JPH11318467A - Shortened type dystrophin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new gene which has rod repeat construct(s) having HindI, HindIV, and rod domain of dystrophin gene, has specific length, and is useful, for example, as a medicine for gene therapy against myotrophia dystonica. SOLUTION: This gene has at least one rod repeat construct(s) having HindI, HindIV, and rod domain of dystrophin gene, has a base sequence with a length of 4.5 kb or shorter, or a base sequence hybridizable to the sequence, and can be used, for example, as a medicine for gene therapy against myotrophia dystonica. Using this gene and a medium for introducing the gene for gene therapy against myotrophia dystonica allows gene therapy against myotrophia dystonica with causing less immunological reaction. This gene is obtained by constructing a rod-shortened type dystrophin cDNA which overcame the length limitation (4.6 kb) for gene introduction into skeletal muscle by further shortening rod domain using human mini-dystrophin gene (6.3 kb).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a nucleotide sequence having at least one hinge 1, hinge 4 and rod domain of a dystrophin gene and having a length of 4.5 kb or less, or a nucleotide sequence thereof. A gene for treating muscular dystrophy having a base sequence capable of hybridizing to the above, a therapeutic agent for muscular dystrophy comprising these genes, a gene transfer medium for gene therapy for muscular dystrophy comprising an adeno-associated virus (AAV) vector or a lentivirus vector, Adeno-associated virus (AAV) vector, lentivirus vector or adenovirus vector comprising the following genes: and a therapeutic agent for muscular dystrophy comprising these vectors.

[0002]

2. Description of the Related Art A severe hereditary muscular disease having an X-linked recessive inheritance pattern and a high incidence of the disease (one out of every 3,500 birth boys) has a Duchenne type (Du).
channel type) muscular dystrophy (DMD) (Emery, "Duchenne Muscular Dystrophy", 2nd edition, University of Oxford [Emery, AEH (1993) Duchenne Muscul
ar Dystrophy, 2nd ed., Oxford University Press, O
xford.]), positional cloning (pos
As a result of itional cloning, the dystrophin gene (14 kb), which is the causative gene, was isolated (Koenig et al., Cell, 50, 509-517 (1987)).
[Koenig, M., Hoffman, EP, Bertelson, CJ, Monac
o, AP, Feener, C. and Kunkel, LM, (1987) Cel
1, 50, 509-517]), and studies on the relationship between genetic abnormalities and pathological conditions, including the involvement of dystrophin-binding proteins, are underway.

However, dystrophin, which is deficient in skeletal muscle of DMD patients, is a membrane-associated cytoskeletal protein (Zubrzicker-Gallen et al., Nature 333).
Volume, pp. 466-469 (1988) [Zubrzycka-Gaar
n, EE, Bulman, DE, Karpati, G., Burghes, AH
M., Belfall, B., Klamut, HJ, Talbot, J., Hodges,
RS, Ray, PN and Worton, RG (1988) Nature 33
3, 466-469.] And Arahata et al., Nature, 33
3, 861-863 (1988) [Arahata, K.,
Ishiura, S., Ishiguro, T., Tsukahara, T., Suhara,
Y., Eguchi, C., Ishihara T., Nonaka, I., Ozawa, E.
and Sugita H. (1988) Nature 333, 861-863.]), it is difficult to anticipate drug treatment, and one-third of those who develop prenatal diagnosis because of mutations at the maternal egg cell level. Not necessarily valid.

Accordingly, gene therapy is being considered. In order to establish gene therapy for muscular dystrophy, a method with high efficiency and a wide safety margin for skeletal muscle is desired. Until now, studies using highly infectious adenovirus vectors have been actively conducted (Lagot et al., Nature, 361, 647-650 (1993)).
[Ragot, T., Vincent, N., Chafey, P., Vigne, E., G
ilgenkrantz, H., Couton, D., Cartaud, J., Briand,
P., Kaplan, J.-C., Perricaudet, M. and Kahn, A. (19
93) Nature 361, 647-650.], Banson et al., Nature Genetics, 5, 130-134 (199).
3 years) [Vincent, N., Ragot, T., Gilgenkrantz, H.,
Couton, D., Chafey, P., Gregoire, A., Briand, P.,
Kaplan, J.-C., Kahn, A. and Perricaudet, M. (1993)
Nature Genet. 5, 130-134.], Dekonik et al., Proceedings of National Academy of Sciences USA, 93, 3570-3574 (1
996) [Deconinck, N., Ragot, T., Marfichal,
G., Perricaudet, M. and Gillis, JM (1996) Proc.
Natl. Acad. Sci. USA 93, 3570-3574.] And Axady et al., Gene Therapy, Vol. 7, pp. 129-140 (1996) [Acsadi, G., Lochmiiller, H., Jani,
A., Huard, l., Massie, B., Prescott, S., Simonea
u, M., Petrof, BJ and Karpati, G. (1996) Hum.Ge
ne Ther. 7, 129-140.]).

[0005] However, the first generation adenovirus vector is limited to 7.5 kb in length of the gene that can be introduced.
The transgene is not integrated into the chromosome, but has the problem that the antigenicity of the vector is high (Axady et al., Gene Therapy, Vol. 7, pp. 129-140 (1996).
Acsadi, G., Lochmiiller, H., Jani, A., Huar
d, l., Massie, B., Prescott, S., Simoneau, M., Pet
rof, BJ and Karpati, G. (1996) Hum. Gene Ther. 7,
129-140.]).

The dystrophin molecule can be structurally divided into four regions from the N-terminus: an actin-binding domain, a rod domain, a cysteine-rich domain, and a C-terminal domain (Koenig et al., Cell, 53).
Volume, pp. 219-228 (1988) [Koenig, M., Mo
naco, AP and Kunkel, LM (1988) Cell 53, 219-22
8.]).

[0007] Of these, three regions excluding the rod domain are domains necessary for linking the plasma membrane and the actin filament (Hemmings et al., Journal Cell Biology, 116, 1369-1380 (1).
992) [Hemmings, L., Kuhlman, PA and Critchl
ey, DR (1992) J. Cell Biol. 116, 1369-1380.],
And Suzuki et al., European Journal of Biochemistry, 220, 283-292 (19
1994) [Suzuki, A., Yoshida M., Hayashi, K., Mizu
no, Y., Hagiwara, Y. and Ozawa, E. (1994) Eur.
Biochem. 220,283-292.]).

[0008] The rod domain (consisting of 24 repeats and a hinge structure) represents 76% of the dystrophin molecule.
And high homology with spectrin, it has been expected to be related to the membrane lining structure. However, gene deletion in this region may cause clinically mild symptoms of Becker muscular dystrophy (BMD). (Beggs et al., Journal Human Genetics, 49
Vol. 54-67 (1991) [Beggs, AH, Hoffm
an, EP, Snyder, JR, Arahata, K., Specht, L., Sh
apiro, F., Angelini, C., Sugita, H. and Kunkel, L.
M. (1991) Am. J. Hum. Genet. 49, 54-67.]). Indeed, very mild BMD patients have been reported that have lost about 60% of the rod domain (England et al., Nature, 343, 180-182 (1990)).
[England, SB, Nicholson, LVB, Johnson, MA,
Forrest, SM, Love, DR, Zubrzycka-Gaarn, EE,
Bulman, DE, Harris, JB and Davies, KE (1990)
Nature 343, 180-182.]).

[0009] With the emergence of such patients, a 6.3 kb mini-dystrophin gene lacking 60% of the rod domain was cloned and introduced into a mdx mouse as a transgene, or a first-generation adenovirus. It has been shown that when incorporated into a viral vector and introduced into mdx mouse skeletal muscle, muscular dystrophy findings are improved (Lago et al., Nature, 361).
Volume, pages 647-650 (1993) [Ragot, T., Vin
cent, N., Chafey, P., Vigne, E., Gilgenkrantz, H.,
Couton, D., Cartau, J., Briand, P., Kaplan, J.-
C., Perricaudet, M. and Kahn, A. (1993) Nature 36
1, 647-650.], Vanson et al., Nature Genet, 5, 130-134 (1993) [Vincent,
N., Ragot, T., Gilgenkrantz, H., Couton, D., Chafe
y, P., Gregoire, A., Briand, P., Kaplan, J.-C., Ka
hn, A. and Perricaudet, M. (1993) Nature Genet. 5,
130-134.], Dekonik et al., Proceeding of National Academy of Sciences USA,
93, 3570-3574 (1996) [Deconi
nck, N., Ragot, T., Marfichal, G., Perricaudet, M.
and Gillis, JM (1996) Proc. Natl. Acad. Sci. USA
93, 3570-3574.], And Axadie et al., Therapy, 7, 129-140 (1996) [Acsadi,
G., Lochmiiller, H., Jani, A., Huard, l., Massie,
B., Prescott, S., Simoneau, M., Petrof, BJ and K
arpati, G. (1996) Hum. Gene Ther. 7, 129-14
0.]).

[0010] Studies have been conducted in two directions on the antigenicity of the vector possessed by the combination of the mini-dystrophin gene and the first-generation adenovirus vector and the restriction on the length of the gene that can be integrated. one,
A new generation of adenovirus vectors (gut-less adenovirus ve) from which all adenovirus protein genes have been removed
ctor). This method not only reduced the antigenicity of the vector, but also allowed recombination of long genes of 35 kb or less (Kochanek et al., Proceeding.
Of National Academy of Sciences US
A, 93, 5731-5736 (1996) [Ko
chanek, S., Clemens, PR, Mitani, K., Chen, H.-
H., Chan, S. and Caskey, CT (1996) Proc. Natl. Ac
ad. Sci. USA 93, 5731-5736.]). However, problems remain such that a helper virus necessary for the production of the vector is mixed into the final product, and at present, the lacZ gene is required as a marker for titer measurement.

[0011] Another direction is the development of new viral vectors with lower antigenic residence. Recently, adeno-associated virus (A) has been used as a vector capable of long-term stable gene transfer into skeletal muscle by integration into chromosome.
AV) vectors have been developed and have been shown to have low antigenicity (Fisher et al., Nature Medicine, 3, 306-312 (1997) [Fisher,
KJ, Jooss, K., Alston, J., Yang, Y., Haecker, S.
E., High, K., Pathak, R., Raper, SE and Wilson, J.
M. (1997) Nature Med. 3, 306-312.]). However, the problem with combining this vector with the dystrophin gene is that the transgene is limited to only 4.5 kb (Ferally et al., Nature).
Medicine, 3, 1295-1297 (1997)
Year) [Ferrari, FK, Xiao, X., McCarty, D. and Samu
lski, RJ (1997) Nature Med. 3, 1295-1297.]).

[0012]

An object of the present invention is to overcome these problems and to establish a method for introducing a dystrophin gene into skeletal muscle.

The present inventors constructed a truncated dystrophin gene in which the rod portion of the mini-dystrophin gene was further deleted in order to obtain a functional dystrophin gene of the minimum size applicable to other viral vectors. did. Next, the truncated dystrophin gene was inserted into an adenovirus vector to allow cultured skeletal muscle cells to mature
dystrophin-binding protein (D
It was examined whether the expression of (AP) recovered.

The present inventors have also introduced an adenovirus vector in which the lacZ gene has been recombined into cultured skeletal muscle cells and adult mouse skeletal muscle, and obtained a CAG promoter (Niwa et al., Gene, 108, 193-200). Page (1991) [Niwa, H., Yamamura, K. and Miyazak
i, J. (1991) Gene 108, 193-200.]), which produces the highest gene expression and, with the introduction of adenovirus, produces an immune response to adenovirus proteins and transgene products, It was revealed that the strain was different depending on the strain of the mouse. These results suggest that the first-generation adenovirus vector, which has many problems in direct application to gene therapy, is an excellent method for gene transfer into cultured cells and adult mouse skeletal muscle. It was used as an expression assay for the type dystrophin gene.

[0015]

SUMMARY OF THE INVENTION The present invention comprises at least one hinge 1, hinge 4 of the dystrophin gene and at least one rod repeat structure of the rod domain.
The present invention relates to a gene for treating muscular dystrophy having a base sequence having the following length or a base sequence capable of hybridizing to the base sequence. The gene of the present invention may have two or more rod domain rod repeat structures. Furthermore, the gene of the present invention is a cysteine-rich gene.
The present invention relates to a gene that may further have a domain, an actin-binding domain, and / or a C-terminal domain. In addition, the present invention relates to a therapeutic agent for muscular dystrophy comprising these genes.

The present invention also relates to the adeno-associated virus (A
AV) a gene transfer medium for muscular dystrophy gene therapy comprising a vector. That is, the present invention is characterized in that an adeno-associated virus (AAV) vector with low antigenicity is used as a gene transfer medium for gene therapy of muscular dystrophy. The present invention relates to a gene transfer medium for gene therapy of muscular dystrophy, wherein the adeno-associated virus (AAV) vector contains any of the above-mentioned genes of the present invention.

Further, the present invention relates to a vector containing any of the aforementioned genes of the present invention, preferably an adeno-associated virus (AAV) vector, an adenovirus vector or a lentivirus vector. The present invention also relates to a therapeutic agent for muscular dystrophy comprising the above-described vector.

Regarding gene transfer to skeletal muscle, AA
Although V vectors have several advantages, overcoming the problem of transgene length limitation (4.6 kb) requires having a small and functional dystrophin gene. The mini-dystrophin gene (6.3 kb) that has been used in the research so far has greatly exceeded the limit of introduction. Therefore, the construction of a minimal dystrophin gene that is long enough to be incorporated into an AAV vector and effective for treatment was assumed.

The full-length dystrophin gene encodes an actin-binding domain, a rod domain, a cysteine-rich domain, and a C-terminal domain from the N-terminus. Using the human mini dystrophin gene (6.3 kb) having eight rod repeats, the present inventors constructed six types of rod shortened dystrophin cDNAs in which the rod domain is further shortened (FIG. 1).
A). All constructs retain an N-terminal actin-binding domain, a cysteine-rich domain, and a C-terminal domain.

Designed ΔDysAX2, AX1
1, AH3 and M3 are respectively three, three, two,
It has one rod repeat and both hinge 1 and hinge 4. In these four truncated dystrophins, a putative triple-helical structure of rod repeats at the fusion site (Koenig et al., Journal of Biological Chemistry, 265, 4560-4566).
Page (1990) [Koenig, M. and Kunkel, LM (199
0) cDNA was designed to maintain J. Biol. Chem. 265, 4560-4566.]) (FIG. 1B). On the other hand, ΔD
ysH1 and H4 have no rod repeats,
Each has either hinges 1 or 4 (Fig. 1
A, FIG. 1C). The nucleotide sequences of primers and oligonucleotides used for construction of these cDNAs are as follows:
This is shown in Table 1 of Example 1 described later.

The N constructed by the present inventors including the hinge 1
A truncated dystrophin gene ΔDys having a C-terminus including the terminus and hinge 4 and having only one rod repeat
M3 was confirmed to have a function of improving the phenotype of muscular dystrophy by an experiment of introduction into skeletal muscle of neonatal mdx mouse. For dystrophin structurally smaller than ΔDysM3, that is, for the dystrophin gene lacking all of the rod domain but retaining hinge 1 and hinge 4, it is localized on the plasma membrane as dystrophin, but improves muscular dystrophy findings I can't. It is also known that the small dystrophin Dp71 from the latter half of the hinge 4 to the C-terminal rather worsens the findings of muscular dystrophy. Thus, to date, ΔDysM3 is considered to be the smallest dystrophin functional unit.

Next, the construction of these dystrophin genes will be described. That is, human mini dystrophin c
NotI / SalI of 6.3 kb DNA gene
The fragment was cloned into the plasmid pBluescriptII (SK
+) (Not available from Stratagene)
/ SalI site to create plasmid pBSBMD.

The resulting PCR fragment amplified with the plasmid pBSBMD and the primers F1 / R1 (see Table 1) or F2 / R2 (see Table 1) is cut with AflII / XhoI and then inserted into the AflII / XhoI site of pBSBMD. And pBSΔDysAX2 or pBSΔ, respectively.
DysAX11 was produced. Next, the template pBSBMD
And PCR amplified with primers F4 / R4 (see Table 1)
After cutting the product with MunI / HindIII, pBSB
Inserted into MunI / HindIII site of MD, pBSΔ
DysM3 was prepared. Subsequently, oligonucleotide F
The fragment prepared by the anne ring of 3 / R3 (see Table 1) was used to ligate the AflII / HindIII sites of pBSBMD to prepare pBSΔDysAH3. These inserts were designed to maintain the rod repeat triple helical structure when ligated. The amino acid sequence of the ligated rod repeat is shown in FIG.

As a result, ΔDysAX2, AX11, A
H3 and M3 retain an N-terminal actin binding domain, a cysteine rich domain and a C-terminal domain,
It also has both 3, 3, 2, 1 rod repeats and hinges 1 and 4 respectively. ΔDysH1 and ΔDy
The two plasmids with the cDNA for sH4 are pBSΔ
It was prepared from DysM3 (FIG. 1A). One EcoO
To remove the 109I site, pBSΔDysM3 was replaced with Ap
cut with aI, smoothed, self-ligated,
pBSΔDysM3b was prepared. PBSΔDy of template
The PCR product amplified using sM3 and primer F5 / R5 (see Table 1) was used for EcoT22I / EcoO109I.
After digestion with pBSΔDysM3b,
22I / EcoO109I site and insert pBSΔDy
sH1 was prepared.

For the production of pBSΔDysH4, pB
Two types of PCR reactions were separately performed using SΔDysM3 as a template and primers F5 / R6 (see Table 1) or F6 / R7 (see Table 1). Two types of PC obtained
Using the mixture of R products as a template, primer F5 / R7
A second PCR reaction was performed using (see Table 1). The resulting fragment was digested with EcoRV, and then digested with pBSΔ.
Inserted between two EcoRV sites in DysM3.
The amino acid sequence of the connecting region is shown in FIG. Δ obtained
DysH1 and H4 have no rod repeats and have either hinges 1 or 4, respectively (A in FIG. 1).

FIG. 1 shows the construction of truncated dystrophin genes with various numbers of rod repeats. FIG. 1A is a list of human full-length dystrophin gene, mini-dystrophin gene and newly prepared truncated dystrophin cDNA. ΔDysAX2,
To construct ΔDysAX, ΔDysAH3 and ΔDysM3, the central rod domain of the minidystrophin cDNA was cut with the restriction enzymes shown to the right of each construct. In order to reconstruct the rod repeat structure, both ends obtained using a PCR amplified fragment or a synthetic DNA fragment were ligated. ΔDysH1 and ΔDy
In order to construct sH4, ΔDysM3 was digested with the restriction enzymes shown, and then both ends were ligated using a PCR amplified fragment. The dotted line indicates the connection. The size of the cDNA and the estimated molecular weight of truncated dystrophin are shown on the right. The actin binding domain is a dotted box, the rod domain is an open box (each repeat is shown as one box), the cysteine rich domain is a hatched box, and the C-terminal domain is Illustrated with shaded boxes. Black boxes indicate hinges. The hinge is described in M. Koenig and L.A. M. Kunkel
According to the description.

FIG. 1B shows ΔDysAX2 (AX2),
ΔDysAX11 (AX11), ΔDysAH3 (AH
3) shows the amino acid sequences of reconstructed rod repeats in ΔDysM3 (M3). The vertical line indicates the connection site. Triangles and dotted lines indicate the location of gaps and deletions to optimize rod repeat alignment (M. Koe).
nig and L.M. M. Kunkel). CS1 and CS
2 shows the consensus sequence of 24 repeats of dystrophin. CS1 replaces the amino acids found in at least 8 of the 24 beats with CS
2 indicates an amino acid found in 5, 6 or 7 ribets.

FIG. 1C shows ΔDysH1 (H1) and ΔDysH1 (H1).
Amino acid sequence Δ of connecting region in DysH4 (H4)
In DysH1 (H1), hinge 1 is directly connected to the cysteine-rich domain. In ΔDysH4 (H4),
The actin binding domain is directly connected to hinge 4. Hinge 1
(T) (star) is a North American XLC
M was mutated to alanine (A) in the family. The dotted line under hinge 4 indicates the WW domain; of the WW domain,
The most conserved amino acids are underlined.

Next, a method for introducing each truncated dystrophin cDNA obtained by the above method into an adenovirus vector will be described. COS-TPC method (Miyake et al., Proceeding of National Academy of Sciences USA, 93, 1320-
1324 (1996) [Miyake, S., Makimura,
M., Kanegae, Y., Harada, S., Sato, Y., Takamori,
K., Tokuda, C. and Saito, I. (1996) Proc. Natl. Aca
d. Sci. USA 93, 1320-1324.]), an E1-substituted recombinant adenovirus vector expressing each truncated dystrophin can be produced.

