KR20100059766A - Recombinant expression vector system for variants of coagulation factor viii and von willebrand factor - Google Patents

Abstract

PURPOSE: An expression system of blood coagulation factor VIII and mutated von Willebrand factor is provided to enhance stabilization and the activation efficiencies of blood coagulation factor VIII. CONSTITUTION: A mutated von Willebrand factor(vWF28) has an amino acid of sequence number 4 in which exons 29-46 are deleted. A mutated vWF28 gene has a nucleotide of sequence number 3. An animal cell expression vector contains a gene encoding the vWF28. The animal cell expression vector is lentivirus vector. The expression vector further contains a gene encoding human coagulation factor VIII in which B domain is deleted. A pharmaceutical composition for treating and preventing hemophilia contains the expression vector or the mutated vWF28, and human blood coagulation factor VIII.

Description

Recombinant expression vector system for variants of coagulation factor VIII and von Willebrand factor

The present invention relates to an expression system of the 8th blood coagulation factor and the von Willebrand factor variant, and more particularly, the mutant von Willebrand, which significantly increases the stabilization and activation efficiency of the 8th blood coagulation factor even though the exon is deleted and the size is greatly reduced. The present invention relates to a vector system capable of simultaneously expressing the factor and the 8th blood coagulation factor useful for the treatment of hemophilia.

Hemophilia A is a congenital blood clotting disease related to X-chromosome, which causes a disorder of blood clotting when bleeding due to a deficiency of the 8th coagulation factor. Symptoms include frequent bleeding in muscles, bones, stomach or urinary tract, and swelling or pain associated therewith. Currently, treatment is performed by supplementing a coagulation factor from the outside, but this has to be administered throughout the life, resulting in many difficulties in terms of economy as well as the hassle of daily life. In addition, there is a problem to be careful about secondary infection during administration.

The 8th blood coagulation factor is a 180Kb gene located in the X chromosome and is a glycoprotein with several domains of A1-A2-B-A3-C1-C2, and its synthesis is mostly performed in the liver. Until now, many studies have been attempted to transmit the 8th blood coagulation cognition, but the size of the 8th blood coagulation factor gene is not expressed or secreted. Followed. Among the structures of the 8th blood coagulation factor, the B domain is composed of one large exon, and has a structure that is highly glycosylated in asparagin, serin, and threonine residues. According to recent functional studies, this is not an essential domain for procoagulant activity, and thus it has been found that removal of this part has no problem in the formation of functional molecules of the 8th coagulation factor. When the B-domain-removed blood coagulation factor 8 (BDD-FVIII) was expressed in cells, the unstable coagulation factor 8 mRNA structure and the reaction of ER chaperones were overcome, so that a large number of mRNA levels could be secured. In BDD-FVIII, when 226 amino acids were left toward the N-terminus and 6 consensus sites of N-linked glycosylaton were placed, the secretion of coagulation factor 8 was remarkably increased.

When hemophilia A is approached genetically, the target cells are bone marrow cells, and stem cells or progenitor cells are most suitable among bone marrow cells. A lentivirus-based vector is used as a gene transfer medium, and when the cell is infected, the vector is expressed by intercalating the gene material into the chromosome of the infected cell, thus enabling stable and continuous expression. In the case of other types of viruses, such as the existing Moloney murine leukemia virus, there is a problem in targeting and infecting cells such as stem cells or progenitor cells because they are only infected with differentiating cells. In addition, in the case of adenovirus, it is possible to express a large amount of protein, but it is not possible to continuously express it because it is continuously diluted according to the differentiation of cells.

Therefore, the necessity of a safe and continuous delivery method of the 8th blood coagulation factor is emphasized, and method development is therefore required. Lentiviral vector is capable of infecting not only dividing cells but also almost all non-dividing cells, and is inserted into the chromosome of cells after infection, allowing stable expression for a long time. Therefore, if the 8th blood coagulation factor is developed through a lentivirus-based vector, it is very useful in gene therapy.

The von Willebrand factor (vWF) is an important factor for the smooth activation of the 8th blood coagulation factor in the blood coagulation effect during bleeding in the body. vWF is a blood glycoprotein that binds to the 8th blood coagulation factor and prevents the 8th blood coagulation factor from being decomposed in the blood. In addition, when there is a wound on the blood endothelial cells, it binds to collagen or binds to platelets and plays a large role in blood coagulation. This vWF consists of a domain of D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2, of which the D'-D3 portion binds to the 8th blood coagulation factor. The vWF is a 250 kDa protein, and the gene size is about 9 kb. Therefore, it is impossible to insert vWF into the lentiviral vector to help the role of the 8th blood coagulation factor. Accordingly, by studying the minimum part necessary for the role of the 8th blood coagulation factor in the domain structure of vWF, it was found that vWF up to exon 32 of the gene functions most efficiently. A lentivirus-based vector was prepared by adding the 8th blood coagulation factor followed by the internal ribosomal entry site (IRES) and von Willebrand factor. The invention of a viral vector that expresses the 8th blood coagulation factor and von Willebrand factor when infected with cells by expressing its vector and proteins essential for the generation of lentivirus, gag-pol, env, tat, and rev, respectively, was completed. This has never been attempted before, and it is highly valued for its usefulness in gene therapy and hemophilia research in the future.

Accordingly, an object of the present invention is to provide a mutant von Willebrand factor that significantly increases the stabilization and activation efficiency of the 8th blood coagulation factor even though the exon is deleted and the size is greatly reduced.

In addition, an object of the present invention is to provide a vector that continuously and stably expresses the 8th blood coagulation factor and von Willebrand factor, respectively, in cells.

The ultimate object of the present invention is to succeed in gene therapy in hemophilia A due to the expression of these factors, in that it expresses not only the 8th coagulation factor, but also the von Willebrand factor essential for its function. It is distinct from other existing inventions.

The above object of the present invention is to generate a VSV-G pseudotyped lentivirus expressing the 8th blood coagulation factor and the von Willebrand factor using a lentivirus-based vector, and then infect the cells to obtain the 8th blood coagulation factor in various types of cells. Is to effectively express and measure its activity. This was finally confirmed by quantifying and measuring the degree to which the 8th blood coagulation factor activated the 10th blood coagulation factor together with the ninth coagulation factor activated.

According to one aspect of the present invention, the present invention provides a mutant von Willebrand factor (vWF23) having the amino acid sequence of SEQ ID NO: 2 in which exon 24-46 in the von Willebrand factor is deleted.

According to another aspect of the present invention, the present invention provides a mutant von Willy Brandt factor (vWF23) gene having a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 2. Preferably, the gene is characterized by having the nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides a mutant von Willebrand factor (vWF28) having the amino acid sequence of SEQ ID NO: 4 in which exons 29-46 in the von Willebrand factor are deleted.

According to another aspect of the present invention, the present invention provides a mutant von Willy Brand factor (vWF28) gene having a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 4. Preferably, the gene is characterized by having a nucleotide sequence of SEQ ID NO: 3.

According to another aspect of the present invention, the present invention provides a mutant von Willebrand factor (vWF32) having the amino acid sequence of SEQ ID NO: 6 in which exons 33-46 in the von Willebrand factor are deleted.

According to another aspect of the present invention, the present invention provides a mutant von Willy Brandt factor (vWF32) gene having a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 6. Preferably, the gene is characterized by having the nucleotide sequence of SEQ ID NO: 5.

According to another aspect of the present invention, the present invention provides an animal cell expression vector comprising a gene encoding the mutant von Willy Brandt factor (vWF23, vWF28 or vWF32) according to the present invention.

In the present invention, the animal cell expression vector includes any non-viral (plasmid or liposome) or viral vector that can be expressed by delivering the gene in an animal cell, but preferably retrovirus, lentivirus, adeno It may be a viral vector such as a virus or an adeno-associated virus, more preferably a lentiviral vector. In FIG. 12 of the present invention, pvEx23, pvEx28 and pvEx32, which are lentiviral vectors expressing vWF23, vWF28 or vWF32, are disclosed.

In the present invention, the animal cell expression vector further includes a gene encoding a human blood coagulation factor VIII (Factor VIII) in which the B domain is deleted in addition to the gene encoding the mutant von Willy Brand factor (vWF23, vWF28 or vWF32). Characterized in that. In this case, two components of the active ingredient for hemophilia treatment can be expressed in one vector at the same time.

In the present invention, preferably, the human blood coagulation factor VIII from which the B domain is deleted is characterized in that it has an amino line sequence of SEQ ID NO: 8, and the gene has a nucleotide sequence of SEQ ID NO: 7 It is characterized by that.

In the present invention, an animal cell expression vector capable of simultaneously expressing a mutant von Willybrand factor (vWF23, vWF28 or vWF32) and a human blood coagulation factor VIII (Factor VIII) in which the B domain is deleted is any non-viral (plasmid) Or liposome) or a viral vector is also included, preferably a retrovirus, a lentivirus, an adenovirus, a viral vector such as an adenovirus, and more preferably a lentiviral vector. 13 of the present invention discloses a pvBDD.FVIII.ires.vWex32 lentiviral vector, a bicistronic expression system in which the two genes are linked by an internal ribosome entry site (IRS).

According to another aspect of the present invention, the present invention is packaged by transfecting a packaging cell with a lentiviral vector capable of expressing the mutant von Willy Brandt factor of the present invention or the human coagulation factor 8 in which the B domain is deleted. Provides lentiviral particles.

In the present invention, the packaging cells may be any packaging cells such as 293T cells and HT1080 cells capable of packaging the lentiviral vector of the present invention to produce lentiviral particles, but preferably 293T cells.

In the present invention, it is characterized in that the lentiviral vector of the present invention is coat-transfected with pGag-pol, pRev, pTat, and pVSV-G to the packaging cells to make the lentiviral particles. In the examples of the present invention, a split gene expression system was used to produce a safer virus. In other words, only gag-pol, tat, rev, and VSV-G, which are essential factors for viral production, were expressed, but they were all transported to other vectors to lower the probability of recombination.

According to another aspect of the present invention, the present invention is a pharmaceutical for the treatment and prevention of hemophilia using the animal cell expression vector of the present invention or the mutant von Willy Brandt factor expressed therefrom and the human blood coagulation factor 8 deleted from the B domain Provide the appropriate composition.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for treating and preventing hemophilia containing the lentiviral particles according to the present invention as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present inventors have developed a system that simultaneously expresses the 8th blood coagulation factor and the mutant von Willebrand factor in a lentiviral-based vector. Specifically, the present invention consists of the steps of confirming the expression and activity of the 8th blood coagulation factor in a lentiviral-based system and the steps of identifying the essential domain of von Willebrand factor for the activity of the 8th coagulation factor and expressing them together. .

