CN112575034A - Product for treating hemophilia A and application - Google Patents

Abstract

The application discloses a product for treating hemophilia A and application thereof. The products include expression cassettes for the BDD FVIII gene, recombinant vectors comprising the expression cassettes, recombinant viruses, particularly recombinant lentiviruses, recombinant bacteria, or recombinant cells. The promoter of the BDD FVIII gene in the expression cassette comprises the PF4 promoter. The lentiviral vector containing the PF4 promoter and the BDD FVIII gene expression cassette constructed by the invention can ensure that the BDD FVIII gene is specifically expressed in platelets derived from cytoplasm shedding of megakaryocytes from CD34+ cells through the PF4 promoter, thereby playing a blood coagulation function and being applicable to gene therapy of hemophilia A of all ages.

Description

Product for treating hemophilia A and application

Technical Field

The invention relates to the field of gene therapy, in particular to a product for treating hemophilia A and application thereof.

Background

Hemophilia is an X-linked recessive hereditary hemorrhagic disease, and is classified into hemophilia a (hemophilia a) and hemophilia B (hemophilia B). Hemophilia a is a deficiency of coagulation factor viii (fviii) caused by a gene mutation. FVIII gene is located at the end of the long arm of the X chromosome: (Xq28) encoding a synthetic peptide chain arranged in the manner of A1-A2-B-A3-C1-C2. The major types of mutations that are closely related to the production of inhibitors in severe hemophiliacs include large fragment deletions, nonsense mutations, inversion of intron 22, followed by small fragment deletions and insertions, missense mutations, and the like. The incidence of hemophilia is not population or regional. In the male population, the incidence of hemophilia a is about 1/5000, accounting for 80% -85% of all hemophilia populations; the incidence of hemophilia B is about 1/25000, accounting for 15% -20% of all hemophilia populations.

The main treatment for hemophilia a is replacement therapy, preferably a recombinant FVIII preparation or a virus inactivated blood-derived FVIII preparation, and cryoprecipitate or fresh frozen plasma or the like can be selected only in the absence of the above conditions. Either a genetically recombinant FVIII preparation or a virally inactivated blood-derived FVIII preparation induces production of inhibitors in the patient's body. In recent years, there has been a trend toward increasing inhibitors of acquired blood coagulation factor viii (fvii)/blood coagulation factor ix (fxi) (also known as acquired hemophilia) or pooled inhibitors in hemophilia patients. The former is an anti-FVIII/FIX autoantibody produced by a non-hemophiliac patient, and the latter is an anti-FVIII/FIX alloantibody produced by a hemophiliac patient who has received an infusion of an exogenous blood coagulation factor product. The incidence of inhibitors for patients with severe type A hemophilia is about 30 percent, and the incidence of inhibitors for patients with non-severe type A is 3 to 13 percent; patients with hemophilia B range from 1% to 6%. Even with the use of standardized immune tolerance Induction Therapy (ITI), the success rate of the ITI for hemophilia a inhibitor positive patients is 70%, the success rate of the ITI for hemophilia B inhibitor positive patients is only 30%, and there is a risk of anaphylaxis and irreversible kidney damage.

The gene therapy for hemophilia a is to directly transfect a vector carrying a modified FVIII gene, such as adeno-associated virus (AAV), into a human body, allowing direct synthesis of FVIII in cells in the body. FVIII mRNA is 9kb in length, encodes a precursor polypeptide of 2351 amino acids, and after a signal peptide of 19 residues at the N-terminal is removed, the mature protein contains 2332 amino acids. FVIII cDNAs are too long to be loaded into vectors such as AVV. The B domain of FVIII is not essential for the activity and function of FVIII in vivo. Deletion of the B domain resulted in a 17-fold increase in mRNA levels and a 30% increase in secreted protein relative to full-length wild-type FVIII. There have been numerous organizations that have developed corresponding research and development for knock-outs of the B domain and shortened linker substitutions.

FVIII (BDD FVIII) genes knocked out by B structural domain loaded and modified by adeno-associated virus (AAV) are directly input into a human body, and after the AAV enters liver cells, the BDD FVIII is synthesized and released, so that the treatment purpose is achieved.

WO/2017/083764 discloses a method for treating hemophilia A by using Adenovirus Associated Virus (AAV) vector carrying FVIII-BDD gene, which shows that the expression amount of FVIII protein can be improved in mammalian cells. However, the biggest problem with AAV for treating hemophilia a is the presence of AAV antibodies in about 30-40% of the population. It is estimated that the serological positive rate of neutralizing antibodies to AAV1 in healthy humans is about 67%, AAV2 72%, AAV5 40%, AAV6 46%, AAV8 38%, and AAV 9. Based on the homology of the AAV capsid structure, antibodies directed against one AAV serotype may cross-react with other AAV. In addition, AAV exists primarily in free form within the cell, and cell division will dilute AAV-loaded genes and ultimately lead to diminished therapeutic effect. Since adult liver cells are mostly in the resting stage, gene therapy for hemophilia using AAV mostly targets the liver as AAV organ and is limited to adult patients. AAV gene therapy has not been practical for children with hemophilia a.

CN108795986A discloses a hemophilia a lentiviral vector modified at the splice donor site 5' of the pTYF lentiviral vector, further comprising FVIII and/or FVIII-BDD genes. The patent mainly relates to the modification of lentivirus, so that the lentivirus loses the self-replication capacity and the safety of gene therapy is improved. The promoter sequence utilized in the patent is EF1 alpha, and can realize the increase of the expression amount of the BDD FVIII gene in cells, but EF1 alpha is not a tissue-specific promoter and cannot effectively control the expression of the BDD FVIII in vivo.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a product for treating hemophilia A and application thereof, wherein the product comprises an expression cassette of a BDD FVIII gene, a recombinant vector containing the expression cassette, a recombinant virus, especially a recombinant lentivirus, a recombinant bacterium or a recombinant cell. The promoter of the BDD FVIII gene in the expression cassette comprises the PF4 promoter. The PF4 promoter enables the BDD FVIII gene to be specifically and highly expressed in recombinant lentiviruses, megakaryocytes and hematopoietic stem cells.

The invention adopts the following technical scheme:

in a first aspect, the present invention provides a recombinant vector for treating hemophilia a comprising an expression cassette co-expressed in tandem of the PF4 promoter and the BDD FVIII gene;

i.e., the recombinant vector comprises an expression cassette for the BDD FVIII gene, wherein the promoter for the BDD FVIII gene comprises the PF4 promoter.

In one embodiment, the platelet factor 4 promoter is the human PF4 promoter (hPF4 promoter) and has the sequence shown in SEQ ID No.1 or a nucleotide sequence at least 80% homologous, preferably at least 85% homologous, and more preferably at least 95% homologous thereto.

In one embodiment, the hPF4 promoter is further optimized to have a sequence as shown in SEQ ID NO.2 or a nucleotide sequence at least 80% homologous, preferably at least 85% homologous, and more preferably at least 95% homologous thereto.

In one embodiment, the hPF4 promoter is further optimized to have a sequence as shown in SEQ ID NO.3 or a nucleotide sequence at least 80% homologous, preferably at least 85% homologous, and more preferably at least 95% homologous thereto.

In one example, the hPF4 promoter can be replaced by the rat PF4 promoter (rPF4), the sequence of which is shown in patent PCT/US 91/07233.

In one embodiment, the hPF4 promoter can also be replaced by other species, such as the mouse PF4 promoter (mPF4 promoter).

In one embodiment, the hPF4 promoter can also be replaced by the GPIba promoter, the ITGA2B promoter, the CD68 promoter, the c-mpl promoter, and the like.

In one embodiment, the sequence of the PF4 promoter may be more or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 600 or more nucleic acid sequences than SEQ ID nos. 1-3.

The nucleotide sequence of the BDD FVIII gene is shown in SEQ ID NO.4, or the nucleotide sequence which has at least 80% homology, preferably at least 85% homology, and further preferably at least 95% homology with the BDD FVIII gene;

the amino acid sequence coded by the BDD FVIII gene is shown in SEQ ID NO.16, or the amino acid sequence which has at least 80 percent of homology, preferably at least 85 percent of homology, and further preferably at least 95 percent of homology with the BDD FVIII gene;

