A humanized human-pig chimeric uricase chimeric mutant and its application

Abstract

The present invention discloses a humanized human-porcine chimeric uricase chimeric mutant. The amino acid sequence of the mutant is shown in SEQ ID NO. 1. The mutant has excellent uric acid level reduction and high homology with human uricase, providing a new option for drug treatment of hyperuricemia.

Description

Technical Field

[0001] The invention belongs to the field of medical biology and biopharmaceutical technology, and particularly relates to a humanized human-pig chimeric uricase chimeric mutant and an application thereof. Background Art

[0002] Uricase (uricase; EC 1.7.3.3; Uox) is an important enzyme in the purine metabolic pathway, which catalyzes uric acid to produce allantoin, which is easily soluble in water and easily excreted by the kidneys. During the evolutionary process, the human uricase gene was inactivated due to the continuous accumulation of nonsense mutations and missense mutations, resulting in uric acid becoming the final product of purine metabolism in the human body. When the uric acid concentration in the human body exceeds the threshold of human kidney excretion, it will lead to hyperuricemia. Long-term hyperuricemia can cause a series of complications such as gout, such as hypertension, stroke, chronic kidney disease, etc. Therefore, more and more medical experts have begun to study the treatment of hyperuricemia.

[0003] So far, uricosuric drugs, uric acid synthesis inhibitors and recombinant uricase (Uox) preparations are the three types of drugs routinely used in clinical treatment of hyperuricemia. However, uricosuric drugs and uric acid synthesis inhibitors have certain limitations in clinical treatment. They cannot cure patients with tophi and gout, and long-term use of these drugs can cause patients to suffer from a series of adverse reactions such as liver and kidney damage. There are currently two types of recombinant uricase preparations on the market, including (A method of recombinantly expressing the Aspergillus flavus uricase gene by cloning the cDNA of the Aspergillus flavus uricase gene in the Saccharomyces cerevisiae expression system) and (A PEG-modified recombinant pig-baboon chimeric uricase), due to its excellent ability to reduce uric acid levels and dissolve tophi, it has shown good therapeutic prospects for patients with hyperuricemia. However, the immunogenicity of recombinant protein drugs still limits the development of therapeutic uricase drugs. Methods for restoring the biological activity of human uricase have been proven to be difficult, so the best way to reduce the immunogenicity of uricase is to humanize the amino acid sequence of functionally active uricase. The simplest method at present is to humanize a uricase based on a functionally active mammalian uricase, such as several chimeric uricases that have been reported, such as pig-human (PHC) and dog-human (CHC) chimeric uricases, which have high homology and biological activity with the amino acid sequence of human uricase. Now, constructing a therapeutic chimeric uricase with a high homology to the amino acid sequence of human uricase has a very bright application prospect in the clinical treatment of hyperuricemia. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a humanized human-porcine chimeric uricase chimeric mutant.

[0005] Another object of the present invention is to provide an application of the above-mentioned humanized human-porcine chimeric uricase chimeric mutant.

[0006] The purpose of the present invention is achieved by the following technical solution: A humanized human-porcine chimeric uricase chimeric mutant, the amino acid sequence of which is as follows:

[0007]

[0008] The nucleic acid molecule encoding the above humanized human-porcine chimeric uricase chimeric mutant is preferably as follows:

[0009]

[0010] The humanized human-porcine chimeric uricase mutant can be obtained by chemical preparation, or by cloning a nucleic acid molecule encoding the humanized human-porcine chimeric uricase mutant into an expression vector, and then expressing and purifying it.

[0011] The expression vector includes a prokaryotic vector and a eukaryotic vector.

[0012] The eukaryotic vector is preferably pMal-c4x.

[0013] The use of the humanized human-porcine chimeric uricase chimeric mutant in the preparation of a drug for treating hyperuricemia.

[0014] The use of the above nucleic acid molecule in the preparation of a drug for treating hyperuricemia preferably comprises the following steps: cloning the above nucleic acid molecule into an expression vector, expressing and purifying to obtain a humanized human-porcine chimeric uricase chimeric mutant; or cloning the above nucleic acid molecule downstream of a eukaryotic promoter to obtain a nucleic acid fragment that can express uricase in the human body.

[0015] Compared with the prior art, the present invention has the following advantages and effects:

[0016] The human-porcine chimeric mutant uricase provided by the present invention has excellent ability to reduce uric acid levels and is highly homologous to human uricase, thus providing a new option for drug treatment of hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the construction of human-pig chimeric uricase.

[0018] Figure 2 This is a comparison of the amino acid sequences of uricases from different species.

[0019] Figure 3 It is a solubility analysis result diagram of each chimeric uricase mutant engineered bacteria; wherein, lane M is a protein marker, lane C is uninduced whole bacteria, lane W is induced whole bacteria, lane s is the broken supernatant, and lane P is the broken precipitate.

