CN114181318B - Recombinant adeno-associated virus for tissue-specific expression of IDUA fusion protein penetrating blood brain barrier and application thereof - Google Patents

Recombinant adeno-associated virus for tissue-specific expression of IDUA fusion protein penetrating blood brain barrier and application thereof Download PDF

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CN114181318B
CN114181318B CN202111315401.2A CN202111315401A CN114181318B CN 114181318 B CN114181318 B CN 114181318B CN 202111315401 A CN202111315401 A CN 202111315401A CN 114181318 B CN114181318 B CN 114181318B
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idua
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杨阳
晋秀
魏于全
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Sichuan University
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Abstract

The invention discloses a recombinant adeno-associated virus for tissue-specific expression of IDUA fusion proteins penetrating through blood brain barrier and application thereof. The human IDUA fusion protein is a connecting peptide connected IDUA protein and a melanin transferrin peptide, and the amino acid sequence of the melanin transferrin peptide is shown as SEQ ID NO. 18; the connecting peptide is selected from (EAAAK) n N=any integer from 1 to 5. We also constructed liver-specific IDUA fusion protein gene expression vector, and through three plasmid transfection, packaging and preparation of AAV8 virus. The MTfp-L-IDUA fusion protein expressed and secreted by the AAV8.TBG. MTfp-hIDUA virus vector can efficiently penetrate through the blood brain barrier after entering the blood system, reach the brain tissue, improve the nervous system diseases, reverse the cell damage caused by lysosome storage in the brain tissue, and improve the cognitive function of a disease model.

Description

Recombinant adeno-associated virus for tissue-specific expression of IDUA fusion protein penetrating blood brain barrier and application thereof
Technical Field
The invention belongs to the field of biological medicine, and relates to a recombinant adeno-associated virus for tissue-specific expression of IDUA fusion proteins penetrating through blood brain barrier and application thereof.
Background
Mucopolysaccharidosis type I (mucopolysaccharidosis type I, MPS I) is an autosomal recessive rare inherited metabolic disorder, a serious metabolic disorder caused by a-L-iduronidase deficiency resulting from IDUA (α -L-iduronidase enzyme, IDUA) gene mutation can result in excessive accumulation of glycosaminoglycans in all organs and tissues in the body, thus endangering life. The accumulation of glycosaminoglycans results in multiple system diseases and different clinical manifestations, mainly including cardiomyopathy, hepatosplenomegaly, upper airway obstruction and progressive neurological diseases. MPS I IS classified as mild (Scheie syndrome or MPS IS; MIM # 607016), moderate (Hurler-Scheie syndrome or MPS IH/S; MIM # 607015) and severe (Hurler syndrome or MPS-IH; MIM # 607014) according to the severity of the disease.
Laroninase (laroniase) was approved by the U.S. food and drug administration and the national drug administration for long-term treatment of MPS I in 2003 and 2020, respectively, and is currently the only ERT drug approved worldwide for treatment of MPS I. However, laroninase is a recombinant protein that produces neutralizing antibodies in vivo; most importantly, larceny enzyme cannot penetrate the blood brain barrier and cannot improve neurological disease symptoms in MPS I patients. Through recombinant adeno-associated viral vector (Adeno associated viral, AAV) gene therapy, IDUA genes are specifically expressed in liver cells or brain tissues, and the IDUA genes have great potential in an animal model of MPS I diseases. The nervous system disease phenotype in the disease mice, cats and dogs model can be significantly improved by intrathecal injection. Since the expressed wild-type IDUA enzyme does not have the ability to penetrate the blood brain barrier, intrathecally injected AAV genes are not effective for treating peripheral organ diseases. Likewise, systemic intravenous administration only ameliorates the disease in peripheral organs, but is ineffective against neurological disease.
The blood brain barrier (Blood brain barriar, BBB) presents a continuing challenge for the effective delivery of therapeutic drugs for brain diseases. Studies have shown that drugs conjugated to antibodies that bind to specific receptors on brain endothelial cells can cross the blood brain barrier. This suggests that the use of ligands for brain endothelial cell receptors as carriers for therapeutic drugs may facilitate the passage of the drug into brain tissue through brain capillary endothelial cells in the blood brain barrier. Melanin transferrin (MTf) belongs to the family of transferrin proteins. The melanin transferrin has 37-39% protein sequence homology with human serum transferrin and human lactoferrin. Melanin transferrin exists in vivo in two forms: membrane proteins attached to the cell surface by glycosyl phosphatidylinositol anchors and free soluble forms in serum. Soluble melanin transferrin is located on the surface of normal brain endothelial cells and is able to cross brain capillary endothelium. Karkan et al demonstrated that the brain delivery of soluble melanin-coupled-drug was 10-fold higher than the free drug control and reduced brain tumor growth (PMCID: PMC 2424243). Nounou et al found that melanin transferrin-coupled trastuzumab reduced the number of Her2 positive breast cancer metastases in the brain by 68% (PMCID: PMC 5267937). Thom et al induced significant and long lasting analgesia by peripheral administration by coupling interleukin 1 receptor antagonist using a 12 amino acid peptide fragment of melanin transferrin (PMCID: PMC 6775589).
Specific asa technologies, ltd, filed a series of patents such as CN201280040630.0, CN 20138048133. X, CN201480021574.5, CN201580022655.1, CN201910827251.X, etc., claiming fragments of human p97 (melanin transferrin) polypeptide having Blood Brain Barrier (BBB) transport activity, as well as conjugates of p97 with small molecule drugs, antibodies or antigen binding fragments, siRNA, etc. But no conjugate of p97 with alpha-L-iduronidase has been disclosed. The linkage between proteins or polypeptides may result in altered conformation of the tertiary structural protein due to altered length of the primary structural sequence, potentially resulting in altered or lost activity. Thus, it is unexpected whether p97 and α -L-iduronidase affect the respective activities.
Disclosure of Invention
The object of the present invention is to address the above-mentioned deficiencies of the prior art by providing human IDUA fusion proteins.
The second object of the present invention is to provide a coding gene, an expression cassette, a vector containing the coding gene, and a host cell of the human IDUA fusion protein.
It is a third object of the present invention to provide recombinant adeno-associated viral vectors which tissue-specifically express IDUA fusion proteins.
The fourth object of the present invention is to provide the coding gene of human IDUA fusion protein, expression frame, vector containing the coding gene, host cell and recombinant adenovirus application.
The aim of the invention can be achieved by the following technical scheme:
a human IDUA fusion protein, characterized in that: the peptide is connected with IDUA protein and melanin transferrin peptide, and the amino acid sequence of the melanin transferrin peptide is shown as SEQ ID NO. 18; the connecting peptide is selected from (EAAAK) n N=any integer from 1 to 5.
As a preferred aspect of the present invention, the linker peptide is (EAAAK) 3 As shown in SEQ ID NO. 19.
As a preferred aspect of the present invention, the human IDUA fusion protein is: IDUA protein-linker peptide-melanin transferrin peptide fragment or melanin transferrin peptide fragment-linker peptide-IDUA protein; the amino acid sequence is shown as SEQ ID NO.20 or SEQ ID NO. 21.
As a further preferred aspect of the present invention, the nucleotide sequence of the encoding gene of the human IDUA fusion protein is shown as SEQ ID NO.9 or SEQ ID NO. 10.
The coding gene of the human IDUA fusion protein.
As a preferable mode of the invention, the coding gene of the human IDUA fusion protein is subjected to codon optimization, and the nucleotide sequence is shown as SEQ ID NO.9 or SEQ ID NO. 10.
An IDUA fusion protein expression cassette, which consists of an enhancer-promoter-target gene sequence-polyA signal, wherein the enhancer is selected from human apolipoprotein hepatic cotrol region bp enhancer sequence, alpha micro/bikunin precursor enhancer, human transthystatin enhancer or human apolipoprotein E enhancer; the promoter is selected from hepatocyte specific human TTR promoter, human hAAT promoter or TBG promoter; a gene of interest selected from the group consisting of genes encoding the human IDUA fusion protein of claim 5 or 6; the polyA signal sequence is selected from SV40 PolyA, bGH polyA, hGH polyA or rBG polyA sequences; the enhancer, the promoter, the target gene sequence and the polyA are connected through a bond or a nucleotide connecting sequence.
As a preferable mode of the invention, the enhancer is selected from alpha micro/bikunin precursor enhancers, and the sequence of the enhancer is shown as SEQ ID NO. 1; the promoter is selected from TBG promoter, and the sequence is shown as SEQ ID NO. 2; the polyA is selected from bGH ployA, and the sequence is shown as SEQ ID NO. 4.
As a preferred mode of the invention, the nucleotide sequence of the IDUA protein expression frame is shown as SEQ ID NO.11 and SEQ ID NO. 12.
A vector, which is characterized by comprising the coding gene of the human IDUA fusion protein or the IDUA fusion protein expression frame of the invention.
As a preferred aspect of the invention, the vector is selected from any one of the following recombinant adeno-associated viral vector serotypes: AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-LK03 or AAVAnc80d, preferably AAV3B, AAV5, AAV8 or AAV9.
As a preferred aspect of the present invention, the backbone vector sequence of the vector is shown in SEQ ID NO. 13.
A host cell comprising a vector of the invention, or a gene encoding a human IDUA fusion protein of the invention or an IDUA fusion protein expression cassette of the invention integrated into its chromosome.
As a preferred aspect of the invention, the host cell is a mammalian cell, preferably a HEK293 cell, a Huh7 cell, a CHO cell or an Sf9 cell.
The recombinant adeno-associated virus for expressing IDUA fusion protein specifically is prepared by co-transfecting HEK293 cells with REP protein of AAV, CAP protein expression plasmid of selected serotypes, auxiliary plasmid and the vector of the invention.
As a preferred aspect of the present invention, the REP protein and CAP protein expression plasmid of the AAV is selected from pAAV2/8; the helper plasmid is selected from pAdΔF6.
The coding gene of the human IDUA fusion protein, the IDUA fusion protein expression frame, the vector and the recombinant adeno-associated virus are applied to the preparation of medicines for treating mucopolysaccharidosis type I diseases.
A pharmaceutical formulation comprising a vector of the invention or a recombinant adeno-associated virus of the invention, and a pharmaceutically acceptable carrier or excipient.