The respective truncated dystrophin cDNAs, ΔDysAX2, AX11, A
H3, M3, H1 and H4 were converted to cassette cosmid pAX
CAwt (Kanegae et al., Nucleic Acid Research, 23, 3816-3821 (1995) [Ka
negae, Y., Lee, G., Sato, Y., Tanaka, M., Nakai,
M., Sakaki., T., Sugano, S. and Saito, I. (1995) N
ucl. Acids Res. 23, 3816-3821.]) (Niwa et al., Gene, 108, 193-200 (1991)) [Niwa, H., Yamamura, K. and Miyazak.
i, J. (1991) Gene 108, 193-200.]). This expression unit can be expressed in vitro (in vitro, supra, by Niwa et al.) And in vivo (Araki et al., Proceeding of National Academy of Sciences USA, 92, 160-164 (1995)). [Araki, K., Araki, M., Miyazaki, J.
and Vassalli, P. (1995) Proc. Natl. Acad. Sci. USA
92, 160-164.]).

The production of each recombinant adenovirus was performed using 29
In three cells, the obtained cosmid and Ad5 dlx
(Saito et al., Journal of Virology, 5
4, 711-719 (1985) [Saito, I., O.
ya, Y., Yamamoto, K., Yuasa, T. and Shimojo, H. (1
985) J. Virol. 54, 711-719.]) Was performed by homologous recombination with the DNA-terminal protein complex. The resulting recombinant adenovirus vector was transformed into AxCAΔDy
s and the method already described (Kanegae et al., Japanese Journal of Medical Sciences)
Biology, 47, 157-166 (1994)
Year) [Kanegae, Y., Makimura, M. and Saito, I. (199
4) Jpn. J. Med. Sci. Biol. 47,157-166.]), Growth, purification and titration were performed. Each AxCAΔDys was treated with phosphate buffered saline containing 10% glycerol (PB
In S), it was stored at -80 ° C.

The expression of truncated dystrophin in cultured skeletal muscle cells using the recombinant adenovirus vector of the present invention was confirmed as follows. That is, in order to check that truncated dystrophin is transcribed and translated correctly,
AxCAΔDy was added to mouse skeletal muscle cell line C2C12 cells.
s was infected and Western plot analysis was performed. C2
100 moi of each recombinant adenovirus in C12 cells
And then induced differentiation by exchanging the culture medium. Three days after infection, cells were collected. The whole cell extract (20 μg / lane) was separated by SDS-PAGE (5% acrylamide), transferred to a PVDF membrane, and reacted with the monoclonal antibody DYS2. This antibody reacts with the last 17 amino acids of dystrophin. The result is shown in FIG. Lane 1 in FIG. 2 is from uninfected C2C12 cells, lane 2 is with AxCAΔDysAX2, and lane 3 is with AxCAΔDysAX1
1, and lane 4 is AxCAΔDysA
H3 was used, and lane 5 was AxCAΔDys
M3 was used, and lane 6 was AxCAΔDys
H1 was used, and lane 7 was AxCAΔDys
H4 was used. The MW in FIG. 2 is the molecular weight (k
Da) is shown.

Each truncated dystrophin gene showed the expected size (FIG. 2, lanes 2-6).
However, ΔDysH4 appeared at a position larger than expected (103 kDa) (FIG. 2, lane 7). AxCAΔDys
The product of H4 (FIG. 2, lane 7) was slower in mobility than expected. Endogenous dystrophin was not detected in cultured skeletal muscle cells. This is because the cells did not differentiate into fully mature myotubes. When comparing the amount of truncated dystrophin, ΔDysM3 showed the highest expression level. These results indicated that the recombinant AxCAΔDys effectively infects cultured skeletal muscle cells, expresses truncated dystrophin under the control of the CAG promoter, and furthermore that ΔDysM3 protein is most stably expressed.

Furthermore, the truncated dystrophin of the present invention using a recombinant adenovirus vector can be used in vivo.
In vivo), recombinant AxCAΔDys was transformed with mature md to determine if it was stably expressed in muscle fibers.
The mouse was directly introduced into the tibialis anterior muscle (TA) of the x mouse, and subjected to immunohistological analysis (FIG. 3 (photograph replacing the drawing)). Recombinant adenovirus was introduced directly into the tibialis anterior muscle of adult mdx mice. The amount of the introduced vector was determined in Example 4 described later.
In Table 2 below. Seven days after the injection, TA was removed from the mouse, and dystrophin antibody staining was performed using a frozen section and a rabbit polyclonal antibody anti-C. This antibody recognizes the C-terminus of dystrophin.

B10 in FIG. 3 is normal mature C57BL / 10
3 is a non-introduced mdx mouse, AX2 in FIG. 3 is AxCAΔDysA × 2, AX11 in FIG. 3 is AxCAΔDysAX11, and FIG.
AH3 is AxCAΔDysAH3, and M3 in FIG.
Is AxCAΔDysM3, and H1 in FIG.
ΔDysH1, and H4 in FIG. 3 is AxCAΔDysH
4 is used. The bar in the photograph indicates the scale, and indicates that the length of the bar is 100 μm.

As previously reported, HE staining detected strong infiltration of mononuclear cells and necrosis of muscle fibers by the recombinant adenovirus. Dystrophin-positive fibers are
They tended to appear in groups around the damaged area. All truncated dystrophins, except for ΔDysH1, are also considered when examined on one and the same slide.
It was more strongly expressed on the plasma membrane than dystrophin of C57BL / 10 mice in normal controls. The proportion of dystrophin-positive fibers was clearly higher than the revertant fibers found in mdx skeletal muscle. Furthermore, a P23a antibody against the 19th rod repeat of dystrophin (Yoshida et al., Journal of Biochemistry,
108, 748-752 (1990) [Yoshida,
M. and Ozawa, E. (1990) J. Biochem. 108, 748-75
2.]) was used to confirm that the dystrophin-positive fibers were not revertant fibers.

In the skeletal muscle into which AxCAΔDysM was introduced, although the intensity of dystrophin immunostaining varied greatly between fibers, strong immunofluorescence was consistently observed in skeletal muscle into which AxCAΔDysM3 had been introduced (FIG. 3). ). In contrast, in skeletal muscle transfected with AxCAΔDysH1, the signal of dystrophin intestinal fibers at the plasma membrane was weak and discontinuous.

In order to evaluate the effect of each truncated dystrophin on skeletal muscle of mdx mice, the present inventors
From each of the skeletal muscles into which AxCAΔDys had been introduced, three regions in which clusters were formed were picked up, and the number of lines expressing truncated dystrophin and the intensity of dystrophin immunofluorescence were separately evaluated. The results are summarized in Table 2. These results suggest that truncated dystrophin with both a short rod domain and hinges 1 and 4 can effectively localize to the plasma membrane. As seen in ΔDysH1, deletion of hinge 4 resulted in greatly reduced localization to the plasma membrane.

Next, recovery of the expression of dystrophin-binding protein (DAP) in the plasma membrane was examined. To evaluate the function of dystrophin as a key molecule for forming dystrophin-DAP complexes, we examined the expression of DAPs in the plasma membrane of mdx skeletal muscle after introduction of AxCAΔDys. AxCAΔDysM
In order to confirm the recovery of dystrophin-binding protein in the plasma membrane of mdx skeletal muscle injected with No.3, gene transfer and antibody staining were performed by the method described in FIG. FIG. 4 (a photograph replacing the drawing) shows the result. In mdx mice transfected with AxCAΔDysM3, dystrophin-expressing muscle fibers were strongly stained with antibodies against β-dystroglycan, α-sarcoglycan and α1-syntrophin. DAP was negative for dystrophin negative fibers (star in FIG. 4). In mdx skeletal muscle injected with AxCAΔDysH1, the signal of dystrophin-positive fibers at the plasma membrane was extremely weak. In such fibers, DAP was not detected at the plasma membrane. The bar in the photograph indicates the scale, and indicates that the length of the bar is 50 μm.

In mdx skeletal muscle, the expression of DAPs is reduced (Oendick et al., Journal of Cell Biology, 115, 1685-1694 (1991) [Ohlendieck, K. and Campbell, KP.
(1991) J. Cell Biol. 115, 1685-1694.]) (FIG. 4), compared to AxCAΔDy other than AxCAΔDysH1.
In the skeletal muscle into which s was introduced, the expression of DAPs at the plasma membrane was significantly restored in dystrophin-positive fibers. Interestingly, the intensity of immunofluorescence of DAPs was similar regardless of the expression level of dystrophin. However, in mdx skeletal muscle into which AxCAΔDysH1 was introduced, it was difficult to detect immunofluorescence of DAPs along the plasma membrane. In particular, mdx with AxCAΔDysH1 introduced
Β-dystroglycan and α-sarcoglycan signals were extremely low in stromal dystrophin-positive fibers. These results indicate that truncated dystrophin expressed on plasma membranes other than ΔDysH1 can effectively replace DAP on the plasma membrane.
s expression was found to be restored.

When these recombinant adenovirus vectors are introduced into mature mouse skeletal muscle, long-term expression of the gene transfer product cannot be expected due to the antigenicity of the virus vector. However, in gene transfer into neonatal mice, immune tolerance is established. Therefore, AxCAΔDysM3 among recombinant adenovirus vectors incorporating the truncated dystrophin gene was introduced into neonatal mdx mouse skeletal muscle and expressed for a long time. By letting
We examined whether the phenotype of muscular dystrophy could be improved.

One week after birth, 6 μl of a mixture of AxCAΔDysM3 and AxCALacZ was directly introduced into the central part of the gastrocnemius muscle of one hind limb of the mdx mouse. Four weeks later, the skeletal muscle of the gastrocnemius muscle of the hind limb was taken out and subjected to H & E staining, X-Gal staining and dystrophin staining. As a result, X-Gal staining was performed on the gastrocnemius muscle group of the hind limb to which the adenovirus was injected to confirm the introduction of the adenovirus vector, and the superficial digital flexor muscle (flexor digitorum) of the gastrocnemius muscle group was observed.
superficialis), the highest percentage of transgenic fibers was observed. When dystrophin immunostaining was performed on the β-Gal positive region, dystrophin was expressed in most of the fibers.
H & E staining was performed on the same part to observe it in detail. As a result, the superficial digital flexor muscle (flexor digitorum superfi
cialis), the number of degenerative and necrotic images of muscle and the number of central nucleus fibers were significantly reduced.

By infecting cultured skeletal muscle cell line C2C12 and skeletal muscle of mature mdx mice with a recombinant adenovirus vector incorporating the truncated dystrophin gene, the present inventors have obtained the truncated dystrophin designed according to the present invention. As a result of examining whether or not it is stably expressed in muscle cells, the combination of an adenovirus vector with a wide infectious area and a strong CAG promoter in skeletal muscle has led to The expression of truncated dystrophin could be compared.

ΔDy having only one rod repeat
sM3 showed the highest expression in vitro (in vitro). Clemens, etc.
Three truncated dystrophins with frame deletions (3.
0, 4.4 and 5.7 kb deletions) (Clemens et al., Human Gene Therapy, 6, 1477).
-1485 (1995) [Clemens, PR, Krause,
TL, Chan, S., Korb, KE, Graham, FL and Cask
ey, CT (1995) Hum. Gene Ther. 6, 1477-1485.]).
These have 15, 10 or 6 rod repeats. They showed in transfection experiments on cultured skeletal muscle cells that the production of these dystrophins was not solely determined by the size of the deletion.
We have also concluded that the stability of truncated dystrophin with a deletion in the rod domain is independent of the number of rods. These results were consistent with the findings in BMD patients that the size of the deletion was not related to either dystrophin production or the severity of symptoms.

When AxCAΔDys was introduced into skeletal muscle of adult mdx mice, ΔDysM3 was also expressed as effectively as truncated dystrophin with more rod repeats. The frequency of myofibers expressing dystrophin tended to be proportional to the viral load administered. In addition, although the correct higher-order structure of ΔDysM3 has not been determined, it appears to be involved in the stability in skeletal muscle of adult mice.

AxCAΔDysH1 and AxCAΔDys
For H4, as well as other AxCAΔDys, a large amount of virus was introduced into skeletal muscle of mature mdx mice, but their expression was clearly lower than other ΔDys. This may be because both ΔDysH1 and ΔDysH4 completely lack rod repeats. In particular, the expression of ΔDysH1 lacking hinge 4 was extremely low. Hinge 4 has a “WW domain” (Schodor et al., Journal of Biological Chemistry, 270, 14733-14741 (199)
5 years) [Sudol, M., Bork, P., Einbond, A ,, Kastury,
K., Druck, T., Negrini, M., Huebner, K. and Lehman,
D. (1995) J. Biol. Chem. 270, 14733-14741.] And the domain is the XP of β-dystroglycan.
It has been proposed that dystrophin molecules are anchored to the plasma membrane by binding to the PXY motif (Einbond et al., Feves Letters, 384, 1
-8 (1996) [Einbond, A. and Sudol, M. (1
996) FEBS Letters 384, 1-8.]). Thus, we speculated that ΔDysH1 was destabilized due to reduced binding to β-dystroglycan.

ΔDysH4 lacks hinge 1.
The importance of the hinge 1 region has recently been pointed out. In a North American X-linked dilated cardiomyopathy kindred, hinge 1
A missense mutation was identified in the region and it was assumed that the structure of the dystrophin molecule was altered (Orti-Lopetz et al., Circulation, 95, 2434-).
2440 (1997) [Ortiz-Lopez, R., Li, H.,
Su, J., Goytia, V. and Towbin, JA (1997) Circula
tion 95, 2434-2440.]). For this reason, ΔDy
It was considered that the loss of hinge 1 may be involved in the reduction of sH4 expression.

Transgenic studies of mini-dystrophin cDNA (Wells et al., Human Molecular Genetics, Vol. 4, pp. 1245-1250 (1995) [Wells, DJ, Wells, KE, ASante,
EA, Turner, G., Sunada, Y., Campbell, KP, Walsh,
FS and Dickson, G. (1995) Hum. Mol. Genet. 4, 1
245-1250.]), Even a small truncated dystrophin such as ΔDysM3 could accumulate DAPs in mdx mouse skeletal muscle if the C-terminal domain was retained. That is, according to the experiments of the present invention, truncated dystrophin having both hinge 4 and cysteine-rich domain was effectively DA
It has been demonstrated that Ps accumulates at the plasma membrane. But,
Of note, restoration of DAPs does not necessarily mean preventing or reducing the development of muscular dystrophy.

Recovery of DAPs at the plasma membrane may not be sufficient to improve dystrophin function.
Dp71, one of the molecular species of dystrophin, lacking the rod domain and the N-terminal actin binding domain
In experiments where the gene was transfected into mdx mice as a transgene, there was no effective improvement in the muscular dystrophy phenotype, despite the complete recovery of DAPs (Cox et al., Nature Genetics). 8, Vol. 333-339 (1994) [Cox,
GA, Sunada, Y., Campbell, KP and Chamberlain,
JS (1994) Nature Genet. 8, 333-339.] And Greenberg et al., Nature Genetics, 8
Vol. 340-344 (1994) [Greenberg, D.
S., Sunada, Y., Campbell, KP, Yaffe, D. and Nude
l, U. (1994) Nature Genet. 8, 340-344.]).

On the other hand, Chamber Rain (Chamber Rain)
lain) et al. constructed a series of truncated dystrophin genes, and introduced them into a mdx mouse as a transgene. When they examined the genes, they had an N-terminal side up to hinge 1 and a C-terminal side up to hinge 4 or lower. It has been revealed that dystrophin of a partially deleted type is stably expressed in the membrane, but the phenotype of muscular dystrophy is not improved. From this viewpoint, in order to prove the function of truncated dystrophin ΔDysM3 in vivo, an experiment capable of long-term expression is required.

Indeed, the present inventors examined the effect of the adenovirus vector encoding ΔDysM3 four weeks after introducing the adenovirus vector into the skeletal muscle of neonatal mdx mice. It has been found that central nucleus fibers, which are indicators of muscle regeneration resulting from decreased muscle degeneration and muscle regeneration, almost disappear.
Further experiments with transgenic mice are needed, as longer expression systems may be needed to determine whether this truncated dystrophin improves the muscular dystrophy phenotype Might be.

In the present invention, the shortened dystrophin holding at least one rod repeat is a mature mdx
It was shown to be effectively expressed in mouse skeletal muscle. As already evident, first generation adenovirus vectors provoke a strong immune response in the host.

Therefore, in gene therapy for muscular dystrophy, use of a new vector that does not induce an immune response in a host and provides long-term expression of a transgene is being studied in the future. In particular, the adeno-associated virus (AAV) vector has the advantage that stable expression can be expected in skeletal muscle by incorporating the transgene into the chromosome.

However, the number of genes that can be inserted into this vector was limited to 4-4.5 kb. Therefore,
Regarding the dystrophin gene, it is impossible to insert not only a 14 kb full-length gene but also a 6.3 kb mini dystrophin gene. The truncated dystrophin gene obtained by the present invention, in particular, a 3.7 kb ΔDysM3cDN holding only one rod repeat
A is a very good gene to insert into an adeno-associated virus vector.

As is clear from the above results, the gene for treating muscular dystrophy of the present invention has at least one hinge 1, hinge 4 of the dystrophin gene and a rod repeat structure of the rod domain, and is 4.5 kb or less. , Or a base sequence capable of hybridizing to the base sequence. The gene of the present invention only needs to have one rod repeat structure of the rod domain, but may have two or more, preferably two or three, in some cases. It is preferable that these rod repeat structures have exactly the same base sequence.However, even if a part of the rod repeat structure is substituted with another base sequence, another base sequence is added, May be deleted.

The gene of the present invention further comprises a cysteine
Those having a rich domain, an actin-binding domain, and a C-terminal domain are preferred. CD of the present invention
The AN may have a total length of 4.5 kb or less, preferably 4.2 kb or less, more preferably 4.0 kb or less, and still more preferably 3.7 kb or less.

The gene of the present invention can be used as a therapeutic agent for muscular dystrophy. As a method for introducing the gene of the present invention into a patient, a conventionally used method can be used, but it is preferable to incorporate the gene of the present invention into an adeno-associated virus (AAV) vector. A known method can be adopted as the introduction method.

The present invention also relates to the adeno-associated virus (A
AV) a gene transfer medium for muscular dystrophy gene therapy comprising a vector. The adeno-associated virus (AAV) vector has been used as a gene transfer vehicle for gene therapy of other diseases, but the present invention has revealed for the first time that it can be used as a gene transfer vehicle for muscular dystrophy gene therapy. This is a new use of vector. The gene transfer medium for muscular dystrophy gene therapy preferably contains any of the above-described genes of the present invention, but the gene transfer medium for gene therapy of muscular dystrophy of the present invention is not limited thereto. is not.

The adeno-associated virus (AAV) vector of the present invention contains any of the aforementioned genes of the present invention. The method for introducing the gene of the present invention into an adeno-associated virus (AAV) vector is not particularly limited, and the gene can be introduced by a method commonly used by those skilled in the art. The adenovirus vector of the present invention can be produced by introducing any of the above-described genes of the present invention into an adenovirus vector by a conventional method.

The therapeutic agent for muscular dystrophy comprising the adenovirus of the present invention can be used in the same manner as a conventional gene therapy method using a virus.

[0061]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (Construction of Rod Truncated Dystrophin Gene) Six types of dystrophin genes in which the rod domain was further shortened were constructed by the following method (see FIG. 1A). First, the human mini dystrophin cDNA
(Asukadi et al., Nature, 352, 615-81
8 (1991) [Acsadi, G., Dickson, G., Love,
DR, Jani, A., Walsh, FS, Gurusinghe, A., Wolf
f, TA, and Davies, KE (1991) Nature 352, 815-8
18.]) of the NotI / SalI fragment of pBluescriptII (SK +) (Strata
gene) at the NotI / SalI site and pBS
BMD was produced. Next, AX2, AX11, AH3,
C of truncated dystrophin (ΔDys) named M3
Four plasmids with DNA were made as shown below. Table 1 shows the nucleotide sequences of primers and oligonucleotides used for cDNA construction.

[0063]

[Table 1]

In Table 1, the DNA sequences of the synthetic oligonucleotides used for the construction of truncated dystrophin Oligonucleotides F3 and R3 were used to anneal directly to form DNA fragments. Other oligonucleotides were used as primers for the PCR reaction. The underline indicates the nucleotide sequence of human dystrophin cDNA (GeneBank accession number).
r M18533). PBSBMD of template
And a PCR fragment amplified with primers F1 / R2 or F2 / R2 cut with AflII / XhoI,
Insert into the AflII / XhoI site of BMD,
pBSΔDysAX2 or pBSΔDysAX11 was prepared. Template pBSBMD and primer F4 / R4
Was digested with MunI / HindIII, and inserted into the MunI / HindIII site of pBSBMD to prepare pBSΔDysM3. The fragment generated by the annealing of the oligonucleotide F3 / R3 was used to ligate the AflII / HindIII sites of pBSBMD to generate pBSΔDysAH3.

