Catalytic antibody mutant for degrading helicobacter pylori urease as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a catalytic antibody mutant for degrading helicobacter pylori urease as well as a preparation method and application of the catalytic antibody mutant. A wild type helicobacter pylori urease degrading catalytic antibody Ua15-L (WT) is used as a starting antibody, a higher-performance helicobacter pylori urease degrading catalytic antibody mutant is obtained through rational design, experiments prove that the 48th amino acid and the 64th amino acid of the I48C-G64C mutant are mutated, isoleucine and glycine are mutated into cysteine respectively, and the mutant is converted into cysteine. A disulfide bond is formed between the two, so that the thermal stability of the catalytic antibody is improved. On the basis, mutations of L9E, L54R and T14Q-I15P are introduced, so that the solubility and the expression quantity of the protein are increased. Compared with a wild type catalytic antibody for degrading helicobacter pylori urease, the mutant 1 (L9E-I48C-G64C) has the advantages that the Tm value is increased by 6 DEG C, the solubility is increased by 81%, and the expression quantity is increased by 4 times; and the time for hydrolyzing half of the urease fragment at 37 DEG C is shortened by 52% compared with that of the wild type.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a catalytic antibody mutant for degrading Helicobacter pylori urease, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Catalytic antibodies are antibodies that not only bind to specific antigens but also catalyze the corresponding chemical reactions. The first catalytic antibody was discovered in 1989 from the plasma of patients with bronchial asthma. Subsequently, multiple catalytic antibodies were found in individuals with various diseases or in health. In addition, catalytic antibodies can be obtained by immunization with immunogens or transition state analogs. The effect of catalytic antibodies can be further enhanced by directed evolution or rational design.

[0004] Helicobacter pylori is the only bacterium that can survive in the highly acidic environment of the human stomach. A large number of studies have confirmed that Helicobacter pylori is the main cause of chronic gastritis, gastric ulcers and duodenal ulcers, and is closely related to the occurrence of gastric cancer. The light chain of the catalytic antibody UA15-L can cleave the peptide bond of Helicobacter pylori urease, thereby inhibiting the spread of Helicobacter pylori. The light chain variable region (VL) performs protease functions, while the light chain constant region (CL) helps maintain solubility and thermal stability and therefore does not play a direct role. Removing the light chain constant region can reduce the protein size by half, effectively increasing the drug potency of the same protein mass and providing advantages for drug delivery. Compared with the complete antibody or the entire light chain, the VL of UA15-L (i.e., UA15-VL) exhibits relatively poor thermal stability and solubility, which hinders its therapeutic application. Therefore, it is necessary to enhance the thermal stability and solubility of this catalytic antibody. Summary of the invention

[0005] In order to overcome the above problems, the present invention provides a catalytic antibody mutant for degrading Helicobacter pylori urease and a preparation method and application thereof.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a catalytic antibody mutant for degrading Helicobacter pylori urease is provided, wherein any one or more sites selected from the following group are mutated:

[0008] L9E, T14Q, I15P, I48C, G64C, L54R;

[0009] Among them, the amino acid residue numbering adopts SEQ ID NO.1 (amino acid sequence of the wild-type catalytic antibody that degrades Helicobacter pylori urease (Ua15-L (WT)), MGSSHHHHHHSSGLVPRGSHMDVVMTQTPLTLSVTIGQPASISCKSSQSLLDS DGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK).

[0010] The catalytic antibody mutant for degrading Helicobacter pylori urease is mutated based on the wild-type catalytic antibody for degrading Helicobacter pylori urease shown above, and the catalytic antibody mutant for degrading Helicobacter pylori urease is selected from the mutants in the following group:

[0011] Mutant 1: L9E-I48C-G64C; (amino acid sequence is shown in SEQ ID NO.2);

[0012] The amino acid sequence shown in SEQ ID NO.2 is:

[0013] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0014] Mutant 2: L9E-T14Q-I15P-I48C-G64C; (amino acid sequence is shown in SEQ ID NO.3);

[0015] The amino acid sequence shown in SEQ ID NO.3 is:

[0016] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVQPGQPASISCKSSQS LLDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKIS RVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0017] Mutant 3: L9E-I48C-G64C-L54R. (amino acid sequence is shown in SEQ ID NO. 4);

[0018] The amino acid sequence shown in SEQ ID NO.4 is:

[0019] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKRDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0020] The second aspect of the present invention provides a polynucleotide encoding the catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect.

[0021] The third aspect of the present invention provides a recombinant expression vector, which contains the polynucleotide described in the second aspect.

