A 3a-hydroxysteroid dehydrogenase, gene, kit, expression vector

Abstract

The application provides a 3alpha-hydroxysteroid dehydrogenase, a gene, a kit, a catalyst, an expression vector and a recombinant bacterium, wherein the 3alpha-hydroxysteroid dehydrogenase is subjected to substitution modification at positions 59, 85, 135 and 233 of the amino acid sequence of the wild-type 3alpha-hydroxysteroid dehydrogenase of the genus Pseudomonas, and is respectively substituted as C59Y, V85L, D135E and S233R, and the amino acid sequence is shown as SEQ ID NO. 1. The application solves the technical problem that the 3alpha-HSD enzyme activity obtained after extraction, purification or expression through genetic engineering is not high, and the catalytic efficiency in the process of enzymatic detection of bile acid is low, and achieves the technical effect of improving the 3alpha-HSD catalytic activity and further improving the detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, and more specifically, to a 3α-hydroxysteroid dehydrogenase, gene, kit, and expression vector. Background Technology

[0002] Bile acids are the main organic components of bile and are a collective term for several structurally similar steroid acids. Their primary metabolic process is completed by the liver. Early methods for determining bile acids mainly involved gas chromatography and high-performance liquid chromatography (HPLC). However, these methods were cumbersome, time-consuming, and required large amounts of serum samples, limiting their clinical application. Modern clinical research shows that changes in blood bile acid concentrations can effectively detect whether hepatocellular diseases have occurred and whether liver function is impaired. Therefore, developing a simple and quick bile acid detection method is of paramount importance.

[0003] In recent years, enzymatic reagents for the detection of bile acids have been developed for clinical use. Enzymatic detection is simple to operate, highly sensitive, and low in cost. Combined with the use of fully automated biochemical analyzers, it allows for rapid routine testing. The principle of enzymatic detection is as follows: bile acids are specifically oxidized by 3α-hydroxysteroid dehydrogenase (3α-HSD) and Thio-NAD+ to generate 3-ketosteroids and Thio-NADH. 3-ketosteroids continue to generate bile acids and NAD+ in the presence of 3α-HSD and NADH, and this cycle repeats, amplifying trace amounts of total bile acids. The concentration of bile acids is determined by measuring the absorbance change of the generated Thio-NADH.

[0004] The existing problems are as follows: 3α-HSD, a key raw material in total bile acid detection reagents, is currently mainly derived from *Pseudomonas testosteroneae* extraction or genetic engineering expression. However, the extraction and purification steps are cumbersome, resulting in low yields and high production costs. Furthermore, the wild-type 3α-HSD obtained through genetic engineering expression exhibits low catalytic activity and poor thermal stability, leading to low kit sensitivity and poor stability during transportation and use. In summary, the 3α-HSD enzyme activity obtained after extraction, purification, or genetic engineering expression is not high, resulting in low catalytic efficiency during enzymatic detection, and therefore needs improvement. Summary of the Invention

[0005] This invention solves the technical problem that the 3α-HSD enzyme obtained after extraction, purification or genetic engineering expression has low activity, resulting in low catalytic efficiency in the enzymatic detection of bile acids. It achieves the technical effect of improving the catalytic activity of 3α-HSD and thus improving the detection efficiency.

[0006] To address the aforementioned problems, this invention provides a 3α-hydroxysteroid dehydrogenase. The 3α-hydroxysteroid dehydrogenase has substitution modifications at positions 59, 85, 135, and 233 of the amino acid sequence of wild-type Pseudomonas spp. The substitutions are C59Y, V85L, D135E, and S233R, respectively. The amino acid sequence is shown in SEQ ID NO.1.