in one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/between 759aa and 760aa with a nucleotide/amino acid sequence as shown in SEQ ID NO.5, SEQ ID NO.18 or with at least 80% homology thereto, preferably at least 85% homology thereto, further preferably at least 95% homology thereto, based on the sequence as shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/between 759aa and 760aa with a nucleotide/amino acid sequence as shown in SEQ ID NO.6, SEQ ID NO.19 or with at least 80% homology thereto, preferably at least 85% homology thereto, further preferably at least 95% homology thereto, based on the sequence as shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/759 aa and 760aa with a nucleotide/amino acid sequence as shown in SEQ ID NO.8, SEQ ID NO.21 or with at least 80%, preferably at least 85%, further preferably at least 95% homology thereto, based on the sequence as shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/between 759aa and 760aa with a nucleotide/amino acid sequence as shown in SEQ ID NO.9, SEQ ID NO.22 or with at least 80% homology thereto, preferably at least 85% homology thereto, further preferably at least 95% homology thereto, based on the sequence as shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/759 aa and 760aa with a nucleotide/amino acid sequence as shown in SEQ ID NO.10, SEQ ID NO.23 or with at least 80%, preferably at least 85%, further preferably at least 95% homology thereto, based on the sequence as shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/between 759aa and 760aa with the nucleotide/amino acid sequence shown in SEQ ID NO.11, SEQ ID NO.24 or with at least 80% homology thereto, preferably at least 85% homology thereto, further preferably at least 95% homology thereto, based on the sequence shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/between 759aa and 760aa with the nucleotide/amino acid sequence shown in SEQ ID NO.12, SEQ ID NO.25 or with at least 80% homology thereto, preferably at least 85% homology thereto, further preferably at least 95% homology thereto, based on the sequence shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/between 759aa and 760aa with the nucleotide/amino acid sequence shown in SEQ ID NO.13, SEQ ID NO.26 or with at least 80% homology thereto, preferably at least 85% homology thereto, further preferably at least 95% homology thereto, based on the sequence shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/759 aa and 760aa with the nucleotide/amino acid sequence shown in SEQ ID NO.14, SEQ ID NO.27 or with at least 80%, preferably at least 85%, further preferably at least 95% homology thereto, based on the sequence shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is substituted between 2277bp and 2278 bp/between 759aa and 760aa with the nucleotide/amino acid sequence shown in SEQ ID NO.15, SEQ ID NO.28 or with at least 80% homology thereto, preferably at least 85% homology thereto, further preferably at least 95% homology thereto, based on the sequence shown in SEQ ID NO.4, SEQ ID NO. 16.

In one embodiment, the BDD FVIII sequence is based on the sequence shown in SEQ ID NO.4, SEQ ID NO.16, between 2277bp and 2278 bp/759 aa and 760aa substituted with the nucleotide/amino acid sequence shown in SEQ ID NO.7 and deleted 2278-2289bp/SEQ ID NO.20 and deleted 760-763aa or with at least 80%, preferably at least 85%, more preferably at least 95% homology thereto.

In one embodiment, the BDD FVIII sequence can be further codon optimized for higher protein expression. The optimized nucleotide sequence is shown as SEQ ID NO.17 or nucleotide with at least 80% homology, preferably at least 85% homology, and further preferably at least 95% homology, and the BDD FVIII amino acid sequence is shown as SEQ ID NO.29 or amino acid sequence with at least 80% homology, preferably at least 85% homology, and further preferably at least 95% homology. The expression amount of the optimized BDD FVIII protein can be increased by about 100 percent and above.

In one example, the BDD FVIII sequence may be an engineered FVIII sequence not mentioned in this patent, including B region knockout and ligation, other region genetic modifications, codon optimization, etc., CpG reduced BDD FVIII as shown in patent WO 2017/075619 a1, or BDD FVIII as shown in patent WO 2017/074526 a1, or codon optimized BDD FVIII-SQ as shown in patent WO 2015/038625, or BDD FVIII sequence as shown in WO 2017/083764, etc.

In one embodiment, the BDD FVIII sequence may be further linked to other protein peptide fragments to form a fusion protein, such as VWFSPD2-BDD FVIII fusion protein sequence shown in patent WO 2014/066663 a 1.

In one embodiment, the 1 st to 57bp (signal peptide sequence) of the sequence shown in SEQ ID NO.4 is replaced by a nucleotide sequence shown in SEQ ID NO.30 or having at least 80% homology thereto, preferably at least 85% homology thereto, further preferably at least 95% homology thereto; 1-19aa (signal peptide sequence) of the sequence shown in SEQ ID NO.16 is substituted by an amino acid sequence shown in SEQ ID NO.31 or having at least 80% homology thereto, preferably at least 85% homology thereto, and more preferably at least 95% homology thereto.

In one example, portions/total nucleotide sequence of PF 4-promoter-BDD FVIII composition can be obtained by chemical synthesis and/or overlap extension PCR.

In one example, the 5 'and 3' ends of the PF4-BDD FVIII compositions may be supplemented with the corresponding sequences of restriction sites XcmI, SalI, respectively, for insertion into the corresponding lentiviral vector plasmids.

In one example, the PF4-BDD FVIII composition (SEQ ID No.1, SEQ ID No.4) can be inserted into prrlsin. cppt. PGK-GFP. wpre (Plasmid #12252) with PGK promoter and GFP sequence deleted to form PF4-BDD FVIII loaded lentiviral vector Plasmid-1, the nucleotide sequence of PF4-BDD FVIII composition is shown in SEQ ID No.32, and the structure of prrlsin. cppt. pf4-BDD FVIII. wpre-1 is shown in fig. 1.

In one example, the PF4-BDD FVIII composition (SEQ ID No.1, SEQ ID No.17) can be inserted into prrlsin. cppt. PGK-GFP. wpre (Plasmid #12252) with PGK promoter and GFP sequences knocked out to form PF4-BDD FVIII loaded lentiviral vector Plasmid-2, the PF4-BDD FVIII composition nucleotide sequence is shown in SEQ ID No.33, and the prrlsin. pf4-BDD FVIII. wpre-2 structure is shown in fig. 1.

In one embodiment, the PF4-BDD FVIII sequence may be followed by a PPT- Δ U3-R-rabbit-globin polyadenylation signal sequence.

In one embodiment, the PF4-BDD FVIII sequence may be followed by a Δ U3-R-BGHpa sequence.

In one example, the PF4-BDD FVIII sequence can also be inserted into other lentiviral vector plasmids not mentioned in this patent.

The recombinant vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a simian viral vector, a vaccinia viral vector, a Sendai viral vector, an EB viral vector or a herpes simplex viral vector, and a sleeping beauty transposon system vector; preferably, a lentiviral vector, more preferably, the lentiviral vector is obtained by inserting the PF4 promoter and the BDD FVIII gene between the XcmI and SalI sites of vector pRRLSIN.

In a second aspect, the present invention provides any one of the following 1) to 2) biomaterials:

1) an expression cassette as described in any of the above,

2) a recombinant virus comprising 1) said expression cassette, preferably said recombinant virus is a recombinant lentivirus,

3) the recombinant cell or the recombinant bacterium containing the expression cassette 1), preferably, the recombinant cell is a hematopoietic stem cell or a megakaryocyte.

The recombinant bacteria can be engineering bacteria and are used for propagating, storing or detecting the expression cassette;

the recombinant cell may be a cell for lentivirus packaging purification, such as a mammalian cell, preferably a HEK293T cell;

the recombinant cell can be a megakaryocyte such as a Dami cell, and is used for identifying the expression of the BDD FVIII gene in the recombinant lentivirus,

the recombinant cells may be hematopoietic stem cells for use in gene therapy of haemophilia a in a patient after reinfusion.

In one example, lentiviral vectors (e.g., lentiviral vector-1, lentiviral vector-2, etc.) loaded with PF4-BDD FVIII gene were able to stably express BDD FVIII in Dami cells and their secreted supernatant via the corresponding transfection procedure.

In one example, mononuclear cells in cord blood, bone marrow, mobilized peripheral blood, or peripheral blood are sorted by CD34 magnetic bead monoclonal antibodies to obtain purified CD34+ cells.

In one example, lentiviral vectors (e.g., lentiviral vector-1, lentiviral vector-2, etc.) loaded with PF4-BDD FVIII gene were capable of stably expressing BDD FVIII in CD34+ cell-derived megakaryocytes and platelets formed by cytoplasmic shedding thereof via a corresponding transfection step;

in one embodiment, the lentiviral vector loaded with the PF4-BDD FVIII gene can also transfect stem cells such as pluripotent stem cells (iPS), Mesenchymal Stem Cells (MSC), adipose-derived stem cells (ADSCs) and the like.

In a third aspect, the invention provides the use of said recombinant vector, such as said lentiviral vector, said expression cassette, said recombinant lentivirus, said recombinant cell, for expressing the BDD FVIII gene or for the manufacture of a medicament and/or an agent for the treatment of haemophilia A,

preferably, the BDD FVIII expressing gene is specifically expressed in platelets derived from cytoplasmic shedding of CD34+ cell-derived megakaryocytes.

In one example, mouse bone marrow hematopoietic stem cells such as Sca + cells transfected with a lentiviral vector loaded with the PF4-BDD FVIII gene are capable of correcting the coagulation deficiencies of hemophilia A mice.

In one example, stem cells such as CD34+, iPS, transfected with a lentiviral vector loaded with PF4-BDD FVIII gene, can be used for gene therapy of hemophilia a.

The invention has the beneficial effects that:

the invention constructs a lentiviral vector containing a PF4 promoter and a BDD FVIII gene expression cassette, and the vector enables the BDD FVIII gene to be specifically expressed in platelets formed by the cytoplasm shedding of megakaryocytes from CD34+ cells through the PF4 promoter, thereby playing a blood coagulation function and being applicable to the gene therapy of the hemophilia A of all ages.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is a schematic diagram of the structure of vector pRRL-PF4-BDD FVIII-WPRE-1/2.

FIG. 2 shows the detection of CD34 by Western Blot⁺Relative expression levels of BDD FVIII protein in the cell colonies, wherein, indicates a significant difference compared to the control group under the same conditions (P < 0.05), indicates a very significant difference compared to the control group under the same conditions (P < 0.01), and indicates a very significant difference compared to the control group under the same conditions (P < 0.001).