On the other hand, the cD of ΔDysH1 and ΔDysH4
The two plasmids with NA are pBSΔDysM3
(See FIG. 1A). First, EcoO109
To remove one I site, pBSΔDysM3 was replaced with Apa
After cutting with I, smoothing, self-ligation and pBS
ΔDysM3b. A template pBSΔDysM3;
PCR product amplified using primer F5 / R5
After digestion with EcoT22I / EcoO109I, pB
EcoT22I / EcoO109I of SΔDysM3b
Into the site to create pBSΔDysH1. pBS
To prepare ΔDysH4, two types of PCR reactions were performed using pBSΔDysM3 as a template and primer F5
Separately using / R6 or F6 / R7. A second PCR reaction was performed using a mixture of the obtained two types of PCR products as a template and primer F5 / R7.
The resulting fragment was digested with EcoRV, and then digested with pBS.
Inserted between two EcoRV sites in ΔDysM3. The amino acid sequence of the connecting region is shown in FIG. 1B and FIG. 1C.

The obtained four kinds of M3, AX11, AX2,
And the nucleotide sequences of AH3 cDNA are shown in SEQ ID NOs: 1, 3, 5, and 7, respectively, in the sequence listing. In addition, two types of H1
And the nucleotide sequences of the H4 cDNAs are shown in SEQ ID NOs: 9 and 11 in the sequence listing, respectively. The amino acid sequences encoded by these cDNAs are represented by SEQ ID NOs: 2, 4, 6,
8, 10, and 12 respectively.

Example 2 (Preparation of Recombinant Adenovirus Vector Expressing Truncated Dystrophin) An E1-substituted recombinant adenovirus vector expressing each truncated dystrophin was prepared by the COS-TPC method. Each truncated dystrophin cDNA, Δ
DysAX2, AX11, AH3, M3, H1 and H4
Was inserted into the CAG expression unit of the cassette cosmid pAXCAwt. This expression unit shows strong expression in vitro (in vitro) and in vivo (in vivo). The production of each recombinant adenovirus vector was performed in 293 cells using the obtained cosmid and Ad5.
This was done by homologous recombination between dlx and the DNA-terminal protein complex. The obtained recombinant adenovirus vector was named AxCAΔDys, and was subjected to proliferation, purification, and titration by the methods described above. Each Ax
CAΔDys was stored at −80 ° C. in phosphate buffered saline (PBS) containing 10% glycerol.

Example 3 (Gene transfer into cultured skeletal muscle cells using adenovirus vector) One of the subclones of C2C12 myoblasts (Yoshida et al.
Journal of Cell Biology, 132, 8
18-193 (1996) [Yoshida, S., Fujisawa
-Sehara, A., Taki, T., Arai, K. and Nabeshima, Y.
(1996) J. Cell Biol. 132, 181-193.]) (About 1.0 ×
10 ⁵ ) on a 6 cm collagen-coated dish, and DMEM containing 20% (vol / vol) calf serum.
For 1 day. AxCAΔDys in myoblasts
00 plaque-forming unit / cell (pla
que-forming unit (pfu) / cel
1 (moi)), and the growth tower was
And 5% (vol / vol) of horse serum. After 3 days, cells were harvested and SDS-PAGE lysis buffer (15% SDS, 70 mM Tris-H
Cl pH 6.8, 5% β-mercaptoethanol (β
-Mercatoethanol), 10 mM EDT
A).

20 μg of the cell lysate was added to 5 lanes per lane.
% SDS-PAGE and separated by PVDF membrane (Immob
illon (TM), Millipore). Dystrophin monoclonal antibody DYS2 (Novo
castra). This antibody recognizes the last 17 amino acids of human dystrophin. The immune complex on the plot was compared with peroxidase-labeled rabbit anti-mouse IgG1 (Zymed) and ECL.
Western blotting detection reagent (Amersha
m).

FIG. 2 shows the results. Except for ΔDysH4,
Each truncated dystrophin showed the expected size. In comparison of the expression levels of truncated dystrophin, Δ
DysM3 showed the highest expression level. These results indicate that the recombinant AxCAΔDys effectively infects cultured skeletal muscle cells and expresses truncated dystrophin under the control of the CAG promoter.

Example 4 (In vivo gene transfer of mdx into mouse skeletal muscle using adenovirus vector) Prior to in vivo (in vivo) gene transfer, in order to remove glycerol, AxCAΔDys stock in PB
CHROMA SPIN ^™ column (Cl
ontech). 50 μl of the purified AxCAΔDys solution was directly injected into the anterior muscle of the left leg of a 12-16 week old mdx mouse using a 27-gauge needle (intramuscular injection).
did. The amount of each vector introduced and the results are shown in Table 2 below.

[0072]

[Table 2]

Table 2 shows the amount of vector used and the amount of truncated dystrophin cDNA obtained using adenovirus vector.
9 shows the results of quantitative analysis when dx skeletal muscle was introduced. In Table 2, "*" indicates the percentage of dystrophin-positive fibers in the selected region, and "**" indicates the result of evaluating the signal intensity of dystrophin at the plasma membrane from 0 to +++. One week later, skeletal muscle was removed and frozen in liquid nitrogen cooled isopentane. From a mdx skeletal muscle that had undergone gene transfer, and a non-transgenic mdx skeletal muscle and a normal control C57BL / 10 skeletal muscle, a 6-μm section was prepared on the same slide, air-dried, and then fixed with acetone for 10 minutes did.

Immunohistological staining was performed using the following antibodies. Rabbit polyclonal antibody that recognizes the most C-terminal 25 amino acids of dystrophin (anti-C, obtained from Dr. Nonomura), 19
Rabbit polyclonal antibody recognizing amino acids 2360 to 2409 of dystrophin corresponding to the second rod repeat (P23a, obtained from Dr. M. Yoshida) (Yoshida et al., Journal of Biochemistry, 108, 748-752). (199
0) [Yoshida, M. and Ozawa, E. (1990) J. Bioche
m. 108, 748-752.]), a goat polyclonal antibody against β-dystroglycan, a rabbit polyclonal antibody against α-sarcoglycan (Y. Wakayama).
a) Obtained from Dr. (Wakayama et al., Anals of Neurology, 39, 217-223 (199)
6 years) [Wakayama, Y., Inoue, M., Murahashi, M., Sh
ibuya, S., Jimi, T., Kojima, H. and Oniki, H. (199
6) Ann. Neurol. 39, 217-223.]), Amino acids 191 to 206 of α1-syntrophin (Peter et al., Neuroreport, Vol. 5, pp. 1577-1580 (1994).
Years] [Peters, MF, Kramarcy, NR, Sealock, R. an
d Froehner, SC (1994) NeuroReport 5, 1577-158
0.]) (Kameya (S.I.
(Kameya). ).

The primary antibody was a FITC-labeled goat anti-rabbit IgG (Tago Immunological).
s) or rabbit anti-goat IgG (Organon
Teknika). The result is a laser scanning confocal imaging system M
Recording was performed using RC-1000 (Bio-Rad).

FIG. 3 shows the results. As a result, truncated dystrophin (ΔDysAX2, AX11, AH3 and M3) having a short rod domain and both hinges 1 and 4
Suggests that it can be effectively localized to the plasma membrane.
The missing arrow in hinge 4 seen in ΔDysH1 resulted in a significant decrease in localization to the plasma membrane.

Example 5 (Restoring Expression of Dystrophin-Binding Protein in Plasma Membrane) To evaluate the function of dystrophin as a key molecule for forming a dystrophin-DAP complex, Ax was evaluated.
The expression of DAPs in the plasma membrane of mdx skeletal muscle after introduction of CAΔDys was examined. In mdx skeletal muscle, DAPs
(Orlendick et al., Journal of Cell Biology, 115, 1685-1)
694 (1991) [Ohlendieck, K. and Campbel
l, KP (1991) J. Cell Biol. 115, 1685-1694.])
In contrast, in skeletal muscle into which AxCAΔDys other than AxCAΔDysH1 was introduced, expression of DAPs in the plasma membrane of dystrophin-positive fibers was significantly restored (see FIG. 4).

Example 6 (In Vivo Gene Transfer into Skeletal Muscles of Neonatal mdx Mice) One day after birth, mdx mice had an A in the middle gastrocnemius muscle of one hind leg.
6 μl of a mixture of xCAΔDysM3 and AxCALacZ
Was introduced directly. Four weeks later, the skeletal muscle of the gastrocnemius muscle of the hind limb was taken out and subjected to H & E staining, X-Gal staining and dystrophin staining. As a result, X-Gal staining was performed on the gastrocnemius muscle group of the hind limb on the side where the adenovirus was injected in order to confirm the introduction of the adenovirus vector.
Flexor digitorum superfici of the gastrocnemius muscle group
alis), the highest percentage of transgenic fibers was found. When dystrophin immunostaining was performed on the β-Gal positive region, dystrophin was expressed in most of the fibers. When the same portion was subjected to H & E staining and observed in detail, as compared with the superficial digital flexor muscle (flexor digitorum superficialis) on the non-introduction side, the degeneration / necrosis image of the muscle and the number of central nucleus fibers were significantly reduced.

[0079]

By using the gene of the present invention and the gene transfer medium for gene therapy of muscular dystrophy, gene therapy of muscular dystrophy with less immune response can be performed.

[0080]