[0020] Figure 4 It is the SDS-PAGE diagram of each fusion protein uricase purified by MBP column and Ni-NTA column; wherein, lane M is a protein marker, lane Load is the supernatant of Ni-NTA column purification after enzyme cleavage, lane FT is the flow-through of Ni-NTA column purification, lane 10mM Maltose is the product obtained by elution with maltose, and lane 300mM Imidazole is the product obtained by elution with 300mM imidazole.

[0021] Figure 5 It is an SDS-PAGE image of the active uricase purified after SUMO protease cleavage; wherein, lane Pre-zymedigestion is the fusion protein before cleavage, lane Load is the supernatant of Ni-NTA column purification after cleavage, lane FT is the flow-through of Ni-NTA column purification, and lane Imidazole is 50mM imidazole eluent, 100mM imidazole eluent, and 300mM imidazole eluent, respectively.

[0022] Figure 6 This is a graph showing the change in substrate OD value of the reaction between fusion protein uricase and uric acid over time. DETAILED DESCRIPTION

[0023] The present invention will be described in further detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0024] Since porcine uricase (wPU) has the highest activity among mammalian functional uricases and its amino acid sequence has a high degree of sequence homology (87.5%) with human uricase (dHU), the amino acid sequence of wPU is most suitable for humanization. Both the wild-type porcine uricase (wPU) and human uricase (dHU) sequences contain 8 exons. In addition, during the evolution of uricase, nonsense and missense mutations mainly occur in exons 1-6. At the same time, literature has shown that exons 5 or 6 are essential for the functional activity of uricase. When exons 5 or 6 of wPU are replaced with the corresponding dHU exons, the enzyme activity is completely lost.

[0025] In this study, we retained exons 5-6 of wPU and replaced exons 1-4 and 7-8 with the corresponding exons of dHU to construct H 1-4 P 5-6 H 7-8The human-pig chimeric uricase has a homology of up to 94.41%, but we found through functional activity verification that this chimera does not have functional activity. So we used homologous multiple sequence alignment technology to search for amino acid residues that have a potential effect on uricase activity in exons 1-4. We found that there are 5 functional amino acids that may have a great impact on the functional activity of the enzyme and 5 functional amino acids that have a greater impact on the enzyme activity. They were all mutated into amino acid residues corresponding to wPU through site-directed mutagenesis technology. Finally, through functional activity verification, a human-pig chimeric mutant uricase with excellent uric acid level reduction and high homology was obtained, named mutant 7, which provides an option for future drug treatment of hyperuricemia. See the embodiment for the specific process.

[0026] The sources of reagents and materials used are as follows:

[0027] The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0028] 10×Fastdigest Green Buffer, endonuclease BamHI, and endonuclease Hind III were from ThermoFisher Scientific, with catalog numbers 00322002, ER0051, and 00276838, respectively.

[0029] PrimerSTAR Mix and 10× loading buffer were purchased from Takara with catalog numbers AL52849A and AK71253A, respectively.

[0030] Goldview comes from biosharp, product number BS357A.

[0031] The PCR product recovery kit and plasmid small-scale extraction kit were from Magen, with the catalog numbers DJC30-01 and PJI08-01, respectively.

[0032] T4 DNA Ligase (T4 DNA ligase) and 10×T4 DNA Ligase Buffer are from NEW ENGLANDBioLabs, with catalog numbers 1181608 and 10115407 respectively.

[0033] Example 1: Construction of human-porcine uricase chimera

[0034] (1) During the evolution of uricase, nonsense mutations and missense mutations mainly occurred in exons 1-6. At the same time, studies have shown that when exons 5 or 6 of wPU are replaced with the corresponding human uricase exons, the functional activity of the chimera is completely lost, indicating that exons 5 and 6 are essential for the functional activity of the enzyme. Therefore, we retained exons 5 and 6 in wPU and constructed a highly humanized human porcine uricase H 1-4 P5-6H7-8 chimera, such as Figure 1 Human-pig chimeric uricase H 1-4 P 5-6 H 7-8 The homology with dHU amino acid sequence is 94.41%. 1-4 P 5-6 H 7-8 Chimeras are not biologically active.