[0022] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect or the chromosome has the polynucleotide described in the second aspect integrated therein or expresses the catalytic antibody mutant for degrading Helicobacter pylori urease described in the first aspect.

[0023] A fifth aspect of the present invention provides a method for preparing the above-mentioned catalytic antibody mutant for degrading Helicobacter pylori urease, comprising:

[0024] Cultivating the host cell described in the fourth aspect to express the catalytic antibody mutant that degrades Helicobacter pylori urease; and isolating and purifying to obtain the catalytic antibody mutant that degrades Helicobacter pylori urease.

[0025] The sixth aspect of the present invention provides the use of the catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect in the preparation of a drug for improving, preventing or treating Helicobacter pylori infection.

[0026] The seventh aspect of the present invention provides a drug for improving, preventing or treating Helicobacter pylori infection, comprising the catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect.

[0027] The eighth aspect of the present invention provides a pharmaceutical composition, comprising the catalytic antibody mutant for degrading Helicobacter pylori urease according to the first aspect and one or more pharmaceutically acceptable carriers.

[0028] The beneficial effects of the present invention are:

[0029] The present invention provides a catalytic antibody mutant for degrading Helicobacter pylori urease and its preparation method and application. Specifically, the present invention uses the wild-type catalytic antibody for degrading Helicobacter pylori urease as the starting antibody, and uses bioinformatics and protein engineering technology for engineering transformation to obtain a catalytic antibody mutant for degrading Helicobacter pylori urease. It has been experimentally verified that the I48C-G64C mutant has mutations in the 48th and 64th amino acids, respectively mutated from isoleucine and glycine to cysteine, forming a disulfide bond between the two, which helps to improve the thermal stability of the catalytic antibody, but the mutation significantly reduces the solubility and expression of the catalytic antibody. On this basis, additional mutations L9E, L54R and T14Q-I15P are introduced to increase protein solubility and hope to restore protein expression without sacrificing thermal stability. Compared with the wild-type catalytic antibody for degrading Helicobacter pylori urease, the mutant 1L9E-I48C-G64C increases Tm by 6°C, solubility by 81%, and expression by 4 times. The mutant 1L9E-I48C-G64C also showed better urease fragment hydrolysis activity at 37°C than the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 To map Tmp to the 3D structure of UA15-VL;

[0032] Figure 2 is the variation of effective contact area between I48 and G64 with temperature;

[0033] Figure 3 The thermal stability of Ua15-L (WT) and its mutants, A is Ua15-L (WT), B is mutant 4I48C-G64C, C is mutant 1L9E-I48C-G64C, D is mutant 2L9E-T14Q-I15P-I48C-G64C and E is mutant 3L9E-I48C-G64C-L54R;

[0034] Figure 4is the proteolytic activity of Ua15-L (WT) and its mutants, A is the hydrolytic activity of Ua15-L (WT) at 25°C, B is the hydrolytic activity of Ua15-L (WT) at 37°C, C is the hydrolytic activity of mutant 1L9E-I48C-G64C at 25°C, D is the hydrolytic activity of mutant 1L9E-I48C-G64C at 37°C, E is the hydrolytic activity of mutant 2L9E- T14Q-I15P-I48C-G64C is the hydrolysis activity at 25°C, F is the hydrolysis activity of mutant 2L9E-T14Q-I15P-I48C-G64C at 37°C, G is the hydrolysis activity of mutant 3L9E-I48C-G64C-L54R at 25°C, and H is the hydrolysis activity of mutant 3L9E-I48C-G64C-L54R at 37°C. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0037] A first typical embodiment of the present invention provides a catalytic antibody mutant for degrading Helicobacter pylori urease, wherein any one or more sites selected from the following group are mutated:

[0038] L9E, T14Q, I15P, I48C, G64C, L54R;

[0039] Among them, the amino acid residue numbering adopts SEQ ID NO.1 (amino acid sequence of the wild-type catalytic antibody that degrades Helicobacter pylori urease (Ua15-L (WT)), MGSSHHHHHHSSGLVPRGSHMDVVMTQTPLTLSVTIGQPASISCKSSQSLLDS DGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK).

[0040] The catalytic antibody for degrading Helicobacter pylori urease in the present invention only contains the light chain UA15-L of the Ua15 catalytic antibody, and does not contain the heavy chain of the catalytic antibody for degrading Helicobacter pylori urease. Studies have shown that the heavy chain of the Ua15 catalytic antibody has no activity in degrading Helicobacter pylori urease.

