CN115109790A

CN115109790A - Recombinant a-L-iduronate prase and preparation method thereof

Info

Publication number: CN115109790A
Application number: CN202110306525.8A
Authority: CN
Inventors: 王冀姝; 夏文娟; 李玲玲; 魏婷婷; 饶易坤; 彭璐佳; 任亚芳; 郭建云; 张维
Original assignee: Beijing Jude Pharmaceutical Technology Co ltd
Current assignee: Beijing Jude Pharmaceutical Technology Co ltd
Priority date: 2021-03-23
Filing date: 2021-03-23
Publication date: 2022-09-27
Also published as: WO2022199243A1

Abstract

The present invention discloses an isolated nucleotide sequence comprising a signal peptide sequence and a nucleotide sequence encoding a-L-iduronidase, wherein the signal peptide sequence is not the native signal peptide sequence of a-L-iduronidase. By replacing a natural signal peptide with an artificial signal peptide, the expression level of a-L-iduronidase can be increased, and a protein with a single cleavage point and correct post-translational modification (N-glycosylation phosphorylation) can be generated, and can cross cell membranes and exert enzymatic activity in cells.

Description

Recombinant a-L-iduronate prase and preparation method thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a recombinant a-L-iduronate prolidase and a preparation method thereof.

Background

Mucopolysaccharidosis (MPS), a rare disease, is primarily due to a deficiency in the hydrolytic enzymes required in lysosomes to degrade mucopolysaccharides, resulting in a large mucopolysaccharidosis in the tissue. Mucopolysaccharidosis type I (MPS I) is a metabolic disorder syndrome caused by deficiency of Alpha-L-Iduronidase (Alpha-L-Iduronidase), the most severe subtype of the disease, also known as Hurler syndrome, usually dies at about 10 years old, and clinically, it is manifested by various manifestations such as low intelligence, ugly face, hepatosplenomegaly, skeletal disorders, cardiovascular disorders, corneal opacity and deafness. At present, besides symptomatic treatment, bone marrow transplantation and gene therapy are also available, but the treatment process is complex, the risk is high, and the treatment effect is not exact. The clinical recombinase replacement therapy is the only drug therapy with definite curative effect at present, is small in risk by intravenous injection, needs lifelong treatment and is high in cost.

Aldurazyme is a recombinant Alpha-L-Iduronidase co-developed by Genzyme and BioMarin for enzyme replacement therapy of MPS-I, and is currently the only commercially available specific drug for the treatment of MPS I.

The a-L-iduronidase is present in lysosomes in mammalian cell cytoplasm and is undetectable in normal human serum. The amino acid sequence of the protein has an atypical signal peptide sequence (1-27: MRPLRPRAALLALLASLL AAPPVAPAE) [ Unit https:// www.uniprot.org/Uniprot/P35475], however, analysis by signal peptide prediction software [ SignalP ] shows that the signal peptide is cleaved at least three positions [ FIG. 10], and the signal peptide is cleaved at positions other than amino acids 27-28. Therefore, the secretion process guided by the signal peptide of a-L-iduronidase is not a classical protein secretion process from the endoplasmic reticulum to the outside of the cell after translation, and may be a process that mainly guides the translated protein from the endoplasmic reticulum to lysosomes. Since the 90 s of the 20 th century, attempts have been made to extract natural enzymes from various tissues, and it has been found that amino termini of enzymes derived from various tissues are greatly different [ elements et al, 1989 ]. This finding also indirectly confirms that the so-called 'secretory signal peptide' of a-L-iduronidase is not a signal peptide of a typical secretory protein. Nevertheless, Neufeld discovered in 1968 that a-L-iduronidase was still secreted from cells and taken up by cells with genetic defects in the periphery, alleviating the cytoplasmic mucopolysaccharide storage phenotype [ Fratantoni et al, 1968], which was a foundational finding that has subsequently been used to facilitate the genetic engineering of a-L-iduronidase and for clinical therapy.

Further studies have shown that a-L-iduronidase is subject to a number of post-translational modifications, in addition to the usual N-glycosylation, also phosphorylation of two mannose sugar groups (bisphophorylated oligomanosidic glyco-can). The phosphorylation of Mannose residues at amino acids 347 and 390, which are essential for substrate recognition [ Maita et al, 2013], 311 and 390, are key modifications for binding to the 6-phospho-Mannose receptor (M6-phosphate receptor, M6PR) and achieving cellular internalization, crossing the cell membrane by M6PR, entering the cytosol from outside the cell, and then entering the lysosome by M6 PR. Thus, it is considered that post-translational modification is indispensable for the a-L-iduronidase to exert normal activity. Since the process by which these specific post-translational modifications (phosphorylation of N-glycosylation) are formed is still unclear, it is hypothesized that they may be formed during secretion.