[0035] (2) In order to restore human porcine uricase H 1-4 P 5-6 H 7-8 We investigated the functional activity of chimeras, human-pig chimeric uricase H 1-4 P 5-6 H 7-8 The amino acid residues in exons 1-4 of the enzyme that are unfavorable for the functional activity of the enzyme. 1-4 P 5-6 H 7-8 The amino acid sequences of chimeras and human uricase (dHU), three primate functional uricases (baboon, rhesus monkey and crab-eating monkey), four mammal functional uricases (pig (wPU), dog (Canine), cattle (bovin) and sheep (Sheep)) and three reported functional uricase chimeras (pig-human (PHC), pig-baboon (PBC) and dog-human (CHC)) were aligned using Cluster Omega. The results of the multiple sequence alignment highlighted the inconsistent amino acid residues in the exon 1-4 sequences of the inactive uricase and the functionally active uricase. Figure 2 As shown in the figure, we were surprised to find that in exons 1-4, there were 5 different amino acid residues between non-functional uricase and functional uricase, which were located at the 24th, 89th, 112th, 119th and 121st amino acid residues. Therefore, we speculated that these five amino acid residues might be the cause of human porcine uricase H 1-4 P 5-6 H 7-8The reason for the loss of biological activity of the chimera. During the evolution of uricase, the primate uricase is inferior to the mammalian uricase in catalyzing uric acid. In addition, we further found that at the 7th, 89th, 91st, 115th, and 120th amino acid residues, the primate and non-functional human porcine uricase H 1-4 P 5-6 H 7-8 The chimera has the same amino acid residues as human uricase (dHU), but different from the corresponding amino acid residues of functional uricase. We speculate that the amino acid residues at these five sites may be the cause of the low functional activity of primate uricase, which may also affect H 1-4 P 5- 6H 7-8 Functional activity of chimeric uricase. Finally, we used site-directed mutagenesis to mutate the following 10 amino acid residues and constructed 7 mutants, as shown in Table 1. Finally, through functional activity verification, we observed whether these mutants could restore human porcine uricase H after site-directed mutagenesis. 1-4 P 5-6 H 7-8 Biological activity of the chimeras.

[0036] (3) H 1-4 P 5-6 H 7-8 The sequence was connected to the lytic tag SUMO sequence (positions 131-424 of MH394329.1), and then constructed on the pMal-c4x plasmid with a maltose binding protein MBP tag and commissioned to Sangon Biotech (Shanghai) Co., Ltd. (Sangon Biotech) for synthesis; the cDNA sequences of dHU, wPU and PBC were delivered to Sangon Biotech for synthesis according to the same construction method as a control group; among them, the target sequence was cloned into the expression vector using BamHI and HindIII.

[0037] SUMO-H 1-4 P 5-6 H 7-8 The sequence is shown below, where the underlined part is the SUMO sequence (1-294), H 1-4 P 5- 6H 7-8 6×His at the C-terminus:

[0038]

[0039] The sequence of dHU is shown below. When constructed into the pMal-c4x expression vector, it is the same as SUMO-H 1-4 P 5-6 H 7-8 As shown, a SUMO sequence is connected to the 5′ end and a 6×His is connected to the 3′ end:

[0040]

[0041]

[0042] The sequence of wPU (positions 11-925 of NM-214270) is shown below. When constructed into the pMal-c4x expression vector, it was used in the same manner as SUMO-H 1-4 P 5-6 H 7-8 As shown, a SUMO sequence is connected to the 5′ end and a 6×His is connected to the 3′ end:

[0043]

[0044] The sequence of PBC is shown below. When constructed into the pMal-c4x expression vector, it is the same as SUMO-H 1-4 P 5-6 H 7-8 As shown, a SUMO sequence is connected to the 5′ end and a 6×His is connected to the 3′ end:

[0045]

[0046] Example 2: Construction of chimeric mutants of human porcine uricase

[0047] (1) According to H 1-4 P 5-6 H 7-8 Based on the chimeric nucleotide sequence, site-directed mutagenesis was performed using overlapping PCR, and the constructed mutants are shown in Table 1.

[0048] Table 1 The mutation positions of amino acids and nucleotides are counted only from uricase itself

[0049]

[0050] Note: Mutant 1 is the Chimeric mutant 1 in the figure of the specification, and the same applies to other mutants.

[0051] Nucleotide sequence of mutant 6 (including C-terminal 6×His):

[0052]

[0053] Nucleotide sequence of mutant 7 (including C-terminal 6×His):

[0054]

[0055] The required primer sequences are shown in Table 2:

[0056] Table 2

[0057]

[0058] Note: GGATCC is the BamHI restriction site, AAGCTT is the HindIII restriction site, and CM is the abbreviation for Chimeric mutant.

[0059] (2) Overlapping PCR: mutant 1 was used as a sample to illustrate the preparation process of the mutant. pMal-c4x-SUMO-H was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. 1-4 P 5-6 H 7-8 Plasmid was used as template, and four primers CM-1-F1, CM-1-F2, CM-1-R1 and CM-1-R2 were used to construct the linear fragment of mutant 1 by overlapping PCR using a gene amplification instrument (purchased from Hangzhou Langji Scientific Instrument Co., Ltd., A100), with restriction sites of BamH I (N-terminus) and Hind III (C-terminus), respectively.

[0060] PCR reaction system: upstream primer (10 pM) 2 μL, downstream primer (10 pM) 2 μL, template DNA 2 μL, PrimerSTAR Mix 25 μL, ddH2O 19 μL.

[0061] PCR reaction conditions: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 3 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 32-35 cycles; final extension at 72°C for 10 min; insulation at 4°C∞.