The beneficial effects are that:
the invention designs and reforms IDUA protein, which passes through (EAAAK) at N end and C end of IDUA protein respectively 3 The protein connecting peptide is connected with an amino acid peptide segment (DSSHAFTLDELR) of melanin transferrin, which are sequentially named as MTfp-L-IDUA and IDUA-L-MTfp, and the modified IDUA fusion protease activity is found to be unchanged. Furthermore, the selection of the linker peptide has a great influence on the activity of IDUA fusion proteins. Use (GGS) 3 Connection peptide such as XTEN, GSAGSAAGSGEF or SIVAQLSRPDPA, IDUA fusion protease activity is significantly reduced, while (EAAAK) 3 The connecting peptide can promote the activity of IDUA fusion protease. In the blood brain barrier in-vitro cell model, MTfp-L-IDUA fusion protein can penetrate vascular endothelial cells with high efficiency. On this basis, we constructed liver-specific IDUA fusion protein gene expression vectors, and packaged and prepared AAV8 viruses (named AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp and AAV8.Tbg. MTfp-hIDUA, respectively) by three plasmid transfection. In MPS I mice model, AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp and AAV8.TBG. MTfp-hIDUA virus vector can express and secrete IDUA in hepatocytes, IDUA-L-MTfp or MTfp-L-IDUA fusion protein to blood, and reach peripheral organs (including heart, liver, spleen, kidney, lung, etc.) through blood circulation by means of intravenous systemic administration, improve IDUA protease activity in peripheral organ tissues, reduce accumulation of glycosaminoglycan in tissues to normal level, and reverse cell injury caused by lysosome accumulation of peripheral tissues. In addition, MTfp-L-IDUA fusion protein expressed and secreted by AAV8.TBG. MTfp-hIDUA virus vector can efficiently penetrate through blood brain barrier after entering blood system, reach brain tissue, improve nervous system diseases, reverse cell damage caused by lysosome storage in brain tissue, and improve cognitive function of disease model. The research results show that the technical proposal is expected to solve the bottleneck faced in the treatment of MPS I diseases, and shows hugeGreat potential for drug development.
Drawings
FIG. 1pAAV. TBG. HIDUA. BGH, pAAV. TBG. HIDUA-MTfp. BGH and pAAV. TBG. MTfp-hIDUA. BGH plasmid maps
FIG. 2 detection of plasmid-expressed IDUA, MTfp-L-IDUA and IDUA-L-MTfp fusion protease Activity
FIG. 3 comparison of the IDUA fusion protease Activity of different connecting peptides
FIG. 4IDUA, MTfp-L-IDUA and IDUA-L-MTfp fusion proteins penetrating in vitro blood brain barrier model detection
FIG. 5AAV8.TBG. HIDUA, AAV8.TBG. HIDUA-MTfp and AAV8.TBG. MTfp-hIDUA Virus genome Structure FIG. 6AAV8 Gene therapy restores IDUA enzyme Activity in serum of MPS I disease model
FIG. 7AAV8 Gene therapy restoring IDUA enzyme Activity in peripheral tissues and brain tissues of MPS I disease model
FIG. 8AAV8 Gene therapy reduces glycosaminoglycan accumulation in peripheral tissue and brain tissue of MPS I disease model
FIG. 9AAV8 Gene therapy reverse lysosomal storage injury in MPS I disease model
FIG. 10AAV8 Gene therapy improves skeletal development in MPS I disease model
FIG. 11AAV8 Gene therapy to restore nerve function in MPS I disease model
Detailed Description
EXAMPLE 1 design of human IDUA and IDUA fusion protein, codon optimization and construction of expression plasmid vector
The invention designs two IDUA fusion proteins of IDUA-L-MTfp and MTfp-L-IDUA based on human IDUA protein (SEQ ID NO. 17), through protein connecting peptide (EAAAKEAAAKEAAAK, amino acid sequence is shown as SEQ ID NO.19, coding sequence is shown as SEQ ID NO. 7) and connecting amino acid peptide segment (MTfp, DSSHAFTLDELR, amino acid sequence is shown as SEQ ID NO.18, coding sequence is shown as SEQ ID NO. 8) of melanin transferrin to C end (named IDUA-L-MTfp protein, SEQ ID NO. 20) or N end (named MTfp-L-IDUA protein, SEQ ID NO. 21) of IDUA protein. Then, codon optimization is performed according to amino acid sequences of IDUA, IDUA-L-MTfp and MTfp-L-IDUA proteinsAnd (3) carrying out chemical reaction to obtain the nucleotide sequence with optimized codons shown as SEQ ID NO.3, SEQ ID NO.9 and SEQ ID NO. 10. Full gene synthesis wild type and codon optimized IDUA, MTfp-L-IDUA and IDUA-L-MTfp gene sequences were cloned by In-Fusion ligation into backbone plasmid vectors (SEQ ID No. 13) containing 5'aav ITR sequence (SEQ ID No. 5), alpha micro/bikunin precursor enhancer (SEQ ID No. 1), TBG promoter (SEQ ID No. 2), bGH polyA (SEQ ID No. 4) and 3' aav ITR sequence (SEQ ID No. 6), respectively, obtaining pAAV.TBG.hIDUAwt.bGH, pAAV.TBG.hIDUAwt-mtfp.bgh and paav.tbg.mtfp-hduawt.bgh plasmids expressing wild type human IDUA proteins, and pAAV.TBG.hIDUA.bGH, pAAV.TBG.hIDUA-mtfp.bgh and paav.tbg.mtfp-hdua.bgh expressing codon optimized human IDUA proteins, respectively, the vectors expressing codon optimized human IDUA were as shown In fig. 1A-C, additionally replaced with several gene encoding sequences of the following gene sequences of the plasmid encoding the wild type human IDUA protein: (GGS) 3 The linker peptide, XTEN linker peptide, GSAGSAAGSGEF linker peptide, SIVAQLSRPDPA linker peptide, respectively pAAV. TBG. MTfp- (GGS) 3 -hIDUA.bGH, pAAV.TBG.MTfp-XTEN-hIDUA.bGH, pAAV.TBG.MTfp-GSA-hIDUA.bGH and pAAV. TBG. MTfp-SIV-hIDUA.bGH plasmids.
EXAMPLE 2 detection of plasmid-expressed IDUA, MTfp-L-IDUA and IDUA-L-MTfp fusion protease Activity
The pAAV.TBG.hIDUAwt.bGH, pAAV.TBG.hIDUAwt-MTfp.bGH, pAAV.TBG.MTfp-hIDUAwt.bGH, pAAV.TBG.hIDUA.bGH, pAAV.TBG.hIDUA-MTfp.bgh and pAAV.TBG.MTfp-hIDUA.bgh plasmids of example 1 were transfected by PEImax to mouse hepatocytes H2.35 or human hepatocytes Huh7 cultured in 6-well plates at the same mass (2. Mu.g) and the cell supernatants were collected by centrifugation 48 hours after transfection. Protease activity in cell transfected supernatants was detected by IDUA enzyme activity assay, which was performed as follows: mu.L of the cell transfection supernatant was diluted to 0.1mL of sterile water, and then mixed with 0.1mL of 100mmol/L of 4MU-iduronide (buffer: 0.15mol/L sodium chloride, 0.05% Triton-X100, 0.1mol/L sodium acetate, pH 3.58) and incubated at 37℃for 1-3 hours. After addition of 2ml of stop buffer (290 mmol/l glycine, 180mmol/l sodium citrate, pH 10.9.), the fluorescence absorbance was measured by a multifunctional microplate reader.A 4MU standard curve was established and the enzyme activity was calculated by calculating the 4MU released by fluorescence absorbance. The unit of enzyme activity is expressed as nmol/ml/h. The enzyme activity of pAAV. TBG. HIDUAwt. BGH plasmid transfected group was 3.88.+ -. 0.88nmol/ml/h (n=3); pAAV TBG. MTfp-hIDUAwt. BGH plasmid transfected group enzyme activity was 10.11+ -2.30 nmol/ml/h (n=3); the enzyme activity of pAAV. TBG. HIDUAwt-MTfp. BGH plasmid transfected group was 44.55.+ -. 3.77nmol/ml/h (n=3); the enzyme activity of the pAAV TBG. HIDUA. BGH plasmid transfected group was 32.15.+ -. 5.66nmol/ml/h (n=3); pAAV TBG. MTfp-hIDUA. BGH plasmid transfected group enzyme activity was 63.66+ -5.97 nmol/ml/h (n=3); the enzyme activity of the pAAV TBG. HIDUA-MTfp. BGH plasmid transfected group was 37.19.+ -. 5.64nmol/ml/h (n=3). The experimental result shows that the codon optimization of IDUA protein can obviously improve the protein expression level and further improve the IDUA enzyme activity. Furthermore, by (EAAAK) 3 The connecting peptide is fused with MTfp peptide at N or C end of IDUA protein, and does not affect IDUA protein expression and activity (figure 2).
Example 3 Effect of different connecting peptides on MTfp-L-IDUA fusion protease Activity
pAAV.TBG.hIDUA.bGH, pAAV.TBG.MTfp-hIDUA.bGH, pAAV.TBG.MTfp- (GGS) constructed in example 1 3 the-hIDUA.bGH, pAAV.TBG.MTfp-XTEN-hIDUA.bGH, pAAV.TBG.MTfp-GSA-hIDUA.bGH and pAAV. TBG. MTfp-SIV-hIDUA.bGH plasmids were transfected at the same mass (2. Mu.g) by PEImax into human hepatocytes Huh7 cultured in 6-well plates, and the cell supernatants were collected by centrifugation 48 hours after transfection. Protease activity in cell transfected supernatants was detected by IDUA enzyme activity assay. pAAV TBG. MTfp-hIDUA. BGH plasmid transfected group enzyme activity 92.18 + -8.30 nmol/ml/h (n=3); pAAV TBG MTfp- (GGS) 3 The enzyme activity of the hIDUA.bGH plasmid transfected group was 2.06.+ -. 0.77nmol/ml/h (n=3); the enzyme activity of pAAV TBG. MTfp-XTEN-hIDUA. BGH plasmid transfected group was 4.91+ -0.99 nmol/ml/h (n=3); pAAV TBG. MTfp-GSA-hIDUA. BGH plasmid transfected group enzyme activity was 10.87+ -0.85 nmol/ml/h (n=3); the enzyme activity of pAAV TBG. MTfp-SIV-hIDUA. BGH plasmid transfected group was 0.79.+ -. 0.34nmol/ml/h (n=3). The experimental results show that the use (GGS) 3 Connecting peptides such as XTEN, GSAGSAAGSGEF and SIVAQLSRPDPA, and the IDUA fusion protease activity is significantly reduced, while (EAAAK) 3 The linker peptide may enhance IDUA fusion protease activity (fig. 3).