Based on the complex post-translational modification of a-L-iduronidase (N-glycosylation phosphorylation), the first generation recombinase (Aldurazyme) carefully utilized its own native signal peptide and was produced by DNA recombination technology in CHO cells under medium conditions of 10% FBS and harvested from the culture supernatant. But the yield was low, about 20-40. mu.g/day/10E 7 cells. Low expression levels present many challenges for subsequent purification; especially if the native signal peptide of a-L-iduronidase is used, it may result in inconsistent amino termini, greatly limiting the purity and homogeneity of the recombinase.

Therefore, those skilled in the art have made efforts to develop a recombinant a-L-iduronate-prase capable of increasing the expression level of a-L-iduronate-prase and a method for producing the same.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention provides a recombinant a-L-iduronidase capable of increasing the expression level of the a-L-iduronidase, and a method for preparing the same.

To achieve the above objects, one aspect of the present invention provides an isolated nucleotide sequence comprising a signal peptide sequence and a nucleotide sequence encoding a-L-iduronidase, wherein the signal peptide sequence is not the native signal peptide sequence of a-L-iduronidase.

Furthermore, the signal peptide is selected from the sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

Furthermore, the nucleotide sequence of the coding a-L-iduronidase is shown in SEQ ID No. 9.

Optionally, the isolated nucleotide sequence is selected from the group consisting of:

1) SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8;

2) a nucleotide sequence having at least more than 80% homology with SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8;

3) the codon for the polypeptide is degenerate with the coding part of the nucleotide sequence in 1) or 2) and codes for a nucleotide sequence of a-L-iduronidase.

In a second aspect the present invention provides a recombinant a-L-iduronidase comprising an a-L-iduronidase and a heterologous signal peptide capable of being cleaved at a single site.

Alternatively, the amino acid sequence is selected from the following sequences:

1) SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13;

2) an amino acid sequence having at least 95% homology with SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13.

Further, the recombinant a-L-iduronidase has glycosylation modifications at positions 311, 390 and 426.

Further, glycosylation is modified to GlcNAc (2) Man (7) P (2).

Further, cellular internalization of recombinant a-L-iduronidase is mediated by M6 PR.

In a third aspect of the invention, there is provided a process for the preparation of a-L-iduronidase for eukaryotic expression using an isolated nucleotide sequence as described above.

Alternatively, the isolated nucleotide sequence may be preceded by a KOZARK sequence, constructed into a eukaryotic expression vector, and then transferred into a eukaryotic expression system for expression.

Optionally, the KOZARK sequence is GCCGCCACCATGC.

Alternatively, eukaryotic expression is expression in mammalian cells.

Alternatively, the mammalian cell is CHOK1SV GS-KO.

Further, the preparation method comprises the following steps: constructing a recombinant eukaryotic expression vector containing the isolated nucleotide sequence; transferring the recombinant eukaryotic expression plasmid into an eukaryotic expression system for eukaryotic expression; collecting supernatant, and purifying to obtain the alpha-L-iduronidase.

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

Alternatively, the purification is a three-step purification including affinity chromatography, hydrophobic interaction chromatography, and ion exchange chromatography.

Alternatively, affinity chromatography is a linear gradient elution using agarose gel to obtain an eluate containing the protein of interest. Wherein the elution buffer is buffer B, 20mM PB,2M NaCl, pH5.3.

Optionally, hydrophobic interaction chromatography employs a hydrophobic chromatography column for removing hetero-proteins, polymers; adding 4M NaCl to a final concentration of 2M before sample loading, adjusting the pH to 5-6 by using 1M NaOH, and isocratically eluting the target protein by using an elution buffer solution. Wherein the elution buffer is: 20mM PB 0.15M NaCl pH 5.5.

Alternatively, ion exchange chromatography uses a salt tolerant cation column, and after sample loading and equilibration, the target protein is eluted with an elution buffer in a linear gradient of 0-70%. Wherein the washing buffer is 20mM PB 1M NaCl pH5.5.

Optionally, the purified solution is concentrated by ultrafiltration using a 30kDa membrane to a phosphate buffer containing 0.0001% Tween 80, pH 5.5.

The heterologous signal peptide in the invention is used, not only the expression quantity of the recombinant alpha-L-iduronate prase is improved, but also the protein with single cutting point and correct post-translational modification (N-glycosylation phosphorylation) can be generated, and the heterologous signal peptide can cross cell membranes and exert the enzymatic activity in cells.

The conception, the specific steps, and the technical effects produced by the present invention will be further described in conjunction with the accompanying drawings to fully understand the objects, the features, and the effects of the present invention.

