KR100863381B1 - Cellular Permissivity Factor for Viruses, and Uses Thereof - Google Patents

Abstract

The present invention provides methods and compositions related to viral growth, in particular the production of host cells that are proliferatively tolerant against swine genital respiratory syndrome (PRRS) virus.

Permissible virus propagation, swine genital respiratory syndrome virus (PRRSV), CD163, Asparyridae, Arteriviridae

Description

Cellular Permissivity Factor for Viruses, and Uses thereof

The present invention provides methods and compositions relating to the production of host cells that are proliferative to virus growth for Asfarviridae and Arteriviridae and viruses.

Asparbi University

Asparviridae is an octahedral enveloped viral family that consists of a single molecule of linear double-stranded DNA of about 150000-190000 nucleotides in length. Name of and is derived from the African swine fever and related viruses (A frican S wine ever F and R elated Virus). African swine fever virus (ASFV) is a type of the genus Asfivirus and is the only member of the family. Recently, it has been implicitly assumed that swine CD163 polypeptide is a cellular receptor for African swine fever virus (ASFV) (Sanchez-Torres et al., 2003).

Arteri corruption

Arteriviridae viruses include equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and monkey hemorrhagic fever virus (SHFV). Arteriviruses of greatest economic importance are Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

PRRSV

Swine genital respiratory syndrome (PRRS) is one of the most economically important diseases of pigs. This syndrome appeared almost simultaneously in North America and Western Europe in the late 1980s and then spread to become endemic in major swine producers in Europe, Asia and the Americas. The pathogen of PRRS is a virus named PRRS virus or PRRSV. For both European and North American PRRS, the disease is genital insufficiency (late lactation, stillbirth, and mummies) in swine and gilt, high mortality in piglets, and pigs of all ages. Is characterized by respiratory disease. This disease has been the subject of recent review ([Mengeling and Lager, 2000]; [Murtaugh et al., 2002]; [Nodelijk, 2002]; [Plagemann, 2003]).

In pigs, PRRSV infection is limited to a subset of cells of the monocyte / macrophage lineage. Fully differentiated porcine alveolar macrophage (PAM) cells are the primary target cells for viral replication (Duan et al., 1997a; Duan et al., 1997b). Immortalization of PAM cells is a technical challenge and upon success has resulted in cell lines that are not proliferative for PRRS virus growth (Weingartl et al., 2002). PRRS virions are specifically bound by macrophages and incorporated into clathrin-coated pit by endocytosis. Release from endocytic vesicles requires acidic pH (Nauwynck et al., 1999). Initial binding of virions is mediated by the interaction of viral matrix proteins with heparin sulfate glycosaminoglycans (Delputte et al., 2002). Incorporation can be facilitated by membrane glycoproteins of 210 or 220 kDa, incubating PAM cells with monoclonal antibodies against these proteins blocks infection with the PRRS virus (Duan et al., 1998); Wissink et al., 2003). The 210 kDa glycoprotein has recently been identified as sialoadhesin, a member of the siglec family of sialic acid binding immunoglobulin-type lectins (Pensaert et al., 2003). Transfection of a nonproliferative PK-15 (pig kidney) cell line with porcine sialorhehesin conferred the ability to incorporate PRRSV particles, but was still clearly blocked at the time of removing the coating, whereby virion was introduced into the cellular vesicles. But nucleocapsid degradation and vesicle membrane fusion did not proceed. No viral genes were expressed and transfected PK-15 cells did not become proliferative to the PRRS virus (Vanderheijden et al., 2003). To the best of the inventors' knowledge, transfection with cyaladhesin did not appear to be sufficient to convert any PRRSV non-proliferative cell lines to the PRRSV-proliferative phenotype.

Apart from the primary pig cells of the monocyte / macrophage lineage, the only other cell type known to be proliferative for growth of PRRSV in cell culture is the immortalized monkey kidney cell line MA-104 (Chladek et al., 1998 ) And derivatives such as MARC-145 (Kim et al., 1993) and CL-2621. It is not known why one such specific cell line is proliferative, but other mammalian cell lines are nonproliferative. PRRS viruses specifically bind to many different cell types but do not initiate infection ([Kreutz, 1998]; [Therrien et al., 2000]). In MARC-145 cells, the incorporation of the virus by endocytosis and subsequent desorption at low pH appears to mimic similar events in PAM cells (Kreutz and Ackermann, 1996). However, many monoclonal antibodies that bind to porcine sialoradhesin did not detect homologous proteins on the surface of MARC-145 cells (Duan et al., 1998; Wissink et al., 2003). This suggests that MARC-145 cells may use divergent members of the same protein family or entirely different receptors.

Current PRRSV vaccines are propagated on monkey cell lines, which have potentially dangerous activity. Using monkey cell lines for vaccine production has the potential to introduce important primate viruses into swine cell lines for which xenotransplantation purposes are intended. Since pigs are increasingly being studied as a source of xenotransplantation organs for humans, introducing primate cell lines into swine populations can ultimately be dangerous for humans receiving xenotransplanted organs. Therefore, it would be wise to avoid using monkey cell lines in swine vaccine formulations. Thus, it would be desirable to identify or produce non-monkey cells or cell lines capable of supporting PRRSV replication. For this purpose, it is essential to identify the gene product (s) that may be responsible for conferring proliferative tolerance for PRRSV replication as seen in PAM cells as well as certain monkey cell lines. Once such gene products have been identified, non-proliferative cells can be made proliferative by transfecting essential genes into non-proliferative cells, thereby providing a wider production line for vaccines.

A laboratory reported that tetraspanin protein CD151 from MARC-145 cells confers proliferative tolerance for PRRS virus when transfected into non-proliferative BHK-21 cells (Kapil and Shanmukhappa, 2003). [Shanmukhappa and Kapil, 2001]. This observation has not yet been confirmed in an independent laboratory. We describe herein an unrelated polypeptide that confers proliferative tolerance for PRRS virus when transfected into non-proliferative cells.

Cited References

Chladek, D. W., Harris, L. L., and Gorcyca, D. E. Method of growing and attenuating a viral agent associated with mystery swine disease. Boehringer Ingelheim Animal Health, Inc. 677,585 [US 5,840,563], 1-24. 11-24-1998. USA. 7-9-1996].

Reference Type: Patent

Dea, S., Gagnon, C. A., Mardassi, H., Pirzadeh, B., and Rogan, D. (2000). Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates [Review]. Arch. Virol. 145, 659-688.

Delputte, P. L., Vanderheijden, N., Nauwynck, H. J., and Pensaert, M. B. (2002). Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages. J. Virol. 76, 4312-4320.

Duan, X., Nauwynck, H. J., and Pensaert, M. B. (1997a). Effects of origin and state of differentiation and activation of monocytes / macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV). Arch. Virol. 142, 2483-2497].

Duan, X., Nauwynck, H. J., and Pensaert, M. B. (1997b). Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV). Vet. Microbiol. 56, 9-19].

Duan, X. B., Nauwynck, H. J., Favoreel, H. W., and Pensaert, M. B. (1998). Identification of a putative receptor for porcine reproductive and respiratory syndrome virus on porcine alveolar macrophages. J. Virol. 72, 4520-4523.

Graversen, J. H., Madsen, M., and Moestrup, S. K. (2002). CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma. [Review] [19 refs]. International Journal of Biochemistry & Cell Biology 34, 309-314.

Gronlund J. Vitved L. Lausen M. Skjodt K. Holmskov U. Cloning of a novel scavenger receptor cysteine-rich type I transmembrane molecule (M160) expressed by human macrophages. Journal of Immunology 165 (11): 6406-6415, 2000].

Kapil, S. and Shanmukhappa, K. Host susceptibility factor (s) for porcine reproductive and respiratory syndrome virus and uses in swine breeding, as a target for antiviral compounds, and development of a non-simian recombinant cell line for propagation of the virus. none. US 2003/0186236 A1, 1-45. 10-2-2003. USA. 1-28-2002].

Reference Type: Patent

Kim, H. S., Kwang, J., Yoon, I. J., Joo, H. S., and Frey, M. L. (1993). Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogenous subpopulation of MA-104 cell line. Arch. Virol. 133, 477-483.

Kreutz, L. C. (1998). Cellular membrane factors are the major determinants of porcine reproductive and respiratory syndrome virus tropism. Virus Res. 53, 121-128].

Kreutz, L. C. and Ackermann, M. R. (1996). Porcine reproductive and respiratory syndrome virus enters cells through a low pH-dependent endocytic pathway. Virus Res. 42,137-147].

Mengeling, W. L. and Lager, K. M. (2000). A brief review of procedures and potential problems associated with the diagnosis of porcine reproductive and respiratory syndrome. Veterinary Research 31, 61-69].

Meulenberg, J. J. M. (2000). PRRSV, the virus. Veterinary Research 31, 11-21].

Murtaugh, M. P., Xiao, Z. G., and Zuckermann, F. (2002). Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection [Review]. Viral Immunology 15, 533-547.

Neuwynck, H. J., Duan, X., Favoreel, H. W., Van Oostveldt, P., and Pensaert, M. B. (1999). Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis. J. Gen. Virol. 80, 297-305].

Nodelijk, G. (2002). Porcine Reproductive and Respiratory Syndrome Virus (PRRS) with special reference to clinical aspects and diagnosis-A review [Review]. Vet. Quart. 24, 95-100].

Pensaert, M., Nauwynck, H., and Vanderheijden, N. Nucleic acid encoding polypeptide involved in cellular entrance of the PRRS virus. Akzo Nobel N. V. and Universiteit Gent. WO 03/010200 A2, 1-24. 2-6-2003. 7-18-2002.

Reference Type: Patent

Philippidis, P., Mason, J. C., Evans, B. J., Nadra, I., Taylor, K. M., Haskard, D. O., and Landis, R. C. (2004). Hemoglobin scavenger receptor CD163 mediates interleukin-10 release and hemeoxygenase-1 synthesis-Antiinflammatory monocyte-macrophage responses in vitro, in resolving skin blisters in vivo, and after cardiopulmonary bypass surgery. Circulation Research 94, 119-126].

Plugmann, P. G. W. (2003). Porcine reproductive and respiratory syndrome virus: Origin hypothesis. Emerging Infectious Diseases 9, 903-908].

Ritter, M., Buechler, C., Langmann, T., and Schmitz, G. (1999). Genomic organization and chromosomal localization of the human CD163 (M130) gene: a member of the scavenger receptor cysteine-rich superfamily. Biochemical & Biophysical Research Communications 260, 466-474.

Sanchez-Torres, C., Gomez-Puertas, P., Gomez-del-Moral, M., Alonso, F., Escribano, J. M., Ezquerra, A., and Dominguez, J. (2003). Expression of porcine CD163 on monocytes / macrophages correlates with permissiveness to African swine fever infection. Arch. Virol. 148, 2307-2323.

Shanmukhappa, K. and Kapil, S. (2001). Cloning and identification of MARC-145 cell proteins binding to 3'UTR and partial nucleoprotein gene of porcine reproductive and respiratory syndrome virus. Adv. Exp. Med. Biol. 494, 641-646.

Snijder, E.J. and Meulenberg, J. J. M. (2001). Arteriviruses. In Fields Virology, D. M. Knipe, P. M. Howley, D. E. Griffin, M. A. Martin, R. A. Lamb, B. Roizman, and S. E. Straus, eds. Philadelphia: Lippincott Williams & Wilkins, pp. 1205-1220.

Therrien, D., St Pierre, Y., and Dea, S. (2000). Preliminary characterization of protein binding factor for porcine reproductive and respiratory syndrome virus on the surface of permissive and non-permissive cells. Arch. Virol. 145, 1099-1116.

Vanderheijden, N., Delputte, P. L., Favoreel, H. W., Vandekerckhove, J., Van Damme, J., van Woensel, P. A., and Nauwynck, H. J. (2003). Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages. J. Virol. 77, 8207-8215.

Weingartl, H. M., Sabara, M., Pasick, J., van Moorlehem, E., and Babiuk, L. (2002). Continuous porcine cell line developed from alveolar macrophages-Partial characterization and virus susceptibility. J. Virol. Methods 104, 203-216.

Wissink, E. H. J., van Wijk, H. A. R., Pol, J. M. A., Godeke, G. J., van Rijn, P. A., Rottier, P. J. M., and Meulenberg, J. J. M. (2003). Identification of porcine alveolar macrophage glycoproteins involved in infection of porcine respiratory and reproductive syndrome virus. Arch. Virol. 148, 177-187.

Summary of the Invention

The present invention includes a method of promoting infection of one or more cells by a virus selected from the group consisting of Arteriviridae and Asparviridae, the method comprising causing an increase in CD163 polypeptide expression in the cells. . In a preferred embodiment, CD163 is bound to the membrane. In one embodiment, the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment, the virus is PRRSV. In another embodiment, the virus is equine arteritis virus (EAV). In another embodiment, the virus is African swine fever virus (ASFV).

Increased expression of the CD163 polypeptide can be achieved by methods such as the introduction of exogenous nucleic acids encoding the CD163 polypeptide, which may include transfection of a carrier of a polynucleotide comprising a polynucleotide encoding the CD163 polypeptide, Electroporation and fusion are included, but are not limited to these. Increased expression can also be achieved by induction of endogenous CD163 expression by chemical treatment.

This method can make PRRS proliferative cells previously non-PRRSV-prone tolerable. The method may also include making one or more cells that did not previously express a CD163 polypeptide into cells induced to express the CD163 polypeptide.

The cells in a preferred embodiment are animal cells. It may be a vertebrate or invertebrate cell. The cell may be a mammalian cell. The cell or cell line may be an insect cell. The cell may be a BHK21 cell. The cells may be derived from porcine kidney cells. The cell or cell line may be derived from cat kidney cells. The cell or cell line may be, but is not limited to, BHK-21, NLST-1, NLFK-1, Vero or RL cells. The PRRSV can be a European or North American phenotype.

As mentioned above, increased expression of the CD163 polypeptide may be achieved by methods including, but not limited to, transformation, electroporation, and fusion of carriers of polynucleotides comprising polynucleotides encoding the CD163 polypeptide. Any CD163 polypeptide is implemented. Preference is given to those containing transmembrane regions. Exemplary CD163 polypeptides are selected from the group consisting of polynucleotides listed below.

One such polynucleotide is SEQ ID NO: 2 and at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96%, 97%, 98%, Polynucleotides encoding polypeptides having 99% identity.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 2 with up to 20 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 2 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 1.

One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 99% identity with the polypeptide set forth in SEQ ID NO: 14.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 14 with up to 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 14 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 14.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 13.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 24 with up to two conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 24.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 23.

One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 96%, 97%, 98%, or 99% identity with the polypeptide set forth in SEQ ID NO: 27.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 27 with up to 20 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 27 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 27.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 26.

One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 98%, 99% identity with the polypeptide set forth in SEQ ID NO: 32.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 32 with up to 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 32 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 32.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 31.

One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99% identity with the polypeptide set forth in SEQ ID NO: 34.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 34 with up to 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 34 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 34.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 33.

One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99% identity with the polypeptide set forth in SEQ ID NO: 36.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 36 with up to 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 36 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 36.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 35.

One such polynucleotide encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the polypeptide set forth in SEQ ID NO: 42. Polynucleotides.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 42 with up to 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 42 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 42.

One such polynucleotide comprises a polynucleotide having the sequence set forth in SEQ ID NO: 41.

One such polynucleotide encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the polypeptide set forth in SEQ ID NO: 44. Polynucleotides.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 44 with up to 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 44 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 44.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 43.

One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, 99% identity with the polypeptide set forth in SEQ ID NO: 46.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 46 with up to 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 46 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 46.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 45.

One such polynucleotide encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the polypeptide set forth in SEQ ID NO: 48. Polynucleotides.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 48 with up to 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide different from SEQ ID NO: 48 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 48.

One such polynucleotide includes a polynucleotide having the sequence set forth in SEQ ID NO: 47.

The method for promoting infection described above may further comprise producing a culture of the virus.

The invention also includes cultures isolated by the methods described above.

Any of the above described methods may further comprise producing a PRRS or other viral vaccine. The vaccine can be a dead vaccine or an attenuated live vaccine.

The invention also encompasses cells or cell lines in which the ability of one or more cells to be infected by a virus selected from the group consisting of Arteriviridae and Asparviridae results in increased CD163 polypeptide expression in said cells.

In a preferred embodiment, the CD163 polypeptide comprises a transmembrane region. In one embodiment, the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment, the virus is PRRSV. In another embodiment, the virus is equine arteritis virus (EAV). In another embodiment, the virus is African swine fever virus (ASFV).

The cells or cell lines of the invention may have previously been PRRSV proliferative and become PRRSV proliferable by causing increased CD163 polypeptide expression in said cells or cell lines.

Cells or cell lines of the invention include cells or cell lines that do not express a CD163 polypeptide but are induced to express a CD163 polypeptide.

The cells in a preferred embodiment are animal cells. These may be vertebrate or invertebrate cells. The cell may be a mammalian cell. The cell or cell line may be an insect cell or cell line. The cell may be a BHK21 cell. The cells may be derived from porcine kidney cells. The cell or cell line may be derived from cat kidney cells. The cell may be, but is not limited to, BHK-21, NLST-1, NLFK-1, Vero or RL cells. PRRSV may be North American or European.

