Constitutive expression plasmid vector for high expression of xanthine oxidase and application of constitutive expression plasmid vector

Abstract

The invention belongs to the technical field of gene engineering, and discloses a constitutive expression plasmid vector for high expression of xanthine oxidase and application of the constitutive expression plasmid vector. The plasmid vector, as a heterologous expression system, can be used for efficiently expressing the recombinant xanthine oxidase derived from the cellulomicrobe TH20 in Pseudomonas putida KT2440. Specifically, a replication starting point of pUCP18 is replaced by a replication starting point of pSC101 with a low copy number, and a constitutive expression plasmid pPegP119 is constructed, so that the metabolic burden is relieved. A TH20 XOD gene cluster is cloned into the vector to construct a recombinant strain, and xanthine oxidase can be efficiently produced at low cost. According to the invention, the high-efficiency expression of the xanthine oxidase is realized in Pseudomonas putida KT2440 through a gene recombination technology, and the maximum expression quantity of the xanthine oxidase can be realized without adding an inducer in the expression.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a constitutive expression plasmid vector for high expression of xanthine oxidase and its application. Background Technology

[0002] Xanthine oxidase (XOD) is widely distributed in organisms and is a key enzyme in the purine metabolism pathway. It catalyzes the oxidation of hypoxanthine to xanthine, which is further oxidized to uric acid. Currently, XOD has extensive applications in the medical diagnostic field, primarily for the detection of purine compounds such as xanthine, hypoxanthine, and hypoxanthine nucleoside (inosine), as well as for the auxiliary detection of guanine and guanosine. It can also be used to determine the activity of 5'-nucleoside phosphorylase, thus making it suitable for the detection of hepatobiliary tumors. XOD is also widely used to detect serum inorganic phosphorus levels (hyperphosphatemia) and superoxide dismutase (SOD) activity. In addition, xanthine oxidase has applications in disease treatment, food testing, industrial catalysis, and environmental protection, making it an important biocatalyst.

[0003] Natural xanthine oxidase is mainly derived from milk, but the quality of xanthine oxidase products extracted from milk is unstable, and the yield is low, far from meeting market demand. Currently, the main XOD products used in the market are derived from Rhodococcus (…). R.erythropolis The recombinant xanthine oxidase product has a long growth cycle, and the inducer is very expensive, resulting in high production costs. The main reason for the difficulty in heterologous expression of this protein is that there are significant differences in the XOD gene clusters among different species, and the different assembly processes and coenzyme molecule integration mechanisms of different microorganisms make it very difficult for prokaryotes to heterologously express XOD protein.

[0004] Therefore, developing a more efficient and economical XOD heterologous expression system has great market value. Summary of the Invention

[0005] To address the problems of heavy metabolic burden, high induction cost, low heterologous expression efficiency, and insufficient product activity in the production of xanthine oxidase in existing technologies, this invention provides a high-activity, low-cost XOD preparation scheme without inducers through a combination design of low-copy plasmid modification, constitutive promoter optimization, and specific gene cluster and host adaptation. At the same time, it simplifies the purification process, ensures product purity, and meets the stringent requirements of medical diagnostic scenarios such as uric acid detection.

[0006] The first objective of this invention is to provide a constitutive expression plasmid vector for high expression of xanthine oxidase, which, as a heterologous expression system, can... Pseudomonas putidaKT2440 efficiently expresses recombinant xanthine oxidase derived from the genus *Ceratophyllum* TH20. Specifically, the constitutive expression plasmid pPegP119 was constructed by replacing the pUCP18 origin of replication with the low-copy-number pSC101 origin of replication to reduce the metabolic burden. The TH20 XOD gene cluster was cloned into this vector to construct a recombinant strain, enabling low-cost and high-efficiency production of xanthine oxidase. This invention utilizes gene recombination technology to achieve high efficiency in... Pseudomonas putida KT2440 has achieved highly efficient expression of xanthine oxidase, which can achieve maximum expression level of xanthine oxidase without the addition of an inducer.

