A xanthine transporter mutant and use thereof

Abstract

This invention discloses a xanthine transporter mutant and its applications, belonging to the field of biotechnology. Using live bacterial cells obtained from recombinant Escherichia coli Nissle 1917 containing xanthine transporter protein cultured in M9 medium as the transporter, and xanthine as the substrate, this invention reduces the extracellular xanthine concentration. Animal experiments showed that gavage administration of the xanthine transporter mutant effectively reduced the xanthine content in the intestine and serum uric acid levels in hyperuricemic mouse models. The invention also discloses that the transport activity of the xanthine transporter mutant changes; in a reaction system containing M9 medium with a final xanthine concentration of 5-20 μg / mL, the transport efficiency of xanthine substrate reaches 83%-100%, and the maximum conversion rate of the substrate is increased by 110-120% compared to the wild type. This invention provides a new treatment method and direction for the treatment of hyperuricemia and various chronic diseases caused by high uric acid.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a xanthine transporter mutant and its applications. Background Technology

[0002] Xanthine is a product of the purine degradation pathway in the human body. Under the action of xanthine oxidase, it is further converted into uric acid, making it a direct precursor to uric acid in the body. Uric acid, as the end product of purine metabolism, cannot be further degraded in the human body. When the concentration of uric acid in the blood reaches or exceeds 6.8 mg / dL, there is a significant risk of developing hyperuricemia and an increased incidence of related diseases, including diabetes, cardiovascular disease, hypertension, and kidney disease. Current drugs for treating hyperuricemia mainly fall into three categories: those that inhibit uric acid production, those that promote uric acid dissolution, and those that increase uric acid excretion in the kidneys. Clinically used drugs for treating hyperuricemia include allopurinol and febuxostat, which target uric acid synthesis, or probenecid and benzbromarone, which promote uric acid excretion. Treatment with these drugs is often accompanied by liver and kidney damage and other toxic side effects.

[0003] Allopurinol drugs, which inhibit xanthine oxidase to lower uric acid, cause a large accumulation of xanthine in the kidneys, requiring the body to excrete large amounts of xanthine and placing a burden on the kidneys. Therefore, the level of xanthine in the body directly affects the level of high uric acid. Thus, the emergence of a new drug that can regulate high uric acid levels is crucial to solving the problems existing in current technologies. Summary of the Invention

[0004] The purpose of this invention is to provide a xanthine transporter mutant and its applications to address the problems existing in the prior art. Through molecular modification techniques, a mutant xanthine transporter was screened. Using xanthine as a substrate, this mutant exhibits significantly increased activity compared to the wild-type xanthine transporter, and can regulate the transport performance of the xanthine transporter, significantly reducing extracellular xanthine content. When this engineered bacterium was applied to a mouse model, it effectively reduced the xanthine content in the mouse intestine and the level of serum uric acid.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a xanthine transporter (XanQ) mutant (XanQ) m The amino acid sequence of the xanthine transporter mutant is shown in SEQ ID NO.6.

[0007] The present invention also provides a gene encoding the xanthine transporter mutant described above, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0008] The present invention also provides a recombinant vector containing the aforementioned gene.

[0009] The present invention also provides recombinant engineered bacteria comprising the recombinant vector described above. More preferably, recombinant engineered bacteria are constructed using E. coli Nissle1917 (EcN) as the host bacterium.

[0010] This invention can be constructed by linking the above-mentioned xanthine transporter mutant to other vectors using conventional methods in the art. The recombinant vector of this invention is not limited, as long as it can maintain replication ability in prokaryotic host cells. The vector can be any other conventional plasmid in the art, such as pET-28a-type inducible plasmids. The expression plasmid of this invention can be obtained by the following method: inserting the XanQ mutant gene product into the multiple cloning site region of the vector pWT-021a to construct the recombinant expression plasmid pWT021a-XanQ of this invention. m .