In the present invention

a) providing a sample containing nucleic acid from a test cell or cell line;

b) determining the amount of the polynucleotide or complement thereof that encodes the CD163 polypeptide in the sample

An increased amount of polynucleotide encoding the CD163 polypeptide compared to a control sample derived from a control cell or cell line known to not support the propagation of the virus, indicating a propensity of the test cell or cell line to support replication of the virus. Included are methods for measuring the propensity of test cells or cell lines to allow infection by a virus selected from the group consisting of Arteriviridae and Asparviridae.

In one embodiment, the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment, the virus is PRRSV. In another embodiment, the virus is equine arteritis virus (EAV). In another embodiment, the virus is African swine fever virus (ASFV).

The amount of polynucleotide encoding a CD163 polypeptide can be determined by hybridization.

The amount of polynucleotide encoding the CD163 polypeptide can be determined by PCR.

In the present invention

(a) providing a sample containing a polypeptide from a test cell or cell line;

(b) determining the amount of CD163 polypeptide in the sample

Wherein the increased amount of CD163 polypeptide is indicative of the propensity of the test cell or cell line to support replication of the virus as compared to a control sample derived from a control cell or cell line known to not support the proliferation of the virus. Also included are methods for measuring the propensity of test cells or cell lines to allow infection by a virus selected from the group consisting of terriviridae and aspartyridae.

In one embodiment, the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment, the virus is PRRSV. In another embodiment, the virus is equine arteritis virus (EAV). In another embodiment, the virus is African swine fever virus (ASFV).

In one embodiment, determining the amount of polypeptide is accomplished by contacting the CD163 polypeptide with an antibody specific for the CD163 polypeptide under conditions that the antibody binds to the CD163 polypeptide.

In the present invention

a) providing a sample containing nucleic acid from the pig tested;

b) determining the amount of the polynucleotide or complement thereof that encodes the CD163 polypeptide in the sample

Wherein the increased amount of polynucleotide encoding the CD163 polypeptide indicates a propensity to be infected by the virus of the test pig compared to a control sample derived from a pig known to be resistant to viral infection. Included are methods for determining propensity of swine to be infected by a virus selected from the group consisting of large and aspartic ridae.

In one embodiment, the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment, the virus is PRRSV. In another embodiment, the virus is equine arteritis virus (EAV). In another embodiment, the virus is African swine fever virus (ASFV).

In one embodiment, determining the amount of polynucleotide is achieved by hybridization. In another embodiment, determining the amount of polynucleotide is accomplished by PCR.

In the present invention

(a) providing a sample containing a polypeptide from a pig tested;

(b) determining the amount of CD163 polypeptide in the sample

Wherein the increased amount of CD163 polypeptide indicates a propensity to be infected by said virus in test pigs as compared to a control sample derived from a pig known to be resistant to viral infection. Included are methods for measuring propensity of a pig to be infected by a virus selected from the group consisting of.

In one embodiment, the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment, the virus is PRRSV. In another embodiment, the virus is equine arteritis virus (EAV). In another embodiment, the virus is African swine fever virus (ASFV).

In one embodiment, determining the amount of polypeptide is accomplished by contacting the CD163 polypeptide with an antibody specific for the CD163 polypeptide under conditions that the antibody binds to the CD163 polypeptide.

Also included in the present invention are isolated polypeptides selected from the group consisting of the polypeptides described below.

Therefore, in the present invention, SEQ ID NO: 2 and at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Also included are isolated polypeptides having an identity of.

One such polypeptide is a polypeptide different from SEQ ID NO: 2 with up to 20 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 2 with up to 10 conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 2.

Thus, the invention also includes isolated polypeptides having at least 99% identity with the polypeptides set forth in SEQ ID NO: 14.

One such polypeptide is a polypeptide different from SEQ ID NO: 14 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 14 with up to 10 conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 14.

Thus, the invention also encompasses isolated polypeptides that differ from SEQ ID NO: 24 with up to two conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 24.

Thus, the invention also encompasses isolated polypeptides having at least 96%, 97%, 98%, or 99% identity with the polypeptides set forth in SEQ ID NO: 27.

One such polypeptide is a polypeptide different from SEQ ID NO: 27 with up to 20 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 27 with up to 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 27.

Thus, the invention also encompasses isolated polypeptides having at least 98% and 99% identity with the polypeptides set forth in SEQ ID NO: 32.

One such polypeptide is a polypeptide different from SEQ ID NO: 32 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 32 with up to 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 32.

Thus, the invention also encompasses isolated polypeptides having at least 95%, 96%, 97%, 98%, 99% identity with the polypeptide set forth in SEQ ID NO: 34.

One such polypeptide is a polypeptide different from SEQ ID NO: 34 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 34 with up to 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 34.

Thus, the present invention also encompasses isolated polypeptides having at least 95%, 96%, 97%, 98%, 99% identity with the polypeptides set forth in SEQ ID NO: 36.

One such polypeptide is a polypeptide different from SEQ ID NO: 36 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 36 with up to 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 36.

The invention also includes isolated polypeptides having at least 95%, 96%, 97%, 98%, 99% identity with the polypeptides set forth in SEQ ID NO: 38.

One such polypeptide is a polypeptide different from SEQ ID NO: 38 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 38 with up to 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 40.

Thus, the invention also encompasses isolated polypeptides having at least 95%, 96%, 97%, 98%, 99% identity with the polypeptide set forth in SEQ ID NO: 40.

One such polypeptide is a polypeptide different from SEQ ID NO: 40 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 40 with up to 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 40.

Thus, the present invention also provides an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the polypeptide described in SEQ ID NO: 42. Included.

One such polypeptide is a polypeptide different from SEQ ID NO: 42 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 42 with up to 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 42.

Thus, the present invention also provides an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the polypeptide described in SEQ ID NO: 44. Included.

One such polypeptide is a polypeptide different from SEQ ID NO: 44 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 44 with up to 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 44.

Thus, the present invention also provides an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the polypeptide described in SEQ ID NO: 46. Included.

One such polypeptide is a polypeptide different from SEQ ID NO: 46 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 46 with up to 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 46.

Thus, the present invention also provides an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the polypeptide described in SEQ ID NO: 48. Included.

One such polypeptide is a polypeptide different from SEQ ID NO: 48 with up to 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide different from SEQ ID NO: 48 with up to 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 48.

Also included in the present invention are isolated CD163 polynucleotides selected from the group consisting of the polynucleotides counted below.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 1 or 5,

(b) at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83 with the polypeptide of SEQ ID NO: 2 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96%, 97%, 98%, Or a polynucleotide encoding a polypeptide having 99% identity and / or similarity,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 2,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 12 or 13,

(b) a polynucleotide encoding a polypeptide having at least 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 14,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 14,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 22 or 23,

(b) a polynucleotide encoding the polypeptide of SEQ ID NO: 24,

(d) a polynucleotide that is the complement of any of (a) or (b)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 25 or 26,

(b) a polynucleotide encoding a polypeptide having at least 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 27,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 27,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 30 or 31,

(b) a polynucleotide encoding a polypeptide having at least 98% or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 32,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 32,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 33,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 34,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 34,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 35,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 36,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 36,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 37,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 38,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 38,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 39,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 40,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 40,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 41,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 42,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 42,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 43,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 44,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 44,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 45,

(b) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 46 Coding polynucleotides,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 46,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Therefore, in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 47,

(b) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 48 Coding polynucleotides,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 49,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Thus, the present invention also encompasses CD163 polypeptides having deleted transmembrane regions.

Thus, the present invention also encompasses polynucleotides that encode a CD163 polypeptide from which a transmembrane region has been deleted.

In addition to the above, the present invention, as an additional aspect, includes all embodiments of the invention within a narrower scope in any way than the variants specifically mentioned above.

Brief description of sequence listing

SEQ ID NO: 1 -cDNA sequence encoding porcine susCD163v1

SEQ ID NO: 2 -expected amino acid sequence of porcine susCD163v1

SEQ ID NO: 3 -cDNA sequence, Genbank Accession No. AJ311716

SEQ ID NO: 4 -expected amino acid sequence derived from Genbank accession number AJ311716

SEQ ID NO: 5 cDNA sequence of susCD163v1 containing flanking (non-coding) sequence

SEQ ID NO: 6-11 -Primer sequence

SEQ ID NO: 12 cDNA sequence encoding porcine susCD163v2 containing flanking (non-coding) sequence.

SEQ ID NO: 13 -cDNA sequence encoding porcine susCD163v2

SEQ NO: 14 -Expected amino acid sequence of porcine susCD163v2

SEQ ID NO: 15-16 -Primer sequence

SEQ ID NO: 17 cDNA sequence encoding human CD163v2 containing a flanking (non-coding) sequence.

SEQ ID NO: 18 -cDNA sequence encoding human CD163v2.

SEQ ID NO: 19 -Expected amino acid sequence of human CD163v2

SEQ ID NO: 20-21 -Primer sequence

SEQ NO-22 cDNA sequence encoding mouse CD163v2 containing flanking (non-coding) sequence

SEQ ID NO: 23 cDNA sequence encoding mouse CD163v2.

SEQ NO: 24 -expected amino acid sequence of mouse CD163v2

SEQ NO: 25 -cDNA sequence encoding mouse CD163v3 containing flanking (non-coding) sequence

SEQ NO: 26 -cDNA sequence encoding mouse CD163v3

SEQ NO: 27 -Expected amino acid sequence of mouse CD163v3

SEQ ID NO: 28-29 -primer sequence

SEQ NO: 30 -cDNA sequence encoding MARC-145 CD163v3 containing flanking (non-coding) sequence

SEQ ID NO: 31 -cDNA sequence encoding MARC-145 CD163v3

SEQ NO: 32 -Expected amino acid sequence of MARC-145 CD163v3

SEQ NO: 33 -cDNA sequence encoding Vero cell CD163v2 transcript

SEQ NO: 34 -Expected Amino Acid Sequence of Vero Cell CD163v2

SEQ NO: 35 cDNA sequence encoding Vero cell CD163v3 transcript

SEQ ID NO: 36 -expected amino acid sequence of Vero cell CD163v3

SEQ ID NO: 37 -cDNA sequence encoding Vero cell CD163v4 transcript

SEQ ID NO: 38 -Expected amino acid sequence of Vero cell CD163v4

SEQ ID NO: 39 -cDNA sequence encoding Vero cell CD163v5 transcript

SEQ NO: 40 -Expected amino acid sequence of Vero cell CD163v5

SEQ ID NO: 41 -cDNA sequence encoding Vero cell CD163v6 transcript

SEQ NO: 42 -Expected amino acid sequence of Vero cell CD163v6

SEQ ID NO: 43 -cDNA sequence of Vero cell CD163v7 transcript

SEQ ID NO: 44 -Prospective amino acid sequence of Vero cell CD163v7

SEQ NO: 45 cDNA sequence encoding a dog CD163v2 transcript

SEQ ID NO: 46 -expected amino acid sequence of dog CD163v2

SEQ NO: 47 -cDNA sequence of the dog CD163v3 transcript

SEQ ID NO: 48 -Prospective amino acid sequence of CD163v3

Figure 1 Schematic Comparison of susCD163v1 and AJ311716

Figure 2 Amino acid sequence alignment of susCD163v1 (SEQ ID NO: 2) and AJ311716 (SEQ ID NO: 4)

Figure 3 Nucleotide sequence alignment of susCD163v1 and AJ311716

4 Generation and ligation of DNA fragments to place CD163 immediately after the RSV promoter. Plasmids were digested with Dra III or Drd I and then blunt-terminated with Klenow enzyme. After clean up, the plasmid was digested with Not I. Gel purification yielded DNA fragments, which were then ligated using the sticky Not I terminus. Promoters from RSV (pRSV) and SV40 (pSV40) are indicated by arrows.

Figure 5 Map of pCDNA3.1 Directional V5 / His / TOPO Cloning Vector

6 Three BHK / CMV / v1 cell lines, # 3, # 5, and # 12, and non-proliferative BHK cell lines were infected with PRRSV isolate P129 and stained with SDOW17-FITC. Panel A shows nonproliferative BHK21 cell clones. Panel B shows BHK / CMV / v1 clone # 3. Panel C shows BHK / CMV / v1 clone # 5. Panel D shows BHK / CMV / v1 clone # 12.

7 Three BHK / RSV / v1 cell lines, # 2, # 3, and # 4 were infected with PRRSV isolate P129 and stained with SDOW17-FITC. Panel A shows BHK / RSV / v1 clone # 2. Panel B shows BHK / RSV / v1 clone # 3. Panel C shows BHK / RSV / v1 clone # 4.

Fig. 8 Feline kidney cell line stably expressing swine CD163v1, showing PRRSV plaques. Cell lines NLFK-CMV-susCD163v1-G4F and NLFK-CMV-susCD163v1-G4L, both passage 4, were infected with P129 isolates of North American PRRSV and incubated for 6 days. Monolayers were fixed with 80% acetone and stained with monoclonal antibody SDOW17-FITC. Phase contrast microscopy (right) shows localized regions of viral CPE (plaques) and FA detection (left) shows co-localized viral nucleocapsid antigens.

9 Four FK / RSV / v1 cell lines, # 1, # 2, # 3, and # 4 were infected with PRRSV isolate P129 and stained with monoclonal antibody SDOW17-FITC. Panel A shows FK / RSVvl # 1 cell clones. Panel B shows FK / RSV / v1 clone # 2. Panel C shows FK / RSV / v1 clone # 3. Panel D shows FK / RSV / v1 # 4.

10 PK-CMV-susCD163v1-A10 cells of passage 19 infected with PRRSV isolate P129. Left: Monolayers were fixed with 80% acetone and stained with FITC-conjugate monoclonal antibody SDOW17 (Rural Technologies Inc) specific for PRRSV nucleocapsids. Right: Same well under bright field illumination showing cell distribution.

11 BHK-CMVScript-susCD163v2-A9 of passage 17 infected with PRRSV isolate P129. Monolayers were fixed with 80% acetone and stained with FITC-conjugate monoclonal antibody SDOW17 (Rural Technologies Inc) specific for PRRSV nucleocapsids.

12 Three representative examples of BHK / RSV / v2 cell lines. Cells were infected with PRRSV isolate P129 and then stained with SDOW17-FITC. Panel A shows cell line BHK / RSV / v2 # 1, panel B shows cell line BHK / RSV / v2 # 34 and panel C shows cell line BHK / RSV / v2 # 47.

13 FK-cDNA3.1D-humCD163v2-A6 of passage 15 infected with PRRSV isolate P129. Monolayers were then fixed with 80% acetone and stained with FITC-conjugate monoclonal antibody SDOW17 (Rural Technologies Inc) specific for PRRSV nucleocapsids.

14 The amount of progeny PRRSV produced by four recombinant cell lines stably expressing susCD163v1 and MARC-145 cells was determined using NVSL 94-3 isolates of PRRSV in growth curve experiments. Samples harvested at 12 hour intervals were titrated on MARC-145 cell monolayers.

15 Flow cytometry analysis of PRRSV infection in the presence of CD163 specific antibody. BHK-21 cells expressing MARC-145 CD163 from transient transfection were incubated with CD163 specific antibody or normal goat IgG (NGS) and infected with GFP-expressing PRRSV. Each data point represents the result of triple wells.

16 Flow cytometry analysis of PRRSV infection in the presence of CD163 specific antibody. NLFK cells stably expressing human CD163 were incubated with CD163 specific antibody or normal goat IgG (NGS) and transfected with GFP expressing PRRSV. 24 hours after infection, the percentage of infected cells expressing GFP was determined. Each data point represents the result from a single well of the cell.

17 Graphical illustration of six alternative splicing variants of CD163 mRNA recovered from Vero cells. The six variants differ in the presence or absence of three exons named E6, E105, and E83. Exons E6 and E105 are multiples of three in length, so that in the absence no change in the reading frame is caused. On the other hand, the result of the absence of E83 shifts the reading frame and produces an alternative amino acid sequence at the carboxy terminus of the protein (indicated by the hatched pattern in the figure). Hydrophobic transmembrane (TM) region is coded in E105.

18 PK-RSVScript-susCD163v2 # 9 cells infected with PRRSV isolate P129. Undiluted supernatants from PAM infected with PRRSV isolate P201 were used to infect PK-RSVScript-susCD163v2 # 9 cells. After incubation for 2 days, the cells were fixed and stained with monoclonal antibody SDOW17 as described in Example 11.

19 FK-RSVScript-susCD163v2 # 51 cells infected with PRRSV isolate P129. Undiluted supernatant from PAM infected with PRRSV isolate P201 was used to infect FK-RSVScript-susCD163v2 # 51 cells. Two days after infection, cells were fixed with acetone and stained with monoclonal antibody SDOW17 as described in Example 11.

20 Infection of PK-RSVScript-susCD163v2 clone # 9 cells with PRRSV isolate P201. Panel A shows monolayers of cells infected with PRRSV P201 at passage 1 24 hours after infection. Panel B shows monolayers of cells 2 days after infection with passage-free supernatant PRRSV P201.

21 NLFK parental cells and one subclone of FK-cDNA3.1D-humCD163v2-A6 were tested for CD163 expression. Cells were fixed in 80% acetone and reacted with goat anti-human CD163 (R & D System, 1: 200) for 1 hour and then washed with PBS. For visualization, donkey anti-goat IgG (Biodesign Inc, 1: 100) conjugated with FITC was used. As shown in FIG. 21A, no specific fluorescence was detected in NLFK parental cells. Most of the FK.A6.A2 subclones showed good fluorescence staining, indicating the presence of CD163 (FIG. 21B).

