A recombinant a-l-iduronidase and a method for preparing the same

Abstract

The application discloses an isolated nucleotide sequence, comprising a signal peptide sequence and an a-L-iduronate pyruvase-encoding nucleotide sequence, wherein the signal peptide sequence is not a natural signal peptide sequence of the a-L-iduronate pyruvase. By replacing the natural signal peptide with an artificial signal peptide, the expression amount of the a-L-iduronate pyruvase can be improved, a protein with a single cleavage point and correct post-translational modification (N-glycosylation phosphorylation) can be produced, the protein can cross the cell membrane, and the enzyme activity of the protein can be exerted in the cell.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a recombinant aL-iduronase and its preparation method. Background Technology

[0002] Mucopolysaccharidosis (MPS) is a rare disease primarily caused by a deficiency of hydrolytic enzymes in lysosomes required to degrade mucopolysaccharides, leading to the accumulation of large amounts of mucopolysaccharides in tissues. MPS type I (MPS I) is a metabolic disorder syndrome caused by a deficiency of alpha-L-iduronidase. The most severe subtype, also known as Hurler syndrome, often results in death around the age of 10. Clinically, it presents with a variety of symptoms including intellectual disability, facial disfigurement, hepatosplenomegaly, skeletal abnormalities, cardiovascular disease, corneal opacity, and deafness. Currently, in addition to symptomatic treatment, bone marrow transplantation and gene therapy are available, but these treatments are complex, high-risk, and have uncertain efficacy. Recombinant enzyme replacement therapy is currently the only proven effective drug therapy. Administered intravenously, it carries low risk but requires lifelong treatment and is very expensive.

[0003] Aldurazyme is a recombinant alpha-L-Iduronidase jointly developed by Genzyme and BioMarin for enzyme replacement therapy of MPS-I. It is also currently the only commercially available specific drug for the treatment of MPS-I.

[0004] aL-iduronase is present in lysosomes in the cytoplasm of mammalian cells and is undetectable in normal human serum. Its amino acid sequence contains an atypical signal peptide sequence (1-27: MRPLRPRAALLALLASLL AAPPVAPAE) [Uniprot https: / / www.uniprot.org / uniprot / P35475]; however, signal peptide prediction software [SignalP] analysis shows that its signal peptide has at least three cleavage sites. Figure 10Furthermore, the signal peptide cleavage site is not at amino acids 27-28. Therefore, the secretion process guided by the signal peptide of aL-iduronase is not the classic post-translational secretion process from the endoplasmic reticulum to the extracellular space, but rather likely primarily guides the translated protein from the endoplasmic reticulum to the lysosome. Since the 1990s, attempts have been made to extract natural enzymes from different tissues, revealing significant differences in the amino termini of enzymes from different tissue sources [Clements et al., 1989]. This discovery indirectly confirms that the so-called 'secretory signal peptide' of aL-iduronase is not the typical signal peptide of a secretory protein. Nevertheless, in 1968, Neufeld discovered that aL-iduronase can still be secreted from cells and taken up by surrounding genetically defective cells, alleviating the phenotype of cytoplasmic mucopolysaccharide accumulation [Fratantoni et al., 1968]. This discovery became a foundational finding that later promoted the use of genetic engineering to produce aL-iduronase for clinical treatment.

[0005] Further research revealed several post-translational modifications on aL-iduronase, including common N-glycosylation and two bisphosophorylated oligomannosidic glycans. Glycosylation at amino acid 347 is essential for substrate recognition [Maita et al., 2013], while bisphosophorylated oligomannosidic glycans at amino acids 311 and 390 are key modifications for binding to the mannose-6-phosphate receptor (M6PR) and achieving internalization. The M6PR allows the enzyme to cross the cell membrane, enter the cytoplasm from the extracellular space, and then enter the lysosome. Therefore, post-translational modifications are considered indispensable for the normal activity of aL-iduronase. Since the formation process of these specific post-translational modifications (N-glycosylation and phosphorylation) remains unclear, it is speculated that they may occur during secretion.

[0006] Based on the complex post-translational modifications (N-glycosylation and phosphorylation) of aL-iduronase, the first-generation recombinant enzyme (Aldurazyme) cautiously utilized its own natural signal peptide and was produced in CHO cells under 10% FBS medium conditions via DNA recombination technology, obtained from the culture supernatant. However, its yield was low, approximately 20-40 μg / day / 10E7 cells. This low expression level presented many challenges for subsequent purification; especially if the natural signal peptide of aL-iduronase was used, it could lead to inconsistent amino terms, greatly limiting the purity and homogeneity of the recombinant enzyme.

[0007] Therefore, those skilled in the art are dedicated to developing a recombinant aL-iduronase that can increase the expression level of aL-iduronase and its preparation method. Summary of the Invention

[0008] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a recombinant aL-iduronase that can improve the expression level of aL-iduronase and its preparation method.

[0009] To achieve the above objectives, one aspect of the present invention provides an isolated nucleotide sequence comprising a signal peptide sequence and a nucleotide sequence encoding aL-iduronase, wherein the signal peptide sequence is not the natural signal peptide sequence of aL-iduronase.

[0010] Furthermore, the signal peptide is selected from sequences such as those shown in SEQ ID No:1, SEQ ID No:2, SEQ ID No:3 or SEQ ID No:4.

[0011] Furthermore, the nucleotide sequence encoding aL-iduronase is shown in SEQ ID No:9.

[0012] Optionally, the isolated nucleotide sequence is selected from the following sequences:

[0013] 1) SEQ ID No:5, SEQ ID No:6, SEQ ID No:7 or SEQ ID No:8;

[0014] 2) A nucleotide sequence that has at least 80% homology with SEQ ID No:5, SEQ ID No:6, SEQ ID No:7 or SEQ ID No:8;

[0015] 3) The codon encoding the polypeptide is degenerate from the coding portion of the nucleotide sequence in 1) or 2), and encodes the nucleotide sequence of aL-iduronase.

[0016] A second aspect of the present invention provides a recombinant aL-iduronase, comprising aL-iduronase and a heterologous signal peptide, the heterologous signal peptide being cleaved at a single site.

[0017] Optionally, its amino acid sequence is selected from the following sequences:

[0018] 1) SEQ ID No:10, SEQ ID No:11, SEQ ID No:12 or SEQ ID No:13;

[0019] 2) An amino acid sequence that has at least 95% homology with SEQ ID No:10, SEQ ID No:11, SEQ ID No:12 or SEQ ID No:13.

[0020] Furthermore, the recombinant aL-iduronase has glycosylation modifications at positions 311, 390, and 426.

[0021] Furthermore, the glycosylation modification is GlcNAc(2)Man(7)P(2).

[0022] Furthermore, the internalization of recombinant aL-iduronase is mediated by M6PR.

[0023] A third aspect of the present invention provides a method for preparing aL-iduronase, using the isolated nucleotide sequence as described above for eukaryotic expression.

[0024] Optionally, the KOZARK sequence is added before the isolated nucleotide sequence, constructed into a eukaryotic expression vector, and then transferred into a eukaryotic expression system for expression.

[0025] Optionally, the KOZARK sequence is GCCGCCACCATGC.

[0026] Alternatively, eukaryotic expression may be performed in mammalian cells.

[0027] Optionally, the mammalian cells are CHOK1SV GS-KO.

[0028] Further, the preparation method includes: constructing a recombinant eukaryotic expression vector containing the isolated nucleotide sequence; transferring the recombinant eukaryotic expression plasmid into a eukaryotic expression system for eukaryotic expression; collecting the supernatant and purifying it to obtain the aL-iduronidase.

[0029] Further purification includes one or more of affinity chromatography, hydrophobic interaction chromatography, and ion exchange chromatography.

[0030] Optionally, purification can be performed in three steps, including affinity chromatography, hydrophobic interaction chromatography, and ion exchange chromatography.

[0031] Optionally, affinity chromatography involves linear gradient elution using an agarose gel to obtain an eluent containing the target protein. The elution buffer is buffer B, consisting of 20 mM PB, 2 M NaCl, and pH 5.3.

[0032] Optionally, hydrophobic interaction chromatography uses a hydrophobic column to remove impurities such as proteins and polymers. Before sample loading, 4M NaCl is added to a final concentration of 2M, and the pH is adjusted to 5-6 with 1M NaOH. The target protein is then eluted isocratically with elution buffer. The elution buffer is: 20mM PB + 0.15M NaCl + pH 5.5.

[0033] Optionally, ion exchange chromatography uses a salt-tolerant cation exchange column. After sample loading and equilibration, the target protein is eluted with elution buffer at a linear gradient of 0-70%. The elution buffer is 20 mM PB + 1 M NaCl, pH 5.5.

[0034] Optionally, the purified solution is concentrated by 30 kDa membrane ultrafiltration and then replaced with phosphate buffer containing 0.0001% Tween 80 pH 5.5.

[0035] The use of the heterologous signal peptide in this invention not only increases the expression level of recombinant aL-iduronase, but also produces a protein with a single cleavage site and correct post-translational modification (N-glycosylation and phosphorylation), which can cross the cell membrane and exert enzymatic activity within the cell.

[0036] The following will further explain the concept, specific steps, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0037] Figure 1 This is an electrophoresis diagram of cell supernatant proteins expressing five different signal peptides of aL-iduronase in mammalian cells, as shown in Example 2 of this invention.

[0038] Figure 2 It is a first-level LC-MS map of the N-terminal sequence analysis of commercial Aldurazyme;

[0039] Figure 3 This is a first-order LC-MS spectrum of the N-terminal sequence of ASP-1-Laronidase prepared in Example 3 of this invention;

[0040] Figure 4 (A) is a secondary LC-MS / MS spectrum of N-glycosylation phosphorylation analysis of commercial Aldurazyme; (B) is the theoretical molecular weight of the peptide fragment ions matched in this secondary LC-MS / MS spectrum (mass deviation less than 20 ppm).

[0041] Figure 5(A) is a secondary LC-MS / MS spectrum of N-glycosylation phosphorylation analysis of ASP-1-Laronidase prepared in Example 3 of the present invention; (B) is the theoretical molecular weight of the polypeptide fragment ions matched in this secondary LC-MS / MS spectrum (mass deviation less than 20 ppm);

[0042] Figure 6 This is a schematic diagram of 4-methylumbelliferone, a product with a fluorescent signal, produced by the degradation of the substrate Alpha-L tetrahydroxyepoxyvalerate-4-methylumbelliferate by aL-iduronase.