[0041] The catalytic antibody mutant for degrading Helicobacter pylori urease is mutated based on the wild-type catalytic antibody for degrading Helicobacter pylori urease shown above, and the catalytic antibody mutant for degrading Helicobacter pylori urease is selected from the mutants in the following group:

[0042] Mutant 1: L9E-I48C-G64C; (amino acid sequence is shown in SEQ ID NO.2);

[0043] The amino acid sequence shown in SEQ ID NO.2 is:

[0044] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0045] Mutant 2: L9E-T14Q-I15P-I48C-G64C; (amino acid sequence is shown in SEQ ID NO.3);

[0046] The amino acid sequence shown in SEQ ID NO.3 is:

[0047] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVQPGQPASISCKSSQS LLDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKIS RVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0048] Mutant 3: L9E-I48C-G64C-L54R. (amino acid sequence is shown in SEQ ID NO. 4);

[0049] The amino acid sequence shown in SEQ ID NO.4 is:

[0050] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKRDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0051] Preferably, the amino acids at positions 48 and 64 in mutants 1 to 3 are mutated from isoleucine and glycine to cysteine, respectively, and a disulfide bond is formed between the two.

[0052] The 48th and 64th amino acids in mutants 1 to 3 mutated from isoleucine and glycine to cysteine, respectively, and a disulfide bond was formed between the two. The construction of the disulfide bond helps to improve the thermal stability of the catalytic antibody that degrades Helicobacter pylori urease.

[0053] Mutant 1L9E-I48C-G64C, mutant 2L9E-T14Q-I15P-I48C-G64C and mutant 3L9E-I48C-G64C-L54R can increase the solubility of catalytic antibodies that degrade Helicobacter pylori urease.

[0054] Mutant 1L9E-I48C-G64C, mutant 2L9E-T14Q-I15P-I48C-G64C and mutant 3L9E-I48C-G64C-L54R can increase the expression level of catalytic antibodies that degrade Helicobacter pylori urease.

[0055] The mutant 1L9E-I48C-G64C can enhance the activity of the catalytic antibody for degrading Helicobacter pylori urease in catalyzing the degradation of Helicobacter pylori urease.

[0056] A second typical embodiment of the present invention provides a polynucleotide encoding the catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect.

[0057] A third typical embodiment of the present invention provides a recombinant expression vector, wherein the recombinant expression vector contains the polynucleotide described in the second aspect above.

[0058] In one or more embodiments, the recombinant expression vector is obtained by effectively connecting the above-mentioned polynucleotide to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid, a phage, a cosmid or an artificial chromosome; the viral vector may include an adenoviral vector, a retroviral vector or an adeno-associated viral vector, and the artificial chromosome includes a bacterial artificial chromosome, a phage P1-derived vector, a yeast artificial chromosome or a mammalian artificial chromosome; preferably, the expression vector is a plasmid, and in a specific embodiment of the present invention, the plasmid is pET28a.

[0059] A fourth typical embodiment of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect or the chromosome integrated with the polynucleotide described in the second aspect or expresses the catalytic antibody mutant for degrading Helicobacter pylori urease described in the first aspect.

[0060] In one or more embodiments, the host cell can be a prokaryotic cell or a eukaryotic cell.

[0061] Preferably, the host cell is any one or more of a bacterial cell or a fungal cell;

[0062] wherein the bacterial cell is any species within the genus Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus;

[0063] Further preferably, the bacterial cell is Escherichia coli (such as Escherichia coli BL21 (DE3)), Agrobacterium tumefaciens (such as GV3101), Agrobacterium rhizogenes, Bacillus subtilis, Bacillus cereus or Pseudomonas sp.

[0064] The fungal cells include yeast (such as Pichia pastoris) and the like.

[0065] A fifth typical embodiment of the present invention provides a method for preparing the above-mentioned catalytic antibody mutant for degrading Helicobacter pylori urease, comprising:

[0066] Cultivating the host cell described in the fourth aspect to express the catalytic antibody mutant that degrades Helicobacter pylori urease; and isolating and purifying to obtain the catalytic antibody mutant that degrades Helicobacter pylori urease.

[0067] A sixth typical embodiment of the present invention provides use of the catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect in the preparation of a drug for improving, preventing or treating Helicobacter pylori infection.

[0068] A seventh typical embodiment of the present invention provides a drug for improving, preventing or treating Helicobacter pylori infection, comprising the catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect.

[0069] An eighth typical embodiment of the present invention provides a pharmaceutical composition, comprising the catalytic antibody mutant for degrading Helicobacter pylori urease according to the first aspect and one or more pharmaceutically acceptable carriers.

[0070] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant but is not limited thereto.