General definition

Cells and cell lines may be "viral proliferative" or "viral proliferative". For example, cells or cell lines that are virus proliferative may allow for viral infection, subsequent replication and virus production. Cells or cell lines that are incapable of viral propagation cannot tolerate viral infection, subsequent replication and virus production. Cell lines that are already somewhat proliferative can be made more proliferative by the methods of the present invention.

Arteriviridae refers to the family of enveloped, positive stranded RNA viruses belonging to the genus Nidovirales . This family includes mouse lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV), monkey hemorrhagic fever virus (SHFV), and PRRSV.

Asparviridae is an octahedral enveloped viral family that consists of a single molecule of linear double-stranded DNA of about 150000-190000 nucleotides in length. The family name is derived from African swine fever and related viruses. African swine fever virus (ASFV) is a type of the aspirovirus genus and is the only member of the family.

The term "PRRSV" or PRRS virus refers to both European and North American PRRS virus phenotypes. Within each phenotype, isolates typically share at least 85% nucleotide identity. However, between phenotypes, the sequence identity level is only about 60%.

The PRRS virus is a member of the Arteriviridae family. The genome of the arterivirus is a positive polar single stranded RNA between 12 and 16 kb in length, capped at the 5 'end and polyadenyled at the 3' end. More than two thirds of the genome is dedicated to open reading frames (ORFs) 1a and 1b, which encode the non-structural functions of the virus. ORF1b is an extension of ORF1a and is the result of ribosomal frameshifting. ORFs 1a and 1b are translated directly from genomic RNA. This large polypeptide product is cleaved by viral protease to yield 12 or 13 separate smaller peptides. The remaining ORF, which encodes a viral structural protein, is expressed from a series of 3 'co-terminal subgenomic RNAs (sgRNAs). sgRNAs are produced by discontinuous transcription of negative strand RNA such that a common 5 'leader sequence is fused to each transcript. Major structural proteins are nucleocapsids (encoded by N, ORF7), matrix proteins (encoded by M, ORF6), and major envelope glycoproteins (encoded by GP5, ORF5). The remaining proteins GP4 (ORF4), GP3 (ORF3), GP2 (ORF2a), and E (ORF2b) are a few structural proteins of virion, and their function has not yet been elucidated. The molecular biology of PRRSV has been the subject of a recent review ([Dea et al., 2000]; [Meulenberg, 2000]; [Snijder and Meulenberg, 2001]).

As used herein, the term “CD163 polypeptide” refers to a protein encoded by a mammalian CD163 gene, including allelic variants containing conservative or non-conservative changes. CDNA sequences encoding porcine CD163 polypeptide have been reported (Genbank Accession No. AJ311716). CDNA sequences encoding the mouse CD163 polypeptide (Genbank Accession No. AF274883), as well as numerous human variants exemplified by Genbank Accession Nos. AAH51281 and CAA80543, have also been reported. The inventors encode pig, human, mouse, dog and African green monkey CD163 polypeptides and comprise SEQ ID NOs: 1, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, Polynucleotides comprising the sequences set forth in 39, 41, 42, 43, 45 and 47 are reported herein. "CD163 polypeptide" is a member of the scavenger receptor cysteine-rich (SRCR) family of transmembrane glycoproteins and is thought to be expressed exclusively on monocytes and macrophages. One identified role of CD163 is to inhibit oxidative tissue damage after hemolysis by consuming the hemoglobin: haptoglobin complex by endocytosis. Subsequent interleukin-10 release and the synthesis of heme oxygenase-1 result in anti-inflammatory and cytoprotective effects (Philippidis et al., 2004; Graversen et al., 2002). The human CD163 gene spans 35 kb on chromosome 12 and consists of 17 exons and 16 introns. It is known that many isoforms of CD163 polypeptides are produced by alternative splicing, including membrane binding type, cytoplasmic type and secretion type (Ritter et al., 1999). Particular preference is given to isotypes comprising the transmembrane domain.

The transmembrane domain is characterized by polypeptide fragments of larger sequences exposed on both sides of the membrane. The cytoplasmic and extracellular domains are separated by one or more segments spanning the membrane, across the hydrophobic environment of the lipid bilayer. Sections spanning the membrane generally consist of amino acid residues with nonpolar side chains in the form of alpha helices. Fragments containing about 20-30 hydrophobic residues are long enough to span the membrane with alpha helices, which can often be identified by hydrotherapy plots. The predicted transmembrane domains of SEQ ID NOs: 2 and 14 are shown in bold in the specification. Determining whether other CD163 sequences have similar sequence characteristics is readily determined by inspection or numerical therapy plots of the sequences. SEQ ID NOs: 37-40 show variant CD163 proteins and their coding nucleic acids, which do not contain the transmembrane domain.

"Polynucleotide" as used below generally refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” can include single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, and single stranded. Hybrid molecules, including DNA and RNA, which may be, or more typically, double-stranded or a mixture of single-stranded and double-stranded regions, are included without limitation. "Polynucleotide" also refers to RNA or DNA or triple-stranded regions comprising both RNA and DNA. The term "polynucleotide" also includes DNA or RNA containing one or more modified bases, and DNA or RNA whose backbone has been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; Thus, "polynucleotide" includes chemical forms of DNA, RNA, as well as viruses and cells, as well as chemically, enzymatically or metabolically modified forms of polynucleotides typically found in nature. "Polynucleotides" also include relatively short polynucleotides, often referred to as oligonucleotides.

As used below, “polypeptide” refers to any peptide or protein comprising amino acids linked to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains commonly referred to as peptides, oligopeptides or oligomers and longer chains generally referred to as proteins. Polypeptides may comprise amino acids other than amino acids encoded by the 20 genes. “Polypeptides” include amino acid sequences modified by natural processes, such as post-translational processing, or chemical modification techniques well known in the art. Such modifications are well described in basic textbooks and more detailed thesis, as well as many research literature. Modifications can occur anywhere within the polypeptide, including peptide backbones, amino acid side chains and amino or carboxy termini. It will be appreciated that the same type of modification may be present at the same or varying degrees at several sites within a given polypeptide. In addition, certain polypeptides may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination and may be cyclic with or without branching. Cyclic, branched and branched cyclic polypeptides can be produced from posttranslational natural processes or can be prepared by synthetic methods. Modified or modified forms include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives Bond attachment, covalent attachment of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glyco On proteins mediated by silylation, GPI anchor formation, hydroxylation, iodide, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfated, tRNA Amino acid additions such as arginylation, and ubiquitination (see, eg, Proteins-Structure and Molecular Properties, 2nd Ed., TE Creighton, WH Freeman and Comp). any, New York, 1993]; [Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Postranslational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, 1983] Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182: 626-646 and Rattan et al., "Protein Synthesis: Post-translational Modifications and Aging", Ann NY Acad; Sci (1992) 663: 4842).

As used herein, "isolated" means altered from natural state by human manipulation. If an "isolated" composition or material appears in nature, it has been changed, removed, or changed and removed from its original environment. For example, a polynucleotide or polypeptide that is naturally present in a living animal is not "isolated", but the same polynucleotide or polypeptide that is separated from its natural state of coexistent material is "isolated" when the term is used herein. " Thus, “isolated” as used herein and understood in the art, refers to an isolated from the original cellular environment in which the polypeptide or nucleic acid is normally found, whether or not it refers to an “isolated” polynucleotide or polypeptide. I understand. Thus, as used herein, by way of example only, a transgenic animal or recombinant cell line constructed from a polynucleotide of the invention uses an “isolated” nucleic acid. Overall isolated chromosomes from natural host cells from which the polynucleotides are originally derived are specifically excluded from the definition of isolated polynucleotides of the present invention.

In the description below, we will often use the term “identity” or similarity as applied to the amino acid sequence of a polypeptide. The percent amino acid sequence “identity” with respect to the polypeptide aligns the target sequence with the candidate sequence, introduces a gap if necessary to achieve maximum percentage sequence identity, and targets when any conservative substitutions are not considered part of the sequence identity. A percentage of amino acid residues in a candidate sequence that is identical to a residue in a sequence is defined herein. Percent sequence identity is determined by conventional methods. See, for example, BLASTP 2.2.6 [Tatusova TA and TL Madden, "BLAST 2 sequences-a new tool for comparing protein and nucleotide sequences." (1999) FEMS Microbiol Lett. 174: 247-250.

Briefly, as mentioned above, the two amino acid sequences are aligned such that the alignment score is optimized using a gap opening penalty of 10, a gap extension penalty of 0.1, and the "blosum62" scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad). Sci. USA 89: 10915-10919.1992).

Percent identity is then calculated as follows:

Total number of identical matches / (length of longer sequence + number of gaps introduced into longer sequence to align two sequences) x 100

The percentage sequence "similarity" (often referred to as "homology") for the polypeptides of the invention aligns the target sequence with the candidate sequence and introduces a gap if necessary to achieve maximum percentage sequence identity (as described above). And any percentage of amino acid residues in the candidate sequence that is identical to the residues in the target sequence when any conservative substitution is considered part of the sequence identity.

Total number of identical match and conservative substitutions / (length of longer sequence + number of gaps introduced into longer sequence to align two sequences) × 100

Amino acids can be classified according to their physical properties and their contribution to secondary and tertiary protein structures. Conservative substitutions are understood in the art as one amino acid being substituted with another amino acid having similar properties.

Exemplary conservative substitutions are shown in Tables 1, 2, and 3 below.

Conservative Substitution I Side chain features amino acid Aliphatic Nonpolar Polarity-Electroless Polarity-Charge Aromatic Others  G A P I L V C S T M N Q D E K R H F W Y N Q D E

Alternatively, conservative amino acids may be selected from Lehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY: NY (1975), pp. 71-77.

Conservative Substitution II Side chain features amino acid Nonpolar (hydrophobic) A. Aliphatic B. Aromatic C. Sulfur-containing D. Boundary chargeless-polar A. Hydroxyl B. Amide C. Sulphhydryl D. Boundary positive charge (basic) Negative charge (acidic)  A L I V P F W M G S T Y N Q C G K R H D E

As another alternative, exemplary conservative substitutions are shown in Table 3 below.

Conservative Substitution III Original residue Exemplary Substitution Ala (A) Arg (R) Asn (N) Asp (D) Cys (C) Gln (Q) Glu (E) His (H) Ile (I) Leu (L) Lys (K) Met (M) Phe ( F) Pro (P) Ser (S) Thr (T) Trp (W) Tyr (Y) Val (V) Val, Leu, Ile Lys, Gln, Asn Gln, His, Lys, Arg Glu Ser Asn Asp Asn, Gln, Lys, Arg Leu, Val, Met, Ala, Phe Ile, Val, Met, Ala, Phe Arg, Gln, Asn Leu, Phe, Ile Leu, Val, Ile, Ala Gly Thr Ser Tyr Trp, Phe, Thr, Ser Ile, Leu, Met, Phe, Ala

Methods of Producing Viruses and Host Cells of the Invention

The present invention provides a method of modifying the production of a virus that is a member of the Arteriviridae and Asparticidae families in a cell, comprising causing the cell to express a CD163 polypeptide. The method may include making the cell proliferative incapable of virus proliferation, or may involve making the cell more proliferative to the virus.

In one embodiment, the virus, which is a member of the Arteriviridae or Asparviridae family, is from a group consisting of LDV of mouse, equine arteritis virus (EAV), monkey hemorrhagic fever virus (SHFV), PRRSV of pig and ASFV of pig Is selected.

In a preferred embodiment, the virus is PPRSV.

The present invention provides a cell line; Causing the cell line to express a CD163 polypeptide; Infecting the cell line with a virus; And providing a cell culture of the virus that is a member of the Arteriviridae or Asparridae family, comprising the step of causing the cell line to produce virus progeny.

In one embodiment, the virus, which is a member of the Arteriviridae or Asparviridae family, is from a group consisting of LDV of mouse, equine arteritis virus (EAV), monkey hemorrhagic fever virus (SHFV), PRRSV of pig and ASFV of pig Is selected.

In a preferred embodiment, the virus is PPRSV.

All these methods use cells and cell lines that express the CD163 polypeptide. CD163 can be promoted or increased by methods involving the introduction of exogenous nucleic acids into cells. Such cells may comprise polynucleotides or vectors in a manner that allows expression of the encoded CD163 polypeptide.

The polynucleotide encoding CD163 can be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein-coding region, or in a viral vector. Methods of introducing exogenous nucleic acids, which are well known and commonly practiced in the art, into host cells include transformation, transfection, electroporation, nuclear injection, or liposomes, micelles, ghost cells and protoplasts. Fusion to a carrier is included. Host cell systems of the present invention include invertebrate and vertebrate cell systems. Host cells include insect cells, porcine kidney (PK) cells, cat kidney (FK) cells, porcine testes (ST) cells, African green monkey kidney cells (MA-104, MARC-145, VERO, and COS cells), Chinese Hamster ovary (CHO) cells, baby hamster kidney cells, human 293 cells, and mouse 3T3 fibroblasts may be included, but are not limited to these. Insect host cell culture systems can also be used for expression of CD163 polypeptide. In another embodiment, the CD163 polypeptide is expressed using the Drosophila expression system.

Selection of an appropriate expression vector for expression of a CD163 polypeptide will naturally depend on the particular host cell to be used and is within the skill of one in the art. Examples of suitable expression vectors include pSport and pcDNA3 (Invitrogen), pCMV-Script (Stratagene), and pSVL (Pharmacia Biotech). Expression vectors for use in mammalian host cells can include transcriptional and translational control sequences derived from the viral genome. Commonly used promoter sequences and modifier sequences that can be used in the present invention include human cytomegalovirus (CMV), Rous sarcoma virus (RSV), adenovirus 2, polyoma virus, and monkey Included but not limited to virus 40 (SV40). Methods for constructing mammalian expression vectors are described, eg, in Okayama and Berg (Mol. Cell. Biol. 3: 280 (1983)); Cosman et al. (Mol. Immunol. 23: 935 (1986)); Cosman et al. (Nature 312: 768 (1984)); EP-A-0367566; And WO 91/18982.

Because CD163 sequences are known to be present in cells from various species, endogenous genes can be modified to allow or increase the expression of CD163 polypeptides. By replacing the naturally-occurring CD163 promoter in whole or in part with a wholly or partially heterologous promoter such that the cell expresses CD163 at higher levels, the cell can be modified to provide increased expression (eg, for homologous recombination). due to). The heterologous promoter is inserted in a manner that is operably linked to the endogenous CD163 coding sequence. See, eg, PCT International Publication WO 94/12650, PCT International Publication WO 92/20808, and PCT International Publication WO 91/09955. In addition to heterologous promoter DNA, amplifiable marker DNA (eg, ada , dhfr , and multifunctional cad genes encoding carbamyl phosphate synthase, aspartate transcarbamylase , and dehydrorotase ) and / Or intron DNA can be inserted with heterologous promoter DNA. Once linked to the CD163 coding sequence, the CD163 coding sequence is amplified together in the cell by amplification of the marker DNA by standard selection methods.

CD163 expression can also be induced by chemical treatment. Phorbol esters, in particular phorbol myristyl acetate (PMA), are particularly preferred means of activating one or more isozymes of protein kinase C (PKC), a localized membrane receptor, and increasing CD163 expression. Other methods of mobilizing intracellular calcium are also implemented.

Vaccine production

The methods described above can be used to produce any virus that is a member of the Arteriviridae or Asparviridae family for the purpose of vaccine production or diagnosis.

In one embodiment, the virus that is a member of the Arteriviridae family is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), monkey hemorrhagic fever virus (SHFV), and PRRSV of pigs.

In a preferred embodiment, the virus is PRRSV.

Vaccine production

The methods described above can be used to produce viruses for the purpose of vaccine production or diagnosis.

Killed (inactivated) vaccines or live vaccines can be produced. Thus, to prepare live vaccines, virus isolates or attenuated or mutant variants thereof are grown in cell culture. The virus is harvested according to methods well known in the art. The virus is then concentrated and frozen and stored at -70 ° C or freeze-dried and stored at 4 ° C. Prior to vaccination, the virus is administered in a suitable dosage (about 10 ³ to 10 ⁸ tissue culture infectious dose / ml (TCID ₅₀ / ml)) with a pharmaceutically acceptable carrier such as saline solution, and optionally adjuvant Mix.

The vaccine produced may also include inactivated or killed vaccines comprising viruses grown by the methods of the invention. Inactivated vaccines can be prepared by methods well known in the art. For example, once a virus has propagated to high titers, it will be apparent to those skilled in the art that viral antigenic masses can be obtained by methods well known in the art. For example, viral antigenic mass can be obtained by dilution, concentration or extraction. All these methods have been used to obtain appropriate viral antigenic masses to produce vaccines. The virus is then inactivated by treatment with formalin, betapropriolactone (BPL), binary ethyleneimine (BEI), or other methods known to those skilled in the art. The inactivated virus is then mixed with a pharmaceutically acceptable carrier such as saline solution, and optionally with adjuvant. Examples of adjuvants include, but are not limited to, aluminum hydroxide, oil-in-water and water-in-oil emulsions, AMPHIGEN, saponins such as QuilA, and polypeptide adjuvants including interleukin, interferon and other cytokines.

Inactivation by formalin is performed by mixing the virus suspension with 37% formaldehyde and a final formaldehyde concentration of 0.05%. The virus-formaldehyde mixture is mixed by constant stirring at room temperature for about 24 hours. The inactivated virus mixture is then tested for residual live virus by analyzing for growth on appropriate cell lines.