[0062] (3) Agarose gel electrophoresis of target gene: Weigh 0.30g agarose and dissolve it in 30mL 1×TAE (Tris-base, Na2EDTA·2H2O and glacial acetic acid) solvent, heat in a microwave oven for 2min, cool to 60℃, add 2μL Goldview, mix well and add to the gel mold, prepare 1% agarose gel for electrophoresis. Take 50μL of the prepared sample, add 5μL 10× loading buffer, mix well and add to the agarose gel injection hole, stop electrophoresis after 120V voltage for 30min. Place the agarose gel in a UV instrument, turn on the UV lamp and observe clear DNA bands at the target position. Use a knife to cut out the target band from the agarose gel and recover the target fragment by gel.

[0063] (4) Recovering the target gene from agarose gel: Use a gel DNA mini-recovery kit (Magen, DJK03-01) to recover and extract the target gene to obtain the mutant 1 gene fragment.

[0064] (5) Double restriction enzyme digestion reaction of vector and target gene: BamH I and Hind III endonucleases were used to digest the pMal-c4x vector and mutant 1 gene fragment. The reaction system and conditions were as follows:

[0065] 10×Fastdigest Green Buffer 2μL, mutant 1 gene fragment or pMal-c4x 1μg, BamHI 1μL, HindIII 1μL, ddH2O make up to 20μL.

[0066] Incubate in a 37°C water bath for 1 h.

[0067] (6) Purification of vector and target gene after enzyme digestion: Use PCR product recovery kit for purification after enzyme digestion.

[0068] (7) Enzymatic ligation of vector and target gene: Use T4 DNA ligase to ligate the pMal-c4x vector and the mutant 1 gene fragment.

[0069] The reaction system conditions are as follows:

[0070] pMal-c4x 10ng after restriction digestion, mutant 1 gene fragment 80ng after restriction digestion, T4 DNA Ligase 1μL, 10×T4 DNA Ligase Buffer 2μL, dd H2O to make up to 20μL.

[0071] Incubate the enzyme in a 37°C water bath for 1 h.

[0072] (8) Transformation of mutant 1 recombinant plasmid: 10 μL of the above T4 ligase ligation product was mixed with 50 μL of DH5 α competent E. coli (Beijing Qingke Biotechnology Co., Ltd., 1DA22347) bacterial solution and then placed on ice for 30 min. Then, the mixture was quickly transferred to ice and placed in a 42°C water bath for 45 s and then placed on ice for 2 min. 600 μL of LB medium (purchased from Huankai Microbiology Technology Co., Ltd.) was added and cultured in a shaker at 37°C and 220 rpm for 1 h. Next, 50 μL of the cultured bacterial solution was spread on an LB solid medium plate with a final concentration of 50 μg / mL ampicillin Amp (Solarbio, 429V031) resistance. The solid plate was placed upright in a 37°C incubator for 30 min and then inverted for overnight culture. The next day, 5 single clones were picked from the plate with a toothpick and placed in the PCR reaction system. Colony PCR was performed using the above CM-1-F1 and CM-1-R2 primers. After PCR is completed, electrophoresis is performed using the same agarose gel electrophoresis method as above to observe whether there is a positive band at the target position.

[0073] The colony PCR reaction system was as follows: 1 μL CM-1-F1 (10 pM), 1 μL CM-1-R2 (10 pM), one single clone, 5 μL PrimerSTAR Mix, and 3 μL ddH2O.

[0074] Positive transformants were picked and placed in 10 mL LB medium containing 50 μg / mL Amp, and cultured overnight at 37°C and 220 rpm in a shaker (ISF-4-W, AdolfKuhner AG (Switzerland)). The next day, plasmids were extracted using a plasmid mini-extraction kit, and sequencing was commissioned to Sangon Biotech. After sequencing, the sequencing results were compared using the Blast function in NCBI, and the positive transformant plasmids with correct sequencing results were transformed into BL21 Escherichia coli competent cells (purchased from Shanghai Ruichu Biotechnology Co., Ltd., R09203-30T) by heat shock method.

[0075] Take 50μL BL21 E. coli competent cells out of the -80℃ refrigerator, place them on ice for 5 minutes to melt, mix them with 100ng plasmid, place them on ice for 30 minutes, then heat shock them in a 42℃ water bath for 45s, continue to place them on ice for 2 minutes, then quickly add 600μL LB non-resistant medium, and culture them in a shaker at 37℃ and 220rpm for 1 hour. After 1 hour, take 100μL of culture solution and spread it on the LB solid plate containing 50μg / mL Amp resistance, place the coated solid plate upright in a 37℃ incubator for 30 minutes, and then invert it for overnight culture. On the next day, a single colony on the plate was picked and inoculated into 10 mL of LB medium containing 50 μg / mL Amp resistance, and cultured in a shaker at 37°C and 220 rpm for 8 h. After 8 h, the bacterial solution was frozen at a volume ratio of bacterial solution: glycerol (purchased from Tianjin Yongda Chemical Reagent Co., Ltd.) (v:v) = 7′3, transferred to a bacterial freezing tube, and stored in a -80°C refrigerator to obtain a bacterial solution containing pMal-c4x-SUMO-Chimeric mutant 1.