[0007] The second objective of this invention is to provide a recombinant strain for high expression of xanthine oxidase, which requires no induction, has high expression levels, a simple affinity purification process, high purification yield, low production cost, and recombinant xanthine oxidase with specific activity similar to currently commercially available xanthine oxidases, making it suitable for a variety of applications.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A constitutive expression plasmid vector for high expression of xanthine oxidase, the nucleotide sequence of which is shown in SEQ ID NO.1; The constitutive expression plasmid vector is constructed by replacing the origin of replication of the basic backbone plasmid pUCP18 with the low copy number origin of replication pSC101, and is named pPegP119.

[0009] Preferably, the constitutive expression plasmid vector is composed of a pSC101 replication origin containing a RepA protein coding sequence, a J23119 constitutive promoter, an RBS sequence, an XOD gene cluster, and a terminator. The XOD gene cluster is derived from the genus TH20 of Microbes fibrobacterium, including the XodBA gene cluster or the XodCBA gene cluster. The XodBA gene cluster includes xodb and xodA Gene; The XodCBA gene cluster includes xodC, xodB and xodA Gene.

[0010] Preferably, the specific preparation process of the constitutive expression plasmid vector is as follows: S1. Replace the ampicillin resistance cassette (sites 95-1062) in the basic backbone plasmid pUCP18 with the P119-RBS-MCS fragment to obtain the intermediate plasmid pUCP18P119. S2. Replace the pBR322 origin of replication sequence (1232-1860 sites) in the intermediate plasmid pUCP18P119 with the origin of replication of the pSC101 plasmid containing the RepA protein sequence with a low copy number, and finally obtain the pPegP119 plasmid. The low copy number is 6 copies / cell.

[0011] Preferably, the nucleotide sequence of the P119-RBS-MCS fragment is shown in SEQ ID NO.2; The nucleotide sequence of the origin of replication of the pSC101 plasmid containing the RepA protein sequence is shown in SEQ ID NO.3.

[0012] A recombinant expression vector for high expression of xanthine oxidase, wherein the recombinant expression vector is obtained by cloning not less than one XOD gene cluster into the constitutive expression plasmid vector, thereby obtaining multiple recombinant expression vectors containing xanthine oxidase expression cassettes. The xanthine oxidase expression cassette consists of a promoter and an XOD gene cluster connected downstream of the promoter.

[0013] Preferably, the recombinant expression vector is pPegP119XodBA or pPegP119XodCBA.

[0014] A recombinant strain for high expression of xanthine oxidase is obtained by transforming the constitutive plasmid vector into the host bacteria and then screening. The host bacteria is Pseudomonas putida KT2440.

[0015] Application of a recombinant strain that highly expresses xanthine oxidase in the preparation of xanthine oxidase and / or hypoxanthine oxidase.

[0016] Application of a recombinant strain that highly expresses xanthine oxidase in the preparation of reagents for detecting xanthine and / or hypoxanthine concentrations.

[0017] A xanthine oxidase that can be highly expressed was obtained by expression from the recombinant strain described above.

[0018] Compared with the prior art, the present invention has at least the following technical effects: This invention provides a constitutive expression plasmid vector for high expression of xanthine oxidase. This plasmid vector, as a heterologous expression system, can... Pseudomonas putidaKT2440 efficiently expresses recombinant xanthine oxidase derived from the genus *Ceratophyllum* TH20. Specifically, the constitutive expression plasmid pPegP119 was constructed by replacing the pUCP18 origin of replication with the low-copy-number pSC101 origin of replication to reduce the metabolic burden. The TH20 XOD gene cluster was cloned into this vector to construct a recombinant strain, enabling low-cost and high-efficiency production of xanthine oxidase. This invention utilizes gene recombination technology to achieve high efficiency in... Pseudomonas putida KT2440 has achieved highly efficient expression of xanthine oxidase, which can achieve maximum expression level of xanthine oxidase without the addition of an inducer.