[0007] Compared with existing technologies, the technical effects achieved by this technical solution are as follows: Using the wild-type 3α-HSD gene sequence of *Pseudomonas* as a template, this invention performs error-prone PCR random mutations to construct a 3α-HSD mutant library. Through specific enzyme digestion, the mutant library fragments are ligated into the PET-22b plasmid and transformed into *E. coli* BL21(DE32). After initial plate screening and shake-flask screening, the 3α-HSD with optimal activity and thermostability, along with its recombinant expression strain, are obtained. The amino acid sequence of this 3α-HSD is shown in SEQ ID. No. 1, and the enzyme sequence contains four mutant amino acids: C59Y, V85L, D135E, and S233R. The 3α-hydroxysteroid dehydrogenase obtained by this invention exhibits improved activity and thermostability, with a specific enzyme activity of 130 U / mg for catalyzing androstenedione dehydrogenation. After treatment at 60℃ for 30 min, the enzyme activity retention rate reaches 72.3%. Moreover, this enzyme can be recombinantly expressed using E. coli genetically engineered bacteria. The culture method of the engineered bacteria is simple, the expression level is high, the purification is simple, and the recovery rate is high, which makes the production cost of the enzyme low and enables rapid mass production.

[0008] The present invention also provides a gene whose nucleotide sequence encodes the 3α-hydroxysteroid dehydrogenase of the above examples, the nucleotide sequence being as shown in SEQ ID NO.2 or a nucleotide sequence with a codon synonymous mutation obtained by substituting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.2.

[0009] Compared with existing technologies, the technical effects achieved by this technical solution are as follows: After DNA sequencing, the nucleotide coding sequence of this enzyme, as shown in SEQ ID No. 2, shows six nucleotide sequence mutations compared to the original gene sequence: G→A at position 176, G→C at position 253, T→A at position 405, T→A at position 456, G→C at position 540, and C→G at position 699. Among these, mutations at positions 456 and 540 are synonymous base mutations and do not result in changes to the encoded amino acids. The gene provided by this invention possesses all the beneficial effects of the aforementioned 3α-hydroxysteroid dehydrogenase, which will not be elaborated further here.

[0010] The present invention also provides a kit for the in vitro detection of total bile acid content, comprising the 3α-hydroxysteroid dehydrogenase of the above examples.

[0011] Compared with existing technologies, the technical effects achieved by this solution are as follows: The 3α-hydroxysteroid dehydrogenase provided by this invention can be used in an in vitro total bile acid detection kit. This kit not only demonstrates good accuracy, precision, and linearity in total bile acid detection, but also reduces enzyme dosage and reagent costs due to its high catalytic activity. Furthermore, improved enzyme stability enhances the stability of the kit. The kit exhibits excellent performance indicators after 11 days of accelerated heat storage at 37°C and 18 months of storage at 4°C.

[0012] The present invention also provides a catalyst whose active ingredient comprises the 3α-hydroxysteroid dehydrogenase of the above examples.

[0013] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The catalyst provided by the present invention has all the beneficial effects of the above-mentioned 3α-hydroxysteroid dehydrogenase, which will not be repeated here.

[0014] The present invention also provides an expression vector containing the genes described above.

[0015] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The expression vector provided by this invention has all the beneficial effects of the above-mentioned genes, which will not be repeated here.

[0016] The present invention also provides a recombinant bacterium, which is transformed by the expression vector of the above examples, and the recombinant bacterium transformed by the expression vector is selected from Escherichia coli.

[0017] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The recombinant bacteria provided by this invention have all the beneficial effects of the above expression vector, which will not be repeated here. Attached Figure Description

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0019] Figure 1 The linear relationship of 3α-HSD provided in the embodiments of the present invention in the total bile acid enzyme cycling assay kit.

