Fusion protease, construction method and application

Abstract

The invention relates to the field of enzyme and protein structures, in particular to fusion protease as well as a construction method and application thereof. The construction method comprises the following steps: carrying out gene recombination on the FpGalNAcDeAc and the FpGalNase, and carrying out prokaryotic expression and purification, so as to obtain the FpGalNAcDeAc-FpGalNase or the FpGalNase-FpGalNAcDeAc. The construction method comprises the following steps: carrying out gene recombination on the FpGalNAcDeAc and the FpGalNase; the invention describes that under the conditions of specific buffer solution, incubation time and enzyme concentration, A type red blood cells can be converted into universal O type red blood cells with maximum efficiency. The structure of galactosaminase is analyzed by using a cryoelectron microscope, and acetylgalactosamine deacetylase and galactosaminase are subjected to fusion expression, so that more efficient erythrocyte type conversion is carried out. The method can be used for rapidly, efficiently and safely carrying out type-A erythrocyte type conversion, and has the advantages of high protease yield, low working concentration, simple and convenient conversion operation, universal conditions, low cost and the like.

Description

Technical Field

[0001] The present invention relates to the field of enzyme and protein structure, in particular to a fusion protease, a construction method and an application thereof. Background Art

[0002] More than 118 million blood donations are needed each year to meet the global transfusion demand (Solvent Extraction & Ion Exchange 2015, 33 (3), 224-235.). Correct blood type matching is essential for safe transfusion, because incompatible blood transfusions can trigger complement activation and red blood cell hemolysis, which may lead to transfusion-related death of patients (Transfusion Clinique et Biologique 2019, 26 (2), 116-124.). In the blood type system, ABO antigens are the most immunogenic. Individuals with blood types A, B, and O express A, B, and H antigens on their red blood cell membranes, respectively (Vox Sang 1967, 12 (5), 321-328.). It is worth noting that type O red blood cells lack immunogenic sugars, such as α-N-acetylgalactose (GalNAc) in the A antigen and galactose (Gal) in the B antigen. This lack of antigens prevents type O red cells from agglutinating with the anti-A or anti-B antibodies of type A and B individuals, and type O individuals are therefore known as "universal donors." An adequate supply of type O red cells is essential for emergency transfusions, especially in situations where the recipient's blood type is unknown or donors are limited, such as in battlefield settings, pediatric transfusions, or when blood is in short supply.

[0003] O-type red blood cells can be generated from type A or type B cells by enzymatic removal of GalNAc or Gal molecules (Science 1982, 215 (4529), 168-170.). Historically, the conversion of type B blood to type O blood has attracted more research attention than the conversion of type A blood to type O blood, because there are multiple subtypes of A antigen, such as type A1 and type A2 (Vox Sang 1989, 56 (1), 1-20.). In 1980, scientists such as Lenny used α-galactosidase from green coffee beans to catalyze the removal of the terminal antigen Gal, and for the first time converted type B blood to type O. Clinical studies have shown that the safety and effectiveness of red blood cells converted from type B to type O are comparable to those of natural type O red blood cells. However, the high enzyme concentration required (1-2 g enzyme per unit of red blood cells) is a major obstacle to the widespread clinical application of this technology (Science 1982, 215(4529), 168-170.), which highlights the need for more efficient enzymes to promote the production of universal O-type red blood cells.

[0004] Recent studies have found more efficient α-N-acetylgalactosaminidase (α-GalNAcases) and α-galactosidase from bacterial sources (Nat Biotechnol 2007, 25 (4), 454-464.; Nat Microbiol 2024, 9 (5), 1176-1188.; Nat Microbiol 2019, 4 (9), 1475-1485.). α-GalNAcase from Elizabethkingia meningosepticum and α-galactosidase from Bacteriodesfragilis were found to require about 60 mg and about 2 mg per unit of type A and type B red blood cells, respectively (Nat Biotechnol 2007, 25 (4), 454-464.). Recently, the enzymes AmGH36A and AmGH110A from Akkermansia muciniphila were shown to be highly efficient in cleaving A1, extended A antigen, and B antigen, requiring 18 mg and 8 mg per unit of red blood cells, respectively. However, type O red blood cells generated from type A red blood cells by AmGH36A still showed some cross-reactivity with type O plasma (Nat Microbiol 2024, 9(5), 1176-1188.).