Inactivation by BEI is performed by mixing the virus suspension of the present invention with 0.1 M BEI (2-bromo-ethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM. After the virus-BEI mixture is mixed by constant stirring at room temperature for about 48 hours, 1.0 M sodium thiosulfate is added at a final concentration of 0.1 mM. Continue mixing for an additional 2 hours. Inactivated virus mixtures are tested for residual live virus by assaying for growth on appropriate cell lines.

Virus proliferative cells that have become capable of expressing CD163 can also be used to quantify live virus. Two common methods well known to those skilled in the art are plaque assay and limit dilution assay.

The CD163-expressing cell line of the invention can be used to grow viruses for the purpose of producing viral antigens for diagnostic kits. For example, lysates (optionally purifying viral particles or extracting selected viral proteins) from uninfected cells can be coated onto an ELISA plate to detect and quantify antibodies to viruses in pig serum.

After isolation of viral proteins optionally, live or inactivated viruses grown on CD163-expressing cells can be used to immunize the animals to produce polyclonal, monospecific or monoclonal antibodies. In turn these antibodies can be used as the basis for diagnostic assays for the detection and quantification of viruses in porcine serum and other biological samples.

Assay of the Invention

The present invention determines the propensity of an animal to be infected by a virus that is a member of the Arteriviridae or Asparticidae family or a cell line that supports the replication of a virus that is a member of the Arteriviridae or Asparyidae family. Provide a method. Samples from each source are obtained and analyzed for expression of CD163. The level of CD163 gene expression can be compared with that of a control known to not support replication of the virus.

In the case of animals, the sample may be any sample comprising a sample nucleic acid molecule or protein, and any body expressing CD163, including but not limited to alveolar macrophages, cultured cells, biopsies, or other tissue preparations. Can be obtained from tissue. Expression levels can be assessed at the level of mRNA or produced protein, or both. In a preferred embodiment, the members of the Arteriviridae or Asparviridae family of viruses consist of LDV of mouse, equine arteritis virus (EAV), monkey hemorrhagic fever virus (SHFV), PRRSV of pig, and ASFV of pig Is selected from.

Nucleic Acid Based Analysis

The method of determining CD163 levels can be based on nucleic acids as mentioned above. CD163-derived nucleic acid may be present in solution or on a solid support. In some embodiments, it may be used as an array element either alone in a microarray or in combination with other array element molecules. Methods based on nucleic acids can be used to isolate DNA or RNA from a sample, followed by any known CD163 coding sequence or SEQ ID NO: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, PCR amplification or hybridization using specific primers derived from those specifically disclosed in 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 is generally required. DNA or RNA can be isolated from a sample according to any of a number of methods well known to those skilled in the art. For example, methods for purifying nucleic acids are described in Tijssen, P. (1993) Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Elsevier, New York, NY. have. In one preferred embodiment, total RNA is isolated using TRIZOL Total RNA Isolation Reagent (Life Technologies, Inc., Gaithersburg Md.), And mRNA is raised using d (T) column chromatography or glass beads. Isolate. When sample nucleic acid molecules are amplified, it is desirable to include low abundance transcripts to amplify the sample nucleic acid molecules and maintain the relative abundance of the original sample. RNA can be amplified in vitro, in situ, or in vivo (see US Pat. No. 5,514,545 to Eberwine).

It is also advantageous to include a control in the sample to ensure that the amplification and labeling procedure does not change the actual distribution of nucleic acid molecules in the sample. For this purpose, the sample is spiked with an amount of control nucleic acid molecule that is predetermined as detectable upon hybridization to the arrayed complementary nucleic acid molecule, and the composition of the nucleic acid molecule specifically hybridizes with the arrayed control nucleic acid molecule. Reference nucleic acid molecules. After hybridization and processing, the hybridization signal obtained should accurately reflect the amount of arrayed control nucleic acid added to the sample.

Prior to hybridization, it may be desirable to fragment the sample nucleic acid molecule. Fragmentation improves hybridization by minimizing cross-hybridization to secondary structure and other sample nucleic acid molecules or non-complementary nucleic acid molecules in a sample. Fragmentation can be performed by mechanical or chemical means.

Cover

Sample nucleic acid molecules or probes can be labeled with one or more labeling moieties so that hybridized arrayed / sample nucleic acid molecule complexes can be detected. Labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical, or chemical means. Labeled moieties include radioisotopes such as (32) P, (33) P or (35) S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, magnetic labels, linked linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and receptors, and the like. Preferred fluorescent markers include Cy3 and Cy5 fluorophores (Amersham Pharmacia Biotech, Piscataway N.J.).

Hybridization

The nucleic acid molecule sequence of SEQ ID NO: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 or in the art Other CD163 coding sequences and fragments thereof can be used in various hybridization techniques for various purposes. Hybridization probes can be designed or derived from any mammalian CD163 sequence, but SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39 The sequences disclosed in, 41, 43, 45 and 47 can be used. Such probes can be prepared from highly specific regions or conserved motifs and used in protocols to quantify CD163 messages, allelic variants, or related sequences. Hybridization probes of the present invention may be DNA or RNA, herein described as any mammalian CD163 sequence or SEQ ID NO: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30 Or genomic sequences comprising promoters, enhancers and introns of mammalian genes. Hybridization or PCR probes can be produced using oligolabeling, nick translation, end-labeling, or PCR amplification in the presence of labeled nucleotides. Vectors containing nucleic acid sequences can be used to add RNA polymerase and labeled nucleic acid molecules to produce mRNA probes in vitro. This procedure can be performed using commercially available kits such as those provided by Amersham Pharmacia Biotech.

The stringency of hybridization is determined by the G + C content, salt concentration, and temperature of the probe. In particular, stringency can be increased by decreasing salt concentration or raising the hybridization temperature. In solutions used in some hybridizations based on membranes, the addition of organic solvents such as formamide causes the reaction to occur at lower temperatures. Hybridization can be performed with low stringency at 60 ° C. with 5 × SSC containing a buffer such as 1% sodium dodecyl sulfate (SDS), which allows hybridization complexes to form between nucleotides containing some mismatches. Subsequent washings are performed at higher stringency with 0.2 × SSC containing 0.1% SDS at 45 ° C. (medium stringency) or 68 ° C. (high stringency). At high stringency, the hybridization complex will remain stable only where the nucleic acid sequence is almost completely complementary. In some hybridizations based on membranes, preferably 35%, most preferably 50% of formamide can be added to the hybridization solution to reduce the temperature at which hybridization is carried out, other detergents such as Sarkosyl or Triton X-100 and Background signals may be reduced by the use of blocking agents such as salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and include Ausubel, supra and Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.

Exemplary highly stringent hybridization conditions include: hybridization at 42 ° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1M NaCl, 10% dextran sulfate, and 0.1 × SSC and 1% SDS Wash twice at 60 ° C. for 30 min in a washing solution. Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10, it is understood in the art that changes in temperature and buffer, or salt concentration, can achieve the same stringent conditions. Modifications in hybridization conditions can be determined experimentally or can be accurately calculated based on the length of the probe and the percentage of guanosine / cytosine (GC) base pairs. Hybridization conditions are described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51].

Hybridization specificity can be assessed by comparing hybridization of specificity-control nucleic acid molecules to specificity-control sample nucleic acid molecules added to the sample in known amounts. Arrayed specificity-control nucleic acid molecules may have one or more sequence mismatches as compared to the corresponding nucleic acid molecules arrayed. In this manner, it is possible to determine whether only the arrayed complementary nucleic acid molecules hybridized to the sample nucleic acid molecule or whether a mismatched hybrid duplex was formed.

Hybridization reactions can be performed in an absolute or differential hybridization format. In the absolute hybridization format, nucleic acid molecules from one sample hybridize to molecules in the microarray format, and the signal detected after hybridization complex formation correlates with the level of nucleic acid molecules in the sample. In the differential hybridization format, differential expression of a set of genes in two biological samples is analyzed. For differential hybridization, nucleic acid molecules from both biological samples are prepared and labeled with different labeling moieties. A mixture of two labeled nucleic acid molecules is added to the microarray. The microarray is then analyzed under conditions in which release from two different labels is detectable separately. Molecules in the microarray hybridized with substantially the same number of nucleic acid molecules from both biological samples provide distinct combined fluorescence (Shalon et al .; PCT publication WO95 / 35505). In a preferred embodiment, the label is a fluorescent marker with distinguishable emission spectra such as Cy3 and Cy5 fluorophores.

After hybridization, the microarray is washed to remove unhybridized nucleic acid molecules and to detect complex formation between hybridizable array elements and nucleic acid molecules. Methods of detecting complex formation are well known to those skilled in the art. In a preferred embodiment, the nucleic acid molecule is labeled with a fluorescent label and the measurement of fluorescence levels and patterns indicative of complex formation is achieved by fluorescence microscopy, preferably confocal fluorescence microscopy.

In differential hybridization experiments, nucleic acid molecules from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. The fluorescence signals are detected separately with different optical amplifiers set up to detect specific wavelengths. Relative abundance / expression levels of nucleic acid molecules in two or more samples are obtained.

Typically, microarray fluorescence intensities can be normalized to account for variations in hybridization intensities when two or more microarrays are used under similar test conditions. In a preferred embodiment, the individual arrayed sample nucleic acid molecule complex hybridization intensities are normalized using the intensity derived from the internal standardization control contained on each microarray.

Polypeptide-based Assays

The present invention provides methods and reagents for detecting and quantifying CD163 polypeptide. Such methods may include analytical biochemical methods such as electrophoresis, mass spectroscopy, chromatography, etc., or various immunological methods such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, Western blotting ( western blotting), affinity capture mass spectroscopy, biological activity, and other methods described below and apparent to those skilled in the art upon review of this specification.

Immunoassay

The invention also provides methods of detecting CD163 polypeptide (ie, immunoassay) using one or more anti-CD163 antibody reagents. Assays used herein are assays using antibodies that specifically bind to CD163 polypeptides or epitopes, which are broadly defined herein and specifically include fragments, chimeras and other binding agents.

Many well-established immunological binding assay formats suitable for practicing the present invention are known (see, eg, US Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, eg, Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds. (1991); See Harlow and Lane, supra [Chapter 14], and Ausubel et al., Supra [Chapter 11]. Typically, immunological binding assays (or immunoassays) use "captures" that bind specifically to an analyte and often immobilize it on a solid phase. In one embodiment, the capture agent is a moiety that specifically binds a CD163 polypeptide or sequence, such as an anti-CD163 antibody.

In general, the CD163 gene product to be analyzed is detected directly or indirectly using a detectable label. The particular label or detectable group used in the assay is generally not a critical aspect of the present invention, unless it significantly interferes with the specific binding of the antibody (s) used in the assay. The label may be attached covalently to the capture agent (eg anti-CD163 antibody) or may be attached to a third moiety that specifically binds to the CD163 polypeptide, such as another antibody.

The present invention provides methods and reagents for competitive and noncompetitive immunoassays for detecting CD163 polypeptides. Noncompetitive immunoassays are assays in which the amount of analyte captured (in this case CD163) is directly measured. One such assay is a monoclonal based two-site immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on CD163 polypeptide. For example, for background information see Maddox et al., 1983, J. Exp. Med., 158: 1211. In one “sandwich” assay, a capture agent (eg, an anti-CD163 antibody) is bound and immobilized directly on a solid substrate. This immobilized antibody then captures any CD163 polypeptide present in the test sample. The immobilized CD163 polypeptide can then be labeled in this way, ie by binding a second anti-CD163 antibody with a label. Alternatively, although the second CD163 antibody may be unlabeled, a labeled third antibody specific for an antibody of the species from which the second antibody is derived may be bound to the second antibody. Alternatively, the second antibody can be modified with a detectable moiety such as biotin, to which a third labeled molecule such as enzyme-labeled streptavidin can specifically bind.

In a competitive assay, a CD163 polypeptide present in a sample by measuring the amount of added (exogenous) CD163 polypeptide substituted (or competitively removed) from a capture agent (eg, CD163 antibody) by the CD163 polypeptide present in the sample. The amount of is indirectly measured. The hapten inhibition assay is another example of a competitive assay. In this assay, the CD163 polypeptide is immobilized on a solid substrate. After a known amount of CD163 antibody is added to the sample, the sample is contacted with the immobilized CD163 polypeptide. In this case, the amount of anti-CD163 antibody bound to the immobilized CD163 polypeptide is inversely proportional to the amount of CD163 polypeptide present in the sample. The amount of immobilized antibody can be detected by detecting a fixed fraction of the antibody or a fraction of the antibody remaining in solution. In this aspect, the detection can be direct when the antibody is labeled or indirect when the label is bound to a molecule that specifically binds to the antibody as described above.

Other assay formats based on antibodies

The invention also provides reagents and methods for detecting and quantifying the presence of CD163 polypeptide in a sample by using an immunoblot (Western blot) format. Another immunoassay is so called "side flow chromatography". In a noncompetitive version of lateral flow chromatography, the sample moves across the substrate, for example by capillary action, and meets a labeled mobile antibody that binds to the analyte to form a conjugate. The conjugate then moves across the substrate and encounters a second, immobilized antibody that binds to the analyte. Thus, immobilized analytes are detected by detecting labeled antibodies. In the competitive version of lateral flow chromatography, the labeled version of the analyte moves across the carrier and competes with the unlabeled analyte for binding to the immobilized antibody. The greater the amount of analyte in the sample, the less binding by the labeled analyte, and thus the weaker the signal. See, eg, US Pat. No. 5,622,871 (May et al.) And US Pat. No. 5,591,645 (Rosenstein).

Depending on the assay, various components, including antigens, target antibodies, or anti-cathepsin S antibodies can be bound to a solid surface or support (ie, substrate, membrane, or filter paper). Numerous methods are known in the art for immobilizing biomolecules to various solid surfaces. For example, solid surfaces may include membranes (eg, nitrocellulose), microtiter dishes (eg, PVC, polypropylene, or polystyrene), test tubes (glass or plastic), dipsticks (eg, , Glass, PVC, polypropylene, polystyrene, latex, etc.), microcentrifuge tubes, or glass or plastic beads. Desired components may be covalently attached or non-covalently attached through nonspecific binding.

A wide range of natural and synthetic organic and inorganic polymers can be used as the material for the solid surface. Exemplary polymers include polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly (ethylene terephthalate), rayon, nylon, poly (vinylbutyrate), polyvinylidene difluoride (PVDF ), Silicone, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like. Other materials that can be used include paper, glass, ceramics, metals, metalloids, semiconductor materials, cement, and the like. In addition, gel forming materials such as proteins (eg gelatin), lipopolysaccharides, silicates, agarose and polyacrylamides can be used. Also suitable are polymers which form several aqueous phases, such as dextran, polyalkylene glycols or surfactants such as phospholipids, long chain (12-24) alkyl ammonium salts and the like. If the solid surface is porous, various pore sizes may be used depending on the nature of the system.

Mass spectroscopy

The mass of a molecule can often be used as an identifier of the molecule. Thus, protein analytes can be identified using methods of mass spectrometry. In mass spectroscopy, the mass can be measured by determining the time required for the ionized analyte to travel through the flight tube and be detected by the ion detector. One method of mass spectrometry for proteins is matrix-assisted laser desorption ionization mass spectrometry (MALDI). In MALDI, the analyte is mixed with an energy absorption matrix material that absorbs energy at the laser wavelength and is placed on the surface of the probe. When the laser is directed to the matrix, the analyte is desorbed from the probe surface, ionized and detected by an ion detector. See, eg, US Pat. No. 5,118,937 to Hillenkamp et al.

Another method of mass spectrometry for proteins is described in US Pat. No. 5,719,060 (Hutchens and Yip). In one such method, referred to as Surfaces Enhanced for Affinity Capture ("SEAC"), a solid phase affinity reagent, such as an antibody or metal ion, that binds specifically or nonspecifically to an analyte is used to interact with other substances in the sample. Isolate the analyte. The captured analyte is then desorbed from the solid phase, for example by laser energy, ionized and detected by a detector.

Nucleic Acids of the Invention

The examples disclose the discovery of several novel CD163 polynucleotides of the inventors. The present invention includes such novel CD163 polynucleotides. The present invention provides several isolated novel polynucleotides (eg, both DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single and double-stranded), including splicing variants, encoding novel CD163 polypeptides. to provide. The inventors have encoded pig, mouse, human, dog and African green monkey CD163 polypeptides and found SEQ ID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, Isolated new polynucleotides comprising the sequences described in 43, 45, and 47 are reported herein.

By disclosing SEQ ID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47, a number of It should be understood that a method is provided. For example, a probe can be generated from a sequence disclosed in SEQ ID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 By screening the mouse, human, dog and African green monkey cDNA or genomic libraries, the overall sequence 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, It would be possible to obtain 37, 39, 41, 43, 45 and 47, or genomic equivalents thereof. Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989). Also provided by the sequences disclosed in, for example, SEQ ID NOs: 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 Those skilled in the art will readily recognize that it is possible to generate appropriate primers for PCR amplification to obtain the overall sequence indicated. (See, eg, PCR Technology, HA Erlich, ed., Stockton Press, New York, 1989); PCR Protocols: A Guide to Methods and Applications, MA Innis, David H. Gelfand, John J. Sninsky, and Thomas J. White, eds., Academic Press, Inc., New York, 1990.].