[0019] This recombinant strain for high expression of xanthine oxidase has the advantages of high expression levels without induction, simple affinity purification process, high purification yield, low production cost, and recombinant xanthine oxidase with specific activity similar to currently commercially available xanthine oxidases, making it suitable for a variety of applications. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the construction process of the pPegP119 plasmid vector in a specific implementation embodiment; Figure 2 This is a schematic diagram of SDS-PAGE protein electrophoresis analysis of the recombinant expression vector in a specific implementation. Detailed Implementation

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the accompanying drawings is only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0022] One specific embodiment of the present invention is as follows: To address the problems of complex and inefficient xanthine oxidase preparation methods, this invention provides a constitutive expression plasmid vector, recombinant strain, recombinant expression vector, and preparation process for high expression of xanthine oxidase.

[0023] This method constructs a new recombinant plasmid by combining two different xanthine oxidase gene clusters (XodBA or XodCBA) with the pPegP119 vector, and then introduces it into the vector. Pseudomonas putida Expression was performed in KT2440. After expression, the expression was further purified by affinity chromatography, concentrated, and lyophilized.

[0024] This method has the advantages of short cell growth cycle, no induction required, high expression level, simple purification process, and low production cost.

[0025] A specific method for preparing a constitutive expression plasmid vector for high expression of xanthine oxidase includes the following steps: Figure 1 As shown, 1. Preparation of recombinant plasmids The pUCP18 plasmid (from the literature Schweizer, HP:) was used. Escherichia-Pseudomonas The ampicillin resistance cassette (sites 95-1062) in shuttle vectors derived from pUC18 / 19, Gene, 97, 109-121 (1991), deposited in our laboratory, was replaced with the P119-RBS-MCS fragment to obtain the intermediate plasmid pUCP18P119. The nucleotide sequence of the replaced P119-RBS-MCS fragment is shown in SEQ ID NO.2.

[0026] Subsequently, the pBR322 origin of replication sequence (positions 1232-1860) of the intermediate plasmid pUCP18P119 was replaced with the origin of replication of the pSC101 plasmid and the RepA protein sequence, finally obtaining the pPegP119 plasmid (nucleotide sequence shown in SEQ ID NO. 1). The nucleotide sequence is shown in SEQ ID NO. 3.

[0027] 2. Based on the XOD gene cluster XodCBA of the genus *Fibromys* TH20 (containing... xodC, xodB and xodA The gene (GenBank ID: CP020857.1) is a synthetic gene cluster XodCBA with an N-terminal 6×His tag.

[0028] The synthesis was performed by Nanjing Gene Technology Co., Ltd. The synthesized sequence was cloned into the pUC18 plasmid to construct the pUC18CsXodCBA vector.

[0029] 3. Design primers for PCR amplification. The PCR primers are as follows: 3.1 XodCBA gene cluster amplification: (using pUC18CsXodCBA as a template) SEQ ID NO.4: XodCBA-F: 5'-GAGGAATTAACCCAGAATGCTCCACATCGTCGACC-3' SEQ ID NO.5: XodCBA-R: 5'-GCGTTCTGATTTAACTAGTGATGTGTGATGGTGA-3' 3.2 XodBA gene cluster amplification: (using pUC18CsXodCBA as a template) SEQ ID NO.6: XodBA-F: 5'-GAGGAATTAACCCAGAATGGACCTGGGCACCGT-3' SEQ ID NO.7: XodBA-R: 5'-GCGTTCTGATTTAACTAGTGATGTGTGATGGTGA-3' 3.3 Amplification of vector pPegP119: SEQ ID NO.8: PPegP119-F: 5'-TAGTTAAATCAGAACGCAGAAG-3' SEQ ID NO.9: PPegP119-R: 5'-TCTGGGTTAATTCCTCCTG-3' 4. PCR was performed according to standard procedures. In a sterile 200 μl centrifuge tube, add 5 μl Taq buffer (10×), 5 μl dNTPs, 2.5 μl F primer (10 pmol / μl), 2.5 μl R primer (10 pmol / μl), 1–10 ng template DNA, and sterile water to a final volume of 50 μl. Mix well and place in a PCR instrument (Eppendorf). Set the PCR cycling parameters as follows: denaturation at 94°C for 30–40 s, annealing at 55°C for 1 min, extension at 72°C for 5 min, for a total of 30–35 cycles. After the reaction, store at 10°C.