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 construction of Lentiviral vectors

1. The nucleotide sequences of the PGK promoter and GFP gene on pRRLSIN.cPPT.PGK-GFP.WPRE vector Plasmid (Plasmid #12252) were excised using restriction enzymes XcmI and SalI, and the vector backbone was recovered;

2. obtaining a nucleotide sequence composition 1(SEQ ID NO.32) or a nucleotide sequence composition 2(SEQ ID NO.33) containing a PF4 promoter (SEQ ID NO.1) and BDD FVIII (SEQ ID NO.4 or SEQ ID NO.17) by chemical synthesis and/or overlap extension PCR, and respectively connecting the obtained nucleotide sequence compositions with the vector skeleton obtained in the step 1 to obtain two recombinant vectors: pRRL-PF4-BDD FVIII-WPRE-1 and pRRL-PF4-BDD FVIII-WPRE-2, the basic structures of which are shown in FIG. 1, and which differ in sequence only at the position of BDD FVIII.

Example 2 packaging and purification of Lentiviral vectors

1. Taking a 10cm culture dish as an example, planting HEK293T cells in a DMEM medium containing 10% fetal bovine serum; when the virus grows to 70-80% of fusion degree, starting to package the virus;

2. removing the original culture medium by suction, adding 50% volume of fresh culture medium, and continuing incubation;

3. according to pRRL-PF4-BDD FVIII-WPRE-1/2: psPAX 2: PMD2.0G is 8: 6: 2, packaging the lentiviral vector by adopting a calcium transfer method;

4. after 12h, changing the liquid;

5. after 36h, collecting cell suspension, filtering by a 0.44 mu m filter membrane, and centrifuging at 50000g for 2 h;

6. and collecting the precipitate, namely the PF4-BDD FVIII loaded lentiviral vector, wherein the lentiviral vector comprises the sequence shown in SEQ ID NO.32 as the lentiviral vector-1, and the lentiviral vector comprises the sequence shown in SEQ ID NO.33 as the lentiviral vector-2.

Example 3 preparation of genetically modified Dami cells

1. 1640+ 10% FBS + L-Glutamin medium to logarithmic growth phase;

2. sucking cells, washing, and blowing and mixing Dami cells uniformly by adopting X-VIVO10+ 10% FBS +4ug/ml Polybrene culture medium according to the proportion of 2 multiplied by 10⁶Planting at 20 μ g/cm per well²In the retroNectin-coated six-well plate, cells are divided into 3 groups, and the group 1 is a control group without adding a lentiviral vector; group 2 is lentivirus vector-1 transfection group; group 3 is the lentiviral vector-2 transfection group.

3. After 2h, adding a PF4-BDD FVIII loaded lentiviral vector to the cell suspension at an MOI of 5-50; after 24h, adding the same amount of lentiviral vector again;

4. after 24h, the cells were aspirated, washed, centrifuged, and conditioned to 1X 10 by adding 1640+ 10% FBS + L-Glutamin medium⁶Planting in a new six-hole plate; continuously culturing for 48 h;

5. and sucking the supernatant for later use.

Example 4 BDD FVIII assay in genetically modified Dami cell culture supernatant

FVIII: C in the supernatant was assayed using a Coatest VIII: C/4Kit (Diacharma, Franklin, OH, USA) using an Afstyla (single chain coagulation factor for treating haemophilia A) as a standard curve. Group 2, group 3 had significantly higher FVIII: C compared to the control group, and group 3 was higher than group 2. The results are shown in Table 1.

TABLE 1

Group of	FVIII: C assay results (U/ml)
		Group 1	0
Group 2	6.63±1.23
		Group 3	13.36±2.53

The above results show that: in the recombinant cells of Dami cells infected by the lentiviral vector-1 or the lentiviral vector-2, the protein expression level of FVIII is obviously higher than that of a control group not infected by the lentivirus, and the protein expression level of FVIII in the recombinant cells infected by the lentiviral vector-2 is obviously higher than that of the recombinant cells infected by the lentiviral vector-1.

Example 5 genetically modified CD34⁺Preparation of

1. Collecting peripheral blood, umbilical cord blood, or bone marrow, and separating with Meitian and whirlwind CD34 Microbead KitPurified CD34⁺A cell;

2. according to a 1-4X 10⁶Per ml, CD34 collected⁺The cells were seeded in X-VIVO 20 medium containing 100ng/ml IL-3, 100ng/ml TPO, 100ng/ml SCF, 100ng/ml FLT-L3 at 37 ℃ with 5% CO₂Incubating for 12-48 hours in the environment;

3. collection of CDs 34⁺Cells at 1-4X 10⁶Per ml, it was planted at 20. mu.g/cm²In the retroNectin-coated six-well plate, cells are divided into 3 groups, and the group 1 is a control group without adding a lentiviral vector; group 2 is lentivirus vector-1 transfection group; group 3 lentiviral vector-2 transfection group;

4. to each cell suspension was added the corresponding lentiviral vector at 37 ℃ with 5% CO at MOI of 10-100₂Incubation in the environment;

5. after 24h, each set of cells was collected, washed, counted and prepared for further processing.

Example 6 genetically modified CD34⁺Cell colony formation

1. 500-^TMC Staingkit for CFU-Mk for CFU-MK (megakaryocyte colony) culture.

2. Culturing for 12-14 days, after staining, picking MK and other colonies in each group of culture dish, washing, centrifuging, wherein group 1-1 is control group-non-MK, group 1-2 is control group-MK; group 2-1 is lentivector-1 transfection group-non-MK, group 2-2 is lentivector-1 transfection group-MK; the group 3-1 is lentivirus vector-2 transfection group-non-MK, the group 3-2 is lentivirus vector-2 transfection group-MK, and the next detection is carried out.

Example 7 BDD FVIII detection in various lines of colonies

1. Collecting the cells of each group in example 6, and extracting total protein respectively;

2. BDD FVIII expression in each group of cells was detected using Western Blot of FVIII antibody, and the expression level of BDD FVIII relative to GAPDH in each group of cells was calculated using GAPDH as internal reference.

The results are shown in FIG. 2, with essentially no BDD FVIII expression in the non-MK colonies of groups 1-1 and the MK colonies of groups 1-2, a small amount of BDD FVIII expression is seen in groups 2-1, 3-1, while BDD FVIII expression is significantly enhanced in groups 2-2, 3-2, and significantly higher in groups 3-2 than in groups 2-2.

Example 8 Gene therapy in hemophilia A mice

1. Lentiviral vector-1 and Lentiviral vector-2 were transfected into hemophilia A mouse-derived bone marrow Sca⁺Hematopoietic stem cells;

2. bone marrow Sca after transfection⁺Hematopoietic stem cells are transplanted into the A hemophilia mice which are pretreated by radiation through tail vein injection respectively, and the transplantation is carried out on the A hemophilia mice by 0.5 multiplied by 10⁶Only, as group 2, group 3; control group was untransfected bone marrow Sca⁺Hematopoietic stem cells were transplanted into hemophilia a mice, group 1; each group of 5 hemophilia a mice;

3. after 1-3 months, the mouse tail-breaking test is performed. Mice in group 1 died due to excessive blood loss caused by failure of endogenous blood coagulation function after tail cutting, and mice in group 2 and group 3 survived due to endogenous blood coagulation after tail cutting. The results are shown in Table 2.

TABLE 2

Group of	Survival rate of hemophilia a mice
		Group
1	0
		Group 2	100％
Group 3	100％

The above description is only an example of the present application, and the protection scope of the present application is not limited by these specific examples, but is defined by the claims of the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.