Example 4 detection of IDUA, MTfp-L-IDUA and IDUA-L-MTfp fusion proteins by in vitro blood brain barrier model penetration
The pAAV.TBG.hIDUA.bGH, pAAV.TBG.hIDUA-MTfp.bGH and pAAV.TBG.MTfp-hIDUA.bGH plasmids of example 1 were transfected at the same mass (2. Mu.g) by PEImax into human hepatoma cells Huh7 cultured in 6-well plates, and the cell culture supernatants were concentrated by centrifugation 48 hours after transfection for use. An in vitro blood brain barrier model was established using immortalized human brain capillary endothelial cells hCMEC/D3 (Millipore, # SCC 066): hCMEC/D3 cells were first seeded on top of type I collagen-coated Transwell cells at a density of 1.2X10 5 cells/cm 2 The ECM complete medium was added for continuous culture for 3-5 days. The tightness of hCMEC/D3 monolayers was examined using a sodium fluorescein permeability test. Before the start of the blood brain barrier penetration experiment, the prepared hCMEC/D3 monolayer cells were washed with PBS, then added to serum free ECM medium and placed in a 37 ℃ incubator for preheating. Then, 4. Mu.l of the cell transfection supernatant was added to the Transwell chamber, and incubated at 37℃for 1 hour, 2 hours, and the culture medium in the Transwell chamber was taken for 4 hours to measure the IDUA enzyme activity. The results of each group penetrating the hCMEC/D3 in vitro blood brain barrier model are as follows: the pAAV. TBG. HIDUA. BGH transfected group was incubated for 1 hour with an enzyme activity of 0.88+ -0.30 nmol/ml/h, for 2 hours with an enzyme activity of 1.05+ -0.35 nmol/ml/h, and for 4 hours with an enzyme activity of 1.58+ -0.07 nmol/ml/h; the pAAV. TBG. HIDUA-MTfp. BGH transfected group was incubated for 1 hour with an enzyme activity of 1.10+ -0.47 nmol/ml/h, for 2 hours with an enzyme activity of 1.18+ -0.29 nmol/ml/h, and for 4 hours with an enzyme activity of 1.77+ -0.70 nmol/ml/h; the pAAV TBG. MTfp-hIDUA. BGH transfected group was incubated for 1 hour with an enzyme activity of 1.81.+ -. 0.35nmol/ml/h, 2 hours with an enzyme activity of 2.60.+ -. 0.41nmol/ml/h, and 4 hours with an enzyme activity of 3.84.+ -. 0.07nmol/ml/h. The above experimental results show that MTfp-L-IDUA and IDUA-L-MTfp fusion proteins are capable of penetrating the blood brain barrier model in vitro, compared with the natural human IDUA protein, wherein MTfp-L-IDUA is the most efficient in penetrating the blood brain barrier model (FIG. 4).
EXAMPLE 5AAV8 Virus preparation and purification
The method for packaging and purifying recombinant AAV virus is reported by Martin Lock et al, and adopts PEIpro to express REP of AAV2 and CAP protein of AAV8 (pAAV 2/8) and auxiliaryCo-transfection of HEK293 cell packaging with helper plasmid (pAdΔF6) and AAV packaging cis-plasmid (pAAV.TBG.hIDUA.bGH, pAAV.TBG.hIDUA-MTfp. BGH or pAAV. TBG. MTfp-hIDUA. BGH), respectively, preparation of virus AAV8.TBG. HIDUA (FIG. 5a, SEQ ID NO. 14), AAV8.TBG. HIDUA-MTfp (FIG. 5b, SEQ ID NO. 15) and AAV8.TBG. MTfp-hIDUA (FIG. 5c, SEQ ID NO. 16) viruses, after 144h transfection, cell culture supernatants were harvested, after concentration of virus liquid by tangential filtration, AAV8 virus was purified by ultracentrifugation using an iodixanol ultracentrifugation gradient, AAV8 virus was collected, purified by centrifugation using Amicon Ultra 100K to remove salts, and finally AAV8 virus was placed in 20mM Tris (pH8.0), 1mM MgCl 2 200mM NaCl and 0.001% PF68 preparation. The prepared AAV8 virus is used for detecting the purity of the virus by SDS-PAGE staining, detecting the endotoxin content by limulus reagent, and determining the virus titer by a Taqman fluorescent quantitative (qPCR) probe method for the gene therapy research of a subsequent MPS I disease model.
EXAMPLE 6AAV8 Gene therapy restoration of IDUA enzyme Activity in serum of MPS I disease model
A mouse model of MPS I disease (Idua-W392X, stock No: 017681) was purchased from Jackson laboratories, USA. MPS I mice, 4-6 weeks old, were injected with the same dose of AAV8 virus by tail vein (3 x10 11 vg per mouse), serum was isolated from blood taken at various time points after injection, and IDUA enzyme activity in the serum was measured. The results showed that after injection of AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp and aav8.Tbg. MTfp-hdua virus, IDUA enzyme activity in MPS I disease mice reached a maximum at the third week, after which it was slightly decreased and stabilized for a duration of the study (week 16 after treatment) (fig. 6). IDUA activity in serum of MPS I untreated mice was 0.23±0.04nmol/ml/h (n=7) at 16 weeks post-treatment; IDUA activity in serum of wild-type control mice was 3.94±0.30nmol/ml/h (n=7); serum of mice in aav8.Tbg. Hdua treated group had IDUA activity of 8904±5003nmol/ml/h (n=8); serum of mice in the AAV8.TBG. MTfp-hIDUA treatment group had IDUA activity of 11488+ -3430 nmol/ml/h (n=9); serum IDUA activity in aav8.Tbg. Hdua-MTfp treated mice was 3806±1081nmol/ml/h (n=9). The above results show that AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp or AAV8.TBG. MTfp-hIDUA virus vectors can be specifically expressed in liver cell tissueHuman IDUA protein, MTfp-L-IDUA or IDUA-L-MTfp fusion protein is secreted into blood.
EXAMPLE 7AAV8 Gene therapy restoration and improvement of IDUA Activity in peripheral and brain tissues of MPS I disease model
At 16 weeks post-treatment, the treated mice in example 6 were sacrificed, peripheral organs (heart, liver, spleen, lung and kidney) and brain tissues (olfactory bulb, cortex, striatum, hippocampus, cerebellum, thalamus, brainstem) were collected, the tissues were homogenized in lysate (0.9% nacl,0.2% triton-X100, pH 3.5), repeatedly freeze-thawed 3-5 times, and protein quantification was performed by BCA kit after centrifugation to collect the tissue lysate, and then IDUA enzyme activity in the tissue lysate was detected. The results showed that both AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp and AAV8.TBG. MTfp-hIDUA increased IDUA activity to normal levels in heart, liver, spleen, lung and kidney tissues following treatment in peripheral tissues (FIG. 7 a). In brain tissue, after aav8.Tbg. Hidua treatment, there was no significant difference in IDUA activity in all brain tissues versus untreated group (n=8, p > 0.05); aav8.Tbg. Hidua-MTfp treatment only increased IDUA activity in olfactory bulb and cortical brain tissue (n=9, p < 0.05); aav8.Tbg. Mtfp-hdua treatment increased IDUA activity in all brain tissues (olfactory bulb p <0.05; cortical p <0.01; striatal p <0.01; hippocampal p <0.05; cerebellum p <0.05; thalamus and brainstem p <0.01; n=9) (fig. 7 b). The results show that after the AAV8.TBG. MTfp-hIDUA virus vector specifically expresses and secretes MTfp-L-IDUA fusion protein in liver cell tissues and enters blood circulation, the AAV8.TBG. MTfp-hIDUA virus vector not only can improve IDUA activity in peripheral tissues, but also can efficiently penetrate through blood brain barrier and enter brain tissues, and can improve IDUA enzyme activity in the brain tissues.
Example 8AAV8 Gene therapy to reduce glycosaminoglycan accumulation in peripheral and brain tissues of MPS I disease model
Glycosaminoglycan levels of the tissue lysis supernatants of example 7 were tested using a Blyscan Glycosaminoglycan kit (Biocolor, carrickfugus, UK). The results showed that both AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp and AAV8.TBG. MTfp-hIDUA reduced glycosaminoglycans to normal levels in heart, liver, spleen, lung and kidney tissues following treatment in peripheral tissues (FIG. 8 a). Furthermore, there was no significant difference in glycosaminoglycan levels in all brain tissues after aav8.Tbg. Hidua treatment compared to untreated group (n=8, p > 0.05); aav8.Tbg. Hidua-MTfp treatment reduced glycosaminoglycan accumulation in parts of brain tissue, such as striatal tissue (p <0.01, n=9), hippocampal tissue (p <0.05, n=9), and cerebellum (p <0.05, n=9); aav8.Tbg. Mtfp-hIDUA treatment reduced glycosaminoglycan accumulation in all brain tissues (olfactory bulb p <0.05; striatal p <0.001; hippocampal p <0.001; cerebellum p <0.01; thalamus and brainstem p <0.05; n=9) (fig. 8 b). At week 16 post-treatment, the glycosaminoglycan content in the urine of mice was examined, and the results showed that both AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp and AAV8.TBG. MTfp-hIDUA reduced glycosaminoglycan in urine to normal levels after treatment (FIG. 8 c). In addition, the treated mice in example 6 were sacrificed, peripheral organs (heart, liver, spleen, lung and kidney) and brain tissues were collected, paraffin-embedded and sectioned, and histological analysis by alcian blue staining revealed that the glycosaminoglycan storage amount in heart, liver, spleen, lung and kidney tissues was reduced after treatment with AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp and aav8.Tbg. MTfp-hIDUA, and the glycosaminoglycan storage amount in brain tissues was restored to normal level after treatment with aav8.Tbg. MTfp-hIDUA (fig. 8 d). The results show that after the AAV8.TBG. MTfp-hIDUA virus vector specifically expresses and secretes MTfp-L-IDUA fusion protein in liver cell tissues and enters blood circulation, the AAV8.TBG. MTfp-hIDUA virus vector not only can reduce the accumulation of glycosaminoglycan in peripheral tissues, but also can efficiently penetrate through blood brain barrier to enter brain tissues and reduce the accumulation of glycosaminoglycan in brain tissues.