[0020] Figure 2 The linear relationship of 3α-HSD applied to the total bile acid enzyme cycling assay kit after heat storage, as provided in the embodiments of the present invention. Detailed Implementation

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0022] This invention provides a 3α-steroid dehydrogenase (3α-HSD) with enhanced activity and thermostability. Based on the wild-type 3α-HSD gene sequence of *Pseudomonas* genus as a template, error-prone PCR random mutations were performed to construct a 3α-HSD mutant library. The mutant library fragments were ligated into the PET-22b plasmid via specific enzyme digestion and transformed into *E. coli* BL21(DE32). After initial plate screening and shake-flask screening, a 3α-HSD strain with enhanced activity and thermostability and its recombinant expression strain were obtained. The enzyme's specific activity for catalyzing androgen is 130 U / mg, and the enzyme activity retention rate is 72.3% after treatment at 60℃ for 30 min. The amino acid sequence of the 3α-HSD is shown in SEQ ID. No. 1, and the nucleotide sequence encoding the enzyme is shown in SEQ ID. No. 2.

[0023] The 3α-steroid dehydrogenase of the present invention with enhanced activity and stability is obtained through the following pathway:

[0024] (1) Using the 3α-HSD gene sequence of Pseudomonas spp. as a template, error-prone PCR amplification mutations were performed to establish a random mutant library of 3α-HSD.

[0025] (2) The obtained 3α-HSD mutant library was digested with specific restriction enzymes and ligated with ligase, ligated into the PET22b vector, and introduced into the expression strain E. coli BL21(DE32) for recombinant expression.

[0026] (3) The plates of the recombinant expression strains were heat-treated, and five 3α-HSD mutant strains with improved thermal stability were obtained through high-throughput screening.

[0027] (4) Five 3α-HSD mutant strains with improved thermal stability were expressed and purified in shake flasks, and their specific enzyme activity was measured. One 3α-HSD strain with improved activity and thermal stability was selected.

[0028] (5) The selected mutant 3α-HSD strain was recombinantly expressed, purified, and recovered. Its specific enzyme activity and thermostability were further determined. The specific enzyme activity catalyzing the dehydrogenation of androstenedione was determined to be 130 U / mg. After the enzyme solution was treated at 60℃ for 30 min, the enzyme activity retention rate was 72.3%.

[0029] DNA sequencing revealed the enzyme's nucleotide coding sequence as shown in SEQ ID No. 2. Compared to the original gene sequence, six nucleotide mutations were observed: G→A at position 176, G→C at position 253, T→A at position 405, T→A at position 456, G→C at position 540, and C→G at position 699. Mutations at positions 456 and 540 were synonymous base mutations and did not alter the encoded amino acid. The other four mutations resulted in amino acid sequence changes: Cys→Tyr at position 59, Val→Leu at position 85, Asp→Glu at position 135, and Ser→Arg at position 233. The resulting thermostable 3α-HSD amino acid sequence is shown in SEQ ID No. 1.

[0030] Furthermore, this enzyme can be applied to a total bile acid assay kit for non-disease diagnosis and treatment. This kit demonstrates good accuracy, precision, and linearity in total bile acid detection, and the reagents exhibit excellent stability. After 11 days of accelerated storage at 37°C and 18 months at 4°C, the kit's performance indicators remained excellent.

[0031] The present invention also provides a catalyst whose active ingredient comprises the 3α-hydroxysteroid dehydrogenase of the above examples.

[0032] The present invention also provides an expression vector containing the gene of the above example, the gene encoding the nucleotide sequence of the 3α-HSD of the above example.

[0033] The present invention also provides a recombinant bacterium, which is transformed by the expression vector of the above examples, and the recombinant bacterium transformed by the expression vector is selected from Escherichia coli.

[0034] Example 1: Error-prone PCR amplification of 3α-hydroxysteroid dehydrogenase and preparation of a gene mutation library

[0035] Primer sequences were designed based on the 3α-HSD sequence in Pseudomonas:

[0036] Forward primer: 5'-TACAGGCAATAGCGTTAATG-3',

[0037] Reverse primer: 5'-AGTCTTCCAGAACCGCGCGT-3'

[0038] Using the primers described above, amplification was performed using error-prone PCR. The error-prone PCR amplification system was as follows:

[0039] 10* Amplification Buffer 20μl

[0040] dNTP mixture 30 μM

[0041] Primer 60pM

[0042] Template 0.5μg

[0043] Error-prone Taq DNA polymerase 2.5 U / L

[0044] Mg 2+ 0.15mM

[0045] Mn 2+ 6mM

[0046] Add double-distilled water to 200 μl.