[0071] The pharmaceutical composition is used for improving, preventing or treating Helicobacter pylori infection.

[0072] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0073] Example 1

[0074] Mutant 4: I48C-G64C (amino acid sequence as shown in SEQ ID NO.5). The amino acids at positions 48 and 64 were mutated from isoleucine and glycine to cysteine, respectively. The genome of the Ua15-L (WT) strain was used as a DNA template, and PCR amplification was performed using primer pairs I48C-F / I48C-R and G64C-F / G64C-R, respectively:

[0075] I48C-F: 5'-TGTTATCTGGTTAGCAAACTGGATAGCGG-3';

[0076] I48C-R:5'-CAGACGTTTA GGGCTCTGAC CTG-3'.

[0077] G64C-F: 5'-TGTAGCGGCAGCGGTACCGATTT-3';

[0078] G64C-R: 5'-GGTAAAACGATTCTGGCACAC CGC-3'.

[0079] Mutant 4: I48C-G64C amino acid sequence (shown in SEQ ID NO.5) is:

[0080] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPLTLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0081] Mutant 1: L9E-I48C-G64C; (amino acid sequence is shown in SEQ ID NO.2); using the genome of the I48C-G64C strain as a DNA template, PCR amplification was performed using primer pair L9E-F / L9E-R respectively:

[0082] 9E-F:5'-GAAACCCTGAGCGTGACAATTGGTC-3';

[0083] 9E-R:5'-CGGCGTCTGGGTCATAACAACAT-3'.

[0084] Mutant 3: L9E-I48C-G64C-L54R (amino acid sequence as shown in SEQ ID NO.4); Mutant 1: Based on L9E-I48C-G64C, the amino acid at position 54 was mutated from leucine to arginine. The genome of the L9E-I48C-G64C strain was used as a DNA template, and PCR amplification was performed using primers L54R-F / L54R-R respectively:

[0085] L54R-F: 5'-CGTGATAGCGGTGTGCCAGATCGTTTTAC-3';

[0086] L54R-R: 5'-TTTGCTAACCAGATAACACAGACGTTTAGGG-3'.

[0087] Mutant 2: L9E-T14Q-I15P-I48C-G64C; (amino acid sequence as shown in SEQ ID NO.3); Mutant 1: Based on L9E-I48C-G64C, the amino acids at positions 14 and 15 are mutated, from threonine and isoleucine to glutamine and proline, respectively. The genome of the L9E-I48C-G64C strain was used as a DNA template, and PCR amplification was performed using primers T14Q-I15P-F / T14Q-I15P-R:

[0088] T14Q-I15P-F: 5'-CAGCCGGGTCAGCCGGCAAGCATTAGTTG-3';

[0089] T14Q-I15P-R: 5'-CACGCTCAGG GTTCCGGC-3'.

[0090] The PCR amplification system was as follows: 25 μL of 2×Pimer Star Max DNA mixed enzyme solution, 20 ng of DNA template, 1 μL of each primer (10 μM), and 22.5 μL of distilled water, with a total volume of 50 μL.

[0091] Amplification conditions were as follows: pre-denaturation at 98°C for 3 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 70°C for 15 seconds, and extension at 72°C for 2 minutes and 30 seconds (20 cycles), with the annealing temperature decreasing by 0.5°C in each cycle, and extension at 72°C for 3 minutes and 30 seconds (1 cycle); denaturation at 98°C for 10 seconds, annealing at 66°C for 15 seconds, and extension at 72°C for 2 minutes and 30 seconds (13 cycles); extension at 72°C for 3 minutes and 30 seconds (1 cycle).

[0092] The PCR amplification product was digested with restriction endonucleases Nco I and Xho I, and connected with plasmid pET28a digested with the same enzymes. The ligation product was transferred into DH5α competent cells, ice-bathed for 30 minutes, heat-shocked at 42°C for 90 seconds, immediately placed on ice for 5 minutes, added with 500 μL LB liquid medium, incubated at 200 rpm and 37°C for 1 hour. The transformed cells were coated on LB solid medium plates containing kanamycin resistance (final concentration of 50 μg / mL), cultured overnight at 37°C, four single clones were picked for sequencing, and the expression plasmid with correct sequencing was transformed into Escherichia coli BL21 (DE3), and the expression engineering bacteria of the mutant were successfully constructed.