[0043] Figure 7 This is a graph showing the enzyme activity results of ASP-1-Laronidase and commercially available Aldurazyme in Example 4 of this invention;

[0044] Figure 8 This is a Western blotting diagram illustrating the efficiency of ASP-1-Laronidase and commercially available Aldurazyme entering HEK293 cells under M6P presence and absence conditions. HEK293 cells were cultured for 3 days in media containing 0 μg / ml ASP-1-Laronidase and Aldurazyme (lane 1), 10 μg / ml ASP-1-Laronidase (lane 2), 10 μg / ml Aldurazyme (lane 4), 10 μg / ml ASP-1-Laronidase + 10 mM / L M6P (lane 3), and 10 μg / ml Aldurazyme + 10 mM / L M6P (lane 5). Lysis was then collected, and 50 ng of total protein was analyzed by Western blotting. St: (ASP-1-Laronidase);

[0045] Figure 9 This is a comparison diagram of the cell internalization experiment of ASP-1-Laronidase and commercially available Aldurazyme in Example 6 of this invention;

[0046] Figure 10 The signal peptide prediction software [SignalP] was used to analyze the cleavage positions of atypical signal peptide sequences in the amino acid sequence of aL-iduronase. Detailed Implementation

[0047] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0048] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, all reagents used were commercially available or publicly available.

[0049] In this invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids.

[0050] According to one specific embodiment of the present invention, an artificial signal peptide sequence is added before the encoding nucleotide sequence of aL-iduronase to form a recombinant nucleotide sequence, and recombinant aL-iduronase is obtained by eukaryotic expression using the recombinant nucleotide sequence. In one specific embodiment of the present invention, the four artificial signal peptides used (sequences shown in SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, or SEQ ID No:4) can effectively improve the expression level of aL-iduronase and obtain aL-iduronase with a single cleavage site and correct post-translational modification (N-glycosylation phosphorylation).

[0051] Example 1: Synthesis of 5 plasmids for expressing αL-iduronase of 5 different signal peptides.

[0052] Laronidase plasmids containing four different signal peptides (ASP-1, SEQ ID No:1; ASP-2, SEQ ID No:2; ASP-3, SEQ ID No:3; ASP-4, SEQ ID No:4) and laronidase plasmids containing natural signal peptides (NSPs) were synthesized by Genewiz. The nucleotide sequences of the enzymes containing artificial signal peptides are shown in SEQ ID No:5 (ASP-1), SEQ ID No:6 (ASP-2), SEQ ID No:7 (ASP-3), and SEQ ID No:8 (ASP-4); the nucleotide sequence of the enzyme containing the natural signal peptide is shown in SEQ ID No:14, and the amino acid sequence is shown in SEQ ID No:15. The vector used was pZD, and its nucleotide sequence is shown in SEQ ID No:16.

[0053] Example 2: Expression and quantification of αL-iduronase of 5 different signal peptides in mammalian cells.

[0054] 2.1 aL-iduronase expressing five different signal peptides in mammalian cells

[0055] Plasmids were extracted from ASP-1-Laronidase-pZD, ASP-2-Laronidase-pZD, ASP-3-Laronidase-pZD, ASP-4-Laronidase-pZD, and NSP-Laronidase-pZD, premixed with PEI at a ratio of 1:5, and transfected into HEK293 cells. After 5 days, the supernatant was collected, cell debris was removed, and the pH was adjusted to 5.5 with 1M phosphate. 25 μL of each expression supernatant was added to 5 μL of protein loading buffer, and the mixture was boiled at 95°C for 5 minutes. A 12% SDS-protein electrophoresis was then performed.

[0056] See protein electrophoresis results Figure 1 Electrophoresis results of cell supernatants expressing Laronidase using five different signal peptides clearly show that the expression level of Laronidase using four heterologous signal peptides is higher than that using the natural signal peptide NSP.

[0057] 2.2 Quantitative analysis of supernatant

[0058] The RP-HPLC method can be used to determine the content of laronidase in samples.

[0059] The RP-HPLC quantitative method was performed on an Agilent HPLC system using the Waters Empower3 network-based HPLC workstation control system for data acquisition and analysis. An ACE 5C4-300 HPLC column (250 x 4.6 mm I.D.) was used for detection. The run time for each sample was 30 minutes. The flow rate was 0.8 mL / min, the column temperature was 75 °C, and the detection UV absorbance was 215 nm. Mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid and 10% acetonitrile, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. In the Agilent HPLC system, the system was first equilibrated with 20% buffer B for 3 minutes. At 3.1 minutes, the proportion of buffer B was increased to 31% (i.e., the initial proportion of buffer B in the elution range was X%*), and isocratic elution was performed for 6 minutes. The target protein was eluted with a linear gradient of 31%–43% buffer B over 15 minutes (i.e., a linear change in buffer B from X% to [X+12]% over 15 minutes). The column was flushed with 90% B for 4 minutes before rebalancing.

[0060] The results are shown in Table 2. Comparison of the four heterologous signal peptides with NSP-Laronidase showed that the expression levels of Asp-1-Laronidase, Asp-2-Laronidase, Asp-3-Laronidase, and Asp-4-Laronidase were all significantly increased. Specifically, the expression levels of Asp-1-Laronidase and Asp-4-Laronidase were 3.38 times and 3.59 times that of NSP-Laronidase, respectively.

[0061] Table 2. Transient expression of five different signal peptides of laronidase in HEK293 cells - UPLC quantification.

[0062] ASP-1-Laronidase 12.5 ASP-2-Laronidase 9.6 ASP-3-Laronidase 6.9 ASP-4-Laronidase 13.3 NSP-Laronidase 3.7

[0063] Example 3: Mass spectrometry N-terminal analysis and N-glycosylation phosphorylation analysis of ASP-1-Laronidase protein

[0064] 3.1 A suitable KOZAK sequence (GCCGCCACCATGC, SEQ ID NO: 17) was added before the ASP-1-Laronidase sequence. The ASP-1-Laronidase plasmid was synthesized by Genewiz using the plasmid provided by Lonza as the vector.

[0065] 3.2 Using the Lonza CHOK1SV GS-KO cell line (https: / / go2.lonza.com / Biologics_CHOK1SV.html), single-clone construction and screening were performed according to the construction and screening procedures provided by Lonza. The obtained stable single-clone cell lines were then expressed according to the culture methods provided by Lonza. Nine days after expression, the cell supernatant was harvested and purified to obtain high-purity ASP-1-Laronidase protease.

[0066] The purification process involved three steps: affinity chromatography, hydrophobic interaction chromatography, and ion exchange chromatography. Specifically, the cell culture supernatant was adjusted to pH 5.3 with 1M phosphate and clarified by deep membrane filtration. The filtered supernatant was then loaded onto a Blue Sepharose 6Fast Flow (Cytiva 17-0948-01) column equilibrated with 20mM PB, 0.15M NaCl, and pH 5.3. After loading, the column was washed with equilibration buffer until baseline stability was achieved to remove most non-specifically bound impurities (such as host proteins and host DNA). Finally, a linear gradient elution was performed using 0-80% buffer B (20mM PB, 2M NaCl, pH 5.3), and the elution peaks containing the target protein were analyzed by SDS-PAGE and combined.

[0067] The Blue Sephaorse 6Fast flow collection solution was purified using a hydrophobic chromatography column (such as Cytiva Phenyl Sepharose Fast Flow (High Sub), Butyl Sepharose 4Fast Flow, Tosoh Butyl-650M, Phenyl-5PW, or Borglon Phenyl Bestarose FF (HS)) to remove impurities such as proteins and polymers. Before loading, 4M NaCl was added to a final concentration of 2M, and the pH was adjusted to 5.5 with 1M NaOH. The solution was then loaded onto a hydrophobic chromatography column equilibrated with 20mM PB, 2M NaCl, and pH 5.5. After loading, the solution was eluted to baseline using equilibration buffer. The target protein was eluted isocratically with 20mM PB, 0.15M NaCl, and pH 5.5. The eluent containing the target protein was collected.

[0068] ASP-1-Laronidase was further purified using salt-tolerant cation exchange columns (such as Sulfate-650F (Tosoh), POROS XS (Thermo Scientific), Eshmuno-CPS (Merck), etc.). The hydrophobic chromatography collection buffer was loaded onto a salt-tolerant cation exchange column equilibrated with 20 mM PB, 0.15 M NaCl, and pH 5.5. After loading, the sample was eluted with equilibration buffer until the baseline stabilized. Finally, the target protein was eluted with a linear gradient of 20 mM PB, 1 M NaCl, and pH 5.5 from 0-70%. The eluent was collected. The final sample was concentrated using 30 kDa membrane ultrafiltration and the buffer was replaced with phosphate buffer containing 0.0001% Tween 80 pH 5.5.

[0069] 3.3 ASP-1-Laronidase and commercially available Aldurazyme were sent to Chia Tai Health Technology Co., Ltd. for N-terminal protein analysis using LC-MS / MS. The results are shown in Table 3. Figure 2 , Figure 3 , Figure 4 , Figure 5 .

[0070] Table 3. LC-MS / MS of N-terminal peptides generated by trypsin digestion of commercially available Aldurazyme and ASP-1-Laronidase.

[0071]

[0072] From Table 3, Figures 2-5 As can be seen from the LC-MS / MS results, the location of the ASP-1 signal peptide cleavage after intracellular protein processing is consistent with that of the commercially available Aldulazyme.

[0073] In this embodiment, the IDUA gene expressing laronidase was selected as shown in SEQ ID No:9. The protein expressed by this gene differs from that of the commercially available Aldurazyme at the 8th amino acid position (excluding the signal peptide). In this embodiment, laronidase is 8Q, while the commercially available Aldurazyme is 8H. This is based on the polymorphism of the IDUA gene, with the former accounting for approximately 85% of the population.

[0074] 3.4 The high-purity protein was sent to Chia Tai Health Technology Co., Ltd. for N-glycosylation and phosphorylation analysis. The results are shown in Table 4.

[0075] Table 4. LC-MS / MS quantification results of glycopeptides hydrolyzed by chymotrypsin in the samples.

[0076]

[0077] As shown in Table 4, the glycosylation modifications of commercialized Aldurazyme and ASP-1-Laronidase are basically the same.

[0078] Among them, the modification sites at positions 311, 390, and 426 are the same as those at positions 311, 390, and 426 of Laronidase, which does not contain a signal peptide.

[0079] Example 4 Enzyme activity identification of ASP-1-Laronidase and commercially available Aldurazyme

[0080] 4.1 Prepare a sodium acetate solution with a pH of 3.5 as a reaction buffer.