Drawings

FIG. 1 is an electrophoretogram of cell supernatant protein expressed by mammalian cells of a-L-iduronidase of 5 different signal peptides according to example 2 of the present invention;

FIG. 2 is a primary LC-MS spectrum of N-terminal sequence analysis of a commercial Aldurazyme;

FIG. 3 is a primary LC-MS spectrum of N-terminal sequence analysis of ASP-1-Laronidase prepared in example 3 of the present invention;

FIG. 4(A) is a secondary LC-MS/MS spectrum of a commercial N-glycosylation phosphorylation assay of Aldurazyme; (B) is the theoretical molecular weight of the polypeptide fragment ion matched (mass deviation less than 20ppm) in this secondary LC-MS/MS profile;

FIG. 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 ion matched (mass deviation less than 20ppm) in this secondary LC-MS/MS profile;

FIG. 6 is a schematic diagram of the degradation of Alpha-L tetrahydroxy epoxy-4-methylumbelliferyl valerate, a substrate by Alpha-L-iduronate to produce the product 4-methylumbelliferone (4-methylumbelliferone) with a fluorescent signal;

FIG. 7 is a graph showing the results of an experiment of the enzyme activities of ASP-1-Laronidase and commercial Aldura zyme in example 4 of the present invention;

FIG. 8 is a Western Blotting identification of the efficiency of ASP-1-Laronidase with commercial Aldura zyme in HEK293 in the presence and absence of M6P; HEK293 cells were cultured for 3 days in medium containing 0. mu.g/ml ASP-1-Laronidase and Aldura zyme (lane 1), 10. mu.g/ml ASP-1-Laronidase (lane 2), 10. mu.g/ml Aldura zyme (lane 4), 10. mu.g/ml ASP-1-Laronidase +10mM/L M6P (lane 3), 10. mu.g/ml Aldura zyme +10mM/L M6P (lane 5), then harvested for lysis and Western Blotting assay using 50ng total protein. St: (ASP-1-Laronidase);

FIG. 9 is a graph comparing the cell internalization experiments of ASP-1-Laronidase according to example 6 of the present invention with commercial Aldura zyme;

FIG. 10 is a diagram for analyzing cleavage positions of an atypical signal peptide sequence in the amino acid sequence of a-L-iduronidase using signal peptide prediction software [ SignalP ].

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: conditions described in a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The reagents used are commercially available or publicly available reagents unless otherwise specified.

In the present invention, various vectors known in the art, such as commercially available vectors, including plasmids and the like, can be used.

According to a specific embodiment of the invention, an artificial signal peptide sequence is added before a coding nucleotide sequence of the a-L-iduronidase to form a recombinant nucleotide sequence, and the recombinant nucleotide sequence is used for eukaryotic expression to obtain the recombinant a-L-iduronidase. In one embodiment of the invention, the artificial signal peptide (SEQ ID No:1, SEQ ID No:2, SEQ ID No:3 or SEQ ID No:4) in the sequence 4 can be used for well improving the expression level of the a-L-iduronidase, and the a-L-iduronidase with single cleavage point and correct post-translational modification (N-glycosylation phosphorylation) can be obtained.

EXAMPLE 1 Synthesis of 5 plasmids a-L-iduronate Prolase for expression of 5 different Signal peptides

Laronidase plasmids with 4 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 Peptide (NSP) are synthesized by Jinwei Zhi company, wherein the nucleotide sequences of the enzymes with artificial signal peptides are shown as SEQ ID No:5 (with ASP-1), SEQ ID No:6 (with ASP-2), SEQ ID No:7 (with ASP-3) and SEQ ID No:8 (with ASP-4); the nucleotide sequence of the enzyme with the natural signal peptide is shown as SEQ ID No. 14, and the amino acid sequence is shown as SEQ ID No. 15. The vector used was pZD, the nucleotide sequence of which is shown in SEQ ID No. 16.

EXAMPLE 25 mammalian cell expression of a-L-iduronidase with different Signal peptides and culture supernatant quantification

2.1 mammalian cells express 5 different signal peptides of a-L-iduronate prase

Extracting plasmids from ASP-1-Laronidase-pZD, ASP-2-Laronidase-pZD, ASP-3-Laronidase-pZD, ASP-4-Laronidase-pZD and NSP-Laronidase-pZD, and mixing the plasmids with PEI 1: 5, and transfected into HEK293 cells. After 5 days, the supernatant was collected, cell debris removed, and the supernatant was adjusted to pH5.5 with 1M phosphoric acid. 25ul of each expression supernatant was taken, 5ul of protein loading buffer was added, and the sample was boiled at 95 ℃ for 5 minutes. Run 12% SDS-protein electrophoresis.

Protein electrophoresis results are shown in figure 1, and it can be seen visually from the electrophoresis results of cell supernatants expressing Laronidase by 5 different signal peptides that the expression level of Laronidase by using four heterologous signal peptides is improved compared with the expression level of natural signal peptide NSP.

2.2 quantification of supernatant

The RP-HPLC method can be used for content determination of Laronidase samples.