[Sequence List] SEQ ID NO: 1 Length of array: 3748 Array type: Number of nucleic acid strands: Both topologies: Type of linear sequence: Features of cDNA to mRNA sequence: active-site sequence CGGCCGCTCT AGAGGATCCC CGGGTACCGA GCTCGAATTC CGGAACTCCC GGAGAAAAAC 60 GAATAGGAAA AACTGAAGTG TTACTTTTTT TAAAGCTGCT GAAGTTTGTT GGTTTCTCAT 120 TGTTTTTAAG CCTACTGGAG CAATAAAGTT TGAAGAACTT TTACCAGGTT TTTTTTATCG 180 CTGCCTTGAT ATACACTTTT CAAAATGCTT TGGTGGGAAG AAGTAGAGGA CTGTTATGAA 240 AGAGAAGATG TTCAAAAGAA AACATTCACA AAATGGGTAA ATGCACAATT TTCTAAGTTT 300 GGGAAGCAGC ATATTGAGAA CCTCTTCAGT GACCTACAGG ATGGGAGGCG CCTCCTAGAC 360 CTCCTCGAAG GCCTGACAGG GCAAAAACTG CCAAAAGAAA AAGGATCCAC AAGAGTTCAT 420 GCCCTGAACA ATGTCAACAA GGCACTGCGG GTTTTGCAGA ACAATAATGT TGATTTAGTG 480 AATATTGGAA GTACTGACAT CGTAGATGGA AATCATAAAC TGACTCTTGG TTTGATTTGG 540 AATATAATCC TCCACTGGCA GGTCAAAAAT GTAATGAAAA ATATCATGGC TGGATTGCAA 600 CAAACCAACA GTGAAAAGAT TCTCCTGAGC TGGGTCCGAC AATCAACTCG TAATTATCCA 660 CAGGTTAATG TAATCAACTT CACCACCAGC TGGTCTGATG GCCTGGCTTT GAATGCTCTC 720 ATCCATAGTC ATAGGCCAGA CCTATTTGAC TGGAATAGTG TGGTTTGCCA GCAGTCAGCC 780 ACACAACGAC TGGAACATGC ATTCAACATC GCCAGATATC AATTAGGCAT AGAGAAACTA 840 CT CGATCCTG AAGATGTTGA TACCACCTAT CCAGATAAGA AGTCCATCTT AATGTACATC 900 ACATCACTCT TCCAAGTTTT GCCTCAACAA GTGAGCATTG AAGCCATCCA GGAAGTGGAA 960 ATGTTGCCAA GGCCACCTAA AGTGACTAAA GAAGAACATT TTCAGTTACA TCATCAAATG 1020 CACTATTCTC AACAGATCAC GGTCAGTCTA GCACAGGGAT ATGAGAGAAC TTCTTCCCCT 1080 AAGCCTCGAT TCAAGAGCTA TGCCTACACA CAGGCTGCTT ATGTCACCAC CTCTGACCCT 1140 ACACGGAGCC CATTTCCTTC ACAGCATTTG GAAGCTCCTG AAGACAAGTC ATTTGGCAGT 1200 TCATTGATGG AGAGTGAAGT AAACCTGGAC CGTTATCAAA CAGCTTTAGA AGAAGTATTA 1260 TCGTGGCTTC TTTCTGCTGA GGACACATTG CAAGCACAAG GAGAGATTTC TAATGATGTG 1320 GAAGTGGTGA AAGACCAGTT TCATACTCAT GAGGGGTACA TGATGGATTT GACAGCCCAT 1380 CAGGGCCGGG TTGGTAATAT TCTACAATTG GGAAGTAAGC TGATTGGAAC AGGAAAATTA 1440 TCAGAAGATG AAGAAACTGA AGTACAAGAG CAGATGAATC TCCTAAATTC AAGATGGAAG 1500 CTTCTGCAGG TGGCCGTCGA GGACCGAGTC AGGCAGCTGC ATGAAGCCCA CAGGGACTTT 1560 GGTCCAGCAT CTCAGCACTT TCTTTCCACG TCTGTCCAGG GTCCCTGGGA GAGAGCCATC 1620 TCGCCAAACA AAGTGCCCTA CTATATCAAC CACGAGACTC AAACAACTTG CTGGGACCAT 1680 CCCAAAATGA CAGAGCTCTA CCAGTCTTTA GCTGACCTGA ATAATGTCAG ATTCTCAGCT 1740 TATAGGACTG CCATGAAACT CCGAAGACTG CAGAAGGCCC TTTGCTTGGA TCTCTTGAGC 1800 CTGTCAGCTG CATGTGATGC CTTGGACCAG CACAACCTCA AGCAAAATGA CCAGCCCATG 1860 GATATCCTGC AGATTATTAA TTGTTTGACC ACTATTTATG ACCGCCTGGA GCAAGAGCAC 1920 AACAATTTGG TCAACGTCCC TCTCTGCGTG GATATGTGTC TGAACTGGCT GCTGAATGTT 1980 TATGATACGG GACGAACAGG GAGGATCCGT GTCCTGTCTT TTAAAACTGG CATCATTTCC 2040 CTGTGTAAAG CACATTTGGA AGACAAGTAC AGATACCTTT TCAAGCAAGT GGCAAGTTCA 2100 ACAGGATTTT GTGACCAGCG CAGGCTGGGC CTCCTTCTGC ATGATTCTAT CCAAATTCCA 2160 AGACAGTTGG GTGAAGTTGC ATCCTTTGGG GGCAGTAACA TTGAGCCAAG TGTCCGGAGC 2220 TGCTTCCAAT TTGCTAATAA TAAGCCAGAG ATCGAAGCGG CCCTCTTCCT AGACTGGATG 2280 AGACTGGAAC CCCAGTCCAT GGTGTGGCTG CCCGTCCTGC ACAGAGTGGC TGCTGCAGAA 2340 ACTGCCAAGC ATCAGGCCAA ATGTAACATC TGCAAAGAGT GTCCAATCAT TGGATTCAGG 2400 TACAGGAGTC TAAAGCACTT TAATTATGAC ATCTGCCAAA GCTGCTTTTT TTCTGGTCGA 2460 GTTGCAAAAG GCCATAAAAT GCACTATCCC ATGGTGGAAT ATTGCACTCC GACTACATCA 2520 GGAGAAGATG TTCGA GACTT TGCCAAGGTA CTAAAAAACA AATTTCGAAC CAAAAGGTAT 2580 TTTGCGAAGC ATCCCCGAAT GGGCTACCTG CCAGTGCAGA CTGTCTTAGA GGGGGACAAC 2640 ATGGAAACTC CCGTTACTCT GATCAACTTC TGGCCAGTAG ATTCTGCGCC TGCCTCGTCC 2700 CCTCAGCTTT CACACGATGA TACTCATTCA CGCATTGAAC ATTATGCTAG CAGGCTAGCA 2760 GAAATGGAAA ACAGCAATGG ATCTTATCTA AATGATAGCA TCTCTCCTAA TGAGAGCATA 2820 GATGATGAAC ATTTGTTAAT CCAGCATTAC TGCCAAAGTT TGAACCAGGA CTCCCCCCTG 2880 AGCCAGCCTC GTAGTCCTGC CCAGATCTTG ATTTCCTTAG AGAGTGAGGA AAGAGGGGAG 2940 CTAGAGAGAA TCCTAGCAGA TCTTGAGGAA GAAAACAGGA ATCTGCAAGC AGAATATGAC 3000 CGTCTAAAGC AGCAGCACGA ACATAAAGGC CTGTCCCCAC TGCCGTCCCC TCCTGAAATG 3060 ATGCCCACCT CTCCCCAGAG TCCCCGGGAT GCTGAGCTCA TTGCTGAGGC CAAGCTACTG 3120 CGTCAACAC AAAGGCCGCC TGGAAGCCAG GATGCAAATC CTGGAAGACC ACAATAAACAG 3180 CTGGAGTCA CAGTTACACA GGCTAAGGCA GCTGCTGGAG CAACCCCAGG CAGAGGCCAAA 3240 GTGAATGGC ACAACGGTGT CCTCTCCTTC TACCTCTCTA CAGAGGTCCG ACAGCAGTCAG 3300 CCTATGCTG CTCCGAGTGG TTGGCAGTCA AACTTCGGAC TCCATGGGTG AGGAAGATCTT 3360 CTCAGTCCT CCCCAGGACA C AAGCACAGG GTTAGAGGAG GTGATGGAGC AACTCAACAAC 3420 TCCTTCCCT AGTTCAAGAG GAAGAAATAC CCCTGGAAAG CCAATGAGAG AGGACACAATG 3480 TAGGAAGTC TTTTCCACAT GGCAGATGAT TTGGGCAGAG CGATGGAGTC CTTAGTATCAG 3540 TCATGACAG ATGAAGAAGG AGCAGAATAA ATGTTTTACA ACTCCTGATT CCCGCATGGTT 3600 TTTATAATA TTCATACAAC AAAGAGGATT AGACAGTAAG AGTTTACAAG AAATAAATCTA 3660 TATTTTTGT GAAGGGTAGT GGTATTATAC TGTAGATTTC AGTAGTTTCT AAGTCTGTTAT 3720 GTTTTGTTG GGGATCCTCT AGAGTCGA 3748 SEQ ID NO: 2 Length of array: 1092 Array type: Amino acid topology: Type of linear sequence: Protein sequence Met Leu Trp Trp Glu Glu Val Glu Asp Cys Tyr Glu Arg Glu Asp 15 Val Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser 30 Lys Phe Gly Lys Gln His Ile Glu Asn Leu Phe Ser Asp Leu Gln 45 Asp Gly Arg Arg Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln 60 Lys Leu Pro Lys Glu Lys Gly Ser Thr Arg Val His Ala Leu Asn 75 Asn Val Asn Lys Ala Leu Arg Val Leu Gln Asn Asn Asn Val Asp 90 Leu Val Asn Ile Gly Ser Thr Asp Ile Val Asp GlyA sn His Lys 105 Leu Thr Leu Gly Leu Ile Trp Asn Ile Ile Leu His Trp Gln Val 120 Lys Asn Val Met Lys Asn Ile Met Ala Gly Leu Gln Gln Thr Asn 135 Ser Glu Lys Ile Leu Leu Ser Trp Val Arg Gln Ser Thr Arg Asn 150 Tyr Pro Gln Val Asn Val Ile Asn Phe Thr Thr Ser Trp Ser Asp 165 Gly Leu Ala Leu Asn Ala Leu Ile His Ser His Arg Pro Asp Leu 180 Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala Thr Gln Arg 195 Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly Ile Glu 210 Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys 225 Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro 240 Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Met Leu Pro 255 Arg Pro Pro Lys Val Thr Lys Glu Glu His Phe Gln Leu His His 270 Gln Met His Tyr Ser Gln Gln Ile Thr Val Ser Leu Ala Gln Gly 285 Tyr Glu Arg Thr Ser Ser Pro Lys Pro Arg Phe Lys Ser Tyr Ala 300 Tyr Thr Gln Ala Ala Tyr Val Thr Thr Ser Asp Pro Thr Arg Ser 315 Pro Phe Pro Ser Gln His Leu Glu Ala Pro Glu Asp Lys Ser Phe 330 Gly Ser Ser Leu Met Glu Ser Glu Val Asn Leu Asp Arg Tyr Gln 345 Thr Ala Leu Glu Glu Val Leu Ser Trp Leu Leu Ser Ala Glu Asp 360 Thr Leu Gln Ala Gln Gly Glu Ile Ser Asn Asp Val Glu Val Val 375 Lys Asp Gln Phe His Thr His Glu Gly Tyr Met Met Asp Leu Thr 390 Ala His Gln Gly Arg Val Gly Asn Ile Leu Gln Leu Gly Ser Lys 405 Leu Ile Gly Thr Gly Lys Leu Ser Glu Asp Glu Glu Thr Glu Val 420 Gln Glu Gln Met Asn Leu Leu Asn Ser Arg Trp Lys Leu Leu Gln 435 Val Ala Val Glu Asp Arg Val Arg Gln Leu His Glu Ala His Arg 450 Asp Phe Gly Pro Ala Ser Gln His Phe Leu Ser Thr Ser Val Gln 465 Gly Pro Trp Glu Arg Ala Ile Ser Pro Asn Lys Val Pro Tyr Tyr 480 Ile Asn His Glu Thr Gln Thr Thr Cys Trp Asp His Pro Lys Met 495 Thr Glu Leu Tyr Gln Ser Leu Ala Asp Leu Asn Asn Val Arg Phe 510 Ser Ala Tyr Arg Thr Ala Met Lys Leu Arg Arg Leu Gln Lys Ala 525 Leu Cys Leu Asp Leu Leu Ser Leu Ser Ala Ala Cys Asp Ala Leu 540 Asp Gln His Asn Leu Lys Gln Asn Asp Gln Pro Met Asp Ile Leu 555 Gln Ile Ile Asn Cys Leu Thr Thr Ile Tyr Asp Arg Leu Glu Gln 570 Glu His Asn Asn Leu Val Asn Val Pro Leu Cys Val Asp Met Cys 585 Leu Asn Trp Leu Leu Asn Val Tyr Asp Thr Gly Arg Thr Gly Arg 600 Ile Arg Val Leu Ser Phe Lys Thr Gly Ile Ile Ser Leu Cys Lys 615 Ala His Leu Glu Asp Lys Tyr Arg Tyr Leu Phe Lys Gln Val Ala 630 Ser Ser Thr Gly Phe Cys Asp Gln Arg Arg Leu Gly Leu Leu Leu 645 His Asp Ser Ile Gln Ile Pro Arg Gln Leu Gly Glu Val Ala Ser 660 Phe Gly Gly Ser Asn Ile Glu Pro Ser Val Arg Ser Cys Phe Gln 675 Phe Ala Asn Asn Lys Pro Glu Ile Glu Ala Ala Leu Phe Leu Asp 690 Trp Met Arg Leu Glu Pro Gln Ser Met Val Trp Leu Pro Val Leu 705 His Arg Val Ala Ala Ala Glu Thr Ala Lys His Gln Ala Lys Cys 720 Asn Ile Cys Lys Glu Cys Pro Ile Ile Gly Phe Arg Tyr Arg Ser 735 Leu Lys His Phe Asn Tyr Asp Ile Cys Gln Ser Cys Phe Phe Ser 750 Gly Arg Val Ala Lys Gly His Lys Met His Tyr Pro Met Val Glu 765 Tyr Cys Thr Pro Thr Thr Ser Gly Glu Asp Val Arg Asp Phe Ala 780 Lys Val Leu Lys Asn Lys Phe Arg Thr Lys Arg Tyr Phe Ala Lys 795 His Pro Arg Met Gly Tyr Leu Pro Val Gln Thr Val Leu Glu Gly 810 Asp Asn Met Glu Thr Pro Val Thr Leu Ile Asn Phe Trp Pro Val 825 Asp Ser Ala Pro Ala Ser Ser Pro Gln Leu Ser His Asp Asp Thr 840 His Ser Arg Ile Glu His Tyr Ala Ser Arg Leu Ala Glu Met Glu 855 Asn Ser Asn Gly Ser Tyr Leu Asn Asp Ser Ile Ser Pro Asn Glu 870 Ser Ile Asp Asp Glu His Leu Leu Ile Gln His Tyr Cys Gln Ser 885 Leu Asn Gln Asp Ser Pro Leu Ser Gln Pro Arg Ser Pro Ala Gln 900 Ile Leu Ile Ser Leu Glu Ser Glu Glu Arg Gly Glu Leu Glu Arg 915 Ile Leu Ala Asp Leu Glu Glu Glu Asn Arg Asn Leu Gln Ala Glu 930 Tyr Asp Arg Leu Lys Gln Gln His Glu His Lys Gly Leu Ser Pro 945 Leu Pro Ser Pro Pro Glu Met Met Pro Thr Ser Pro Gln Ser Pro 960 Arg Asp Ala Glu Leu Ile Ala Glu Ala Lys Leu Leu Arg Gln His 975 Lys Gly Arg Leu Glu Ala Arg Met Gln Ile Leu Glu Asp His Asn 990 Lys Gln Leu Glu Ser Gln Leu His Arg Leu Arg Gln Leu Leu Glu 1005 Gln Pro Gln Ala Glu Ala Lys Val Asn Gly Thr Thr Val Ser Ser1020 Pro Ser Thr Ser Leu Gln Arg Ser Asp Ser Ser Gln Pro Met Leu 1035 Leu Arg Val Val Gly Ser Gln Thr Ser Asp Ser Met Gly Glu Glu 1050 Asp Leu Leu Ser Pro Pro Gln Asp Thr Ser Thr Gly Leu Glu Glu 1065 Val Met Glu Gln Leu Asn Asn Ser Phe Pro Ser Ser Arg Gly Arg 1080 Asn Thr Pro Gly Lys Pro Met Arg Glu Asp Thr Met 1092 SEQ ID NO: 3 Sequence length: 4402 Array type: Number of nucleic acid strands: Both topologies: Type of linear sequence: Features of cDNA to mRNA sequence: active-site sequence CGGCCGCTCT AGAGGATCCC CGGGTACCGA GCTCGAATTC CGGAACTCCC GGAGAAAAAC 60 GAATAGGAAA AACTGAAGTG TTACTTTTTT TAAAGCTGCT GAAGTTTGTT GGTTTCTCAT 120 TGTTTTTAAG CCTACTGGAG CAATAAAGTT TGAAGAACTT TTACCAGGTT TTTTTTATCG 180 CTGCCTTGAT ATACACTTTT CAAAATGCTT TGGTGGGAAG AAGTAGAGGA CTGTTATGAA 240 AGAGAAGATG TTCAAAAGAA AACATTCACA AAATGGGTAA ATGCACAATT TTCTAAGTTT 300 GGGAAGCAGC ATATTGAGAA CCTCTTCAGT GACCTACAGG ATGGGAGGCG CCTCCTAGAC 360 CTCCTCGAAG GCCTGACAGG GCAAAAACTG CCAAAAGAAA AAGGATCCAC AAGAGTTCAT 420 GCCCTGAACA ATGTCAACAA GGCACTGCGG GTTTTGCAGA ACAATAATGT TGATTTAGTG 480 AATATTGGAA GTACTGACAT CGTAGATGGA AATCATAAAC TGACTCTTGG TTTGATTTGG 540 AATATAATCC TCCACTGGCA GGTCAAAAAT GTAATGAAAA ATATCATGGC TGGATTGCAA 600 CAAACCAACA GTGAAAAGAT TCTCCTGAGC TGGGTCCGAC AATCAACTCG TAATTATCCA 660 CAGGTTAATG TAATCAACTT CACCACCAGC TGGTCTGATG GCCTGGCTTT GAATGCTCTC 720 ATCCATAGTC ATAGGCCAGA CCTATTTGAC TGGAATAGTG TGGTTTGCCA GCAGTCAGCC 780 ACACAACGAC TGGAACATGC ATTCAACATC GCCAGATATC AATTAGGCAT AGAGAAACTA 840 CT CGATCCTG AAGATGTTGA TACCACCTAT CCAGATAAGA AGTCCATCTT AATGTACATC 900 ACATCACTCT TCCAAGTTTT GCCTCAACAA GTGAGCATTG AAGCCATCCA GGAAGTGGAA 960 ATGTTGCCAA GGCCACCTAA AGTGACTAAA GAAGAACATT TTCAGTTACA TCATCAAATG 1020 CACTATTCTC AACAGATCAC GGTCAGTCTA GCACAGGGAT ATGAGAGAAC TTCTTCCCCT 1080 AAGCCTCGAT TCAAGAGCTA TGCCTACACA CAGGCTGCTT ATGTCACCAC CTCTGACCCT 1140 ACACGGAGCC CATTTCCTTC ACAGCATTTG GAAGCTCCTG AAGACAAGTC ATTTGGCAGT 1200 TCATTGATGG AGAGTGAAGT AAACCTGGAC CGTTATCAAA CAGCTTTAGA AGAAGTATTA 1260 TCGTGGCTTC TTTCTGCTGA GGACACATTG CAAGCACAAG GAGAGATTTC TAATGATGTG 1320 GAAGTGGTGA AAGACCAGTT TCATACTCAT GAGGGGTACA TGATGGATTT GACAGCCCAT 1380 CAGGGCCGGG TTGGTAATAT TCTACAATTG GGAAGTAAGC TGATTGGAAC AGGAAAATTA 1440 TCAGAAGATG AAGAAACTGA AGTACAAGAG CAGATGAATC TCCTAAATTC AAGATGGGAA 1500 TGCCTCAGGG TAGCTAGCAT GGAAAAACAA AGCAATTTAC ATAGAGTTTT AATGGATCTC 1560 CAGAATCAGA AACTGAAAGA GTTGAATGAC TGGCTAACAA AAACAGAAGA AAGAACAAGG 1620 AAAATGGAGG AAGAGCCTCT TGGACCTGAT CTTGAAGACC TAAAACGCCA AGTACAACAA 1680 CATAAGGTGC TTCAAGAAGA TCTAGAACAA GAACAAGTCA GGGTCAATTCT CTCACTCAC 1740 ATGGTGGTGG TAGTTGATGA ATCTAGTGGA GATCACGCAA CTGCTGCTTTG GAAGAACAA 1800 CTTAAGGAGG TCAATACTGA GTGGGAAAAA TTGAACCTGC ACTCCGCTGAC TGGCAGAGA 1860 AAAATAGATG AGACCCTTGA AAGACTCCAG GAACTTCAAG AGGCCACGGAT GAGCTGGAC 1920 CTCAAGCTGC GCCAAGCTGA GGTGATCAAG GGATCCTGGC AGCCCGTGGGC GATCTCCTC 1980 ATTGACTCTC TCCAAGATCA CCTCGAGAAA GTCAAGGCAC TTCGAGGAGAA ATTGCGCCT 2040 CTGAAAGAGA ACGTGAGCCA CGTCAATGAC CTTGCTCGCC AGCTTACCACT TTGGGCATT 2100 CAGCTCTCAC CGTATAACCT CAGCACTCTG GAAGACCTGA ACACCAGATGG AAGCTTCTG 2160 CAGGTGGCCG TCGAGGACCG AGTCAGGCAG CTGCATGAAG CCCACAGGGAC TTTGGTCCA 2220 GCATCTCAGC ACTTTCTTTC CACGTCTGTC CAGGGTCCCT GGGAGAGAGCC ATCTCGCCA 2280 AACAAAGTGC CCTACTATAT CAACCACGAG ACTCAAACAA CTTGCTGGGAC CATCCCAAA 2340 ATGACAGAGC TCTACCAGTC TTTAGCTGAC CTGAATAATG TCAGATTCTCA GCTTATAGG 2400 ACTGCCATGA AACTCCGAAG ACTGCAGAAG GCCCTTTGCT TGGATCTCTTG AGCCTGTCA 2460 GCTGCATGTG ATGCCTTGGA CCAGCACAAC CTCAAGCAAA ATGACCAGCCC ATGGATATC 2520 CTGCAGATTA TTAAT TGTTT GACCACTATT TATGACCGCC TGGAGCAAGAG CACAACAAT 2580 TTGGTCAACG TCCCTCTCTG CGTGGATATG TGTCTGAACT GGCTGCTGAAT GTTTATGAT 2640 ACGGGACGAA CAGGGAGGAT CCGTGTCCTG TCTTTTAAAA CTGGCATCATT TCCCTGTGT 2700 AAAGCACATT TGGAAGACAA GTACAGATAC CTTTTCAAGC AAGTGGCAAGT TCAACAGGA 2760 TTTTGTGACC AGCGCAGGCT GGGCCTCCTT CTGCATGATT CTATCCAAATT CCAAGACAG 2820 TTGGGTGAAG TTGCATCCTT TGGGGGCAGT AACATTGAGC CAAGTGTCCGG AGCTGCTTC 2880 CAATTTGCTA ATAATAAGCC AGAGATCGAA GCGGCCCTCT TCCTAGACTGG ATGAGACTG 2940 GAACCCCAGT CCATGGTGTG GCTGCCCGTC CTGCACAGAG TGGCTGCTGCA GAAACTGCC 3000 AAGCATCAGG CCAAATGTAA CATCTGCAAA GAGTGTCCAA TCATTGGATTC AGGTACAGG 3060 AGTCTAAAGC ACTTTAATTA TGACATCTGC CAAAGCTGCT TTTTTTCTGGT CGAGTTGCA 3120 AAAGGCCATA AAATGCACTA TCCCATGGTG GAATATTGCA CTCCGACTACA TCAGGAGAA 3180 GATGTTCGAG ACTTTGCCAA GGTACTAAAA AACAAATTTC GAACCAAAAGG TATTTTGCG 3240 AAGCATCCCC GAATGGGCTA CCTGCCAGTG CAGACTGTCT TAGAGGGGGAC AACATGGAA 3300 ACTCCCGTTA CTCTGATCAA CTTCTGGCCA GTAGATTCTG CGCCTGCCTCG TCCCCTCAG 3360 CTTTCACACG ATGATACTCA TTCACGCATT GAACATTATG CTAGCAGGCTA GCAGAAATG 3420 GAAAACAGCA ATGGATCTTA TCTAAATGAT AGCATCTCTC CTAATGAGAGC ATAGATGAT 3480 GAACATTTGT TAATCCAGCA TTACTGCCAA AGTTTGAACC AGGACTCCCCC CTGAGCCAG 3540 CCTCGTAGTC CTGCCCAGAT CTTGATTTCC TTAGAGAGTG AGGAAAGAGGG GAGCTAGAG 3600 AGAATCCTAG CAGATCTTGA GGAAGAAAAC AGGAATCTGC AAGCAGAATAT GACCGTCTA 3660 AAGCAGCAGC ACGAACATAA AGGCCTGTCC CCACTGCCGT CCCCTCCTGAA ATGATGCCC 3720 ACCTCTCCCC AGAGTCCCCG GGATGCTGAG CTCATTGCTG AGGCCAAGCTA CTGCGTCAA 3780 CACAAAGGCC GCCTGGAAGC CAGGATGCAA ATCCTGGAAG ACCACAATAAA CAGCTGGAG 3840 TCACAGTTAC ACAGGCTAAG GCAGCTGCTG GAGCAACCCC AGGCAGAGGCC AAAGTGAAT 3900 GGCACAACGG TGTCCTCTCC TTCTACCTCT CTACAGAGGT CCGACAGCAGT CAGCCTATG 3960 CTGCTCCGAG TGGTTGGCAG TCAAACTTCG GACTCCATGG GTGAGGAAGAT CTTCTCAGT 4020 CCTCCCCAGG ACACAAGCAC AGGGTTAGAG GAGGTGATGG AGCAACTCAAC