[0076] The construction process of mutants 2-5 was the same as above. At the same time, mutants 6 and 7 were delivered to Sangon Biotech Co., Ltd. for synthesis, and bacterial solutions containing pMal-c4x-SUMO-Chimeric mutant 2, pMal-c4x-SUMO-Chimeric mutant 3, pMal-c4x-SUMO-Chimeric mutant 4, pMal-c4x-SUMO-Chimeric mutant 5, pMal-c4x-SUMO-Chimericmutant 6, and pMal-c4x-SUMO-Chimeric mutant 7 were obtained, respectively.

[0077] Example 3: Preparation of recombinant human-porcine chimeric uricase and its mutants by E. coli expression system

[0078] (1) Taking mutant 1 as an example, the expression of MBP-SUMO-Chimericmutant1 fusion protein is demonstrated

[0079] Small-scale expression: Inoculate the frozen bacterial solution into 10 mL of LB medium containing 50 μg / mL Amp resistance at a 1% inoculum volume, and activate overnight at 37°C and 220 rpm in a shaker. The next morning, inoculate the activated seed solution into 10 mL of LB medium containing 50 μg / mL Amp resistance at a 1% (v / v) inoculum volume, and culture in a shaker at 37°C / 220 rpm until OD = 0.6-0.8, add IPTG (Bio FRoxx, EZ6789D139) at a final concentration of 1 mM, and continue shaking culture for 10 hours.

[0080] After induction, measure the OD 600 Value. Taking the same cell amount as the standard, collect 2 portions of induced bacterial liquid, resuspend the bacterial liquid with 1×PBS buffer, wash 3 times and centrifuge, and finally resuspend the two bacterial bodies with 1mL of 1×PBS. One of them is named as the whole bacteria after induction, and the other is crushed by ultrasonic crusher (purchased from Ningbo Xinzhi Biotechnology Co., Ltd., UP-250). The crusher parameters are set as follows: 10% power, total crushing time 5min, crushing 3s and stopping 4s, until the bacterial liquid is clear and transparent and can pass light. After the crushing is completed, centrifuge at 10000rpm for 1min, collect the crushed supernatant, resuspend the bottom precipitate with 1×PBS, wash once, and resuspend with 1mL of 1×PBS.

[0081] SDS-PAGE test for bacterial expression: The collected components (whole bacteria without induction (negative control), whole bacteria after induction, supernatant and precipitate) were subjected to 10% SDS-PAGE precast gel (purchased from Invitrogen TM , NP0306BOX) for SDS-PAGE. Take 80 μL of each component and mix with 20 μL 5× loading buffer, mix well and boil in a 100°C metal bath for 10 min, take 25 μL of the prepared sample and add it to the precast gel well, perform electrophoresis at 70 V for 30 min to allow the sample to electrophoresed to the separation gel, adjust the voltage to 120 V and continue electrophoresis until the indicator reaches the bottom and stop electrophoresis.

[0082] The PAGE gel after electrophoresis was soaked in Coomassie Brilliant Blue solution (Solarbio, 419B025), placed on a shaker for staining for 1 hour, and then destained with a decolorizing solution until clear protein bands can be seen. SDS-PAGE electrophoresis detection proved that MBP-SUMO-Chimericmutant1 obtained a considerable expression level after induction, and the expression was soluble. Figure 3 The results of SDS-PAGE solubility analysis of MBP-SUMO-Chimeric mutant 1.

[0083] (2) Purification of MBP-SUMO-Chimeric mutant 1 fusion protein using MBP column and Ni-NTA column

[0084] The bacteria were induced in a 1L shake flask containing 300mL TB medium in the same manner as above. The bacterial solution after induction culture was centrifuged (4000rpm, 30min) to collect the bacteria, weigh the wet weight of the bacteria, and resuspend the bacteria at a ratio of wet weight of bacteria (m): MBP binding buffer (20mM tris-HCL, 200mM NaCl, 1mM EDTA, 1mM DTT, pH7.4) (v) = 1:10. After ultrasonic disruption, the bacterial solution was centrifuged at the maximum speed for 40min in a refrigerated high-speed centrifuge (purchased from Bechman (USA)), and the supernatant of the disrupted bacterial solution was collected and filtered through a 0.45μm filter membrane (Miiiex-GP, SLGPR33RB).