[0047] The PCR reaction conditions were: 92℃ for 5 min; 92℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min, 35 cycles; 72℃ for 10 min.

[0048] 5 μl of the PCR amplification product was subjected to agarose gel electrophoresis. The target product band was observed at 750-800 bp, confirming the PCR amplification band. The PCR amplification product was purified and recovered using a DNA recovery kit to obtain the gene fragment. The fragment was digested with NdeI and XhoI enzymes, ligated into PET22b using T4 ligase, and transformed into E. coli BL21(DE32) expression strain. The transformed strain was plated on LB agar plates containing 100 mg / L AMP and incubated overnight at 37°C to obtain a series of 3α-HSD error-prone PCR random mutant expression single colonies for subsequent screening.

[0049] Example 2: Initial screening of mutant strains on plates

[0050] The single clones of the randomly mutant expression strain obtained in Example 1 were transferred from LB plates to LB plates containing a final concentration of 0.4 mM IPTG and 100 mg / L AMP using the stamp method, and cultured at 25°C for 16 h to fully induce the expression of 3α-HSD.

[0051] Cell lysis buffer was sprayed onto single-colony plates expressing 3α-HSD. The cell lysis buffer consisted of 50 mM PB, 0.6 g / L CTAB, 50 mM KCl, 10 mM MgCl2, 0.4 g / L sodium deoxycholate, and pH 7.6. The plates were then placed in a 60°C oven for 30 min.

[0052] The colorimetric reaction solution was evenly sprayed onto the treated plates to determine the enzyme activity retention rate of single colonies. The enzyme activity reaction solution consisted of: 40 mM Tris-HCl, 1 mM NAD, 1 mM androstenedione, 0.5% Triton X-100, 0.1% NTB, 10 U / ml DI, and pH 8.0. After reacting for 10 minutes, the color reaction around each single colony was observed visually. Five single colonies with a clear reaction and a deep purple color were selected for subsequent rescreening.

[0053] Example 3: Shake-flask rescreening of mutant strains

[0054] Five monoclonal colonies obtained from the initial plate screening in Example 2 were inoculated into 5 ml of LB liquid medium and cultured at 37°C until the OD reached 1. They were then transferred to 200 ml of LB liquid medium and, when the OD reached 0.8, IPTG with a final concentration of 0.4 mM was added. The culture was then induced at 25°C for 16 h.

[0055] After centrifuging the induced fermentation broth at 3000 rpm for 30 min, collect the precipitated cells, add 10 times the volume of cell lysis buffer, and lyse for 2 h. Then, centrifuge at 8000 rpm for 40 min, and slowly pass the supernatant through the IDA-Ni packing material by gravity. The packing material and target protein are then washed using washing buffer 1 and washing buffer 2, respectively. The cell lysis buffer consisted of 50 mM PB, 1% SDS, 100 U / ml lysozyme, and 0.5% Triton X-100, pH 7.5; washing buffer 1 consisted of 50 mM PB, 300 mM NaCl, and 30 mM imidazole, pH 7.5; washing buffer 2 consisted of 50 mM PB, 300 mM NaCl, and 250 mM imidazole, pH 7.5.

[0056] The collected target protein was analyzed for protein concentration using the BCA method, and enzyme activity was determined using an enzyme activity reaction solution. The enzyme activity reaction solution consisted of 50 mM Tris-HCl, 2 mM NAD, 2 mM androstenedione, 1% Triton X-100, 0.2% NTB, 15 U / ml DI, and pH 8.0. Based on the protein concentration determined by the BCA method, the enzyme solution was diluted to 0.1 g / L using washing buffer 2. Then, 100 μl of the solution was added to a 2 ml enzyme activity reaction system, and the reaction was incubated at 37°C for 10 min. The reaction was then terminated by adding 100 μl of 100% TCA, and the absorbance was measured at 546 nm using a spectrophotometer. Using 3α-HSD as a standard, the production of 1 μM androstenedione within 1 min of catalyzing androstenedione was defined as one enzyme activity unit. The enzyme activity per mg of protein was calculated, yielding the specific enzyme activity of five mutant 3α-HSD strains. The specific enzyme activity data are shown in Table 1.