[0093] WT and all mutants were recombinantly expressed and purified in the same manner as follows:

[0094] Inoculate fresh colonies into 1L LB medium containing 100g / mL Kan. Culture the cells in a shaker at 37°C (about 220rpm) until the optical density of OD600 reaches 0.6-0.8. Then add 1mM concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) to induce recombinant protein expression at 18°C ​​for 24 hours. Place the induced bacterial solution into a centrifuge bottle, centrifuge at 2600g, 4°C, for 30 minutes, resuspend the bacterial solution with an appropriate amount of Binding buffer, lyse with ultrasonic treatment, and centrifuge at 13000rpm, 4°C for 30 minutes. Then filter the supernatant using a 0.45-micron pore size filter membrane. After this, the protein is passed through a pre-equilibrated Ni-NTA column using Purification was performed using a pure chromatography system (GE Healthcare, Chicago, USA). Non-target proteins bound to the column were washed with a buffer solution (20 mM Tris, 300 mM NaCl, pH 8.0). Finally, the target protein was eluted by applying a linear gradient of imidazole (20-500 mM). The protein was concentrated to less than 5 mL using an ultrafiltration tube with a molecular weight cutoff of 3 KDa and then filtered through a pre-equilibrated Superdex 75 molecular sieve column (Cytiva, Shanghai, China). The protein was further purified using a pure chromatography system (GE Healthcare, Chicago, USA). The buffer system was 20 mM Tris, 80 mM NaCl, pH 8.0. The purity of the protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein concentration was measured by using a Nanophotometer NP80 Microvolume spectrophotometer (Implen, Germany). The extinction coefficient of the purified protein was 17210 M -1 cm -1 280nm. Store at -20℃.

[0095] Example 2

[0096] Since the 3D structure of the catalytic antibody UA15-VL is not available, alphafold2 was used to predict the protein structure model from its sequence. Most protein regions have high predicted local distance difference test (pLDDT) scores, indicating high overall structure prediction accuracy, except for the C-terminal residues, which have low pLDDTs, indicating that these residues are poorly predicted. Although the global melting temperature is usually obtained experimentally during the protein folding-unfolding process, MD simulations at multiple temperatures can provide information about protein unfolding hotspots, described by local melting temperatures ( Figure 1 ). A lower local melting temperature indicates that this region is unstable and therefore tends to unfold first as the temperature increases.

[0097] A series of MD simulations were performed for UA15-VL in the temperature range of 23.2 °C (250 K) to 326.8 °C (600 K), with two independent MD runs (15 ns each) at each temperature. If two residues were separated by more than four amino acids in the sequence and their closest distance in the starting structure was less than (between heavy atoms), the two residues are considered to be in contact with each other. A total of 207 contact points were identified, and the C-atom distances between each pair of contacting residues were recorded at each temperature and the average was calculated. Such distances are generally larger at higher temperatures due to unfolding. The distances are converted to effective contacts, ce, and contacts are quantitatively compared using the equation ce = exp(r / r0), where r is the distance between the two C atoms and r0 is a constant This equation ensures that as the distance increases, the effective contact decreases and the change in ce becomes smaller for larger distances. ce is averaged over the MD snapshots of the two runs and is obtained as <ce>, where <> represents the ensemble average. Then, the effective contact at different temperatures is scaled to ces(Ti) =<ce(Ti)> / <ce(T0)> ,in<ce(T0)> is the average contact at the lowest temperature (23.2°C) of the MD simulation. Therefore, ces at the lowest MD temperature is equal to 1. In this work, the average of the four lowest temperatures is used. <ce>The value is used as the denominator to minimize numerical noise. ces is plotted against temperature and fitted to a sigmoid function ( Figure 2 ).

[0098]

[0099] Where a, b and c are three fitting parameters, a corresponds to the ces value at the lowest temperature. b reflects the rate of decrease of ces, and c is the temperature at which ces equals a / 2. The temperature at which ces = 0.5 is defined as the local melting temperature Tmp of the contact pair. If the ces of a pair is greater than 0.5 even at the highest simulation temperature (326.8°C), its Tmp cannot be accurately extracted. Nevertheless, a value of 326.8°C is assigned to the Tmp of the pair, corresponding to the lower limit of this residue pair. The Tmps of different residue pairs are listed in Table 1. To simplify the data, the Tmp of the pair is evenly distributed to the two constituent residues. If a residue forms multiple contact pairs, the lowest Tmp is selected for that residue. Map the residue-specific Tmp to the 3D structure of the enzyme to directly view the weak points ( Figure 1 ).

[0100] Figure 1 To map Tmp to the 3D structure of UA15-VL. Figure 1 In the figure, the color gradient changes from red through white to blue as Tmp increases. Regions with lower Tmp values ​​tend to unfold first at high temperatures. Untouched residues are shown in gray. The residues involved in the designed mutations are labeled and their side chains are represented in stick form. In addition, the three complementarity determining regions (CDRs) are labeled.