[0085] MBP column purification of fusion protein: The bacterial supernatant was subjected to MBP column affinity chromatography using an AKTA purifier (purchased from GE). First, the MBP column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA026010) was equilibrated with MBP binding buffer at a flow rate of 2 mL / min for 10 column volumes, and the sample was loaded at a flow rate of 2 mL / min; after the loading was completed, the MBP column was equilibrated with MBP binding buffer at a flow rate of 2 mL / min for 10 column volumes; then the fusion protein was eluted with MBP eluent (20 mM tris-HCL, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 mM maltose, pH 7.4) at a flow rate of 2 mL / min, and the elution was stopped after the UV peak was completed; then the MBP medium was washed with 0.5 M NaOH at a flow rate of 2 ml / min for 10 column volumes; then the Ni medium was washed with ultrapure water at a flow rate of 2 mL / min for 10 column volumes; finally, the MBP medium was washed with 20% ethanol at a flow rate of 2 mL / min for 10 column volumes to preserve the column.

[0086] The fusion protein was purified by Ni-NTA column: The eluted protein sample of the MBP column was concentrated to 10 mL by ultrafiltration using an ultrafiltration tube (milipore, 36100101), and then the ultrafiltration tube was continued to ultrafilter, and the system was replaced with the fusion protein Ni-NTA column equilibrium buffer (100mM Tris-HCL, 300mM NaCl, pH10.28), and the replacement was completed by ultrafiltration 7 times. Subsequently, the protein solution of the replaced system was subjected to Ni-NTA affinity chromatography using an AKTA purifier (purchased from GE). After the purifier was loaded with Ni filler, first, the Ni medium was equilibrated with Ni column equilibration solution (100mM Tris-HCL, 300mM NaCl, pH10.28) at 2mL / min for 10 column volumes; then the enzyme cleavage reaction mixture was loaded at 2mL / min; after loading, the Ni column was equilibrated with Ni-NTA binding buffer at a flow rate of 2mL / min for 10 column volumes; then, Ni-NTA elution buffer (50mM, 300mM, and 500mM imidazole dissolved in 100mM tris-HCL, 300mM NaCl, pH10.28 binding buffer) at a flow rate of 2 mL / min to elute the target protein, 50 mM and 500 mM imidazole to elute the impurities, and 300 mM imidazole to elute the target protein; then, 0.5 M NaOH was used to clean the Ni medium at a flow rate of 2 ml / min for 10 column volumes; then, ultrapure water was used to clean the Ni medium at a flow rate of 2 mL / min for 10 column volumes; finally, 20% ethanol was used to clean the Ni medium at a flow rate of 2 mL / min, and the column was preserved after cleaning for 10 column volumes.

[0087] SDS-PAGE was used to check the purification results: precast gel was used for electrophoresis, and the prepared sample components (supernatant after crushing, MBP column purification flow-through, MBP column elution sample and Ni-NTA column 300mM imidazole elution sample) were mixed with 20μL 5× loading buffer respectively. After mixing, the mixture was placed in a 100℃ metal bath and boiled for 10 min. 25μL of the prepared sample was added to the precast gel well, and the sample was electrophoresed at 70V for 30min to allow the sample to be electrophoresed to the separation gel. Then the voltage was adjusted to 120V and the electrophoresis was continued until the indicator reached the bottom and the electrophoresis was stopped.

[0088] The PAGE gel after electrophoresis was soaked in Coomassie Brilliant Blue solution, placed on a shaker for staining for 1 hour, and then destained with a decolorizing solution until clear protein bands can be seen. SDS-PAGE electrophoresis detection proved that the MBP eluent eluted the target protein with a purity of more than 90%. Figure 4 The results of SDS-PAGE purification and analysis of MBP-SUMO-Chimeric mutant 1 fusion protein.

[0089] Determination of target protein concentration: The purified protein solution was concentrated by ultrafiltration using an ultrafiltration tube to a final volume of 5 mL. 1 mL per tube was dispensed into 5 1.5 mL centrifuge tubes, and the protein concentration was determined using the BCA method.

[0090] According to Pierce TM The protein concentration was determined by BCA protein quantification kit (Thermo Fisher Scientific, WL336484), and the standard (bovine serum albumin BSA) was prepared as follows.

[0091] Table 3

[0092]

[0093] First, calculate the required volume of AB solution (A solution and B solution are mixed at a volume ratio of 1:50). In a 96-well plate, each sample requires 3 secondary wells, and the AI ​​standard requires 27 wells. Each well requires 100μL of AB solution, so the volume of AB solution required for the standard is 2700μL. Assuming there are X samples, there are 3X wells, and each well requires 100μL of AB solution. The volume of AB solution required for the sample is 300XμL, and the total volume of AB solution required is 2700μL+300XμL; the standard AI and x samples are spotted into the wells along the order of AI in the 96-well plate, and 3 secondary wells are set. After the spotting is completed, add AB solution with a spray gun, adding 100μL to each well, and the total volume of AB solution required is 2700μL+300XμL; incubate in a 37℃ oven for 30 minutes away from light; use a UV spectrophotometer to measure its OD 562 Absorbance value; according to the OD of the standard 562 Make a standard curve based on the absorbance value; substitute the sample value into the standard curve to calculate the concentration of the sample.