[0057] Table 1: Specific enzyme activities of the 3α-HSD mutant strain

[0058]

[0059] Comparing the specific enzyme activity data in Table 1, it can be seen that mutant strain 4 has the highest specific enzyme activity. Mutant strain 4 is preferred, that is, the 3α-HSD mutant strain with improved activity and stability is obtained.

[0060] Example 4: Determination of enzyme activity and thermostability of mutant strains

[0061] The mutant strain No. 4 obtained in Example 3 was inoculated into 20 ml of 2YT liquid medium and cultured at 37°C until the OD reached 1.2. Then, it was transferred to 500 ml of 2YT liquid medium and cultured at 37°C until the OD reached 0.8. Then, 0.5 mM IPTG was added and induced overnight at 23°C.

[0062] The fermentation broth was centrifuged at 5000 rpm for 30 min to collect the cells. Eight times the volume of lysis buffer was added, and the cells were sonicated for 20 min. After centrifugation at 8400 rpm for 50 min, the supernatant was collected. The supernatant was then slowly passed through the IDA-Ni packing material by gravity. The packing material was then washed with washing buffer 1 and washing buffer 2 to elute impurities and the target protein, respectively. The cell lysis buffer consisted of 50 mM Tris-HCl, 1.2% SDS, 50 U / ml lysozyme, and 1% Triton X-100, pH 8.0. Washing buffer 1 consisted of 50 mM Tris-HCl, 500 mM NaCl, and 50 mM imidazole, pH 8.0. Washing buffer 2 consisted of 50 mM Tris-HCl, 500 mM NaCl, and 300 mM imidazole, pH 8.0.

[0063] The collected target protein was analyzed for protein concentration using the BCA method, and enzyme activity was determined using an enzyme activity reaction solution. The enzyme activity reaction solution consisted of 20 mM Tris-HCl, 5 mM NAD, 5 mM androstenedione, 1.2% Triton X-100, 0.3% NTB, 20 U / ml DI, and pH 8.0. Based on the protein concentration determined by the BCA method, the enzyme solution was diluted to 0.05 g / L using washing buffer 2. Then, 100 μl was added to 1 ml of the enzyme activity reaction system, and the reaction was carried out at 37°C for 10 min. The reaction was then terminated by adding 100 μl of 100% TCA. The absorbance at 546 nm was measured using a spectrophotometer, and the enzyme activity per mg of protein was calculated, yielding the specific enzyme activity of mutant 4 3α-HSD, which was further determined to be 130 U / mg.

[0064] The 3α-HSD enzyme solution was heat-treated in a water bath at 60°C for 30 min, and the retained enzyme activity was determined using the same method. The retained enzyme activity was compared with the control enzyme activity, and the enzyme activity retention rate was determined to be 72.3%.

[0065] Example 5: Performance validation of 3α-HSD in a bile acid assay kit

[0066] The 3α-steroid dehydrogenase solution prepared in Example 4 is added to a total bile acid cyclic enzymatic kit, and indicators such as accuracy, precision and linear relationship of the kit are analyzed. The kit consists of reagent R1 and reagent R2. The components of R1 are: 20 mM Tris-HCl, 1 g / L Thio-NAD, 0.1% TritonX-100, pH 8.0; the components of R2 are: 50 mM Tris-HCl, 5 g / L NADH, 10 KU / L 3α-HSD, 0.05% TritonX-100, pH 9.0.