[0101] It is not surprising that most hot spots are located in or near the flexible CDRs (Kabat numbering: CDR1, K24N34; CDR2, L50S56; and CDR3, W89T97) with low Tmp. However, CDRs are very important for the functional realization of UA15-VL. Therefore, mutations in CDRs should be avoided to prevent perturbation of protein function. As shown in Table 1 (and Figure 1 ), after excluding residues in the CDR region, the I48G64 pair has the lowest Tmp. In other words, it initiates the unfolding of the framework region (FR) of UA15-VL according to MD simulations. By strengthening the interactions in this hotspot, contacts can in principle be maintained at high temperatures.

[0102] Table 1 Tmps of different residue pairs

[0103]

[0104]

[0105]

[0106] The local melting temperatures Tmp of different residue pairs. The residue pairs that comprise the CDRs are in italics, and the other residue pairs are in normal font.

[0107] Example 3

[0108] Design of disulfide bonds to improve the thermal stability of antibodies that degrade Helicobacter pylori urease:

[0109] A disulfide bond is a simple interaction that prevents local unfolding. DbD2 predicted several potential disulfide bonds, including C3-C26, C6-C99, C14-C17, C23-C88, C32-C91, C34-C49, and C48-C64. The C23-C88 disulfide bond already exists in WT UA15-VL. Disulfide bonds C3-C26, C32-C91, and C34-C49 involve residues in CDR1, where the nucleophilic catalytic residue S27a is located. Mutations in these regions may impair antibody function. The C14-C17 disulfide bond connects two residues that are too close. The C6-C99 disulfide bond is far away from the hotspot, and only the C48-C64 disulfide bond is located in the I48-G64 hotspot region. To see if the C48-C64 disulfide bond does improve the thermal stability of the protein, Ua15-L (WT) and mutant 4I48C-G64C protein samples were prepared. Their melting temperatures were measured using the differential scanning calorimetry (DSC) method. Ua15-L (WT) showed two melting temperatures, Tm1 = 41.5 ± 0.3 ° C and Tm2 = 53.2 ± 0.7 ° C ( Figure 3 The mutant I48C-G64C also showed two melting temperatures, Tm1 = 51.1 ± 0.4 °C and Tm2 = 57.9 ± 0.3 °C ( Figure 3 Figure 2 (B) shows that the disulfide bonds increase Tm1 and Tm2 by 9.6°C and 4.7°C, respectively. The increase in melting temperature suggests that the additional disulfide bonds help stabilize the protein and prevent unfolding at high temperatures.

[0110] Example 4

[0111] Adjusting the solubility and expression level of catalytic antibodies that degrade Helicobacter pylori urease:

[0112] During the preparation of catalytic antibodies against Helicobacter pylori urease, it was observed that the protein solution became turbid when it was concentrated using a concentrator. The solubility of Ua15-L (WT) was about 1.1 ± 0.1 mg / mL in a buffer of 20 mM Tris, 80 mM NaCl (pH 7.0), which was relatively low (Table 2). The introduction of the I48C-G64C mutation further reduced the solubility to 0.4 ± 0.1 mg / mL. High protein solubility is a prerequisite for potential therapeutic applications. In order to enhance protein solubility without sacrificing stability, a combined FoldX and CamSol approach was used, the former for stability prediction and the latter for solubility prediction. This approach was used to identify mutations (above I48C-G64C) that may have favorable solubility and stability properties, including L9E, L54R and T14Q, I15P. First, the mutant 1L9E-I48C-G64C was tested experimentally, and its solubility was 2.0±0.2 mg / mL, which was 4 times higher than that of the I48C-G64C mutant and 82% higher than that of Ua15-L (WT). The mutant 1L9E-I48C-G64C also showed two melting temperatures, Tm1=47.5±0.6℃ and Tm2=63.6±0.7℃ ( Figure 3 (C) Tm1 is about 3°C ​​lower than that of the I48C-G64C mutant, but Tm2 is about 6°C higher, indicating that the L9E mutation has a rather complex effect on the thermal stability of the protein.