RP-HPLC quantitative method on Agilent's HPLC high performance liquid system, the network version of Waters' Empower3 liquid workstation control system, data acquisition and analysis used. HPLC chromatography column for detection of ACE 5C4-300,250X4.6mm I.D. using ACE. The run method time for each sample was 30 minutes. The flow rate was 0.8ml/min, the column temperature was 75 ℃ and the UV absorption 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 an Agilent HPLC system, first equilibrate with 20% buffer B for 3 minutes. At 3.1 min, the buffer B ratio was increased to 31% (i.e., the initial buffer B ratio in the elution zone was X%), and the elution was carried out isocratically for 6 min. The elution of the target protein was eluted with a 31% -43% buffer B linear gradient over 15 min (i.e., the linear change of buffer B was from X% to [ X +12 ]%) over 15 min. The column was washed with 90% B for 4 minutes before re-equilibrating the column.

The results are shown in Table 2, and the Laronidase and NSP-Laronidase of 4 heterologous signal peptides are compared, and the expression levels of Asp-1-Laronidase, Asp-2-Laronidase, Asp-3-Laronidase and Asp-4-Laronidase are obviously improved. Wherein the expression levels of Asp-1-Laronidase and Asp-4-Laronidase respectively reach 3.38 times and 3.59 times of NSP-Laronidase.

Table 25 transient expression of Laronidase for different Signal peptides in HEK 293-UPLC quantification

Name (R)	Concentration ug/ml
		ASP-1-Laronidase	12.5
ASP-2-Laronidase	9.6
		ASP-3-Laronidase	6.9
ASP-4-Laronidase	13.3
		NSP-Laronidase	3.7

Example 3 Mass Spectrometry N-terminal analysis and N-glycosylation phosphorylation analysis of ASP-1-Laronidase protein

3.1 ASP-1-Laronidase was synthesized by Kinzhi corporation using a plasmid provided in Lonza as a vector, to which a suitable KOZAK sequence (GCCGCCACCATGC, SEQ ID NO:17) was added before the ASP-1-Laronidase sequence.

3.2 monoclonal construction and selection were carried out using cell line CHOK1SV GS-KO (https:// go2.Lonza. com/Biologics _ CHOK1SV. html) from Lonza, according to the construction and selection program provided in line with Lonza. The obtained stable monoclonal cell line was then expressed according to the culture method provided by Lonza. After 9 days of expression, cell supernatant was harvested and purified to obtain high-purity ASP-1-Laronidase protease.

Wherein, the purification is three-step purification including affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography. The method specifically comprises the following steps: adjusting pH of the cell culture supernatant to 5.3 with 1M phosphoric acid, and clarifying by membrane deep filtration; the supernatant was filtered and loaded onto a Blue Sepharose 6Fast Flow (Cytiva 17-0948-01) column equilibrated with 20mM PB 0.15M NaCl pH5.3, and after loading, washed with an equilibration buffer until the baseline was stabilized to remove most of non-specifically bound impurities (e.g., host proteins, host DNA, etc.), and finally subjected to linear gradient elution with 0-80% buffer B (20mM PB,2M NaCl, pH5.3), analyzed by SDS-PAGE, and the elution peaks containing the target protein were combined.

The Blue Sepharose 6Fast Flow pool is purified by a hydrophobic column (e.g., Cytiva Phenyl Sepharose Fast Flow (High Sub) Butyl Sepharose 4Fast Flow, Butyl-650M available from Tosoh, Phenyl-5PW, Phenyl Bestarose FF (HS) available from Bogelon) to remove hetero-proteins, multimers, etc. Adding 4M NaCl to a final concentration of 2M before loading the collection solution, adjusting pH to 5.5 with 1M NaOH, loading the collection solution onto a hydrophobic chromatography column balanced with 20mM PB 2M NaCl pH5.5, eluting with an equilibrium buffer solution until the balance of a base line is achieved, isocratically eluting the target protein with 20mM PB 0.15M NaCl pH5.5, and collecting the eluate containing the target protein.

Further purifying ASP-1-Laronidase with salt-tolerant cation column (such as Sulfate-650F (Tosoh), POROS XS (Thermo Scientific), Eshmuno-CPS (Merck)). Loading the collected liquid from the hydrophobic chromatography onto a salt-tolerant cation column balanced by 20mM PB 0.15M NaCl pH5.5, eluting with the balanced liquid until the baseline is stable, and finally eluting the target protein with 20mM PB 1M NaCl pH5.5 according to a linear gradient of 0-70%. Collecting the eluent. The final sample was concentrated by ultrafiltration using a 30kDa membrane in a buffer containing 0.0001% Tween 80pH 5.5 phosphate.

3.3 ASP-1-Laronidase and commercial Aldura zyme were sent to great health Corp and protein N-terminal analysis was performed using LC-MS/MS. The results are shown in Table 3, FIG. 2, FIG. 3, FIG. 4, FIG. 5.