Sequence listing

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<210> 3

<211> 315

<212> DNA

<213> Artificial sequence

<400> 3

gtcccagcca aggcagctgc ccagagcctt ttctttcttt ctttctttct ttcttttctt 60

ttcttttttt tttttttttt tttttttttt tttttttttt ttttttttgg taatcttggc 120

tggccagaac ccaagtcttc ccagtactat cttagtttcc gcaccgcagt tcctcggtgt 180

ccacttcagg cttccggact ggaaggacag ccgggaataa aacgtgccgg cgaggctcag 240

gagtcattgg ccacagagac ccagcccgag tttcccatcg cactgagcac tgagggggat 300

ccaccggtcg ccacc 315

<210> 4

<211> 4332

<212> DNA

<213> Artificial sequence

<400> 4

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagagaa 2280

ataactcgta ctactcttca gtcagatcaa gaggaaattg actatgatga taccatatca 2340

gttgaaatga agaaggaaga ttttgacatt tatgatgagg atgaaaatca gagcccccgc 2400

agctttcaaa agaaaacacg acactatttt attgctgcag tggagaggct ctgggattat 2460

gggatgagta gctccccaca tgttctaaga aacagggctc agagtggcag tgtccctcag 2520

ttcaagaaag ttgttttcca ggaatttact gatggctcct ttactcagcc cttataccgt 2580

ggagaactaa atgaacattt gggactcctg gggccatata taagagcaga agttgaagat 2640

aatatcatgg taactttcag aaatcaggcc tctcgtccct attccttcta ttctagcctt 2700

atttcttatg aggaagatca gaggcaagga gcagaaccta gaaaaaactt tgtcaagcct 2760

aatgaaacca aaacttactt ttggaaagtg caacatcata tggcacccac taaagatgag 2820

tttgactgca aagcctgggc ttatttctct gatgttgacc tggaaaaaga tgtgcactca 2880

ggcctgattg gaccccttct ggtctgccac actaacacac tgaaccctgc tcatgggaga 2940

caagtgacag tacaggaatt tgctctgttt ttcaccatct ttgatgagac caaaagctgg 3000

tacttcactg aaaatatgga aagaaactgc agggctccct gcaatatcca gatggaagat 3060

cccactttta aagagaatta tcgcttccat gcaatcaatg gctacataat ggatacacta 3120

cctggcttag taatggctca ggatcaaagg attcgatggt atctgctcag catgggcagc 3180

aatgaaaaca tccattctat tcatttcagt ggacatgtgt tcactgtacg aaaaaaagag 3240

gagtataaaa tggcactgta caatctctat ccaggtgttt ttgagacagt ggaaatgtta 3300

ccatccaaag ctggaatttg gcgggtggaa tgccttattg gcgagcatct acatgctggg 3360

atgagcacac tttttctggt gtacagcaat aagtgtcaga ctcccctggg aatggcttct 3420

ggacacatta gagattttca gattacagct tcaggacaat atggacagtg ggccccaaag 3480

ctggccagac ttcattattc cggatcaatc aatgcctgga gcaccaagga gcccttttct 3540

tggatcaagg tggatctgtt ggcaccaatg attattcacg gcatcaagac ccagggtgcc 3600

cgtcagaagt tctccagcct ctacatctct cagtttatca tcatgtatag tcttgatggg 3660

aagaagtggc agacttatcg aggaaattcc actggaacct taatggtctt ctttggcaat 3720

gtggattcat ctgggataaa acacaatatt tttaaccctc caattattgc tcgatacatc 3780

cgtttgcacc caactcatta tagcattcgc agcactcttc gcatggagtt gatgggctgt 3840

gatttaaata gttgcagcat gccattggga atggagagta aagcaatatc agatgcacag 3900

attactgctt catcctactt taccaatatg tttgccacct ggtctccttc aaaagctcga 3960

cttcacctcc aagggaggag taatgcctgg agacctcagg tgaataatcc aaaagagtgg 4020

ctgcaagtgg acttccagaa gacaatgaaa gtcacaggag taactactca gggagtaaaa 4080

tctctgctta ccagcatgta tgtgaaggag ttcctcatct ccagcagtca agatggccat 4140

cagtggactc tcttttttca gaatggcaaa gtaaaggttt ttcagggaaa tcaagactcc 4200

ttcacacctg tggtgaactc tctagaccca ccgttactga ctcgctacct tcgaattcac 4260

ccccagagtt gggtgcacca gattgccctg aggatggagg ttctgggctg cgaggcacag 4320

gacctctact ga 4332

<210> 5

<211> 42

<212> DNA

<213> Artificial sequence

<400> 5

agcttctccc agaatccacc agtcttgaaa cgccatcaac gg 42

<210> 6

<211> 30

<212> DNA

<213> Artificial sequence

<400> 6

agcttctccc agaatccacc agtcttgaaa 30

<210> 7

<211> 72

<212> DNA

<213> Artificial sequence

<400> 7

agcttctccc agaattcaag acaccctagc actaggcaaa agcaatttaa tgccaccaca 60

attccagaaa at 72

<210> 8

<211> 63

<212> DNA

<213> Artificial sequence

<400> 8

agcttctccc agaattcaag acacccttct caaaacccac cagtcttgaa acgccatcaa 60

cgg 63

<210> 9

<211> 51

<212> DNA

<213> Artificial sequence

<400> 9

agcttctccc agaattcaag acacccttct caaaacccac cagtcttgaa a 51

<210> 10

<211> 48

<212> DNA

<213> Artificial sequence

<400> 10

agcttctccc agaattcaag acaccaggcg tatcgttatc gtcgcggc 48

<210> 11

<211> 36

<212> DNA

<213> Artificial sequence

<400> 11

agcttctccc agaattcaag acaccaggcg tatggc 36

<210> 12

<211> 93

<212> DNA

<213> Artificial sequence

<400> 12

agcttctccc agaatgccac taatgtgtct aacaacagca acaccagcaa tgacagcaat 60

gtgtctccac cagtcttgaa acgccatcaa cgg 93

<210> 13

<211> 81

<212> DNA

<213> Artificial sequence

<400> 13

agcttctccc agaatgccac taatgtgtct aacaacagca acaccagcaa tgacagcaat 60

gtgtctccac cagtcttgaa a 81

<210> 14

<211> 708

<212> DNA

<213> Artificial sequence

<400> 14

agcttctccc agaattcaag acaccctagc actaggcaaa agcaatttaa tgccaccaca 60

attccagaaa atgacataga gaagactgac ccttggtttg cacacagaac acctatgcct 120

aaaatacaaa atgtctcctc tagtgatttg ttgatgctct tgcgacagag tcctactcca 180

catgggctat ccttatctga tctccaagaa gccaaatatg agactttttc tgatgatcca 240

tcacctggag caatagacag taataacagc ctgtctgaaa tgacacactt caggccacag 300

ctccatcaca gtggggacat ggtatttacc cctgagtcag gcctccaatt aagattaaat 360

gagaaactgg ggacaactgc agcaacagag ttgaagaaac ttgatttcaa agtttctagt 420

acatcaaata atctgatttc aacaattcca tcagacaatt tggcagcagg tactgataat 480

acaagttcct taggaccccc aagtatgcca gttcattatg atagtcaatt agataccact 540

ctatttggca aaaagtcatc tccccttact gagtctggtg gacctctgag cttgagtgaa 600

gaaaataatg attcaaagtt gttagaatca ggtttaatga atagccaaga aagttcatgg 660

ggaaaaaatg tatcgacccg tccaccagtc ttgaaacgcc atcaacgg 708

<210> 15

<211> 678

<212> DNA

<213> Artificial sequence

<400> 15

agcttctccc agaattcaag acaccctagc actaggcaaa agcaatttaa tgccaccaca 60

attccagaaa atgacataga gaagactgac ccttggtttg cacacagaac acctatgcct 120

aaaatacaaa atgtctcctc tagtgatttg ttgatgctct tgcgacagag tcctactcca 180

catgggctat ccttatctga tctccaagaa gccaaatatg agactttttc tgatgatcca 240

tcacctggag caatagacag taataacagc ctgtctgaaa tgacacactt caggccacag 300

ctccatcaca gtggggacat ggtatttacc cctgagtcag gcctccaatt aagattaaat 360

gagaaactgg ggacaactgc agcaacagag ttgaagaaac ttgatttcaa agtttctagt 420

acatcaaata atctgatttc aacaattcca tcagacaatt tggcagcagg tactgataat 480

acaagttcct taggaccccc aagtatgcca gttcattatg atagtcaatt agataccact 540

ctatttggca aaaagtcatc tccccttact gagtctggtg gacctctgag cttgagtgaa 600

gaaaataatg attcaaagtt gttagaatca ggtttaatga atagccaaga aagttcatgg 660

ggaaaaaatg tatcgtca 678

<210> 16

<211> 1443

<212> PRT

<213> Artificial sequence

<400> 16

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser

755 760 765

Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys

770 775 780

Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg

785 790 795 800

Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg

805 810 815

Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg

820 825 830

Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu

835 840 845

Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn

850 855 860

Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp

865 870 875 880

Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe

885 890 895

Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu

900 905 910

Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp

915 920 925

Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys

930 935 940

Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser

945 950 955 960

Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro

965 970 975

Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr

980 985 990

Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg

995 1000 1005

Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys

1010 1015 1020

Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu

1025 1030 1035 1040

Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu

1045 1050 1055

Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His

1060 1065 1070

Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn

1075 1080 1085

Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala

1090 1095 1100

Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly

1105 1110 1115 1120

Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu

1125 1130 1135

Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly

1140 1145 1150

Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly

1155 1160 1165

Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val

1170 1175 1180

Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala

1185 1190 1195 1200

Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr

1205 1210 1215

Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly

1220 1225 1230

Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His

1235 1240 1245

Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro

1250 1255 1260

Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys

1265 1270 1275 1280

Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile

1285 1290 1295

Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala

1300 1305 1310

Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn

1315 1320 1325

Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp

1330 1335 1340

Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys

1345 1350 1355 1360

Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser

1365 1370 1375

Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys

1380 1385 1390

Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu

1395 1400 1405

Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp

1410 1415 1420

Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln

1425 1430 1435 1440

Asp Leu Tyr

<210> 17

<211> 4413

<212> DNA

<213> Artificial sequence

<400> 17

atgcagatcg agttgagtac gtgtttcttc ctctgcctgc tgcgattttg tttctccgct 60