[0094] (3) Taking MBP-SUMO-Chimeric mutant 6 as an example, the Ni-NTA column purification of mutant 6 after fusion protease cleavage is described

[0095] Take 10 mg of MBP-SUMO-Chimeric mutant 6 fusion protein purified by MBP column and dilute it to 100 mL with Ni-NTA binding buffer (100 mM Tris, 50 mM NaCl, pH = 10.28) after digestion, and the final concentration is 0.1 mg / mL. Use SUMO protease (prepared according to the literature "Li Shijie et al. High-efficiency expression and purification of SUMO protease Ulp1 and preparation of scFv through His-SUMO tag. China Biotechnology, 2018, 38 (3): 51-61") in a 4℃ chromatography cabinet for digestion reaction, with a mass ratio of 1:3000 (SUMO protease: fusion protein). Add 3.3 μg SUMO protease to 100 mL of protein diluent, mix well and place in a 4℃ chromatography cabinet for digestion overnight.

[0096] After the digestion is completed, the digestion reaction mixture is subjected to Ni-NTA affinity chromatography using an AKTA purifier (purchased from GE). After the purifier is equipped with Ni filler, first, the Ni medium is equilibrated with Ni column equilibration solution (100mM Tris-HCL, 50mM NaCl, pH10.28) at 2mL / min for 10 column volumes; then the digestion reaction mixture is loaded at 2mL / min; after loading, the Ni column is equilibrated with Ni-NTA binding buffer at a flow rate of 2mL / min for 10 column volumes; then the target protein is eluted with Ni-NTA elution buffer (50mM, 100mM, and 300mM imidazole are dissolved in 100mM Tris-HCL, 50mM NaCl, pH10.28, respectively) at a flow rate of 2mL / min, 50mM and 100mM imidazole elute the label and impurities, and 300mM imidazole elutes the target protein; then 0.5M The Ni medium was cleaned with NaOH at a flow rate of 2 mL / min for 10 column volumes; then with ultrapure water at a flow rate of 2 mL / min for 10 column volumes; finally, the Ni medium was cleaned with 20% ethanol at a flow rate of 2 mL / min, and the column was preserved after cleaning for 10 column volumes.

[0097] SDS-PAGE test of Ni-NTA column purification results after fusion protease cleavage: Use precast gel (purchased from Invitrogen, EC6025BOX) for electrophoresis. The collected components (enzyme cleavage mixture, purification flow-through, 50mM imidazole eluent, 100mM imidazole eluent, 300mM imidazole eluent) were mixed with 20μL 5× loading buffer, mixed and placed in a 100℃ metal bath for 10min, 25μL of the prepared sample was added to the precast gel well, and the sample was electrophoresed to the separation gel at 70V for 30min, and the voltage was adjusted to 120V to continue electrophoresis until the indicator reached the bottom and stopped electrophoresis.

[0098] Soak the PAGE gel after electrophoresis in Coomassie Brilliant Blue solution, place it on a shaker for 1 hour, and then decolorize it with a decolorizing solution until clear protein bands can be seen. SDS-PAGE electrophoresis test proves (such as Figure 5 As shown), the MBP-SUMO tag exists in the flow-through, 50mM and 100mM imidazole can elute the host impurities, and the target protein mutant 6 is eluted in 300mM imidazole with a purity of more than 90%.

[0099] Example 4: dHU-wPU enzyme activity assay

[0100] 1) Prepare substrate solution:

[0101] Uric acid buffer: weigh 2.0122 g of borax (purchased from Guangzhou Xincheng Fine Chemical Factory, 304131) in a beaker, add deionized water and continue heating and stirring until dissolved, adjust the pH value to 8.6, make up to 100 mL, filter and sterilize with a 0.45 μm filter membrane, and store at room temperature after filtration;

[0102] Uric acid reaction solution: weigh 0.0840 g of uric acid white powder (aladdin, J1929193) into a beaker, add 50 mM sodium tetraborate solution (pH 8.6) and stir continuously until dissolved, dilute to 100 mL and store at room temperature, the final concentration is 5 mM;

[0103] 2) Enzyme activity determination method:

[0104] Uricase can catalyze uric acid into allantoin, which is more water-soluble. Since uric acid has a special absorption peak at 293nm, and its product has no absorption peak at this wavelength, according to the drawn uric acid standard curve, different concentrations of uric acid correspond to different absorbance values. As uricase decomposes the substrate, the decrease in absorbance at 293nm is regularly detected to calculate the enzyme activity.

[0105] The wavelength of the UV spectrophotometer was adjusted to 293 nm, and the temperature of the water bath circulation system was adjusted to 42 °C and kept constant. The uric acid buffer was used as a blank control for zero adjustment, and then 3 mL of 100 μM uric acid solution was added to the cuvette, followed by 100 μL of 50 μg mL -1 After the uricase protein solution was quickly mixed, the absorbance was measured at 293 nm and the reading was taken every 10 seconds; the uricase activity was calculated according to the enzyme activity calculation formula of the substrate consumption method.