TABLE 3 commercial Aldurazyme and ASP-1-Laronidase enzymatic hydrolysis to N-terminal polypeptide LC-MS/MS Using Trypsin

As is clear from Table 3 and FIGS. 2 to 5, the LC-MS/MS results show that the cleavage site of the signal peptide of the ASP-1 signal peptide used after intracellular processing of the protein is consistent with that of commercial Aldulanzyme.

Wherein, when the IDUA gene expressing Laronidase is selected in this example, the selected gene is shown in SEQ ID No. 9, the protein expressed by the gene is different from commercial Aldurazyme in the 8 th position (without signal peptide) of the amino acid sequence, Laronidase in this example is 8Q, and commercial Aldurazyme is 8H. This is based on the polymorphic consideration of the IDUA gene, which accounts for about 85% of the population.

3.4 the high purity protein was sent to Zhengda health company for N-glycosylation phosphorylation analysis, and the results are shown in Table 4.

TABLE 4 sample quantitation of glycopeptide LC-MS/MS Using chymotrypsin hydrolysis

As can be seen from Table 4, the glycosylation modifications of commercial Aldura zyme and ASP-1-Laronidase were substantially the same.

Wherein, the 311 th, 390 th and 426 th sites of the modification site are the 311 th, 390 th and 426 th sites of Laronidase containing no signal peptide.

Example 4 identification of enzyme Activity of ASP-1-Laronidase with commercial Aldura zyme

4.1 sodium acetate solution at pH3.5 was prepared as the reaction buffer.

4.2 Glycine solution with pH10.7 was prepared as stop solution.

4.3 stock solutions of 4mg/mL substrate (Alpha-L tetrahydroxyepoxypentanoic acid-4-methylumbelliferyl ester) were prepared in DMSO and stored at-20 ℃.

4.4 the substrate stock was diluted to 800. mu.M with reaction buffer for use.

4.5 ASP-1-Laronidase was diluted to 2 μ M with commercial Aldura zyme in 0.9% sodium chloride solution, gradiently 10-fold diluted to 0.0002 μ M, and a blank (i.e., 0.9% sodium chloride solution without enzyme) was added.

4.6 taking 25 mul of substrate, 25 mul of enzyme and sodium chloride solution diluted to different concentrations in 4.5, uniformly mixing in a 96-hole fluorescence detection plate, preserving the temperature for 20 minutes at 37 ℃, and adding 200 mul of stop solution to stop the reaction.

4.7 placing the 96-hole fluorescence detection plate into an enzyme-labeling instrument for detection, and setting the parameters of the enzyme-labeling instrument as follows: excitation 360/40nm, emission 460/40 nm.

ASP-1-Laronidase and commercial Aldura zyme can decompose the substrate Alpha-L tetrahydroxy epoxy valeric acid-4-methylumbelliferyl ester to produce 4-methylumbelliferone (4-methylumbelliferone) with a fluorescent signal, as shown in FIG. 6.

ASP-1-Laronidase and commercial Aldura zyme with the same molar concentration and volume are used for reacting with a substrate with the same system, volume and concentration, the generated product is subjected to fluorescence value detection, and the magnitude of a fluorescence signal value directly shows the enzyme activity. The results are shown in FIG. 7, which shows that ASP-1-Laronidase and commercial Aldura zyme have equivalent enzyme activities at the same molar concentration and volume.

Example 5 internalization of ASP-1-Laronidase with commercial Aldura zyme was mediated by M6PR

5.1 Day 0

5.1.1 digestion of HEK293 cells in T25 flasks with 500ul of pancreatin for 1-2 min;

5.1.2 Add 5ml DMEM (10% FBS +25ug/ml G418), blow open the cells, mix;

5.1.3 cell count, then 0.2X 10 cells ⁶ 5 bottles of/ml, 5ml/T25 bottles;

5.1.43-4 h later, 4 flasks of cells from 5.1.3 were taken and 10ug/ml ASP-1-Laronidase (without M6P), Aldura zyme (without M6P), ASP-1-Laronidase (with 10mM/L M6P), and Aldura zyme (with 10mM/L M6P) were added.

5.2 Day 3

5.2.1 trypsinizing the treated HEK293 cells cultured for 3 days, adding 500ul of pancreatin per T25 bottle for 1-2 min;

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

5.2.3 based on counts, calculate 3 x 10 ⁶ Volume of individual cells. Centrifuging the cells in the corresponding volume, removing the culture medium, and adding PBS for washing for 2 times;

5.2.4 lysis by ultrasonication and measurement of total protein concentration by BCA kit;

and 5.2.5, taking 50ug of the ultrasonically-crushed protein to carry out Western blotting detection. The protein samples are separated by SDS-PAGE, transferred to a PVDF membrane (Roche) by a semidry method, closed, sequentially added with a primary antibody (sheet Anti-Laronidase antibody) and a secondary antibody (peroxidase Anti-sheet IgG) for warm bath, and finally added with a developing solution for developing and photographing, wherein the results are shown in figure 8.