AACTCCTTC 4080 CCTAGTTCAA GAGGAAGAAA TACCCCTGGA AAGCCAATGA GAGAGGACACA ATGTAGGAA 4140 GTCTTTTCCA CATGGCAGAT GATTTGGGCA GAGCGATGGA GTCCTTAGTAT CAGTCATGA 4200 CAGATGAAGA AGGAGCAGAA TAAATG TTTT ACAACTCCTG ATTCCCGCATG GTTTTTATA 4260 ATATTCATAC AACAAAGAGG ATTAGACAGT AAGAGTTTAC AAGAAATAAAT CTATATTTT 4320 TGTGAAGGGT AGTGGTATTA TACTGTAGAT TTCAGTAGTT TCTAAGTCTGT TATTGTTTT 4380 GTTGGGGATC CTCTAGAGTC GA 4 Length of array: 1310 Array types: Amino acid topology: Type of linear sequence: Protein sequence Met Leu Trp Trp Glu Glu Val Glu Asp Cys Tyr Glu Arg Glu Asp 15 Val Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser 30 Lys Phe Gly Lys Gln His Ile Glu Asn Leu Phe Ser Asp Leu Gln 45 Asp Gly Arg Arg Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln 60 Lys Leu Pro Lys Glu Lys Gly Ser Thr Arg Val His Ala Leu Asn 75 Asn Val Asn Lys Ala Leu Arg Val Leu Gln Asn Asn Asn Val Asp 90 Leu Val Asn Ile Gly Ser Thr Asp Ile Val Asp Gly Asn His Lys 105 Leu Thr Leu Gly Leu Ile Trp Asn Ile Ile Leu His Trp Gln Val 120 Lys Asn Val Met Lys Asn Ile Met Ala Gly Leu Gln Gln Thr Asn 135 Ser Glu Lys Ile Leu Leu Ser Trp Val Arg Gln Ser Thr Arg Asn 150 Tyr Pro Gln Val Asn Val Ile Asn Phe Thr Thr Ser Trp Ser Asp 165 Gly Leu Ala Leu Asn Ala Leu Ile His Ser His Arg Pro Asp Leu 180 Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala Thr Gln Arg 195 Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly Ile Glu 210 Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys 225 Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro 240 Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Met Leu Pro 255 Arg Pro Pro Lys Val Thr Lys Glu Glu His Phe Gln Leu His His 270 Gln Met His Tyr Ser Gln Gln Ile Thr Val Ser Leu Ala Gln Gly 285 Tyr Glu Arg Thr Ser Ser Pro Lys Pro Arg Phe Lys Ser Tyr Ala 300 Tyr Thr Gln Ala Ala Tyr Val Thr Thr Ser Asp Pro Thr Arg Ser 315 Pro Phe Pro Ser Gln His Leu Glu Ala Pro Glu Asp Lys Ser Phe 330 Gly Ser Ser Leu Met Glu Ser Glu Val Asn Leu Asp Arg Tyr Gln 345 Thr Ala Leu Glu Glu Val Leu Ser Trp Leu Leu Ser Ala Glu Asp 360 Thr Leu Gln Ala Gln Gly Glu Ile Ser Asn Asp Val Glu Val Val 375 Lys Asp Gln Phe His Thr His Glu Gly Tyr Met Met Asp Leu Thr 390 Ala His Gln Gly Arg Val Gly Asn Ile Leu Gln Leu Gly Ser Lys 405 Leu Ile Gly Thr Gly Lys Leu Ser Glu Asp Glu Glu Thr Glu Val 420 Gln Glu Gln Met Asn Leu Leu Asn Ser Arg Trp Glu Cys Leu Arg 435 Val Ala Ser Met Glu Lys Gln Ser Asn Leu His Arg Val Leu Met 450 Asp Leu Gln Asn Gln Lys Leu Lys Glu Leu Asn Asp Trp Leu Thr 465 Lys Thr Glu Glu Arg Thr Arg Lys Met Glu Glu Glu Pro Leu Gly 480 Pro Asp Leu Glu Asp Leu Lys Arg Gln Val Gln Gln His Lys Val 495 Leu Gln Glu Asp Leu Glu Gln Glu Gln Val Arg Val Asn Ser Leu 510 Thr His Met Val Val Val Val Asp Glu Ser Ser Gly Asp His Ala 525 Thr Ala Ala Leu Glu Glu Gln Leu Lys Glu Val Asn Thr Glu Trp 540 Glu Lys Leu Asn Leu His Ser Ala Asp Trp Gln Arg Lys Ile Asp 555 Glu Thr Leu Glu Arg Leu Gln Glu Leu Gln Glu Ala Thr Asp Glu 570 Leu Asp Leu Lys Leu Arg Gln Ala Glu Val Ile Lys Gly Ser Trp 585 Gln Pro Val Gly Asp Leu Leu Ile Asp Ser Leu Gln Asp His Leu 600 Glu Lys Val Lys Ala Leu Arg Gly Glu Ile Ala Pro Leu Lys Glu 615 Asn Val Ser His Val Asn Asp Leu Ala Arg Gln Leu Thr Thr Leu 630 Gly Ile Gln Leu Ser Pro Tyr Asn Leu Ser Thr Leu Glu Asp Leu 645 Asn Thr Arg Trp Lys Leu Leu Gln Val Ala Val Glu Asp Arg Val 660 Arg Gln Leu His Glu Ala His Arg Asp Phe Gly Pro Ala Ser Gln 675 His Phe Leu Ser Thr Ser Val Gln Gly Pro Trp Glu Arg Ala Ile 690 Ser Pro Asn Lys Val Pro Tyr Tyr Ile Asn His Glu Thr Gln Thr 705 Thr Cys Trp Asp His Pro Lys Met Thr Glu Leu Tyr Gln Ser Leu 720 Ala Asp Leu Asn Asn Val Arg Phe Ser Ala Tyr Arg Thr Ala Met 735 Lys Leu Arg Arg Leu Gln Lys Ala Leu Cys Leu Asp Leu Leu Ser 750 Leu Ser Ala Ala Cys Asp Ala Leu Asp Gln His Asn Leu Lys Gln 765 Asn Asp Gln Pro Met Asp Ile Leu Gln Ile Ile Asn Cys Leu Thr 780 Thr Ile Tyr Asp Arg Leu Glu Gln Glu His Asn Asn Leu Val Asn 795 Val Pro Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu Asn Val 810 Tyr Asp Thr Gly Arg Thr Gly Arg Ile Arg Val Leu Ser Phe Lys 825 Thr Gly Ile Ile Ser Leu Cys Lys Ala His Leu Glu Asp Lys Tyr 840 Arg Tyr Leu Phe Lys Gln Val Ala Ser Ser Thr Gly Phe Cys Asp 855 Gln Arg Arg Leu Gly Leu Leu Leu His Asp Ser Ile Gln Ile Pro 870 Arg Gln Leu Gly Glu Val Ala Ser Phe Gly Gly Ser Asn Ile Glu 885 Pro Ser Val Arg Ser Cys Phe Gln Phe Ala Asn Asn Lys Pro Glu 900 Ile Glu Ala Ala Leu Phe Leu Asp Trp Met Arg Leu Glu Pro Gln 915 Ser Met Val Trp Leu Pro Val Leu His Arg Val Ala Ala Ala Glu 930 Thr Ala Lys His Gln Ala Lys Cys Asn Ile Cys Lys Glu Cys Pro 945 Ile Ile Gly Phe Arg Tyr Arg Ser Leu Lys His Phe Asn Tyr Asp 960 Ile Cys Gln Ser Cys Phe Phe Ser Gly Arg Val Ala Lys Gly His 975 Lys Met His Tyr Pro Met Val Glu Tyr Cys Thr Pro Thr Thr Ser 990 Gly Glu Asp Val Arg Asp Phe Ala Lys Val Leu Lys Asn Lys Phe 1005 Arg Thr Lys Arg Tyr Phe Ala Lys His Pro Arg Met Gly Tyr Leu 1020 Pro Val Gln Thr Val Leu Glu Gly Asp Asn Met Glu Thr Pro Val 1035 Thr Leu Ile Asn Phe Trp Pro Val Asp Ser Ala Pro Ala Ser Ser 1050 Pro Gln Leu Ser His Asp Asp Thr His Ser Arg Ile Glu His Tyr 1065 Ala Ser Arg Leu Ala Glu Met Glu Asn Ser Asn Gly Ser Tyr Leu 1080 Asn Asp Ser Ile Ser Pro Asn Glu Ser Ile Asp Asp Glu His Leu 1095 Leu Ile Gln His Tyr Cys Gln Ser Leu Asn Gln Asp Ser Pro Leu 1110 Ser Gln Pro Arg Ser Pro Ala Gln Ile Leu Ile Ser Leu Glu Ser 1125 Glu Glu Arg Gly Glu Leu Glu Arg Ile Leu Ala Asp Leu Glu Glu 1140 Glu Asn Arg Asn Leu Gln Ala Glu Tyr Asp Arg Leu Lys Gln Gln 1155 His Glu His Lys Gly Leu Ser Pro Leu Pro Ser Pro Pro Glu Met 1170 Met Pro Thr Ser Pro Gln Ser Pro Arg Asp Ala Glu Leu Ile Ala 1185 Glu Ala Lys Leu Leu Arg Gln His Lys Gly Arg Leu Glu Ala Arg 1200 Met Gln Ile Leu Glu Asp His Asn Lys Gln Leu Glu Ser Gln Leu 1215 His Arg Leu Arg Gln Leu Leu Glu Gln Pro Gln Ala Glu Ala Lys 1230 Val Asn Gly Thr Thr Val Ser Ser Pro Ser Thr Ser Leu Gln Arg 1245 Ser Asp Ser Ser Gln Pro Met Leu Leu Arg Val Val Gly Ser Gln 1260 Thr Ser Asp Ser Met Gly Glu Glu Asp Leu Leu Ser Pro Pro Gln 1275 Asp Thr Ser Thr Gly Leu Glu Glu Val Met Glu Gln Leu Asn Asn 1290 Ser Phe Pro Ser Ser Arg Gly Arg Asn Thr Pro Gly Lys Pro Met 1305 Arg Glu Asp Thr Met *** 1310 SEQ ID NO: 5 Sequence length: 4402 Array type: Number of nucleic acid strands: Both topologies: Type of linear sequence: Features of cDNA to mRNA sequence: active-site sequence CGGCCGCTCT AGAGGATCCC CGGGTACCGA GCTCGAATTC CGGAACTCCC GGAGAAAAAC 60 GAATAGGAAA AACTGAAGTG TTACTTTTTT TAAAGCTGCT GAAGTTTGTT GGTTTCTCAT 120 TGTTTTTAAG CCTACTGGAG CAATAAAGTT TGAAGAACTT TTACCAGGTT TTTTTTATCG 180 CTGCCTTGAT ATACACTTTT CAAAATGCTT TGGTGGGAAG AAGTAGAGGA CTGTTATGAA 240 AGAGAAGATG TTCAAAAGAA AACATTCACA AAATGGGTAA ATGCACAATT TTCTAAGTTT 300 GGGAAGCAGC ATATTGAGAA CCTCTTCAGT GACCTACAGG ATGGGAGGCG CCTCCTAGAC 360 CTCCTCGAAG GCCTGACAGG GCAAAAACTG CCAAAAGAAA AAGGATCCAC AAGAGTTCAT 420 GCCCTGAACA ATGTCAACAA GGCACTGCGG GTTTTGCAGA ACAATAATGT TGATTTAGTG 480 AATATTGGAA GTACTGACAT CGTAGATGGA AATCATAAAC TGACTCTTGG TTTGATTTGG 540 AATATAATCC TCCACTGGCA GGTCAAAAAT GTAATGAAAA ATATCATGGC TGGATTGCAA 600 CAAACCAACA GTGAAAAGAT TCTCCTGAGC TGGGTCCGAC AATCAACTCG TAATTATCCA 660 CAGGTTAATG TAATCAACTT CACCACCAGC TGGTCTGATG GCCTGGCTTT GAATGCTCTC 720 ATCCATAGTC ATAGGCCAGA CCTATTTGAC TGGAATAGTG TGGTTTGCCA GCAGTCAGCC 780 ACACAACGAC TGGAACATGC ATTCAACATC GCCAGATATC AATTAGGCAT AGAGAAACTA 840 CT CGATCCTG AAGATGTTGA TACCACCTAT CCAGATAAGA AGTCCATCTT AATGTACATC 900 ACATCACTCT TCCAAGTTTT GCCTCAACAA GTGAGCATTG AAGCCATCCA GGAAGTGGAA 960 ATGTTGCCAA GGCCACCTAA AGTGACTAAA GAAGAACATT TTCAGTTACA TCATCAAATG 1020 CACTATTCTC AACAGATCAC GGTCAGTCTA GCACAGGGAT ATGAGAGAAC TTCTTCCCCT 1080 AAGCCTCGAT TCAAGAGCTA TGCCTACACA CAGGCTGCTT ATGTCACCAC CTCTGACCCT 1140 ACACGGAGCC CATTTCCTTC ACAGCATTTG GAAGCTCCTG AAGACAAGTC ATTTGGCAGT 1200 TCATTGATGG AGAGTGAAGT AAACCTGGAC CGTTATCAAA CAGCTTTAGA AGAAGTATTA 1260 TCGTGGCTTC TTTCTGCTGA GGACACATTG CAAGCACAAG GAGAGATTTC TAATGATGTG 1320 GAAGTGGTGA AAGACCAGTT TCATACTCAT GAGGGGTACA TGATGGATTT GACAGCCCAT 1380 CAGGGCCGGG TTGGTAATAT TCTACAATTG GGAAGTAAGC TGATTGGAAC AGGAAAATTA 1440 TCAGAAGATG AAGAAACTGA AGTACAAGAG CAGATGAATC TCCTAAATTC AAGATGGGAA 1500 TGCCTCAGGG TAGCTAGCAT GGAAAAACAA AGCAATTTAC ATAGAGTTTT AATGGATCTC 1560 CAGAATCAGA AACTGAAAGA GTTGAATGAC TGGCTAACAA AAACAGAAGA AAGAACAAGG 1620 AAAATGGAGG AAGAGCCTCT TGGACCTGAT CTTGAAGACC TAAAACGCCA AGTACAACAA 1680 CATAAGGTGC TTCAAGAAGA TCTAGAACAA GAACAAGTCA GGGTCAATTC TCTCACTCAC 1740 ATGGTGGTGG TAGTTGATGA ATCTAGTGGA GATCACGCAA CTGCTGCTTT GGAAGAACAA 1800 CTTAAGGTAT TGGGAGATCG ATGGGCAAAC ATCTGTAGAT GGACAGAAGA CCGCTGGGTT 1860 CTTTTACAAG ACATCCTTCT CAAATGGCAA CGTCTTACTG AAGAACAGTG CCTTTTTAGT 1920 GCATGGCTTT CAGAAAAAGA AGATGCAGTG AACAAGATTC ACACAACTGG CTTTAAAGAT 1980 CAAAATGAAA TGTTATCAAG TCTCGAGAAA GTCAAGGCAC TTCGAGGAGA AATTGCGCCT 2040 CTGAAAGAGA ACGTGAGCCA CGTCAATGAC CTTGCTCGCC AGCTTACCAC TTTGGGCATT 2100 CAGCTCTCAC CGTATAACCT CAGCACTCTG GAAGACCTGA ACACCAGATG GAAGCTTCTG 2160 CAGGTGGCCG TCGAGGACCG AGTCAGGCAG CTGCATGAAG CCCACAGGGA CTTTGGTCCA 2220 GCATCTCAGC ACTTTCTTTC CACGTCTGTC CAGGGTCCCT GGGAGAGAGC CATCTCGCCA 2280 AACAAAGTGC CCTACTATAT CAACCACGAG ACTCAAACAA CTTGCTGGGA CCATCCCAAA 2340 ATGACAGAGC TCTACCAGTC TTTAGCTGAC CTGAATAATG TCAGATTCTC AGCTTATAGG 2400 ACTGCCATGA AACTCCGAAG ACTGCAGAAG GCCCTTTGCT TGGATCTCTT GAGCCTGTCA 2460 GCTGCATGTG ATGCCTTGGA CCAGCACAAC CTCAAGCAAA ATGACCAGCC CATGGATATC 2520 CTGCAGATTA TTAAT TGTTT GACCACTATT TATGACCGCC TGGAGCAAGA GCACAACAAT 2580 TTGGTCAACG TCCCTCTCTG CGTGGATATG TGTCTGAACT GGCTGCTGAA TGTTTATGAT 2640 ACGGGACGAA CAGGGAGGAT CCGTGTCCTG TCTTTTAAAA CTGGCATCAT TTCCCTGTGT 2700 AAAGCACATT TGGAAGACAA GTACAGATAC CTTTTCAAGC AAGTGGCAAG TTCAACAGGA 2760 TTTTGTGACC AGCGCAGGCT GGGCCTCCTT CTGCATGATT CTATCCAAAT TCCAAGACAG 2820 TTGGGTGAAG TTGCATCCTT TGGGGGCAGT AACATTGAGC CAAGTGTCCG GAGCTGCTTC 2880 CAATTTGCTA ATAATAAGCC AGAGATCGAA GCGGCCCTCT TCCTAGACTG GATGAGACTG 2940 GAACCCCAGT CCATGGTGTG GCTGCCCGTC CTGCACAGAG TGGCTGCTGC AGAAACTGCC 3000 AAGCATCAGG CCAAATGTAA CATCTGCAAA GAGTGTCCAA TCATTGGATT CAGGTACAGG 3060 AGTCTAAAGC ACTTTAATTA TGACATCTGC CAAAGCTGCT TTTTTTCTGG TCGAGTTGCA 3120 AAAGGCCATA AAATGCACTA TCCCATGGTG GAATATTGCA CTCCGACTAC ATCAGGAGAA 3180 GATGTTCGAG ACTTTGCCAA GGTACTAAAA AACAAATTTC GAACCAAAAG GTATTTTGCG 3240 AAGCATCCCC GAATGGGCTA CCTGCCAGTG CAGACTGTCT TAGAGGGGGA CAACATGGAA 3300 ACTCCCGTTA CTCTGATCAA CTTCTGGCCA GTAGATTCTG CGCCTGCCTC GTCCCCTCAG 3360 CTTTCACACG ATGATACTCA TTCACGCATT GAACATTATG CTAGCAGGCT AGCAGAAATG 3420 GAAAACAGCA ATGGATCTTA TCTAAATGAT AGCATCTCTC CTAATGAGAG CATAGATGAT 3480 GAACATTTGT TAATCCAGCA TTACTGCCAA AGTTTGAACC AGGACTCCCC CCTGAGCCAG 3540 CCTCGTAGTC CTGCCCAGAT CTTGATTTCC TTAGAGAGTG AGGAAAGAGG GGAGCTAGAG 3600 AGAATCCTAG CAGATCTTGA GGAAGAAAAC AGGAATCTGC AAGCAGAATA TGACCGTCTA 3660 AAGCAGCAGC ACGAACATAA AGGCCTGTCC CCACTGCCGT CCCCTCCTGA AATGATGCCC 3720 ACCTCTCCCC AGAGTCCCCG GGATGCTGAG CTCATTGCTG AGGCCAAGCT ACTGCGTCAA 3780 CACAAAGGCC GCCTGGAAGC CAGGATGCAA ATCCTGGAAG ACCACAATAA ACAGCTGGAG 3840 TCACAGTTAC ACAGGCTAAG GCAGCTGCTG GAGCAACCCC AGGCAGAGGC CAAAGTGAAT 3900 GGCACAACGG TGTCCTCTCC TTCTACCTCT CTACAGAGGT CCGACAGCAG TCAGCCTATG 3960 CTGCTCCGAG TGGTTGGCAG TCAAACTTCG GACTCCATGG GTGAGGAAGA TCTTCTCAGT 4020 CCTCCCCAGG ACACAAGCAC AGGGTTAGAG GAGGTGATGG AGCAACTCAA CAACTCCTTC 4080 CCTAGTTCAA GAGGAAGAAA TACCCCTGGA AAGCCAATGA GAGAGGACAC AATGTAGGAA 4140 GTCTTTTCCA CATGGCAGAT GATTTGGGCA GAGCGATGGA GTCCTTAGTA TCAGTCATGA 4200 CAGATGAAGA AGGAGCAGAA TAAATG TTTT ACAACTCCTG ATTCCCGCAT GGTTTTTATA 4260 ATATTCATAC AACAAAGAGG ATTAGACAGT AAGAGTTTAC AAGAAATAAA TCTATATTTT 4320 TGTGAAGGGT AGTGGTATTA TACTGTAGAT TTCAGTAGTT TCTAAGTCTG TTATTGTTTT 4380 GTTGGGGATC CTCTAGAGTC GA 6 Length of array: 1310 Array types: Amino acid topology: Type of linear sequence: Protein sequence Met Leu Trp Trp Glu Glu Val Glu Asp Cys Tyr Glu Arg Glu Asp 15 Val Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser 30 Lys Phe Gly Lys Gln His Ile Glu Asn Leu Phe Ser Asp Leu Gln 45 Asp Gly Arg Arg Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln 60 Lys Leu Pro Lys Glu Lys Gly Ser Thr Arg Val His Ala Leu Asn 75 Asn Val Asn Lys Ala Leu Arg Val Leu Gln Asn Asn Asn Val Asp 90 Leu Val Asn Ile Gly Ser Thr Asp Ile Val Asp Gly Asn His Lys 105 Leu Thr Leu Gly Leu Ile Trp Asn Ile Ile Leu His Trp Gln Val 120 Lys Asn Val Met Lys Asn Ile Met Ala Gly Leu Gln Gln Thr Asn 135 Ser Glu Lys Ile Leu Leu Ser Trp Val Arg Gln Ser Thr Arg Asn 150 Tyr Pro Gln Val Asn Val Ile Asn Phe Thr Thr Ser Trp Ser Asp 165 Gly Leu Ala Leu Asn Ala Leu Ile His Ser His Arg Pro Asp Leu 180 Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala Thr Gln Arg 195 Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly Ile Glu 210 Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys 225 Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro 240 Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Met Leu Pro 255 Arg Pro Pro Lys Val Thr Lys Glu Glu His Phe Gln Leu His His 270 Gln Met His Tyr Ser Gln Gln Ile Thr Val Ser Leu Ala Gln Gly 285 Tyr Glu Arg Thr Ser Ser Pro Lys Pro Arg Phe Lys Ser Tyr Ala 300 Tyr Thr Gln Ala Ala Tyr Val Thr Thr Ser Asp Pro Thr Arg Ser 315 Pro Phe Pro Ser Gln His Leu Glu Ala Pro Glu Asp Lys Ser Phe 330 Gly Ser Ser Leu Met Glu Ser Glu Val Asn Leu Asp Arg Tyr Gln 345 Thr Ala Leu Glu Glu Val Leu Ser Trp Leu Leu Ser Ala Glu Asp 360 Thr Leu Gln Ala Gln Gly Glu Ile Ser Asn Asp Val Glu Val Val 375 Lys Asp Gln Phe His Thr His Glu Gly Tyr Met Met Asp Leu Thr 390 Ala His Gln Gly Arg Val Gly Asn Ile Leu Gln Leu Gly Ser Lys 405 Leu Ile Gly Thr Gly Lys Leu Ser Glu Asp Glu Glu Thr Glu Val 420 Gln Glu Gln Met Asn Leu Leu Asn Ser Arg Trp Glu Cys Leu Arg 435 Val Ala Ser Met Glu Lys Gln Ser Asn Leu His Arg Val Leu Met 450 Asp Leu Gln Asn Gln Lys Leu Lys Glu Leu Asn Asp Trp Leu Thr 465 Lys Thr Glu Glu Arg Thr Arg Lys Met Glu Glu Glu Pro Leu Gly 480 Pro Asp Leu Glu Asp Leu Lys Arg Gln Val Gln Gln His Lys Val 495 Leu Gln Glu Asp Leu Glu Gln Glu Gln Val Arg Val Asn Ser Leu 510 Thr His Met Val Val Val Val Asp Glu Ser Ser Gly Asp His Ala 525 Thr Ala Ala Leu Glu Glu Gln Leu Lys Val Leu Gly Asp Arg Trp 540 Ala Asn Ile Cys Arg Trp Thr Glu Asp Arg Trp Val Leu Leu Gln 555 Asp Ile Leu Leu Lys Trp Gln Arg Leu Thr Glu Glu Gln Cys Leu 570 Phe Ser Ala Trp Leu Ser Glu Lys Glu Asp Ala Val Asn Lys Ile 585 His Thr Thr Gly Phe Lys Asp Gln Asn Glu Met Leu Ser Ser Leu 600 Glu Lys Val Lys Ala Leu Arg Gly Glu Ile Ala Pro Leu Lys Glu 615 Asn Val Ser His Val Asn Asp Leu Ala Arg Gln Leu Thr Thr Leu 630 Gly Ile Gln Leu Ser Pro Tyr Asn Leu Ser Thr Leu Glu Asp Leu 645 Asn Thr Arg