The eighth blood coagulation factor of the present invention is a form in which the B domain is removed from its domain to further increase the secretion of the eighth blood coagulation factor (BDD-FVIII). Among the structures of the 8th blood coagulation factor, the B domain is composed of one large exon, and has a structure that is highly glycosylated in asparagin, serin, and threonine residues. According to recent functional studies, this is not an essential domain for procoagulant activity, and thus it has been found that removal of this part has no problem in the formation of functional molecules of the 8th coagulation factor. When the B-domain-removed blood coagulation factor 8 (BDD-FVIII) was expressed in cells, the unstable coagulation factor 8 mRNA structure and the reaction of ER chaperones were overcome, so that a large number of mRNA levels could be secured.

In BDD-FVIII, when 226 amino acids were left toward the N-terminus and 6 consensus sites of N-linked glycosylaton were placed, the secretion of coagulation factor 8 was remarkably increased. In addition, almost all non-dividing cells can be infected, and stable expression for a long time is possible by being inserted into the chromosome of the cell after infection. Therefore, if the 8th blood coagulation factor is developed through a lentivirus-based vector, it is very useful in gene therapy.

The mutant von Willebrand factor of the present invention removes some of the entire exons so that only the D1-D2-D'-D3 domain (vWF23), the D1-D2-D'-D3-A1 domain (vWF28), or D1 -D2-D'-D3-A1-A2 domain only (vWF32) is included. This domain has a binding site to the 8th blood coagulation factor, the 8th blood coagulation factor and the binding site of platelets to GP1b, or the 8th blood coagulation factor, the binding site of platelets to GP1b and collagen.

In the present invention, the expression of von Willebrand factor is required to maximize the activity of the 8th blood coagulation factor. This is to protect against factors that inactivate the 8th blood coagulation factor such as thrombin in vivo, and also to help the stable structure of the 8th blood coagulation factor. However, when the 8th coagulation factor is expressed externally together with the full-lengh von Willebrand factor, they co-localize in the cell, resulting in the result that the von Willebrand factor inhibits the secretion of the 8th coagulation factor. Therefore, the function of the eighth blood coagulation factor is maximized, and a mutant von Willebrand factor in the form of leaving only a portion that does not inhibit secretion is preferable.

Other mammalian expression vectors may be used as carriers for the expression of the 8th blood coagulation factor and von Willebrand factor of the present invention, but lentiviral-based vectors are used for cells that are not easy to transfer genes, such as stem cells and hematopoietic progenitor cells. It is more advantageous to use. However, in the case of a lentiviral-based vector, there is a limitation on the size of the expressed gene. The size of the B-domain deleted blood coagulation factor 8 (BDD.FVIII) is 4.4 kb, and the size of the von Willebrand factor up to the A2 domain is 5.6 kb. Therefore, these genes can be expressed without significant loss of viral titer, and in order to increase viral titer, the envelope protein of lentivirus can be produced by pseudotyping with VSV-G, and then the virus can be concentrated.

The animal cell expression vector of the present invention may include a promoter derived from eukaryotic or prokaryotic cells capable of inducing the transcription of foreign genes in the animal cell, and the promoter region may also include a regulatory element for increasing and inhibiting transcription. have. Suitable promoters include the cytomegalovirus class early promoter (pCMV), the mouse sarcoma virus long terminal repeat promoter (pRSV) and the SP6, T3 or T7 promoter. An enhancer upstream of the promoter or a terminator sequence downstream of the coding region may be optionally included in the vector of the present invention to facilitate expression. In addition, the vector of the present invention may contain an additional nucleic acid sequence such as a polyadenylate sequence, a localized sequence, or a signal sequence sufficient for the cell to efficiently and effectively process the protein expressed by the nucleic acid of the vector. Examples of preferred polyadenylate sequences are SV40 early site polyadenylate sites (CV Hall et al., J. Molec. App. Genet. 2, 101 (1983)) and SV40 late site polyadenylate sites (S. Carswell and JC Alwine, Mol. Cell Biol. 9, 4248 (1989)). Such additional sequences are inserted into the vector and are operably linked to the promoter sequence if transcription is desired, or to the initiation and processing sequences if additional translation and processing are desired. The insertion sequence can be placed at any position in the vector. The term “operably linked” is used to describe the linkage between a gene sequence and a promoter or other regulatory or processing sequence. As a result, transcription of the gene sequence is related by operative linkage with the promoter sequence, translation of the gene sequence is related by operably linking with the translational control sequence, and post-transcription processing of the gene sequence is operatively linked with the processing sequence. It is related by becoming.

Standard techniques for constructing the vector of the present invention are well known to those skilled in the art and can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989). Various strategies are useful for joining segments of DNA, the choice of which depends on the nature of the ends of the DNA segments and is easily made by skilled technicians.

Examples of lentiviruses usable in the present invention include HIV-1 and HIV-2, SIV, FIV, BLV, EIAV, CEV, and visna viruses. Of these, HIV and SIV are preferred for use in gene therapy. Human immunodeficiency virus type 1 (HIV-1) belongs to a small lineage within retroviruses, lentiviruses, and, like other members of these lineages, HIV can infect dormant cells. This makes lentiviruses an attractive vector for gene therapy.

HIV-1-derived vectors are most often used as gene carriers due to their ability to infect dividing and non-dividing cells with their cytoplasmic and nuclear entry proteins (Kohn, 2001). This ability is often thought to be due to the various properties of the vectors, which generate nuclear localization signals in a variety of multiple virion proteins and triple-stranded'DNA flaps' in the reverse-transcribed genome. It includes a central polypurine tract (Zennou et al., 2000). As a result of these properties, bioengineered HIV-1 can infect hematopoietic progenitor cells very effectively at a fairly low MOI (Park and Choi, 2004). The first consideration with regard to the use of lentiviral vectors as a tool for gene therapy is that the transfer vector is derived from HIV-1. However, all viral components required for viral replication were removed from the viral vector used in previous studies (Hofmann et al., 1999), and the transfer vector finally contained less than 5% of the HIV-1 genome (Kohn, 2001). . Another barrier to using lentiviral vectors is that they are limited to the size of the transferred genes. The vWF contains 52 exons with a cDNA size of approximately 9 Kb, which exceeds the size limit of most lentiviral vectors. In the present invention, the present inventors successfully introduced vWF cDNA into a lentiviral vector (Figs. 1 and 2). In the preparation and preparation of the lentivirus, the present inventors replaced the env of HIV-1 with the VSV-G protein. VSV-G mediates viral invasion into cells through membrane fusion rather than through specific cell surface receptor proteins, resulting in a remarkably widening of the host range (Hofmann et al., 1999). More importantly, VSV-G confers structural stability during ultracentrifugation, enabling concentration of viruses with high titers with little loss of infectivity (Burns et al., 1993; Hofmann et al., 1999). ). By utilizing these properties of VSV-G, the present inventors successfully prepared and concentrated vEx52, resulting in a transduction efficiency 6 times or more higher with only 1/100 of the volume of the lentiviral supernatant (FIG. 3). These results were analyzed by FACS and clearly observed under fluorescence: a significantly greater amount of eGFP was observed in cells transduced with vEx52 enriched than vEx52 which was not enriched (FIG. 4 ). A recent study by De Meyer et al. has shown the incorporation of long vWF cDNA into a lentiviral vector, and hematogenous endothelial obtained from dogs with VWD type 3 to develop gene therapy for von Willybrand disease type 3 (VWD type 3). Transduction of cells (blood-outgrowth endothelial cells, BOECs) was included (De Meyer et al., 2006). However, since concentrating the virus of low titer requires additional time and a difficult process, it cannot prove ideal for practical application in the treatment of hemophilia A. Therefore, the present inventors attempted to reduce the size of the vWF cDNA inserted into the lentiviral vector. We removed the domains of vWF, leaving only a minimal site for the interaction between vWF and FVIII. Mature vWF is composed of the D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2 domains. FVIII binds to the D'-D3 domain, and the A1 domain binds to platelet glycoprotein Ib, heparin and collagen. This facilitates the aggregation of platelets and aids in adhesion to the site of damage to blood vessels. The vWF gene is located on chromosome 12 and includes 52 exons having 178,000 bases. The present inventors removed exon 24-46 to prepare pRex23 and pvEx23, leaving only the site that binds to FVIII. FVIII binds to vWF within 272 amino acid residues located at the amino terminus of vWF (Sadler, 1998). In addition, the present inventors prepared pRex28 and pvEx28, which had exons 29-46 removed, leaving a platelet binding site in addition to the FVIII binding site. The platelet binding site on vWF is located in the A1 domain (Sadler, 1998). When pvEx23, pvEx28 and pvEx52 were packed in lentivirus, the viral preparations obtained from pvEx23 and pvEx28 were significantly more than those obtained from pvEx52. In general, the viral titer of unconcentrated vEx52 is 2×10 ⁴ to 4×10 ⁴ particles/ml (see FIG. 3), whereas the titers of vEx23 and vEx28 are ^{between 1×10 5} and 3×10 ⁵ particles/ml. Was in (Fig. 5). In Figures 3 and 5, the transduction efficiency of the three viruses can be compared with a graph (histogram). When 500 μl of vEx23, vEx28, and vEx52 were used to transduce Jurkat cells, 35.02%, 26.30% and 4.64% of cells were respectively positive for eGFP. Therefore, the present inventors were able to improve viral titer and transduction efficiency by removing domains in vWF, which are hardly needed for interaction with FVIII, and thus reduce the packing size. When functional FVIII was measured in the supernatant by transfecting pRex23, pRex28 and pRex52 into 293T cells, pRex23 and pRex28 showed lower FVIII activity than that observed with pRex52, a full-length vWF. However, when using the viral system, the supernatant obtained from cells transduced with vEx28 showed higher secreted BDD.FVIII activity than that obtained from vEx52 (FIG. 6 ). This may be because the large size of the full-length vWF limits the packing and expression efficiency of vWF. However, the inventors could not determine whether the expression of FVIII was altered by vWF, whether vEx28 increased the secreted level of FVIII expressed in the supernatant, and whether this effect was probably due to the protection of the BDD.FVIII structure. This is consistent with the observation that more FVIII activity was detected in cells in the absence of vWF (Kaufman et al., 1998). Another indicator that vWF stabilizes FVIII is the fact that FVIII decomposes rapidly in the absence of vWF (Over et al., 1978), while FVIII is eliminated more slowly in the presence of vWF (Tuddenham et al., 1982). . Taking a closer look at the properties of vWF and FVIII, both proteins may have been designed to provide powerful genetic tools in repairing FVIII-deficient cells.