[0067] The kit accuracy test is carried out as follows: after the kit is normally calibrated, an assigned quality control is tested, the measured value is compared with the quality control value, and an accuracy within a deviation of ±5% is considered qualified. The precision test is carried out as follows: after the kit is normally calibrated, high-value samples and low-value samples are tested continuously for 20 times respectively, the CV is calculated after statistical analysis, and a CV ≤ 2% is considered qualified. The linearity test is carried out as follows: after the kit is normally calibrated, a 150 μM linear high-value sample is mixed with normal saline at the ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10, regression analysis is performed on the measured values and theoretical values, and R 2 ≥0.995 is considered qualified. The accuracy and precision test results are shown in Table 2, and the linear relationship test results are shown in Figure 1 .

[0068] Table 2: Accuracy and precision test results of 3α-HSD applied in total bile acid enzymatic recycling detection kit

[0069]

[0070]

[0071] It can be seen from Table 2 that the deviations of low-value quality control and high-value quality control are 2.47% and 1.13% respectively, therefore, the accuracy of the kit is qualified; the CVs of low-value samples and high-value samples are 1.24% and 0.834% respectively, therefore, the precision of the kit is qualified. It can be seen from Figure 1 that the linear regression equation is Y=1.007X-0.3227, R 2 = 0.9996, therefore, the kit has a good linear relationship for detection.

[0072] Example 6: Performance verification of 3α-HSD in bile acid detection kit after heat storage

[0073] The 3α-steroid dehydrogenase solution prepared in Example 4 was added to the total bile acid cycling enzymatic assay kit. The accuracy, precision, and linearity of the kit after heat storage were analyzed to assess its stability. The kit includes reagents R1 and R2. R1 consists of 20 mM Tris-HCl, 1.3 g / L Thio-NAD, and 0.15% Triton X-100, pH 8.2; R2 consists of 100 mM Tris-HCl, 10 g / L NADH, 25 KU / L 3α-HSD, and 0.5% Triton X-100, pH 8.8. Reagents R1 and R2 were placed at 37°C for accelerated storage for 11 days. After the heat storage period, the accuracy, precision, and linearity were measured under normal calibration, using the same methods as in Example 5. The results are shown in Table 3.

[0074] Table 3: Accuracy and precision determination results of 3α-HSD in the total bile acid oxidase cycling assay kit after heat storage.

[0075]

[0076]

[0077] As shown in Table 3, the deviations of the low-value and high-value quality control samples after heat storage were -2.47% and -0.528%, respectively; therefore, the accuracy of the kit is acceptable. The CVs of the low-value and high-value samples were 1.39% and 0.595%, respectively; therefore, the precision of the kit is acceptable. Figure 2 As can be seen from the data, the linear regression equation is Y = 0.9948X + 1.055, R0 2 =0.9993, therefore, the linearity of the kit detection is good.

[0078] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A 3α-hydroxysteroid dehydrogenase, characterized in that, The 3α-hydroxysteroid dehydrogenase described herein has undergone substitution modifications at positions 59, 85, 135, and 233 of the amino acid sequence of the wild-type Pseudomonas 3α-hydroxysteroid dehydrogenase, which are respectively replaced by C59Y, V85L, D135E, and S233R, and the amino acid sequence is shown in SEQ ID NO.

1.

2. A gene characterized in that, The nucleotide sequence of the gene encodes the 3α-hydroxysteroid dehydrogenase as described in claim 1, wherein the nucleotide sequence is as shown in SEQ ID NO.2 or is a nucleotide sequence with a codon synonymous mutation obtained by substituting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.

2.

3. A kit for the in vitro detection of total bile acid content, characterized in that, It contains the 3α-hydroxysteroid dehydrogenase as described in claim 1.

4. A catalyst, characterized in that, Its active ingredient contains the 3α-hydroxysteroid dehydrogenase as described in claim 1.

5. An expression carrier, characterized in that, It contains the gene as described in claim 2.

6. A recombinant bacterium, characterized in that, The recombinant bacteria are transformed by the expression vector as described in claim 5, and the recombinant bacteria transformed by the expression vector are selected from Escherichia coli.

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