[0011] The present invention also provides the use of the xanthine transport mutant, the gene, the recombinant vector, or the recombinant engineered bacteria in the preparation of drugs that regulate the transport performance of xanthine transporter proteins.

[0012] Preferably, the regulation of xanthine transporter performance refers to the ability to efficiently transport extracellular xanthine into the cell.

[0013] Preferably, the method for efficiently transporting extracellular xanthine into the cell includes the following steps:

[0014] Live bacterial cells were obtained by culturing recombinant engineered bacteria containing the xanthine transporter mutant described above.

[0015] Using the live bacterial cells as transporters and extracellular xanthine as a substrate, a reaction system is constructed to carry out a transport reaction, so as to efficiently transport extracellular xanthine into the cell and reduce the extracellular xanthine content.

[0016] This invention involves the routine cultivation of recombinant engineered bacteria containing the XanQ mutant sequence to achieve the expression of the XanQ mutant. The culture medium used is any medium in the art that can enable the transformant to grow and produce the XanQ of this invention; preferably, M9 liquid medium is selected, the components of which are: sodium chloride 3.0 g / L, potassium dihydrogen phosphate 3.0 g / L, disodium hydrogen phosphate 6.0 g / L, ammonium sulfate 0.8 g / L, magnesium sulfate 0.25 g / L, glucose 7.20 g / L, calcium chloride 0.002 g / L, with ultrapure water as the solvent and pH 7.0. There are no special limitations on the cultivation method and conditions, as long as the transformant can grow and produce cells. More preferably, the recombinant engineered bacteria designed in this invention (such as recombinant EcN) are inoculated into M9 liquid medium containing 100 μg / mL streptomycin sulfate and cultured at 37°C until OD... 600A pH of 0.4-0.7 allows for the expression of the XanQ mutant protein of the present invention during bacterial growth without the need for additional inducers. More preferably, the live bacterial cells are resuspended in the M9 liquid medium and then mixed with the substrate to form a reaction system, which is then co-incubated at 37°C for 1 hour to complete the transport reaction.

[0017] Preferably, the final concentration of the extracellular xanthine is 5-20 μg / mL. The bacterial concentration in the system is OD0.05. 600 =0.6.

[0018] This invention also provides the use of the xanthine transporter mutant, the gene, the recombinant vector, or the recombinant engineered bacteria in any of the following:

[0019] (1) Application in the preparation of drugs that reduce xanthine content in animals or humans;

[0020] (2) Application in the preparation of drugs that reduce blood uric acid levels in animals or humans.

[0021] The present invention discloses the following technical effects:

[0022] This invention screened for the xanthine transporter (XanQ) from *Escherichia coli* str. K-12 substr. MG1655 and obtained a XanQ mutant through molecular modification. Experiments showed that using live recombinant engineered bacteria containing the XanQ mutant as the transporter and xanthine as the substrate, the extracellular xanthine content was significantly reduced in the M9 medium reaction system. This indicates that the xanthine transport activity of the obtained xanthine mutant changes, and mutation at a specific site can significantly promote xanthine transport activity. Furthermore, in the M9 medium reaction system, OD... 600 When adjusted to 0.6, under optimal co-incubation time, the transport efficiency for substrates of 5-20 μg / mL reaches 83%-100%, representing a 110-120% increase in maximum substrate transport rate compared to the wild type. In mice administered xanthine via gavage, the engineered bacteria significantly reduced fecal xanthine excretion within 1 hour, lowering it to control levels. In a mouse model of hyperuricemia, it effectively reduced serum uric acid levels to control levels. The XanQ mutant disclosed in this invention can regulate the transport performance of xanthine transporters, reducing extracellular xanthine content and lowering intestinal xanthine content in mice to levels comparable to the normal group. In hyperuricemic mice, it reduces serum uric acid levels to normal levels. This invention provides a new treatment method and direction for hyperuricemia and various chronic diseases caused by high uric acid. Attached Figure Description

[0023] Figure 1The results show the measurement of xanthine transport in the mouse intestine by the mutant recombinant bacteria;