[0081] 4.2 Prepare a glycine solution with a pH of 10.7 as the stop solution.

[0082] 4.3 Prepare a 4 mg / mL stock solution of substrate (Alpha-L tetrahydroxyepoxyvalerate-4-methylumbelliferate) using DMSO and store at -20°C.

[0083] 4.4 Dilute the substrate stock solution to 800 μM with reaction buffer for later use.

[0084] 4.5 ASP-1-Laronidase and commercially available Aldurazyme were diluted to 2 μM in 0.9% sodium chloride solution, serially diluted 10-fold to 0.0002 μM, and a blank control (i.e., 0.9% sodium chloride solution without enzyme) was added.

[0085] 4.6 Take 25 μl of substrate and 25 μl of enzyme and sodium chloride solution diluted to different concentrations in 4.5 and mix them in a 96-well fluorescent detection plate. After incubating at 37°C for 20 minutes, add 200 μl of stop solution to terminate the reaction.

[0086] 4.7 Place the 96-well fluorescence detection plate into the microplate reader for detection. Set the microplate reader parameters as follows: excitation 360 / 40nm, emission 460 / 40nm.

[0087] ASP-1-Laronidase and commercially available Aldurazyme can decompose the substrate Alpha-L-tetrahydroxyepoxyvalerate-4-methylumbelliferone to produce a fluorescently signaling product, 4-methylumbelliferone. Figure 6 As shown.

[0088] ASP-1-Laronidase and commercially available Aldurazyme were reacted with substrates of the same molar concentration and volume using the same reaction conditions. The resulting products were then analyzed for fluorescence intensity; the magnitude of the fluorescence signal directly reflects the enzyme activity. The detection results are shown below. Figure 7 As shown, the results indicate that ASP-1-Laronidase and commercially available Aldurazyme have comparable enzyme activities at the same molar concentration and volume.

[0089] Example 5: The internalization of both ASP-1-Laronidase and commercially available Aldurazyme is mediated by M6PR.

[0090] 5.1 Day 0

[0091] 5.1.1 Digest HEK293 cells in T25 flasks with 500 μL of trypsin for 1-2 min;

[0092] 5.1.2 Add 5 ml of DMEM (10% FBS + 25 ug / ml G418), blow the cells apart, and mix well;

[0093] 5.1.3 Perform cell counting, then passage the cells into 0.2 × 10⁻⁶ cells. 6 / ml, 5ml / T25 bottles, 5 bottles in total;

[0094] 5.1.4 After 3-4 hours, take the four flasks of cells from 5.1.3 and add 10ug / ml of ASP-1-Laronidase (without M6P), Aldurazyme (without M6P), ASP-1-Laronidase (containing 10mM / L M6P), and Aldurazyme (containing 10mM / L M6P) respectively.

[0095] 5.2 Day 3

[0096] 5.2.1 Digest HEK293 cells cultured for 3 days after the above treatment with trypsin. Add 500 μL of trypsin to each T25 flask and digest for 1-2 min.

[0097] 5.2.2 Add 3 ml of DMEM (10% FBS + 25 ug / ml G418), blow the cells apart, mix well, and count;

[0098] 5.2.3 Based on the counting, calculate 3 * 10 6 The volume of cells was taken. The corresponding volume was centrifuged, the culture medium was discarded, and the cells were washed twice with PBS.

[0099] 5.2.4 The protein was lysed using sonication, and the total protein concentration was measured using a BCA assay kit;

[0100] 5.2.5 Take another 50 μg of sonicated protein for Western blotting. The protein samples were first separated by SDS-PAGE, then transferred to a PVDF membrane (Roche) using a semi-dry method. After blocking, primary antibody (Sheep anti-Laronidase antibody) and secondary antibody (Anti-Sheep IgG) were added sequentially, followed by incubation. Finally, chromogenic buffer was added, and the samples were developed and photographed. The results are shown below. Figure 8 As shown.

[0101] like Figure 8 As shown, in the presence of M6P, after culturing HEK293 cells in medium containing ASP-1-Laronidase or commercially available Aldurazyme for 3 days, neither ASP-1-Laronidase nor Aldurazyme was detected in the HEK293 cell lysates. This indicates that the internalization of both is mediated by M6PR.

[0102] Example 6: Identification of the intracellularization efficiency of ASP-1-Laronidase and commercially available Aldurazyme in HEK293 cells.

[0103] 6.1 HEK293 cells were cultured at a rate of 0.2 × 10⁻⁶. 6 The cells were seeded at a density of 1 ml into 6-well plates, with a volume of 2 ml per well.

[0104] 6.2 ASP-1-Laronidase and commercially available Aldurazyme were added to the above HEK293 culture medium at concentrations of 40 μg / ml, 20 μg / ml, 10 μg / ml, 0.055 μg / ml, and 0.0275 μg / ml, respectively, and incubated on a shaker.

[0105] 6.3 After incubation for 2.5 hours, approximately 2 ml of cells were transferred to new centrifuge tubes, centrifuged at 200g for 5 minutes, and resuspended in PBS. This washing process was repeated three times. At this point, the internalization process of ASP-1-Laronidase and commercially available Aldurazyme was terminated. The washed cells were then lysed in 300 μl of cell lysis buffer (Thermo / #78501). The resulting cell lysate can be considered a crude enzyme solution of ASP-1-Laronidase or Aldurazyme.

[0106] 6.4 The concentration of total protein in the above crude enzyme solution was quantified using a BCA kit.

[0107] 6.5 Prepare a sodium acetate solution with a pH of 3.5 as a reaction buffer.

[0108] 6.6 Dilute the substrate stock solution (Alpha-L tetrahydroxyepoxyvalerate-4-methylumbelliferate) described in 4.3 to 360 μM using reaction buffer for later use.

[0109] 6.7 Preparation of untreated (i.e., culture medium never containing enzymes) HEK293 cell lysates. Take 15 ml of 0.2 × 10⁻⁶ cells... 6 Transfer / ml of HEK293 cells to a new centrifuge tube, centrifuge at 200g for 5 minutes, resuspend the cells with PBS, repeat the washing 3 times, and then add 7ml of cell lysis buffer for lysis.

[0110] 6.8 The product (4-methylumbelliferone) was diluted with a mixture (50% untreated HEK293 cell lysate as described in 6.7 + 50% reaction buffer) to 1.6 μg / ml, 0.8 μg / ml, 0.4 μg / ml, 0.2 μg / ml, 0.1 μg / ml, 0.05 μg / ml, and 0.025 μg / ml, and a blank control was set up.

[0111] 6.9 Add 50 μl of the crude enzyme solution from 6.3 and 50 μl of 360 μM substrate to a 96-well fluorescence detection plate, and simultaneously add 100 μl of the product dilution solution described in 6.8 to the same 96-well fluorescence detection plate.

[0112] 6.10 Place the 96-well fluorescent detection plate into a microplate reader to detect the amount of fluorescent product generated. Perform kinetic analysis for 1.5 hours, detecting once per minute. (Microplate reader parameters are set as follows: 37℃, excitation 360 / 40nm, emission 460 / 40nm.)

[0113] 6.11 The readings from 3 to 16 minutes were ultimately selected for calculating enzyme activity units. The amount of fluorescent product generated in the early stage of the reaction is directly proportional to time. To minimize error, readings from 3 to 16 minutes were selected for the final result calculation.

[0114] In simple terms, the process involves adding the same concentrations of ASP-1-Laronidase and commercially available Aldurazyme to the same number of HEK293 cells and incubating for 2.5 hours. After this time, some proteins will be internalized into the HEK293 cells. The cells are then centrifuged and washed three times to remove any enzymes that have not been internalized. The cells are then lysed with the same volume of cell lysis buffer to obtain cell lysates (i.e., crude enzyme solutions of ASP-1-Laronidase or Aldurazyme). Since other conditions are kept constant, the enzyme activity of the crude enzyme solution is positively correlated with the amount of enzyme internalized into the cells. Therefore, comparing the enzyme activity of the crude enzyme solution reflects the degree of cell internalization. A background control (without added enzyme) was excluded from the calculations.

[0115] like Figure 9 As shown, the intracellularization rate of ASP-1-Laronidase was not significantly different from that of commercially available Aldurazyme.