[0005] Another dual enzyme system from Flavonifractor plautii uses N-acetylgalactosamine deacetylase (FpGalNAcDeAc) and galactosidase (FpGalNase), which work together to efficiently cut the A antigen, thereby achieving efficient conversion of type A red blood cells to type O red blood cells. FpGalNAcDeAc first removes the N-acetyl group of GalNAc to generate galactosamine, and then FpGalNase cuts the galactosamine to expose the H antigen (Nat Microbiol 2019, 4 (9), 1475-1485.). Although the synergistic effect of these two enzymes has great potential, the clinical application of this enzyme system has not yet been explored, and there is an urgent need to design more efficient enzyme variants to improve clinical practicality. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention can be achieved through the following technical solutions:

[0007] A method for constructing a fusion protease comprises: performing gene recombination on FpGalNAcDeAc and FpGalNase, and obtaining FpGalNAcDeAc-FpGalNase or FpGalNase-FpGalNAcDeAc through prokaryotic expression and purification.

[0008] Furthermore, the specific steps of the gene recombination are:

[0009] S1. According to the amino acid sequences of FpGalNAcDeAc and FpGalNase, the forward and backward primers required for PCR amplification were designed and synthesized respectively;

[0010] S2. Clone the DNA fragments of FpGalNAcDeAc and FpGalNase from Fusobacterium prausnitzii, separate the PCR products by agarose gel electrophoresis, and recover the target fragments by DNA gel recovery kit;

[0011] S3, after sequencing by Sanger method, the sequence was compared with the sequence published by NCBI;

[0012] S4, codon optimization of the amino acids of the correctly sequenced target fragment was performed, 5'NcoI and 3'XhoI were added, and the gene was cloned into the vector pET-25b through 5'NcoI and 3'XhoI using the gene recombination method, and His6 was used as a purification tag;

[0013] S5. The constructed plasmid was transformed into BL21 E. coli and the expression of fusion protein was induced by IPTG.

[0014] S6. After the bacteria are lysed, the protein is purified using a Ni column and a gel chromatography column in sequence to obtain a high-yield, high-purity recombinant protein.

[0015] Furthermore, the sequence of the FpGalNAcDeAc is SEQ ID NO.1.

[0016] Furthermore, the sequence of the FpGalNase is SEQ ID NO.2.

[0017] A fusion protease is constructed by the construction method of the fusion protease described above.

[0018] A fusion protease is used to convert type A red blood cells into type O red blood cells.

[0019] Furthermore, the specific operation steps for converting type A red blood cells into type O red blood cells are as follows: after incubating FpGalNAcDeAc-FpGalNase or FpGalNase-FpGalNAcDeAc fusion protein with type A red blood cells for a certain period of time, type A red blood cells can be converted into type O red blood cells.

[0020] A reagent containing the above-mentioned fusion protease, which is used for converting type A red blood cells into type O red blood cells.

[0021] Beneficial effects of the present invention:

[0022] 1. The proteases FpGalNAcDeAc and FpGalNase prepared by the present invention can achieve nearly 100% antigen conversion efficiency and A-to-O red blood cell type conversion at extremely low concentrations after incubation with type A red blood cells in optimized incubation time, enzyme concentration and buffer system, and there is almost no abnormal change in the morphology and function of red blood cells after the conversion.

[0023] 2. This invention analyzed the cryo-electron microscopy structure of FpGalNase for the first time, determined the catalytic center site and verified its activity, providing a theoretical basis for the redesign of the two proteases.

[0024] 3. The fusion protein FpGalNAcDeAc-FpGalNase or FpGalNase-FpGalNAcDeAc prepared by the present invention has nearly 100% A-to-O conversion at lower working concentrations and incubation times, indicating that it has higher catalytic activity, showing the necessity of enzyme redesign and its importance to clinical transfusion applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is a research schematic diagram of the present application "Structural basis of an enzyme FpGalNase for converting type A red blood cells and preparation and application of a fusion protein for achieving efficient A-to-O blood type conversion by its combined use with FpGalNAcDeAc."

[0027] Figure 2 Schematic diagram of the overview of A, B and H antigens on red blood cells and the conversion of different antigens under enzyme catalysis.