acccggagat attacttggg cgccgtagaa ctcagctggg actatatgca gagcgacctg 120

ggggagctcc ctgtagatgc tcggtttcca ccacgcgtgc ctaagtcatt ccctttcaat 180

actagtgttg tgtacaagaa gaccttgttt gtagagttca ccgaccatct tttcaacatc 240

gccaaacccc gaccaccatg gatgggactg ctgggaccca ctattcaggc cgaggtgtac 300

gacaccgtgg taatcactct gaagaacatg gcaagccacc ctgtctcact gcacgctgtt 360

ggagtgtcat actggaaggc cagtgaaggc gccgaatatg acgaccagac ctctcaacgg 420

gagaaggagg atgataaggt attcccaggc ggaagccata cttacgtctg gcaggtcctt 480

aaggagaatg gacccatggc ctcagatcca ctttgtctca cctattctta cctgtctcac 540

gtggaccttg tcaaggatct caacagtgga ctcatcggtg ctctcctggt ctgccgagag 600

ggatcactcg ccaaggaaaa gacgcaaact ctccacaaat tcattctgct ctttgccgtc 660

tttgacgagg gaaaatcatg gcacagcgaa acgaagaact ctcttatgca ggatagggat 720

gctgcatccg ccagagcgtg gcctaagatg cacacagtca acggatacgt gaatcgctct 780

cttccggggc tcatagggtg tcatagaaag tccgtgtact ggcatgtaat tggaatggga 840

acaaccccag aagtgcattc catattcctg gagggacata cttttttggt tagaaaccac 900

agacaggcct ccctcgaaat atcccccatc actttcctga ccgcccagac attgctgatg 960

gatctcgggc aattcctttt gagctgtcat atcagctctc accagcacga cggaatggaa 1020

gcttacgtaa aggttgacag ttgcccagag gaaccacagc tgcgcatgaa aaacaacgag 1080

gaagccgagg actatgacga tgacttgaca gacagcgaaa tggatgtggt gcggtttgac 1140

gatgataaca gccctagttt tattcagatc cgctccgtag cgaaaaagca tcccaaaacc 1200

tgggtgcact acattgcagc tgaggaagaa gactgggact acgccccact ggttttggcg 1260

ccagatgacc gaagttacaa gagccagtac ctgaataacg gcccacagag aatcgggcgc 1320

aaatataaga aggtcagatt tatggcttac accgatgaga cattcaagac aagagaggcc 1380

attcagcatg aatccggcat tctgggtcct cttctgtacg gcgaagtcgg agacacactg 1440

ctgattatct ttaaaaatca ggcctcaagg ccatacaaca tctacccaca tggaattacc 1500

gatgtacggc ctctttatag ccgaaggctt cctaaaggag tgaagcatct gaaagatttt 1560

ccgatattgc cgggagagat attcaagtac aagtggacgg tcaccgtgga agatggccca 1620

acaaagagtg acccccggtg tctcacaagg tattactctt cattcgttaa tatggagagg 1680

gatttggcat ccggcctgat tggaccactg ctgatttgtt acaaggagtc tgtcgatcag 1740

agaggaaacc agatcatgag cgataaacgc aatgttatcc tcttctctgt tttcgacgag 1800

aatcggtctt ggtatctgac cgaaaatatc cagagattcc tgccaaaccc ggctggtgta 1860

cagcttgaag atcccgaatt tcaagccagc aatattatgc acagcatcaa cggatacgtc 1920

tttgacagtc ttcagttgtc cgtgtgtctg catgaggtgg cctactggta cattcttagc 1980

atcggcgctc agaccgattt ccttagtgtg ttcttttctg ggtacacatt caagcacaaa 2040

atggtatacg aggacaccct gacacttttc ccctttagtg gtgagactgt ttttatgtcc 2100

atggagaacc ccggactctg gatcctgggc tgccacaaca gtgacttccg caacaggggt 2160

atgactgcat tgctgaaagt tagcagctgt gataagaata ccggcgacta ttacgaggac 2220

tcctatgaag acatctccgc ctatctgttg agcaaaaaca atgcaataga acccagaagc 2280

tttagccaga atgctacaaa tgtgagcaac aacagcaaca ccagtaacga cagtaacgtg 2340

tctccacctg tgctcaagga gatcacacgg acaacgctgc aatcagatca ggaggagatt 2400

gattatgacg acaccataag cgtcgagatg aagaaggagg atttcgacat ttacgacgag 2460

gatgagaacc aatcacccag aagtttccag aagaagaccc gccactattt tatcgcagcg 2520

gtcgaaaggc tgtgggatta tggcatgtct agcagtccac acgtgctgcg aaaccgcgca 2580

cagtctggct ctgttcctca atttaagaag gtcgtattcc aggaatttac cgatgggtca 2640

ttcacccaac cattgtatag aggggaactt aatgaacatc tgggacttct tggcccctac 2700

attcgagcag aggtggagga taatattatg gtaacgttcc gaaatcaagc gtcaagaccc 2760

tactccttct actccagcct cattagttat gaagaggacc aacgccaagg agctgagcca 2820

aggaaaaact tcgtgaagcc caatgagacg aagacctact tttggaaagt gcagcatcac 2880

atggctccaa caaaagacga gttcgattgc aaagcatggg cttacttcag tgacgtggac 2940

ctcgaaaagg acgtgcattc aggacttatc gggcctctgc ttgtctgcca taccaacacc 3000

ctgaaccctg cccatggaag gcaggtaacc gttcaggagt tcgctctctt ttttacaatt 3060

tttgatgaga cgaagagctg gtacttcacc gaaaacatgg aaaggaactg tagggcaccc 3120

tgcaatatcc agatggaaga tcccaccttt aaggagaatt acaggtttca cgctatcaac 3180

ggttacataa tggataccct tcctgggctg gttatggctc aagatcagcg gattagatgg 3240

tatcttctgt ccatggggtc aaacgaaaac attcatagta ttcattttag tggacacgtg 3300

tttaccgtgc gcaaaaaaga agagtacaag atggctctgt acaatctgta tcccggtgtt 3360

ttcgaaactg tcgagatgct tccttctaag gccgggatct ggcgagtcga atgtcttata 3420

ggtgaacact tgcatgccgg tatgagcacc ctgttcctgg tatacagcaa caagtgccag 3480

actccacttg gaatggccag cggtcacata agggattttc aaatcaccgc ttccggacag 3540

tacggccagt gggctccaaa gctggcccga cttcattact ccgggtcaat caatgcttgg 3600

tccactaaag agcccttttc atggatcaaa gtggatctgc ttgccccgat gatcattcat 3660

ggcatcaaga cacagggagc acgacagaaa tttagctctc tctacatcag ccagtttatt 3720

atcatgtata gtctggatgg aaaaaagtgg cagacctaca ggggaaattc aactgggacc 3780

ttgatggtgt tcttcggtaa cgtggattcc agtggcataa agcataacat ctttaaccct 3840

ccgattattg cacgctatat taggttgcac cctacacatt atagcattcg ctctactctg 3900

cggatggaac ttatgggctg tgacctgaat tcttgttcca tgccacttgg catggaaagt 3960

aaggccatca gtgacgctca gatcacggct tctagttatt tcaccaatat gtttgctacc 4020

tggtcacctt ctaaggccag actccatctc cagggcagat ccaacgcttg gagaccacag 4080

gtgaacaatc ctaaggagtg gctgcaagtg gatttccaga agacaatgaa ggtgaccgga 4140

gtcacaaccc aaggagtgaa gtcactgctg acttcaatgt atgtaaagga gttcctgatc 4200

agctcaagtc aagatggtca tcagtggaca ctgtttttcc agaatggcaa ggttaaagtg 4260

tttcagggaa atcaggactc tttcactccc gtcgtgaata gtctggaccc tccgcttctt 4320

accaggtatt tgcgcattca cccccagtct tgggtccatc aaattgctct gagaatggaa 4380

gttcttggtt gtgaggctca ggatctgtac tga 4413

<210> 18

<211> 14

<212> PRT

<213> Artificial sequence

<400> 18

Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Artificial sequence

<400> 19

Ser Phe Ser Gln Asn Pro Pro Val Leu Lys

1 5 10

<210> 20

<211> 24

<212> PRT

<213> Artificial sequence

<400> 20

Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg Gln Lys Gln Phe

1 5 10 15

Asn Ala Thr Thr Ile Pro Glu Asn

20

<210> 21

<211> 21

<212> PRT

<213> Artificial sequence

<400> 21

Ser Phe Ser Gln Asn Ser Arg His Pro Ser Gln Asn Pro Pro Val Leu

1 5 10 15

Lys Arg His Gln Arg

20

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence

<400> 22

Ser Phe Ser Gln Asn Ser Arg His Pro Ser Gln Asn Pro Pro Val Leu

1 5 10 15

Lys

<210> 23

<211> 16

<212> PRT

<213> Artificial sequence

<400> 23

Ser Phe Ser Gln Asn Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly

1 5 10 15

<210> 24

<211> 12

<212> PRT

<213> Artificial sequence

<400> 24

Ser Phe Ser Gln Asn Ser Arg His Gln Ala Tyr Gly

1 5 10

<210> 25

<211> 31

<212> PRT

<213> Artificial sequence

<400> 25

Ser Phe Ser Gln Asn Ala Thr Asn Val Ser Asn Asn Ser Asn Thr Ser

1 5 10 15

Asn Asp Ser Asn Val Ser Pro Pro Val Leu Lys Arg His Gln Arg

20 25 30

<210> 26

<211> 27

<212> PRT

<213> Artificial sequence

<400> 26

Ser Phe Ser Gln Asn Ala Thr Asn Val Ser Asn Asn Ser Asn Thr Ser

1 5 10 15

Asn Asp Ser Asn Val Ser Pro Pro Val Leu Lys

20 25

<210> 27

<211> 236

<212> PRT

<213> Artificial sequence

<400> 27

Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg Gln Lys Gln Phe

1 5 10 15

Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys Thr Asp Pro Trp

20 25 30

Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn Val Ser Ser Ser

35 40 45

Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro His Gly Leu Ser

50 55 60

Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe Ser Asp Asp Pro

65 70 75 80

Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser Glu Met Thr His

85 90 95

Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val Phe Thr Pro Glu

100 105 110

Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly Thr Thr Ala Ala

115 120 125

Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser Thr Ser Asn Asn

130 135 140

Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala Gly Thr Asp Asn

145 150 155 160

Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His Tyr Asp Ser Gln

165 170 175

Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro Leu Thr Glu Ser

180 185 190

Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp Ser Lys Leu Leu

195 200 205

Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp Gly Lys Asn Val

210 215 220

Ser Thr Arg Pro Pro Val Leu Lys Arg His Gln Arg

225 230 235

<210> 28

<211> 226

<212> PRT

<213> Artificial sequence

<400> 28

Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg Gln Lys Gln Phe

1 5 10 15

Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys Thr Asp Pro Trp

20 25 30

Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn Val Ser Ser Ser

35 40 45

Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro His Gly Leu Ser

50 55 60

Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe Ser Asp Asp Pro

65 70 75 80

Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser Glu Met Thr His

85 90 95

Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val Phe Thr Pro Glu

100 105 110

Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly Thr Thr Ala Ala

115 120 125

Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser Thr Ser Asn Asn

130 135 140

Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala Gly Thr Asp Asn

145 150 155 160

Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His Tyr Asp Ser Gln

165 170 175

Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro Leu Thr Glu Ser

180 185 190

Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp Ser Lys Leu Leu

195 200 205

Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp Gly Lys Asn Val

210 215 220

Ser Ser

225

<210> 29

<211> 1470

<212> PRT

<213> Artificial sequence

<400> 29

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Ser Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ala Thr Asn Val