Example 9AAV8 Gene therapy to reverse lysosomal storage injury in MPS I disease model
Accumulation of glycosaminoglycans in tissues results in the formation of characteristic microscopic lysosomal vacuoles. The treated mice in example 6 were sacrificed, peripheral organs (heart, liver, spleen, lung and kidney) and brain tissues were collected, paraffin-embedded and sectioned, and then histologically analyzed by H & E staining. The results showed that a significant reduction in vacuolated cells was detected in peripheral tissues both for AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp and AAV8.TBG. MTfp-hIDUA treatment, and that a significant improvement in purkinje cell vacuolation was also observed following AAV8.TBG. MTfp-hIDUA treatment (FIG. 9). The results show that the AAV8.TBG. MTfp-hIDUA virus vector can specifically express and secrete MTfp-L-IDUA fusion protein in liver cell tissue to enter blood circulation, and can reverse lysosomal storage injury in peripheral tissues and brain tissues of mice with diseases.
Example 10AAV8 Gene therapy improving skeletal development in MPS I disease model
Mucopolysaccharidosis type I can lead to bone dysplasia, for example, wang et al observe thickening of the zygomatic arch and distortion of femur length and width in MPS I disease mice. The thickness of the forehead cheekbone and femur of the mice in example 6 was measured by microcomputer tomography (Micro computed tomography, micro-CT) at 16 weeks post-treatment. The results showed that AAV8.TBG.hIDUA, AAV8.TBG.hIDUA-MTfp and AAV8.TBG. MTfp-hIDUA significantly improved the zygomatic arch and femur phenotypes after treatment, restoring their thickness to normal levels (FIGS. 10a, b, c, d).
EXAMPLE 11AAV8 Gene therapy restoring neurological function in MPS I disease model
Mice in example 6 were subjected to a DMP (Delayed-stacking-to-place, DMP) dry maze test 12 weeks after treatment. The DMP dry maze was a circular platform (diameter=122 cm, thickness=1.2 cm) with 40 holes. An escape tube is secured under an aperture to allow mice to escape the platform. The position of the escape hole is changed every day. Each of the four walls has a visual cue attached for the mouse to use for spatial navigation. To start the experiment, the mice were placed at the edge of the platform at a distance from the escape aperture and covered with an opaque funnel. After a delay of about 30 seconds, the tonal noise (2 khz,85 db) was turned on and the transmission box was immediately removed, exposing the mice to intense light (1200 lux). To cope with these uncomfortable situations, the mouse spontaneously searches for and drills into the escape hole. Mice were evaluated in four trials every day for six consecutive days, with a maximum escape time limit of 3 minutes. Data were collected and analyzed using the ANY-size program. After 6 days of testing and training, the average escape latency of normal cognitive wild type mice was reduced from 175 seconds to 62 seconds. In contrast, the average escape latency of untreated MPS I mice slowly decreased from 160 seconds to 117 seconds, indicating the presence of a cognition deficit phenotype. The mean escape latency of MPS I mice receiving aav8.Tbg. Hidua treatment slowly decreased from 162 seconds to 115 seconds; the escape rate of mice receiving AAV8.TBG. HIDUA-MTfp treatment and AAV8.TBG. MTfp-hIDUA treatment was significantly increased, rapidly decreasing from 167 seconds to 87 seconds, and from 159 seconds to 61 seconds, respectively. Wherein aav8.Tbg. Mtfp-hIDUA treated mice were not significantly different from wild-type mice (fig. 11). The results show that after AAV8.TBG. MTfp-hIDUA gene therapy, the AAV8.TBG. MTfp-hIDUA virus vector specifically expresses secreted MTfp-L-IDUA fusion protein in liver cell tissues, can penetrate through blood brain barrier into brain tissues with high efficiency, thereby obviously improving the nerve function of MPS I disease model and enhancing cognitive function.
Sequence listing
<110> university of Sichuan
<120> recombinant adeno-associated virus expressing IDUA fusion protein penetrating blood brain barrier specifically and application thereof
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 206
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
aggttaattt ttaaaaagca gtcaaaagtc caagtggccc ttggcagcat ttactctctc 60
tgtttgctct ggttaataat ctcaggagca caaacattcc agatccaggt taatttttaa 120
aaagcagtca aaagtccaag tggcccttgg cagcatttac tctctctgtt tgctctggtt 180
aataatctca ggagcacaaa cattcc 206
<210> 2
<211> 410
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tgcatgtata atttctacag aacctattag aaaggatcac ccagcctctg cttttgtaca 60
actttccctt aaaaaactgc caattccact gctgtttggc ccaatagtga gaactttttc 120
ctgctgcctc ttggtgcttt tgcctatggc ccctattctg cctgctgaag acactcttgc 180
cagcatggac ttaaacccct ccagctctga caatcctctt tctcttttgt tttacatgaa 240
gggtctggca gccaaagcaa tcactcaaag ttcaaacctt atcatttttt gctttgttcc 300
tcttggcctt ggttttgtac atcagctttg aaaataccat cccagggtta atgctggggt 360
taatttataa ctaagagtgc tctagttttg caatacagga catgctataa 410
<210> 3
<211> 1962
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgagacccc tgcggcctag agccgccctg ctggccctgc tggccagcct gctggcagca 60
ccccctgtgg caccagcaga ggccccccac ctggtgcacg tggacgcagc cagagccctg 120
tggccactgc ggagattctg gaggtccacc ggcttttgcc caccactgcc acactctcag 180
gcagaccagt acgtgctgag ctgggatcag cagctgaacc tggcatatgt gggagcagtg 240
ccacacaggg gcatcaagca ggtgcggaca cactggctgc tggagctggt gaccacaaga 300
ggcagcaccg gcaggggcct gtcctacaac ttcacacacc tggacggcta tctggatctg 360
ctgcgggaga atcagctgct gcccggcttt gagctgatgg gctccgcctc tggccacttc 420
accgactttg aggataagca gcaggtgttc gagtggaagg acctggtgag ctccctggcc 480
aggcgctaca tcggcagata tggcctggcc cacgtgagca agtggaactt tgagacctgg 540
aatgagcctg accaccacga cttcgataac gtgtccatga caatgcaggg ctttctgaat 600
tactatgatg cctgctccga gggactgaga gcagcatctc ctgccctgag gctgggagga 660
ccaggcgata gcttccacac ccctccaaga tctccactga gctggggcct gctgaggcac 720
tgtcacgacg gcaccaactt ctttacaggc gaggccggcg tgagactgga ttacatctct 780
ctgcaccgga agggcgccag atctagcatc agcatcctgg agcaggagaa ggtggtggcc 840
cagcagatca ggcagctgtt cccaaagttt gccgacaccc ccatctacaa tgacgaggca 900
gatccactgg tgggatggtc cctgccacag ccatggaggg ccgatgtgac atatgccgcc 960
atggtggtga aagtgatcgc ccagcaccag aacctgctgc tggccaatac cacatctgcc 1020
ttcccttacg ccctgctgag caacgacaat gccttcctgt cctatcaccc tcacccattt 1080
gcccagcgca ccctgacagc ccggtttcag gtgaacaata cccgcccacc tcacgtgcag 1140
ctgctgagga agcccgtgct gacagcaatg ggactgctgg ccctgctgga cgaggagcag 1200
ctgtgggcag aggtgtccca ggcaggaacc gtgctggatt ctaatcacac agtgggcgtg 1260
ctggcctccg cccaccgccc acagggacca gcagacgcct ggagggcagc cgtgctgatc 1320
tatgccagcg acgataccag agcccaccct aacaggtccg tggccgtgac actgaggctg 1380
aggggagtgc caccaggacc tggactggtg tacgtgaccc gctatctgga caatggactg 1440
tgcagcccag atggagagtg gcggagactg ggccggcccg tgttcccaac agcagagcag 1500
tttaggagga tgagggcagc agaggatccc gtggcagcag caccaaggcc tctgccagca 1560
ggcggcaggc tgaccctgcg ccctgccctg aggctgccat ccctgctgct ggtgcacgtg 1620
tgcgcaaggc cagagaagcc tccaggacag gtgacccggc tgagagccct gcctctgaca 1680
cagggccagc tggtgctggt gtggtctgac gagcacgtgg gcagcaagtg tctgtggacc 1740
tacgagatcc agttctctca ggatggcaag gcctataccc ccgtgtcccg caagcccagc 1800
accttcaacc tgttcgtgtt tagcccagat acaggcgccg tgagcggatc ctacagggtg 1860
cgcgccctgg actattgggc aagaccaggc cctttctccg atccagtgcc ctacctggag 1920
gtgcctgtgc caaggggccc accttctcca ggaaatcctt ga 1962
<210> 4
<211> 208
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60
tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120
tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180
gggaagagaa tagcaggcat gctgggga 208
<210> 5
<211> 168
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct tgtagttaat gattaacccg ccatgctact tatctacg 168
<210> 6
<211> 168
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt 60
ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 120
acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcag 168
<210> 7
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gaggccgctg ctaaagaggc tgccgccaaa gaagccgccg ctaag 45
<210> 8
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gactcctctc acgccttcac cctggacgag ctgcgg 36
<210> 9
<211> 2043
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atgagacccc tgcggcctag agccgccctg ctggccctgc tggccagcct gctggcagca 60
ccccctgtgg caccagcaga ggccccccac ctggtgcacg tggacgcagc cagagccctg 120
tggccactgc ggagattctg gaggtccacc ggcttttgcc caccactgcc acactctcag 180
gcagaccagt acgtgctgag ctgggatcag cagctgaacc tggcatatgt gggagcagtg 240
ccacacaggg gcatcaagca ggtgcggaca cactggctgc tggagctggt gaccacaaga 300