[0028] Figure 3This is the identification diagram of red blood cell surface antigens after the double enzymes are incubated with various types of red blood cells for a certain period of time.

[0029] Figure 4 These are the flow cytometry detection graphs and statistical graphs of A antigen and H antigen on the surface of red blood cells after incubation of double enzymes with type A red blood cells in different buffers.

[0030] Figure 5 The flow cytometric detection graph and statistical graph of A antigen and H antigen on the surface of red blood cells after incubation of double enzymes with type A red blood cells at different concentrations.

[0031] Figure 6 Gel chromatography purification peak diagram and SDS-PAGE gel diagram of fusion proteins FpGalNAcDeAc-FpGalNase and FpGalNase-FpGalNAcDeAc.

[0032] Figure 7 The flow cytometric detection graphs and statistical graphs of A antigen and H antigen on the surface of erythrocytes after incubation of type A erythrocytes with the fusion proteins FpGalNAcDeAc-FpGalNase and FpGalNase-FpGalNAcDeAc at different concentrations.

[0033] Figure 8 This is a diagram showing the identification of red blood cell surface antigens after the fusion proteins FpGalNAcDeAc-FpGalNase and FpGalNase-FpGalNAcDeAc were incubated with type A red blood cells.

[0034] Fig. 9 Schematic diagram of the cryo-electron microscopy structure of FpGalNase.

[0035] Fig.10 The flow cytometry detection graph and statistical graph of A antigen and H antigen on the surface of red blood cells after incubation of FpGalNase mutant with type A red blood cells.

[0036] Fig.11 This is a diagram showing the identification of red blood cell surface antigens after the FpGalNase mutant was incubated with type A red blood cells for a certain period of time. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The core requirement for blood transfusion in clinical treatment is the correct matching of blood types. Individuals with A, B and O blood types express A and H, B and H, and H antigens on the surface of their red blood cell membranes, respectively. Antigen molecules are composed of one or more linear or branched carbohydrate chains, and blood type specificity is determined by the nature and connection of the monosaccharides at the end of the carbohydrate chain. For A and B antigen molecules, the immunodominant is bound to the penultimate galactose residue, which is the end of the H antigen, with an a-1,2-glycosidic bond. The two terminate in α-1,3-n-acetylgalactosamine (GalNAc) or galactose (Gal), respectively, resulting in A, B or H blood type specificity ( Figure 2 ). On the other hand, because the H antigen lacks the terminal immunodominant GalNAc or Gal sugar of human immunity, it does not agglutinate with the anti-B antigen antibodies in the plasma of type A blood individuals or the anti-A antigen antibodies in the plasma of type B blood individuals, and can be safely transfused to individuals of any ABO blood type. Therefore, type O blood donors are called "universal blood donors." Theoretically, under the catalytic action of specific proteases, type O red blood cells can be produced from type A or type B cells of another ABO blood type, thereby solving emergency blood transfusion situations such as emergency blood transfusions of unknown blood types and insufficient reserves of type O blood. The FpGalNAcDeAc and FpGalNase dual enzyme system can highly efficiently convert type A red blood cells into type O red blood cells. By fusion expression of FpGalNAcDeAc and FpGalNase, a fusion protease that is more efficient than the combined action of the two enzymes is obtained.

[0039] Example 1: Identification of the conversion of erythrocyte antigens by the prepared FpGalNAcDeAc and FpGalNase dual enzymes

[0040] Randomly select 20 samples of type A, type B, type AB and type O red blood cells from healthy people, take 200μl of red blood cells, centrifuge at 1000g for 5 minutes, wash twice with saline, and then wash three times with the selected enzyme buffer solution, and then incubate with FpGalNAcDeAc and FpGalNase enzymes in the selected buffer solution at 37℃ for a certain period of time. The reaction system is 200μl (RBC volume 1% to 40%). After the enzymatic reaction, wash the red blood cells twice with saline, and take a certain volume of enzymatically hydrolyzed RBC to prepare a 0.5% to 3% red blood cell saline suspension. The ABO blood type antigen test card (microcolumn gel) was numbered, and the red blood cell specimens to be tested were added to the first to second microtubes or the third to fourth microtubes or the fifth to sixth microtubes, 50 μl in each tube, and immediately centrifuged for 5 minutes (900 rpm for 2 minutes, 1500 rpm for 3 minutes) using a card-specific centrifuge (centrifuge (TD-3A model, Changchun, China) produced by Changchun Boyan Scientific Instrument Co., Ltd.), and the results were observed with the naked eye. The specific antigen-antibody complex formed by the red blood cell antigen and the corresponding antibody (anti-A or anti-B) in the microcolumn gel floats on the surface of the gel or in the gel, which is a positive reaction. The red blood cell antigen to be tested has no corresponding antibody binding, no specific antigen-antibody complex appears, and the red blood cells sink to the tip of the microcolumn gel, which is a negative reaction. Hemolysis is the appearance of clear and transparent red in the microcolumn gel pores. Double group (mixed agglutination) is that the agglutinated red blood cells are located at the top of the gel after centrifugation, while the negative red blood cells sink to the tip of the microcolumn gel.