DNA polynucleotides of the invention include cDNA, and DNA that has been chemically synthesized in whole or in part, and is also intended to include allelic variants thereof. Allelic variants are modified forms of wild-type gene sequences, and the modifications result from recombination during chromosomal separation, or exposure to conditions that cause gene mutations. Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants resulting from in vitro manipulation).

DNA encoding the novel CD163 polypeptide is set forth in SEQ ID NOs: 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47. Those skilled in the art will appreciate that the DNA of the present invention may be a double-stranded molecule, eg, SEQ ID NOs: 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 The sequences described in SEQ ID NO: 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, according to the Watson-Crick base pair law for DNA It will be readily understood to include molecules having with complementary molecules ("non-coding strands" or "complements") having sequences deduced from 45, and 47. As is well known in the art, by the well-known synonyms of universal genetic code, SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37 Pigs, mice, and Africans of SEQ ID NO: 2, 14, 24, 27 and 32, 34, 36, 38, 40, 42, 44, 46, 48 that differ in sequence from the polynucleotides of 39, 41, 43, 45, and 47 Other polynucleotides encoding the green monkey CD163 polypeptide are also embodied by the present invention. Accordingly, the present invention embodies such other DNA and RNA molecules that encode the polypeptides of SEQ ID NOs: 2, 14, 24, 27 and 32 upon expression. Once the amino acid residue sequences encoding the porcine CD163 polypeptide have been identified, it is possible to describe all such coding RNA and DNA sequences with the knowledge of all three letter codons for each particular amino acid residue. DNA and RNA molecules other than those specifically disclosed herein, which are simply characterized by changes in codons for specific amino acids, are therefore within the scope of the present invention.

Tables of amino acids and their abbreviations, symbols, and codons are shown in Table 4 below.

As is well known in the art, codons consist of three letter sequences of nucleotides in an mRNA and its corresponding cDNA molecule. Codons are characterized by base uracil (U) when present in an mRNA molecule, but are characterized by base thymidine (T) when present in DNA. Simple changes in codons for the same amino acid residues in a polynucleotide will not change the sequence or structure of the encoded polypeptide. In the case of a phrase where a particular three nucleotide sequence is said to "code" any particular amino acid, it is apparent to those skilled in the art that the table provides a means to identify that particular nucleotide. For example, if a particular three nucleotide sequence encodes threonine, it is disclosed in the table that the possible three letter sequences are ACA, ACG, ACC and ACU (ACT if DNA).

Therefore, in the present invention

(a) the susCD163v1 polynucleotide sequence set forth in SEQ ID NOs: 1 and 5,

(b) at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83 with the polypeptide of SEQ ID NO: 2 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96%, 97%, 98%, Or a polynucleotide encoding a polypeptide having 99% identity and / or similarity,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 2,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Also included are isolated polynucleotides comprising a.

Also in the present invention

(a) the susCD163v2 polynucleotide sequence set forth in SEQ ID NO: 12 or 13,

(b) a polynucleotide encoding a polypeptide having at least 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 14,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 14,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the mouse CD63v2 polynucleotide sequence set forth in SEQ ID NO: 22 or 23,

(b) a polynucleotide encoding the polypeptide of SEQ ID NO: 24,

(c) a polynucleotide that is the complement of any of (a) or (b)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the mouse CD163v3 polynucleotide sequence set forth in SEQ ID NO: 25 or 26,

(b) a polynucleotide encoding a polypeptide having at least 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 27,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 27,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the African green monkey CD163v2 polynucleotide sequence set forth in SEQ ID NO: 30 or 31,

(b) a polynucleotide encoding a polypeptide having at least 98% or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 32,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 32,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 33,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 34,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 34,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 35,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 36,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 36,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 37,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 38,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 38,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 39,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 40,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 40,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 41,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 42,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 42,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 43,

(b) a polynucleotide encoding a polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 44,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 44,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 45,

(b) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 46 Coding polynucleotides,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 46,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

Also in the present invention

(a) the polynucleotide sequence set forth in SEQ ID NO: 47,

(b) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 48 Coding polynucleotides,

(c) a polynucleotide encoding the polypeptide of SEQ ID NO: 49,

(d) a polynucleotide that is the complement of any of (a), (b) or (c)

Isolated polynucleotides comprising a.

The polynucleotide sequences provided by the present invention allow for the large scale expression of encoded polypeptides by techniques well known in the art and practiced routinely. The polynucleotides of the present invention are also polynucleotides encoding related porcine CD163v1 polypeptides, such as allelic variants and species, by well known techniques including Southern and / or Northern hybridization and polymerase chain reaction (PCR). Allow identification and isolation of the fuselage.

Knowledge of the information of any of the CD163 sequences disclosed herein identifies genomic DNA sequences encoding CD163 regulatory sequences, such as promoters, effectors, enhancers, inhibitors, etc., through the use of Southern hybridization or polymerase chain reaction (PCR). It also makes it possible to do.

As mentioned in the section entitled “Assays of the Invention”, polynucleotides of the invention are also useful in hybridization assays to detect the ability of cells to express CD163 or to measure CD163 expression levels. The polynucleotides of the present invention may also be the basis of diagnostic methods useful for determining the animal's susceptibility to viral infection as described above.

The disclosure herein for full length polynucleotides encoding CD163 polypeptides makes the fragments of full length polynucleotides readily available to those skilled in the art. The present invention therefore provides a unique fragment of a CD163 coding polynucleotide comprising at least 15 consecutive nucleotides (including any integer in between) over the length of the full length sequence of the polynucleotide encoding CD163 disclosed herein. Because the polynucleotides (including fragments) of the invention comprise sequences unique to a particular CD163-encoding polynucleotide sequence, they are only (ie "specifically" ") Will hybridize. Sequences unique to the polynucleotides of the present invention are recognizable through sequence comparisons to other known polynucleotides, and through the use of alignment programs routinely used in the art, such as those available in public sequence databases. You can check it. One such sequence is also recognizable from Southern hybridization assays that determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that allows for their detection, including radioactivity, fluorescence and enzymatic labeling.

One or more unique fragment polynucleotides (or other CD163 polynucleotides as discussed above) are used to detect the presence of a polynucleotide encoding CD163, or to detect variations in the polynucleotide sequence encoding CD163. It can be included in the kit. Anti-sense polynucleotides that recognize and hybridize to polynucleotide sequences encoding CD163 are also available by the present invention. Full length and fragment anti-sense polynucleotides are provided. Fragment anti-sense molecules of the invention include those that (i) specifically recognize and hybridize to the CD163 variant disclosed herein (determined by sequence comparison of DNA encoding CD163 with DNA encoding other known molecules). ). Identification of sequences unique to new CD163-encoding polynucleotides can be inferred through the use of any publicly available sequence database, and / or through the use of commercially available sequence comparison programs. Uniqueness of selected sequences in the entire genome can be further demonstrated by hybridization analysis. After identification of the desired sequence, isolation via restriction digestion or amplification using any of a variety of polymerase chain reaction techniques well known in the art can be performed. Anti-sense polynucleotides are particularly involved in regulating the expression of CD163 by cells expressing CD163 mRNA.

Antisense nucleic acids (preferably 10-20 base pairs of oligonucleotides) capable of specifically binding to CD163 expression control sequences or CD163 RNA are introduced into cells (e.g., by viral vectors or colloidal dispersion systems such as liposomes). ). Antisense nucleic acids bind to porcine CD163 target nucleotide sequences in cells to prevent transcription or translation of the target sequence. Phosphorothioate and methylphosphonate antisense oligonucleotides are particularly embodied in the therapeutic use according to the invention. Antisense oligonucleotides may be further modified with a poly-L-lysine, transferrin polylysine, or cholesterol moiety at the 5 'end. Inhibition of porcine CD163 expression at the transcriptional or translational level is useful for generating cellular or animal models for diseases characterized by abnormal porcine CD163 expression or as a therapeutic modality.

As mentioned in more detail above, the nucleic acid of the present invention comprises a vector comprising the polynucleotide of the present invention. Such vectors are useful for amplifying polynucleotides in a host cell, for example, to produce useful amounts of polynucleotides. In another embodiment, the vector is an expression vector in which the polynucleotide of the invention is operably linked to a polynucleotide comprising an expression control sequence. Such vectors are useful for recombinant production of polypeptides of the invention.

As also mentioned above, the present invention provides host cells that have been transformed or transfected (stable or transiently) with a polynucleotide of the invention or a vector of the invention. As mentioned above, such host cells are useful for the production of viruses and for the production of vaccines.

The invention also provides an isolated CD163 polypeptide encoded by the novel polynucleotides of the invention.

Polypeptides of the Invention

The Examples disclose the discovery of several novel CD163 polypeptides by the inventors. The present invention includes such novel CD163 polypeptides set forth in SEQ ID NOs: 2, 14, 19, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and 48.

Thus, the present invention includes an isolated polynucleotide comprising a susCD163v1 polypeptide having the sequence set forth in SEQ ID NO: 2.

In the present invention, the polypeptide of SEQ ID NO: 2 and at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96%, 97%, 98% Or polypeptides having 99% identity and / or similarity.

Thus, the invention includes an isolated polynucleotide comprising a susCD163v2 polypeptide having the sequence set forth in SEQ ID NO: 14.

The invention also includes polypeptides having at least 99% identity and / or similarity with the susCD163v2 polypeptide set forth in SEQ ID NO: 14.

Also included in the present invention are mouse CD163v2 polypeptides having the sequence set forth in SEQ ID NO: 24.

Also included in the present invention are mouse CD163v3 polypeptides having the sequence set forth in SEQ ID NO: 27.

The invention also includes polypeptides having at least 96%, 97%, 98%, or 99% identity and / or similarity with the polypeptides set forth in SEQ ID NO: 27.

The present invention also encompasses polypeptides having the sequence set forth in SEQ ID NO: 32.

Also included in the present invention are polypeptides having at least 98% or 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 32.

The present invention also encompasses polypeptides having the sequence set forth in SEQ ID NO: 34.

The present invention also encompasses polypeptides having at least 95%, 96%, 97%, 98%, 99% identity and / or similarity with the polypeptides set forth in SEQ ID NO: 34.

The present invention also encompasses polypeptides having the sequence set forth in SEQ ID NO: 36.

The present invention also encompasses polypeptides having at least 95%, 96%, 97%, 98%, 99% identity and / or similarity to the polypeptides set forth in SEQ ID NO: 36.

The present invention also encompasses polypeptides having the sequence set forth in SEQ ID NO: 38.

The present invention also encompasses polypeptides having at least 95%, 96%, 97%, 98%, 99% identity and / or similarity with the polypeptides set forth in SEQ ID NO: 39.

The present invention also encompasses polypeptides having the sequence set forth in SEQ ID NO: 40.

Also included in the present invention are polypeptides having at least 95%, 96%, 97%, 98%, 99% identity and / or similarity with the polypeptide set forth in SEQ ID NO: 40.

Also included in the present invention are polypeptides having the sequence set forth in SEQ ID NO: 42.

In the present invention, a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity and / or similarity with the polypeptide described in SEQ ID NO: 42 Included.

The present invention also encompasses polypeptides having the sequence set forth in SEQ ID NO: 44.

In the present invention, a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity and / or similarity with the polypeptide described in SEQ ID NO: 44 Included.

Also included in the present invention are polypeptides having the sequence set forth in SEQ ID NO: 46.

In the present invention, a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity and / or similarity with the polypeptide described in SEQ ID NO: 46 Included.

The present invention also encompasses polypeptides having the sequence set forth in SEQ ID NO: 48.

In the present invention, a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity and / or similarity with the polypeptide described in SEQ ID NO: 48 Included.

Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving the host cells of the invention. The use of mammalian host cells is contemplated to provide post-translational modifications (eg, glycosylation, truncation, lipidation, and phosphorylation) that may be necessary to provide optimal biological activity to the recombinant expression products of the invention. . Glycosylated and non-glycosylated forms of novel CD163 polypeptides are included.

Overexpression in eukaryotes and prokaryotic hosts as described above promotes the isolation of CD163 polypeptides. Thus, in the present invention, isolated CD163 as described in SEQ ID NO: 2, 14, 19, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, 48, including polypeptides with a label and tag attached thereto. Polypeptides and variants and conservative amino acid substitutions are included.

The present invention includes "labeled" novel CD163 polypeptides. The term "labeled" refers to an enzyme (eg, horseradish peroxidase, beta-glucuronidase, alkaline phosphatase, and beta-D-galactosidase), fluorescent labels (e.g., That any suitable detectable group, including fluorescein, luciferase, and radiolabel (eg, ¹⁴ C, ¹²⁵ I, ³ H, ³² P, and ³⁵ S), is conjugated or covalently bound As used to refer to. Methods of labeling a variety of compounds including proteins, peptides and antibodies are well known. See, eg, Morrison, Methods in Enzymology 32b, 103 (1974); Syvanen et al., J. Biol. Chem. 284, 3762 (1973); Bolton and Hunter, Biochem. J. 133, 529 (1973). The term labeled may also include polypeptides having a covalently attached amino acid tag as discussed below.

In addition, the novel CD163 polypeptides of the invention may be indirectly labeled. Such labeling involves covalently adding the moiety to the polypeptide and then coupling the added moiety to a label or labeled compound that exhibits specific binding to the added moiety. Possible indirect labeling includes biotinylation of the peptide followed by binding of avidin coupled to one of the labeling groups. Another example is the incubation of a radiolabeled antibody specific for a histidine tag with a CD163 polypeptide comprising a polyhistidine tag. Due to the significant affinity of the antibody for the tag, the net effect is that the radioactive antibody binds to the polypeptide.

The invention also includes variants (or analogs) of the novel CD163 protein. In one example, insert variants are provided wherein one or more amino acid residues are supplemented with a new CD163 amino acid sequence. Insertion may be located at one or both ends of the protein or may be placed within an internal region of the new CD163 protein amino acid sequence. Insertion variants having additional residues at one or both ends can include, for example, fusion proteins and proteins with amino acid tags or labels. Insertion variants include novel CD163 polypeptides, wherein one or more amino acid residues are added to the CD163 acid sequence or biologically active fragment thereof.

Thus insertional variants may also include fusion proteins in which the amino and / or carboxy terminus of the novel CD163 polypeptide is fused to another polypeptide. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tag; Influenza HA Tag Polypeptides and Antibodies 12CA5 [Field et al., Mol. Cell. Biol., 8: 2159-2165 (1988); c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)), and simple herpes virus glycoprotein D (gD) Tacks and antibodies thereof (Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)). Other tag polypeptides include Flag-peptides (Hopp et al., BioTechnology, 6: 1204-1210 (1988)); KT3 epitope peptides (Martin et al., Science, 255: 192-194 (1992)); Alpha-tubulin epitope peptides [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991); And T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397 (1990). In addition, a tag may be attached to the CD163 polypeptide with enzyme proteins such as peroxidase and alkaline phosphatase.

In another aspect, the invention provides deletion variants in which one or more amino acid residues in a novel CD163 polypeptide have been removed. The deletion may occur at one or both ends of the new CD163 polypeptide, or one or more residues in the new CD163 amino acid sequence may be removed. Thus, deletion variants include all fragments of the novel CD163 polypeptide.

CD163 polypeptides contain a transmembrane or membrane anchor region. It should be understood that such transmembrane domains are useful for assisting in targeting heterologous proteins to the membrane when expressed in the context of heterologous proteins. It should also be understood that it may be advantageous to delete some transmembrane domains to enhance the purification or solubility of the protein. Transmembrane deletion variants of CD163 and polynucleotides encoding them have potential value as antiviral therapeutics. Such variants are particularly disclosed herein as SEQ ID NOs: 37-40.

The present invention also encompasses variants of the above-mentioned polypeptides, ie polypeptides changed from a reference sequence by conservative amino acid substitutions.

Exemplary conservative substitutions are described in Tables 1, 2 and 3 of this section entitled “Definitions”.

In situations where it is desirable to partially or completely isolate the novel CD163 polypeptide, purification can be accomplished using standard methods well known to those skilled in the art. Such methods include, but are not limited to, electrophoresis followed by electrolysis, various types of chromatography (immunity, molecular sieve, and / or ion exchange), and / or high pressure liquid chromatography. In some cases, it may be desirable to use two or more such methods for complete purification.

Purification of the novel CD163 polypeptide can be accomplished using a variety of techniques. If the polypeptide is synthesized to contain hexahistidine (CD163 / hexaHis) or other small peptides such as FLAG (Eastman Kodak Co., New Haven, Conn.) Or myc (Invitrogen, Carlsbad, Calif.) At the carboxyl or amino terminus, The polypeptide can be purified essentially in a one step process by passing the solution directly through the column matrix to the polypeptide (ie, a monoclonal antibody that specifically recognizes CD163) or through an affinity column with high affinity for the tag. For example, polyhistidine binds to nickel with great affinity and specificity, and therefore an affinity column of nickel (such as a Qiagen Registered ™ nickel column) can be used for purification of CD163 / polyHis. (See, eg, Ausubel et al., Eds., Current Protocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, New York [1993]).

Even when novel CD163 polypeptides are prepared without a label or tag that facilitates purification, the novel CD163 of the present invention can be purified by immunoaffinity chromatography. To accomplish this, antibodies specific for the CD163 polypeptide can be prepared by means well known in the art.

Antibodies generated against the novel CD163 polypeptides of the invention can be obtained by administering polypeptides or epitope-containing fragments, analogs, or cells to animals, preferably non-human animals, using routine protocols. For the preparation of monoclonal antibodies, any technique known in the art can be used to provide antibodies produced by continuous cell line cultures. Examples include Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., Pg. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), including various techniques.