[0030] 5. The amplified fragments were recovered separately using a DNA recovery kit (Qingke Company DNA Recovery Kit), and the recovered gene fragments and pUCP18 fragments were further ligated using Gibson ligation (following the method of the Nanjing Novizan Company Gibson Ligation Kit). The recovered fragments were then transformed into [unclear - possibly a specific product or service] using standard methods. E. coli DH5 competent cells were screened for positive clones on LB agar plates containing 25 μg / mL gentamicin, and then transferred to liquid culture medium for plasmid extraction and sequencing. The recombinant plasmids pPegP119XodBA and pPegP119XodCBA were finally obtained.

[0031] 6. Recombinant plasmids in Pseudomonas putida Electroconversion and expression in KT2440 6.1 Cultured using LB medium Pseudomonas putida KT2440 to OD 600With nm=0.6~0.75, the cultured bacterial cells were centrifuged at 4°C for 10 minutes at 5000 r / min to collect the bacterial cells.

[0032] 6.2 Discard the supernatant and wash three times with 15 mL of pre-cooled 10% glucose solution. Concentrate the resulting precipitate 100 times and resuspend it in ice-cold 10% glucose solution.

[0033] 6.3 Add 1 μL of the constructed plasmid to 50 μL of concentrated competent cell suspension, incubate on ice for 30 min, and then perform electroporation using a Bio-RadGene-Pulser II electroporator with a 1 mm electroporation cuvette at 1.5 kV, 200 Ω, and 25 μF. Immediately after electroporation, add 1 mL of SOC medium and incubate at 30°C for 1 hour. Then, plate the mixture onto gentamicin-resistant LB agar plates and incubate at 30°C for 18 hours.

[0034] 6.4 Take a single colony, transfer it to 5 mL of LB liquid medium with the same antibiotic and incubate overnight at 30°C. Then, inoculate it with 100 mL of LB medium with the same antibiotic and incubate at 30°C for 16 hours. Centrifuge at 4°C, 5000 rpm for 15 min to obtain bacterial cells.

[0035] 7. Purification of gene expression products 7.1 Resuspend the bacterial cells in PBS buffer and sonicate them (Ningbo Xinzhi, 200W, 3s lysis, 3s interval) to release intracellular proteins. Centrifuge the lysate (4°C, 10000rpm, 30min) and collect the supernatant.

[0036] 7.2 Prepare the purification buffer according to the following formula: Binding buffer: 20 mM sodium phosphate buffer, 0.1 M sodium chloride, 20 mM imidazole, pH 8.0; Elution buffer: 20mM sodium phosphate buffer, 0.1M sodium chloride, 200mM imidazole, pH 8.0.

[0037] After the buffer solution is prepared, it needs to be pre-filtered and sonicated to remove air bubbles.

[0038] 7.3 Affinity chromatography purification was performed on a GE AKTA using a GE Histrap pre-packed column. The system was first rinsed with deionized water, followed by elution buffer. The column was equilibrated with 5 column volumes of binding buffer, and sample supernatant was added using a sample pump. Washing was continued with binding buffer for at least 10-15 column volumes until a stable baseline was reached or no material was eluent. Elution was performed using either a one-step elution or a linear gradient elution (changing the imidazole concentration gradient). One-step elution was performed for 5 column volumes. After eluent recovery, the solution was dialyzed using a dialysis bag with 20 mM Tris-HCl buffer (20 mM, pH 7.0). The obtained enzyme solution was frozen at -20°C or lyophilized and stored at -20°C for later use.

[0039] 7.4 such as Figure 2 As shown, the purified protein was analyzed by SDS-PAGE electrophoresis. The left side shows the electrophoretic band of pPegP119XodCBA expressed protein, and the right side shows the electrophoretic band of pPegP119XodBA expressed protein.

[0040] The purified sample clearly showed the expression of the target bands, namely the 105kDa and 31kDa proteins, without interference from background proteins.