[0041] The results are as follows ( Figure 3 ): The results of the ABO blood type antigen test card (microcolumn gel) showed that only after the co-incubation of FpGalNAcDeAc and FpGalNase, only the type of A-type red blood cells changed from A-type red blood cells to O-type red blood cells, that is, from A antigen to H antigen, while the surface antigens of red blood cells of other blood types did not change. In addition, no antigen changes were observed when red blood cells of various blood types, including type A, were incubated with FpGalNAcDeAc or FpGalNase alone.

[0042] Example 2: Optimization of buffer

[0043] In order to determine the best incubation system of enzyme and red blood cells, the present invention tests several buffer solutions, and incubates type A red blood cells with 1-50nM FpGalNAcDeAc and FpGalNase in 100μL of different buffer solutions, respectively. The buffers used are saline, glycine buffer (Gly), phosphate-citrate-saline buffer (PCS), 5% glucose sodium chloride solution (5% Glu-Sod) and citric acid-phosphate-glucose buffer (CPD), and phosphate buffer (PBS) is used as the control buffer. The incubation condition is incubated at 37°C for 15 minutes. The incubated system is centrifuged at a speed of 1000g for 5 minutes, and then the cells are subjected to flow cytometry (FACS) to quantify the proportion of A antigen and H antigen on the red blood cell surface to evaluate the maximum conversion efficiency of the protease to red blood cell antigens in different buffers.

[0044] In brief, 5 μL of enzyme-incubated erythrocytes were washed twice with PBS, resuspended in 200 μL PBS to a hematocrit of 0.5% to 3%, and incubated with 1:100 diluted APC-labeled anti-A antibody (Alexa Fluor 647 mouse anti-human blood group A antibody) at 26°C for 30 minutes to detect the remaining A antigen. At the same time, 1:10 diluted FITC-labeled olea agglutinin I (FITC-UEA-I) was used to detect the H antigen on the surface of erythrocytes. All antibody and lectin incubations were performed under light-protected conditions.

[0045] The results are as follows ( Figure 4 ): Combined with FACS results ( Figure 4 a) and statistical results ( Figure 4 b) It can be seen that PBS and saline buffers are conducive to antigen conversion of type A red blood cells, showing the highest conversion rates in all incubation groups (99.50% ± 4.04 and 88.03% ± 13.56), while 5% Glu-Sod and PCS buffers show lower conversion rates (65.90% ± 12.27 and 22.21% ± 13.88), and CPD and Gly buffers show the lowest conversion rates (1.70% ± 2.30 and 0.30% ± 0.25). In view of the wide clinical applicability of PBS, we selected it as the optimal buffer for further enzyme condition optimization.

[0046] Example 3: Optimization of enzyme concentration and time

[0047] Different concentrations of FpGalNAcDeAc and FpGalNase (4nM, 8nM, 16nM and 32nM) were incubated with type A erythrocytes in PBS solution for 5 minutes, 15 minutes, 30 minutes or 60 minutes. After the incubation, the erythrocytes were collected and washed twice with PBS to remove residual enzymes. The treated erythrocytes were then subjected to FACS analysis to evaluate the degree of enzymatic conversion at different concentrations and different incubation times.