ggcagcaccg gcaggggcct gtcctacaac ttcacacacc tggacggcta tctggatctg 360
ctgcgggaga atcagctgct gcccggcttt gagctgatgg gctccgcctc tggccacttc 420
accgactttg aggataagca gcaggtgttc gagtggaagg acctggtgag ctccctggcc 480
aggcgctaca tcggcagata tggcctggcc cacgtgagca agtggaactt tgagacctgg 540
aatgagcctg accaccacga cttcgataac gtgtccatga caatgcaggg ctttctgaat 600
tactatgatg cctgctccga gggactgaga gcagcatctc ctgccctgag gctgggagga 660
ccaggcgata gcttccacac ccctccaaga tctccactga gctggggcct gctgaggcac 720
tgtcacgacg gcaccaactt ctttacaggc gaggccggcg tgagactgga ttacatctct 780
ctgcaccgga agggcgccag atctagcatc agcatcctgg agcaggagaa ggtggtggcc 840
cagcagatca ggcagctgtt cccaaagttt gccgacaccc ccatctacaa tgacgaggca 900
gatccactgg tgggatggtc cctgccacag ccatggaggg ccgatgtgac atatgccgcc 960
atggtggtga aagtgatcgc ccagcaccag aacctgctgc tggccaatac cacatctgcc 1020
ttcccttacg ccctgctgag caacgacaat gccttcctgt cctatcaccc tcacccattt 1080
gcccagcgca ccctgacagc ccggtttcag gtgaacaata cccgcccacc tcacgtgcag 1140
ctgctgagga agcccgtgct gacagcaatg ggactgctgg ccctgctgga cgaggagcag 1200
ctgtgggcag aggtgtccca ggcaggaacc gtgctggatt ctaatcacac agtgggcgtg 1260
ctggcctccg cccaccgccc acagggacca gcagacgcct ggagggcagc cgtgctgatc 1320
tatgccagcg acgataccag agcccaccct aacaggtccg tggccgtgac actgaggctg 1380
aggggagtgc caccaggacc tggactggtg tacgtgaccc gctatctgga caatggactg 1440
tgcagcccag atggagagtg gcggagactg ggccggcccg tgttcccaac agcagagcag 1500
tttaggagga tgagggcagc agaggatccc gtggcagcag caccaaggcc tctgccagca 1560
ggcggcaggc tgaccctgcg ccctgccctg aggctgccat ccctgctgct ggtgcacgtg 1620
tgcgcaaggc cagagaagcc tccaggacag gtgacccggc tgagagccct gcctctgaca 1680
cagggccagc tggtgctggt gtggtctgac gagcacgtgg gcagcaagtg tctgtggacc 1740
tacgagatcc agttctctca ggatggcaag gcctataccc ccgtgtcccg caagcccagc 1800
accttcaacc tgttcgtgtt tagcccagat acaggcgccg tgagcggatc ctacagggtg 1860
cgcgccctgg actattgggc aagaccaggc cctttctccg atccagtgcc ctacctggag 1920
gtgcctgtgc caaggggccc accttctcca ggaaatcctg aggccgctgc taaagaggct 1980
gccgccaaag aagccgccgc taaggactcc tctcacgcct tcaccctgga cgagctgcgg 2040
tga 2043
<210> 10
<211> 2043
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atgagacccc tgcggcctag agccgccctg ctggccctgc tggccagcct gctggcagca 60
ccccctgtgg caccagcaga ggactcctct cacgccttca ccctggacga gctgcgggag 120
gccgctgcta aagaggctgc cgccaaagaa gccgccgcta aggcccccca cctggtgcac 180
gtggacgcag ccagagccct gtggccactg cggagattct ggaggtccac cggcttttgc 240
ccaccactgc cacactctca ggcagaccag tacgtgctga gctgggatca gcagctgaac 300
ctggcatatg tgggagcagt gccacacagg ggcatcaagc aggtgcggac acactggctg 360
ctggagctgg tgaccacaag aggcagcacc ggcaggggcc tgtcctacaa cttcacacac 420
ctggacggct atctggatct gctgcgggag aatcagctgc tgcccggctt tgagctgatg 480
ggctccgcct ctggccactt caccgacttt gaggataagc agcaggtgtt cgagtggaag 540
gacctggtga gctccctggc caggcgctac atcggcagat atggcctggc ccacgtgagc 600
aagtggaact ttgagacctg gaatgagcct gaccaccacg acttcgataa cgtgtccatg 660
acaatgcagg gctttctgaa ttactatgat gcctgctccg agggactgag agcagcatct 720
cctgccctga ggctgggagg accaggcgat agcttccaca cccctccaag atctccactg 780
agctggggcc tgctgaggca ctgtcacgac ggcaccaact tctttacagg cgaggccggc 840
gtgagactgg attacatctc tctgcaccgg aagggcgcca gatctagcat cagcatcctg 900
gagcaggaga aggtggtggc ccagcagatc aggcagctgt tcccaaagtt tgccgacacc 960
cccatctaca atgacgaggc agatccactg gtgggatggt ccctgccaca gccatggagg 1020
gccgatgtga catatgccgc catggtggtg aaagtgatcg cccagcacca gaacctgctg 1080
ctggccaata ccacatctgc cttcccttac gccctgctga gcaacgacaa tgccttcctg 1140
tcctatcacc ctcacccatt tgcccagcgc accctgacag cccggtttca ggtgaacaat 1200
acccgcccac ctcacgtgca gctgctgagg aagcccgtgc tgacagcaat gggactgctg 1260
gccctgctgg acgaggagca gctgtgggca gaggtgtccc aggcaggaac cgtgctggat 1320
tctaatcaca cagtgggcgt gctggcctcc gcccaccgcc cacagggacc agcagacgcc 1380
tggagggcag ccgtgctgat ctatgccagc gacgatacca gagcccaccc taacaggtcc 1440
gtggccgtga cactgaggct gaggggagtg ccaccaggac ctggactggt gtacgtgacc 1500
cgctatctgg acaatggact gtgcagccca gatggagagt ggcggagact gggccggccc 1560
gtgttcccaa cagcagagca gtttaggagg atgagggcag cagaggatcc cgtggcagca 1620
gcaccaaggc ctctgccagc aggcggcagg ctgaccctgc gccctgccct gaggctgcca 1680
tccctgctgc tggtgcacgt gtgcgcaagg ccagagaagc ctccaggaca ggtgacccgg 1740
ctgagagccc tgcctctgac acagggccag ctggtgctgg tgtggtctga cgagcacgtg 1800
ggcagcaagt gtctgtggac ctacgagatc cagttctctc aggatggcaa ggcctatacc 1860
cccgtgtccc gcaagcccag caccttcaac ctgttcgtgt ttagcccaga tacaggcgcc 1920
gtgagcggat cctacagggt gcgcgccctg gactattggg caagaccagg ccctttctcc 1980
gatccagtgc cctacctgga ggtgcctgtg ccaaggggcc caccttctcc aggaaatcct 2040
tga 2043
<210> 11
<211> 3065
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
aggttaattt ttaaaaagca gtcaaaagtc caagtggccc ttggcagcat ttactctctc 60
tgtttgctct ggttaataat ctcaggagca caaacattcc agatccaggt taatttttaa 120
aaagcagtca aaagtccaag tggcccttgg cagcatttac tctctctgtt tgctctggtt 180
aataatctca ggagcacaaa cattccagat ccggcgcgcc agggctggaa gctacctttg 240
acatcatttc ctctgcgaat gcatgtataa tttctacaga acctattaga aaggatcacc 300
cagcctctgc ttttgtacaa ctttccctta aaaaactgcc aattccactg ctgtttggcc 360
caatagtgag aactttttcc tgctgcctct tggtgctttt gcctatggcc cctattctgc 420
ctgctgaaga cactcttgcc agcatggact taaacccctc cagctctgac aatcctcttt 480
ctcttttgtt ttacatgaag ggtctggcag ccaaagcaat cactcaaagt tcaaacctta 540
tcattttttg ctttgttcct cttggccttg gttttgtaca tcagctttga aaataccatc 600
ccagggttaa tgctggggtt aatttataac taagagtgct ctagttttgc aatacaggac 660
atgctataaa aatggaaaga tgttgctttc tgagagacag ctttattgcg gtagtttatc 720
acagttaaat tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca 780
gaccggtgcc accatgagac ccctgcggcc tagagccgcc ctgctggccc tgctggccag 840
cctgctggca gcaccccctg tggcaccagc agaggccccc cacctggtgc acgtggacgc 900
agccagagcc ctgtggccac tgcggagatt ctggaggtcc accggctttt gcccaccact 960
gccacactct caggcagacc agtacgtgct gagctgggat cagcagctga acctggcata 1020
tgtgggagca gtgccacaca ggggcatcaa gcaggtgcgg acacactggc tgctggagct 1080
ggtgaccaca agaggcagca ccggcagggg cctgtcctac aacttcacac acctggacgg 1140
ctatctggat ctgctgcggg agaatcagct gctgcccggc tttgagctga tgggctccgc 1200
ctctggccac ttcaccgact ttgaggataa gcagcaggtg ttcgagtgga aggacctggt 1260
gagctccctg gccaggcgct acatcggcag atatggcctg gcccacgtga gcaagtggaa 1320
ctttgagacc tggaatgagc ctgaccacca cgacttcgat aacgtgtcca tgacaatgca 1380
gggctttctg aattactatg atgcctgctc cgagggactg agagcagcat ctcctgccct 1440
gaggctggga ggaccaggcg atagcttcca cacccctcca agatctccac tgagctgggg 1500
cctgctgagg cactgtcacg acggcaccaa cttctttaca ggcgaggccg gcgtgagact 1560
ggattacatc tctctgcacc ggaagggcgc cagatctagc atcagcatcc tggagcagga 1620
gaaggtggtg gcccagcaga tcaggcagct gttcccaaag tttgccgaca cccccatcta 1680
caatgacgag gcagatccac tggtgggatg gtccctgcca cagccatgga gggccgatgt 1740
gacatatgcc gccatggtgg tgaaagtgat cgcccagcac cagaacctgc tgctggccaa 1800
taccacatct gccttccctt acgccctgct gagcaacgac aatgccttcc tgtcctatca 1860
ccctcaccca tttgcccagc gcaccctgac agcccggttt caggtgaaca atacccgccc 1920
acctcacgtg cagctgctga ggaagcccgt gctgacagca atgggactgc tggccctgct 1980
ggacgaggag cagctgtggg cagaggtgtc ccaggcagga accgtgctgg attctaatca 2040
cacagtgggc gtgctggcct ccgcccaccg cccacaggga ccagcagacg cctggagggc 2100
agccgtgctg atctatgcca gcgacgatac cagagcccac cctaacaggt ccgtggccgt 2160
gacactgagg ctgaggggag tgccaccagg acctggactg gtgtacgtga cccgctatct 2220
ggacaatgga ctgtgcagcc cagatggaga gtggcggaga ctgggccggc ccgtgttccc 2280
aacagcagag cagtttagga ggatgagggc agcagaggat cccgtggcag cagcaccaag 2340
gcctctgcca gcaggcggca ggctgaccct gcgccctgcc ctgaggctgc catccctgct 2400
gctggtgcac gtgtgcgcaa ggccagagaa gcctccagga caggtgaccc ggctgagagc 2460
cctgcctctg acacagggcc agctggtgct ggtgtggtct gacgagcacg tgggcagcaa 2520
gtgtctgtgg acctacgaga tccagttctc tcaggatggc aaggcctata cccccgtgtc 2580
ccgcaagccc agcaccttca acctgttcgt gtttagccca gatacaggcg ccgtgagcgg 2640
atcctacagg gtgcgcgccc tggactattg ggcaagacca ggccctttct ccgatccagt 2700
gccctacctg gaggtgcctg tgccaagggg cccaccttct ccaggaaatc ctgaggccgc 2760
tgctaaagag gctgccgcca aagaagccgc cgctaaggac tcctctcacg ccttcaccct 2820
ggacgagctg cggtgagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca 2880
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 2940
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3000
gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct 3060
gggga 3065
<210> 12
<211> 3065
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
aggttaattt ttaaaaagca gtcaaaagtc caagtggccc ttggcagcat ttactctctc 60
tgtttgctct ggttaataat ctcaggagca caaacattcc agatccaggt taatttttaa 120
aaagcagtca aaagtccaag tggcccttgg cagcatttac tctctctgtt tgctctggtt 180
aataatctca ggagcacaaa cattccagat ccggcgcgcc agggctggaa gctacctttg 240
acatcatttc ctctgcgaat gcatgtataa tttctacaga acctattaga aaggatcacc 300
