MN in urine samples

[0054] 1. Enzymatic hydrolysis

[0055] 20μL urine calibrator + 100μL 0.5M Tris-NaCl (to adjust the weak acid pH of urine to about 7) + aromatic sulfatase (30μL, enzyme activity adjusted to 5U / mL), mix well and place in a 37℃ water bath for 30min; add 100μL acidification solution, mix well and let stand for 10min; add 250μL acylation buffer and mix well.

[0056] 2. Sample testing after treatment

[0057] After the urine samples with different S-MN concentrations were treated with the experimental group (recombinant) and the control group (natural), the content of free MNs was determined by the competition method using the fully automatic chemiluminescence analyzer A2000 Plus (Zhengzhou Antu Biotechnology Co., Ltd.). The results are shown in Table 2 (the luminescence values ​​of different treatments were determined by the chemiluminescence method. The lower the luminescence value of the same sample, the higher the content of free MNs and the more complete the enzymatic hydrolysis effect). From the data in Table 2, it can be seen that the enzymatic hydrolysis effects of recombinant and natural are at the same level.

[0058] Table 2

[0059]

[0060] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nucleic acid molecule encoding an aromatic sulfatase, characterized in that It has: (1) the nucleotide sequence shown in SEQ ID NO: 1; or (2) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (1) but is different from the nucleotide sequence shown in (1) due to the degeneracy of the genetic code; or (3) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (1) or (2), and having the same or similar functions as the nucleotide sequence shown in (1) or (2); or (4) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (1), (2) or (3).

2. A transcription unit of an arylsulfatase, characterized in that include: A promoter, the nucleic acid molecule according to claim 1 and a terminator.

3. A recombinant vector, characterized in that: It comprises a backbone vector and the nucleic acid molecule according to claim 1.

4. The recombinant vector according to claim 3, characterized in that The backbone vector includes at least one of the pET series vectors.

5. A host cell transformed or transfected with the recombinant vector according to claim 3 or 4.

6. The host cell according to claim 5, characterized in that The host cell is a prokaryotic cell.

7. Use of the nucleic acid molecule according to claim 1, the transcription unit according to claim 2, the recombinant vector according to claim 3 or 4, and / or the host cell according to claim 5 or 6 in the preparation of at least one of the following (5) to (7): (5) Reagents or kits for treating sulfate groups in samples; (6) Food additives; (7) Antibacterial, antiviral or anti-tumor drugs.

8. A reagent or a kit, characterized in that The method comprises the nucleic acid molecule according to claim 1, the transcription unit according to claim 2, the recombinant vector according to claim 3 or 4 and / or the host cell according to claim 5 or 6, and an acceptable auxiliary agent.

9. A food additive, characterized in that The invention comprises the nucleic acid molecule according to claim 1, the transcription unit according to claim 2, the recombinant vector according to claim 3 or 6 and / or the host cell according to claim 5 or 6, and a food-acceptable auxiliary material.

10. A drug, characterized in that The invention comprises the nucleic acid molecule according to claim 1, the transcription unit according to claim 2, the recombinant vector according to claim 3 or 6 and / or the host cell according to claim 5 or 6, and pharmaceutically acceptable excipients.

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