[0116] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims. SEQUENCE LISTING <110> Beijing Jude Pharmaceutical Technology Co., Ltd. <120> A recombinant aL-iduronase and its preparation method <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 48 <212> DNA <213> Artificial Sequence <400> 1 atgcctctgc tgctgctgct gcctctgctg tgggctggag ccctcgct 48 <210> 2 <211> 75 <212> DNA <213> Artificial Sequence <400> 2 atggctgctg ctgccatccc tgccctgctg ctgtgcctgc ccctgctgtt cctgctgttt 60 ggctggagca gagcc 75 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <400> 3 atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcc 60 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <400> 4 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 <210> 5 <211> 1932 <212> DNA <213> Artificial Sequence <400> 5 atgcctctgc tgctgctgct gcctctgctg tgggctggag ccctcgctgc tgaagctcct 60 cacctggtcc aggtcgatgc cgcaagagca ctgtggcctc tgcgcaggtt ctggagaagc 120 accggattct gtccacctct gcctcattca caggccgacc agtacgtcct gtcctgggat 180 cagcagctga acctggccta cgttggtgct gtgccacaca gaggcatcaa gcaagttcgc 240 acacattggc tgctggagct ggttacaaca agaggctcaa caggaagagg actgtcctat 300 aacttcacac acctggacgg ctatctcgac ctcctccggg agaatcagct gctcccaggg 360 tttgagctga tgggatcagc aagcggacac tttactgact tcgaggacaa gcaacaagtg 420 tttgagtgga aggatctcgt cagctctctg gcaagaaggt atatcggacg gtatggcctg 480 gcacacgtga gcaagtggaa tttcgaaaca tggaacgagc cagaccatca cgatttcgac 540 aacgttagca tgaccatgca gggatttctc aactactacg atgcctgctc agagggcctg 600 agggcagctt ctccagccct gcgcctgggt ggtcctggcg actctttcca cactccaccc 660 agaagccctc tgtcatgggg gctcctcaga cattgtcacg acggcaccaa tttcttcact 720 ggcgaggctg gtgttcgcct cgattacatt agtctccatc gcaaaggcgc aaggtctagt 780 atatcaatcc tggagcagga gaaagtggtg gcccagcaga tcaggcagct gttccctaag 840 ttcgccgata ctcccatata caatgacgaa gccgatccac tggtcggttg gtccctgcct 900 cagccctgga gagccgatgt gacttacgct gcaatggtcg tgaaagtgat cgcccagcac 960 cagaacctgc tgctggcaaa cactacttct gcattccctt acgcactcct gagcaacgat 1020 aacgctttcc tcagctacca ccctcaccct ttcgcccaac ggaccctgac agctcgcttt 1080 caggtgaata acactaggcc acctcatgtg cagctgctga gaaagcccgt gctcacagca 1140 atgggtctgc tggctctcct ggacgaagag caactgtggg ctgaggtgtc ccaggctggc 1200 accgttctgg atagtaatca cacagtcggc gtgctcgcct ccgcacatcg gcctcaaggc 1260 cctgctgatg cctggagagc cgctgttctc atctatgcta gtgacgacac acgggcacat 1320 ccaaacaggt ccgtggccgt cactctgcgc ctgagaggag tgccaccagg accagggctc 1380 gtctatgtta caagatacct ggataacgga ctgtgtagcc cagacggaga atggcgcagg 1440 ctgggtcgcc cagtctttcc tacagcagag cagttcagga gaatgagagc agctgaggac 1500 cctgtcgcag cagctcccag accactgcct gccggaggtc ggctgacact caggccagcc 1560 ctcagactgc cttctctcct cctcgtgcac gtgtgtgctc ggcctgagaa acctccaggt 1620 caagtcacta ggctgagggc actccctctg acccagggtc agctggtgct ggtttggtca 1680 gatgagcacg ttgggagcaa gtgtctgtgg acttacgaga ttcaattctc acaggacggg 1740 aaggcttata caccagttag ccgcaaacca agcactttca acctctttgt gtttagtcct 1800 gacacaggtg cagtctccgg ctcttacagg gttcgggctc tggactattg ggccagacct 1860 ggacctttct cagacccagt gccctacctg gaagtgccag tccctagagg cccaccttct 1920 cctggaaatc cc 1932 <210> 6 <211> 1959 DNA <213> Artificial Sequence <400> 6 atggctgctg ctgccatccc tgccctgctg ctgtgcctgc ccctgctgtt cctgctgttt 60 ggctggagca gagccgctga agctcctcac ctggtccagg tcgatgccgc aagagcactg 120 tggcctctgc gcaggttctg gagaagcacc ggattctgtc cacctctgcc tcattcacag gccgaccagt acgtcctgtc ctgggatcag cagctgaacc tggcctacgt tggtgctgtg 240 ccacacagag gcatcaagca agttcgcaca cattggctgc tggagctggt cacaacaaga 360. ggctcaacag gaagaggact gtcctataac ttcacacacc tggacggcta tctcgacctc ctccggggaga atcagctgct cccagggttt gagctgatgg gatcagcaag cggacacttt 420 actgacttcg aggacaagca acaagtgttt actgactgagg atctcgtcag ctctctggca agaaggtata tcggacggta tggcctggca cacgtgagca agtggaattt cgaaacatgg 540 aacgagccag accatcacga tttcgacaac gttagcatga ccatgcaggg atttctcaac tactacgatg cctgctcaga gggcctgagg gcagcttctc cagccctgcg cctgggtggt 660 cctggcgact ctttccacac tccacccaga agccctctgt catgggggct cctcagacat 720 tgtcacgacg gcaccaattt cttcactggc gaggctggtg ttcgcctcga ttacattagt 780 ctccatcgca aaggcgcaag gtctagtata tcaatcctgg agcaggaga agtggtggcc 840 cagcagatca ggcagctgtt ccctaagttc gccgatactc ccatatacaa tgacgaagcc 900 gatccactgg tcggttggtc cctgcctcag ccctggagag ccgatgtgac ttacgctgca 960 atggtcgtga aagtgatcgc ccagcaccag aacctgctgc tggcaaacac tacttctgca 1020 ttcccttacg cactcctgag caacgataac gctttcctca gctaccaccc tcaccctttc 1080 gcccaacgga ccctgacagc tcgctttcag gtgaataaca ctaggccacc tcatgtgcag 1140 ctgctgagaa agcccgtgct cacagcaatg ggtctgctgg ctctcctgga cgaagagcaa 1200 ctgtgggctg aggtgtccca ggctggcacc gttctggata gtaatcacac agtcggcgtg 1260 ctcgcctccg cacatcggcc tcaaggccct gctgatgcct ggagagccgc tgttctcatc 1320 tatgctagtg acgacacacg ggcacatcca aacaggtccg tggccgtcac tctgcgcctg 1380 agaggagtgc caccaggacc agggctcgtc tatgttacaa gatacctgga taacggactg 1440 tgtagcccag acggagaatg gcgcaggctg ggtcgcccag tctttcctac agcagagcag 1500 ttcaggagaa tgagagcagc tgaggaccct gtcgcagcag ctcccagacc actgcctgcc 1560 ggaggtcggc tgacactcag gccagccctc agactgcctt ctctcctcct cgtgcacgtg 1620 tgtgctcggc ctgagaaacc tccaggtcaa gtcactaggc tgagggcact ccctctgacc 1680 cagggtcagc tggtgctggt ttggtcagat gagcacgttg ggagcaagtg tctgtggact 1740 tacgagattc aattctcaca ggacgggaag gcttatacac cagttagccg caaaccaagc 1800 actttcaacc tctttgtgtt tagtcctgac acaggtgcag tctccggctc ttacagggtt 1860 cgggctctgg actattgggc cagacctgga cctttctcag acccagtgcc ctacctggaa 1920 gtgccagtcc ctagaggccc accttctcct ggaaatccc 1959 <210> 7 <211> 1944 <212> DNA <213> Artificial Sequence <400> 7 atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcc 60 gctgaagctc ctcacctggt ccaggtcgat gccgcaagag cactgtggcc tctgcgcagg 120 ttctggagaa gcaccggatt ctgtccacct ctgcctcatt cacaggccga ccagtacgtc 180 ctgtcctggg atcagcagct gaacctggcc tacgttggtg ctgtgccaca cagaggcatc 240 aagcaagttc gcacacattg gctgctggag ctggttacaa caagaggctc aacaggaaga 300 ggactgtcct ataacttcac acacctggac ggctatctcg acctctctccg ggagaatcag 360 ctgctcccag ggtttgagct gatgggatca gcaagcggac actttactga cttcgaggac 420 480 cggtatggcc tggcacacgt gagcaagtgg aatttcgaaa catggaacga gccagaccat 540 cacgatttcg acaacgttag catgaccatg cagggatttc tcaactacta cgatgcctgc 600 tcagagggcc tgagggcagc ttctccagcc ctgcgcctgg gtggtcctgg cgactctttc 660 cacatccac ccagaagccc tctgtcatgg gggctcctca gacattgtca cgacggcacc 720 aatttcttca ctggcgaggc tggtgttcgc ctcgattaca ttagtctcca tcgcaaaggc 780 gcaaggtcta gtatatcaat cctggagcag gagaaagtgg tggcccagca gatcaggcag 840 ctgttcccta agttcgccga tactccata tacaatgacg aagccgatcc actggtcggt 900 tggtccctgc ctcagccctg gagagccgat gtgacttacg ctgcaatggt cgtgaaagtg 960 atcgcccagc accagaacct gctgctggca aacactactt ctgcattccc ttacgcactc 1020 ctgagcaacg ataacgcttt cctcagctac caccctcacc ctttcgccca acggaccctg 1080 acagctcgct ttcaggtgaa taacactagg ccacctcatg tgcagctgct gagaaagccc 1140 gtgctcacag caatgggtct gctggctctc ctggacgaag agcaactgtg ggctgaggtg 1200 tcccaggctg gcaccgttct ggatagtaat cacacagtcg gcgtgctcgc ctccgcacat 1260 cggcctcaag gccctgctga tgcctggaga gccgctgttc tcatctatgc tagtgacgac 1320 acacgggcac atccaaacag gtccgtggcc gtcactctgc gcctgagagg agtgccacca 1380 ggaccagggc tcgtctatgt tacaagatac ctggataacg gactgtgtag cccagacgga 1440 gaatggcgca ggctgggtcg cccagtcttt cctacagcag agcagttcag gagaatgaga 1500 gcagctgagg accctgtcgc agcagctccc agaccactgc ctgccggagg tcggctgaca 1560 ctcaggccag ccctcagact gccttctctc ctcctcgtgc acgtgtgtgc tcggcctgag 1620 aaacctccag gtcaagtcac taggctgagg gcactccctc tgacccaggg tcagctggtg 1680 ctggttggt cagatgagca cgttgggagc aagtgtctgt ggacttacga gattcaattc 1740 tcacaggacg ggaaggctta tacaccagtt agccgcaaac caagcacttt caacctcttt 1800 gtgtttagtc ctgacacagg tgcagtctcc ggctcttaca gggttcgggc tctggactat 1860 tgggccagac ctggaccttt ctcagaccca gtgccctacc tggaagtgcc agtccctaga 1920 ggcccacctt ctcctggaaa tccc 1944 <210> 8 <211> 1944 <212> DNA <213> Artificial Sequence <400> 8 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60 gctgaagctc ctcacctggt ccaggtcgat gccgcaagag cactgtggcc tctgcgcagg 120 ttctggagaa gcaccggatt ctgtccacct ctgcctcatt cacaggccga ccagtacgtc 180 ctgtcctggg atcagcagct gaacctggcc tacgttggtg ctgtgccaca cagaggcatc 240 aagcaagttc gcacacattg gctgctggag ctggttacaa caagaggctc aacaggaaga 300 ggactgtcct ataacttcac acacctggac ggctatctcg acctcctccg ggagaatcag 360 ctgctcccag ggtttgagct gatgggatca gcaagcggac actttactga cttcgaggac 420 aagcaacaag tgtttgagtg gaaggatctc gtcagctctc tggcaagaag gtatatcgga 480 cggtatggcc tggcacacgt gagcaagtgg aatttcgaaa catggaacga gccagaccat 540 cacgatttcg acaacgttag catgaccatg cagggatttc tcaactacta cgatgcctgc 600 tcagagggcc tgagggcagc ttctccagcc ctgcgcctgg gtggtcctgg cgactctttc 660 cacactccac ccagaagccc tctgtcatgg gggctcctca gacattgtca cgacggcacc 720 aatttcttca ctggcgaggc tggtgttcgc ctcgattaca ttagtctcca tcgcaaaggc 780 gcaaggtcta gtatatcaat cctggagcag gagaaagtgg tggcccagca gatcaggcag 840 ctgttcccta agttcgccga tactcccata tacaatgacg aagccgatcc actggtcggt 900 tggtccctgc ctcagccctg gagagccgat gtgacttacg ctgcaatggt cgtgaaagtg 960 atcgcccagc accagaacct gctgctggca aacactactt ctgcattccc ttacgcactc 1020 ctgagcaacg ataacgcttt cctcagctac caccctcacc ctttcgccca acggaccctg 1080 acagctcgct ttcaggtgaa