cagcctctgc ttttgtacaa ctttccctta aaaaactgcc aattccactg ctgtttggcc 360
caatagtgag aactttttcc tgctgcctct tggtgctttt gcctatggcc cctattctgc 420
ctgctgaaga cactcttgcc agcatggact taaacccctc cagctctgac aatcctcttt 480
ctcttttgtt ttacatgaag ggtctggcag ccaaagcaat cactcaaagt tcaaacctta 540
tcattttttg ctttgttcct cttggccttg gttttgtaca tcagctttga aaataccatc 600
ccagggttaa tgctggggtt aatttataac taagagtgct ctagttttgc aatacaggac 660
atgctataaa aatggaaaga tgttgctttc tgagagacag ctttattgcg gtagtttatc 720
acagttaaat tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca 780
gaccggtgcc accatgagac ccctgcggcc tagagccgcc ctgctggccc tgctggccag 840
cctgctggca gcaccccctg tggcaccagc agaggactcc tctcacgcct tcaccctgga 900
cgagctgcgg gaggccgctg ctaaagaggc tgccgccaaa gaagccgccg ctaaggcccc 960
ccacctggtg cacgtggacg cagccagagc cctgtggcca ctgcggagat tctggaggtc 1020
caccggcttt tgcccaccac tgccacactc tcaggcagac cagtacgtgc tgagctggga 1080
tcagcagctg aacctggcat atgtgggagc agtgccacac aggggcatca agcaggtgcg 1140
gacacactgg ctgctggagc tggtgaccac aagaggcagc accggcaggg gcctgtccta 1200
caacttcaca cacctggacg gctatctgga tctgctgcgg gagaatcagc tgctgcccgg 1260
ctttgagctg atgggctccg cctctggcca cttcaccgac tttgaggata agcagcaggt 1320
gttcgagtgg aaggacctgg tgagctccct ggccaggcgc tacatcggca gatatggcct 1380
ggcccacgtg agcaagtgga actttgagac ctggaatgag cctgaccacc acgacttcga 1440
taacgtgtcc atgacaatgc agggctttct gaattactat gatgcctgct ccgagggact 1500
gagagcagca tctcctgccc tgaggctggg aggaccaggc gatagcttcc acacccctcc 1560
aagatctcca ctgagctggg gcctgctgag gcactgtcac gacggcacca acttctttac 1620
aggcgaggcc ggcgtgagac tggattacat ctctctgcac cggaagggcg ccagatctag 1680
catcagcatc ctggagcagg agaaggtggt ggcccagcag atcaggcagc tgttcccaaa 1740
gtttgccgac acccccatct acaatgacga ggcagatcca ctggtgggat ggtccctgcc 1800
acagccatgg agggccgatg tgacatatgc cgccatggtg gtgaaagtga tcgcccagca 1860
ccagaacctg ctgctggcca ataccacatc tgccttccct tacgccctgc tgagcaacga 1920
caatgccttc ctgtcctatc accctcaccc atttgcccag cgcaccctga cagcccggtt 1980
tcaggtgaac aatacccgcc cacctcacgt gcagctgctg aggaagcccg tgctgacagc 2040
aatgggactg ctggccctgc tggacgagga gcagctgtgg gcagaggtgt cccaggcagg 2100
aaccgtgctg gattctaatc acacagtggg cgtgctggcc tccgcccacc gcccacaggg 2160
accagcagac gcctggaggg cagccgtgct gatctatgcc agcgacgata ccagagccca 2220
ccctaacagg tccgtggccg tgacactgag gctgagggga gtgccaccag gacctggact 2280
ggtgtacgtg acccgctatc tggacaatgg actgtgcagc ccagatggag agtggcggag 2340
actgggccgg cccgtgttcc caacagcaga gcagtttagg aggatgaggg cagcagagga 2400
tcccgtggca gcagcaccaa ggcctctgcc agcaggcggc aggctgaccc tgcgccctgc 2460
cctgaggctg ccatccctgc tgctggtgca cgtgtgcgca aggccagaga agcctccagg 2520
acaggtgacc cggctgagag ccctgcctct gacacagggc cagctggtgc tggtgtggtc 2580
tgacgagcac gtgggcagca agtgtctgtg gacctacgag atccagttct ctcaggatgg 2640
caaggcctat acccccgtgt cccgcaagcc cagcaccttc aacctgttcg tgtttagccc 2700
agatacaggc gccgtgagcg gatcctacag ggtgcgcgcc ctggactatt gggcaagacc 2760
aggccctttc tccgatccag tgccctacct ggaggtgcct gtgccaaggg gcccaccttc 2820
tccaggaaat ccttgagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca 2880
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 2940
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3000
gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct 3060
gggga 3065
<210> 13
<211> 4292
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180
aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240
caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300
caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360
cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420
ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480
tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540
aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600
tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660
taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720
ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780
gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840
taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900
tgagagacag ctttattgcg gtagtttatc acagttaaat tgctaacgca gtcagtgctt 960
ctgacacaac agtctcgaac ttaagctgca gaccggtgga tccactagtc cagtgtggtg 1020
gaattctgca gatatcgagc tcgctgatca gcctcgactg tgccttctag ttgccagcca 1080
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 1140
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 1200
gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct 1260
ggggactcga gttaagggcg aattcccgat aaggatcttc ctagagcatg gctacgtaga 1320
taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac 1380
tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 1440
gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agccttaatt aacctaattc 1500
actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 1560
ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 1620
cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt 1680
aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 1740
gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca 1800
agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 1860
caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt 1920
tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac 1980
aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc 2040
ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt 2100
aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 2160
tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 2220
caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 2280
ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 2340
gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 2400
aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 2460
ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 2520
atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 2580
gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 2640
gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 2700
atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 2760
aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 2820
actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 2880
aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 2940
tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 3000
ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 3060
agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 3120
tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 3180
aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 3240
gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 3300
atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 3360
gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 3420
gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 3480
tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 3540
accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 3600
ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 3660
cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 3720
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 3780
ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 3840
tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 3900
ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 3960
cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 4020
gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 4080
tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 4140
cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 4200
cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 4260
tatgaccatg attacgccag atttaattaa gg 4292
<210> 14
<211> 3411
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180
aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240
caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300
caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360
cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420
ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480
tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540
aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600
tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660
taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720
ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780
gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840
taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900
tgagagacag ctttattgcg gtagtttatc acagttaaat tgctaacgca gtcagtgctt 960
ctgacacaac agtctcgaac ttaagctgca gaccggtgcc accatgagac ccctgcggcc 1020
tagagccgcc ctgctggccc tgctggccag cctgctggca gcaccccctg tggcaccagc 1080
agaggccccc cacctggtgc acgtggacgc agccagagcc ctgtggccac tgcggagatt 1140
ctggaggtcc accggctttt gcccaccact gccacactct caggcagacc agtacgtgct 1200
gagctgggat cagcagctga acctggcata tgtgggagca gtgccacaca ggggcatcaa 1260