[0106] Uricase activity (U) = (A-A0) × V × 10 6 / (ε×L×T×C(enzyme)×v(enzyme)), where U is the enzyme activity unit (defined as the conversion of 1 μmol·mL per minute under the optimal reaction temperature and optimal buffer pH conditions). -1 =The amount of enzyme required for uric acid, U / mg); A is the absorbance of uric acid at 293 nm at the end of the reaction; A0 is the absorbance of uric acid at 293 nm at the start of the reaction; V is the total volume of the uricase reaction system (L); ε is the molar extinction coefficient of uric acid 1.2×10 4 L·mol -1 cm -1 ; L is the light path of the cuvette (1 cm); T is the reaction time (min); C (enzyme) is the concentration of uricase in the reaction system (0.05 mg·mL -1 ); v(enzyme) is the concentration of uricase in the reaction system (0.1 mL).

[0107] We first reacted the fusion protein uricases purified by MBP column and Ni column with uric acid to preliminarily identify the activity of each uricase. Figure 6 The graphs are the changes of substrate absorbance over time when each fusion protein uricase reacts with uric acid solution. The results show that the chimera H 1-4 P 5-6 H 7-8 When Chimeric mutant 6, Chimeric mutant 7, and two positive controls wPU and PBC reacted with uric acid, the substrate absorbance did not change, indicating that they were inactive; Chimeric mutant 6, Chimeric mutant 7, and two positive controls wPU and PBC all had a good effect on reducing uric acid. Therefore, we measured the specific enzymatic parameters of the four active uricases (Chimeric mutant 6, Chimeric mutant 7, wPU, and PBC) after digestion with SUMO protease.

[0108] 3) Determination of Michaelis constant

[0109] The concentrations of uric acid buffer were 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160 and 180 μmol mL -1 The uric acid substrate was used as the substrate. Under the optimal conditions (temperature, pH), four active uricases (wPU, PBC, Chimeric mutant 6 and Chimeric mutant 7) were reacted with different concentrations of substrate for 3 minutes, and the reaction rates of uricases at different substrate concentrations were measured. The corresponding data of substrate concentration and reaction rate were fitted to the Michaelis-Menten equation using Origin software to obtain the Km value of each uricase. Table 4 shows the enzymatic characteristics of the four active uricases. It can be seen from Table 4 that the two human porcine chimeric mutant uricases (Chimericmutant 6 and Chimeric mutant 7) constructed in the present invention have excellent catalytic activity and homology with humans (4.76 U / mg, 6.33 U / mg and 93.09%, 91.45%, respectively), which are higher than the pig with the highest uricase activity among mammals (4.16 U / mg, 87.5%), and the enzyme activity of Chimeric mutant 7 is higher than that of the marketed recombinant uricase preparation drug Krystexxa (PBC, a PEG-modified recombinant porcine-baboon chimeric uricase).

[0110] Table 4 Enzymatic characteristics of active uricase

[0111]

[0112] ND stands for Not determined

[0113] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A humanized human-porcine chimeric uricase chimeric mutant, characterized in that: The mutant with the amino acid sequence as shown in SEQ ID NO.1 is further mutated as follows: 7N→7D; 24E→24D; 83E→83G; 89A→89T; 91G→91A; 112M→112V; 115I→115V; 119H→119R; 120L→120F and 121G→121E.

2. A nucleic acid molecule encoding the humanized human-porcine chimeric uricase chimeric mutant according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

4. The method for preparing the humanized human-porcine chimeric uricase chimeric mutant according to claim 1, characterized in that The method comprises the following steps: cloning the nucleic acid molecule encoding the humanized human-porcine chimeric uricase chimeric mutant according to claim 1 into an expression vector, expressing and purifying the humanized human-porcine chimeric uricase chimeric mutant.

5. The preparation method according to claim 4, characterized in that: The expression vector is a prokaryotic vector.

6. The preparation method according to claim 5, characterized in that: The prokaryotic vector is pMal-c4x.

7. Use of the humanized human-porcine chimeric uricase chimeric mutant according to claim 1 in the preparation of a drug for treating hyperuricemia.

8. Use of the nucleic acid molecule according to claim 2 or claim 3 in the preparation of a drug for treating hyperuricemia.

9. The use according to claim 8, characterized in that The method comprises the following steps: cloning the nucleic acid molecule according to claim 2 or claim 3 into an expression vector, expressing and purifying to obtain a humanized human-porcine chimeric uricase chimeric mutant.

10. The use according to claim 8, characterized in that The method comprises the following steps: cloning the nucleic acid molecule according to claim 2 or claim 3 downstream of a eukaryotic promoter to obtain a nucleic acid fragment capable of expressing uricase in the human body.

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