Formulations of the pharmaceutical composition of the present invention include those suitable for parenteral (eg, intradermal, subcutaneous, intramuscular, intravenous, intraarterial), oral or inhalation administration. Alternatively, the pharmaceutical formulation of the present invention may be suitable for intramucosal administration (eg, nasal administration) in a subject. The formulation can be simply prepared in a disposable dosage form and can be prepared by any method known in the art.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, severity of disease symptoms, food, administration time, route of administration, excretion rate and response sensitivity. In general, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment. In general, the pharmaceutical compositions of the present invention once about 10 ³ per dose is suitable to be administered in an amount of protein of 10 ⁷ viral particles, or 0.001-100 ㎎ / ㎏.

1 is a schematic diagram of packaging constructs including a mutant vWF gene as an embodiment of the present invention.
2 shows the integration and transcription of vWF from transduced COS-1 cells as an embodiment of the present invention.
Figure 3 shows the concentration (concentration) of vWF-expressing HIV-1 as an embodiment of the present invention.
4 shows transduction by pseudotyped HIV-1 expressing vWF as an embodiment of the present invention.
5 is a schematic diagram of a deletion structure of vWF as an embodiment of the present invention.
6 shows the result of detecting the activity of secreted functional FVIII as an embodiment of the present invention.
7 is a photograph of the result of an expression test of eGFP in order to confirm the expression of the 8th blood coagulation factor as an embodiment of the present invention.
8 is a result of measuring the activity of the 8th blood coagulation factor by each mutant vWF as an embodiment of the present invention by a Chromogenic Assay.
9 is a diagram illustrating a manufacturing process of a Lentivirus vector including the gene of the 8th blood coagulation factor in which the B-domain is deleted as an embodiment of the present invention.
10 is a diagram showing a manufacturing process of a Lentivirus vector including a gene of vWF (von Willebrand Factor) as an embodiment of the present invention.
FIG. 11 is a diagram showing the manufacturing process of pRex23, pRex28, and pRex32, which are vectors containing vWF variants, as an embodiment of the present invention.
12 is a diagram showing the manufacturing process of pvEx23, pvEx28, and pvEx32, which are Lentivirus vectors including genes of vWF variants, as an embodiment of the present invention.
13 shows the manufacturing process of pvBDD.FVIII.vWEx32, a Lentivirus vector including BDD.FVIII and vWF variant genes as an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

Example 1: vector preparation

-GAACCGAAGCTGGTACCT-3

및 5

-GACAGGAGGGGCATTAAATTGCTTTTGCCT-3

를 이용하고, 하향 3‘-영역을 프라이머쌍 5

-TTTAATGCCCCACCAGTCTTGAAACGCCAT-3

및 5

-ATGCTCGCCAATAAGGCATTCCA-3

을 이용하여 PCR 증폭하였고, 다음 이들 산물을 열-변성(denaturation)과 재결합(renaturation)을 거쳐 원하는 결실을 얻었다. 그 산물을 KpnI 및 BglI 로 분해하고 원 pRF8 플라스미드의 KpnI - BglI 내로 서브클로닝하여 pREP7 (Invitrogen, USA)의 RSV 3

LTR 제어하에 B-도메인 결실된(BDD) FVIII cDNA가 삽입된 pREP7-BDD.FVIII를 제조하였다 (참조: The Journal of Gene Medicine, Volume 6, Issue 7 , Pages 760 - 768). 본 발명자는 상기 논문의 저자인 Subrata Banerjee에게서 pREP7-BDD.FVII를 제공받아 사용하였다. 또한, 전장 vWF cDNA (ATCC #59126)를 상기와 마찬가지로 링커를 이용하여 pREP7 vector (Invitrogen, USA)의 NotI 부위에 클로닝하여 pRex52 플라스미드를 얻었다. Full-length FVIII cDNA (ATCC Accession No. 40086) was cloned into the NotI site of the modified pREP7 vector (Invitrogen, USA) using a linker to obtain plasmid pRF8, and then the B-domain was raised to remove most of the B-domain. 5'-region primer pair 5

-GAACCGAAGCTGGTACCT-3

And 5

-GACAGGAGGGGCATTAAATTGCTTTTGCCT-3

And the downward 3'-region primer pair 5

-TTTAATGCCCCACCAGTCTTGAAACGCCAT-3

And 5

-ATGCTCGCCAATAAGGCATTCCA-3

PCR amplification was performed using, and then these products were subjected to heat-denaturation and recombination to obtain a desired deletion. The product was digested with KpnI and BglI and subcloned into KpnI - BglI of the original pRF8 plasmid, and RSV 3 of pREP7 (Invitrogen, USA)

PREP7-BDD.FVIII into which the B-domain deleted (BDD) FVIII cDNA was inserted under LTR control was prepared (see The Journal of Gene Medicine, Volume 6, Issue 7, Pages 760-768). The present inventor received and used pREP7-BDD.FVII from Subrata Banerjee, the author of the paper. In addition, the full length vWF cDNA (ATCC #59126) was cloned into the NotI site of the pREP7 vector (Invitrogen, USA) using a linker in the same manner as above to obtain a pRex52 plasmid.

The Lentivirus backbone (transfer vector) used in the experiment was Journal of Virology, December 1999, p. 10020-10028, Vol. 73, No. The HIV-1 vector pHIvec2.GFP received by Joseph Sodroski, the author of 12, was used (see above). The pHIvec2.GFP was prepared by removing the env and vpu sequences from the v653 rtatpC virus, maintaining the Rev-responsive element, and inserting the eGFP gene (Clontech) behind the IRFS. The vector map of pHIvec2.GFP is the same as that in FIG. 1 from which the vWF gene has been removed.

To make a Lentivirus vector containing the gene of the 8th coagulation factor in which the B-domain is deleted, cDNA of BDD.FVIII (B-Domain-Deleted Coagulation Factor VIII) was obtained from pREP7-BDD.FVIII using NotI, and This was put into the lentivirus backbone using the same enzyme to generate pvBDD.FVIII. The manufacturing process is shown in FIG. 9. Specifically, pRep7-BDD.FVIII and lentivirus backbone were digested with NotI, and the BDD.FVIII fragment from pREP7-BDD.FVIII was ligate to the NotI site of the lentivirus backbone.

To make a Lentivirus vector containing the gene of vWF (von Willebrand Factor), the pRex52 and lentivirus backbone were digested with NotI, and the vWF fragment from pRex52 was ligate to the NotI site of the lentivirus backbone, thereby preparing pvEx52. The manufacturing process is shown in FIG. 10. A detailed map of pvEx52, which is the result of FIG. 10, is disclosed in FIG. 1. The vWF gene is located between the long terminal repeats (LTR) and is fused to IRES-eGFP in a viral vector.

The vWF variants were created using PCR (polymerase chain reaction) in pRex52. Each PCR was reacted with each dNTP 25 mM, each phosphorlyated primers 10 μM, 2 mM Mg ²⁺ , and a DNA template, and amplified with PfuUltraTMII Fusion HS DNA Polymerase. The total volume of PCR was 50 µl and was carried out under the following conditions. At 95℃ for 5 minutes, at 95℃ for 30 seconds, at 52℃ for 30 seconds, and at 72℃ for 30 seconds per 1 Kb, the reaction was repeated 18 times, and at 72℃ for 10 minutes. The sequence of the forward primer for making vWF variants was 5-CGT GATGAGACGCTCCAG-3, the sequence of the reverse primers was 5-TTTTCTGGTGTCAGCACACTG-3 for pRex23, 5-AGGTGCAGGGGAGAGGGT-3 for pRex28, and 5-AGAGCACAGTTTGTGGAG-3 for pRex32. After performing PCR, the amplified product was isolated using a PCR removal kit (Qiagen), and reacted at 15° C. for about 24 hours using a ligase (Takara) to prepare pRex23, pRex28, and pRex32. The manufacturing process is shown in FIG. 11. Ligated mixture was transformed into TOP10.

To make a Lentivirus vector containing the genes of the vWF variants, the pRex23, pRex28 or pRex32 and the lentivirus backbone were digested with NotI, and the vWF fragment from pRex23, pRex28 or pRex32 was ligate to the NotI site of the lentivirus backbone, and pvEx23, pvEx28 and pvEx32 was prepared. The manufacturing process is shown in FIG. 12.

In pvBDD.FVIII, ires-eGFP is attached behind BDD.FVIII, where only the eGFP portion was removed by PCR, and then the vWF variant of pRex32 was added to this site to create pvBDD.FVIII.vWEx32. The manufacturing process is shown in FIG. 13. For transformation, TOP10 was used. In order to express vWF32 together following BDD.FVIII, an IRES (internal ribosomal entry site) sequence was inserted after BDD.FVIII, followed by vWF32. As a result, two proteins can be simultaneously expressed under one promoter, and vWF32 expressed later plays a role in helping the activity and function of BDD.FVIII.

Example 2: Generation of viruses

Vesicular stomatitis G protein (VSV-G) pseudotyped HIV-1 is a method of quinquepartite plasmid transient transfection of 293T (see Park and Choi, 2004 Mol. Cells 17, 297-303) by gag-pol, tat, rev, VSV-G. And by transfection with a transfer vector. The 293T was split into 100 mm plates at 4.5×10 ⁶ 24 hours before transfection, and then 4 hours before transfection, the supernatant was replaced with a culture medium containing 10% FCS and 25 mM HEPES. For transfection, 10 μg of Gag and Pol packaging plasmid, 2 μg of VSV-G plasmid, 1 μg of Tat plasmid, 1 μg of Rev plasmid, and 10 μg of transfer vector were added. Add these DNAs to 62 µl of 2.5 M CaCl ₂ and adjust the volume to 500 µl with water, and then vortex and add 500 µl of 2x HBS (281 mM NaCl, 100 mM hepes, 1.5 mM Na ₂ HPO ₄ pH 7.12 to this mixture). ) Was added and allowed to stand at room temperature for 30 minutes, and then sprinkled on 293T cells. 16 hours after transfection, the supernatant was changed to RPMI in 10 mM HEPES buffer, and the virus generated after 48 hours and released as a supernatant was harvested using a 0.45 μm filter.