755 760 765

Ser Asn Asn Ser Asn Thr Ser Asn Asp Ser Asn Val Ser Pro Pro Val

770 775 780

Leu Lys Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile

785 790 795 800

Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp

805 810 815

Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys

820 825 830

Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly

835 840 845

Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser

850 855 860

Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser

865 870 875 880

Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu

885 890 895

Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr

900 905 910

Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile

915 920 925

Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe

930 935 940

Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His

945 950 955 960

Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe

965 970 975

Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro

980 985 990

Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln

995 1000 1005

Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr

1010 1015 1020

Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro

1025 1030 1035 1040

Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe

1045 1050 1055

His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met

1060 1065 1070

Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn

1075 1080 1085

Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg

1090 1095 1100

Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val

1105 1110 1115 1120

Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val

1125 1130 1135

Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe

1140 1145 1150

Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly

1155 1160 1165

His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp

1170 1175 1180

Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp

1185 1190 1195 1200

Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro

1205 1210 1215

Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser

1220 1225 1230

Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys

1235 1240 1245

Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe

1250 1255 1260

Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro

1265 1270 1275 1280

Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile

1285 1290 1295

Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys

1300 1305 1310

Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile

1315 1320 1325

Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser

1330 1335 1340

Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln

1345 1350 1355 1360

Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met

1365 1370 1375

Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser

1380 1385 1390

Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln

1395 1400 1405

Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn

1410 1415 1420

Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu

1425 1430 1435 1440

Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala

1445 1450 1455

Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr

1460 1465 1470

<210> 30

<211> 63

<212> DNA

<213> Artificial sequence

<400> 30

atgagctccg cagccgggtt ctgcgcctca cgccccgggc tgctgttcct ggggttgctg 60

ctc 63

<210> 31

<211> 31

<212> PRT

<213> Artificial sequence

<400> 31

Met Ser Ser Ala Ala Gly Phe Cys Ala Ser Arg Pro Gly Leu Leu Phe

1 5 10 15

Leu Gly Leu Leu Leu Leu Pro Leu Val Val Ala Phe Ala Ser Ala

20 25 30

<210> 32

<211> 5119

<212> DNA

<213> Artificial sequence

<400> 32

cagccaggca ctaagctttt acaaaaagaa tgcattcaaa tattacttct gcaatttttc 60

caagagtacg agtaatggga aaatagacct tgcgtcctac agcatactgg gtgattgttg 120

gtggagcctg gtaaatacca tccagagact agggaagttt tccacattca ggtgaaattc 180

aaaagagtca tcgcctagag accaaattaa acttattagt agcagaaaat aattgcgaat 240

aacacatgct tcacacacaa agccaccaaa ctcagtagta ataattggta gttgcagctg 300

ggaggttgga aaggaaacag gaactgtcag ggtcctgaaa gcacctactg tgtaagttgc 360

atgatgacag cagagaactc tactgttaat acaacacaaa aaacattcct tttctgactc 420

aagttctgtt tctaactcta caaacgtgct tgtatgtaag ttgaggttgg aggtcccagc 480

caaggcagct gcccagagcc ttttctttct ttctttcttt ctttcttttc ttttcttttt 540

tttttttttt tttttttttt tttttttttt tttttttttt ggtaatcttg gctggccaga 600

acccaagtct tcccagtact atcttagttt ccgcaccgca gttcctcggt gtccacttca 660

ggcttccgga ctggaaggac agccgggaat aaaacgtgcc ggcgaggctc aggagtcatt 720

ggccacagag acccagcccg agtttcccat cgcactgagc actgaggggg atccaccggt 780

cgccaccatg caaatagagc tctccacctg cttctttctg tgccttttgc gattctgctt 840

tagtgccacc agaagatact acctgggtgc agtggaactg tcatgggact atatgcaaag 900

tgatctcggt gagctgcctg tggacgcaag atttcctcct agagtgccaa aatcttttcc 960

attcaacacc tcagtcgtgt acaaaaagac tctgtttgta gaattcacgg atcacctttt 1020

caacatcgct aagccaaggc caccctggat gggtctgcta ggtcctacca tccaggctga 1080

ggtttatgat acagtggtca ttacacttaa gaacatggct tcccatcctg tcagtcttca 1140

tgctgttggt gtatcctact ggaaagcttc tgagggagct gaatatgatg atcagaccag 1200

tcaaagggag aaagaagatg ataaagtctt ccctggtgga agccatacat atgtctggca 1260

ggtcctgaaa gagaatggtc caatggcctc tgacccactg tgccttacct actcatatct 1320

ttctcatgtg gacctggtaa aagacttgaa ttcaggcctc attggagccc tactagtatg 1380

tagagaaggg agtctggcca aggaaaagac acagaccttg cacaaattta tactactttt 1440

tgctgtattt gatgaaggga aaagttggca ctcagaaaca aagaactcct tgatgcagga 1500

tagggatgct gcatctgctc gggcctggcc taaaatgcac acagtcaatg gttatgtaaa 1560

caggtctctg ccaggtctga ttggatgcca caggaaatca gtctattggc atgtgattgg 1620

aatgggcacc actcctgaag tgcactcaat attcctcgaa ggtcacacat ttcttgtgag 1680

gaaccatcgc caggcgtcct tggaaatctc gccaataact ttccttactg ctcaaacact 1740

cttgatggac cttggacagt ttctactgtt ttgtcatatc tcttcccacc aacatgatgg 1800

catggaagct tatgtcaaag tagacagctg tccagaggaa ccccaactac gaatgaaaaa 1860

taatgaagaa gcggaagact atgatgatga tcttactgat tctgaaatgg atgtggtcag 1920

gtttgatgat gacaactctc cttcctttat ccaaattcgc tcagttgcca agaagcatcc 1980

taaaacttgg gtacattaca ttgctgctga agaggaggac tgggactatg ctcccttagt 2040

cctcgccccc gatgacagaa gttataaaag tcaatatttg aacaatggcc ctcagcggat 2100

tggtaggaag tacaaaaaag tccgatttat ggcatacaca gatgaaacct ttaagactcg 2160

tgaagctatt cagcatgaat caggaatctt gggaccttta ctttatgggg aagttggaga 2220

cacactgttg attatattta agaatcaagc aagcagacca tataacatct accctcacgg 2280

aatcactgat gtccgtcctt tgtattcaag gagattacca aaaggtgtaa aacatttgaa 2340

ggattttcca attctgccag gagaaatatt caaatataaa tggacagtga ctgtagaaga 2400

tgggccaact aaatcagatc ctcggtgcct gacccgctat tactctagtt tcgttaatat 2460

ggagagagat ctagcttcag gactcattgg ccctctcctc atctgctaca aagaatctgt 2520

agatcaaaga ggaaaccaga taatgtcaga caagaggaat gtcatcctgt tttctgtatt 2580

tgatgagaac cgaagctggt acctcacaga gaatatacaa cgctttctcc ccaatccagc 2640

tggagtgcag cttgaggatc cagagttcca agcctccaac atcatgcaca gcatcaatgg 2700

ctatgttttt gatagtttgc agttgtcagt ttgtttgcat gaggtggcat actggtacat 2760

tctaagcatt ggagcacaga ctgacttcct ttctgtcttc ttctctggat ataccttcaa 2820

acacaaaatg gtctatgaag acacactcac cctattccca ttctcaggag aaactgtctt 2880

catgtcgatg gaaaacccag gtctatggat tctggggtgc cacaactcag actttcggaa 2940

cagaggcatg accgccttac tgaaggtttc tagttgtgac aagaacactg gtgattatta 3000