gcaggtgcgg acacactggc tgctggagct ggtgaccaca agaggcagca ccggcagggg 1320
cctgtcctac aacttcacac acctggacgg ctatctggat ctgctgcggg agaatcagct 1380
gctgcccggc tttgagctga tgggctccgc ctctggccac ttcaccgact ttgaggataa 1440
gcagcaggtg ttcgagtgga aggacctggt gagctccctg gccaggcgct acatcggcag 1500
atatggcctg gcccacgtga gcaagtggaa ctttgagacc tggaatgagc ctgaccacca 1560
cgacttcgat aacgtgtcca tgacaatgca gggctttctg aattactatg atgcctgctc 1620
cgagggactg agagcagcat ctcctgccct gaggctggga ggaccaggcg atagcttcca 1680
cacccctcca agatctccac tgagctgggg cctgctgagg cactgtcacg acggcaccaa 1740
cttctttaca ggcgaggccg gcgtgagact ggattacatc tctctgcacc ggaagggcgc 1800
cagatctagc atcagcatcc tggagcagga gaaggtggtg gcccagcaga tcaggcagct 1860
gttcccaaag tttgccgaca cccccatcta caatgacgag gcagatccac tggtgggatg 1920
gtccctgcca cagccatgga gggccgatgt gacatatgcc gccatggtgg tgaaagtgat 1980
cgcccagcac cagaacctgc tgctggccaa taccacatct gccttccctt acgccctgct 2040
gagcaacgac aatgccttcc tgtcctatca ccctcaccca tttgcccagc gcaccctgac 2100
agcccggttt caggtgaaca atacccgccc acctcacgtg cagctgctga ggaagcccgt 2160
gctgacagca atgggactgc tggccctgct ggacgaggag cagctgtggg cagaggtgtc 2220
ccaggcagga accgtgctgg attctaatca cacagtgggc gtgctggcct ccgcccaccg 2280
cccacaggga ccagcagacg cctggagggc agccgtgctg atctatgcca gcgacgatac 2340
cagagcccac cctaacaggt ccgtggccgt gacactgagg ctgaggggag tgccaccagg 2400
acctggactg gtgtacgtga cccgctatct ggacaatgga ctgtgcagcc cagatggaga 2460
gtggcggaga ctgggccggc ccgtgttccc aacagcagag cagtttagga ggatgagggc 2520
agcagaggat cccgtggcag cagcaccaag gcctctgcca gcaggcggca ggctgaccct 2580
gcgccctgcc ctgaggctgc catccctgct gctggtgcac gtgtgcgcaa ggccagagaa 2640
gcctccagga caggtgaccc ggctgagagc cctgcctctg acacagggcc agctggtgct 2700
ggtgtggtct gacgagcacg tgggcagcaa gtgtctgtgg acctacgaga tccagttctc 2760
tcaggatggc aaggcctata cccccgtgtc ccgcaagccc agcaccttca acctgttcgt 2820
gtttagccca gatacaggcg ccgtgagcgg atcctacagg gtgcgcgccc tggactattg 2880
ggcaagacca ggccctttct ccgatccagt gccctacctg gaggtgcctg tgccaagggg 2940
cccaccttct ccaggaaatc cttgagagct cgctgatcag cctcgactgt gccttctagt 3000
tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 3060
cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 3120
tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc 3180
aggcatgctg gggactcgag ttaagggcga attcccgata aggatcttcc tagagcatgg 3240
ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga 3300
gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 3360
ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca g 3411
<210> 15
<211> 3492
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180
aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240
caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300
caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360
cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420
ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480
tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540
aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600
tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660
taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720
ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780
gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840
taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900
tgagagacag ctttattgcg gtagtttatc acagttaaat tgctaacgca gtcagtgctt 960
ctgacacaac agtctcgaac ttaagctgca gaccggtgcc accatgagac ccctgcggcc 1020
tagagccgcc ctgctggccc tgctggccag cctgctggca gcaccccctg tggcaccagc 1080
agaggccccc cacctggtgc acgtggacgc agccagagcc ctgtggccac tgcggagatt 1140
ctggaggtcc accggctttt gcccaccact gccacactct caggcagacc agtacgtgct 1200
gagctgggat cagcagctga acctggcata tgtgggagca gtgccacaca ggggcatcaa 1260
gcaggtgcgg acacactggc tgctggagct ggtgaccaca agaggcagca ccggcagggg 1320
cctgtcctac aacttcacac acctggacgg ctatctggat ctgctgcggg agaatcagct 1380
gctgcccggc tttgagctga tgggctccgc ctctggccac ttcaccgact ttgaggataa 1440
gcagcaggtg ttcgagtgga aggacctggt gagctccctg gccaggcgct acatcggcag 1500
atatggcctg gcccacgtga gcaagtggaa ctttgagacc tggaatgagc ctgaccacca 1560
cgacttcgat aacgtgtcca tgacaatgca gggctttctg aattactatg atgcctgctc 1620
cgagggactg agagcagcat ctcctgccct gaggctggga ggaccaggcg atagcttcca 1680
cacccctcca agatctccac tgagctgggg cctgctgagg cactgtcacg acggcaccaa 1740
cttctttaca ggcgaggccg gcgtgagact ggattacatc tctctgcacc ggaagggcgc 1800
cagatctagc atcagcatcc tggagcagga gaaggtggtg gcccagcaga tcaggcagct 1860
gttcccaaag tttgccgaca cccccatcta caatgacgag gcagatccac tggtgggatg 1920
gtccctgcca cagccatgga gggccgatgt gacatatgcc gccatggtgg tgaaagtgat 1980
cgcccagcac cagaacctgc tgctggccaa taccacatct gccttccctt acgccctgct 2040
gagcaacgac aatgccttcc tgtcctatca ccctcaccca tttgcccagc gcaccctgac 2100
agcccggttt caggtgaaca atacccgccc acctcacgtg cagctgctga ggaagcccgt 2160
gctgacagca atgggactgc tggccctgct ggacgaggag cagctgtggg cagaggtgtc 2220
ccaggcagga accgtgctgg attctaatca cacagtgggc gtgctggcct ccgcccaccg 2280
cccacaggga ccagcagacg cctggagggc agccgtgctg atctatgcca gcgacgatac 2340
cagagcccac cctaacaggt ccgtggccgt gacactgagg ctgaggggag tgccaccagg 2400
acctggactg gtgtacgtga cccgctatct ggacaatgga ctgtgcagcc cagatggaga 2460
gtggcggaga ctgggccggc ccgtgttccc aacagcagag cagtttagga ggatgagggc 2520
agcagaggat cccgtggcag cagcaccaag gcctctgcca gcaggcggca ggctgaccct 2580
gcgccctgcc ctgaggctgc catccctgct gctggtgcac gtgtgcgcaa ggccagagaa 2640
gcctccagga caggtgaccc ggctgagagc cctgcctctg acacagggcc agctggtgct 2700
ggtgtggtct gacgagcacg tgggcagcaa gtgtctgtgg acctacgaga tccagttctc 2760
tcaggatggc aaggcctata cccccgtgtc ccgcaagccc agcaccttca acctgttcgt 2820
gtttagccca gatacaggcg ccgtgagcgg atcctacagg gtgcgcgccc tggactattg 2880
ggcaagacca ggccctttct ccgatccagt gccctacctg gaggtgcctg tgccaagggg 2940
cccaccttct ccaggaaatc ctgaggccgc tgctaaagag gctgccgcca aagaagccgc 3000
cgctaaggac tcctctcacg ccttcaccct ggacgagctg cggtgagagc tcgctgatca 3060
gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 3120
ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 3180
cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 3240
gaggattggg aagagaatag caggcatgct ggggactcga gttaagggcg aattcccgat 3300
aaggatcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta 3360
caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga 3420
ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga 3480
gcgagcgcgc ag 3492
<210> 16
<211> 3492
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180
aggaagatcg gaattcgccc ttaagctagc aggttaattt ttaaaaagca gtcaaaagtc 240
caagtggccc ttggcagcat ttactctctc tgtttgctct ggttaataat ctcaggagca 300
caaacattcc agatccaggt taatttttaa aaagcagtca aaagtccaag tggcccttgg 360
cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa cattccagat 420
ccggcgcgcc agggctggaa gctacctttg acatcatttc ctctgcgaat gcatgtataa 480
tttctacaga acctattaga aaggatcacc cagcctctgc ttttgtacaa ctttccctta 540
aaaaactgcc aattccactg ctgtttggcc caatagtgag aactttttcc tgctgcctct 600
tggtgctttt gcctatggcc cctattctgc ctgctgaaga cactcttgcc agcatggact 660
taaacccctc cagctctgac aatcctcttt ctcttttgtt ttacatgaag ggtctggcag 720
ccaaagcaat cactcaaagt tcaaacctta tcattttttg ctttgttcct cttggccttg 780
gttttgtaca tcagctttga aaataccatc ccagggttaa tgctggggtt aatttataac 840
taagagtgct ctagttttgc aatacaggac atgctataaa aatggaaaga tgttgctttc 900
tgagagacag ctttattgcg gtagtttatc acagttaaat tgctaacgca gtcagtgctt 960
ctgacacaac agtctcgaac ttaagctgca gaccggtgcc accatgagac ccctgcggcc 1020
tagagccgcc ctgctggccc tgctggccag cctgctggca gcaccccctg tggcaccagc 1080
agaggactcc tctcacgcct tcaccctgga cgagctgcgg gaggccgctg ctaaagaggc 1140
tgccgccaaa gaagccgccg ctaaggcccc ccacctggtg cacgtggacg cagccagagc 1200
cctgtggcca ctgcggagat tctggaggtc caccggcttt tgcccaccac tgccacactc 1260
tcaggcagac cagtacgtgc tgagctggga tcagcagctg aacctggcat atgtgggagc 1320
agtgccacac aggggcatca agcaggtgcg gacacactgg ctgctggagc tggtgaccac 1380
aagaggcagc accggcaggg gcctgtccta caacttcaca cacctggacg gctatctgga 1440
tctgctgcgg gagaatcagc tgctgcccgg ctttgagctg atgggctccg cctctggcca 1500
cttcaccgac tttgaggata agcagcaggt gttcgagtgg aaggacctgg tgagctccct 1560
ggccaggcgc tacatcggca gatatggcct ggcccacgtg agcaagtgga actttgagac 1620
ctggaatgag cctgaccacc acgacttcga taacgtgtcc atgacaatgc agggctttct 1680
gaattactat gatgcctgct ccgagggact gagagcagca tctcctgccc tgaggctggg 1740
aggaccaggc gatagcttcc acacccctcc aagatctcca ctgagctggg gcctgctgag 1800
gcactgtcac gacggcacca acttctttac aggcgaggcc ggcgtgagac tggattacat 1860
ctctctgcac cggaagggcg ccagatctag catcagcatc ctggagcagg agaaggtggt 1920
ggcccagcag atcaggcagc tgttcccaaa gtttgccgac acccccatct acaatgacga 1980
ggcagatcca ctggtgggat ggtccctgcc acagccatgg agggccgatg tgacatatgc 2040
cgccatggtg gtgaaagtga tcgcccagca ccagaacctg ctgctggcca ataccacatc 2100