taacactagg ccacctcatg tgcagctgct gagaaagccc 1140 gtgctcacag caatgggtct gctggctctc ctggacgaag agcaactgtg ggctgaggtg 1200 tcccaggctg gcaccgttct ggatagtaat cacacagtcg gcgtgctcgc ctccgcacat 1260 cggcctcaag gccctgctga tgcctggaga gccgctgttc tcatctatgc tagtgacgac 1320 acacgggcac atccaaacag gtccgtggcc gtcactctgc gcctgagagg agtgccacca 1380 ggaccagggc tcgtctatgt tacaagatac ctggataacg gactgtgtag cccagacgga 1440 gaatggcgca ggctgggtcg cccagtcttt cctacagcag agcagttcag gagaatgaga 1500 gcagctgagg accctgtcgc agcagctccc agaccactgc ctgccggagg tcggctgaca 1560 ctcaggccag ccctcagact gccttctctc ctcctcgtgc acgtgtgtgc tcggcctgag 1620 aaacctccag gtcaagtcac taggctgagg gcactccctc tgacccaggg tcagctggtg 1680 ctggtttggt cagatgagca cgttgggagc aagtgtctgt ggacttacga gattcaattc 1740 tcacaggacg ggaaggctta tacaccagtt agccgcaaac caagcacttt caacctcttt 1800 gtgtttagtc ctgacacagg tgcagtctcc ggctcttaca gggttcggg tctggactt 1860 tgggccagac ctggacctttt ctcagacccca gtgccctacc tggagtgcc agtccctaga 1920 ggccacctt ctcctggaaa tccc 1944 <210> 9 <211> 1884 <212> DNA <213> Artificial Sequence <400> 9 gctgaagctc ctcacctggt ccaggtcgat gccgcaagg cactgtggcc tctgcgcagg 60 ttctggagaa gcaccggatt ctgtccacct ctgctcatt cacaggccga ccagtacgtc 120 ctgtcctggg atcagcagct gaacctggcc tacgttggtg ctgtgccaca cagaggcatc 180 aagcaagttc gcacattg gctgctggag ctggttacaa caaggctc aacaggaaga 240 ggactgtcct ataacttcac acacctggac ggctatctcg acctcctccg ggagaatcag 300 ctgctcccag ggtttgagct gatgggatca gcaagcggac actttactga ctcgaggac 360 aagcaacaag tgtttgagtg gaaggatctc gtcagctc tggcaagg gtatatcgga 420 cggtatggcc tggcacacgt gagcaagtgg aatttcgaa catggaacga gccagaccat 480 cacgatttcg acaacgttag catgaccatg cagggatttc tcaactacta cgatgcctgc 540 tcagagggcc tgagggcagc ttctccagcc ctgcgcctgg gtggtcctgg cgactctttc 600 cacactccac ccagaagccc tctgtcatgg gggctcctca gacattgtca cgacggcacc 660 aatttcttca ctggcgaggc tggtgttcgc ctcgattaca ttagtctcca tcgcaaaggc 720 gcaaggtcta gtatatcaat cctggagcag gagaaagtgg tggcccagca gatcaggcag 780 ctgttcccta agttcgccga tactcccata tacaatgacg aagccgatcc actggtcggt 840 tggtccctgc ctcagccctg gagagccgat gtgacttacg ctgcaatggt cgtgaaagtg 900 atcgcccagc accagaacct gctgctggca aacactactt ctgcattccc ttacgcactc 960 ctgagcaacg ataacgcttt cctcagctac caccctcacc ctttcgccca acggaccctg 1020 acagctcgct ttcaggtgaa taacactagg ccacctcatg tgcagctgct gagaaagccc 1080 gtgctcacag caatgggtct gctggctctc ctggacgaag agcaactgtg ggctgaggtg 1140 tcccaggctg gcaccgttct ggatagtaat cacacagtcg gcgtgctcgc ctccgcacat 1200 cggcctcaag gccctgctga tgcctggaga gccgctgttc tcatctatgc tagtgacgac 1260 acacgggcac atccaaacag gtccgtggcc gtcactctgc gcctgagagg agtgccacca 1320 ggaccagggc tcgtctatgt tacaagatac ctggataacg gactgtgtag cccagacgga 1380 gaatggcgca ggctgggtcg cccagtcttt cctacagcag agcagttcag gagaatgaga 1440 gcagctgagg accctgtcgc agcagctccc agaccactgc ctgccggagg tcggctgaca 1500 ctcaggccag ccctcagact gccttctctc ctcctcgtgc acgtgtgtgc tcggcctgag 1560 aaacctccag gtcaagtcac taggctgagg gcactccctc tgacccaggg tcagctggtg 1620 ctggtttggt cagatgagca cgttgggagc aagtgtctgt ggacttacga gattcaattc 1680 tcacaggacg ggaaggctta tacaccagtt agccgcaaac caagcacttt caacctcttt 1740 gtgtttagtc ctgacacagg tgcagtctcc ggctcttaca gggttcgggc tctggactat 1800 tgggccagac ctggaccttt ctcagaccca gtgccctacc tggaagtgcc agtccctaga 1860 ggcccacctt ctcctggaaa tccc 1884 <210> 10 <211> 644 <212> PRT <213> Artificial Sequence <400> 10 Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala 1 5 10 15 Ala Glu Ala Pro His Leu Val Gln Val Asp Ala Ala Arg Ala Leu Trp 20 25 30 Pro Leu Arg Arg Phe Trp Arg Ser Thr Gly Phe Cys Pro Pro Leu Pro 35 40 45 His Ser Gln Ala Asp Gln Tyr Val Leu Ser Trp Asp Gln Gln Leu Asn 50 55 60 Leu Ala Tyr Val Gly Ala Val Pro His Arg Gly Ile Lys Gln Val Arg 65 70 75 80 Thr His Trp Leu Leu Glu Leu Val Thr Thr Arg Gly Ser Thr Gly Arg 85 90 95 Gly Leu Ser Tyr Asn Phe Thr His Leu Asp Gly Tyr Leu Asp Leu Leu 100 105 110 Arg Glu Asn Gln Leu Leu Pro Gly Phe Glu Leu Met Gly Ser Ala Ser 115 120 125 Gly His Phe Thr Asp Phe Glu Asp Lys Gln Gln Val Phe Glu Trp Lys 130 135 140 Asp Leu Val Ser Ser Leu Ala Arg Arg Tyr Ile Gly Arg Tyr Gly Leu 145 150 155 160 Ala His Val Ser Lys Trp Asn Phe Glu Thr Trp Asn Glu Pro Asp His 165 170 175 His Asp Phe Asp Asn Val Ser Met Thr Met Gln Gly Phe Leu Asn Tyr 180 185 190 Tyr Asp Ala Cys Ser Glu Gly Leu Arg Ala Ala Ser Pro Ala Leu Arg 195 200 205 Leu Gly Gly Pro Gly Asp Ser Phe His Thr Pro Pro Arg Ser Pro Leu 210 215 220 Ser Trp Gly Leu Leu Arg His Cys His Asp Gly Thr Asn Phe Phe Thr 225 230 235 240 Gly Glu Ala Gly Val Arg Leu Asp Tyr Ile Ser Leu His Arg Lys Gly 245 250 255 Ala Arg Ser Ser Ile Ser Ile Leu Glu Gln Glu Lys Val Val Ala Gln 260 265 270 Gln Ile Arg Gln Leu Phe Pro Lys Phe Ala Asp Thr Pro Ile Tyr Asn 275 280 285 Asp Glu Ala Asp Pro Leu Val Gly Trp Ser Leu Pro Gln Pro Trp Arg 290 295 300 Ala Asp Val Thr Tyr Ala Ala Met Val Val Lys Val Ile Ala Gln His 305 310 315 320 Gln Asn Leu Leu Leu Ala Asn Thr Thr Ser Ala Phe Pro Tyr Ala Leu 325 330 335 Leu Ser Asn Asp Asn Ala Phe Leu Ser Tyr His Pro His Pro Phe Ala 340 345 350 Gln Arg Thr Leu Thr Ala Arg Phe Gln Val Asn Asn Thr Arg Pro Pro 355 360 365 His Val Gln Leu Leu Arg Lys Pro Val Leu Thr Ala Met Gly Leu Leu 370 375 380 Ala Leu Leu Asp Glu Glu Gln Leu Trp Ala Glu Val Ser Gln Ala Gly 385 390 395 400 Thr Val Leu Asp Ser Asn His Thr Val Gly Val Leu Ala Ser Ala His 405 410 415 Arg Pro Gln Gly Pro Ala Asp Ala Trp Arg Ala Ala Val Leu Ile Tyr 420 425 430 Ala Ser Asp Asp Thr Arg Ala His Pro Asn Arg Ser Val Ala Val Thr 435 440 445 Leu Arg Leu Arg Gly Val Pro Pro Gly Pro Gly Leu Val Tyr Val Thr 450 455 460 Arg Tyr Leu Asp Asn Gly Leu Cys Ser Pro Asp Gly Glu Trp Arg Arg 465 470 475 480 Leu Gly Arg Pro Val Phe Pro Thr Ala Glu Gln Phe Arg Arg Met Arg 485 490 495 Ala Ala Glu Asp Pro Val Ala Ala Ala Pro Arg Pro Leu Pro Ala Gly 500 505 510 Gly Arg Leu Thr Leu Arg Pro Ala Leu Arg Leu Pro Ser Leu Leu Leu 515 520 525 Val His Val Cys Ala Arg Pro Glu Lys Pro Pro Gly Gln Val Thr Arg 530 535 540 Leu Arg Ala Leu Pro Leu Thr Gln Gly Gln Leu Val Leu Val Trp Ser 545 550 555 560 Asp Glu His Val Gly Ser Lys Cys Leu Trp Thr Tyr Glu Ile Gln Phe 565 570 575 Ser Gln Asp Gly Lys Ala Tyr Thr Pro Val Ser Arg Lys Pro Ser Thr 580 585 590 Phe Asn Leu Phe Val Phe Ser Pro Asp Thr Gly Ala Val Ser Gly Ser 595 600 605 Tyr Arg Val Arg Ala Leu Asp Tyr Trp Ala Arg Pro Gly Pro Phe Ser 610 615 620 Asp Pro Val Pro Tyr Leu Glu Val Pro Val Pro Arg Gly Pro Pro Ser 625 630 635 640 Pro Gly Asn Pro <210> 11 <211> 653 <212> PRT <213> Artificial Sequence <400> 11 Met Ala Ala Ala Ala Ile Pro Ala Leu Leu Leu Cys Leu Pro Leu Leu 1 5 10 15 Phe Leu Leu Phe Gly Trp Ser Arg Ala Ala Glu Ala Pro His Leu Val 20 25 30 Gln Val Asp Ala Ala Arg Ala Leu Trp Pro Leu Arg Arg Phe Trp Arg 35 40 45 Ser Thr Gly Phe Cys Pro Pro Leu Pro His Ser Gln Ala Asp Gln Tyr 50 55 60 Val Leu Ser Trp Asp Gln Gln Leu Asn Leu Ala Tyr Val Gly Ala Val 65 70 75 80 Pro His Arg Gly Ile Lys Gln Val Arg Thr His Trp Leu Leu Glu Leu 85 90 95 Val Thr Thr Arg Gly Ser Thr Gly Arg Gly Leu Ser Tyr Asn Phe Thr 100 105 110 His Leu Asp Gly Tyr Leu Asp Leu Leu Arg Glu Asn Gln Leu Leu Pro 115 120 125 Gly Phe Glu Leu Met Gly Ser Ala Ser Gly His Phe Thr Asp Phe Glu 130 135 140 Asp Lys Gln Gln Val Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala 145 150 155 160 Arg Arg Tyr Ile Gly Arg Tyr Gly Leu Ala His Val Ser Lys Trp Asn 165 170 175 Phe Glu Thr Trp Asn Glu Pro Asp His His Asp Phe Asp Asn Val Ser 180 185 190 Met Thr Met Gln Gly Phe Leu Asn Tyr Tyr Asp Ala Cys Ser Glu Gly 195 200 205 Leu Arg Ala Ala Ser Pro Ala Leu Arg Leu Gly Gly Pro Gly Asp Ser 210 215 220 Phe His Thr Pro Pro Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His 225 230 235 240 Cys His Asp Gly Thr Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu 245 250 255 Asp Tyr Ile Ser Leu His Arg Lys Gly Ala Arg Ser Ser Ile Ser Ile 260 265 270 Leu Glu Gln Glu Lys Val Val Ala Gln Gln Ile Arg Gln Leu Phe Pro 275 280 285 Lys Phe Ala Asp Thr Pro Ile Tyr Asn Asp Glu Ala Asp Pro Leu Val 290 295 300 Gly Trp Ser Leu Pro Gln Pro Trp Arg Ala Asp Val Thr Tyr Ala Ala 305 310 315 320 Met Val Val Lys Val Ile Ala Gln His Gln Asn Leu Leu Leu Ala Asn 325 330 335 Thr Thr Ser Ala Phe Pro Tyr Ala Leu Leu Ser Asn Asp Asn Ala Phe 340 345 350 Leu Ser Tyr His Pro His Pro Phe Ala Gln Arg Thr Leu Thr Ala Arg 355 360 365 Phe Gln Val Asn Asn Thr Arg Pro Pro His Val Gln Leu Leu Arg Lys 370 375 380 Pro Val Leu Thr Ala Met Gly Leu Leu Ala Leu Leu Asp Glu Glu Gln 385 390 395 400 Leu Trp Ala Glu Val Ser Gln Ala Gly Thr Val Leu Asp Ser Asn His 405 410 415 Thr Val Gly Val Leu Ala Ser Ala His Arg Pro Gln Gly Pro Ala Asp 420 425 430 Ala Trp Arg Ala Ala Val Leu Ile Tyr Ala Ser Asp Asp Thr Arg Ala 435 440 445 His Pro Asn Arg Ser Val Ala Val Thr Leu Arg Leu Arg Gly Val Pro 450 455 460 Pro Gly Pro Gly Leu Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu 465 470 475 480 Cys Ser Pro Asp Gly Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro 485 490 495 Thr Ala Glu Gln Phe Arg Arg Met Arg Ala Ala Glu Asp Pro Val Ala 500 505 510 Ala Ala Pro Arg Pro Leu Pro Ala Gly Gly Arg Leu Thr Leu Arg Pro 515 520 525 