Example 3: viral titration

NIH3T3 cells 3 x 10 ⁵ cells were put into 60 cm ² dishes, and after 20 hours, several volumes of virus were added to the cells. At this time, the total volume was 2 ml of culture media, and polybrene was added at a concentration of 2 μg/ml. After 6 hours, the virus was removed, and the cells were washed with DMEM containing 2% FCS to completely remove the virus, and then the cells were placed in an incubator. After 2 days, the cells were removed using 0.25% trypsin, washed with 1x PBS, and fixed with 3.7% formaldehyde. ^{The percentage of eGFP +} emitted from infected cells was read using FACScan (Becton Dickinson Immunocytometry Systems) and CellQuest program (Becton Dickinson), and the virus titer was calculated using the following formula. (2 × ^{number of cells × eGFP +} percent of number of cells) ÷ amount of virus.

Example 4: Concentration of virus ( Concentration )

The generated virus was placed in a polyallomer tube and centrifuged at 4° C. for 1.5 hours at 50,000 xg in a SW28 rotor. After dissolving the pellets in a small volume of medium, the tube was covered with parafilm and left at 4°C for 24 hours. For long-term storage, concentrated virus was placed at -80°C. Figure 3 shows the concentration (concentration) of vWF-expressing HIV-1 as an embodiment of the present invention. 500 μl unconcentrated (center) and 5 μl concentrated (right) vWF-expressing lentiviral supernatant were used to transduce Jurkat cells. The fraction of eGFP+ cells in the transduced cells was determined by flow cytometry.

Example 5: Transduction of cells ( Transduction )

After counting the cells on a hemocytometer, the desired number of cells was spread on a 24-well plate or a 6-well plate. The virus supernatant was added to the cells and added to the desired MOI. At this time, the total volume was adjusted to the medium to the desired volume, and at this time, polybrene was added at a concentration of 2 µg/ml. Infection was carried out at 37° C. for 6 hours in the presence of _{5% CO 2.} After infection, the cells were washed with medium. 5 is a schematic diagram of a deletion structure of vWF as an embodiment of the present invention. A. From pRex52 and lentiviral vectors, respectively, pRex23 and pvEx23 were constructed by removing exons 24-46, and pRex28 and pvEx28 were prepared by removing exons 29-46. B. vEx23 and vEx28 were produced from pvEx23 and pvEx28, respectively, and 500 μl of virus supernatant was used to transduce Jurkat cells. The proportion of transduced cells was analyzed by FACS.

Example 6: DNA Wow RNA Separation

Genomic DNA was separated with 500 μl lysis buffer (0.1 M Tris HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, and 100 μg/ ml protease K). This was precipitated with isopropanol and washed with 70% Ethanol. RNA was obtained with Trizol (Invitrogen), and cDNA was synthesized using ImPromII (Promega). PCR was amplified with Pfu (Solgent) using 1x reaction buffer, 25 mM each dNTP, 10 μM each primer, 2 mM Mg ^{2 +} , and a DNA template to make a total volume of 50 μl. 2 shows the integration and transcription of vWF from transduced COS-1 cells as an embodiment of the present invention. COS-1 cells were transduced with lentivirus expressing vWF. A. The introduction of the transduced vWF gene was detected by PCR for 421-bp vWF and 227-bp LTR in genomic DNA of transduced COS-1 cells. B. cDNA was prepared from transduced cells and amplified with primer pairs specific for vWF, LTR, and GAPDH with 421-, 227- and 187-bp, respectively. vEx52: transduced with vEx52, eGFP: transduced with eGFP-expressing lentivirus.

Example 7: Plasmid transfection ( Transfection )

DNA to be transfected (pRex23, pRex28, and pRex32) was put in 50 µl of 150 mM NaCl based on a 12-well pate, vortexed, and then spin down. PEI of 3 times the volume of DNA was added to this mixture, then vortexed and spin down again. After standing at room temperature for 10 minutes, it was carefully dropped onto the cells.

Example 8: Cell immunostaining method ( Immunocytochemistry )

Transduced cells were grown on glycogen-coated coverslipsas in 6-well tissue culture plates. The cells were fixed with cooled 100% methanol, washed with TBS [50mM Tris-HCl (pH7.4), 150 mM NaCl], quenched in 0.1% sodium borohydride in fresh TBS for 5 minutes, and then 3 with TBS for 5 minutes. Washed twice. Cells were blocked with blocking buffer (10% horse serum, 10% bovine serum albumin, 0.02% NaN ₃ in 1x PBS) for 60 minutes and washed with TBS for 5 minutes. The primary vWF antibody (Abcam) was diluted with 1% BSA in TBS and incubated overnight with cells at 4°C. After washing the cells three times with TBS for 5 minutes, they were labeled with a secondary goat-anti-mouse IgG TRITC antibody (Santa Cruz) in 1% BSA in TBS for 30 minutes at room temperature covered with aluminum foil. Then, they were washed three times with TBS for 5 minutes and mounted on slides using a ProLong antifade kit (Cell Signaling).

4 shows transduction by pseudotyped HIV-1 expressing vWF as an embodiment of the present invention. A. 293T cells were transfected with a plasmid containing viral components for viral production, including a transfer vector containing vWF-IRES-eGFP. The eGFP obtained from the transfer vector was visualized under a fluorescence microscope at 10× magnification. B. Jurkat cells were visualized with an optical microscope and a fluorescence microscope. C. Jurkat cells were transduced with 500 μl of unenriched vWF-expressing pseudotype HIV-1 and visualized under a microscope. D. Jurkat cells were transduced with 5 μl of 160-fold concentrated vWF-expressing pseudotype HIV-1, and visualized in white and fluorescence. E. COS-1 cells were transduced with vWF-pseudotype HIV-1 at 0.5 MOI. eGFP was visualized under a fluorescence microscope (left). vWF was visualized by staining VWF with antibody and TRITC (center). The detected vWF was derived from the transferred gene, not from COS-1 cells (right).

BDD.FVIII was made excluding the B domain from the sequence of the 8th blood coagulation factor and inserted into a lentiviral vector. For Lentivirus expression, lenvirius expressing BDD.FVIII was produced by expressing BDD.FVIII in 293T cells together with gag, pol, VSV-G, tat, and rev. When the cells were infected with this, BDD.FVIII was expressed in the cells, and its expression was finally confirmed by measuring the activity of the 8th coagulation factor. Virus generation and infection were indirectly confirmed by expression of eGFP in the vector (FIG. 7). 7 is a photograph of the result of an expression test of eGFP in order to confirm the expression of the 8th blood coagulation factor as an embodiment of the present invention (corresponding to FIG. 4A). Virus generation and infection were indirectly confirmed by expression of eGFP.

Example 9: Factor VIII Activity measurement experiment

Activated FVIII activity (FVIII:C) was quantified with Coatest VIII:C/4 kit (DiaPharm). Phospholipids, 100 mg/l ciprofloxacin, and 5 times the volume of factor IXa and factor X were mixed, and 50 μl of this was added to 96-well microtiter plates. 25 µl of cell culture supernatant was added thereto, and incubated at 37C for 5 minutes. After adding 25 µl of 0.025 mol/L CaCl2 and incubating at 37C for 10 minutes, 50 µl of S-2765 and I-2581 were added and reacted again at 37C for 10 minutes. The reaction was stopped with 20% acetic acid and the activity of factor 8 was measured at 405 nm. A standard curve was created and quantified using a known concentration of recombinant human FVIII.

6 shows the result of detecting the activity of secreted functional FVIII as an embodiment of the present invention. A. 293T cells were transfected with pREP7-BDD.FVIII with pRex23, pRex28 or pRex52. The supernatant of the transduced cells was collected and quantitatively analyzed for FVIII activity. B. K562 cells were transduced with vEx23, vEx28 or vEx52 with HIV-1-BDD.FVIII at 1.5 MOI. The supernatant of the transduced cells was collected and screened for FVIII activity. Data are expressed as the mean±standard error (means±SE) of at least 3 independent experiments. C. RT-PCR was performed with RNAs obtained from transduced cells. The B-domain-deleted FVIII showed 1.1 Kb of preparation. vEx23: transduced with vEx23, vEx28: transduced with vEx28, vEx52: transduced with vEx52.

Example 10: PMA With or without processing Factor VIII Activity measurement experiment

The domain of the von Willebrand factor was removed from the exon 23, exon 28, and exon 32 to the C-terminus and named pRex23, pRex28, pRex32, and the full length vWF is pRex52. This was when pREP7-BF was transfected with pRex32 as a result of finding the von Willebrand factor vector with the highest activity of the 8th coagulation factor secreted when expressed with pREP7-BF carrying BDD.FVIII. (Fig. 8). 8 is a result of measuring the activity of the 8th blood coagulation factor by each mutant vWF as an embodiment of the present invention by a Chromogenic Assay. After transfecting pREP7, pREP7-BF and pRex23, pREP7-BF and pRex28, pREP7-BF and pRex32, and pREP7-BF and pRex52 into HeLa cells, the functional activity of FVIII secreted as supernatant was measured. 8A is a measurement of the activity of the 8th blood coagulation factor in a normal state (no wound). All of them showed an increase in activity compared to the basal level, with 32 being the largest. Fig. 8b shows that PMA (phorbol ester) was treated to artificially create a wounded state, all of which induced FVIII secretion by at least three times and showed a significant difference compared to the basal level. Although it is not overexpressed normally (a low level is usually preferable for reasons of antibody formation, etc.), the result of large induction of FVIII secretion in a wounded state has very important significance in the clinical practice of hemophilia.

Experiment result 1. Lentivirus produce

In order to prepare pvEx52 (LTRs), 8.8 Kb von Willebrand factor cDNA was cloned between two long terminal repeats (LTRs) of HIV-1-derived lentivirus (Fig. 1). ). The vWF cDNA was isolated from vW-8 (ATCC #59126) using Not I and then cloned into a lentiviral vector. By fusing the vWF gene with IRES-eGFP, the enhanced green fluorescence protein as an indirect indicator of virus production after transfection into 293T cells along with other viral genes required for packaging viral particles. , eGFP) enabled the use of (Parolin et al., 1996; Yee et al., 1994). The packing vector, including gag and pol under the control of the CMV promoter, individually expressed tat and rev of HIV-1 under the control of the CMV promoter. The present inventors used vesicular stomatitis virus G protein (VSV-G) instead of HIV-1 env (Hofmann et al., 1999).