tgccttccct tacgccctgc tgagcaacga caatgccttc ctgtcctatc accctcaccc 2160
atttgcccag cgcaccctga cagcccggtt tcaggtgaac aatacccgcc cacctcacgt 2220
gcagctgctg aggaagcccg tgctgacagc aatgggactg ctggccctgc tggacgagga 2280
gcagctgtgg gcagaggtgt cccaggcagg aaccgtgctg gattctaatc acacagtggg 2340
cgtgctggcc tccgcccacc gcccacaggg accagcagac gcctggaggg cagccgtgct 2400
gatctatgcc agcgacgata ccagagccca ccctaacagg tccgtggccg tgacactgag 2460
gctgagggga gtgccaccag gacctggact ggtgtacgtg acccgctatc tggacaatgg 2520
actgtgcagc ccagatggag agtggcggag actgggccgg cccgtgttcc caacagcaga 2580
gcagtttagg aggatgaggg cagcagagga tcccgtggca gcagcaccaa ggcctctgcc 2640
agcaggcggc aggctgaccc tgcgccctgc cctgaggctg ccatccctgc tgctggtgca 2700
cgtgtgcgca aggccagaga agcctccagg acaggtgacc cggctgagag ccctgcctct 2760
gacacagggc cagctggtgc tggtgtggtc tgacgagcac gtgggcagca agtgtctgtg 2820
gacctacgag atccagttct ctcaggatgg caaggcctat acccccgtgt cccgcaagcc 2880
cagcaccttc aacctgttcg tgtttagccc agatacaggc gccgtgagcg gatcctacag 2940
ggtgcgcgcc ctggactatt gggcaagacc aggccctttc tccgatccag tgccctacct 3000
ggaggtgcct gtgccaaggg gcccaccttc tccaggaaat ccttgagagc tcgctgatca 3060
gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 3120
ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 3180
cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 3240
gaggattggg aagagaatag caggcatgct ggggactcga gttaagggcg aattcccgat 3300
aaggatcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa tcattaacta 3360
caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga 3420
ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga 3480
gcgagcgcgc ag 3492
<210> 17
<211> 653
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
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
His 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> 18
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg
1 5 10
<210> 19
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
1 5 10 15
<210> 20
<211> 680
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
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
His 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 Glu Ala Ala
645 650 655
Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Asp Ser Ser His
660 665 670
Ala Phe Thr Leu Asp Glu Leu Arg
675 680
<210> 21
<211> 680
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
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 Asp Ser Ser His Ala
20 25 30
Phe Thr Leu Asp Glu Leu Arg Glu Ala Ala Ala Lys Glu Ala Ala Ala
35 40 45
Lys Glu Ala Ala Ala Lys Ala Pro His Leu Val His Val Asp Ala Ala
50 55 60
Arg Ala Leu Trp Pro Leu Arg Arg Phe Trp Arg Ser Thr Gly Phe Cys
65 70 75 80
Pro Pro Leu Pro His Ser Gln Ala Asp Gln Tyr Val Leu Ser Trp Asp
85 90 95
Gln Gln Leu Asn Leu Ala Tyr Val Gly Ala Val Pro His Arg Gly Ile
100 105 110
Lys Gln Val Arg Thr His Trp Leu Leu Glu Leu Val Thr Thr Arg Gly
115 120 125
Ser Thr Gly Arg Gly Leu Ser Tyr Asn Phe Thr His Leu Asp Gly Tyr
130 135 140
Leu Asp Leu Leu Arg Glu Asn Gln Leu Leu Pro Gly Phe Glu Leu Met
145 150 155 160
Gly Ser Ala Ser Gly His Phe Thr Asp Phe Glu Asp Lys Gln Gln Val
165 170 175
Phe Glu Trp Lys Asp Leu Val Ser Ser Leu Ala Arg Arg Tyr Ile Gly
180 185 190
Arg Tyr Gly Leu Ala His Val Ser Lys Trp Asn Phe Glu Thr Trp Asn
195 200 205
Glu Pro Asp His His Asp Phe Asp Asn Val Ser Met Thr Met Gln Gly
210 215 220
Phe Leu Asn Tyr Tyr Asp Ala Cys Ser Glu Gly Leu Arg Ala Ala Ser
225 230 235 240
Pro Ala Leu Arg Leu Gly Gly Pro Gly Asp Ser Phe His Thr Pro Pro
245 250 255
Arg Ser Pro Leu Ser Trp Gly Leu Leu Arg His Cys His Asp Gly Thr
260 265 270
Asn Phe Phe Thr Gly Glu Ala Gly Val Arg Leu Asp Tyr Ile Ser Leu
275 280 285
His Arg Lys Gly Ala Arg Ser Ser Ile Ser Ile Leu Glu Gln Glu Lys
290 295 300
Val Val Ala Gln Gln Ile Arg Gln Leu Phe Pro Lys Phe Ala Asp Thr
305 310 315 320
Pro Ile Tyr Asn Asp Glu Ala Asp Pro Leu Val Gly Trp Ser Leu Pro
325 330 335
Gln Pro Trp Arg Ala Asp Val Thr Tyr Ala Ala Met Val Val Lys Val
340 345 350
Ile Ala Gln His Gln Asn Leu Leu Leu Ala Asn Thr Thr Ser Ala Phe
355 360 365
Pro Tyr Ala Leu Leu Ser Asn Asp Asn Ala Phe Leu Ser Tyr His Pro
370 375 380
His Pro Phe Ala Gln Arg Thr Leu Thr Ala Arg Phe Gln Val Asn Asn
385 390 395 400
Thr Arg Pro Pro His Val Gln Leu Leu Arg Lys Pro Val Leu Thr Ala
405 410 415
Met Gly Leu Leu Ala Leu Leu Asp Glu Glu Gln Leu Trp Ala Glu Val
420 425 430
Ser Gln Ala Gly Thr Val Leu Asp Ser Asn His Thr Val Gly Val Leu
435 440 445
Ala Ser Ala His Arg Pro Gln Gly Pro Ala Asp Ala Trp Arg Ala Ala
450 455 460
Val Leu Ile Tyr Ala Ser Asp Asp Thr Arg Ala His Pro Asn Arg Ser
465 470 475 480
Val Ala Val Thr Leu Arg Leu Arg Gly Val Pro Pro Gly Pro Gly Leu
485 490 495
Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly Leu Cys Ser Pro Asp Gly
500 505 510
Glu Trp Arg Arg Leu Gly Arg Pro Val Phe Pro Thr Ala Glu Gln Phe
515 520 525
Arg Arg Met Arg Ala Ala Glu Asp Pro Val Ala Ala Ala Pro Arg Pro
530 535 540
Leu Pro Ala Gly Gly Arg Leu Thr Leu Arg Pro Ala Leu Arg Leu Pro
545 550 555 560
Ser Leu Leu Leu Val His Val Cys Ala Arg Pro Glu Lys Pro Pro Gly
565 570 575
Gln Val Thr Arg Leu Arg Ala Leu Pro Leu Thr Gln Gly Gln Leu Val
580 585 590
Leu Val Trp Ser Asp Glu His Val Gly Ser Lys Cys Leu Trp Thr Tyr
595 600 605
Glu Ile Gln Phe Ser Gln Asp Gly Lys Ala Tyr Thr Pro Val Ser Arg
610 615 620
Lys Pro Ser Thr Phe Asn Leu Phe Val Phe Ser Pro Asp Thr Gly Ala
625 630 635 640
Val Ser Gly Ser Tyr Arg Val Arg Ala Leu Asp Tyr Trp Ala Arg Pro
645 650 655
Gly Pro Phe Ser Asp Pro Val Pro Tyr Leu Glu Val Pro Val Pro Arg
660 665 670
Gly Pro Pro Ser Pro Gly Asn Pro
675 680

Claims (15)

1. A human IDUA fusion protein, characterized in that: the peptide is connected with IDUA protein and melanin transferrin peptide, and the amino acid sequence of the melanin transferrin peptide is shown as SEQ ID NO. 18; the connection sequence of the human IDUA fusion protein is as follows: melanin transferrin peptide fragment-connecting peptide-IDUA protein; the amino acid sequence of the human IDUA fusion protein is shown as SEQ ID NO. 21.
2. The gene encoding the human IDUA fusion protein of claim 1.
3. The coding gene according to claim 2, wherein the coding gene of the human IDUA fusion protein is codon optimized, and the nucleotide sequence is shown in SEQ ID No. 10.
4. An IDUA fusion protein expression frame is characterized in that the nucleotide sequence of the IDUA fusion protein expression frame is shown in SEQ ID NO. 12.
5. A vector comprising a gene encoding the human IDUA fusion protein of claim 2 or 3 or the IDUA fusion protein expression cassette of claim 4.
6. The vector according to claim 5, characterized in that said vector is selected from any one of the following recombinant adeno-associated viral vector serotypes: AAV1, AAV2, AAV3B, AAV, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-LK03, or AAVAnc80d.
7. The vector according to claim 6, wherein said vector is selected from any one of the following recombinant adeno-associated viral vector serotypes: AAV3B, AAV, AAV8 or AAV9.
8. The vector according to claim 7, characterized in that the backbone vector sequence of the vector is shown in SEQ ID NO. 13.
9. A host cell comprising the vector of any one of claims 5-8, or a coding gene for the human IDUA fusion protein of claim 2 or 3 or the IDUA fusion protein expression cassette of claim 4 integrated into its chromosome as an exogenous source.
10. The host cell of claim 9, wherein the host cell is a mammalian cell.
11. The host cell according to claim 9, characterized in that the host cell is a HEK293 cell, a Huh7 cell, a CHO cell or an Sf9 cell.
12. A recombinant adeno-associated virus for tissue-specific expression of IDUA fusion protein, characterized in that it is prepared by co-transfecting a HEK293 cell with a REP protein of AAV and CAP protein expression plasmid of selected serotype, helper plasmid and vector according to any one of claims 5-8.
13. The recombinant adeno-associated virus of claim 12, wherein the expression plasmid for REP protein and CAP protein of AAV is selected from the group consisting of pAAV2/8; the helper plasmid is selected from pAdΔF6.
14. Use of the gene encoding the human IDUA fusion protein of claim 2 or 3, the IDUA fusion protein expression cassette of claim 4, the vector of any one of claims 5-8, the recombinant adeno-associated virus of claim 12 or 13 in the preparation of a medicament for treating mucopolysaccharidosis type I disease.
15. A pharmaceutical formulation, characterized in that it comprises the vector of any one of claims 5-8 or the recombinant adeno-associated virus of claim 12 or 13, and a pharmaceutically acceptable carrier or excipient.
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