Ala Leu Arg Leu Pro Ser Leu Leu Leu Val His Val Cys Ala Arg Pro 530 535 540 Glu Lys Pro Pro Gly Gln Val Thr Arg Leu Arg Ala Leu Pro Leu Thr 545 550 555 560 Gln Gly Gln Leu Val Leu Val Trp Ser Asp Glu His Val Gly Ser Lys 565 570 575 Cys Leu Trp Thr Tyr Glu Ile Gln Phe Ser Gln Asp Gly Lys Ala Tyr 580,585,590 Thr Pro Val Ser Arg Lys Pro Ser Thr Phe Asn Leu Phe Val Phe Ser 595,600,605 Pro Asp Thr Gly Ala Val Ser Gly Ser Tyr Arg Val Arg Ala Leu Asp 610 615 620 Tyr Trp Ala Arg Pro Gly Pro Phe Ser Asp Pro Val Pro Tyr Leu Glu 625,630,635,640 Val Pro Val Pro Arg Gly Pro Pro Ser Pro Gly Asn Pro 645,650 <210> 12 <211> 648 <212> PRT <213> Artificial Sequence <400> 12 Put Tyr Arg Put Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Glu Ala Pro His Val Gln Val Asp Ala Ala 20 25 30 Arg Ala Leu Trp Pro Leu Arg Arg Phe Trp Arg Ser Thr Gly Phe Cys 35 40 45 Pro Pro Leu Pro His Ser Gln Ala Asp Gln Tyr Val Leu Ser Trp Asp 50 55 60 Gln Gln Leu Asn Leu Ala Tyr Val Gly Ala Val Pro His Arg Gly Ile 65 70 75 80 Lys Gln Val Arg Thr His Trp Leu Leu Glu Leu Val Thr Thr Arg Gly 85 90 95 Ser Thr Gly Arg Gly Leu Ser Tyr Asn Phe Thr His Leu Asp Gly Tyr 100 105 110 Leu Asp Leu Leu Arg Glu Asn Gln Leu Leu Pro Gly Phe Glu Leu Met 115 120 125 Gly Ser Ala Ser Gly His Phe Thr Asp Phe Glu Asp Lys Gln Gln Val 130 135 140 Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala Arg Arg Tyr Ile Gly 145 150 155 160 Arg Tyr Gly Leu Ala His Val Ser Lys Trp Asn Phe Glu Thr Trp Asn 165 170 175 Glu Pro Asp His His Asp Phe Asp Asn Val Ser Met Thr Met Gln Gly 180 185 190 Phe Leu Asn Tyr Tyr Asp Ala Cys Ser Glu Gly Leu Arg Ala Ala Ser 195 200 205 Pro Ala Leu Arg Leu Gly Gly Pro Gly Asp Ser Phe His Thr Pro Pro 210 215 220 Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His Cys His Asp Gly Thr 225 230 235 240 Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu Asp Tyr Ile Ser Leu 245 250 255 His Arg Lys Gly Ala Arg Ser Ser Ile Ser Ile Leu Glu Gln Glu Lys 260 265 270 Val Val Ala Gln Gln Ile Arg Gln Leu Phe Pro Lys Phe Ala Asp Thr 275 280 285 Pro Ile Tyr Asn Asp Glu Ala Asp Pro Leu Val Gly Trp Ser Leu Pro 290 295 300 Gln Pro Trp Arg Ala Asp Val Thr Tyr Ala Ala Met Val Val Lys Val 305 310 315 320 Ile Ala Gln His Gln Asn Leu Leu Leu Ala Asn Thr Thr Ser Ala Phe 325 330 335 Pro Tyr Ala Leu Leu Ser Asn Asp Asn Ala Phe Leu Ser Tyr His Pro 340 345 350 His Pro Phe Ala Gln Arg Thr Leu Thr Ala Arg Phe Gln Val Asn Asn 355 360 365 Thr Arg Pro Pro His Val Gln Leu Leu Arg Lys Pro Val Leu Thr Ala 370 375 380 Met Gly Leu Leu Ala Leu Leu Asp Glu Glu Gln Leu Trp Ala Glu Val 385 390 395 400 Ser Gln Ala Gly Thr Val Leu Asp Ser Asn His Thr Val Gly Val Leu 405 410 415 Ala Ser Ala His Arg Pro Gln Gly Pro Ala Asp Ala Trp Arg Ala Ala 420 425 430 Val Leu Ile Tyr Ala Ser Asp Asp Thr Arg Ala His Pro Asn Arg Ser 435 440 445 Val Ala Val Thr Leu Arg Leu Arg Gly Val Pro Pro Gly Pro Gly Leu 450 455 460 Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu Cys Ser Pro Asp Gly 465 470 475 480 Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro Thr Ala Glu Gln Phe 485 490 495 Arg Arg Met Arg Ala Ala Glu Asp Pro Val Ala Ala Ala Pro Arg Pro 500 505 510 Leu Pro Ala Gly Gly Arg Leu Thr Leu Arg Pro Ala Leu Arg Leu Pro 515 520 525 Ser Leu Leu Leu Val His Val Cys Ala Arg Pro Glu Lys Pro Pro Gly 530 535 540 Gln Val Thr Arg Leu Arg Ala Leu Pro Leu Thr Gln Gly Gln Leu Val 545 550 555 560 Leu Val Trp Ser Asp Glu His Val Gly Ser Lys Cys Leu Trp Thr Tyr 565 570 575 Glu Ile Gln Phe Ser Gln Asp Gly Lys Ala Tyr Thr Pro Val Ser Arg 580 585 590 Lys Pro Ser Thr Phe Asn Leu Phe Val Phe Ser Pro Asp Thr Gly Ala 595 600 605 Val Ser Gly Ser Tyr Arg Val Arg Ala Leu Asp Tyr Trp Ala Arg Pro 610 615 620 Gly Pro Phe Ser Asp Pro Val Pro Tyr Leu Glu Val Pro Val Pro Arg 625 630 635 640 Gly Pro Pro Ser Pro Gly Asn Pro 645 <210> 13 <211> 648 <212> PRT <213> Artificial Sequence <400> 13 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Ala Glu Ala Pro His Leu Val Gln Val Asp Ala Ala 20 25 30 Arg Ala Leu Trp Pro Leu Arg Arg Phe Trp Arg Ser Thr Gly Phe Cys 35 40 45 Pro Pro Leu Pro His Ser Gln Ala Asp Gln Tyr Val Leu Ser Trp Asp 50 55 60 Gln Gln Leu Asn Leu Ala Tyr Val Gly Ala Val Pro His Arg Gly Ile 65 70 75 80 Lys Gln Val Arg Thr His Trp Leu Leu Glu Leu Val Thr Thr Arg Gly 85 90 95 Ser Thr Gly Arg Gly Leu Ser Tyr Asn Phe Thr His Leu Asp Gly Tyr 100 105 110 Leu Asp Leu Leu Arg Glu Asn Gln Leu Leu Pro Gly Phe Glu Leu Met 115 120 125 Gly Ser Ala Ser Gly His Phe Thr Asp Phe Glu Asp Lys Gln Gln Val 130 135 140 Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala Arg Arg Tyr Ile Gly 145 150 155 160 Arg Tyr Gly Leu Ala His Val Ser Lys Trp Asn Phe Glu Thr Trp Asn 165 170 175 Glu Pro Asp His His Asp Phe Asp Asn Val Ser Met Thr Met Gln Gly 180 185 190 Phe Leu Asn Tyr Tyr Asp Ala Cys Ser Glu Gly Leu Arg Ala Ala Ser 195 200 205 Pro Ala Leu Arg Leu Gly Gly Pro Gly Asp Ser Phe His Thr Pro Pro 210 215 220 Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His Cys His Asp Gly Thr 225 230 235 240 Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu Asp Tyr Ile Ser Leu 245 250 255 His Arg Lys Gly Ala Arg Ser Ser Ile Ser Ile Leu Glu Gln Glu Lys 260 265 270 Val Val Ala Gln Gln Ile Arg Gln Leu Phe Pro Lys Phe Ala Asp Thr 275 280 285 Pro Ile Tyr Asn Asp Glu Ala Asp Pro Leu Val Gly Trp Ser Leu Pro 290 295 300 Gln Pro Trp Arg Ala Asp Val Thr Tyr Ala Ala Met Val Val Lys Val 305 310 315 320 Ile Ala Gln His Gln Asn Leu Leu Leu Ala Asn Thr Thr Ser Ala Phe 325 330 335 Pro Tyr Ala Leu Leu Ser Asn Asp Asn Ala Phe Leu Ser Tyr His Pro 340 345 350 His Pro Phe Ala Gln Arg Thr Leu Thr Ala Arg Phe Gln Val Asn Asn 355 360 365 Thr Arg Pro Pro His Val Gln Leu Leu Arg Lys Pro Val Leu Thr Ala 370 375 380 Met Gly Leu Leu Ala Leu Leu Asp Glu Glu Gln Leu Trp Ala Glu Val 385 390 395 400 Ser Gln Ala Gly Thr Val Leu Asp Ser Asn His Thr Val Gly Val Leu 405 410 415 Ala Ser Ala His Arg Pro Gln Gly Pro Ala Asp Ala Trp Arg Ala Ala 420 425 430 Val Leu Ile Tyr Ala Ser Asp Asp Thr Arg Ala His Pro Asn Arg Ser 435 440 445 Val Ala Val Thr Leu Arg Leu Arg Gly Val Pro Pro Gly Pro Gly Leu 450 455 460 Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu Cys Ser Pro Asp Gly 465 470 475 480 Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro Thr Ala Glu Gln Phe 485 490 495 Arg Arg Met Arg Ala Ala Glu Asp Pro Val Ala Ala Ala Pro Arg Pro 500 505 510 Leu Pro Ala Gly Gly Arg Leu Thr Leu Arg Pro Ala Leu Arg Leu Pro 515 520 525 Ser Leu Leu Leu Val His Val Cys Ala Arg Pro Glu Lys Pro Pro Gly 530 535 540 Gln Val Thr Arg Leu Arg Ala Leu Pro Leu Thr Gln Gly Gln Leu Val 545 550 555 560 Leu Val Trp Ser Asp Glu His Val Gly Ser Lys Cys Leu Trp Thr Tyr 565 570 575 Glu Ile Gln Phe Ser Gln Asp Gly Lys Ala Tyr Thr Pro Val Ser Arg 580 585 590 Lys Pro Ser Thr Phe Asn Leu Phe Val Phe Ser Pro Asp Thr Gly Ala 595 600 605 Val Ser Gly Ser Tyr Arg Val Arg Ala Leu Asp Tyr Trp Ala Arg Pro 610 615 620 Gly Pro Phe Ser Asp Pro Val Pro Tyr Leu Glu Val Pro Val Pro Arg 625 630 635 640 Gly Pro Pro Ser Pro Gly Asn Pro 645 <210> 14 <211> 1959 <212> DNA <213> Artificial Sequence(Artificial Sequence) <400> 14 atgcgtcccc tgcgccccg cgccgcgctg ctggcgctcc tggcctcgct cctggccgcg cccccggtgg ccccggctga agctcctcac ctggtccagg tcgatgccgc aagagcactg 120 tggcctctgc gcaggttctg gagaagcacc ggattctgtc cacctctgcc tcattcacag gccgaccagt acgtcctgtc ctgggatcag cagctgaacc tggcctacgt tggtgctgtg 240 ccacacagag gcatcaagca agttcgcaca cattggctgc tggagctggt cacaacaaga 360. ggctcaacag gaagaggact gtcctataac ttcacacacc tggacggcta tctcgacctc ctccggggaga atcagctgct cccagggttt gagctgatgg gatcagcaag cggacacttt 420 actgacttcg aggacaagca acaagtgttt actgactgagg atctcgtcag ctctctggca agaaggtata tcggacggta tggcctggca cacgtgagca agtggaattt cgaaacatgg 540 aacgagccag accatcacga tttcgacaac gttagcatga ccatgcaggg atttctcaac tactacgatg cctgctcaga gggcctgagg gcagcttctc cagccctgcg cctgggtggt 660 cctggcgact ctttccacac tccacccaga agccctctgt catgggggct cctcagacat 720 tgtcacgacg gcaccaattt cttcactggc gaggctggtg ttcgcctcga ttacattagt 780 ctccatcgca aaggcgcaag gtctagtata tcaatcctgg agcaggagaa agtggtggcc 840 cagcagatca ggcagctgtt ccctaagttc gccgatactc ccatatacaa tgacgaagcc 900 gatccactgg tcggttggtc cctgcctcag ccctggagag ccgatgtgac ttacgctgca 960 atggtcgtga aagtgatcgc ccagcaccag aacctgctgc tggcaaacac tacttctgca 1020 ttcccttacg cactcctgag caacgataac gctttcctca gctaccaccc tcaccctttc 1080 gcccaacgga ccctgacagc tcgctttcag gtgaataaca ctaggccacc tcatgtgcag 1140 ctgctgagaa agcccgtgct cacagcaatg ggtctgctgg ctctcctgga cgaagagcaa 1200 ctgtgggctg aggtgtccca ggctggcacc gttctggata gtaatcacac agtcggcgtg 1260 ctcgcctccg cacatcggcc tcaaggccct gctgatgcct ggagagccgc tgttctcatc 1320 tatgctagtg acgacacacg ggcacatcca aacaggtccg tggccgtcac tctgcgcctg 1380 agaggagtgc caccaggacc agggctcgtc tatgttacaa gatacctgga taacggactg 1440 tgtagcccag acggagaatg gcgcaggctg ggtcgcccag tctttcctac agcagagcag 1500 ttcaggagaa tgagagcagc tgaggaccct gtcgcagcag ctcccagacc actgcctgcc 1560 ggaggtcggc tgacactcag gccagccctc agactgcctt ctctcctcct cgtgcacgtg 1620 tgtgctcggc ctgagaaacc tccaggtcaa gtcactaggc tgagggcact ccctctgacc 1680 cagggtcagc tggtgctggt