[0024] Figure 2 The results show the determination of uric acid levels in mouse blood. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] The XanQ mutant xanthine transporter disclosed in this invention was developed using primers designed and synthesized targeting the original XanQ sequence. These primers were obtained from wild-type *Escherichia coli* (*Escherichia coli* str. K-12 substr. MG1655) and ligated into the pWT-021a plasmid. The plasmid was first transformed into *E. coli* DH5α for plasmid amplification, and then further transformed into EcN for gene expression. Site-directed mutagenesis was used to mutate the nucleotides of XanQ. The resulting amplified product was purified and first introduced into *E. coli* DH5α, ultimately expressed in EcN. Mutants with enhanced transport activity were screened by testing their transport activity, resulting in improved xanthine transport activity. These mutants were then applied to mice to achieve in vivo xanthine transport and ultimately reduce serum uric acid levels.

[0031] To further illustrate the implementation of the above scheme, a more detailed description is provided below using specific embodiments.

[0032] The following examples involve some of the culture media:

[0033] The final concentration of LB medium was: LB Broth 25 g / L, agar 3 g / L, ultrapure water as solvent, pH 7.0.

[0034] The final concentration of LB liquid medium was: LB Broth 25 g / L, solvent was ultrapure water, pH 7.0.

[0035] The final concentration of M9 medium consists of: sodium chloride 3.0 g / L, potassium dihydrogen phosphate 3.0 g / L, disodium hydrogen phosphate 6.0 g / L, ammonium sulfate 0.8 g / L, magnesium sulfate 0.25 g / L, glucose 7.20 g / L, calcium chloride 0.002 g / L, with ultrapure water as the solvent and a pH of 7.0.

[0036] Example 1: Construction of XanQ recombinant bacteria

[0037] From the NCBI database, the xanthine transporter (XanQ) gene (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2) from *Escherichia coli* str.K-12substr.MG1655 (NCBI number AAC75920.2) was selected. Primers were designed and synthesized from the wild-type *E. coli* genome, replacing the multiple cloning site (MCS) sequence of pWT-021a. The primers were then transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing a final concentration of 100 μg / mL streptomycin sulfate, and incubated overnight at 37°C. Positive clones were then picked, identified, and sequenced.

[0038] The verified positive monoclonal strain was inoculated into 5 mL of LB liquid medium containing 100 μg / mL streptomycin sulfate and cultured overnight at 37°C. After plasmid extraction, the recombinant expression vector was transformed into EcN competent cells to obtain the recombinant strain EcN / pWT021a-XanQ, which was the parent strain. This strain was plated on LB agar plates containing 100 μg / mL streptomycin sulfate and cultured overnight at 37°C. Positive clones were then picked and identified and sequenced. The verified positive monoclonal strain was inoculated into 5 mL of LB liquid medium containing 100 μg / mL streptomycin sulfate and cultured overnight at 37°C. Then, 50% glycerol was added at a 1:1 volume ratio and stored at -80°C for later use.

[0039] Table 1 Primers used for constructing the wild-type XanQ expression vector.

[0040]

[0041] SEQ ID NO.1:

[0042]

[0043] SEQ ID NO.2:

[0044] MSDINHAGSDLIFELEDRPPFHQALVGAITHLLAIFVPMVTPALIVGAALQLSAETTAYLVSMAMIASGIGTWLQVNRYGIVGSGLLSIQSVNFSFVTVMIALGSSMKSDGFHEELI MSSLLGVSFVGAFLVVGSSFILPYLRRVITPTVSGIVVLMIGLSLIKVGIIDFGGGFAAKSSGTFGNYEHLGVGLLVLIVVIGFNCCRSPLLRMGGIAIGLCVGYIASLCLGMVDFS SMRNLPLITIPHPFKYGFSFSFHQFLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAVGSLPLTTFAQNNGVIQMTGVASRYVGRTIAVMLVILG LFPMIGGFFTTIPSAVLGGAMTLMFSMIAIAGIRIIITNGLKRRETLIVATSLGLGLGVSYDPEIFKILPASIYVLVENPICAGGLTAILLNIILPGGYRQENVLPGITSAEEMD*.