[0041] 7.5 The purified XOD was quantified using the BCA method. A standard curve was plotted using bovine serum albumin (BSA) as a standard, and the total protein content of the sample was calculated accordingly.

[0042] Enzyme activity assay was performed using the antipyrine colorimetric method: the sample to be tested was mixed with a colorimetric solution (containing 50 mM Tris-HCl, pH 8.4, 1 mM 4-aminoantipyrine, 6 mM phenol and 7000 U / L horseradish peroxidase), reacted at 37°C for 20 minutes, and then the reaction was terminated by boiling in a water bath for 5 minutes. The absorbance of the product was then measured at 508 nm using a spectrophotometer.

[0043] To accurately quantify enzyme activity, XOD standard (specific activity: 10 U / mg) purchased from Toyobo was used as a positive control in the experiment.

[0044] Finally, by integrating protein concentration and enzyme activity data, the specific activity of purified XOD was found to be approximately 25 U / mg.

[0045] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A constitutive expression plasmid vector for high expression of xanthine oxidase, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.1; The constitutive expression plasmid vector is constructed by replacing the origin of replication of the basic backbone plasmid pUCP18 with the low copy number origin of replication pSC101, and is named pPegP119.

2. The constitutive expression plasmid vector for high expression of xanthine oxidase according to claim 1, characterized in that, The constitutive expression plasmid vector consists of a pSC101 replication origin containing the RepA protein coding sequence, a J23119 constitutive promoter, an RBS sequence, an XOD gene cluster, and a terminator. The XOD gene cluster is derived from the genus TH20 of Microbes fibrobacterium, including the XodBA gene cluster or the XodCBA gene cluster; The XodBA gene cluster includes xodB and xodA Gene; The XodCBA gene cluster includes xodC, xodB and xodA Gene.

3. The constitutive expression plasmid vector for high expression of xanthine oxidase according to claim 2, characterized in that, The specific preparation process of the constitutive expression plasmid vector is as follows: S1. Replace the ampicillin resistance cassette (sites 95-1062) in the basic backbone plasmid pUCP18 with the P119-RBS-MCS fragment to obtain the intermediate plasmid pUCP18P119. S2. Replace the pBR322 origin of replication sequence (1232-1860 sites) in the intermediate plasmid pUCP18P119 with the origin of replication of the pSC101 plasmid containing the RepA protein sequence with a low copy number, and finally obtain the pPegP119 plasmid. The low copy number is 6 copies / cell.

4. The constitutive expression plasmid vector for high expression of xanthine oxidase according to claim 3, characterized in that, The nucleotide sequence of the P119-RBS-MCS fragment is shown in SEQ ID NO.2; The nucleotide sequence of the origin of replication of the pSC101 plasmid containing the RepA protein sequence is shown in SEQ ID NO.

3.

5. A recombinant expression vector for high expression of xanthine oxidase, characterized in that, The recombinant expression vector is obtained by cloning not less than one XOD gene cluster into the constitutive expression plasmid vector as described in claim 1, thereby obtaining multiple recombinant expression vectors containing xanthine oxidase expression frames. The xanthine oxidase expression cassette consists of a promoter and an XOD gene cluster connected downstream of the promoter; The XOD gene cluster is XodBA or XodCBA.

6. The recombinant expression vector for high expression of xanthine oxidase according to claim 5, characterized in that, The recombinant expression vectors are pPegP119XodBA and pPegP119XodCBA.

7. A recombinant strain for high expression of xanthine oxidase, characterized in that, The constitutive plasmid vector as described in claim 1 is transformed into the host bacteria, and then screened to obtain the desired result. The host bacteria is Pseudomonas putida KT2440.

8. The use of a recombinant strain for high expression of xanthine oxidase as described in claim 7 in the preparation of xanthine oxidase and / or hypoxanthine oxidase.

9. The use of the recombinant strain for high expression of xanthine oxidase as described in claim 7 in the preparation of a reagent for detecting xanthine and / or hypoxanthine concentrations.

10. A xanthine oxidase that can be highly expressed, characterized in that, It is obtained by expression of the recombinant strain as described in claim 7.

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