[0113] Starting from the L9E-I48C-G64C mutation, either L54R or T14Q-I15P mutations were added. The solubility of mutant 2 L9E-T14Q-I15P-I48C-G64C was 2.9 ± 0.4 mg / mL, which is approximately 2.7 times that of Ua15-L (WT) and 1.5 times that of mutant 1 L9E-I48C-G64C, consistent with CamSol solubility predictions. However, the T14Q-I15P mutation reduced the melting temperature Tm1 to 42.8 ± 0.9 °C and Tm2 to 59.7 ± 0.5 °C ( Figure 3 D), indicating that thermal stability is compromised. The L54R mutation (above L9E-I48C-G64C) increased the protein solubility to 2.6±0.3 mg / mL, compared to 2.0±0.2 mg / mL for mutant 1 L9E-I48C-G64C. Measurements of protein thermal stability showed that mutant 3 L9E-I48C-G64C-L54R mutant had only one melting temperature, Tm=52.5±0.2°C, which was about 5°C higher than the Tm1 of the L9E-I48C-G64C mutant and about 1°C higher than the I48C-G64C mutant ( Figure 3 Middle E).

[0114] Table 2 Solubility and expression of Ua15-L (WT) and its mutants

[0115]

[0116] a Represents the solubility measured in a buffer of 20 mM Tris, 80 mM NaCl, pH 7.0, at 4°C.

[0117] In addition to protein thermal stability and solubility, high protein expression is beneficial for experimental purposes in drug development and future applications because it reduces the economic cost of the drug. The yield of wild-type UA15-VL prepared in E. coli was relatively low, with 0.1 mg of protein per liter of growth medium after purification. The double mutant I48C-G64C with an additional disulfide bond reduced the protein amount to 0.07 mg / L. The triple mutant L9E-I48C-G64C increased the protein yield to 0.5 mg / L. The quadruple mutant L9E-I48C-G64C-L54R increased the yield to approximately 0.6 mg / L, while the pentamutant L9E-T14Q-I15P-I48C-G64C reduced the protein yield to approximately 0.3 mg / L. It appears that L9E-I48C-G64C and L9E-I48C-G64C-L54R have different thermal stability ( Figure 3 ), solubility, and expression (Table 2).

[0118] Example 5

[0119] Proteolytic activity of Ua15-L (WT) and its mutants:

[0120] The urease of Helicobacter pylori is a hexameric enzyme consisting of six α subunits and six β subunits. UA15-VL targets the β subunit and is able to cleave the peptide bond between Y241 and D242. A fluorescent protein ligand consisting of CFP-pep-YFP, where pep corresponds to the peptide sequence NHALDVADKYDVQVAIHTDT (from N232 to T251) in the urease β subunit, was used to test the proteolytic activity of catalytic antibodies and their mutants that degrade H. pylori urease. The cleavage of the peptide bond reduced the resonance energy transfer (FRET) between CFP and YFP, resulting in an increase in cyan fluorescence and a decrease in yellow fluorescence. The cleavage reaction showed a biphasic reaction spectrum, which is a unique property of some catalytic antibodies. A sigmoid function was proposed to fit the reaction curve, and the parameter T1 / 2 was obtained, which corresponds to the time point when half of the substrate is converted to the product. The T1 / 2 of Ua15-L (WT) at 25°C was 1600 ± 30 min ( Figure 4 ). All three mutants, L9E-I48C-G64C, L9E-I48C-G64C-L54R, and L9E-T14Q-I15P-I48C-G64C, showed T1 / 2 comparable to that of Ua15-L (WT), indicating that the mutations had little effect on catalysis. This is not surprising, as all mutation sites were carefully chosen away from the active site. In contrast, at 37°C, significantly larger changes were observed for the different mutants. Wild-type UA15-VL exhibited a T1 / 2 of 2170 ± 290 min, 36% higher than its activity at 25°C, suggesting that the activity may be reduced due to its poor thermostability (activity measurement temperature was close to the protein Tm1 of 41.5°C). In contrast, mutant 1L9E-I48C-G64C reduced its T1 / 2 to 1050 ± 50 minutes, showing about 46% higher efficiency than its activity at 25 °C and almost twice the activity of the wild type at 37 °C, which is attributed to its improved thermostability. Mutant 2L9E-T14Q-I15P-I48C-G64C significantly increased its T1 / 2 to 3360 ± 300 minutes, while mutant 3L9E-I48C-G64C-L54R slightly increased its T1 / 2 to 1650 ± 350 minutes. Therefore, the activity measurements clearly showed that mutant 1L9E-I48C-G64C exhibited superior performance than other mutants at 37 °C.