[0045] Example 2: Obtaining the XanQ mutant and constructing recombinant bacteria containing the mutant

[0046] 1. Construction of mutant libraries

[0047] Using the recombinant expression vector pWT021a-XanQ obtained in Example 1 as a template, the mutant sequence was obtained through site-directed mutagenesis, and primers for site-directed saturation mutagenesis of phenylalanine at position 94 were designed (see Table 2).

[0048] Table 2 shows the primers used for the site-directed saturation mutagenesis of phenylalanine at position 94.

[0049]

[0050] The amplification system was as follows: 50 μL reaction system: 25 μL 2×Taq Master Mix, 20 μL ddH2O, 1 μL each of forward and reverse primers, 1 μL DNA template, 1 μL high-fidelity enzyme and 1 μL dNTP.

[0051] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 5 min, followed by temperature cycling at 95℃ for 20 s, 56℃ for 20 s, and extension at 72℃ for 30 s at 1 kb intervals, for a total of 34 cycles. The final temperature was 72℃ for 10 min, with a termination temperature of 10℃. PCR products were subjected to 1.5% agarose gel electrophoresis, and after UV analysis to determine band size, the gel was excised and recovered. DNA was quantified using a microplate reader. The recombinant reaction system consisted of 12 μL: 6 μL homologous recombinase, 5 μL target gene, and 1 μL plasmid backbone. The reaction was carried out at 50℃ for 30 min and kept on ice. 10 μL of the recombinant plasmid was added to DH5α competent cells, incubated on ice for 30 min, then heat-shocked in a 42℃ water bath for 90 s, and placed on ice for 2-3 min. 600 μL of sterile LB broth was added to EP tubes in a clean bench and activated at 37℃ and 220 rpm for 45 min. The activated bacterial culture was evenly spread on LB agar plates containing 100 μg / mL streptomycin sulfate and incubated overnight at 37°C to obtain a mutant library at the XanQ94 site.

[0052] After screening for single-point positive mutants, the following method was used: using the plasmid of the strain with the best transport efficiency as the amplification template, amplification primers for the mutant were designed as shown in Table 3, and the mutant gene was amplified by PCR. The mutant gene was then transformed into *E. coli* DH5α competent cells. After correct sequencing, the plasmid was extracted, and the recombinant expression vector was transformed into *EcN* competent cells, ultimately obtaining the double-mutant recombinant strain with the best xanthine transport efficiency.

[0053] Table 3 Primers used to amplify mutants

[0054]

[0055]

[0056]

[0057] 2. Screening for positive mutants

[0058] Add 200 μL of LB liquid medium containing 100 μg / mL streptomycin sulfate to each well of a 96-well plate. Pick single clones from the XanQ94 mutant library and inoculate them into the 96-well plates. Incubate at 37°C for 3 hours for initial activation. Then, inoculate 1% of the medium into each well of a 24-well plate containing 800 μL of M9 medium containing 100 μg / mL streptomycin sulfate. Incubate at 37°C for 12–14 hours. Centrifuge, collect the cells, and resuspend them in M9 medium on OD. 600 They were in agreement to conduct research on the transport of extracellular xanthine.

[0059] The reaction system was 1 mL, containing xanthine as the substrate at a final concentration of 15 μg / mL, and the final concentration OD... 600Bacterial cells with a pH of 0.6 were cultured in M9 medium at pH 7.0-7.2 and incubated at 37°C for 30 min. The supernatant was collected by centrifugation and filtered through a 0.22 μm microporous membrane. The filtrate was further subjected to high-performance liquid chromatography (HPLC) to determine the extracellular substrate transport efficiency. Results for some mutants are shown in Table 4. Positive clones with improved substrate transport efficiency were screened and sent to a sequencing company for sequencing. Sequencing analysis revealed the optimal single mutant to be F94Y (mutating phenylalanine at amino acid position 94 of SEQ ID NO. 2 to tyrosine), with its amino acid sequence shown in SEQ ID NO. 4 and nucleotide sequence shown in SEQ ID NO. 3. This is the recombinant strain EcN / pWT021a-XanQ. F94Y .