If a new CD163 polypeptide without a tag is prepared and the antibody is not available, other well known preparative procedures can be used. Such procedures include, but are not limited to, ion exchange chromatography, molecular sieve chromatography, HPLC, natural gel electrophoresis in combination with gel elution, and preparative isoelectric focusing (“Isoprime” machine / technique, Hoefer Scientific). In some cases, increased purity may be achieved by combining two or more of these techniques.

It is to be understood that the definition of a polypeptide of the invention is intended to include polypeptides containing modifications other than insertions, deletions or substitutions of amino acid residues. For example, modifications can be natural covalent bonds, including, for example, chemical bonds with polymers, lipids, other organic and inorganic moieties.

Antibodies

Specific antibodies specific for novel CD163 or fragments thereof (eg, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional / bispecific antibodies, humanized antibodies, human antibodies, and polypeptides of the invention Complementarity Determining Regions (CDRs) -grafted antibodies), which include a compound comprising a CDR sequence that recognizes itself, are also embodied by the invention.

The term “specific” when used to describe an antibody of the invention refers to the variable region of the antibody of the invention exclusively recognizing and binding to a CD163 polypeptide (ie, a local sequence between novel CD163 and other known polypeptides). Despite the possibility of identity, homology or similarity, CD163 polypeptides can be distinguished from other known polypeptides by measurable differences in binding affinity). It is understood that specific antibodies can also interact with other proteins (eg, Staphylococcus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the antibody variable region, particularly sequences within the constant region of the molecule. Will be understood. Screening assays for determining the binding specificity of the antibodies of the invention are well known in the art and are routinely performed. For a comprehensive discussion of such an assay, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6]. Antibodies that recognize and bind fragments of the CD163 polypeptide of the invention are also embodied, provided that the antibody is most specific for the novel CD163 polypeptide. Antibodies of the invention can be produced using any method well known in the art and practiced routinely. Non-human antibodies can be humanized by any method known in the art. In one method, non-human CDRs are inserted into a human antibody or consensus antibody backbone sequence. Additional changes can then be introduced into the antibody backbone to adjust for affinity or immunogenicity.

Antibodies of the invention are useful for diagnostic purposes for detecting or quantifying CD163, as well as for the purification of CD163. Kits comprising an antibody of the invention for any of the purposes described herein are also embodied. In general, the kits of the invention also include a control antigen to which the antibody is immunospecific.

The invention is further illustrated by the following examples, but is not limited thereto.

Example  1: pig CD163 Transient transfection into Inability to proliferate Cell line PRRS  Grants proliferation tolerance for viral infections.

Total mRNA from primary porcine alveolar macrophage cells was used to build cDNA libraries in plasmid pCMV-Sport6.1 (Invitrogen), and cDNA was cloned between the Eco RV and Not I sites. Members of this library received the PRRS-proliferative phenotype when isolated and transiently transfected into the BHK-21 (Baby Hamster Kidney) cell line. Cells were grown at 37 ° C. in 5% CO ₂ atmosphere in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% Fetal Bovine Serum (FBS). Cell cultures were transiently transfected using 10.0 μl of Lipofectamine 2000 (Invitrogen) and 2.0 μg of plasmid. Double monolayers were transfected with negative control plasmid pPAMB. This plasmid was pCMV-Sport6.1 without inserts. Transfection efficiency was monitored by green fluorescent protein (GFP) expressed by the plasmid. About 24 hours after transfection, the monolayers were infected with PRRS virus of North American (Isolation P129) or European (Isolation 96V198) phenotype. For detection of PRRS replication, monolayers were fixed with 80% acetone about 24 hours after infection and incubated with FITC-conjugate monoclonal antibody SDOW17 (Rural Technologies Inc.) for about 1 hour. This monoclonal antibody is specific for the PRRS virus nucleocapsid expressed from open reading frame 7. Nikon TE 300 inverted fluorescence microscopy with 10 × objective lens was used to photograph the monolayers containing FITC positive cells and the negative control tomography.

The transfected cells were found to be proliferative to both the North American (Isolation P129) and European (Isolation 96V198) phenotypes of PRRSV. Expression of viral genes could be detected in a number of transfected BHK cells and progeny virus was readily detectable in supernatant. Proliferation tolerance was not conferred in control transfections with insert-free vectors or inadequate plasmids.

Sequencing of inserts in functional plasmids using the Big Dye Terminator Version 1.0 Sequence Reaction Kit (Applied Biosystems, Foster City, Calif.) And Applied Biosystems 3730 DNA Analyzer (Applied Biosystems) was performed using the published porcine CD163 gene cDNA (Genbank Accession No. AJ311716). Very homologous genes were shown. The cDNA identified by the inventors contained additional 5 'and 3' untranslated regions as compared to AJ311716, and different open reading frames in three ways: (1) internal deletion of 738 bp near the 5 'end, (2 A) 15 bp stretch at the 5 'end to the upstream ATG codon, and (3) a change of 16 nucleotides expected to result in a change of 10 amino acids. Nucleotide sequence identity between the sequences was 99.4%. The alignment of the newly discovered porcine CD163 sequence with the previously reported sequence AJ311716 is shown in FIGS. 1 and 2. The new pig CD163 variant was named "susCD163v1".

Example  2: plasmid pCMVsusCD163v1 Build

Construction of the plasmid pCMVsusCD163v1 was performed as follows. Functional clones identified in the primary swine macrophage cDNA library to confer PRRSV proliferation tolerance were identified by primers 5'DS-CD163 (SEQ ID NO: 6) (5'-CGGAATT CCGCGG ATGTAATAATACAAGAAGA-3 ') and 3'CD163 (SEQ ID NO: 7). (5'CCG CTCGAG TAGTCCAGGTCTTCATCAAGGTATCTT-3 ') served as a template for PCR amplification of CD163 inserts containing 5' and 3 'untranslated regions. Primer 5'DS-CD163 incorporates the Sac II restriction site at the 5 'end of the CD163 insert and primer 3'CD163 incorporates the Xho I restriction site at the 3' end of the insert (underlined). Reactions containing 190 ng of plasmid template were amplified using Platinum Pfx DNA polymerase (Invitrogen Cat # 11708-013) according to the manufacturer's instructions. The reaction was heated to 94 ° C. for 2 minutes, followed by 35 cycles of 20 seconds at 94 ° C., 30 seconds at 55 ° C., and 3.5 minutes at 68 ° C., followed by terminal elongation at 72 ° C. for 7 minutes. The resulting PCR product was purified using Qiaquick PCR Purification Kit (Qiagen Catalog # 28104), digested with restriction enzymes Sac II and Xho I, and the resulting fragments gelled using the Qiaquick Gel Extraction Kit (Qiagen Catalog # 28704). Purified. CD163 PCR fragments were then ligated into plasmid pCMV-Script (Stratagene Catalog # 212220) prepared by digestion with Sac II and Xho I as described above and subsequent gel purification to accommodate digested PCR fragments. The ligation material was transformed into E. coli strain DH5α, recombinants were selected by growth in 50 μg / ml kanamycin and confirmed by recombinant analysis. The resulting plasmid “pCMVsusCD163v1” contains the internally deleted porcine CD163 insert described in Example 1 under the transcriptional control of the eukaryotic CMV promoter, and the neomycin / kanamycin resistance gene under the control of both the eukaryotic and prokaryotic promoters.

Example  3: pRSV - Script  Expression vectors and pRSVsusCD163v1 Build

Plasmid pRc / RSV (Invitrogen) was used as template for PCR amplification of the RSV promoter. RSV promoter sequences were contained within nucleotides 209 to 604 of pRc / RSV. Forward primer PCIRSVLTR (SEQ ID NO: 8) (5'-ACACTCG ACATGT CGATGTACGGGCCAGATATACGCGT-3 ') and reverse primer VSRRTLSAC (SEQ ID NO: 9) (5'-TTCCTTACA GAGCTC GAGGTGCACACCAATGTGGTGAA-3'). Restriction enzymes Pci I and Sac I recognition sites (underlined) were incorporated into 5 'and 3' primers, respectively, for later cloning. PCR was performed using the HotMaster Taq DNA Polymerase Kit (Eppendorf) according to the manufacturer's instructions. The reaction contained 0.9 ng of pRc / RSV plasmid template and 0.3 μM of each primer described above. The reaction was heated to 94 ° C. for 2 minutes, then the cycle of 20 seconds at 94 ° C., 10 seconds at 52 ° C. and 1 minute at 65 ° C. was repeated 30 times. The resulting PCR fragment was digested with restriction enzymes Pci I and Sac I, gel purified and cloned into similarly digested plasmid pCMV-Script (Stratagene) to remove CMV promoter sequences. In the final construct, the RSV promoter was placed immediately upstream of the multiple cloning site and named "pRSV-Script".

The susCD163v1 insert was cloned after the RSV promoter as follows. the susCD163v1 sequences cut by restriction digestion (Kpn I and Sac II) from plasmid pCMVsusCD163v1 and purified gel. This fragment was ligated into the same enzyme and also digested and gel purified pRSV-Script. The ligation mixture was transformed into DH5α Escherichia coli and transformants were selected using 50 μg / ml kanamycin. The clone containing the correct insert was named "pRSVsusCD163v1".

Example  4: pig CD163 cDNA Longer Variant Cloning  And Characterization

Based on the porcine CD163v1 sequence, forward primer 5'CD163NotIlong (SEQ ID NO: 10) (5'CGGTCCGGA GCGGCCGC GATGTAATAATACAAGAAGATTTAAATGG-3 ') using the Lasergene PrimerSelect program (DNASTAR Inc., Madison WI) for amplification of the full length porcine CD163 gene. Reverse primer 3'CD163KpnI (SEQ ID NO: 11) (5'CGGTT GGTACC CAGCAATATTCTTTTTTATTTAATGCC-3 ') was designed. Restriction sites (underlined) for Not I and Kpn I were included in the 5 'and 3' primers, respectively, allowing for easy cloning. Total cellular RNA was prepared from primary alveolar macrophages (PAM) harvested from lung washes of healthy pigs. RNA preparation was performed using the RNeasy mini kit (Qiagen, Valencia, CA). RT-PCR reactions were prepared using the [SuperScript one-step RT-PCR for Long Templates] kit (Invitrogen, Carlsbad, Calif.) And RT-PCR parameters were set as follows: 30 min at 50 ° C., 94 The cycle was repeated 35 times (2 seconds at 94 ° C, 30 seconds at 55 ° C and 4 minutes at 68 ° C) and 10 minutes at 72 ° C. PCR products were analyzed on 0.8% SeaKem GTG Agarose gel. RT-PCR products of various sizes were cut from agarose gels and DNA extracted using GeneClean kit (QBiogene). This RT-PCR product was cloned into pCR2.1-TOPO cloning vector (Invitrogen). Clones were analyzed by restriction enzyme digestion for the presence of inserts. Colonies containing inserts were sequenced using the Big Dye Terminator Version 1.0 Sequence Reaction Kit (Applied Biosystems, Foster City, Calif.) And Applied Biosystems 3730 DNA Analyzer (Applied Biosystems) to confirm sequence reliability. Sequences were edited and assembled using the Lasergene EditSeq and SeqMan programs (DNASTAR Inc., Madison WI). One plasmid with a large insert was named "pCRsusCD163v2" (pCR2.1 containing porcine CD163 variant 2 designated SEQ ID NO: 12). The coding sequence contained in SEQ ID NO: 12 is reproduced and named SEQ ID NO: 13. Sequence analysis showed that this porcine CD163 encodes an amino acid sequence of 1115 amino acids designated SEQ ID NO: 14. Compared to the GenBank swine CD163 sequence (accession number AJ311716), the CD163v2 sequence of the present invention is 98.9% identical at the amino acid level. CD163v2 also has an additional five amino acid residues at the end of the 5 ′ end, extending the open reading frame to the in-frame upstream ATG start codon (like the porcine CD163v1 sequence described in Example 1). Porcine CD163 is 84.3% identical to human CD163 (GenBank accession number Z22968) and 73.7% identical to mouse CD163 (GenBank accession number AF274883) at the amino acid level. The expected signal sequence and transmembrane region of SEQ ID NO: 14 are underlined and in bold, respectively. Determining whether other CD163 sequences have similar sequence characteristics is readily determined by inspection of the sequences.

After restriction enzymes Kpn I and Not I digestion and gel purification, susCD163v2 in pCRsusCD163v2 was released from the pCR2.1 vector. Receptor vector pCMV-script was also excised with the same restriction enzyme pair and susCD163v2 was directionally cloned into pCMV-script. After susCD163v2 was ligated with pCMV-script, the ligated mixture was used to transform STBL 2 E. coli cells (Invitrogen). Restriction digestion analysis revealed that one transfectant contained the CD163 gene, which was named pCMV-script susCD163v2 clone # 3.

Example  5: direct Ligation  And by transfection method RSV  Preparation of Promoter-based Expression Systems

A cloning-based procedure was developed to generate microgram amounts of linear DNA suitable for use in generating stable cell lines expressing CD163 from the RSV promoter (FIG. 4). This procedure involves the isolation and ligation of two pieces of DNA, one containing the neomycin gene and the RSV promoter cassette derived from pRSV-script, and the other containing susCD163v2 coding sequence from pCMVsusCD163v2. Vector plasmid pRSV-Script was linearized upstream of the neomycin gene with Dra III and blunt-terminated with the Klenow fragment of E. coli polymerase. This plasmid was then digested with Not I downstream immediately of the RSV promoter. pCMVsusCD163v2 clones vector sequence was digested with Drd I downstream of the insert in the CD163 was terminated smoothing the Klenow fragment of DNA polymerase. The CD163 coding sequence was released to Not I located upstream immediately near the CD163 coding sequence from the vector. For each plasmid digest, the appropriate fragments were purified from agarose gels. Large scale ligation reactions were performed as follows. About 20 μg of each DNA fragment was incubated with 15 units of T4 DNA ligase in a volume of 600 μl. After the reaction was incubated for 20 minutes at room temperature, an aliquot was removed and the reaction was frozen on dry ice. Aliquots of agarose gel analysis indicated that a significant amount of unligated DNA remained, thus adding another 15 units of ligation enzyme and incubating for an additional 10 minutes at room temperature. After ligation, linear pieces of DNA containing all appropriate elements were purified by agarose gel electrophoresis. Ligation of the two DNA fragments through the sticky Not I terminus placed the 5 'sequence of the CD163 gene downstream of the RSV promoter, which allows for the directed expression of CD163 in mammalian cells. Once isolated, purified DNA was used to transfect various mammalian cell lines.

Example  6: human CD163 cDNA of Cloning  And Characterization

Based on the known human CD163 cDNA sequence (GenBank Accession Number BC051281), forward primer Hu5'Not (SEQ ID NO: 15) (5 'CACC GCGGCCGC GAAGTTATAAATCGCCACCATGAGCAAACTCAGAATGG-3') and reverse primer Hu3'Kpn (SEQ ID NO: 16) using the PrimerSelect program (5'-TGCTCC GGTACC TAGTCCAGGTCTTCATCAAGGTATCTTA-3 '). Restriction sites for Not I and Kpn I (underlined) were incorporated into 5 'and 3' primers, respectively, to facilitate cloning into expression vectors. The sequence CACC was introduced at the 5 'end of the 5' primer to allow directed cloning into the pCDNA3.1D / V5 / His / TOPO vector (catalog # K49001, Invitrogen, see Figure 6). Human CD163 cDNA was amplified from RNA extracted from U937 cell line after stimulation with phorbol 12-myristate 13-acetate (100 ng / ml) for 3 days. Total cellular RNA was prepared using the RNeasy Kit (Qiagen). RT-PCR reaction and sequencing methods were the same as described in Example 4. PCR products were separated from a 0.8% SeaKem agarose gel and extracted from the gel using the GeneClean kit. PCR products were directionally cloned into the pCDNA3.1D / V5 / His / TOPO vector according to the manufacturer's instructions. Two clones with large inserts were sequenced. Sequencing and sequencing methods are described in Example 4. The clone with the correct insert was named "pcDNA3.1D-humCD163v2" and the sequence of the insert was named SEQ ID NO: 17.

The CD163 open reading frame in pCDNA3.1D-humCD163v2 is 1121 residues in length (SEQ ID NO: 18 encoding SEQ ID NO: 19 disclosed below) and 100% identical to Genbank Z22968 (human CD163 cDNA of the same length). The human CD163v2 sequence of the invention is Genbank BC051281 and Z22969 (splicing variants of human CD163) and 42 non-homologous residues in two Genbank sequences replace the seven carboxy terminal residues of the sequence of the invention. And 100% the same. This difference is due to the presence of 83 exons of nucleotides in BC051281 and Z22969, and the frame shift at the 3 'end of the resulting exon. (Law, SK, Micklem, KJ, Shaw, JM, Zhang, XP, Dong, Y., Willis, AC and Mason, DY (1993) A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily.European Journal of Immunology 23 (9), 2320-2325).