As shown in FIG. 8, after 3 days of culture of HEK293 cells in the presence of M6P in medium containing ASP-1-Laronidase or commercial Aldurazyme, no ASP-1-Laronidase or commercial Aldurazyme was detected in the lysates of HEK293 cells. Indicating that cellular internalization of both is mediated by M6 PR.

Example 6 cellular internalization efficiency characterization of ASP-1-Laronidase and commercial Aldura zyme in HEK293 cells

6.1 HEK293 cells were plated at 0.2X 10 ⁶ The density per ml was seeded in 6-well plates with 2ml per well volume.

6.2 ASP-1-Laronidase and commercial Aldura zyme were added to the above HEK293 medium in amounts of 40. mu.g/ml, 20. mu.g/ml, 10. mu.g/ml, 0.055. mu.g/ml, 0.0275. mu.g/ml, respectively, and incubated on a shaker.

6.3 after 2.5h incubation, approximately 2ml of each cell was transferred to a new centrifuge tube, centrifuged at 200g for 5 min, resuspended in PBS and washed 3 times. To this end, the internalization process of ASP-1-Laronidase with commercial Aldura zyme was terminated. The washed cells were lysed by adding 300. mu.l of a cell lysate (Thermo/#78501), and the resulting cell lysate was regarded as a crude enzyme solution of ASP-1-Laronidase or Aldura zyme.

6.4 quantitate the concentration of total protein in the above crude enzyme solution using the BCA kit.

6.5A solution of sodium acetate at pH3.5 was prepared as the reaction buffer.

6.6 the substrate (Alpha-L tetrahydroxyepoxyvalerate-4-methylumbelliferyl) stock solution in 4.3 was diluted to 360. mu.M with reaction buffer for further use.

6.7 preparation of cell lysates of untreated (i.e.no enzyme added to the culture broth) HEK293 cells. Taking 15ml of the mixture with the density of 0.2 multiplied by 10 ⁶ Perml of HEK293 cells into a new centrifuge tube, 200g centrifugation for 5 minutes, heavy suspension of cells with PBS, repeated washing 3 times, adding 7ml of fineThe cell lysate is lysed.

6.8 the product (4-methylumbelliferone) was diluted to 1.6. mu.g/ml, 0.8. mu.g/ml, 0.4. mu.g/ml, 0.2. mu.g/ml, 0.1. mu.g/ml, 0.05. mu.g/ml, 0.025. mu.g/ml with the mixture (50% of the untreated HEK293 cell lysate + 50% reaction buffer described in 6.7) and a blank was set.

6.9 mu.l of the 6.3 medium crude enzyme solution and 50. mu.l of 360. mu.M substrate were added to a 96-well fluorescent assay plate, while 100. mu.L of the product dilution described in 6.8 was added to the same 96-well fluorescent assay plate.

6.10 putting the 96-hole fluorescence detection plate into a microplate reader to detect the generation amount of the fluorescence product, and performing dynamic detection for 1.5 hours once per minute. (the parameters of the microplate reader are set as follows: 37 ℃, excitation 360/40nm and emission 460/40 nm.)

6.11 Final 3-16 minute readings were selected for enzyme activity unit calculation. The generation amount of the fluorescent product at the early stage of the reaction is in direct proportion to the time, the principle of minimum error is considered, and the reading value of 3-16 minutes is selected for calculating the final result.

Briefly, the same known concentration of ASP-1-Laronidase and commercial Aldura zyme were added to the same amount of HEK293 for incubation, and after 2.5 hours, a portion of the protein was internalized into HEK293 cells. At this point the cells were washed 3 times by centrifugation to remove enzymes that were not internalized into the cells. The cells were lysed separately with the same volume of cell lysate to obtain cell lysates (i.e., crude enzyme solutions of ASP-1-Laronidase or Aldura zyme). Due to the same setting of other conditions, the enzyme activity of the crude enzyme solution and the content of the enzyme internalized into the cells show positive correlation. Therefore, the condition of cell internalization can be reflected by comparing the enzyme activity of the crude enzyme solution. Background controls without enzyme were subtracted during the calculation.