[0048] The results are as follows ( Figure 5 ): FACS results ( Figure 5 a) shows that as the enzyme concentration increases from 4nM to 16nM, the conversion rate of A antigen to H antigen increases, reaching a conversion rate of about 99% at 16nM. There was no significant increase in the antigen conversion rate between concentrations of 16nM and 32nM. It is worth noting that when the incubation time was extended from 15 minutes to 60 minutes, the higher incubation concentration did not significantly increase the antigen conversion rate. On the contrary, with long incubation times, especially at a concentration of 8nM, the A-to-H conversion efficiency decreased significantly. A similar phenomenon was also observed at concentrations of 4nM and 16nM, with a decrease in conversion efficiency between 30 minutes and 60 minutes, although this decrease was not statistically significant. Interestingly, this reversal effect was not observed at the highest enzyme concentration of 32nM. Statistically ( Figure 5 b) The results show that the maximum antigen conversion efficiency can be achieved when the enzyme concentration is 16nM and the incubation time is 5 minutes.

[0049] Example 4: Expression and purification of fusion proteins FpGalNAcDeAc-FpGalNase and FpGalNase-FpGalNAcDeAc

[0050] According to the FpGalNAcDeAc and FpGalNase coding sequences obtained by sequencing, the coding sequences of the two were tandemly recombined in different orders to obtain the recombinant sequences FpGalNAcDeAc-FpGalNase and FpGalNase-FpGalNAcDeAc( Figure 6a), insert the recombinant sequence into the pET-21a vector through the 5'NcoI and 3'XhoI restriction sites to construct a recombinant plasmid, and then transform them into Escherichia coli BL21 (DE3), add IPTG to a final concentration of 0.5mM at 25°C to induce the expression of FpGalNAcDeAc-FpGalNase and FpGalNAcDeAc-FpGalNase proteins. After collecting the bacteria, lyse the bacteria and centrifuge, take the lysate supernatant and purify it through a HisTrap HP affinity column, using the His6 sequence inserted at the C-terminus of the sequence as a purification tag. The purified product was further purified using a Superdex 200 10 / 300GL gel chromatography column. After 10% SDS-PAGE gel electrophoresis confirmed that it was the target protein, the protein concentration was determined by the BCA method and stored at -80°C for future use. The SDS-PAGE gel electrophoresis diagram of the purified fusion protein is shown below. Figure 6 As shown in b.

[0051] Example 5: Investigation of the conversion efficiency of fusion protein to antigen A

[0052] According to Example 4, the purified fusion proteins FpGalNAcDeAc-FpGalNase and FpGalNAcDeAc-FpGalNase were incubated with type A erythrocytes at different concentrations (1 nM, 2 nM, and 4 nM) in a PBS solution for 5 minutes, 15 minutes, 30 minutes, or 60 minutes. After the incubation, the erythrocytes were collected and washed twice with PBS to remove residual enzymes. The treated erythrocytes were subjected to FACS analysis to evaluate the degree of enzymatic conversion at different concentrations and different incubation times, and to screen the optimal incubation concentration and time.

[0053] The results are as follows ( Figure 7 ): FpGalNAcDeAc-FpGalNase and FpGalNAcDeAc-FpGalNase showed similar enzymatic activities, and the conversion efficiency of both to A antigen was significantly higher than that of the synergistic effect of the two enzymes ( Figure 7 a). When incubated with 2 nM of the fusion protein FpGalNAcDeAc-FpGalNase or FpGalNAcDeAc-FpGalNase for 5 minutes, the conversion rate of A to H antigen can reach 84.90% and 88.13%, which is 1182 and 122 times higher than that of the double enzyme mixture ( Figure 7 b) However, after 60 min of treatment with FpGalNAcDeAc-FpGalNase and FpGalNAcDeAc-FpGalNase, a reverse reaction was observed.

[0054] Example 6: Identification of the conversion of the prepared fusion protein to type A erythrocyte antigen

[0055] According to Example 5, the purified fusion proteins FpGalNAcDeAc-FpGalNase and FpGalNAcDeAc-FpGalNase were incubated with type A red blood cells at the same molar concentration for 5 minutes, 15 minutes or 30 minutes, respectively. Then, according to the method described in Example 1, the type of red blood cells after incubation with the fusion protein was identified using an ABO blood type antigen detection card (microcolumn gel).

[0056] The results are as follows ( Figure 8 ): At the same molar concentration, the fusion proteins FpGalNAcDeAc-FpGalNase and FpGalNase-FpGalNAcDeAc, like the dual enzyme system, can successfully convert type A red blood cells into type O red blood cells.