Trp Lys Leu Leu Gln Val Ala Val Glu Asp Arg Val 660 Arg Gln Leu His Glu Ala His Arg Asp Phe Gly Pro Ala Ser Gln 675 His Phe Leu Ser Thr Ser Val Gln Gly Pro Trp Glu Arg Ala Ile 690 Ser Pro Asn Lys Val Pro Tyr Tyr Ile Asn His Glu Thr Gln Thr 705 Thr Cys Trp Asp His Pro Lys Met Thr Glu Leu Tyr Gln Ser Leu 720 Ala Asp Leu Asn Asn Val Arg Phe Ser Ala Tyr Arg Thr Ala Met 735 Lys Leu Arg Arg Leu Gln Lys Ala Leu Cys Leu Asp Leu Leu Ser 750 Leu Ser Ala Ala Cys Asp Ala Leu Asp Gln His Asn Leu Lys Gln 765 Asn Asp Gln Pro Met Asp Ile Leu Gln Ile Ile Asn Cys Leu Thr 780 Thr Ile Tyr Asp Arg Leu Glu Gln Glu His Asn Asn Leu Val Asn 795 Val Pro Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu Asn Val 810 Tyr Asp Thr Gly Arg Thr Gly Arg Ile Arg Val Leu Ser Phe Lys 825 Thr Gly Ile Ile Ser Leu Cys Lys Ala His Leu Glu Asp Lys Tyr 840 Arg Tyr Leu Phe Lys Gln Val Ala Ser Ser Thr Gly Phe Cys Asp 855 Gln Arg Arg Leu Gly Leu Leu Leu His Asp Ser Ile Gln Ile Pro 870 Arg Gln Leu Gly Glu Val Ala Ser Phe Gly Gly Ser Asn Ile Glu 885 Pro Ser Val Arg Ser Cys Phe Gln Phe Ala Asn Asn Lys Pro Glu 900 Ile Glu Ala Ala Leu Phe Leu Asp Trp Met Arg Leu Glu Pro Gln 915 Ser Met Val Trp Leu Pro Val Leu His Arg Val Ala Ala Ala Glu 930 Thr Ala Lys His Gln Ala Lys Cys Asn Ile Cys Lys Glu Cys Pro 945 Ile Ile Gly Phe Arg Tyr Arg Ser Leu Lys His Phe Asn Tyr Asp 960 Ile Cys Gln Ser Cys Phe Phe Ser Gly Arg Val Ala Lys Gly His 975 Lys Met His Tyr Pro Met Val Glu Tyr Cys Thr Pro Thr Thr Ser 990 Gly Glu Asp Val Arg Asp Phe Ala Lys Val Leu Lys Asn Lys Phe 1005 Arg Thr Lys Arg Tyr Phe Ala Lys His Pro Arg Met Gly Tyr Leu 1020 Pro Val Gln Thr Val Leu Glu Gly Asp Asn Met Glu Thr Pro Val 1035 Thr Leu Ile Asn Phe Trp Pro Val Asp Ser Ala Pro Ala Ser Ser 1050 Pro Gln Leu Ser His Asp Asp Thr His Ser Arg Ile Glu His Tyr 1065 Ala Ser Arg Leu Ala Glu Met Glu Asn Ser Asn Gly Ser Tyr Leu 1080 Asn Asp Ser Ile Ser Pro Asn Glu Ser Ile Asp Asp Glu His Leu 1095 Leu Ile Gln His Tyr Cys Gln Ser Leu Asn Gln Asp Ser Pro Leu 1110 Ser Gln Pro Arg Ser Pro Ala Gln Ile Leu Ile Ser Leu Glu Ser 1125 Glu Glu Arg Gly Glu Leu Glu Arg Ile Leu Ala Asp Leu Glu Glu 1140 Glu Asn Arg Asn Leu Gln Ala Glu Tyr Asp Arg Leu Lys Gln Gln 1155 His Glu His Lys Gly Leu Ser Pro Leu Pro Ser Pro Pro Glu Met 1170 Met Pro Thr Ser Pro Gln Ser Pro Arg Asp Ala Glu Leu Ile Ala 1185 Glu Ala Lys Leu Leu Arg Gln His Lys Gly Arg Leu Glu Ala Arg 1200 Met Gln Ile Leu Glu Asp His Asn Lys Gln Leu Glu Ser Gln Leu 1215 His Arg Leu Arg Gln Leu Leu Glu Gln Pro Gln Ala Glu Ala Lys 1230 Val Asn Gly Thr Thr Val Ser Ser Pro Ser Thr Ser Leu Gln Arg 1245 Ser Asp Ser Ser Gln Pro Met Leu Leu Arg Val Val Gly Ser Gln 1260 Thr Ser Asp Ser Met Gly Glu Glu Asp Leu Leu Ser Pro Pro Gln 1275 Asp Thr Ser Thr Gly Leu Glu Glu Val Met Glu Gln Leu Asn Asn 1290 Ser Phe Pro Ser Ser Arg Gly Arg Asn Thr Pro Gly Lys Pro Met 1305 Arg Glu Asp Thr Met *** 1310 SEQ ID NO: 7 Length of array: 4075 Array type: Number of nucleic acid strands: Both topologies: Type of linear sequence: Features of cDNA to mRNA sequence: active-site sequence CGGCCGCTCT AGAGGATCCC CGGGTACCGA GCTCGAATTC CGGAACTCCC GGAGAAAAAC 60 GAATAGGAAA AACTGAAGTG TTACTTTTTT TAAAGCTGCT GAAGTTTGTT GGTTTCTCAT 120 TGTTTTTAAG CCTACTGGAG CAATAAAGTT TGAAGAACTT TTACCAGGTT TTTTTTATCG 180 CTGCCTTGAT ATACACTTTT CAAAATGCTT TGGTGGGAAG AAGTAGAGGA CTGTTATGAA 240 AGAGAAGATG TTCAAAAGAA AACATTCACA AAATGGGTAA ATGCACAATT TTCTAAGTTT 300 GGGAAGCAGC ATATTGAGAA CCTCTTCAGT GACCTACAGG ATGGGAGGCG CCTCCTAGAC 360 CTCCTCGAAG GCCTGACAGG GCAAAAACTG CCAAAAGAAA AAGGATCCAC AAGAGTTCAT 420 GCCCTGAACA ATGTCAACAA GGCACTGCGG GTTTTGCAGA ACAATAATGT TGATTTAGTG 480 AATATTGGAA GTACTGACAT CGTAGATGGA AATCATAAAC TGACTCTTGG TTTGATTTGG 540 AATATAATCC TCCACTGGCA GGTCAAAAAT GTAATGAAAA ATATCATGGC TGGATTGCAA 600 CAAACCAACA GTGAAAAGAT TCTCCTGAGC TGGGTCCGAC AATCAACTCG TAATTATCCA 660 CAGGTTAATG TAATCAACTT CACCACCAGC TGGTCTGATG GCCTGGCTTT GAATGCTCTC 720 ATCCATAGTC ATAGGCCAGA CCTATTTGAC TGGAATAGTG TGGTTTGCCA GCAGTCAGCC 780 ACACAACGAC TGGAACATGC ATTCAACATC GCCAGATATC AATTAGGCAT AGAGAAACTA 840 CT CGATCCTG AAGATGTTGA TACCACCTAT CCAGATAAGA AGTCCATCTT AATGTACATC 900 ACATCACTCT TCCAAGTTTT GCCTCAACAA GTGAGCATTG AAGCCATCCA GGAAGTGGAA 960 ATGTTGCCAA GGCCACCTAA AGTGACTAAA GAAGAACATT TTCAGTTACA TCATCAAATG 1020 CACTATTCTC AACAGATCAC GGTCAGTCTA GCACAGGGAT ATGAGAGAAC TTCTTCCCCT 1080 AAGCCTCGAT TCAAGAGCTA TGCCTACACA CAGGCTGCTT ATGTCACCAC CTCTGACCCT 1140 ACACGGAGCC CATTTCCTTC ACAGCATTTG GAAGCTCCTG AAGACAAGTC ATTTGGCAGT 1200 TCATTGATGG AGAGTGAAGT AAACCTGGAC CGTTATCAAA CAGCTTTAGA AGAAGTATTA 1260 TCGTGGCTTC TTTCTGCTGA GGACACATTG CAAGCACAAG GAGAGATTTC TAATGATGTG 1320 GAAGTGGTGA AAGACCAGTT TCATACTCAT GAGGGGTACA TGATGGATTT GACAGCCCAT 1380 CAGGGCCGGG TTGGTAATAT TCTACAATTG GGAAGTAAGC TGATTGGAAC AGGAAAATTA 1440 TCAGAAGATG AAGAAACTGA AGTACAAGAG CAGATGAATC TCCTAAATTC AAGATGGGAA 1500 TGCCTCAGGG TAGCTAGCAT GGAAAAACAA AGCAATTTAC ATAGAGTTTT AATGGATCTC 1560 CAGAATCAGA AACTGAAAGA GTTGAATGAC TGGCTAACAA AAACAGAAGA AAGAACAAGG 1620 AAAATGGAGG AAGAGCCTCT TGGACCTGAT CTTGAAGACC TAAAACGCCA AGTACAACAA 1680 CATAAGGTGC TTCAAGAAGA TCTAGAACAA GAACAAGTCA GGGTCAATTC TCTCACTCAC 1740 ATGGTGGTGG TAGTTGATGA ATCTAGTGGA GATCACGCAA CTGCTGCTTT GGAAGAACAA 1800 CTTAAGGTAT TGAACACCAG ATGGAAGCTT CTGCAGGTGG CCGTCGAGGA CCGAGTCAGG 1860 CAGCTGCATG AAGCCCACAG GGACTTTGGT CCAGCATCTC AGCACTTTCT TTCCACGTCT 1920 GTCCAGGGTC CCTGGGAGAG AGCCATCTCG CCAAACAAAG TGCCCTACTA TATCAACCAC 1980 GAGACTCAAA CAACTTGCTG GGACCATCCC AAAATGACAG AGCTCTACCA GTCTTTAGCT 2040 GACCTGAATA ATGTCAGATT CTCAGCTTAT AGGACTGCCA TGAAACTCCG AAGACTGCAG 2100 AAGGCCCTTT GCTTGGATCT CTTGAGCCTG TCAGCTGCAT GTGATGCCTT GGACCAGCAC 2160 AACCTCAAGC AAAATGACCA GCCCATGGAT ATCCTGCAGA TTATTAATTG TTTGACCACT 2220 ATTTATGACC GCCTGGAGCA AGAGCACAAC AATTTGGTCA ACGTCCCTCT CTGCGTGGAT 2280 ATGTGTCTGA ACTGGCTGCT GAATGTTTAT GATACGGGAC GAACAGGGAG GATCCGTGTC 2340 CTGTCTTTTA AAACTGGCAT CATTTCCCTG TGTAAAGCAC ATTTGGAAGA CAAGTACAGA 2400 TACCTTTTCA AGCAAGTGGC AAGTTCAACA GGATTTTGTG ACCAGCGCAG GCTGGGCCTC 2460 CTTCTGCATG ATTCTATCCA AATTCCAAGA CAGTTGGGTG AAGTTGCATC CTTTGGGGGC 2520 AGTAACATTG AGCCA AGTGT CCGGAGCTGC TTCCAATTTG CTAATAATAA GCCAGAGATC 2580 GAAGCGGCCC TCTTCCTAGA CTGGATGAGA CTGGAACCCC AGTCCATGGT GTGGCTGCCC 2640 GTCCTGCACA GAGTGGCTGC TGCAGAAACT GCCAAGCATC AGGCCAAATG TAACATCTGC 2700 AAAGAGTGTC CAATCATTGG ATTCAGGTAC AGGAGTCTAA AGCACTTTAA TTATGACATC 2760 TGCCAAAGCT GCTTTTTTTC TGGTCGAGTT GCAAAAGGCC ATAAAATGCA CTATCCCATG 2820 GTGGAATATT GCACTCCGAC TACATCAGGA GAAGATGTTC GAGACTTTGC CAAGGTACTA 2880 AAAAACAAAT TTCGAACCAA AAGGTATTTT GCGAAGCATC CCCGAATGGG CTACCTGCCA 2940 GTGCAGACTG TCTTAGAGGG GGACAACATG GAAACTCCCG TTACTCTGAT CAACTTCTGG 3000 CCAGTAGATT CTGCGCCTGC CTCGTCCCCT CAGCTTTCAC ACGATGATAC TCATTCACGC 3060 ATTGAACATT ATGCTAGCAG GCTAGCAGAA ATGGAAAACA GCAATGGATC TTATCTAAAT 3120 GATAGCATCT CTCCTAATGA GAGCATAGAT GATGAACATT TGTTAATCCA GCATTACTGC 3180 CAAAGTTTGA ACCAGGACTC CCCCCTGAGC CAGCCTCGTA GTCCTGCCCA GATCTTGATT 3240 TCCTTAGAGA GTGAGGAAAG AGGGGAGCTA GAGAGAATCC TAGCAGATCT TGAGGAAGAA 3300 AACAGGAATC TGCAAGCAGA ATATGACCGT CTAAAGCAGC AGCACGAACA TAAAGGCCTG 3360 TCCCCACTGC CGTCCCCTCC TGAAATGATG CCCACCTCTC CCCAGAGTCC CCGGGATGCT 3420 GAGCTCATTG CTGAGGCCAA GCTACTGCGT CAACACAAAG GCCGCCTGGA AGCCAGGATG 3480 CAAATCCTGG AAGACCACAA TAAACAGCTG GAGTCACAGT TACACAGGCT AAGGCAGCTG 3540 CTGGAGCAAC CCCAGGCAGA GGCCAAAGTG AATGGCACAA CGGTGTCCTC TCCTTCTACC 3600 TCTCTACAGA GGTCCGACAG CAGTCAGCCT ATGCTGCTCC GAGTGGTTGG CAGTCAAACT 3660 TCGGACTCCA TGGGTGAGGA AGATCTTCTC AGTCCTCCCC AGGACACAAG CACAGGGTTA 3720 GAGGAGGTGA TGGAGCAACT CAACAACTCC TTCCCTAGTT CAAGAGGAAG AAATACCCCT 3780 GGAAAGCCAA TGAGAGAGGA CACAATGTAG GAAGTCTTTT CCACATGGCA GATGATTTGG 3840 GCAGAGCGAT GGAGTCCTTA GTATCAGTCA TGACAGATGA AGAAGGAGCA GAATAAATGT 3900 TTTACAACTC CTGATTCCCG CATGGTTTTT ATAATATTCA TACAACAAAG AGGATTAGAC 3960 AGTAAGAGTT TACAAGAAAT AAATCTATAT TTTTGTGAAG GGTAGTGGTA TTATACTGTA 4020 GATTTCAGTA GTTTCTAAGT CTGTTATTGT TTTGTTGGGG ATCCTCTAGA GTCGA 4075 SEQ ID NO: 8 Length of array: 1201 Array type: Amino acid topology: Type of linear sequence: Protein sequence Met Leu Trp Trp Glu Glu Val Glu Asp Cys Tyr Glu Arg Glu Asp 15 Val Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser 30 Lys Phe Gly Lys Gln His Ile Glu Asn Leu Phe Ser Asp Leu Gln 45 Asp Gly Arg Arg Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln 60 Lys Leu Pro Lys Glu Lys Gly Ser Thr Arg Val His Ala Leu Asn 75 Asn Val Asn Lys Ala Leu Arg Val Leu Gln Asn Asn Asn Val Asp 90 Leu Val Asn Ile Gly Ser Thr Asp Ile Val Asp Gly Asn His Lys 105 Leu Thr Leu Gly Leu Ile Trp Asn Ile Ile Leu His Trp Gln Val 120 Lys Asn Val Met Lys Asn Ile Met Ala Gly Leu Gln Gln Thr Asn 135 Ser Glu Lys Ile Leu Leu Ser Trp Val Arg Gln Ser Thr Arg Asn 150 Tyr Pro Gln Val Asn Val Ile Asn Phe Thr Thr Ser Trp Ser Asp 165 Gly Leu Ala Leu Asn Ala Leu Ile His Ser His Arg Pro Asp Leu 180 Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala Thr Gln Arg 195 Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly Ile Glu 210 Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys 225 Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro 240 Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Met Leu Pro 255 Arg Pro Pro Lys Val Thr Lys Glu Glu His Phe Gln Leu His His 270 Gln Met His Tyr Ser Gln Gln Ile Thr Val Ser Leu Ala Gln Gly 285 Tyr Glu Arg Thr Ser Ser Pro Lys Pro Arg Phe Lys Ser Tyr Ala 300 Tyr Thr Gln Ala Ala Tyr Val Thr Thr Ser Asp Pro Thr Arg Ser 315 Pro Phe Pro Ser Gln His Leu Glu Ala Pro Glu Asp Lys Ser Phe 330 Gly Ser Ser Leu Met Glu Ser Glu Val Asn Leu Asp Arg Tyr Gln 345 Thr Ala Leu Glu Glu Val Leu Ser Trp Leu Leu Ser Ala Glu Asp 360 Thr Leu Gln Ala Gln Gly Glu Ile Ser Asn Asp Val Glu Val Val 375 Lys Asp Gln Phe His Thr His Glu Gly Tyr Met Met Asp Leu Thr 390 Ala His Gln Gly Arg Val Gly Asn Ile Leu Gln Leu Gly Ser Lys 405 Leu Ile Gly Thr Gly Lys Leu Ser Glu Asp Glu Glu Thr Glu Val 420 Gln Glu Gln Met Asn Leu Leu Asn Ser Arg Trp Glu Cys Leu Arg 435 Val Ala Ser Met Glu Lys Gln Ser Asn Leu His Arg Val Leu Met 450 Asp Leu Gln Asn Gln Lys Leu Lys Glu Leu Asn Asp Trp Leu Thr 465 Lys Thr Glu Glu Arg Thr Arg Lys Met Glu Glu Glu Pro Leu Gly 480 Pro Asp Leu Glu Asp Leu Lys Arg Gln Val Gln Gln His Lys Val 495 Leu Gln Glu Asp Leu Glu Gln Glu Gln Val Arg Val Asn Ser Leu 510 Thr His Met Val Val Val Val Asp Glu Ser Ser Gly Asp His Ala 525 Thr Ala Ala Leu Glu Glu Gln Leu Lys Val Leu Asn Thr Arg Trp 540 Lys Leu Leu Gln Val Ala Val Glu Asp Arg Val Arg Gln Leu His 555 Glu Ala His Arg Asp Phe Gly Pro Ala Ser Gln His Phe Leu Ser 570 Thr Ser Val Gln Gly Pro Trp Glu Arg Ala Ile Ser Pro Asn Lys 585 Val Pro Tyr Tyr Ile Asn His Glu Thr Gln Thr Thr Cys Trp Asp 600 His Pro Lys Met Thr Glu Leu Tyr Gln Ser Leu Ala Asp Leu Asn 615 Asn Val Arg Phe Ser Ala Tyr Arg Thr Ala Met Lys Leu Arg Arg 630 Leu Gln Lys Ala Leu Cys Leu Asp Leu Leu Ser Leu Ser Ala Ala 645 Cys Asp Ala Leu Asp Gln His Asn Leu Lys Gln Asn Asp Gln Pro 660 Met Asp Ile Leu Gln Ile Ile Asn Cys Leu Thr Thr Ile Tyr Asp 675 Arg Leu Glu Gln Glu His Asn Asn Leu Val Asn Val Pro Leu Cys 690 Val Asp Met Cys Leu Asn Trp Leu Leu Asn Val Tyr Asp Thr Gly 705 Arg Thr Gly Arg Ile Arg Val Leu Ser Phe Lys Thr Gly Ile Ile 720 Ser Leu Cys Lys Ala His Leu Glu Asp Lys Tyr Arg Tyr Leu Phe 735 Lys Gln Val Ala Ser Ser Thr Gly Phe Cys Asp Gln Arg Arg Leu 750 Gly Leu Leu Leu His Asp Ser Ile Gln Ile Pro Arg Gln Leu Gly 765 Glu Val Ala Ser Phe Gly Gly Ser Asn Ile Glu Pro Ser Val Arg 780 Ser Cys Phe Gln Phe Ala Asn Asn Lys Pro Glu Ile Glu Ala Ala 795 Leu Phe Leu Asp Trp Met Arg Leu Glu Pro Gln Ser Met Val Trp 810 Leu Pro Val Leu His Arg Val Ala Ala Ala Glu Thr Ala Lys His 825 Gln Ala Lys Cys Asn Ile Cys Lys Glu Cys Pro Ile Ile Gly Phe 840 Arg Tyr Arg Ser Leu Lys His Phe Asn Tyr Asp Ile Cys Gln Ser 855 Cys Phe Phe Ser Gly Arg Val Ala Lys Gly His Lys Met His Tyr 870 Pro Met Val Glu Tyr Cys Thr Pro Thr Thr Ser Gly Glu Asp Val 885 Arg Asp Phe Ala Lys Val Leu Lys Asn Lys Phe Arg Thr Lys Arg 900 Tyr Phe Ala Lys His Pro Arg Met Gly Tyr Leu Pro Val Gln Thr 915 Val Leu Glu Gly Asp Asn Met Glu Thr Pro Val Thr Leu Ile Asn 930 Phe Trp Pro Val Asp Ser Ala Pro Ala Ser Ser Pro Gln Leu Ser 945 His Asp Asp Thr His Ser Arg Ile Glu His Tyr Ala Ser Arg Leu 960 Ala Glu Met Glu Asn Ser Asn Gly Ser Tyr Leu Asn Asp Ser Ile 975 Ser Pro Asn Glu Ser Ile Asp Asp Glu His Leu Leu Ile Gln His 990 Tyr Cys Gln Ser Leu Asn Gln Asp Ser Pro Leu Ser Gln Pro Arg 1005 Ser Pro Ala Gln Ile Leu Ile Ser Leu Glu Ser Glu Glu Arg Gly 1020 Glu Leu Glu Arg Ile Leu Ala Asp Leu Glu Glu Glu Asn Arg Asn 1035 Leu Gln Ala Glu Tyr Asp Arg Leu Lys Gln Gln His Glu His Lys 1050 Gly Leu Ser Pro Leu Pro Ser Pro Pro Glu Met Met Pro Thr Ser 1065 Pro Gln Ser Pro Arg Asp Ala Glu Leu Ile Ala Glu Ala Lys Leu 1080 Leu Arg Gln His Lys Gly Arg Leu Glu Ala Arg Met Gln Ile Leu 1095 Glu Asp His Asn Lys Gln Leu Glu Ser Gln Leu His Arg Leu Arg 1110 Gln Leu Leu Glu Gln Pro Gln Ala Glu Ala Lys Val Asn Gly Thr 1125 Thr Val Ser Ser Pro Ser Thr Ser Leu Gln Arg Ser Asp Ser Ser 1140 Gln Pro Met Leu Leu Arg Val Val Gly Ser Gln Thr Ser Asp Ser 1155 Met Gly Glu Glu Asp Leu Leu Ser Pro Pro Gln Asp Thr Ser Thr 1170 Gly Leu Glu Glu Val Met Glu Gln Leu Asn Asn Ser Phe Pro Ser 1185 Ser Arg Gly Arg Asn Thr Pro Gly Lys Pro Met Arg Glu Asp Thr 1200 Met *** 1201 SEQ ID NO: 9 Length of array: 3172 Array type: Number of nucleic acid strands: Both topologies: Type of linear sequence: Features of cDNA to mRNA sequence: active-site sequence CGGCCGCTCT AGAGGATCCC CGGGTACCGA GCTCGAATTC CGGAACTCCC GGAGAAAAAC 60 GAATAGGAAA AACTGAAGTG TTACTTTTTT TAAAGCTGCT GAAGTTTGTT GGTTTCTCAT 120 TGTTTTTAAG CCTACTGGAG CAATAAAGTT TGAAGAACTT TTACCAGGTT TTTTTTATCG 180 CTGCCTTGAT ATACACTTTT CAAAATGCTT TGGTGGGAAG AAGTAGAGGA CTGTTATGAA 240 AGAGAAGATG TTCAAAAGAA AACATTCACA AAATGGGTAA ATGCACAATT TTCTAAGTTT 300 GGGAAGCAGC ATATTGAGAA CCTCTTCAGT GACCTACAGG ATGGGAGGCG CCTCCTAGAC 360 CTCCTCGAAG GCCTGACAGG GCAAAAACTG CCAAAAGAAA AAGGATCCAC AAGAGTTCAT 420 GCCCTGAACA ATGTCAACAA GGCACTGCGG GTTTTGCAGA ACAATAATGT TGATTTAGTG 480 AATATTGGAA GTACTGACAT CGTAGATGGA AATCATAAAC TGACTCTTGG TTTGATTTGG 540 AATATAATCC TCCACTGGCA GGTCAAAAAT GTAATGAAAA ATATCATGGC TGGATTGCAA 600 CAAACCAACA GTGAAAAGAT TCTCCTGAGC TGGGTCCGAC AATCAACTCG TAATTATCCA 660 CAGGTTAATG TAATCAACTT CACCACCAGC TGGTCTGATG GCCTGGCTTT GAATGCTCTC 720 ATCCATAGTC ATAGGCCAGA CCTATTTGAC TGGAATAGTG TGGTTTGCCA GCAGTCAGCC 780 ACACAACGAC TGGAACATGC ATTCAACATC GCCAGATATC AATTAGGCAT AGAGAAACTA 840 CT CGATCCTG AAGATGTTGA TACCACCTAT CCAGATAAGA AGTCCATCTT AATGTACATC 900 ACATCACTCT TCCAAGTTTT GCCTCAACAA