[0060] SEQ ID NO.3:

[0061]

[0062] SEQ ID NO.4:

[0063] MSDINHAGSDLIFELEDRPPFHQALVGAITHLLAIFVPMVTPALIVGAALQLSAETTAYLVSMAMIASGIGTWLQVNRYGIVGSGLLSIQSVNYSFVTVMIALGSSMKSDGFHEELI MSSLLGVSFVGAFLVVGSSFILPYLRRVITPTVSGIVVLMIGLSLIKVGIIDFGGGFAAKSSGTFGNYEHLGVGLLVLIVVIGFNCCRSPLLRMGGIAIGLCVGYIASLCLGMVDFS SMRNLPLITIPHPFKYGFSFSFHQFLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAVGSLPLTTFAQNNGVIQMTGVASRYVGRTIAVMLVILG LFPMIGGFFTTIPSAVLGGAMTLMFSMIAIAGIRIIITNGLKRRETLIVATSLGLGLGVSYDPEIFKILPASIYVLVENPICAGGLTAILLNIILPGGYRQENVLPGITSAEEMD*.

[0064] The screening method for the optimal double mutant strain was as described above. Some double mutant results are shown in Table 5. The optimal double mutant obtained was F94YS88T (i.e., mutating phenylalanine at position 94 of the amino acid sequence of SEQ ID NO.2 to tyrosine, and simultaneously mutating serine at position 88 to threonine). Its amino acid sequence is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.5, which is the recombinant strain EcN / pWT021a-XanQ. F94YS88T .

[0065] SEQ ID NO.5:

[0066] ATGTCTGATATAAACCATGCAGGTTCTGACCTTATATTTGAACTGGAGGATCGCCCTCCCTTTCATCAGGCTCTCGTTGGTGCCATTACCCATCTGTTGGCAATTTTCGTTCCGATGGTAACCCCCGCGTTAATCGTGGGTGCGGCCTTACAGCTTTCCGCTGAAACAACTGCCTATCTTGTTTCTATGGCGATGATCGCCTCTGGTATTGGTACCTGGTTACAAGTAAACCGCTACGGCATCGTCGGTTCTGGCCTACTCaCAATTCAGTCAGTCAATtatTCATTTGTTACGGTCATGATTGCGCTGGGCAGCAGCATGAAAAGCGACGGTTTTCACGAAGAGTTAATCATGTCGTCGCTTCTCGGCGTCTCCTTCGTTGGCGCATTTCTGGTTGTCGGATCTTCATTTATCTTGCCCTATTTACGTCGGGTTATTACGCCTACCGTCAGCGGTATTGTGGTACTGATGATCGGCTTAAGCCTGATTAAAGTCGGCATTATCGATTTTGGTGGAGGATTTGCAGCCAAAAGCAGCGGTACGTTCGGCAATTACGAACATCTCGGCGTTGGTTTATTGGTTTTAATTGTGGTGATCGGCTTTAACTGCTGTCGCAGTCCGTTGCTACGCATGGGAGGGATCGCCATTGGGCTATGTGTCGGCTATATCGCATCGTTATGCCTGGGCATGGTGGATTTCAGCAGTATGCGCAATTTGCCGTTAATCACCATCCCGCATCCGTTCAAATACGGCTTTAGTTTTAGCTTCCATCAGTTCCTGGTGGTTGGCACGATTTATCTGCTTAGCGTGCTGGAAGCAGTCGGCGATATCACCGCCACGGCAATGGTTTCCCGCCGCCCCATTCAGGGGGAAGAGTATCAGTCCCGGCTGAAAGGCGGCGTGCTGGCAGATGGTCTGGTTTCTGTTATCGCCTCCGCTGTCGGTTCATTACCATTAACCACGTTTGCGCAAAATAATGGGGTTATTCAGATGACTGGCGTCGCTTCACGTTATGTCGGGCGAACCATCGCGGTAATGCTGGTTATCCTCGGCTTATTTCCGATGATTGGCGGCTTCTTCACGACCATTCCCTCGGCAGTTCTGGGAGGCGCAATGACGTTGATGTTTTCCATGATTGCCATCGCAGGGATTCGCATCATCATCACCAACGGTTTAAAGCGCCGTGAAACACTTATTGTCGCCACTTCTTTAGGTTTAGGGCTTGGCGTCTCCTACGATCCCGAAATTTTTAAAATATTGCCAGCCTCTATTTATGTATTAGTTGAAAACCCTATTTGTGCTGGCGGGTTAACTGCGATTTTATTAAATATTATCCTCCCTGGTGGCTACCGACAGGAAAACGTTCTGCCTGGTATTACCTCAGCGGAAGAGATGGATTAA.