Experimental result 2. HIV -One False type VSV -G( VSV -G pseudotyped HIV -1) transduction

COS-1 cells were transduced with vEx52 with intact vWF, or with HIV-1-eGFP lentivirus expressing eGFP. Genomic DNA was isolated from the transduced COS-1, and PCR was performed using a primer pair specific for human vWF gene and HIV-1 LTR. vWF could only be amplified in vEx52-transduced cells, whereas LTR could be amplified in both vEx52 and HIV-1-eGFP-transduced cells (Fig. 2A). And RNA was prepared from the transduced cells, the cDNA was synthesized, and PCR was performed for vWF and LTR (FIG. 2B). The result of RT-PCR was consistent with the result of PCR amplification of genomic DNA. vWF was amplified only in vEx52-transduced cells, and LTR was detected in both vEx52 and HIV-1-eGFP transduced cells.

Experimental Results 3. Transduced From lymphoblasts vWF And the expression of vEx52 Concentration of

Jurkat cells were cultured at 2×10 ⁵ and transduced with 500 μl of unconcentrated vEx52 virus. FACS analysis shows that 5.55% of cells are positive for GFP expression (FIG. 3 ). After the virus suspension was concentrated to 160 times by centrifugation at 50,000×g, 5 μl of the concentrated virus was used to transduce the same number of Jurkat cells to obtain a yield of 29.51% eGFP+ (FIG. 3). By concentrating the vEx52 virus, the titer was increased from 2.8×10 ⁴ particles/ml to 2.3×10 ⁷ particles/ml. Transfection of the vector induces lentiviral production, which was indirectly confirmed by the expression of gGFP (Fig. 4A). And transduction and expression of both unenriched or enriched vEx52 were visualized by fluorescence from eGFP expression (BD in FIG. 4). In order to demonstrate vWF expression from vEx52, COS-1 cells were transduced with vEx52 at MOI 0.5 and labeled with human vWF antibody, followed by TRITC staining (E in FIG. 4). And in order to confirm whether the transduced constructs were maintained for an extended period of time, 1×10 ⁵ Jurkat cells were transduced at an MOI of 0.5. After 4 days, 38.27% of cells were positive for eGFP by FACS analysis. In the analysis at 9, 15, 35, 50 and 90 days after transduction, 33.01%, 11.99%, 11.32%, 6.13%, and 5.56% of cells were eGFP+, respectively (data not shown).

Experimental Results 4. Domain-removed vWF Composition of

pRex23 and pRex28 were prepared from pREP7-vWF (fully provided by Dr. Subrata Banerjee) by removing exons 24-46 and 29-46. pvEx23 and pvEx28 were prepared from pvEx52 by the same method (Fig. 5A). The sequences were forward primers 5′-CGTGATGAGACGCTCCAG-3′ and Ex23PR reverse primers 5′-TTTTCTGGTGTCAGCACACTG-3′ for pRex23 and pvEx23, and Ex28PR reverse primer 5′-CAGGTGCAGGGGAGAGG-3′ for pRex28 and pvEx28. It was removed by PCR. Thereafter, pvEx23 and pvEx28 were used to prepare HIV-1 pseudotype VSV-G together with the packing vector, and titrated in Jurkat cells. When 500 μl of viral supernatants of vEx23 and vEx28 were used for transduction, respectively, 35.02% and 26.30% of cells were analyzed as positive for eGFP (FIG. 5B).

Experimental Results 5. Secreted FVIII Functional activity of

To test the effect of domain-depleted vWF on secretion of FVIII, 293T cells were transfected with pREP7-BDD.FVIII with one pRex23, pRex28, or pRex52. 48 hours after transfection, the supernatant was collected and the functional activity of FVIII was screened by chromogenic assays. From transduction with pRex23, pRex28 and pRex52, FVIII activity levels of 28.89±18.86, 107.22±30.64 and 199.44±58.93, respectively, were obtained (Fig. 6). Therefore, the functional activity of secreted FVIII decreased as part of the vWF domain was removed. Next, the present inventors evaluated the effect when the domain-deleted vWF as a component of the vWF lentivirus was transduced. K562 cells were transduced at MOI 1.5 with vBDD.FVIII, a BDD-FVIII expressing HIV-1 with vEx23, vEx28 or vEx52. Expression of the transduced FVIII was confirmed from RT-PCR performed with RNA obtained from the transduced cells (Fig. 6). The FVIII activity in the supernatant of the transduced cells was 28.33± 5.50 for vEx52-transduced cells, and 33.89± 3.93 and 53.33± 9.43 for vEx23- and vEx28-transduced cells, respectively (Fig. 6). These data suggest that the deleted form of vWF, vEx28, is the most efficient at promoting secretion of FVIII via interaction of its minimal essential domains with FVIII. These data indicate that vEx28, a deleted form of vWF, is most effective in inducing the secretion of FVIII through the interaction of FVIII with the minimal essential domains of vWF.

As described above, according to the present invention, by using the mutant von Willebrand factor reduced in size, it is possible to efficiently express the 8th coagulation factor together with the 8th coagulation factor in a viral vector and significantly increase the activity of the 8th coagulation factor. In addition, the viral vector of the present invention can be effectively used for gene therapy for hemophilia treatment. In addition, the simultaneous expression of the 8th coagulation factor and von Willebrand factor of the present invention is very useful in clinical applications such as gene therapy for hemophilia A.