Example  7: mouse CD163 of Cloning  And Characterization

Based on the mouse CD163 sequence of GenBank (AF274883), forward primer Mus-new5 '(SEQ ID NO: 20) (5'-CACCGCGGCCGCCACACGGAGCCATCAAAATCATCAA-3') and reverse primer Mus-new3 '(SEQ ID NO: 21) using the PrimerSelect program (5) '-GGTACCGCGAACAAGCAAACCAATAGCAATATTGTTTAATTCCCTC-3' was devised. Restriction sites for Not I and Kpn I were included in the 5 'and 3' primers, respectively, to facilitate subsequent cloning into other expression vectors. Two days after thioglycolate medium was injected into the peritoneal cavity, mouse peritoneal macrophages were harvested from mice. Total cellular RNA was prepared from peritoneal macrophages using the RNeasy kit. RT-PCR reactants and RT-PCR parameters were the same as described in Example 4 except that the annealing temperature was increased to 60 ° C. and the extension temperature was increased to 72 ° C. PCR products were purified on a 0.8% SeaKem agarose gel and directionally cloned into pCDNA3.1D / V5 / His / TOPO according to the manufacturer's instructions. Several clones with large inserts were identified for further analysis. Plasmids containing inserts (SEQ ID NO: 22) encoding proteins of the same length (1121 amino acids, SEQ ID NO: 24) and having only two amino acids different from Genbank AF274883 (99.8% identity) were identified as "pCDNA3.1D-murCD163v2". It was named.

 Another plasmid "pCDNA3.1D-murCD163v3" was generated containing an insert (SEQ ID NO: 25) containing a mouse CD163 coding sequence (SEQ ID NO: 26) encoding a 1159 amino acid long protein (SEQ ID NO: 27). This is only 3 amino acids different from AF274883 in the first 1107 residues (99.7% identity), but from residue 1108 the sequence is completely split. This is due to the insertion of 82 nucleotides in the cDNA, and an incident reading frame shift downstream of the insertion. As a result, mouse CD163v3 contains 52 amino acids at its carboxy terminus that are not homologous to the 14 carboxy-terminal residues of mouse CD163v2. As described for human CD163, these two alternative versions of “full length” mouse CD163 are the most likely splicing variants of the same gene (Law, SK, Micklem, KJ, Shaw, JM, Zhang, XP, Dong, Y., Willis, AC and Mason, DY (1993) A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily.European Journal of Immunology 23 (9), 2320-2325).

Example  8: MARC -145 CD163 of Cloning  And Characterization

Using forward primer 5'simianCD163 (SEQ ID NO: 28) (5'-CACCGGAATGAGCAAACTCAGAATGG-3 ', based on human CD163) and reverse primer HuCD163-3'Kpn (SEQ ID NO: 29) (5'- TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3') CD163 cDNA was amplified from MARC-145 African green monkey kidney cells. Total cellular RNA was prepared from MARC-145 cells using the RNeasy kit. RT-PCR parameters were the same as described in Example 4. RT-PCR products were directionally cloned into the pCDNA3.1D / V5 / His / TOPO vector according to the manufacturer's instructions. Several clones containing large inserts were analyzed. Clone # 25 was named "pCDNA3.1D-MARC-CD163v2". This new CD163 cDNA from MARC-145 cells is 1116 amino acids in length. Compared with the GenBank database, the MARC-145 CD163 amino acid sequence was 96.3% identical to human CD163 (Genbank Z22968), 84.7% identical to pig CD163 (Genbank AJ311716), and 73.9% identical to mouse CD163 (Genbank AF274883). .

Example  9: Vero  Monkey from cells CD163 of Cloning  And Characterization .

Using forward primer 5'simianCD163 (SEQ ID NO: 28) (5'-CACCGGAATGAGCAAACTCAGAATGG-3 ', based on human CD163) and reverse primer HuCD163-3'Kpn (SEQ ID NO: 29) (5'-TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3') CD163 cDNA was amplified from Vero cells. Total cellular RNA was prepared from Vero cells using the RNeasy kit. RT-PCR parameters were the same as described in Example 4. RT-PCR products were directionally cloned into the pCDNA3.1D / V5 / His / TOPO vector according to the manufacturer's instructions. Eight clones containing large inserts were sequenced and six distinct splicing patterns were identified. This pattern is illustrated graphically in FIG.

The six splicing variants differ in the absence or presence of three exons named E6, E105, and E83. Omission of E6 or E105 does not change the leading frame, while omission of E83 changes the frame. Similar patterns to v2 and / or v3 were also seen in pig, mouse, human and MARC-145 monkey cells. Patterns v4 and v5 are devoid of 105 nucleotides that encode hydrophobic transmembrane regions. This cDNA could not allow BHK cells to be proliferative for PRRSV infection in transient transfection assays, probably because CD163 is secreted rather than bound to the membrane. CD163 molecules without the transmembrane region appear to be nonfunctional as cellular proliferative factor, but are immunogens for direct virus neutralization (similar to neutralizing antibodies) or for induction of anti-CD163 antibodies that block viral infection in host animals. It is possible that they can be useful as.

The longest splicing variant v7 contains all three exons E6, E105, and E83. This novel CD163 cDNA from Vero cells encodes a polypeptide of 1153 amino acids in length. Compared with the GenBank database, the Vero CD163v7 amino acid sequence was 95.4% identical to human CD163 (Genbank Z22968), 83.7% identical to pig CD163 (Genbank AJ311716), and 72.1% identical to mouse CD163 (Genbank AF274883). Nucleotide and amino acid sequences of the six splicing variants identified in Vero cells are provided below (SEQ ID NOs: 33-44).

Example  10: DH82  Dog from cells CD163 of Cloning  And Characterization

Using forward primer 5'simianCD163 (SEQ ID NO: 28) (5'-CACCGGAATGAGCAAACTCAGAATGG-3 ', based on human CD163) and reverse primer HuCD163-3'Kpn (SEQ ID NO: 29) (5'-GCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3') CD163 cDNA was amplified from DH82 cells. Total cellular RNA was prepared from DH82 cells using the RNeasy kit. RT-PCR parameters were the same as described in Example 4. RT-PCR products were directionally cloned into the pCDNA3.1D / V5 / His / TOPO vector according to the manufacturer's instructions. Several clones containing large inserts were analyzed. Several clones containing large inserts were analyzed and they belonged to the v2 or v3 splicing pattern seen in other species. The v2 variant lacks 81 nucleotide exons (E81) compared to the v3 variant, resulting in a shift in the reading frame resulting in an alternative carboxy terminal amino acid sequence. Dog CD163v2 cDNA from DH82 cells encodes a peptide of 1115 amino acids. Compared to the GenBank database, it was 83.9% identical to human CD163 (Genbank Z22968), 85.1% identical to pig CD163 (Genbank AJ311716), and 74.3% identical to mouse CD163 (Genbank AF274883). The nucleotide and amino acid sequences of the two splicing variants identified in DH82 cells are provided below (SEQ ID NOs: 45-48).

Example  11: pCMV - with susCD163v1 Cell lines after transient transfection of PRRSV  It becomes proliferative to infection.

Porcine Kidney (PK032495), Northern Labs Swine Testicular (NLST-1), Northern Labs Dog Kidney (NLDK-1) were obtained from Pfizer Inc., and 5% bovine at 37 ° C. and 5% CO ₂ . Growing in growth medium consisting of fetal serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen Cat # 11965) supplemented with antibiotics. Cell lines Baby Hamster Kidney (BHK21), Northern Labs Cat Kidney (NLFK-1), and Rabbit Lung (RL) were obtained from Pfizer Inc., 10% Fetal Bovine Serum (FBS), 1 at 37 ° C. and 5% CO ₂ . Growing in growth medium consisting of Dulbecco's modified Eagle's medium (DMEM, Invitrogen Cat # 11965) supplemented with mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Vero cells were obtained from Pfizer Inc. and minimally essential medium alpha (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 20 μg / ml gentamicin at 37 ° C. and 5% CO ₂ . Minimum essential medium), Pfizer Inc. formulation). Using Lipofectamine 2000 (Invitrogen Catalog # 11668-027) according to the manufacturer's instructions, cell culture wells (35 mm) containing about 1 × 10 ⁶ cells were placed in 2 μg / well of DMEM without FBS or antibiotics. Transfection with plasmid pCMV-susCD163v1. Cell line RL was transfected with 1.0 μg / well of plasmid pCMV-susCD163v1. A member of the insertless PAM cell cDNA library, designated pPAMB (essentially empty pSport plasmid vector), was used as the negative control plasmid. 24 hours after transfection, wells were aspirated, washed twice with DMEM / 5% FBS, and then infected with North American type PRRSV isolate P129. After the virus was adsorbed in 0.5 ml growth medium for a minimum of 2 hours, additional medium was added to a final volume of 2.0 ml and incubated overnight. Virus was then removed, the wells washed twice with growth medium and fresh growth medium was added (2.0 mL / well). A time point sample of the culture was taken immediately to determine the background level of infectious virus from the inoculum. At least 48 hours after infection, cultures were screened for proliferative tolerance by removing the cultures for analysis of viable viruses and proliferative cells in monolayers were detected by fluorescent antibody assay (FA). Monolayers were fixed with 80% acetone to complete FA and stained with FITC-conjugate monoclonal antibody SDOW17 (Rural Technologies Inc) specific for PRRSV nucleocapsid protein. Viable viruses were titrated by inoculating dilutions of the culture onto MARC-145 cells. Table 5 shows the results of viral infection by FA and the presence of progeny virus for each cell line tested.

Failure to detect progeny virus from some cell lines may be a result of low viral titers in cell culture below the detection limit of the assay. Proliferation tolerance of Vero cells to PRRSV infection was enhanced by expression of susCD163v1. Compared to the time-point measurement of background virus, viral titers increased by almost log2 in Vero cells transfected with pCMV-susCD163v1, but increased in titers below log1 in cells transfected with negative control plasmid pPAMB. After transfection with pCMV-susCD163v1, all cell lines except NLDK-1 were positive in FA for proliferative tolerance for infection with North American type PRRSV isolate P129.

Screening of Various Cell Lines for Proliferative Tolerance on North American PRRSV Isolation P129 After Transient Transfection with pCMV-susCD163v1 or pPAMB Transfected Cell Line Fluorescent Antibody Assay Progeny Virus Produced pCMV-susCD163v1 pPAMB pCMV-susCD163v1 pPAMB BHK21 +++ - +++ - PK032495 + - + - NKFK-1 + - + - NLST-1 + - - - NLDK-1 - - NT NT RL + - - - VeroS ++ + ++ + +++ = Very Positive ++ = Moderately Positive + = Slightly Positive-= Not Detectable NT = Not Tested

Example  12: pCMV - susCD163v1 After transient transfection to the furnace BHK21  Cells european PRRSV  It becomes proliferative to infection.

Cell line baby hamster kidney (BHK21) was obtained from Pfizer Inc. and Dulbecco supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics at 37 ° C. and 5% CO ₂ . Growing in growth medium consisting of modified Eagle's medium (DMEM, Invitrogen catalog # 11965). Using Lipofectamine 2000 (Invitrogen Catalog # 11668-027) according to the manufacturer's instructions, cell culture wells (35 mm) containing about 1 × 10 ⁶ cells were placed in 2 μg / well of DMEM without FBS or antibiotics. Transfection with plasmid pCMV-susCD163v1. 24 hours after transfection, wells were aspirated, washed twice with DMEM / 5% FBS, and then infected with European PRRSV isolate 96V198. Virus was adsorbed for at least 2 hours. Virus was then removed, the wells washed twice with growth medium and fresh growth medium was added (2.0 mL / well). A time point sample of the culture was taken immediately to determine the background level of infectious virus from the inoculum. At least 48 hours after infection, cultures were screened for proliferative tolerance by removing the cultures for analysis of viable viruses and proliferative cells in monolayers were detected by fluorescent antibody assay (FA). Monolayers were fixed with 80% acetone to complete FA and stained with FITC-conjugate monoclonal antibody SDOW17 (Rural Technologies Inc) specific for PRRSV nucleocapsid protein. Viable viruses were titrated by inoculating dilutions of the culture onto MARC-145 cells. As a result of the transient transfection of BKH21 with pCMV-susCD163v1, the cells became proliferable for infection with European-type PRRSV isolate 96V198 and progeny virus was produced.

Example  13: from various animal species CD163  Gene BHK21  Cells PRRS  Allows proliferation for viral infections.

BHK21 cells grown in DMEM (Invitrogen Cat # 11965) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and antibiotics were used in transient transfection experiments. Prior to transfection, cells were washed with OptiMEM (Invitrogen) without serum or other additives. Lipofectamine 2000 (Invitrogen) was used in all transfection experiments according to the manufacturer's protocol. The transfection mixture consisted of 10 μl Lipofectamine 2000 and 2-3 μg DNA per 35 mm well. After overnight incubation, the transfection medium was removed and cells were infected with PRRSV isolate P129. After the infection progressed for 24-48 hours, cells were fixed with 80% acetone and stained with monoclonal antibody SDOW17 conjugated with FITC (Rural Technology Inc., Brookings, SD). Staining of nucleocapsid protein was visualized under fluorescence microscopy. Table 6 shows that transient transfection of BHK21 cells with various D163 genes allows the cells to be proliferative to PRRS virus infection.

Plasmid skeleton CD163 gene PRRSV Infection (FA) pCMV-Script Pig CD163v1 +++ pRSV-Script Pig CD163v1 +++ pcDNA3.1D Pig CD163v2 ++ pcDNA3.1D Human CD163v2 ++ pcDNA3.1D Mouse CD163v3 + pcDNA3.1D African green monkey (MARC-145 cells) CD163v2 +++ pcDNA3.1D Vero Cell CD163v7 +++ pcDNA3.1D DH82 cells CD163v2 +++ +++ = very positive ++ = moderately positive + = slightly positive

Example  14: pCMV - susCD163v1 Generation of Stable PRRSV-Proliferative BHK21 Cell Line Using

BHK-21 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and antibiotics. For transfection, cells were seeded in 6-well plates with about 90% confluence and grown overnight at 37 ° C. in 5% CO ₂ . Cells were transfected with pCMV-susCD163v1 DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. One day after transfection, the cells were trypsinized and seeded again in 96-well plates in serial dilutions. To select stable transfectants, the medium was supplemented with 1 mg / ml geneticin (G418 sulphate, Invitrogen Cat # 10131-027) in the future. Medium was changed every 3-5 days. Plates were incubated until wells with colonies derived from single cells reached confluence, then the plates were trypsinized and seeded into double 96 well plates. One of the 96 well plates was infected with PRRSV isolate P129 and clones that were proliferative to infection were identified by staining with FITC conjugated monoclonal antibody SDOW17. Positive clones were then expanded from the second double plate. To ensure homogeneity, positive cultures were single-cell cloned by limiting dilution. In each cloning, subclones exhibiting robust growth and high PRRSV proliferation tolerance were selected for expansion. Three clones named BHK / CMV / v1 # 3, BHK / CMV / v1 # 5, and BHK / CMV / v1 # 12 (FIG. 6) were selected. These cell lines maintained the proliferative tolerance phenotype up to 20 passages.

Example  15: p RSV - susCD163v1 Generation of Stable PRRSV-Proliferative BHK21 Cell Line Using

BHK-21 cells were cultured as described in Example 14. BHK-21 cells were transfected with pRSVsusCD163v1 using Lipofectamine 2000 as described in Example 14. Cloning of transfected cells and screening for proliferative clones were performed essentially as described in Example 14. Three single cell clones were identified from the original cloning that were proliferative, and they were cloned again two times to attempt to ensure homogeneity and to isolate subclones of higher proliferative tolerance (see FIG. 7). The resulting cell lines were named BHK / RSV / v1, # 2, # 3, and # 4. All these clones maintained a proliferative phenotype up to the highest passage (passage 11 for clone # 2, passage 7 for clone # 3 and passage 5 for clone # 4).

Example  16: pCMV - susCD163v1 Generation of Stable PRRSV-Proliferative Cat Renal Cell Line Using

Parental Northern Lab cat kidney (NLFK) cells were grown in Dulbecco's Modified Eagle's Medium (Invitrogen Catalog # 11965-092) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and antibiotics at 37 ° C. and 5% CO ₂ . I was. Using the Lipofectamine 2000 (Invitrogen Catalog # 11668-027) according to the manufacturer's instructions, several 35 mm wells each containing about 2 × 10 ⁶ cells were transfected with 4 μg / well of pCMV-susCD163v1 in OptiMEM. . After overnight incubation, cells were removed from the substrate using Accutase (Innovative Cell Technologies, Catalog # AT104), diluted in media, and placed in three 96 well plates at three densities (about 2 × 10 ² , 2 ×). 10 ³ , and 2 × 10 ⁴ cells / well). Prior to starting the selection of stable transformants, cells were allowed to settle at 37 ° C. overnight. The following morning, the medium was replaced with 100 μl / well with fresh medium containing 500 μg / ml Geneticin (G418 sulphate, Invitrogen Cat # 10131-027) to select cells expressing neomycin resistance genes. The medium was changed every 2 or 3 days to maintain geneticin efficacy. After 19 days of selection, 26 wells with 70 empty wells and one or more colonies of G418-resistant cells were generated in a 96 well plate (about 200 cells / well) with the lowest initial cell density (using Poisson distribution). , The calculated number / well of resistant cells is 0.3). These 26 wells were split into double wells and allowed to settle overnight. One set of wells was infected with PRRSV isolate P129, incubated for 24 hours, fixed with 80% acetone and stained with the FITC-conjugate monoclonal antibody SDOW17 (Rural Technologies Inc) specific for PRRSV nucleocapsids. . Of the 26 clones, eight contained some cells infected with PRRSV. One of these, named "NLFK-CMV-susCD163v1-G4", was clearly more proliferable than others, and nearly 100% of cells stained positive for viral antigen.