ttggtcagat gagcacgttg ggagcaagtg tctgtggact 1740 tacgagattc aattctcaca ggacgggaag gcttatacac cagttagccg caaaccaagc 1800 actttcaacc tctttgtgtt tagtcctgac acaggtgcag tctccggctc ttacagggtt 1860 cgggctctgg actattgggc cagacctgga cctttctcag acccagtgcc ctacctggaa 1920 gtgccagtcc ctagaggccc accttctcct ggaaatccc 1959 <210> 15 <211> 653 <212> PRT <213> Artificial Sequence <400> 15 Put Arg Pro Leu Arg Pro Arg Ala Ala Leu Leu Ala Leu Leu Ala Ser 1 5 10 15 Leu Leu Ala Ala Pro Pro Val Ala Pro Ala Glu Ala Pro His Leu Val 20 25 30 Gln Val Asp Ala Ala Arg Ala Leu Trp Pro Leu Arg Arg Phe Trp Arg 35 40 45 Ser Thr Gly Phe Cys Pro Pro Leu Pro His Ser Gln Ala Asp Gln Tyr 50 55 60 Val Leu Ser Trp Asp Gln Gln Leu Asn Leu Ala Tyr Val Gly Ala Val 65 70 75 80 Pro His Arg Gly Ile Lys Gln Val Arg Thr His Trp Leu Leu Glu Leu 85 90 95 Val Thr Thr Arg Gly Ser Thr Gly Arg Gly Leu Ser Tyr Asn Phe Thr 100 105 110 His Leu Asp Gly Tyr Leu Asp Leu Leu Arg Glu Asn Gln Leu Leu Pro 115 120 125 Gly Phe Glu Leu Met Gly Ser Ala Ser Gly His Phe Thr Asp Phe Glu 130 135 140 Asp Lys Gln Gln Val Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala 145 150 155 160 Arg Arg Tyr Ile Gly Arg Tyr Gly Leu Ala His Val Ser Lys Trp Asn 165 170 175 Phe Glu Thr Trp Asn Glu Pro Asp His His Asp Phe Asp Asn Val Ser 180 185 190 Met Thr Met Gln Gly Phe Leu Asn Tyr Tyr Asp Ala Cys Ser Glu Gly 195 200 205 Leu Arg Ala Ala Ser Pro Ala Leu Arg Leu Gly Gly Pro Gly Asp Ser 210 215 220 Phe His Thr Pro Pro Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His 225 230 235 240 Cys His Asp Gly Thr Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu 245 250 255 Asp Tyr Ile Ser Leu His Arg Lys Gly Ala Arg Ser Ser Ile Ser Ile 260 265 270 Leu Glu Gln Glu Lys Val Val Ala Gln Gln Ile Arg Gln Leu Phe Pro 275 280 285 Lys Phe Ala Asp Thr Pro Ile Tyr Asn Asp Glu Ala Asp Pro Leu Val 290 295 300 Gly Trp Ser Leu Pro Gln Pro Trp Arg Ala Asp Val Thr Tyr Ala Ala 305 310 315 320 Met Val Val Lys Val Ile Ala Gln His Gln Asn Leu Leu Leu Ala Asn 325 330 335 Thr Thr Ser Ala Phe Pro Tyr Ala Leu Leu Ser Asn Asp Asn Ala Phe 340 345 350 Leu Ser Tyr His Pro His Pro Phe Ala Gln Arg Thr Leu Thr Ala Arg 355 360 365 Phe Gln Val Asn Asn Thr Arg Pro Pro His Val Gln Leu Leu Arg Lys 370 375 380 Pro Val Leu Thr Ala Met Gly Leu Leu Ala Leu Leu Asp Glu Glu Gln 385 390 395 400 Leu Trp Ala Glu Val Ser Gln Ala Gly Thr Val Leu Asp Ser Asn His 405 410 415 Thr Val Gly Val Leu Ala Ser Ala His Arg Pro Gln Gly Pro Ala Asp 420 425 430 Ala Trp Arg Ala Ala Val Leu Ile Tyr Ala Ser Asp Asp Thr Arg Ala 435 440 445 His Pro Asn Arg Ser Val Ala Val Thr Leu Arg Leu Arg Gly Val Pro 450 455 460 Pro Gly Pro Gly Leu Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu 465 470 475 480 Cys Ser Pro Asp Gly Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro 485 490 495 Thr Ala Glu Gln Phe Arg Arg Met Arg Ala Ala Glu Asp Pro Val Ala 500 505 510 Ala Ala Pro Arg Pro Leu Pro Ala Gly Gly Arg Leu Thr Leu Arg Pro 515 520 525 Ala Leu Arg Leu Pro Ser Leu Leu Leu Val His Val Cys Ala Arg Pro 530 535 540 Glu Lys Pro Pro Gly Gln Val Thr Arg Leu Arg Ala Leu Pro Leu Thr 545 550 555 560 Gln Gly Gln Leu Val Leu Val Trp Ser Asp Glu His Val Gly Ser Lys 565 570 575 Cys Leu Trp Thr Tyr Glu Ile Gln Phe Ser Gln Asp Gly Lys Ala Tyr 580 585 590 Thr Pro Val Ser Arg Lys Pro Ser Thr Phe Asn Leu Phe Val Phe Ser 595 600 605 Pro Asp Thr Gly Ala Val Ser Gly Ser Tyr Arg Val Arg Ala Leu Asp 610 615 620 Tyr Trp Ala Arg Pro Gly Pro Phe Ser Asp Pro Val Pro Tyr Leu Glu 625 630 635 640 Val Pro Val Pro Arg Gly Pro Pro Ser Pro Gly Asn Pro 645,650 <210> 16 <211> 4923 <212> DNA <213> Artificial Sequence <400> 16 aggcgtctaa ccagtcacag tcgcaaggta ggctgagcac cgtggcgggc ggcagcgggt 60 ggcggtcggg gttgtttg gcggaggtgc tgctgatgat gtaattaaag taggcggtct 120 tgagacggcg gatggtcgag gtgaggtgtg gcaggcttga gatccagctg ttggggtgag 180 tactccctct caaaagcggg cattacttct gcgctaagat tgtcagtttc caaaaacgag 240 gaggattga tattcacctg gccgatctg gccatacact tgagtgacaa tgacatccac 300 tttgccttc tctccacagg tgtccactcc caggtccaag tttaaacgga tctctagcga 360 attcgcggcc gccaccatgc cgctgctgct actgctgccc ctgctgtggg caggggccct 420 cgctgtggag gttgactcag ctcccggtac agacctggca aagattctgt cagatatgcg 480 ctcccagtat gaggttatgg cagagcagaa tcggaaagac gcagaggcat ggttacttc 540 cagaagaa gagctgaata gggaagtggc agggcataca gagcagctcc agatgagtag 600 gtcagaagtc acagacctga gaagaactct gcaagggctg gaaatcgaac tccagagcca 660 actgagcatg aaggcagctc tggaggacac actcgccgag actgaggcta ggtttggtgc 720 ccagctcgct cacattcagg ctctgataag tggtattgaa gcacagctcg gagatgtgag 780 agctgattct gaacgccaga atcaggata ccagcggctg atggacatca agtctaggct 840 cgaacaagag atcgcaacct accggagcct cctggaaggg caagaagatc actataacaa 900 tctgtccgcc agtaaggtcc tgcaccatca ccatcaccat tgaaagcttg gatcccccga 960 cctcgacctc tggctaataa aggaaattta tttcattgc aatagtgtgt tggaattttt 1020 tgtgtctctc actcgggaagg acatatggga gggcaaatca tttggtcgag atccccggga 1080 tctctagcta gaggatcgat ccccgccccg gacgaactaa acctgactac gacatctctg 1140 ccccttcttc gcggggcagt gcatgtaatc ccttcagttg gttggtacaa cttgccaact 1200 gaaccctaaa cgggtagcat atgcttcccg ggtagtagta tatactatcc agactaaccc 1260 tattcaata gcatatgtta cccaacggga agcatatgct atcgaattag ggttagtaaa agggtcctaa ggaacagcga tgtaggtggg cgggccaaga tagggcgcg attgctgcga 1440. tctggaggac aaattacaca cacttgcgcc tgagcgcca gcacagggtt gttggtcctc atattcacga ggtcgctgag agcacggtgg gctaatgttg ccaagggtag catatactac ccaaatatct ggatagcata tgctatccta atctatatct gggtagcata ggctatccta atctatatct gggtagcata tgctatccta atctatatct gggtagtata tgctatccta atttatatct gggtagcata ggctatccta atctatatct gggtagcata tgctatccta atctatatct gggtagtata tgctatccta atctgtatcc gggtagcata tgctatccta together gggtagtata tgctatccta attatatct gggtagcata tactacccaa atatctggat agcatatgct atcctaatct atcctgggt agcatatgct atcctaatct atatctgggt agcataggct atcctaatct atcctgggt agcatatgct atcctaatct atatctgggt agtatatgct atcctaattt atatctgggt agcataggct atcctaatct atatctgggt agcatatgct atcctaatct atatctgggt agtatatgct atcctaatct 2040 gtatccgggt agcatatgct atcctcatga taagctgtca aacatgagaa ttaattcttg 2100 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 2160 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 2220 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 2280 samaattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttatccctt 2340 ttttgcggca tttgccttc ctgttttgc tcacccagaa acgctggtga aagtaaaaga 2400 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 2460 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 2520 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 2580 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 2640 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 2700 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 2760 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 2820 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac 2880 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 2940 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 3000 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 3060 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 3120 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 3180 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 3240 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 3300 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 3360 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 3420 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 3480 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 3540 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 3600 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 3660 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 3720 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 3780 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 3840 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 3900 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 3960 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 4020 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 4080 gtcagtgagc gaggaagcgt acatttatat tggctcatgt ccaatatgcc cgggatgttg 4140 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 4200 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 4260 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 4320 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 4380 agtgtatcat atgccaagtc cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 4440 gcattatgcc cagtacatga ccttacggga ctttcctact tggcagtaca tctacgtatt 4500 agtcatcgct attaccaagg tgatgcggtt ttggcagtac accaatgggc gtggatagcg 4560 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 4620 gcaccaaaat caacgggact ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat 4680 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca 4740 gatcctcact ctcttccgca tcgctgtctg cgagggccag ctgttgggct cgcggttgag 4800 gacaaactct tcgcggtctt tccagtactc ttggatcgga aacccgtcgg cctccgaacg 4860 gtactccgcc accgagggac ctgagcgagt ccgcatcgac cggatcgga aacctctcga 4920 gaa 4923 <210> 17 <211> 13 <212> DNA <213> Artificial Sequence(Artificial Sequence) <400> 17 gccgccacca tgc