[0121] In this paper, we present a combined computational and experimental study to engineer catalytic antibodies that degrade H. pylori urease to produce catalytic antibodies with high thermal stability, high solubility, and enhanced expression. These properties are relevant to the development of protein drugs, resulting in extended shelf life, improved drug delivery, and reduced cost of drug availability. To improve protein thermal stability, multi-temperature MD simulations were performed to identify unfolding hotspots of catalytic antibodies that degrade H. pylori urease and improve protein thermal stability without interfering with protein function. Therefore, hotspots near CDRs and active sites, which may consist of the D1-H93-S27a catalytic triad, should be avoided. With the help of the DbD2 program, a disulfide bond was designed in one of the hotspots, which increased the protein melting temperature by 7-10°C. The mutations significantly reduced the solubility and expression of the protein. With the help of FoldX and CamSol methods, additional mutations L9E, L54R, and T14Q-I15P were introduced to increase protein solubility and hopefully restore protein expression without sacrificing thermal stability. The combined mutation L9E-I48C-G64C increased Tm1 by 6°C, solubility by 81%, and expression by 4-fold compared to Ua15-L(WT). This mutant showed comparable proteolytic activity to Ua15-L(WT) at 25°C, but due to better stability, the time required for the urease fragment to be hydrolyzed by half at 37°C was 52% shorter than that of the wild type.

[0122] When optimizing protein solubility, it is important not to compromise protein thermal stability. The effects of mutations on protein stability were assessed using FoldX, while the effects on solubility were assessed using CamSol. These two methods provide a list of mutations that are likely to enhance stability and solubility. All experimentally tested mutants increased protein solubility, but only L54R showed a slight improvement in protein thermal stability, indicating that computational methods are not very accurate for stability. This computational strategy could be improved by including more protein thermal stability and solubility prediction methods to come up with a more reliable list of mutations for experimental validation.

[0123] In conclusion, we successfully identified a catalytic antibody mutant L9E-I48C-G64C for degrading H. pylori urease, which has enhanced thermal stability, solubility, expression level and a 52% reduction in the half-time of hydrolysis of the urease fragment at 37°C compared with the wild type.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.< / ce> < / ce>

Claims

1. A catalytic antibody mutant for degrading Helicobacter pylori urease, characterized in that: Any one or more sites selected from the following group are mutated: L9E, T14Q, I15P, I48C, G64C, L54R; among which, the amino acid residue numbering adopts SEQ ID NO.1 (amino acid sequence of wild-type catalytic antibody that degrades Helicobacter pylori urease (Ua15-L (WT)), MGSSHHHHHHSSGLVPRGSHMDVVMTQTPLTLSVTIGQPASISCKSSQSLLDS DGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEA EDLGVYYCWQGTHFPQTFGGGTKLEIK).

2. The catalytic antibody mutant for degrading Helicobacter pylori urease according to claim 1, characterized in that: The catalytic antibody mutant for degrading Helicobacter pylori urease is mutated based on the wild-type catalytic antibody for degrading Helicobacter pylori urease shown above, and the catalytic antibody mutant for degrading Helicobacter pylori urease is selected from the mutants in the following group: Mutant 1: L9E-I48C-G64C; (amino acid sequence is shown in SEQ ID NO.2); Mutant 2: L9E-T14Q-I15P-I48C-G64C; (amino acid sequence is shown in SEQ ID NO.3); Mutant 3: L9E-I48C-G64C-L54R. (the amino acid sequence is shown in SEQ ID NO.4).

3. The catalytic antibody mutant for degrading Helicobacter pylori urease according to claim 2, characterized in that: In mutants 1 to 3, the amino acids at positions 48 and 64 mutated from isoleucine and glycine to cysteine, respectively, and a disulfide bond was formed between the two.

4. A polynucleotide, characterized in that The polynucleotide encodes the catalytic antibody mutant for degrading Helicobacter pylori urease according to any one of claims 1 to 3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell contains the vector according to claim 5 or has the polynucleotide according to claim 4 integrated into its chromosome, or expresses the catalytic antibody mutant for degrading Helicobacter pylori urease according to any one of claims 1 to 3.

7. A method for preparing the above-mentioned catalytic antibody mutant for degrading Helicobacter pylori urease, characterized in that: include: Cultivating the host cell of claim 6, thereby expressing the catalytic antibody mutant that degrades Helicobacter pylori urease; And the catalytic antibody mutant that degrades Helicobacter pylori urease is isolated and purified.

8. Use of the catalytic antibody mutant capable of degrading Helicobacter pylori urease according to any one of claims 1 to 3 in the preparation of a drug for improving, preventing or treating Helicobacter pylori infection.

9. A drug for improving, preventing or treating Helicobacter pylori infection, characterized in that: The invention comprises the catalytic antibody mutant for degrading Helicobacter pylori urease as described in any one of claims 1 to 3.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the catalytic antibody mutant for degrading Helicobacter pylori urease according to any one of claims 1 to 3 and one or more pharmaceutically acceptable carriers.

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