As shown in FIG. 9, the cellular internalization rate of ASP-1-Laronidase was not significantly different from that of commercial Aldura zyme.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

SEQUENCE LISTING

<110> Beijing Datder pharmaceutical science and technology Co., Ltd

<120> a recombinant a-L-iduronate prase and preparation method thereof

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgcctctgc tgctgctgct gcctctgctg tgggctggag ccctcgct 48

<210> 2

<211> 75

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggctgctg ctgccatccc tgccctgctg ctgtgcctgc ccctgctgtt cctgctgttt 60

ggctggagca gagcc 75

<210> 3

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcc 60

<210> 4

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

<210> 5

<211> 1932

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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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

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggctgctg ctgccatccc tgccctgctg ctgtgcctgc ccctgctgtt cctgctgttt 60

ggctggagca gagccgctga agctcctcac ctggtccagg tcgatgccgc aagagcactg 120

tggcctctgc gcaggttctg gagaagcacc ggattctgtc cacctctgcc tcattcacag 180

gccgaccagt acgtcctgtc ctgggatcag cagctgaacc tggcctacgt tggtgctgtg 240

ccacacagag gcatcaagca agttcgcaca cattggctgc tggagctggt tacaacaaga 300

ggctcaacag gaagaggact gtcctataac ttcacacacc tggacggcta tctcgacctc 360

ctccgggaga atcagctgct cccagggttt gagctgatgg gatcagcaag cggacacttt 420

actgacttcg aggacaagca acaagtgttt gagtggaagg atctcgtcag ctctctggca 480

agaaggtata tcggacggta tggcctggca cacgtgagca agtggaattt cgaaacatgg 540

aacgagccag accatcacga tttcgacaac gttagcatga ccatgcaggg atttctcaac 600

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> 7

<211> 1944

<212> DNA

<213> Artificial Sequence (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 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 gggttcgggc tctggactat 1860

tgggccagac ctggaccttt ctcagaccca gtgccctacc tggaagtgcc agtccctaga 1920

ggcccacctt ctcctggaaa tccc 1944

<210> 8

<211> 1944

<212> DNA

<213> Artificial Sequence (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 gggttcgggc tctggactat 1860

tgggccagac ctggaccttt ctcagaccca gtgccctacc tggaagtgcc agtccctaga 1920

ggcccacctt ctcctggaaa tccc 1944

<210> 9

<211> 1884

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gctgaagctc ctcacctggt ccaggtcgat gccgcaagag cactgtggcc tctgcgcagg 60

ttctggagaa gcaccggatt ctgtccacct ctgcctcatt cacaggccga ccagtacgtc 120

ctgtcctggg atcagcagct gaacctggcc tacgttggtg ctgtgccaca cagaggcatc 180

aagcaagttc gcacacattg gctgctggag ctggttacaa caagaggctc aacaggaaga 240

ggactgtcct ataacttcac acacctggac ggctatctcg acctcctccg ggagaatcag 300

ctgctcccag ggtttgagct gatgggatca gcaagcggac actttactga cttcgaggac 360

aagcaacaag tgtttgagtg gaaggatctc gtcagctctc tggcaagaag gtatatcgga 420

cggtatggcc tggcacacgt gagcaagtgg aatttcgaaa 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 (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 (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 (Artificial Sequence)

<400> 12

Met Tyr Arg Met 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 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> 13

<211> 648

<212> PRT

<213> Artificial Sequence (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 tgcgcccccg cgccgcgctg ctggcgctcc tggcctcgct cctggccgcg 60

cccccggtgg ccccggctga agctcctcac ctggtccagg tcgatgccgc aagagcactg 120

tggcctctgc gcaggttctg gagaagcacc ggattctgtc cacctctgcc tcattcacag 180

gccgaccagt acgtcctgtc ctgggatcag cagctgaacc tggcctacgt tggtgctgtg 240

ccacacagag gcatcaagca agttcgcaca cattggctgc tggagctggt tacaacaaga 300

ggctcaacag gaagaggact gtcctataac ttcacacacc tggacggcta tctcgacctc 360

ctccgggaga atcagctgct cccagggttt gagctgatgg gatcagcaag cggacacttt 420

actgacttcg aggacaagca acaagtgttt gagtggaagg atctcgtcag ctctctggca 480

agaaggtata tcggacggta tggcctggca cacgtgagca agtggaattt cgaaacatgg 540

aacgagccag accatcacga tttcgacaac gttagcatga ccatgcaggg atttctcaac 600

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 (Artificial Sequence)

<400> 15

Met 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 (Artificial Sequence)

<400> 16

aggcgtctaa ccagtcacag tcgcaaggta ggctgagcac cgtggcgggc ggcagcgggt 60

ggcggtcggg gttgtttctg gcggaggtgc tgctgatgat gtaattaaag taggcggtct 120

tgagacggcg gatggtcgag gtgaggtgtg gcaggcttga gatccagctg ttggggtgag 180

tactccctct caaaagcggg cattacttct gcgctaagat tgtcagtttc caaaaacgag 240

gaggatttga tattcacctg gcccgatctg gccatacact tgagtgacaa tgacatccac 300

tttgcctttc 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 ggtttacttc 540

cagaacagaa 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 atcaggaata ccagcggctg atggacatca agtctaggct 840