[0057] Example 7: Cryo-EM structure of FpGalNase

[0058] 4.0 μL of purified FpGalNase protein (at concentrations of 0.1 mg / mL and 0.3 mg / mL, respectively) was applied to 1.2 / 1.3 300 mesh GO(Au)Quantifoil grids and 60 s neon ion treated 300 mesh NiTi film grids (Au1.2 / 1.3). These grids were then blotted with filter paper for 2.5 s at 4 °C and 100% humidity at a water absorption force of -9 and -5, respectively. Next, they were quickly frozen by immersion in liquid ethylene using a Vitrobot Mark IV (Thermo Fisher Scientific). The prepared grids were then transferred to a 300 kV TitanKrios transmission electron microscope equipped with a Gatan K3 detector and a GIF Quantum energy filter. Movies were collected in super-resolution counting mode at 105,000x magnification with a calibrated pixel size of The focus range is between -1.0μm and -2.0μm, and the total dose is Use EPU automatic acquisition software.

[0059] The results are as follows ( Fig. 9 ): Due to the high flexibility of the N-terminal (residues 27-43) and C-terminal potential carbohydrate binding moiety (CBM) domains, cryo-EM was successful in determining the structure of FpGalNase only from residues Y44 to P701 at a resolution of ( Fig. 9a). The N-terminal region (residues 27-699) is in a disc-shaped conformation and is subdivided into three parts: the N-terminus (residues 27-279), the central GH36 domain (residues 280-604), and the C-terminus (residues 605-699). Detailed structural analysis showed that the N-terminus is a β-sandwich fold consisting of two antiparallel β-sheets and 17 β-strands ( Fig. 9 b). The largest loop between β10 and β11 extends into the GH36 active site, forming the edge of the active site. Structural similarity searches revealed that the N-domain has strong homology with α-N-acetylgalactosidase (α-GalNAcases) (PDB: 5WZR) and has a Z-score of 17.7 ( Fig. 9 c). However, the N-domain has only 4% sequence identity with these proteins. Similarly, the C-terminus ( Fig. 9 d) also has 23% sequence identity with α-galactosidase (PDB: 3MI6) ( Fig. 9 e). The GH36 domain is characterized by a partially disrupted (α / β)8 tube with additional helices and incomplete secondary structure in the β6-β8 and α5 regions ( Fig. 9 f). Structural similarity analysis showed that the GH36 domain has homology with GH13 family proteins (PDB: 1XSI) and α-galactosidase (PDB: 5M1I), with sequence identities of 13% and 17%, respectively. The catalytic barrel of GH36 is structurally consistent with the catalytic barrels of the GH13 family and α-galactosidase, while FpGalNase adopts a more open conformation ( Fig. 9 g). The overall structure of the N-terminal region of FpGalNase has a low sequence identity with α-galactosidase, but a high structural similarity. FpGalNase and α-galactosidase are specific for galactosamine and galactose, respectively. To determine the active site of FpGalNase, the structure of its N-terminal region was compared with the structure of α-galactosidase from GH36 in complex with raffinose (PDB: 4FNT). Notably, the regions of the two proteins distal to the N-domain (pocket A) show similarities, while the proximal patch (pocket B) shows significant differences ( Fig. 9 h). Detailed investigation of the residues within the active site revealed that FpGalNase shares conserved residues with GH36α-galactosidase in patch A, including W317, D346, D347, W387, K461, and D463 ( Fig. 9 i). However, the non-conserved residues Y464, C513 and D532 constitute another part of the FpGalNase active site ( Fig. 9j), which may play a crucial role in determining the substrate specificity of FpGalNase. The cryo-EM density map and the corresponding atomic coordinates analyzed by the present invention have been uploaded to the Electron Microscopy Database (EMDB) and the Protein Data Bank (PDB), and the accession numbers in EMDB and PDB are EMD-37989 and 8X1B, respectively.