<110> Korea University Industrial & Academic Collaboration Foundation <120> Recombinant expression vector system for variants of coagulation factor VIII and von Willebrand factor <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 3564 <212> DNA <213> Homo sapiens <400> 1 atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60 ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120 gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180 ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240 agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300 accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360 gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420 gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480 ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540 acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600 gaacgggcat ctcctcccag cagctcatgc 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acggggagga cctgcagatg 1500 gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560 tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620 ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680 gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740 gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800 ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860 tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920 gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980 tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040 gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100 tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160 atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220 agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280 agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340 ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400 agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460 tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520 acagtgaaga ttggctgcaa cacttgtgtc tgtcaggacc ggaagtggaa ctgcacagac 2580 catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640 ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700 aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760 tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820 gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880 tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940 tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000 ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060 tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaacg tgatgagacg 3120 ctccaggatg gctgtgatac tcacttctgc aaggtcaatg agagaggaga gtacttctgg 3180 gagaagaggg tcacaggctg cccacccttt gatgaacaca agtgtctggc tgagggaggt 3240 aaaattatga aaattccagg cacctgctgt gacacatgtg aggagcctga gtgcaacgac 3300 atcactgcca ggctgcagta tgtcaaggtg ggaagctgta agtctgaagt agaggtggat 3360 atccactact gccagggcaa atgtgccagc aaagccatgt actccattga catcaacgat 3420 gtgcaggacc agtgctcctg ctgctctccg acacggacgg agcccatgca ggtggccctg 3480 cactgcacca atggctctgt tgtgtaccat gaggttctca atgccatgga gtgcaaatgc 3540 tcccccagga agtgcagcaa gtga 3564 <210> 2 <211> 1187 <212> PRT <213> homo sapiens <400> 2 Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile 1 5 10 15 Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30 Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 35 40 45 Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60 Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys 65 70 75 80 Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu 85 90 95 Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110 Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125 Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140 Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly 145 150 155 160 Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln 165 170 175 Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190 Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser 195 200 205 Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln 210 215 220 Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu 225 230 235 240 Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255 Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270 Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275 280 285 Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys 290 295 300 Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met 305 310 315 320 Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu 325 330 335 Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His 340 345 350 Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365 Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380 Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp 385 390 395 400 Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg 405 410 415 Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys 420 425 430 Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu 435 440 445 Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val 450 455 460 Ala Met Asp Gly Gln Asp Val Gln Leu Pro Leu Leu Lys Gly Asp Leu 465 470 475 480 Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495 Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510 Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525 Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540 Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln 545 550 555 560 Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575 Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590 Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605 Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620 Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val 625 630 635 640 Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645 650 655 Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670 Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685 Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys 690 695 700 Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp 705 710 715 720 Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735 His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750 Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765 Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780 Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met 785 790 795 800 Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815 His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820 825 830 Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr 835 840 845 Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp 850 855 860 Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly 865 870 875 880 Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp 885 890 895 Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys 900 905 910 Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu 915 920 925 Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys 930 935 940 Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg 945 950 955 960 Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975 His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 980 985 990 Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 995 1000 1005 Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn Ser 1010 1015 1020 Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Arg Asp Glu Thr 1025 1030 1035 1040 Leu Gln Asp Gly Cys Asp Thr His Phe Cys Lys Val Asn Glu Arg Gly 1045 1050 1055 Glu Tyr Phe Trp Glu Lys Arg Val Thr Gly Cys Pro Pro Phe Asp Glu 1060 1065 1070 His Lys Cys Leu Ala Glu Gly Gly Lys Ile Met Lys Ile Pro Gly Thr 1075 1080 1085 Cys Cys Asp Thr Cys Glu Glu Pro Glu Cys Asn Asp Ile Thr Ala Arg 1090 1095 1100 Leu Gln Tyr Val Lys Val Gly Ser Cys Lys Ser Glu Val Glu Val Asp 1105 1110 1115 1120 Ile His Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala Met Tyr Ser Ile 1125 1130 1135 Asp Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys Ser Pro Thr Arg 1140 1145 1150 Thr Glu Pro Met Gln Val Ala Leu His Cys Thr Asn Gly Ser Val Val 1155 1160 1165 Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys Ser Pro Arg Lys 1170 1175 1180 Cys Ser Lys 1185 <210> 3 <211> 5508 <212> DNA <213> homo sapiens <400> 3 atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60 ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120 gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180 ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240 agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300 accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360 gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420 gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480 ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540 acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600 gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660 ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720 gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780 ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840 gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900 tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960 tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020 ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080 acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140 aatgaagaat gtccagggga gtgccttgtc acaggtcaat cacacttcaa gagctttgac 1200 aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260 cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320 acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380 ggggcaggag ttgccatgga tggccaggac gtccagctcc ccctcctgaa aggtgacctc 1440 cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500 gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560 tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620 ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680 gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740 gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800 ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860 tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920 gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980 tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040 gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100 tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160 atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220 agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280 agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340 ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400 agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460 tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520 acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa ctgcacagac 2580 catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640 ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700 aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760 tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820 gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880 tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940 tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000 ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060 tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120 tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180 agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240 ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgcctgcttc 3300 tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360 aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420 gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480 gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540 aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600 gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660 cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720 ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct gtatgtggag 3780 gacatctcgg aaccgccgtt gcacgatttc tactgcagca ggctactgga cctggtcttc 3840 ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa ggcctttgtg 3900 gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc cgtggtggag 3960 taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc gtcagagctg 4020 cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac cagcgaggtc 4080 ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc ctcccgcatc 4140 accctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt tgtccgctac 4200 gtccagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg gccccatgcc 4260 aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc cttcgtgctg 4320 agcagtgtgg atgagctgga gcagcaaagg gacgagatcg ttagctacct ctgtgacctt 4380 gcccctgaag cccctcctcc tactctgccc cccgacatgg cacaagtcac tgtgggcccg 4440 gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct ggatgtggcg 4500 ttcgtcctgg aaggatcgga caaaattggt gaagccgact tcaacaggag caaggagttc 4560 atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt cacggtgctg 4620 cagtactcct acatggtgac tgtggagtac cccttcagcg aggcacagtc caaaggggac 4680 atcctgcagc gggtgcgaga gatccgctac cagggcggca acaggaccaa cactgggctg 4740 gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg ggagcaggcg 4800 cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa gaggctgcct 4860 ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca ggagctggag 4920 aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct cccccgagag 4980 gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat ccccaccctc 5040 tcccctgcac ctcgtgatga gacgctccag gatggctgtg atactcactt ctgcaaggtc 5100 aatgagagag gagagtactt ctgggagaag agggtcacag gctgcccacc ctttgatgaa 5160 cacaagtgtc tggctgaggg aggtaaaatt atgaaaattc caggcacctg ctgtgacaca 5220 tgtgaggagc ctgagtgcaa cgacatcact gccaggctgc agtatgtcaa ggtgggaagc 5280 tgtaagtctg aagtagaggt ggatatccac tactgccagg gcaaatgtgc cagcaaagcc 5340 atgtactcca ttgacatcaa cgatgtgcag gaccagtgct cctgctgctc tccgacacgg 5400 acggagccca tgcaggtggc cctgcactgc accaatggct ctgttgtgta ccatgaggtt 5460 ctcaatgcca tggagtgcaa atgctccccc aggaagtgca gcaagtga 5508 <210> 4 <211> 1833 <212> PRT <213> homo sapiens <400> 4 Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile 1 5 10 15 Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30 Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 35 40 45 Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60 Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys 65 70 75 80 Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu 85 90 95 Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110 Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125 Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140 Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly 145 150 155 160 Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln 165 170 175 Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190 Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser 195 200 205 Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln 210 215 220 Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu 225 230 235 240 Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255 Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270 Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275 280 285 Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys 290 295 300 Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met 305 310 315 320 Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu 325 330 335 Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His 340 345 350 Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365 Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380 Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp 385 390 395 400 Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg 405 410 415 Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys 420 425 430 Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu 435 440 445 Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val 450 455 460 Ala Met Asp Gly Gln Asp Val Gln Leu Pro Leu Leu Lys Gly Asp Leu 465 470 475 480 Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495 Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510 Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525 Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540 Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln 545 550 555 560 Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575 Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590 Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605 Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620 Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val 625 630 635 640 Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645 650 655 Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670 Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685 Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys 690 695 700 Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp 705 710 715 720 Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735 His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750 Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765 Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780 Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met 785 790 795 800 Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815 His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820 825 830 Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr 835 840 845 Cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr His Val Cys Asp Ala 850 855 860 Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly Leu 865 870 875 880 Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp Tyr 885 890 895 Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys Gly 900 905 910 Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu Val 915 920 925 Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys Arg 930 935 940 Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg Tyr 945 950 955 960 Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg His 965 970 975 Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val Cys 980 985 990 Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr Ser 995 1000 1005 Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn Ser Trp 1010 1015 1020 Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro Leu Asp Ser 1025 1030 1035 1040 Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln Thr Met Val Asp 1045 1050 1055 Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe Gln Asp Cys Asn Lys 1060 1065 1070 Leu Val Asp Pro Glu Pro Tyr Leu Asp Val Cys Ile Tyr Asp Thr Cys 1075 1080 1085 Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys Phe Cys Asp Thr Ile Ala 1090 1095 1100 Ala Tyr Ala His Val Cys Ala Gln His Gly Lys Val Val Thr Trp Arg 1105 1110 1115 1120 Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg Asn Leu Arg Glu 1125 1130 1135 Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys 1140 1145 1150 Gln Val Thr Cys Gln His Pro Glu Pro Leu Ala Cys Pro Val Gln Cys 1155 1160 1165 Val Glu Gly Cys His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu 1170 1175 1180 Leu Leu Gln Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu Val 1185 1190 1195 1200 Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser 1205 1210 1215 Asp Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu Thr 1220 1225 1230 Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr Asp 1235 1240 1245 Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser Glu Pro 1250 1255 1260 Pro Leu His Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe Leu 1265 1270 1275 1280 Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu Lys 1285 1290 1295 Ala Phe Val Val Asp Met Met Glu Arg Leu Arg Ile Ser Gln Lys Trp 1300 1305 1310 Val Arg Val Ala Val Val Glu Tyr His Asp Gly Ser His Ala Tyr Ile 1315 1320 1325 Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala Ser 1330 1335 1340 Gln Val Lys Tyr Ala Gly Ser Gln Val Ala Ser Thr Ser Glu Val Leu 1345 1350 1355 1360 Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp Arg Pro Glu Ala 1365 1370 1375 Ser Arg Ile Thr Leu Leu Leu Met Ala Ser Gln Glu Pro Gln Arg Met 1380 1385 1390 Ser Arg Asn Phe Val Arg Tyr Gln Gly Leu Lys Lys Lys Lys Val Ile 1395 1400 1405 Val Ile Pro Val Gly Ile Gly Pro His Ala Asn Leu Lys Gln Ile Arg 1410 1415 1420 Leu Ile Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser 1425 1430 1435 1440 Val Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile Val Ser Tyr Leu Cys 1445 1450 1455 Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp Met Ala 1460 1465 1470 Gln Val Thr Val Gly Pro Gly Leu Leu Gly Val Ser Thr Leu Gly Pro 1475 1480 1485 Lys Arg Asn Ser Met Val Leu Asp Val Ala Phe Val Leu Glu Gly Ser 1490 1495 1500 Asp Lys Ile Gly Glu Ala Asp Phe Asn Arg Ser Lys Glu Phe Met Glu 1505 1510 1515 1520 Glu Val Ile Gln Arg Met Asp Val Gly Gln Asp Ser Ile His Val Thr 1525 1530 1535 Val Leu Gln Tyr Ser Tyr Met Val Thr Val Glu Tyr Pro Phe Ser Glu 1540 1545 1550 Ala Gln Ser Lys Gly Asp Ile Leu Gln Arg Val Arg Glu Ile Arg Tyr 1555 1560 1565 Gln Gly Gly Asn Arg Thr Asn Thr Gly Leu Ala Leu Arg Tyr Leu Ser 1570 1575 1580 Asp His Ser Phe Leu Val Ser Gln Gly Asp Arg Glu Gln Ala Pro Asn 1585 1590 1595 1600 Leu Val Tyr Met Val Thr Gly Asn Pro Ala Ser Asp Glu Ile Lys Arg 1605 1610 1615 Leu Pro Gly Asp Ile Gln Val Val Pro Ile Gly Val Gly Pro Asn Ala 1620 1625 1630 Asn Val Gln Glu Leu Glu Arg Ile Gly Trp Pro Asn Ala Pro Ile Leu 1635 1640 1645 Ile Gln Asp Phe Glu Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu 1650 1655 1660 Gln Arg Cys Cys Ser Gly Glu Gly Leu Gln Ile Pro Thr Leu Ser Pro 1665 1670 1675 1680 Ala Pro Arg Asp Glu Thr Leu Gln Asp Gly Cys Asp Thr His Phe Cys 1685 1690 1695 Lys Val Asn Glu Arg Gly Glu Tyr Phe Trp Glu Lys Arg Val Thr Gly 1700 1705 1710 Cys Pro Pro Phe Asp Glu His Lys Cys Leu Ala Glu Gly Gly Lys Ile 1715 1720 1725 Met Lys Ile Pro Gly Thr Cys Cys Asp Thr Cys Glu Glu Pro Glu Cys 1730 1735 1740 Asn Asp Ile Thr Ala Arg Leu Gln Tyr Val Lys Val Gly Ser Cys Lys 1745 1750 1755 1760 Ser Glu Val Glu Val Asp Ile His Tyr Cys Gln Gly Lys Cys Ala Ser 1765 1770 1775 Lys Ala Met Tyr Ser Ile Asp Ile Asn Asp Val Gln Asp Gln Cys Ser 1780 1785 1790 Cys Cys Ser Pro Thr Arg Thr Glu Pro Met Gln Val Ala Leu His Cys 1795 1800 1805 Thr Asn Gly Ser Val Val Tyr His Glu Val Leu Asn Ala Met Glu Cys 1810 1815 1820 Lys Cys Ser Pro Arg Lys Cys Ser Lys 1825 1830 <210> 5 <211> 6075 <212> DNA <213> homo sapiens <400> 5 atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt gccagggacc 60 ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct tttcggaagt 120 gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg cagttacctc 180 ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca gaatggcaag 240 agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt tgtcaatggt 300 accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg gctgtatcta 360 gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt ggccaggatc 420 gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa gacctgcggg 480 ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga agggaccttg 540 acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga acagtggtgt 600 gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat gcagaagggc 660 ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg ccaccctctg 720 gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg tgctgggggg 780 ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca ggagggaatg 840 gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc tggtatggag 900 tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat caatgaaatg 960 tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct ggatgaaggc 1020 ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta ccctcccggc 1080 acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg gatctgcagc 1140 aatgaagaat gtccagggga gtgccttgtc acaggtcaat cacacttcaa gagctttgac 1200 aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga ttgccaggac 1260 cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga cgctgtgtgc 1320 acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa actgaagcat 1380 ggggcaggag ttgccatgga tggccaggac gtccagctcc ccctcctgaa aggtgacctc 1440 cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga cctgcagatg 1500 gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctatgc cgggaagacc 1560 tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac cccctctggg 1620 ctggcggagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg ggactgccag 1680 gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac caggttctcc 1740 gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg tgccgtcagc 1800 ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga cggccgcgag 1860 tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg cgtgcgcgtc 1920 gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt gtacctgcag 1980 tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga ggaatgcaat 2040 gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga gaggggggac 2100 tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca gccagaagac 2160 atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca ctgtaccatg 2220 agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct gtctcatcgc 2280 agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc cgctgacaac 2340 ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct ggagtgcatg 2400 agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca tgagaacaga 2460 tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc ccctggagaa 2520 acagtgaaga ttggctgcaa cacttgtgtc tgtcaggacc ggaagtggaa ctgcacagac 2580 catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac cttcgacggg 2640 ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta ctgcggcagt 2700 aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc ctcagtgaaa 2760 tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt tgacggggag 2820 gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga gtctggccgg 2880 tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca cctgagcatc 2940 tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg gaattttgat 3000 ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga ccctgtggac 3060 tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt gcctctggac 3120 tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga ttcctcctgt 3180 agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc cgagccatat 3240 ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg cgcctgcttc 3300 tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt ggtgacctgg 3360 aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga gaacgggtat 3420 gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg tcagcaccct 3480 gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg ccctccaggg 3540 aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc agtgtgtgag 3600 gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag tgaccctgag 3660 cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg ccaggagccg 3720 ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct gtatgtggag 3780 gacatctcgg aaccgccgtt gcacgatttc tactgcagca ggctactgga cctggtcttc 3840 ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa ggcctttgtg 3900 gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc cgtggtggag 3960 taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc gtcagagctg 4020 cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac cagcgaggtc 4080 ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc ctcccgcatc 4140 accctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt tgtccgctac 4200 gtccagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg gccccatgcc 4260 aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc cttcgtgctg 4320 agcagtgtgg atgagctgga gcagcaaagg gacgagatcg ttagctacct ctgtgacctt 4380 gcccctgaag cccctcctcc tactctgccc cccgacatgg cacaagtcac tgtgggcccg 4440 gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct ggatgtggcg 4500 ttcgtcctgg aaggatcgga caaaattggt gaagccgact tcaacaggag caaggagttc 4560 atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt cacggtgctg 4620 cagtactcct acatggtgac tgtggagtac cccttcagcg aggcacagtc caaaggggac 4680 atcctgcagc gggtgcgaga gatccgctac cagggcggca acaggaccaa cactgggctg 4740 gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg ggagcaggcg 4800 cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa gaggctgcct 4860 ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca ggagctggag 4920 aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct cccccgagag 4980 gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat ccccaccctc 5040 tcccctgcac ctgactgcag ccagcccctg gacgtgatcc ttctcctgga tggctcctcc 5100 agtttcccag cttcttattt tgatgaaatg aagagtttcg ccaaggcttt catttcaaaa 5160 gccaatatag ggcctcgtct cactcaggtg tcagtgctgc agtatggaag catcaccacc 5220 attgacgtgc catggaacgt ggtcccggag aaagcccatt tgctgagcct tgtggacgtc 5280 atgcagcggg agggaggccc cagccaaatc ggggatgcct tgggctttgc tgtgcgatac 5340 ttgacttcag aaatgcatgg tgccaggccg ggagcctcaa aggcggtggt catcctggtc 5400 acggacgtct ctgtggattc agtggatgca gcagctgatg ccgccaggtc caacagagtg 5460 acagtgttcc ctattggaat tggagatcgc tacgatgcag cccagctacg gatcttggca 5520 ggcccagcag gcgactccaa cgtggtgaag ctccagcgaa tcgaagacct ccctaccatg 5580 gtcaccttgg gcaattcctt cctccacaaa ctgtgctctc gtgatgagac gctccaggat 5640 ggctgtgata ctcacttctg caaggtcaat gagagaggag agtacttctg ggagaagagg 5700 gtcacaggct gcccaccctt tgatgaacac aagtgtctgg ctgagggagg taaaattatg 5760 aaaattccag gcacctgctg tgacacatgt gaggagcctg agtgcaacga catcactgcc 5820 aggctgcagt atgtcaaggt gggaagctgt aagtctgaag tagaggtgga tatccactac 5880 tgccagggca aatgtgccag caaagccatg tactccattg acatcaacga tgtgcaggac 5940 cagtgctcct gctgctctcc gacacggacg gagcccatgc aggtggccct gcactgcacc 6000 aatggctctg ttgtgtacca tgaggttctc aatgccatgg agtgcaaatg ctcccccagg 6060 aagtgcagca agtga 6075 <210> 6 <211> 2023 <212> PRT <213> homo sapiens <400> 6 Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile 1 5 10 15 Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30 Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly 35 40 45 Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60 Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys 65 70 75 80 Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu 85 90 95 Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro 100 105 110 Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125 Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly 130 135 140 Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly 145 150 155 160 Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln 165 170 175 Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190 Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser 195 200 205 Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln 210 215 220 Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu 225 230 235 240 Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255 Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270 Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His 275 280 285 Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys 290 295 300 Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met 305 310 315 320 Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu 325 330 335 Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His 340 345 350 Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365 Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys 370 375 380 Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp 385 390 395 400 Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg 405 410 415 Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys 420 425 430 Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu 435 440 445 Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val 450 455 460 Ala Met Asp Gly Gln Asp Val Gln Leu Pro Leu Leu Lys Gly Asp Leu 465 470 475 480 Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495 Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu 500 505 510 Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525 Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540 Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln 545 550 555 560 Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575 Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590 Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605 Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly 610 615 620 Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val 625 630 635 640 Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln 645 650 655 Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu 660 665 670 Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685 Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys 690 695 700 Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp 705 710 715 720 Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735 His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750 Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg Ser Leu Ser Cys Arg 755 760 765 Pro Pro Met Val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780 Gly Leu Glu Cys Thr Lys Thr Cys Gln Asn Tyr Asp Leu Glu Cys Met 785 790 795 800 Ser Met Gly Cys Val Ser Gly Cys Leu Cys Pro Pro Gly Met Val Arg 805 810 815 His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gln 820 825 830 Gly Lys Glu Tyr Ala Pro Gly Glu Thr Val Lys Ile Gly Cys Asn Thr 835 840 845 Cys Val Cys Gln Asp Arg Lys Trp Asn Cys Thr Asp His Val Cys Asp 850 855 860 Ala Thr Cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly 865 870 875 880 Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gln Tyr Val Leu Val Gln Asp 885 890 895 Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg Ile Leu Val Gly Asn Lys 900 905 910 Gly Cys Ser His Pro Ser Val Lys Cys Lys Lys Arg Val Thr Ile Leu 915 920 925 Val Glu Gly Gly Glu Ile Glu Leu Phe Asp Gly Glu Val Asn Val Lys 930 935 940 Arg Pro Met Lys Asp Glu Thr His Phe Glu Val Val Glu Ser Gly Arg 945 950 955 960 Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975 His Leu Ser Ile Ser Val Val Leu Lys Gln Thr Tyr Gln Glu Lys Val 980 985 990 Cys Gly Leu Cys Gly Asn Phe Asp Gly Ile Gln Asn Asn Asp Leu Thr 995 1000 1005 Ser Ser Asn Leu Gln Val Glu Glu Asp Pro Val Asp Phe Gly Asn Ser 1010 1015 1020 Trp Lys Val Ser Ser Gln Cys Ala Asp Thr Arg Lys Val Pro Leu Asp 1025 1030 1035 1040 Ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gln Thr Met Val 1045 1050 1055 Asp Ser Ser Cys Arg Ile Leu Thr Ser Asp Val Phe Gln Asp Cys Asn 1060 1065 1070 Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val Cys Ile Tyr Asp Thr 1075 1080 1085 Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala Cys Phe Cys Asp Thr Ile 1090 1095 1100 Ala Ala Tyr Ala His Val Cys Ala Gln His Gly Lys Val Val Thr Trp 1105 1110 1115 1120 Arg Thr Ala Thr Leu Cys Pro Gln Ser Cys Glu Glu Arg Asn Leu Arg 1125 1130 1135 Glu Asn Gly Tyr Glu Cys Glu Trp Arg Tyr Asn Ser Cys Ala Pro Ala 1140 1145 1150 Cys Gln Val Thr Cys Gln His Pro Glu Pro Leu Ala Cys Pro Val Gln 1155 1160 1165 Cys Val Glu Gly Cys His Ala His Cys Pro Pro Gly Lys Ile Leu Asp 1170 1175 1180 Glu Leu Leu Gln Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu 1185 1190 1195 1200 Val Ala Gly Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro 1205 1210 1215 Ser Asp Pro Glu His Cys Gln Ile Cys His Cys Asp Val Val Asn Leu 1220 1225 1230 Thr Cys Glu Ala Cys Gln Glu Pro Gly Gly Leu Val Val Pro Pro Thr 1235 1240 1245 Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr Val Glu Asp Ile Ser Glu 1250 1255 1260 Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe 1265 1270 1275 1280 Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu 1285 1290 1295 Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg Ile Ser Gln Lys 1300 1305 1310 Trp Val Arg Val Ala Val Val Glu Tyr His Asp Gly Ser His Ala Tyr 1315 1320 1325 Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala 1330 1335 1340 Ser Gln Val Lys Tyr Ala Gly Ser Gln Val Ala Ser Thr Ser Glu Val 1345 1350 1355 1360 Leu Lys Tyr Thr Leu Phe Gln Ile Phe Ser Lys Ile Asp Arg Pro Glu 1365 1370 1375 Ala Ser Arg Ile Thr Leu Leu Leu Met Ala Ser Gln Glu Pro Gln Arg 1380 1385 1390 Met Ser Arg Asn Phe Val Arg Tyr Val Gln Gly Leu Lys Lys Lys Lys 1395 1400 1405 Val Ile Val Ile Pro Val Gly Ile Gly Pro His Ala Asn Leu Lys Gln 1410 1415 1420 Ile Arg Leu Ile Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu 1425 1430 1435 1440 Ser Ser Val Asp Glu Leu Glu Gln Gln Arg Asp Glu Ile Val Ser Tyr 1445 1450 1455 Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp 1460 1465 1470 Met Ala Gln Val Thr Val Gly Pro Gly Leu Leu Gly Val Ser Thr Leu 1475 1480 1485 Gly Pro Lys Arg Asn Ser Met Val Leu Asp Val Ala Phe Val Leu Glu 1490 1495 1500 Gly Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn Arg Ser Lys Glu Phe 1505 1510 1515 1520 Met Glu Glu Val Ile Gln Arg Met Asp Val Gly Gln Asp Ser Ile His 1525 1530 1535 Val Thr Val Leu Gln Tyr Ser Tyr Met Val Thr Val Glu Tyr Pro Phe 1540 1545 1550 Ser Glu Ala Gln Ser Lys Gly Asp Ile Leu Gln Arg Val Arg Glu Ile 1555 1560 1565 Arg Tyr Gln Gly Gly Asn Arg Thr Asn Thr Gly Leu Ala Leu Arg Tyr 1570 1575 1580 Leu Ser Asp His Ser Phe Leu Val Ser Gln Gly Asp Arg Glu Gln Ala 1585 1590 1595 1600 Pro Asn Leu Val Tyr Met Val Thr Gly Asn Pro Ala Ser Asp Glu Ile 1605 1610 1615 Lys Arg Leu Pro Gly Asp Ile Gln Val Val Pro Ile Gly Val Gly Pro 1620 1625 1630 Asn Ala Asn Val Gln Glu Leu Glu Arg Ile Gly Trp Pro Asn Ala Pro 1635 1640 1645 Ile Leu Ile Gln Asp Phe Glu Thr Leu Pro Arg Glu Ala Pro Asp Leu 1650 1655 1660 Val Leu Gln Arg Cys Cys Ser Gly Glu Gly Leu Gln Ile Pro Thr Leu 1665 1670 1675 1680 Ser Pro Ala Pro Asp Cys Ser Gln Pro Leu Asp Val Ile Leu Leu Leu 1685 1690 1695 Asp Gly Ser Ser Ser Phe Pro Ala Ser Tyr Phe Asp Glu Met Lys Ser 1700 1705 1710 Phe Ala Lys Ala Phe Ile Ser Lys Ala Asn Ile Gly Pro Arg Leu Thr 1715 1720 1725 Gln Val Ser Val Leu Gln Tyr Gly Ser Ile Thr Thr Ile Asp Val Pro 1730 1735 1740 Trp Asn Val Val Pro Glu Lys Ala His Leu Leu Ser Leu Val Asp Val 1745 1750 1755 1760 Gln Arg Glu Gly Gly Pro Ser Gln Ile Gly Asp Ala Leu Gly Phe Ala 1765 1770 1775 Val Arg Tyr Leu Thr Ser Glu Met His Gly Ala Arg Pro Gly Ala Ser 1780 1785 1790 Lys Ala Val Val Ile Leu Val Thr Asp Val Ser Val Asp Ser Val Asp 1795 1800 1805 Ala Ala Ala Asp Ala Ala Arg Ser Asn Arg Val Thr Val Phe Pro Ile 1810 1815 1820 Gly Ile Gly Asp Arg Tyr Asp Ala Ala Gln Leu Arg Ile Leu Ala Gly 1825 1830 1835 1840 Pro Ala Gly Asp Ser Asn Val Val Lys Leu Gln Arg Ile Glu Asp Leu 1845 1850 1855 Pro Thr Met Val Thr Leu Gly Asn Ser Phe Leu His Lys Leu Cys Ser 1860 1865 1870 Arg Asp Glu Thr Leu Gln Asp Gly Cys Asp Thr His Phe Cys Lys Val 1875 1880 1885 Asn Glu Arg Gly Glu Tyr Phe Trp Glu Lys Arg Val Thr Gly Cys Pro 1890 1895 1900 Pro Phe Asp Glu His Lys Cys Leu Ala Glu Gly Gly Lys Ile Met Lys 1905 1910 1915 1920 Ile Pro Gly Thr Cys Cys Asp Thr Cys Glu Glu Pro Glu Cys Asn Asp 1925 1930 1935 Ile Thr Ala Arg Leu Gln Tyr Val Lys Val Gly Ser Cys Lys Ser Glu 1940 1945 1950 Val Glu Val Asp Ile His Tyr Cys Gln Gly Lys Cys Ala Ser Lys Ala 1955 1960 1965 Met Tyr Ser Ile Asp Ile Asn Asp Val Gln Asp Gln Cys Ser Cys Cys 1970 1975 1980 Ser Pro Thr Arg Thr Glu Pro Met Gln Val Ala Leu His Cys Thr Asn 1985 1990 1995 2000 Gly Ser Val Val Tyr His Glu Val Leu Asn Ala Met Glu Cys Lys Cys 2005 2010 2015 Ser Pro Arg Lys Cys Ser Lys 2020 <210> 7 <211> 4329 <212> DNA <213> homo sapiens <400> 7 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 accaagaata 2280 actcgtacta ctcttcagtc agatcaagag gaaattgact atgatgatac catatcagtt 2340 gaaatgaaga aggaagattt tgacatttat gatgaggatg aaaatcagag cccccgcagc 2400 tttcaaaaga aaacacgaca ctattttatt gctgcagtgg agaggctctg ggattatggg 2460 atgagtagct ccccacatgt tctaagaaac agggctcaga gtggcagtgt ccctcagttc 2520 aagaaagttg ttttccagga atttactgat ggctccttta ctcagccctt ataccgtgga 2580 gaactaaatg aacatttggg actcctgggg ccatatataa gagcagaagt tgaagataat 2640 atcatggtaa ctttcagaaa tcaggcctct cgtccctatt ccttctattc tagccttatt 2700 tcttatgagg aagatcagag gcaaggagca gaacctagaa aaaactttgt caagcctaat 2760 gaaaccaaaa cttacttttg gaaagtgcaa catcatatgg cacccactaa agatgagttt 2820 gactgcaaag cctgggctta tttctctgat gttgacctgg aaaaagatgt gcactcaggc 2880 ctgattggac cccttctggt ctgccacact aacacactga accctgctca tgggagacaa 2940 gtgacagtac aggaatttgc tctgtttttc accatctttg atgagaccaa aagctggtac 3000 ttcactgaaa atatggaaag aaactgcagg gctccctgca atatccagat ggaagatccc 3060 acttttaaag agaattatcg cttccatgca atcaatggct acataatgga tacactacct 3120 ggcttagtaa tggctcagga tcaaaggatt cgatggtatc tgctcagcat gggcagcaat 3180 gaaaacatcc attctattca tttcagtgga catgtgttca ctgtacgaaa aaaagaggag 3240 tataaaatgg cactgtacaa tctctatcca ggtgtttttg agacagtgga aatgttacca 3300 tccaaagctg gaatttggcg ggtggaatgc cttattggcg agcatctaca tgctgggatg 3360 agcacacttt ttctggtgta cagcaataag tgtcagactc ccctgggaat ggcttctgga 3420 cacattagag attttcagat tacagcttca ggacaatatg gacagtgggc cccaaagctg 3480 gccagacttc attattccgg atcaatcaat gcctggagca ccaaggagcc cttttcttgg 3540 atcaaggtgg atctgttggc accaatgatt attcacggca tcaagaccca gggtgcccgt 3600 cagaagttct ccagcctcta catctctcag tttatcatca tgtatagtct tgatgggaag 3660 aagtggcaga cttatcgagg aaattccact ggaaccttaa tggtcttctt tggcaatgtg 3720 gattcatctg ggataaaaca caatattttt aaccctccaa ttattgctcg atacatccgt 3780 ttgcacccaa ctcattatag cattcgcagc actcttcgca tggagtggat gggctgtgat 3840 ttaaatagtt gcagcatgcc attgggaatg gagagtaaag caatatcaga tgcacagatt 3900 actgcttcat cctactttac caatatgttt gccacctggt ctccttcaaa agctcgactt 3960 cacctccaag ggaggagtaa tgcctggaga cctcaggtga ataatccaaa agagtggctg 4020 caagtggact tccagaagac aatgaaagtc acaggagtaa ctactcaggg agtaaaatct 4080 ctgcttacca gcatgtatgt gaaggagttc ctcatctcca gcagtcaaga tggccatcag 4140 tggactctct tttttcagaa tggcaaagta aaggtttttc agggaaatca agactccttc 4200 acacctgtgg tgaactctct agacccaccg ttactgactc gctaccttcg aattcacccc 4260 cagagttggg tgcaccagat tgccctgagg atggaggttc tgggctgcga ggcacaggac 4320 ctctactga 4329 <210> 8 <211> 1442 <212> PRT <213> homo sapiens <400> 8 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 Ile Thr Arg Thr Thr Leu Gln Ser Asp 755 760 765 Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys 770 775 780 Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser 785 790 795 800 Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu 805 810 815 Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala 820 825 830 Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe 835 840 845 Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu 850 855 860 His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn 865 870 875 880 Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr 885 890 895 Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro 900 905 910 Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 915 920 925 Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala 930 935 940 Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly 945 950 955 960 Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala 965 970 975 His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile 980 985 990 Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn 995 1000 1005 Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys Glu 1010 1015 1020 Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro 1025 1030 1035 1040 Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser 1045 1050 1055 Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val 1060 1065 1070 Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu 1075 1080 1085 Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly 1090 1095 1100 Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met 1105 1110 1115 1120 Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly 1125 1130 1135 Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln 1140 1145 1150 Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser 1155 1160 1165 Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp 1170 1175 1180 Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg 1185 1190 1195 1200 Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser 1205 1210 1215 Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr 1220 1225 1230 Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn 1235 1240 1245 Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr 1250 1255 1260 His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Trp Met Gly Cys Asp 1265 1270 1275 1280 Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser 1285 1290 1295 Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr 1300 1305 1310 Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala 1315 1320 1325 Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe 1330 1335 1340 Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser 1345 1350 1355 1360 Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln 1365 1370 1375 Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val 1380 1385 1390 Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp 1395 1400 1405 Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val 1410 1415 1420 His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp 1425 1430 1435 1440 Leu Tyr