[0067] SEQ ID NO.6:

[0068] MSDINHAGSDLIFELEDRPPFHQALVGAITHLLAIFVPMVTPALIVGAALQLSAETTAYLVSMAMIASGIGTWLQVNRYGIVGSGLLTIQSVNYSFVTVMIALGSSMKSDGFHEELI MSSLLGVSFVGAFLVVGSSFILPYLRRVITPTVSGIVVLMIGLSLIKVGIIDFGGGFAAKSSGTFGNYEHLGVGLLVLIVVIGFNCCRSPLLRMGGIAIGLCVGYIASLCLGMVDFS SMRNLPLITIPHPFKYGFSFSFHQFLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAVGSLPLTTFAQNNGVIQMTGVASRYVGRTIAVMLVILG LFPMIGGFFTTIPSAVLGGAMTLMFSMIAIAGIRIIITNGLKRRETLIVATSLGLGLGVSYDPEIFKILPASIYVLVENPICAGGLTAILLNIILPGGYRQENVLPGITSAEEMD*.

[0069] The high-performance liquid chromatograph used was a Shimadzu LC-2030, and the chromatographic detection conditions were as follows:

[0070] Chromatographic column: Diamonsil Plus 5μm C18-A, 250×4.6mm; mobile phase: 50mmol / L ammonium acetate buffer, pH 4.60, with a ratio of 95:5 (v / v) to acetonitrile; flow rate: 1min / min; wavelength: 254nm; column temperature: 30℃; injection volume: 10μL; retention time: 10min.

[0071] Xanthine standards at concentrations of 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, and 30 μg / mL were prepared and detected by HPLC. Standard curves were established based on the relationship between peak area and concentration of the xanthine standards at different concentrations. The xanthine transport capacity of the strain is represented by B, calculated using the following formula:

[0072]

[0073] Where b1 is the initial xanthine concentration and b2 is the remaining xanthine concentration in the supernatant.

[0074] Table 4. Translocation rates of XanQ and its 94-position mutants

[0075]

[0076] Table 5. Translocation ratios of XanQ double mutations

[0077]

[0078]

[0079] Example 3: Preparation of XanQ wild-type and mutant recombinant bacteria

[0080] 1) Plate culture

[0081] EcN was inoculated into LB medium containing streptomycin sulfate at a final concentration of 100 μg / mL and incubated at 37°C for 12 h for plate activation;

[0082] 2) Liquid activation

[0083] The bacterial culture plates were inoculated into LB liquid medium containing streptomycin sulfate at a final concentration of 100 μg / mL and incubated at 37°C for 8-10 h for liquid activation.