Claims

1. An isolated nucleic acid, characterized in that, The nucleic acid sequence is shown in SEQ ID No:

5.

2. A recombinant aL-iduronase, characterized in that, Its amino acid sequence is shown in SEQ ID No:

10.

3. A method for preparing recombinant aL-iduronase, characterized in that, The isolated nucleic acid as described in claim 1 is used for eukaryotic expression, or the recombinant aL-iduronase as described in claim 2 is expressed in eukaryotically.

4. The preparation method according to claim 3, characterized in that, The KOZARK sequence was added before the isolated nucleic acid sequence, constructed into a eukaryotic expression vector, and then transferred into a eukaryotic expression system for expression.

5. The preparation method according to claim 4, characterized in that, The KOZARK sequence is GCCGCCACCATGC.

6. The preparation method according to claim 3, characterized in that, The eukaryotic expression refers to expression within mammalian cells.

7. The preparation method according to claim 6, characterized in that, The mammalian cells were CHOK1SV GS-KO.

8. The preparation method according to claim 3, characterized in that, include: Construct a recombinant eukaryotic expression vector containing the isolated nucleic acid sequence; transfer the recombinant eukaryotic expression plasmid into a eukaryotic expression system for eukaryotic expression; The supernatant was collected and purified to obtain aL-iduronase.

9. The preparation method according to claim 8, characterized in that, The purification includes one or more of affinity chromatography, hydrophobic interaction chromatography, and ion exchange chromatography.

10. The preparation method according to claim 9, characterized in that, The affinity chromatography involves linear gradient elution using agarose gel to obtain an eluent containing the target protein.

11. The preparation method according to claim 9, characterized in that, The hydrophobic interaction chromatography uses a hydrophobic chromatography column to remove impurities such as proteins and polymers. Before sample loading, 4M NaCl is added to a final concentration of 2M and the pH is adjusted to 5-6 with 1M NaOH. The target protein is eluted isocratically with elution buffer.

12. The preparation method according to claim 9, characterized in that, The ion exchange chromatography uses a salt-tolerant cation column. After sample loading and equilibration, the target protein is eluted with elution buffer at a linear gradient of 0-70%. The elution buffer is 20 mM PB + 1 M NaCl + pH 5.

5.

13. The preparation method according to claim 9, characterized in that, The purified solution was concentrated by 30 kDa membrane ultrafiltration and then transferred to phosphate buffer containing 0.0001% Tween 80 pH 5.5.

Images