GTGAGCATTG AAGCCATCCA GGAAGTGGAA 960 ATGTTGCCAA GGCCACCTAA AGTGACTAAA GAAGAACATT TTCAGTTACA TCATCAAATG 1020 CACTATTCTC AACAGATCAC GGTCAGTCTA GCACAGGGAT ATGAGAGAAC TTCTTCCCCT 1080 AAGCCTCGAT TCAAGAGCTA TGCCTACACA CAGGCTGCTT ATGTCACCAC CTCTGACCCT 1140 ACACGGAGCC CATTTCCTTC ACAGCATTTG GAAGCTCCTG AAGACCGAAG ACTGCAGAAG 1200 GCCCTTTGCT TGGATCTCTT GAGCCTGTCA GCTGCATGTG ATGCCTTGGA CCAGCACAAC 1260 CTCAAGCAAA ATGACCAGCC CATGGATATC CTGCAGATTA TTAATTGTTT GACCACTATT 1320 TATGACCGCC TGGAGCAAGA GCACAACAAT TTGGTCAACG TCCCTCTCTG CGTGGATATG 1380 TGTCTGAACT GGCTGCTGAA TGTTTATGAT ACGGGACGAA CAGGGAGGAT CCGTGTCCTG 1440 TCTTTTAAAA CTGGCATCAT TTCCCTGTGT AAAGCACATT TGGAAGACAA GTACAGATAC 1500 CTTTTCAAGC AAGTGGCAAG TTCAACAGGA TTTTGTGACC AGCGCAGGCT GGGCCTCCTT 1560 CTGCATGATT CTATCCAAAT TCCAAGACAG TTGGGTGAAG TTGCATCCTT TGGGGGCAGT 1620 AACATTGAGC CAAGTGTCCG GAGCTGCTTC CAATTTGCTA ATAATAAGCC AGAGATCGAA 1680 GCGGCCCTCT TCCTAGACTG GATGAGACTG GAACCCCAGT CCATGGTGTG GCTGCCCGTC 1740 CTGCACAGAG TGGCTGCTGC AGAAACTGCC AAGCATCAGG CCAAATGTAA CATCTGCAAA 1800 GAGTGTCCAA TCATTGGATT CAGGTACAGG AGTCTAAAGC ACTTTAATTA TGACATCTGC 1860 CAAAGCTGCT TTTTTTCTGG TCGAGTTGCA AAAGGCCATA AAATGCACTA TCCCATGGTG 1920 GAATATTGCA CTCCGACTAC ATCAGGAGAA GATGTTCGAG ACTTTGCCAA GGTACTAAAA 1980 AACAAATTTC GAACCAAAAG GTATTTTGCG AAGCATCCCC GAATGGGCTA CCTGCCAGTG 2040 CAGACTGTCT TAGAGGGGGA CAACATGGAA ACTCCCGTTA CTCTGATCAA CTTCTGGCCA 2100 GTAGATTCTG CGCCTGCCTC GTCCCCTCAG CTTTCACACG ATGATACTCA TTCACGCATT 2160 GAACATTATG CTAGCAGGCT AGCAGAAATG GAAAACAGCA ATGGATCTTA TCTAAATGAT 2220 AGCATCTCTC CTAATGAGAG CATAGATGAT GAACATTTGT TAATCCAGCA TTACTGCCAA 2280 AGTTTGAACC AGGACTCCCC CCTGAGCCAG CCTCGTAGTC CTGCCCAGAT CTTGATTTCC 2340 TTAGAGAGTG AGGAAAGAGG GGAGCTAGAG AGAATCCTAG CAGATCTTGA GGAAGAAAAC 2400 AGGAATCTGC AAGCAGAATA TGACCGTCTA AAGCAGCAGC ACGAACATAA AGGCCTGTCC 2460 CCACTGCCGT CCCCTCCTGA AATGATGCCC ACCTCTCCCC AGAGTCCCCG GGATGCTGAG 2520 CTCATTGCTG AGGCC AAGCT ACTGCGTCAA CACAAAGGCC GCCTGGAAGC CAGGATGCAA 2580 ATCCTGGAAG ACCACAATAA ACAGCTGGAG TCACAGTTAC ACAGGCTAAG GCAGCTGCTG 2640 GAGCAACCCC AGGCAGAGGC CAAAGTGAAT GGCACAACGG TGTCCTCTCC TTCTACCTCT 2700 CTACAGAGGT CCGACAGCAG TCAGCCTATG CTGCTCCGAG TGGTTGGCAG TCAAACTTCG 2760 GACTCCATGG GTGAGGAAGA TCTTCTCAGT CCTCCCCAGG ACACAAGCAC AGGGTTAGAG 2820 GAGGTGATGG AGCAACTCAA CAACTCCTTC CCTAGTTCAA GAGGAAGAAA TACCCCTGGA 2980 AAGCCAATGA GAGAGGACAC AATGTAGGAA GTCTTTTCCA CATGGCAGAT GATTTGGGCA 2940 GAGCGATGGA GTCCTTAGTA TCAGTCATGA CAGATGAAGA AGGAGCAGAA TAAATGTTTT 3000 ACAACTCCTG ATTCCCGCAT GGTTTTTATA ATATTCATAC AACAAAGAGG ATTAGACAGT 3060 AAGAGTTTAC AAGAAATAAAAA TCTATATTTT TGTGAAGGGT AGTGGTATTA TACTGTAGAT 3120 TTCAGTCTG GG 10 Length of array: 900 Array types: Amino acid topology: Type of linear sequence: Protein sequence Met Leu Trp Trp Glu Glu Val Glu Asp Cys Tyr Glu Arg Glu Asp 15 Val Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser 30 Lys Phe Gly Lys Gln His Ile Glu Asn Leu Phe Ser Asp Leu Gln 45 Asp Gly Arg Arg Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln 60 Lys Leu Pro Lys Glu Lys Gly Ser Thr Arg Val His Ala Leu Asn 75 Asn Val Asn Lys Ala Leu Arg Val Leu Gln Asn Asn Asn Val Asp 90 Leu Val Asn Ile Gly Ser Thr Asp Ile Val Asp Gly Asn His Lys 105 Leu Thr Leu Gly Leu Ile Trp Asn Ile Ile Leu His Trp Gln Val 120 Lys Asn Val Met Lys Asn Ile Met Ala Gly Leu Gln Gln Thr Asn 135 Ser Glu Lys Ile Leu Leu Ser Trp Val Arg Gln Ser Thr Arg Asn 150 Tyr Pro Gln Val Asn Val Ile Asn Phe Thr Thr Ser Trp Ser Asp 165 Gly Leu Ala Leu Asn Ala Leu Ile His Ser His Arg Pro Asp Leu 180 Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala Thr Gln Arg 195 Leu Glu His Ala Phe AsnI le Ala Arg Tyr Gln Leu Gly Ile Glu 210 Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys 225 Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro 240 Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Met Leu Pro 255 Arg Pro Pro Lys Val Thr Lys Glu Glu His Phe Gln Leu His His 270 Gln Met His Tyr Ser Gln Gln Ile Thr Val Ser Leu Ala Gln Gly 285 Tyr Glu Arg Thr Ser Ser Pro Lys Pro Arg Phe Lys Ser Tyr Ala 300 Tyr Thr Gln Ala Ala Tyr Val Thr Thr Ser Asp Pro Thr Arg Ser 315 Pro Phe Pro Ser Gln His Leu Glu Ala Pro Glu Asp Arg Arg Leu 330 Gln Lys Ala Leu Cys Leu Asp Leu Leu Ser Leu Ser Ala Ala Cys 345 Asp Ala Leu Asp Gln His Asn Leu Lys Gln Asn Asp Gln Pro Met 360 Asp Ile Leu Gln Ile Ile Asn Cys Leu Thr Thr Ile Tyr Asp Arg 375 Leu Glu Gln Glu His Asn Asn Leu Val Asn Val Pro Leu Cys Val 390 Asp Met Cys Leu Asn Trp Leu Leu Asn Val Tyr Asp Thr Gly Arg 405 Thr Gly Arg Ile Arg Val Leu Ser Phe Lys Thr Gly Ile Ile Ser 420 Leu Cys Lys Ala His Leu Glu Asp Lys Tyr Arg Tyr Leu Phe Lys 435 Gln Val Ala Ser Ser Thr Gly Phe Cys Asp Gln Arg Arg Leu Gly 450 Leu Leu Leu His Asp Ser Ile Gln Ile Pro Arg Gln Leu Gly Glu 465 Val Ala Ser Phe Gly Gly Ser Asn Ile Glu Pro Ser Val Arg Ser 480 Cys Phe Gln Phe Ala Asn Asn Lys Pro Glu Ile Glu Ala Ala Leu 495 Phe Leu Asp Trp Met Arg Leu Glu Pro Gln Ser Met Val Trp Leu 510 Pro Val Leu His Arg Val Ala Ala Ala Glu Thr Ala Lys His Gln 525 Ala Lys Cys Asn Ile Cys Lys Glu Cys Pro Ile Ile Gly Phe Arg 540 Tyr Arg Ser Leu Lys His Phe Asn Tyr Asp Ile Cys Gln Ser Cys 555 Phe Phe Ser Gly Arg Val Ala Lys Gly His Lys Met His Tyr Pro 570 Met Val Glu Tyr Cys Thr Pro Thr Thr Ser Gly Glu Asp Val Arg 585 Asp Phe Ala Lys Val Leu Lys Asn Lys Phe Arg Thr Lys Arg Tyr 600 Phe Ala Lys His Pro Arg Met Gly Tyr Leu Pro Val Gln Thr Val 615 Leu Glu Gly Asp Asn Met Glu Thr Pro Val Thr Leu Ile Asn Phe 630 Trp Pro Val Asp Ser Ala Pro Ala Ser Ser Pro Gln Leu Ser His 645 4 Asp Asp Thr His Ser Arg Ile Glu His Tyr Ala Ser Arg Leu Ala 660 Glu Met Glu Asn Ser Asn Gly Ser Tyr Leu Asn Asp Ser Ile Ser 675 Pro Asn Glu Ser Ile Asp Asp Glu His Leu Leu Ile Gln His Tyr 690 Cys Gln Ser Leu Asn Gln Asp Ser Pro Leu Ser Gln Pro Arg Ser 705 Pro Ala Gln Ile Leu Ile Ser Leu Glu Ser Glu Glu Arg Gly Glu 720 Leu Glu Arg Ile Leu Ala Asp Leu Glu Glu Glu Asn Arg Asn Leu 735 Gln Ala Glu Tyr Asp Arg Leu Lys Gln Gln His Glu His Lys Gly 750 Leu Ser Pro Leu Pro Ser Pro Pro Glu Met Met Pro Thr Ser Pro 765 Gln Ser Pro Arg Asp Ala Glu Leu Ile Ala Glu Ala Lys Leu Leu 780 Arg Gln His Lys Gly Arg Leu Glu Ala Arg Met Gln Ile Leu Glu 795 Asp His Asn Lys Gln Leu Glu Ser Gln Leu His Arg Leu Arg Gln 810 Leu Leu Glu Gln Pro Gln Ala Glu Ala Lys Val Asn Gly Thr Thr 825 Val Ser Ser Pro Ser Thr Ser Leu Gln Arg Ser Asp Ser Ser Gln 840 Pro Met Leu Leu Arg Val Val Gly Ser Gln Thr Ser Asp Ser Met 855 Gly Glu Glu Asp Leu Leu Ser Pro Pro Gln Asp Thr Ser Thr Gly 870 Leu Glu Glu Val Met Glu Gln Leu Asn Asn Ser Phe Pro Ser Ser 885 Arg Gly Arg Asn Thr Pro Gly Lys Pro Met Arg Glu Asp Thr Met 900 *** SEQ ID NO: 11 Length of array: 3163 Array type: Number of nucleic acid strands: Both topologies: Type of linear sequence: Features of cDNA to mRNA sequence: active-site sequence CGGCCGCTCT AGAGGATCCC CGGGTACCGA GCTCGAATTC CGGAACTCCC GGAGAAAAAC 60 GAATAGGAAA AACTGAAGTG TTACTTTTTT TAAAGCTGCT GAAGTTTGTT GGTTTCTCAT 120 TGTTTTTAAG CCTACTGGAG CAATAAAGTT TGAAGAACTT TTACCAGGTT TTTTTTATCG 180 CTGCCTTGAT ATACACTTTT CAAAATGCTT TGGTGGGAAG AAGTAGAGGA CTGTTATGAA 240 AGAGAAGATG TTCAAAAGAA AACATTCACA AAATGGGTAA ATGCACAATT TTCTAAGTTT 300 GGGAAGCAGC ATATTGAGAA CCTCTTCAGT GACCTACAGG ATGGGAGGCG CCTCCTAGAC 360 CTCCTCGAAG GCCTGACAGG GCAAAAACTG CCAAAAGAAA AAGGATCCAC AAGAGTTCAT 420 GCCCTGAACA ATGTCAACAA GGCACTGCGG GTTTTGCAGA ACAATAATGT TGATTTAGTG 480 AATATTGGAA GTACTGACAT CGTAGATGGA AATCATAAAC TGACTCTTGG TTTGATTTGG 540 AATATAATCC TCCACTGGCA GGTCAAAAAT GTAATGAAAA ATATCATGGC TGGATTGCAA 600 CAAACCAACA GTGAAAAGAT TCTCCTGAGC TGGGTCCGAC AATCAACTCG TAATTATCCA 660 CAGGTTAATG TAATCAACTT CACCACCAGC TGGTCTGATG GCCTGGCTTT GAATGCTCTC 720 ATCCATAGTC ATAGGCCAGA CCTATTTGAC TGGAATAGTG TGGTTTGCCA GCAGTCAGCC 780 ACACAACGAC TGGAACATGC ATTCAACATC GCCAGATATC AATTAGGCAT AGAGAAACTA 840 CT CGATCCTG AAGATGTTGA TACCACCTAT CCAGATAAGA AGTCCATCTT AATGTACATC 900 ACATCACTCT TCCAAGTTTT GCCTCAACAA GTGAGCATTG AAGCCATCCA GGAAGTGGAA 960 GCCCACAGGG ACTTTGGTCC AGCATCTCAG CACTTTCTTT CCACGTCTGT CCAGGGTCCC 1020 TGGGAGAGAG CCATCTCGCC AAACAAAGTG CCCTACTATA TCAACCACGA GACTCAAACA 1080 ACTTGCTGGG ACCATCCCAA AATGACAGAG CTCTACCAGT CTTTAGCTGA CCTGAATAAT 1140 GTCAGATTCT CAGCTTATAG GACTGCCATG AAACTCCGAA GACTGCAGAA GGCCCTTTGC 1200 TTGGATCTCT TGAGCCTGTC AGCTGCATGT GATGCCTTGG ACCAGCACAA CCTCAAGCAA 1260 AATGACCAGC CCATGGATAT CCTGCAGATT ATTAATTGTT TGACCACTAT TTATGACCGC 1320 CTGGAGCAAG AGCACAACAA TTTGGTCAAC GTCCCTCTCT GCGTGGATAT GTGTCTGAAC 1380 TGGCTGCTGA ATGTTTATGA TACGGGACGA ACAGGGAGGA TCCGTGTCCT GTCTTTTAAA 1440 ACTGGCATCA TTTCCCTGTG TAAAGCACAT TTGGAAGACA AGTACAGATA CCTTTTCAAG 1500 CAAGTGGCAA GTTCAACAGG ATTTTGTGAC CAGCGCAGGC TGGGCCTCCT TCTGCATGAT 1560 TCTATCCAAA TTCCAAGACA GTTGGGTGAA GTTGCATCCT TTGGGGGCAG TAACATTGAG 1620 CCAAGTGTCC GGAGCTGCTT CCAATTTGCT AATAATAAGC CAGAGATCGA AGCGGCCCTC 1680 TTCCTAGACT GGATGAGACT GGAACCCCAG TCCATGGTGT GGCTGCCCGT CCTGCACAGA 1740 GTGGCTGCTG CAGAAACTGC CAAGCATCAG GCCAAATGTA ACATCTGCAA AGAGTGTCCA 1800 ATCATTGGAT TCAGGTACAG GAGTCTAAAG CACTTTAATT ATGACATCTG CCAAAGCTGC 1860 TTTTTTTCTG GTCGAGTTGC AAAAGGCCAT AAAATGCACT ATCCCATGGT GGAATATTGC 1920 ACTCCGACTA CATCAGGAGA AGATGTTCGA GACTTTGCCA AGGTACTAAA AAACAAATTT 1980 CGAACCAAAA GGTATTTTGC GAAGCATCCC CGAATGGGCT ACCTGCCAGT GCAGACTGTC 2040 TTAGAGGGGG ACAACATGGA AACTCCCGTT ACTCTGATCA ACTTCTGGCC AGTAGATTCT 2100 GCGCCTGCCT CGTCCCCTCA GCTTTCACAC GATGATACTC ATTCACGCAT TGAACATTAT 2160 GCTAGCAGGC TAGCAGAAAT GGAAAACAGC AATGGATCTT ATCTAAATGA TAGCATCTCT 2220 CCTAATGAGA GCATAGATGA TGAACATTTG TTAATCCAGC ATTACTGCCA AAGTTTGAAC 2280 CAGGACTCCC CCCTGAGCCA GCCTCGTAGT CCTGCCCAGA TCTTGATTTC CTTAGAGAGT 2340 GAGGAAAGAG GGGAGCTAGA GAGAATCCTA GCAGATCTTG AGGAAGAAAA CAGGAATCTG 2400 CAAGCAGAAT ATGACCGTCT AAAGCAGCAG CACGAACATA AAGGCCTGTC CCCACTGCCG 2460 TCCCCTCCTG AAATGATGCC CACCTCTCCC CAGAGTCCCC GGGATGCTGA GCTCATTGCT 2520 GAGGCCAAGC TACTG CGTCA ACACAAAGGC CGCCTGGAAG CCAGGATGCA AATCCTGGAA 2580 GACCACAATA AACAGCTGGA GTCACAGTTA CACAGGCTAA GGCAGCTGCT GGAGCAACCC 2640 CAGGCAGAGG CCAAAGTGAA TGGCACAACG GTGTCCTCTC CTTCTACCTC TCTACAGAGG 2700 TCCGACAGCA GTCAGCCTAT GCTGCTCCGA GTGGTTGGCA GTCAAACTTC GGACTCCATG 2760 GGTGAGGAAG ATCTTCTCAG TCCTCCCCAG GACACAAGCA CAGGGTTAGA GGAGGTGATG 2820 GAGCAACTCA ACAACTCCTT CCCTAGTTCA AGAGGAAGAA ATACCCCTGG AAAGCCAATG 2880 AGAGAGGACA CAATGTAGGA AGTCTTTTCC ACATGGCAGA TGATTTGGGC AGAGCGATGG 2940 AGTCCTTAGT ATCAGTCATG ACAGATGAAG AAGGAGCAGA ATAAATGTTT TACAACTCCT 3000 GATTCCCGCA TGGTTTTTAT AATATTCATA CAACAAAGAG GATTAGACAG TAAGAGTTTA 3060 CAAGAAATAA ATCTATATTT TTGTGAAGGG TAGTGGTATT ATACTGTAGA TTTCAGTAGT 3120 TTCTAGTTCTGTTGATTGGTTGGTTGA 12 Length of array: 897 Array type: Amino acid topology: Type of linear sequence: Protein sequence Met Leu Trp Trp Glu Glu Val Glu Asp Cys Tyr Glu Arg Glu Asp 15 Val Gln Lys Lys Thr Phe Thr Lys Trp Val Asn Ala Gln Phe Ser 30 Lys Phe Gly Lys Gln His Ile Glu Asn Leu Phe Ser Asp Leu Gln 45 Asp Gly Arg Arg Leu Leu Asp Leu Leu Glu Gly Leu Thr Gly Gln 60 Lys Leu Pro Lys Glu Lys Gly Ser Thr Arg Val His Ala Leu Asn 75 Asn Val Asn Lys Ala Leu Arg Val Leu Gln Asn Asn Asn Val Asp 90 Leu Val Asn Ile Gly Ser Thr Asp Ile Val Asp Gly Asn His Lys 105 Leu Thr Leu Gly Leu Ile Trp Asn Ile Ile Leu His Trp Gln Val 120 Lys Asn Val Met Lys Asn Ile Met Ala Gly Leu Gln Gln Thr Asn 135 Ser Glu Lys Ile Leu Leu Ser Trp Val Arg Gln Ser Thr Arg Asn 150 Tyr Pro Gln Val Asn Val Ile Asn Phe Thr Thr Ser Trp Ser Asp 165 Gly Leu Ala Leu Asn Ala Leu Ile His Ser His Arg Pro Asp Leu 180 Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala Thr Gln Arg 195 Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly Ile Glu 210 Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp Lys 225 Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro 240 Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Ala His Arg 255 Asp Phe Gly Pro Ala Ser Gln His Phe Leu Ser Thr Ser Val Gln 270 Gly Pro Trp Glu Arg Ala Ile Ser Pro Asn Lys Val Pro Tyr Tyr 285 Ile Asn His Glu Thr Gln Thr Thr Cys Trp Asp His Pro Lys Met 300 Thr Glu Leu Tyr Gln Ser Leu Ala Asp Leu Asn Asn Val Arg Phe 315 Ser Ala Tyr Arg Thr Ala Met Lys Leu Arg Arg Leu Gln Lys Ala 330 Leu Cys Leu Asp Leu Leu Ser Leu Ser Ala Ala Cys Asp Ala Leu 345 Asp Gln His Asn Leu Lys Gln Asn Asp Gln Pro Met Asp Ile Leu 360 Gln Ile Ile Asn Cys Leu Thr Thr Ile Tyr Asp Arg Leu Glu Gln 375 Glu His Asn Asn Leu Val Asn Val Pro Leu Cys Val Asp Met Cys 390 Leu Asn Trp Leu Leu Asn Val Tyr Asp Thr Gly Arg Thr Gly Arg 405 Ile Arg Val Leu Ser Phe Lys Thr Gly Ile Ile Ser Leu Cys Lys 420 Ala His Leu Glu Asp Lys Tyr Arg Tyr Leu Phe Lys Gln Val Ala 435 Ser Ser Thr Gly Phe Cys Asp Gln Arg Arg Leu Gly Leu Leu Leu 450 His Asp Ser Ile Gln Ile Pro Arg Gln Leu Gly Glu Val Ala Ser 465 Phe Gly Gly Ser Asn Ile Glu Pro Ser Val Arg Ser Cys Phe Gln 480 Phe Ala Asn Asn Lys Pro Glu Ile Glu Ala Ala Leu Phe Leu Asp 495 Trp Met Arg Leu Glu Pro Gln Ser Met Val Trp Leu Pro Val Leu 510 His Arg Val Ala Ala Ala Glu Thr Ala Lys His Gln Ala Lys Cys 525 Asn Ile Cys Lys Glu Cys Pro Ile Ile Gly Phe Arg Tyr Arg Ser 540 Leu Lys His Phe Asn Tyr Asp Ile Cys Gln Ser Cys Phe Phe Ser 555 Gly Arg Val Ala Lys Gly His Lys Met His Tyr Pro Met Val Glu 570 Tyr Cys Thr Pro Thr Thr Ser Gly Glu Asp Val Arg Asp Phe Ala 585 Lys Val Leu Lys Asn Lys Phe Arg Thr Lys Arg Tyr Phe Ala Lys 600 His Pro Arg Met Gly Tyr Leu Pro Val Gln Thr Val Leu Glu Gly 615 Asp Asn Met Glu Thr Pro Val Thr Leu Ile Asn Phe Trp Pro Val 630 Asp Ser Ala Pro Ala Ser Ser Pro Gln Leu Ser His Asp Asp Thr 645 His Ser Arg Ile Glu His Tyr Ala Ser Arg Leu Ala Glu Met Glu 660 Asn Ser Asn Gly Ser Tyr Leu Asn Asp Ser Ile Ser Pro Asn Glu 675 Ser Ile Asp Asp Glu His Leu Leu Ile Gln His Tyr Cys Gln Ser 690 Leu Asn Gln Asp Ser Pro Leu Ser Gln Pro Arg Ser Pro Ala Gln 705 Ile Leu Ile Ser Leu Glu Ser Glu Glu Arg Gly Glu Leu Glu Arg 720 Ile Leu Ala Asp Leu Glu Glu Glu Asn Arg Asn Leu Gln Ala Glu 735 Tyr Asp Arg Leu Lys Gln Gln His Glu His Lys Gly Leu Ser Pro 750 Leu Pro Ser Pro Pro Glu Met Met Pro Thr Ser Pro Gln Ser Pro 765 Arg Asp Ala Glu Leu Ile Ala Glu Ala Lys Leu Leu Arg Gln His 780 Lys Gly Arg Leu Glu Ala Arg Met Gln Ile Leu Glu Asp His Asn 795 Lys Gln Leu Glu Ser Gln Leu His Arg Leu Arg Gln Leu Leu Glu 810 Gln Pro Gln Ala Glu Ala Lys Val Asn Gly Thr Thr Val Ser Ser 825 Pro Ser Thr Ser Leu Gln Arg Ser Asp Ser Ser Gln Pro Met Leu 840 Leu Arg Val Val Gly Ser Gln Thr Ser Asp Ser Met Gly Glu Glu 855 Asp Leu Leu Ser Pro Pro Gln Asp Thr Ser Thr Gly Leu Glu Glu 870 Val Met Glu Gln Leu Asn Asn Ser Phe Pro Ser Ser Arg Gly Arg 885 Asn Thr Pro Gly Lys Pro Met Arg Glu Asp Thr Met *** 897