[0084] 3) Growth and Culture

[0085] The liquid-activated bacterial suspension was inoculated at a volume concentration of 1% into M9 liquid medium containing a final concentration of 100 μg / mL streptomycin sulfate, and incubated at 37°C for 12-14 hours until OD500 was reached. 600 The value is 0.4-0.7, and after centrifugation, the solution is resuspended to OD using an M9 syringe. 600 The values ​​are consistent.

[0086] Example 4: XanQ wild-type and mutant recombinant bacteria transport extracellular xanthine to reduce xanthine content in the reaction system

[0087] This embodiment optimizes the conditions for reducing extracellular xanthine content in the M9 system by using recombinant EcN live bacteria containing the XanQ mutant as a transporter and xanthine as a substrate, as follows:

[0088] 1) Add xanthine as substrate to a 1 mL reaction system at a final concentration of 5 μg / mL and a final concentration of OD... 600 The bacterial cells had a value of 0.6. The reaction system was incubated at 37°C for 30 min. After centrifugation and removal of the supernatant, the residual xanthine content in the supernatant was determined using high-performance liquid chromatography (same as Example 2). The xanthine transport efficiency of wild-type XanQ was 100%; the mutant XanQ... F94YS88T The transport efficiency for xanthine is 100%.

[0089] 2) To a 1 mL reaction system (i.e., the wild-type or mutant recombinant bacteria prepared in Example 3), add xanthine as substrate at a final concentration of 15 μg / mL and a final concentration of OD... 600 The bacterial cells had a value of 0.6. The reaction system was incubated at 37°C for 30 min, and the supernatant was removed by centrifugation. The residual xanthine content in the supernatant was determined using high-performance liquid chromatography (same as in Example 2). The xanthine transport efficiency of wild-type XanQ was 33-35%; the mutant XanQ... F94YS88T The transport efficiency for xanthine is 64-72%.

[0090] 3) Add xanthine as substrate to a 1 mL reaction system, with a final concentration of 15 μg / mL and a final concentration of OD. 600 The bacterial cells had a value of 0.6. The reaction system was incubated at 37°C for 1 hour. After centrifugation and removal of the supernatant, the residual xanthine content in the supernatant was determined using high-performance liquid chromatography (same as Example 2). The xanthine transport efficiency of wild-type XanQ was 40-42%; the mutant XanQ... F94YS88T The xanthine transport efficiency is 80-83%. The transport efficiency of the double mutant is no longer significantly improved, which may be due to the detection limit of the detection method. The xanthine concentration co-incubation experiment can be improved.

[0091] 4) Add xanthine as substrate to a 1 mL reaction system, with a final concentration of 20 μg / mL and a final concentration of OD. 600 Bacterial cells with a value of 1. The reaction system was reacted at 37°C for 1 hour, and the supernatant was removed by centrifugation. The residual xanthine content in the supernatant was detected by high performance liquid chromatography (same as in Example 2). The xanthine transport efficiency of wild-type XanQ was 43-45%; mutant XanQ F94YS88T The transport efficiency for xanthine is 93-95%.

[0092] Example 5: Xanthine transport capacity of engineered bacteria in mouse intestine

[0093] To determine the ability of the mutant recombinant bacteria to transport xanthine in vivo, three groups of mice were set up for experiments. The negative and positive control groups were administered 200 μL of PBS by gavage, while the experimental groups were administered 200 μL of 1.0 × 10⁻⁶ PBS by gavage. 10CFU / mL mutant recombinant bacteria were administered. After 30 minutes, the negative control group was administered 200 μL PBS by gavage, while the positive control and experimental groups were administered 200 μL 20 mM xanthine by gavage. Feces from each group were collected 30 and 60 minutes after the second gavage, and the weight of each feces was measured. 1 mL PBS and grinding beads were added to a grinding tube, and the tube was ground. After grinding, the supernatant was collected by centrifugation at 6000 rpm for 2 minutes and filtered through a 0.22 μm aqueous filter membrane. The filtrate was further analyzed by HPLC as described in Example 2 to determine the xanthine content. The xanthine content per gram of feces was measured, and the results are as follows: Figure 1 As shown.