cgaacaagag atcgcaacct accggagcct cctggaaggg caagaagatc actataacaa 900

tctgtccgcc agtaaggtcc tgcaccatca ccatcaccat tgaaagcttg gatcccccga 960

cctcgacctc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt 1020

tgtgtctctc actcggaagg 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

taattcaata gcatatgtta cccaacggga agcatatgct atcgaattag ggttagtaaa 1320

agggtcctaa ggaacagcga tgtaggtggg cgggccaaga taggggcgcg attgctgcga 1380

tctggaggac aaattacaca cacttgcgcc tgagcgccaa gcacagggtt gttggtcctc 1440

atattcacga ggtcgctgag agcacggtgg gctaatgttg ccaagggtag catatactac 1500

ccaaatatct ggatagcata tgctatccta atctatatct gggtagcata ggctatccta 1560

atctatatct gggtagcata tgctatccta atctatatct gggtagtata tgctatccta 1620

atttatatct gggtagcata ggctatccta atctatatct gggtagcata tgctatccta 1680

atctatatct gggtagtata tgctatccta atctgtatcc gggtagcata tgctatccta 1740

atagagatta gggtagtata tgctatccta atttatatct gggtagcata tactacccaa 1800

atatctggat agcatatgct atcctaatct atatctgggt agcatatgct atcctaatct 1860

atatctgggt agcataggct atcctaatct atatctgggt agcatatgct atcctaatct 1920

atatctgggt agtatatgct atcctaattt atatctgggt agcataggct atcctaatct 1980

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

ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 2340

ttttgcggca ttttgccttc ctgtttttgc 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 cggatcggaa aacctctcga 4920

gaa 4923

<210> 17

<211> 13

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gccgccacca tgc 13

Claims

1. An isolated nucleotide sequence comprising a signal peptide sequence and a nucleotide sequence encoding a-L-iduronidase, wherein the signal peptide sequence is not the native signal peptide sequence of a-L-iduronidase.

2. The nucleotide sequence of claim 1, wherein the signal peptide is selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4.

3. The nucleotide sequence of claim 1, wherein the nucleotide sequence encoding a-L-iduronidase is depicted in SEQ ID No. 9.

4. An isolated nucleotide sequence selected from the group consisting of:

1) SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8;

5. A recombinant a-L-iduronidase comprising an a-L-iduronidase and a heterologous signal peptide, said heterologous signal peptide capable of being cleaved at a single site.

6. A recombinant a-L-iduronidase having an amino acid sequence selected from the group consisting of:

1) SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13;

7. The recombinant a-L-iduronidase according to claim 5 having glycosylation modifications at positions 311, 390, and 426.

8. The recombinant a-L-iduronidase according to claim 6, wherein said glycosylation modification is GlcNAc (2) Man (7) P (2).

9. The recombinant a-L-iduronidase according to claim 5, wherein cellular internalization of said recombinant a-L-iduronidase is mediated by M6 PR.

10. A process for the preparation of a recombinant a-L-iduronidase comprising eukaryotic expression of the isolated nucleotide sequence of any of claims 1-4 or eukaryotic expression of the recombinant a-L-iduronidase of any of claims 5-9.

11. The method of claim 10, wherein the isolated nucleotide sequence is preceded by a KOZARK sequence, and the resulting construct is constructed into a eukaryotic expression vector, which is then transferred into a eukaryotic expression system for expression.

12. The method of claim 11, wherein the KOZARK sequence is GCCGCCACCATGC.

13. The method of claim 10, wherein the eukaryotic expression is in a mammalian cell.

14. The method of claim 13, wherein the mammalian cell is CHOK1SV GS-KO.

15. The method of claim 10, comprising: constructing a recombinant eukaryotic expression vector containing the isolated nucleotide sequence; transferring the recombinant eukaryotic expression plasmid into an eukaryotic expression system for eukaryotic expression; collecting the supernatant, and purifying to obtain the alpha-L-iduronidase.

16. The method of claim 10, wherein the purifying comprises one or more of affinity chromatography, hydrophobic interaction chromatography, and ion exchange chromatography.

17. The method of claim 16, wherein the affinity chromatography is a linear gradient elution using agarose gel to obtain an eluate containing the protein of interest.

18. The method of claim 16, wherein the hydrophobic interaction chromatography employs a hydrophobic chromatography column for removing hetero-proteins, multimers; before sample loading, 4M NaCl is added to reach the final concentration of 2M, the pH value is adjusted to 5-6 by using 1M NaOH, and the target protein is isocratically eluted by using an elution buffer solution.

19. The method of claim 16, wherein the ion exchange chromatography uses a salt tolerant cation column, and after sample loading and equilibration, the target protein is eluted with an elution buffer in a linear gradient of 0-70%. Wherein the washing buffer is 20mM PB 1M NaCl pH5.5.

20. The method of claim 16, wherein the purified solution is concentrated by ultrafiltration using a 30kDa membrane to phosphate buffer containing 0.0001% Tween 80 at pH 5.5.