cgaggacagt tatgaagata tttcagcata cttgctgagt aaaaacaatg ccattgaacc 3060

aagagaaata actcgtacta ctcttcagtc agatcaagag gaaattgact atgatgatac 3120

catatcagtt gaaatgaaga aggaagattt tgacatttat gatgaggatg aaaatcagag 3180

cccccgcagc tttcaaaaga aaacacgaca ctattttatt gctgcagtgg agaggctctg 3240

ggattatggg atgagtagct ccccacatgt tctaagaaac agggctcaga gtggcagtgt 3300

ccctcagttc aagaaagttg ttttccagga atttactgat ggctccttta ctcagccctt 3360

ataccgtgga gaactaaatg aacatttggg actcctgggg ccatatataa gagcagaagt 3420

tgaagataat atcatggtaa ctttcagaaa tcaggcctct cgtccctatt ccttctattc 3480

tagccttatt tcttatgagg aagatcagag gcaaggagca gaacctagaa aaaactttgt 3540

caagcctaat gaaaccaaaa cttacttttg gaaagtgcaa catcatatgg cacccactaa 3600

agatgagttt gactgcaaag cctgggctta tttctctgat gttgacctgg aaaaagatgt 3660

gcactcaggc ctgattggac cccttctggt ctgccacact aacacactga accctgctca 3720

tgggagacaa gtgacagtac aggaatttgc tctgtttttc accatctttg atgagaccaa 3780

aagctggtac ttcactgaaa atatggaaag aaactgcagg gctccctgca atatccagat 3840

ggaagatccc acttttaaag agaattatcg cttccatgca atcaatggct acataatgga 3900

tacactacct ggcttagtaa tggctcagga tcaaaggatt cgatggtatc tgctcagcat 3960

gggcagcaat gaaaacatcc attctattca tttcagtgga catgtgttca ctgtacgaaa 4020

aaaagaggag tataaaatgg cactgtacaa tctctatcca ggtgtttttg agacagtgga 4080

aatgttacca tccaaagctg gaatttggcg ggtggaatgc cttattggcg agcatctaca 4140

tgctgggatg agcacacttt ttctggtgta cagcaataag tgtcagactc ccctgggaat 4200

ggcttctgga cacattagag attttcagat tacagcttca ggacaatatg gacagtgggc 4260

cccaaagctg gccagacttc attattccgg atcaatcaat gcctggagca ccaaggagcc 4320

cttttcttgg atcaaggtgg atctgttggc accaatgatt attcacggca tcaagaccca 4380

gggtgcccgt cagaagttct ccagcctcta catctctcag tttatcatca tgtatagtct 4440

tgatgggaag aagtggcaga cttatcgagg aaattccact ggaaccttaa tggtcttctt 4500

tggcaatgtg gattcatctg ggataaaaca caatattttt aaccctccaa ttattgctcg 4560

atacatccgt ttgcacccaa ctcattatag cattcgcagc actcttcgca tggagttgat 4620

gggctgtgat ttaaatagtt gcagcatgcc attgggaatg gagagtaaag caatatcaga 4680

tgcacagatt actgcttcat cctactttac caatatgttt gccacctggt ctccttcaaa 4740

agctcgactt cacctccaag ggaggagtaa tgcctggaga cctcaggtga ataatccaaa 4800

agagtggctg caagtggact tccagaagac aatgaaagtc acaggagtaa ctactcaggg 4860

agtaaaatct ctgcttacca gcatgtatgt gaaggagttc ctcatctcca gcagtcaaga 4920

tggccatcag tggactctct tttttcagaa tggcaaagta aaggtttttc agggaaatca 4980

agactccttc acacctgtgg tgaactctct agacccaccg ttactgactc gctaccttcg 5040

aattcacccc cagagttggg tgcaccagat tgccctgagg atggaggttc tgggctgcga 5100

ggcacaggac ctctactga 5119

<210> 33

<211> 5200

<212> DNA

<213> Artificial sequence

<400> 33

cagccaggca ctaagctttt acaaaaagaa tgcattcaaa tattacttct gcaatttttc 60

caagagtacg agtaatggga aaatagacct tgcgtcctac agcatactgg gtgattgttg 120

gtggagcctg gtaaatacca tccagagact agggaagttt tccacattca ggtgaaattc 180

aaaagagtca tcgcctagag accaaattaa acttattagt agcagaaaat aattgcgaat 240

aacacatgct tcacacacaa agccaccaaa ctcagtagta ataattggta gttgcagctg 300

ggaggttgga aaggaaacag gaactgtcag ggtcctgaaa gcacctactg tgtaagttgc 360

atgatgacag cagagaactc tactgttaat acaacacaaa aaacattcct tttctgactc 420

aagttctgtt tctaactcta caaacgtgct tgtatgtaag ttgaggttgg aggtcccagc 480

caaggcagct gcccagagcc ttttctttct ttctttcttt ctttcttttc ttttcttttt 540

tttttttttt tttttttttt tttttttttt tttttttttt ggtaatcttg gctggccaga 600

acccaagtct tcccagtact atcttagttt ccgcaccgca gttcctcggt gtccacttca 660

ggcttccgga ctggaaggac agccgggaat aaaacgtgcc ggcgaggctc aggagtcatt 720

ggccacagag acccagcccg agtttcccat cgcactgagc actgaggggg atccaccggt 780

cgccaccatg cagatcgagt tgagtacgtg tttcttcctc tgcctgctgc gattttgttt 840

ctccgctacc cggagatatt acttgggcgc cgtagaactc agctgggact atatgcagag 900

cgacctgggg gagctccctg tagatgctcg gtttccacca cgcgtgccta agtcattccc 960

tttcaatact agtgttgtgt acaagaagac cttgtttgta gagttcaccg accatctttt 1020

caacatcgcc aaaccccgac caccatggat gggactgctg ggacccacta ttcaggccga 1080

ggtgtacgac accgtggtaa tcactctgaa gaacatggca agccaccctg tctcactgca 1140

cgctgttgga gtgtcatact ggaaggccag tgaaggcgcc gaatatgacg accagacctc 1200

tcaacgggag aaggaggatg ataaggtatt cccaggcgga agccatactt acgtctggca 1260

ggtccttaag gagaatggac ccatggcctc agatccactt tgtctcacct attcttacct 1320

gtctcacgtg gaccttgtca aggatctcaa cagtggactc atcggtgctc tcctggtctg 1380

ccgagaggga tcactcgcca aggaaaagac gcaaactctc cacaaattca ttctgctctt 1440

tgccgtcttt gacgagggaa aatcatggca cagcgaaacg aagaactctc ttatgcagga 1500

tagggatgct gcatccgcca gagcgtggcc taagatgcac acagtcaacg gatacgtgaa 1560

tcgctctctt ccggggctca tagggtgtca tagaaagtcc gtgtactggc atgtaattgg 1620

aatgggaaca accccagaag tgcattccat attcctggag ggacatactt ttttggttag 1680

aaaccacaga caggcctccc tcgaaatatc ccccatcact ttcctgaccg cccagacatt 1740

gctgatggat ctcgggcaat tccttttgag ctgtcatatc agctctcacc agcacgacgg 1800

aatggaagct tacgtaaagg ttgacagttg cccagaggaa ccacagctgc gcatgaaaaa 1860

caacgaggaa gccgaggact atgacgatga cttgacagac agcgaaatgg atgtggtgcg 1920

gtttgacgat gataacagcc ctagttttat tcagatccgc tccgtagcga aaaagcatcc 1980

caaaacctgg gtgcactaca ttgcagctga ggaagaagac tgggactacg ccccactggt 2040

tttggcgcca gatgaccgaa gttacaagag ccagtacctg aataacggcc cacagagaat 2100

cgggcgcaaa tataagaagg tcagatttat ggcttacacc gatgagacat tcaagacaag 2160

agaggccatt cagcatgaat ccggcattct gggtcctctt ctgtacggcg aagtcggaga 2220

cacactgctg attatcttta aaaatcaggc ctcaaggcca tacaacatct acccacatgg 2280

aattaccgat gtacggcctc tttatagccg aaggcttcct aaaggagtga agcatctgaa 2340

agattttccg atattgccgg gagagatatt caagtacaag tggacggtca ccgtggaaga 2400

tggcccaaca aagagtgacc cccggtgtct cacaaggtat tactcttcat tcgttaatat 2460

ggagagggat ttggcatccg gcctgattgg accactgctg atttgttaca aggagtctgt 2520

cgatcagaga ggaaaccaga tcatgagcga taaacgcaat gttatcctct tctctgtttt 2580

cgacgagaat cggtcttggt atctgaccga aaatatccag agattcctgc caaacccggc 2640

tggtgtacag cttgaagatc ccgaatttca agccagcaat attatgcaca gcatcaacgg 2700

atacgtcttt gacagtcttc agttgtccgt gtgtctgcat gaggtggcct actggtacat 2760

tcttagcatc ggcgctcaga ccgatttcct tagtgtgttc ttttctgggt acacattcaa 2820

gcacaaaatg gtatacgagg acaccctgac acttttcccc tttagtggtg agactgtttt 2880

tatgtccatg gagaaccccg gactctggat cctgggctgc cacaacagtg acttccgcaa 2940

caggggtatg actgcattgc tgaaagttag cagctgtgat aagaataccg gcgactatta 3000

cgaggactcc tatgaagaca tctccgccta tctgttgagc aaaaacaatg caatagaacc 3060

cagaagcttt agccagaatg ctacaaatgt gagcaacaac agcaacacca gtaacgacag 3120

taacgtgtct ccacctgtgc tcaaggagat cacacggaca acgctgcaat cagatcagga 3180

ggagattgat tatgacgaca ccataagcgt cgagatgaag aaggaggatt tcgacattta 3240

cgacgaggat gagaaccaat cacccagaag tttccagaag aagacccgcc actattttat 3300

cgcagcggtc gaaaggctgt gggattatgg catgtctagc agtccacacg tgctgcgaaa 3360

ccgcgcacag tctggctctg ttcctcaatt taagaaggtc gtattccagg aatttaccga 3420

tgggtcattc acccaaccat tgtatagagg ggaacttaat gaacatctgg gacttcttgg 3480

cccctacatt cgagcagagg tggaggataa tattatggta acgttccgaa atcaagcgtc 3540

aagaccctac tccttctact ccagcctcat tagttatgaa gaggaccaac gccaaggagc 3600

tgagccaagg aaaaacttcg tgaagcccaa tgagacgaag acctactttt ggaaagtgca 3660

gcatcacatg gctccaacaa aagacgagtt cgattgcaaa gcatgggctt acttcagtga 3720

cgtggacctc gaaaaggacg tgcattcagg acttatcggg cctctgcttg tctgccatac 3780

caacaccctg aaccctgccc atggaaggca ggtaaccgtt caggagttcg ctctcttttt 3840