[0094] Liquid chromatography analysis revealed no significant difference in xanthine content in the feces of mice in the negative control group, positive control group, and experimental group 30 minutes after gavage. However, xanthine was detected in the feces of mice 60 minutes after gavage. The xanthine content in the negative control group showed no significant change compared to 30 minutes after gavage, indicating that the level of metabolized xanthine in mice remained at a certain level. In contrast, the xanthine concentration in the feces of the positive control group was significantly different from that of the negative control group, suggesting that a large amount of xanthine was excreted in the feces 60 minutes after gavage. The xanthine content in the feces of the experimental group was not significantly different from that of the negative control group and was significantly lower than that of the positive control group, indicating a significant difference between the two groups. This suggests that the mutant recombinant bacteria effectively transported xanthine in the intestine, even efficiently transporting it to the normal level of xanthine in the mouse intestine.

[0095] Example 6: Engineered bacteria reduce serum uric acid levels in a mouse model of hyperuricemia.

[0096] A mouse model of hyperuricemia was established using the following method: potassium oxonate 600 mg / kg + xanthine 600 mg / kg was administered by gavage for 14 consecutive days. Serum collection was performed as follows: on the last day before administration, mice were fasted overnight but allowed free access to water. One hour after administration, blood was collected by enucleation. The collected whole blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 4°C and 3000 rpm for 20 minutes. The collected serum was then gently removed and analyzed using a uric acid assay kit.

[0097] Five groups of mice were set up in the experiment. The negative control group and the model group were given 200 μL of PBS by gavage. The positive control group was given 5 mg / kg of allopurinol by gavage. Experimental group 1 was given 200 μL of empty vector bacteria by gavage. Experimental group 2 was given 200 μL of mutant bacteria by gavage. 30 min after the first gavage, the negative control group was given 200 μL of PBS by gavage. The model group, experimental group 1, experimental group 2 and positive control group were subjected to drug modeling as shown above.

[0098] On the last day before administration, mice were fasted overnight but allowed free access to water. One hour after administration, blood was collected by enucleation. The collected whole blood was left to stand at room temperature for 30 minutes, then centrifuged at 3000 rpm for 20 minutes at 4°C. The blood was then gently removed, and the serum was collected and the uric acid concentration was determined using a uric acid assay kit.

[0099] like Figure 2 As shown in the figure, the results of the mouse serum uric acid level indicate that after 14 days of drug modeling, the serum uric acid level of the model group mice was more than twice that of the negative control group, indicating that this method can successfully establish a mouse hyperuricemia model within 14 days. Although the serum uric acid level of mice in experimental group 1 was slightly lower than that of the model group, it did not constitute a significant difference. This indicates that EcN itself has a certain xanthine transport capacity, but it is insufficient to balance the purine metabolism abnormalities caused by the drug. In experimental group 2, the serum uric acid level of mice decreased to a level that was significantly different from that of the model group and not significantly different from that of the control group. This indicates that the early administration of the mutant bacteria during the mouse modeling process can play a significant role in the transport of xanthine during metabolism, greatly reducing the precursor of uric acid in the intestine, thereby reducing the serum uric acid level of mice. The positive control group was given allopurinol, a clinical drug for treating hyperuricemia, during the same period. As can be seen from the results in the figure, allopurinol also had a good therapeutic effect in the mouse hyperuricemia model.

[0100] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of a recombinant engineered bacterium in the preparation of a medicament for reducing blood uric acid levels in an animal or human body, characterized in that, The recombinant engineered bacteria contains a recombinant vector, the recombinant vector containing a gene encoding a xanthine transporter mutant, the amino acid sequence of the xanthine transporter mutant is shown in SEQ ID NO.6, and the nucleotide sequence of the gene is shown in SEQ ID NO.5; The recombinant engineered bacteria have the ability to efficiently transport extracellular xanthine into the cell.

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