[0060] Example 8: Determination of the conversion efficiency of mutant enzymes of FpGalNase

[0061] According to embodiment 6, in order to verify the FpGalNase active site determined in the structural model, we designed four kinds of mutations: D463A, Y464A, C513A and D532A, and evaluated the enzyme catalytic activity. After the mutants D463A, Y464A, C513A and D532A obtained by prokaryotic expression and purification were mixed with FpGalNAcDeAc at a certain concentration, they were incubated together with type A erythrocytes in PBS solution, and the incubation time was 5 minutes, 15 minutes or 30 minutes. After the incubation, erythrocytes were collected and washed 2 times with PBS to remove residual enzymes. The erythrocytes after the treatment were subsequently subjected to FACS analysis, and the enzymatic conversion degree at different concentrations and different incubation times was measured to evaluate the mutant catalytic activity.

[0062] The results are as follows ( Fig.10 ): FACS detection showed that compared with the wild-type FpGalNase, the A antigen-H antigen signals of all mutants were reduced to varying degrees, indicating that the enzyme conversion rate of the mutant proteins was significantly reduced, among which Y464A retained reduced catalytic activity (about 6%), while the D463A, C513A and D532A mutants almost completely lost their enzyme activity.

[0063] Example 9: Identification of the conversion of type A erythrocyte antigen by the prepared FpGalNase mutant enzyme

[0064] According to Example 8, the purified mutants D463A, Y464A, C513A and D532A were incubated with type A red blood cells at the same molar concentration for 5 minutes, 15 minutes or 30 minutes, respectively. Then, according to the method described in Example 1, the type of red blood cells after incubation with the mutant proteins was identified using an ABO blood type antigen detection card (microcolumn gel).

[0065] The results are as follows ( Fig.11): At the same molar concentration, only after FpGalNAcDeAc was co-incubated with wild-type FpGalNase, type A red blood cells were successfully converted into type O red blood cells, while after FpGalNAcDeAc was co-incubated with FpGalNase mutants, type A red blood cells were not converted into type O red blood cells. These findings verified that amino acid residues such as D463, Y464, C513 and D532 are key sites in the enzymatic activity center of FpGalNase, which is consistent with the structural analysis of cryo-EM analysis.

[0066] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0067] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for constructing a fusion protease, characterized in that: The construction method comprises: performing gene recombination on FpGalNAcDeAc and FpGalNase, and obtaining FpGalNAcDeAc-FpGalNase or FpGalNase-FpGalNAcDeAc through prokaryotic expression and purification.

2. The method for constructing a fusion protease according to claim 1, characterized in that: The specific steps of the gene recombination are: S1. According to the amino acid sequences of FpGalNAcDeAc and FpGalNase, the forward and backward primers required for PCR amplification were designed and synthesized respectively; S2. Clone the DNA fragments of FpGalNAcDeAc and FpGalNase from Fusobacterium prausnitzii, separate the PCR products by agarose gel electrophoresis, and recover the target fragments by DNA gel recovery kit; S3, after sequencing by Sanger method, the sequence was compared with the sequence published by NCBI; S4, codon optimization of the amino acids of the correctly sequenced target fragment was performed, 5'NcoI and 3'XhoI were added, and the gene was cloned into the vector pET-25b through 5'NcoI and 3'XhoI using the gene recombination method, and His6 was used as a purification tag; S5. The constructed plasmid was transformed into BL21 E. coli and the expression of the fusion protein was induced by IPTG; S6. After the bacteria are lysed, the protein is purified using a Ni column and a gel chromatography column in sequence to obtain a high-yield, high-purity recombinant protein.

3. The method for constructing the fusion protease according to claim 2, characterized in that: The sequence of the FpGalNAcDeAc is SEQ ID NO.

1.

4. The method for constructing the fusion protease according to claim 2, characterized in that: The sequence of the FpGalNase is SEQ ID NO.

2.

5. A fusion protease constructed by the construction method of the fusion protease according to any one of claims 1 to 4.

6. A fusion protease as claimed in claim 5 for converting type A red blood cells into type O red blood cells.

7. The use according to claim 6, characterized in that: The specific operation steps for converting type A red blood cells into type O red blood cells are as follows: after incubating FpGalNAcDeAc-FpGalNase or FpGalNase-FpGalNAcDeAc fusion protein with type A red blood cells for a certain period of time, type A red blood cells can be converted into type O red blood cells.

8. A reagent containing the fusion protease according to claim 5, characterized in that: The reagent is used to convert type A red blood cells into type O red blood cells.

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