tacaattttt gatgagacga agagctggta cttcaccgaa aacatggaaa ggaactgtag 3900

ggcaccctgc aatatccaga tggaagatcc cacctttaag gagaattaca ggtttcacgc 3960

tatcaacggt tacataatgg atacccttcc tgggctggtt atggctcaag atcagcggat 4020

tagatggtat cttctgtcca tggggtcaaa cgaaaacatt catagtattc attttagtgg 4080

acacgtgttt accgtgcgca aaaaagaaga gtacaagatg gctctgtaca atctgtatcc 4140

cggtgttttc gaaactgtcg agatgcttcc ttctaaggcc gggatctggc gagtcgaatg 4200

tcttataggt gaacacttgc atgccggtat gagcaccctg ttcctggtat acagcaacaa 4260

gtgccagact ccacttggaa tggccagcgg tcacataagg gattttcaaa tcaccgcttc 4320

cggacagtac ggccagtggg ctccaaagct ggcccgactt cattactccg ggtcaatcaa 4380

tgcttggtcc actaaagagc ccttttcatg gatcaaagtg gatctgcttg ccccgatgat 4440

cattcatggc atcaagacac agggagcacg acagaaattt agctctctct acatcagcca 4500

gtttattatc atgtatagtc tggatggaaa aaagtggcag acctacaggg gaaattcaac 4560

tgggaccttg atggtgttct tcggtaacgt ggattccagt ggcataaagc ataacatctt 4620

taaccctccg attattgcac gctatattag gttgcaccct acacattata gcattcgctc 4680

tactctgcgg atggaactta tgggctgtga cctgaattct tgttccatgc cacttggcat 4740

ggaaagtaag gccatcagtg acgctcagat cacggcttct agttatttca ccaatatgtt 4800

tgctacctgg tcaccttcta aggccagact ccatctccag ggcagatcca acgcttggag 4860

accacaggtg aacaatccta aggagtggct gcaagtggat ttccagaaga caatgaaggt 4920

gaccggagtc acaacccaag gagtgaagtc actgctgact tcaatgtatg taaaggagtt 4980

cctgatcagc tcaagtcaag atggtcatca gtggacactg tttttccaga atggcaaggt 5040

taaagtgttt cagggaaatc aggactcttt cactcccgtc gtgaatagtc tggaccctcc 5100

gcttcttacc aggtatttgc gcattcaccc ccagtcttgg gtccatcaaa ttgctctgag 5160

aatggaagtt cttggttgtg aggctcagga tctgtactga 5200

Claims (10)

1. A recombinant vector for treating hemophilia a comprising an expression cassette for co-expression of the PF4 promoter and BDD FVIII gene in tandem.

2. The recombinant vector according to claim 1, wherein the nucleotide sequence of the PF4 promoter is represented by SEQ ID No.1 or a nucleotide sequence having at least 80% homology thereto, preferably at least 85% homology thereto, and further preferably at least 95% homology thereto;

preferably, the PF4 promoter is shown as SEQ ID NO.2 nucleotide sequence or a nucleotide sequence which has at least 80% homology, preferably at least 85% homology, and further preferably at least 95% homology with the nucleotide sequence;

preferably, the PF4 promoter is shown as SEQ ID NO.3 nucleotide sequence or a nucleotide sequence at least 80% homologous, preferably at least 85% homologous, and further preferably at least 95% homologous thereto.

3. The recombinant vector according to claim 1 or 2, wherein the nucleotide sequence of the BDD FVIII gene is as shown in SEQ ID No.4, or a nucleotide sequence having at least 80% homology, preferably at least 85% homology, further preferably at least 95% homology thereto;

preferably, the amino acid sequence encoded by the BDD FVIII gene is shown in SEQ ID No.16, or an amino acid sequence having at least 80% homology, preferably at least 85% homology, and further preferably at least 95% homology thereto.

4. The recombinant vector according to claim 1 or 2, wherein the nucleotide sequence of the BDD FVIII gene is inserted between 2277bp and 2278bp on the basis of the sequence shown in SEQ ID No.4 as SEQ ID No.5, SEQ ID No.6, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15 or a nucleotide sequence having at least 80%, preferably at least 85%, and even more preferably at least 95% homology thereto;

or, the nucleotide sequence of the BDD FVIII gene is based on the sequence shown in SEQ ID NO.4, the nucleotide sequence shown in SEQ ID NO.7 is inserted between 2277bp and 2278bp, and 2278-2289bp is deleted, or the nucleotide sequence has at least 80 percent of homology with the BDD FVIII gene, preferably at least 85 percent of homology with the BDD FVIII gene, and further preferably at least 95 percent of homology with the BDD FVIII gene;

preferably, the amino acid sequence encoded by the BDD FVIII gene is inserted between 759aa and 760aa on the basis of the sequence shown in SEQ ID NO.16, and is shown in SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO. 28; or, the amino acid sequence coded by the BDD FVIII gene is based on the sequence shown in SEQ ID NO.16, and the amino acid sequence shown in SEQ ID NO.20 is inserted between 759aa and 760aa, and the amino acid sequence shown in 760-763aa is deleted; or an amino acid sequence having at least 80% homology thereto, preferably at least 85% homology thereto, and further preferably at least 95% homology thereto.

5. The recombinant vector according to claim 1 or 2, wherein the nucleotide sequence of the BDD FVIII gene is represented by SEQ ID No.17, or a nucleotide sequence having at least 80% homology, preferably at least 85% homology, further preferably at least 95% homology thereto,

preferably, the amino acid sequence of the BDD FVIII gene is shown in SEQ ID NO.29 or an amino acid sequence at least 80% homologous, preferably at least 85% homologous, and further preferably at least 95% homologous thereto.

6. The recombinant vector according to claim 1 or 2, wherein the nucleotide sequence of the BDD FVIII gene is the sequence of SEQ ID No.4, bp 1-57, substituted by a nucleotide sequence as shown in SEQ ID No.30 or having at least 80% homology, preferably at least 85% homology, further preferably at least 95% homology thereto;

preferably, the amino acid sequence of the BDD FVIII gene is the sequence shown in SEQ ID No.14, the 1 st to 19aa is replaced by an amino acid sequence shown in SEQ ID No.31 or having at least 80% homology, preferably at least 85% homology, and further preferably at least 95% homology with the amino acid sequence.

7. The recombinant vector according to claim 1 or 2, wherein the sequence of the PF4 promoter after tandem connection with the BDD FVIII gene is shown in SEQ ID No.32 or SEQ ID No.33 or a nucleotide sequence at least 80%, preferably at least 85%, and more preferably at least 95% homologous thereto.

8. The recombinant vector according to any one of claims 1 to 7, wherein the recombinant vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a simian viral vector, a vaccinia viral vector, a Sendai viral vector, an EB viral vector or a herpes simplex viral vector, a sleeping beauty transposon system vector; preferably, the lentiviral vector is a vector in which the PF4 promoter and the BDD FVIII gene are inserted into the vector prrlsin. cppt. PGK-GFP. wpre in place of the PGK promoter and GFP gene.

9. Any one of the following 1) -3) biomaterials:

1) the recombinant vector of claim 1-8 in the expression cassette,

2) a recombinant virus comprising 1) said expression cassette, preferably said recombinant virus is a recombinant lentivirus,

3) the recombinant cell or the recombinant bacterium containing the expression cassette 1), preferably, the recombinant cell is a hematopoietic stem cell or a megakaryocyte.

10. Use of the recombinant vector, the expression cassette, the recombinant virus, the recombinant cell, or the recombinant bacterium according to any one of claims 1 to 9 for expressing a BDD FVIII gene or for the manufacture of a medicament and/or an agent for hemophilia a treatment.

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