[Brief description of the drawings]

FIG. 1 shows the construction of truncated dystrophin genes with various numbers of rod repeats. FIG. 1A shows a list of human full-length dystrophin gene, mini-dystrophin gene and newly prepared truncated dystrophin cDNA. FIG. 1B shows ΔDysAX2 (AX2), ΔDysAX (AX
11), ΔDysAH3 (AH3) and ΔDysM3
FIG. 4 shows the amino acid sequence of the reconstructed rod repeat in (M3). FIG. 1C shows ΔDysH1
(H1) shows the amino acid sequence of the connecting region in ΔDysH4 (H4).

FIG. 2 shows the results of introducing truncated dystrophin cDNA into a mouse skeletal muscle cell line using an adenovirus vector.

FIG. 3 is a photograph instead of a drawing showing the introduction of truncated dystrophin cDNA into skeletal muscle of mdx mice using an adenovirus vector.

FIG. 4. md injected with AxCAΔDysM3.
3 is a photograph instead of a drawing, showing the recovery of dystrophin binding protein in the plasma membrane of x skeletal muscle.

[Procedure amendment]

[Submission date] July 21, 1998

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction contents]

The full-length dystrophin gene encodes an actin-binding domain, a rod domain, a cysteine-rich domain, and a C-terminal domain from the N-terminus. Using the human mini dystrophin gene (6.3 kb) having eight rod repeats, the present inventors have constructed six types of rod-shortened dystrophin cDNAs in which the rod domain is further shortened (FIG.
1 ). All constructs have an N-terminal actin binding domain,
Retaining the cysteine rich domain and the C-terminal domain.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction contents]

Designed ΔDysAX2, AX1
1, AH3 and M3 are respectively three, three, two,
It has one rod repeat and both hinge 1 and hinge 4. In these four truncated dystrophins, a putative triple-helical structure of rod repeats at the fusion site (Koenig et al., Journal of Biological Chemistry, 265, 4560-4566).
(1990) [Koenig, M .; and Kun
kel, L .; M. (1990) J. Am. Biol. Che
m. 265,4560-4566. ]) Was designed (Fig. 5 ). On the other hand, ΔDysH
1 and H4 have no rod repeat and have either hinges 1 or 4, respectively (FIGS. 1 and 2).
6 ). The base sequences of primers and oligonucleotides used for constructing these cDNAs are shown in Table 1 of Example 1 described later.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0023

[Correction method] Change

[Correction contents]

The obtained plasmid pBSBMD and the PCR fragment amplified with the primer F1 / R1 (see Table 1) or F2 / R2 (see Table 1) are digested with AflII / XhoI, and then inserted into the AflII / XhoI site of pBSBMD. And pBSΔDysAX2 or pB, respectively.
SΔDysAX11 was prepared. Next, the template pBSB
MD and P amplified with primers F4 / R4 (see Table 1)
After cutting the CR product with MunI / HindIII,
It was inserted into the MunI / HindIII site of pBSBMD to generate pBSΔDysM3. Subsequently, a fragment prepared by annealing the oligonucleotide F3 / R3 (see Table 1) was used to ligate the AflII / Hi of pBSSBMD.
pBSΔDysAH
3 was produced. These inserts were designed to maintain the rod repeat triple helical structure when ligated. The amino acid sequence of the connecting rods repeat shown in FIG.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0024

[Correction method] Change

[Correction contents]

As a result, ΔDysAX2, AX11, A
H3 and M3 retain an N-terminal actin binding domain, a cysteine rich domain and a C-terminal domain,
It also has both 3, 3, 2, 1 rod repeats and hinges 1 and 4 respectively. ΔDysH1 and ΔDy
The two plasmids with the cDNA for sH4 are pBSΔ
It was prepared from DysM3 (FIG. 1 ). One EcoO10
To eliminate the 9I site, pBSΔDysM3 was replaced with ApaI
, After smoothing, self-ligation, pB
SΔDysM3b was prepared. PBSΔDysM of template
3 and the primer F5 / R5 (see Table 1), the PCR product was digested with EcoT22I / EcoO109I, and then cut with EcoT22 of pBSΔDysM3b.
I / EcoO109I site, pBSΔDysH
1 was produced.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0025

[Correction method] Change

[Correction contents]

For the production of pBSΔDysH4, pB
Two types of PCR reactions were separately performed using SΔDysM3 as a template and primers F5 / R6 (see Table 1) or F6 / R7 (see Table 1). Two types of PC obtained
Using the mixture of R products as a template, primer F5 / R7
A second PCR reaction was performed using (see Table 1). The resulting fragment was digested with EcoRV, and then digested with pBSΔ.
Inserted between two EcoRV sites in DysM3.
FIG. 6 shows the amino acid sequence of the connecting region. ΔDy obtained
sH1 and H4 have no rod repeats and each have either hinges 1 or 4 (FIG. 1 ).

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction contents]

FIGS. 1 , 5, and 6 show the construction of truncated dystrophin genes with various numbers of rod repeats. FIG. 1 is a list of human full-length dystrophin gene, mini-dystrophin gene and newly prepared truncated dystrophin cDNA. ΔDy
sAX2, ΔDysAX, ΔDysAH3 and ΔDys
To construct M3, the central rod domain of the mini-dystrophin cDNA was cut with the restriction enzymes shown to the right of each construct. In order to reconstruct the rod repeat structure, both ends obtained using a PCR amplified fragment or a synthetic DNA fragment were ligated. ΔDysH1
To construct ΔDysH4, ΔDysM3 was digested with the indicated restriction enzymes, and both ends were ligated using a PCR amplified fragment. The dotted line indicates the connection. The size of the cDNA and the estimated molecular weight of truncated dystrophin are shown on the right. The actin binding domain is a dotted box, the rod domain is an open box (each repeat is shown as one box), the cysteine rich domain is a hatched box, and the C-terminal domain is Illustrated with shaded boxes. Black boxes indicate hinges. The hinge is described in M. Koenig and L.A. M. K
Unkel was followed.

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0027

[Correction method] Change

[Correction contents]

FIG. 5 shows ΔDysAX2 (AX2), ΔD
ysAX11 (AX11), ΔDysAH3 (AH3)
And reconstructed rods in ΔDysM3 (M3)
2 shows the amino acid sequence of a repeat. The vertical line indicates the connection site. Triangles and dashed lines indicate the location of gaps and deletions to optimize rod repeat alignment (M. Koen
ig and L.G. M. Kunkel). CS1 and CS2
Indicates the consensus sequence of 24 repeats of dystrophin. CS1 replaces the amino acids found in at least 8 of the 24 beats with CS2
Represents amino acids found in 5, 6 or 7 ribets.

[Procedure amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction contents]

FIG. 6 shows ΔDysH1 (H1) and ΔDy
Amino acid sequence ΔDy of connecting region in sH4 (H4)
In sH1 (H1), hinge 1 is cysteine-rich
Connect directly to the domain. In ΔDysH4 (H4), the actin binding domain is directly connected to hinge 4. Tyrosine (T) at the hinge 1 (star) was mutated to alanine (A) in a North American XLCM pedigree. The dotted line below hinge 4 indicates the WW domain; the most conserved amino acids of the WW domain are underlined.

[Procedure amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0062

[Correction method] Change

[Correction contents]

Example 1 (Construction of Rod Truncated Dystrophin Gene) Six types of dystrophin genes with further shortened rod domains were constructed by the following method (see FIG. 1 ). First, a human mini dystrophin cDNA (Ascady et al., Nature, 352, 615-818 (1991) [Acsadi, G., Dickso).
n, G. Love, D .; R. Jani, A .; , Wa
lsh, F .; S. Gurusinghe, A .; , Wo
lff, T .; A. And Davies, K .; E. FIG.
(1991) Nature 352, 815-81.
8. ])), The 6.3 kb NotI / SalI fragment was transformed into pBluescriptII (SK +) (Strata
gene) at the NotI / SalI site and pBS
BMD was produced. Next, AX2, AX11, AH3,
C of truncated dystrophin (ΔDys) named M3
Four plasmids with DNA were made as shown below. Table 1 shows the nucleotide sequences of primers and oligonucleotides used for cDNA construction.

[Procedure amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0065

[Correction method] Change

[Correction contents]

On the other hand, the cD of ΔDysH1 and ΔDysH4
The two plasmids with NA are pBSΔDysM3
(See FIG. 1 ). First, in order to remove one EcoO109I site, pBSΔDysM3 was cut with ApaI, smoothed, and self-ligated to give pBSΔD
ysM3b. The PCR product amplified using the template pBSΔDysM3 and the primer F5 / R5 was subjected to Ec
After cutting with oT22I / EcoO109I, pBSΔ
It was inserted into the EcoT22I / EcoO109I site of DysM3b to create pBSΔDysH1. pBSΔD
For the production of ysH4, two types of PCR reactions were performed using p
Using BSΔDysM3 as a template, primer F5 / R6
Alternatively, they were performed separately using F6 / R7. 2 obtained
Using a mixture of different PCR products as a template, primer F
A second PCR reaction was performed using 5 / R7. The resulting fragment was digested with EcoRV, and then digested with pBSΔDy.
Inserted between two EcoRV sites in sM3. The amino acid sequence of the connecting region shown in FIGS. 5 and 6.

[Procedure amendment 11]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of the drawings]

FIG. 1 shows the construction of truncated dystrophin genes with various numbers of rod repeats. FIG. 1 shows a list of human full-length dystrophin gene, mini-dystrophin gene and newly prepared truncated dystrophin cDNA.

FIG. 2 shows the results of introducing truncated dystrophin cDNA into a mouse skeletal muscle cell line using an adenovirus vector.

FIG. 3 is a photograph instead of a drawing showing the introduction of truncated dystrophin cDNA into skeletal muscle of mdx mice using an adenovirus vector.

FIG. 4. md injected with AxCAΔDysM3.
3 is a photograph instead of a drawing, showing the recovery of dystrophin binding protein in the plasma membrane of x skeletal muscle.

FIG. 5 shows a short with various numbers of rod repeats.
ΔDysA in the construction of the truncated dystrophin gene
X2 (AX2), ΔDysAX (AX11), ΔDys
Reproduction in AH3 (AH3) and ΔDysM3 (M3)
Shows the amino acid sequence of the constructed rod repeat
It is.

FIG. 6 shows a short with various numbers of rod repeats.
ΔDysH in the construction of the truncated dystrophin gene
1 (H1) and the connecting region in ΔDysH4 (H4)
1 shows the amino acid sequence of

[Procedure amendment 12]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

FIG. 2

FIG. 3

FIG. 4

FIG. 5

FIG. 6

Claims (15)

[Claims] 1. A base sequence having at least one hinge repeat of hinge 1 and hinge 4 and a rod domain of a dystrophin gene and having a length of 4.5 kb or less, or a base capable of hybridizing to the base sequence. A gene for treating muscular dystrophy having a sequence. 2. The gene according to claim 1, which has two or more rod repeat structures of a rod domain. 3. The gene according to claim 1, further comprising a cysteine-rich domain. 4. The gene according to claim 1, further comprising an actin binding domain. 5. The method of claim 1, further comprising a C-terminal domain.
The gene according to any one of claims 4 to 4. 6. The gene according to any one of claims 1 to 5, wherein the gene has a base sequence set forth in SEQ ID NO: 1 of the sequence listing or a base sequence capable of hybridizing to the base sequence. 7. The gene is represented by SEQ ID NO: 3, 5 or 7 in the sequence listing.
The gene according to any one of claims 1 to 5, which has the nucleotide sequence described in (1) or a nucleotide sequence capable of hybridizing to the nucleotide sequence. 8. The gene according to any one of claims 1 to 7, wherein the gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing or a nucleotide sequence capable of hybridizing to the nucleotide sequence. Or the gene described in 9. A gene having a base sequence set forth in SEQ ID NO: 9 or 11 in the sequence listing or a base sequence capable of hybridizing to the base sequence. 10. A therapeutic agent for muscular dystrophy comprising the gene according to any one of claims 1 to 8. 11. A gene transfer medium for gene therapy of muscular dystrophy, comprising an adeno-associated virus (AAV) vector or a lentivirus vector. 12. The gene transfer medium according to claim 7, comprising the gene according to any one of claims 1 to 8. A vector comprising the gene according to any one of claims 1 to 8. 14. The vector may be an adeno-associated virus (AA).
V) The vector according to claim 13, which is a vector, an adenovirus vector, or a lentivirus vector. A therapeutic agent for muscular dystrophy, comprising the vector according to claim 13 or 14.