Claims (13)

Mutant von Willibrand factor (vWF28) having the amino acid sequence of SEQ ID NO: 4, which has deleted exons 29-46 in von Willebrand factor. A mutant von Willibrand factor (vWF28) gene having a nucleotide sequence encoding a protein having an amino acid sequence of SEQ ID NO: 4. The mutant von Willibrand factor (vWF28) gene according to claim 2, wherein the gene has a nucleotide sequence of SEQ ID NO. An animal cell expression vector comprising a gene encoding the mutant von Willibrand factor of claim 1. 5. The animal cell expression vector of claim 4, wherein the animal cell expression vector is a lentiviral vector. 5. The animal cell expression vector according to claim 4, further comprising a gene encoding human blood coagulation factor (Factor VIII) deleted from the B domain. 7. The animal cell expression vector of claim 6, wherein the human coagulation factor 8 having the B domain deleted has an amino acid sequence represented by SEQ ID NO: 8. The animal cell expression vector according to claim 6, which is a pvBDD.FVIII.ires.vWex32 lentivirus vector having a cleavage map of FIG. A lentiviral particle packaged by transfecting the lentiviral vector of claim 5 or 8 into a packaging cell. 10. The lentivirus particle of claim 9, wherein said packaging cell is a 293T cell. The lentiviral particle of claim 9, wherein the lentiviral vector is co-fected with pGag-pol, pRev, pTat, and pVSV-G. A pharmaceutical composition for treating and preventing hemophilia comprising the animal cell expression vector of claim 4 or 6 or a mutant von Willibrand factor expressed therefrom and a human blood coagulation factor 8 deleted from the B domain as an active ingredient. A pharmaceutical composition for treating and preventing hemophilia, comprising the lentiviral particles according to claim 9 as an active ingredient.

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