By cell passage number 5, there was some evidence of phenotypic homogeneity in the NLFK-CMV-susCD163v1-G4 cell line. Thus, starting from a frozen stock of NLFK-CMV-susCD163v1-G4 passage 4, cells were single-cell cloned by limiting dilution in G418-containing medium. Twelve such clones ("A"-"L") were expanded for research. Among these, notable were the clones NLFK-CMV-susCD163v1-G4F and NLFK-CMV-susCD163v1-G4L due to their ability to form isolated plaques (local regions of CPE) when infected with PRRSV isolate P129 (see FIG. 8). ).

Example  17: pRSV - susCD163v1 Generation of Stable PRRSV-Proliferative Cat Renal Cell Line Using

Northern Labs cat kidney (NLFK) cells were grown in minimal essential medium alpha medium (Invitrogen Cat # 12571-071) supplemented with 10% fetal bovine serum and antibiotics at 37 ° C. and 5% CO ₂ . NLFK cells were seeded in 6-well plates with about 90% confluence and attached overnight. The cells were then transfected with plasmid pRSV-susCD163v1 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 24 hours, cells were cloned as described in Example 14. Screening for PRRSV proliferative cell clones was performed as described in Example 14. Four clones were selected from the screening and single cell cloned by two more limit dilutions. Four clones named FK / RSV / v1 # 1, FK / RSV / v1 # 2, FK / RSV / v1 # 3, and FK / RSV / v1 # 4 were selected. These cell lines maintained the PRRSV proliferative tolerance phenotype up to at least 8 passages (see FIG. 9).

Example  18: pCMV - susCD163v1 Generation of Stable PRRSV-Proliferative Porcine Kidney Cell Lines Using

Parental pig kidney (PK032495) cells were obtained from Pfizer Inc. and Dulbecco supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics at 37 ° C. and 5% CO ₂ . Growing in growth medium consisting of modified Eagle's medium (DMEM, Invitrogen catalog # 11965). Using Lipofectamine 2000 (Invitrogen Catalog # 11668-027), tissue culture wells (35 mm) each containing approximately 1 × 10 ⁶ cells were treated with 2 μg / well of FMEM or antibiotic-free DMEM, using the manufacturer's instructions. Transfection with plasmid pCMV-susCD163v1 in the virus. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cell Technologies, Catalog # AT104), and 1.0 mg / genesisin (G418 sulphate, Invitrogen Catalog # 10131-027) was removed. Dilutions were made in growth medium containing ml and seeded in 96-well plates at various densities to ensure recovery of single cell clones after geneticin selection. During geneticin selection, the medium was changed about every 3-5 days. After selection, wells containing single cell clones were expanded into double 96 well plates and incubated until 100% confluence was reached. One set of wells was screened for PRRSV-proliferative tolerance by infection with PRRSV isolate P129 for at least 48 hours. Eleven clones were found to be proliferative to PRRSV. One of these, named “PK-CMV-susCD163v1-A10”, clearly maintained the proliferative tolerance phenotype after numerous passages (see FIG. 10).

Example  19: pCMVScript - susCD163v2 Stable with PRRSV Growth Tolerance BHK21  Generation of cell lines

Parental baby hamster kidney (BHK21) cells were obtained from Pfizer Inc. and supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics at 37 ° C. and 5% CO ₂ . Growing in growth medium consisting of Béco modified eagle medium (DMEM, Invitrogen catalog # 11965). Using Lipofectamine 2000 (Invitrogen Catalog # 11668-027), tissue culture wells (35 mm) each containing approximately 1 × 10 ⁶ cells were treated with 2 μg / well of FMEM or antibiotic-free DMEM, using the manufacturer's instructions. And transfected with pCMVScript-susCD163v2. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cell Technologies, Catalog # AT104), and 1.0 mg / genesisin (G418 sulphate, Invitrogen Catalog # 10131-027) was removed. Dilutions were made in growth medium containing ml and seeded in 96-well plates at various densities to ensure recovery of single cell clones after geneticin selection. During geneticin selection, the medium was changed about every 3-5 days. After selection, wells containing single cell clones were expanded into double 96 well plates and incubated until 100% confluence was reached. One set of wells was screened for proliferative tolerance by infection with PRRSV isolate P129 and incubation for a minimum of 48 hours. Three clones were identified as PRRSV-proliferatively acceptable and one of them, designated “BHK-CMVScript-susCD163v2-A9”, was selected for later study (see FIG. 11).

Example  20: pRSV - susCD163v2 Generation of Stable PRRSV-Proliferative BHK-21 Cell Line Using

BHK-21 cells were cultured as described in Example 14. BHK-21 cells were transfected with the ligation pRSV-susCD163v2 DNA construct described in Example 5 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Subsequent cloning and selection of PRRSV proliferative cell lines was performed as described in Example 14. Of the 336 single cell clones tested, 129 were positive. Several of these cell clones were passaged seven times and they maintained a PRRSV proliferative tolerance phenotype (see FIG. 12). These cell lines were named with BHK / RSV / v2 followed by numeric clone numbers.

Example  21: pCMVScript - susCD163v2 Stable with PRRSV Generation of Proliferatively Acceptable Porcine Kidney Cell Lines

Parental pig kidney (PK032495) cells were obtained from Pfizer Inc. and Dulbecco supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics at 37 ° C. and 5% CO ₂ . Growing in growth medium consisting of modified Eagle's medium (DMEM, Invitrogen catalog # 11965). Using Lipofectamine 2000 (Invitrogen Catalog # 11668-027), tissue culture wells (35 mm) each containing approximately 1 × 10 ⁶ cells were treated with 2 μg / well of FMEM or antibiotic-free DMEM, using the manufacturer's instructions. And transfected with pCMVScript-susCD163v2. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cell Technologies, Catalog # AT104), and 1.0 mg / genesisin (G418 sulphate, Invitrogen Catalog # 10131-027) was removed. Dilutions were made in growth medium containing ml and seeded in 96-well plates at various densities to ensure recovery of single cell clones after geneticin selection. During geneticin selection, the medium was changed about every 3-5 days. After selection, wells containing single cell clones were expanded into double 96 well plates and incubated until 100% confluence was reached. One set of wells was screened for proliferative tolerance by infection with PRRSV isolate P129 and incubation for a minimum of 48 hours. One clone named "PK-CMVScript-susCD163v2-D1" showed a PRRSV-proliferative acceptable phenotype.

Example  22: pcDNA3 .1D- humCD163v2 Generation of Stable PRRSV-Proliferative BHK21 Cell Line Using

Parental baby hamster kidney (BHK21) cells were obtained from Pfizer Inc. and supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics at 37 ° C. and 5% CO ₂ . Growing in growth medium consisting of Béco modified eagle medium (DMEM, Invitrogen catalog # 11965). Using Lipofectamine 2000 (Invitrogen Catalog # 11668-027), tissue culture wells (35 mm) each containing approximately 1 × 10 ⁶ cells were treated with 2 μg / well of FMEM or antibiotic-free DMEM, using the manufacturer's instructions. Transfection with pcDNA3.1D-humCD163v2. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cell Technologies, Catalog # AT104), and 1.0 mg / genesisin (G418 sulphate, Invitrogen Catalog # 10131-027) was removed. Dilutions were made in growth medium containing ml and seeded in 96-well plates at various densities to ensure recovery of single cell clones after geneticin selection. During geneticin selection, the medium was changed about every 3-5 days. After selection, wells containing single cell clones were expanded into double 96 well plates and incubated until 100% confluence was reached. One set of wells was screened for proliferative tolerance by infection with PRRSV isolate P129 and incubation for a minimum of 48 hours. Seven candidate clones were identified as PRRSV-proliferative. There was some evidence of phenotypic homogeneity in each of the seven candidate clones, perhaps because they were not clonal. Thus, candidate clones were single-cell cloned by limiting dilution in G418 containing medium. One single cell clone with apparent PRRS-proliferative tolerance was obtained and named BHK-cDNA3.1D-humCD163v2-H9.

Example  23. pcDNA3 .1D- humCD163v2 Generation of Stable PRRSV-Proliferative Cat Renal Cell Line Using

Parental Northern Lab cat kidney (NLFK) cells were treated with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics at 37 ° C. and 5% CO ₂ . , Invitrogen catalog # 11965). Using Lipofectamine 2000 (Invitrogen Catalog # 11668-027), tissue culture wells (35 mm) each containing approximately 1 × 10 ⁶ cells were treated with 2 μg / well of FMEM or antibiotic-free DMEM, using the manufacturer's instructions. Transfection with pcDNA3.1D-humCD163v2. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cell Technologies, Catalog # AT104), and 500 μg / genesisin (G418 sulphate, Invitrogen Catalog # 10131-027) was removed. Dilutions were made in growth medium containing ml and seeded in 96-well plates at various densities to ensure recovery of single cell clones after geneticin selection. During geneticin selection, the medium was changed about every 3-5 days. After selection, wells containing single cell clones were expanded into double 96 well plates and incubated until 100% confluence was reached. One set of wells was screened for PRRSV-proliferative tolerance by infection with PRRSV isolate P129 for at least 48 hours. Five clones were found to be proliferative. One of these, named “FK-cDNA3.1D-humCD163v2-A6”, clearly showed the proliferative tolerance phenotype (see FIG. 13).

NLFK parental cells and one subclone of FK-cDNA3.1D-humCD163v2-A6 were tested for CD163 expression. Cells were fixed in 80% acetone and reacted with goat anti-human CD163 (R & D System, 1: 200) for 1 hour and then washed with PBS. For visualization, donkey anti-goat IgG (Biodesign Inc, 1: 100) conjugated with FITC was used. As shown in FIG. 21A, no specific fluorescence was detected in NLFK parental cells. Most of the FK.A6.A2 subclones showed good fluorescence staining, indicating the presence of CD163 (FIG. 21B).

Example  24: pcDNA3 .1D- humCD163v2 Generation of Stable PRRSV-Proliferative Porcine Kidney Cell Lines Using

Parental pig kidney (PK032495) cells were obtained from Pfizer Inc. and Dulbecco supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics at 37 ° C. and 5% CO ₂ . Growing in growth medium consisting of modified Eagle's medium (DMEM, Invitrogen catalog # 11965). Using Lipofectamine 2000 (Invitrogen Catalog # 11668-027), tissue culture wells (35 mm) each containing approximately 1 × 10 ⁶ cells were treated with 2 μg / well of FMEM or antibiotic-free DMEM, using the manufacturer's instructions. Transfection with pcDNA3.1D-humCD163v2. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cell Technologies, Catalog # AT104), and 1.0 mg / genesisin (G418 sulphate, Invitrogen Catalog # 10131-027) was removed. Dilutions were made in growth medium containing ml and seeded in 96-well plates at various densities to ensure recovery of single cell clones after geneticin selection. During geneticin selection, the medium was changed about every 3-5 days. After selection, wells containing single cell clones were expanded into double 96 well plates and incubated until 100% confluence was reached. One set of wells was screened for PRRSV-proliferative tolerance by infection with PRRSV isolate P129 for at least 48 hours. Two clones were found to be proliferative. One of these, named “PK-cDNA3.1D-humCD163v2-B11”, clearly showed the PRRSV-proliferative phenotype.

Example  25: Ligation pRSV - Script MARC CD163v2 Generation of Stable PRRSV-Proliferative Cat Renal Cell Line Using

A cloning-based procedure was developed to generate microgram amounts of linear DNA suitable for use in generating stable cell lines expressing CD163 from the RSV promoter (FIG. 4). A similar process was modified to place monkey CD163v2 from MARC-145 cells behind the RSV promoter. This procedure involves the isolation and ligation of two pieces of DNA, one containing the neomycin gene and the RSV promoter cassette derived from pRSV-script, and the other containing the MARC CD163v2 coding sequence from pCDNA3.1D MARC CD163v2. do. The plasmid pRSV-Script vector was linearized with Hind III and Kpn I. The plasmid was first digested with Kpn I and blunt-terminated with a cleno fragment of E. coli DNA polymerase. This plasmid was then digested with Hind III downstream immediately of the RSV promoter. pCDNA3.1D MARC CD163v2 clone was digested with Eco RV downstream of the CD163 insert within the vector sequence and upstream of CD163 with Hind III. CD163 coding sequence was released from the vector. For each plasmid digest, the appropriate fragments were purified from agarose gels. Large scale ligation reactions were performed as follows. About 20 μg of each DNA fragment was incubated with 15 units of T4 DNA ligase in a volume of 600 μl. The reaction was incubated for 1 hour at room temperature. After ligation, linear pieces of DNA containing all appropriate elements were purified by agarose gel electrophoresis. Restriction digestion assays were performed to confirm the reliability of each ligation fragment. Ligation of the two DNA fragments through the sticky Hind III terminus placed the 5 'sequence of the MARC CD163 gene downstream of the RSV promoter, which allows for directed expression of CD163 in mammalian cells. Once isolated, purified DNA was used to transfect various mammalian cell lines.

Northern Labs cat kidney (NLFK) cells were grown in DMEM supplemented with 5% fetal bovine serum and antibiotics at 37 ° C. and 5% CO ₂ . NLFK cells were seeded in 6-well plates at about 90% confluence and allowed to attach overnight. The cells were then transfected with the ligated plasmid pRSV-MARC CD163v2 using Lipofectamine 2000 according to the manufacturer's instructions. After 24 hours, cells were cloned as described in Example 12. Screening for PRRSV proliferative cell clones was performed as described in Example 12. One clone was positive for PRRSV infection and named NLFK-MARC CD163 D4. These D4 clones maintained the PRRSV proliferative tolerance phenotype to 9 passages.

Example  26: CMV  From the promoter susCD163v1 Recombinant to stably express BHK -21 and NLFK  In the cell PRRSV Segregation NVSL  94-3 growth kinetics

The amount of progeny virus produced by PRRSV-infected BHK-21 or NLFK cells stably engineered to express CD163v1 was quantified. Four cell lines expressing susCD163v1, BHK / CMV / susv1 # 3, BHK / CMV / susv1 # 5, BHK / CMV / susv1 # 12, and FK / CMV / susv1 G4 were seeded in 6-well plates with sub-front growth And, after overnight incubation, infected with NVSL 94-3 isolate of PRRSV. MARC-145 cells were included in the experiment for comparison. The cells were infected with the virus at a multiplicity of infection (m.o.i.) of about 0.1. Virus was adsorbed for 60-90 minutes and removed. The cells were washed three times with PBS to remove the remaining virus. An aliquot of 1 ml was harvested from the culture at 12 hour intervals beginning immediately after infection and continuing up to 96 hours. Fresh culture medium was added to the cells at various time points to maintain sufficient culture volume to prevent the cell monolayer from drying out. Culture supernatants were stored at -80 ° C until all samples were collected. The amount of PRRSV present in the culture supernatant was determined on MARC-145 cells by plaque assay. 14 shows that all tested CD163 expressing recombinant cell lines were able to produce progeny PRRSV.

Example  27: anti- CD163  To antibodies PRRSV  Infection Blocking: Transiently Transfected Cells

Using Lipofectamine 2000 as described in Example 14, BHK-21 cells seeded in 24-well plates were transiently transfected with the plasmid pCDNA3.1D-MARC-CD163v2 described in Example 8. After expression of CD163 by overnight incubation, a titration of goat polyclonal antibody (R & D Systems, Catalog # AF1607) specific for human CD163 in PBS was added to the cells in a volume of 100 μl. As control, an equivalent amount of normal goat IgG (R & D Systems, Catalog # AB-108-C) was used. After 1 hour incubation at 37 ° C., the monolayer was infected with about 1 × 10 ⁷ pfu of recombinant P129 strain of PRRSV expressing GFP. After incubating the cell monolayer with anti-CD163 antibody and PRRSV for 1 hour at 37 ° C., the virus inoculum / antibody mixture is aspirated, the cell monolayer is washed once with PBS and 1 ml of growth medium is added to the wells. It was. Cells were incubated at 37 ° C. for 24 hours to allow PRRSV-directed GFP expression. For analysis, cells were trypsinized, resuspended in 500 μl of PBS and analyzed by flow cytometry to count PRRSV infected cells via GFP expression. For flow cytometry, uninfected BHK-21 cells were used to establish a baseline for fluorescence detection and about 100,000 cells were analyzed from each subsequent sample. The results of this analysis, shown in FIG. 15, indicate that the CD163 specific antibody was able to significantly reduce the number of cells infected when compared to cells incubated with normal goat IgG.

Example  28: anti- CD163  By antibody PRRSV  Infection Blocking: Stably Transfected Cells

NLFK cells stably expressing human CD163 (FK-cDNA3.1D-humCD163v2-A6), described in Example 23, were seeded into 24-well plates. After overnight cell attachment, a titration of goat polyclonal antibody (R & D Systems, Catalog # AF1607) specific for human CD163 in PBS was added to the cells in a volume of 100 μl. As control, an equivalent amount of normal goat IgG (R & D Systems, Catalog # AB-108-C) was used. After 1 hour incubation at 37 ° C., the monolayer was infected with about 1 × 10 ⁷ pfu of recombinant P129 strain of PRRSV expressing GFP. After incubating the cell monolayer with anti-CD163 antibody and PRRSV for 1 hour at 37 ° C., the virus inoculum / antibody mixture is aspirated, the cell monolayer is washed once with PBS and 1 ml of growth medium is added to the wells. It was. Cells were incubated at 37 ° C. for 24 hours to allow PRRSV-directed GFP expression. For analysis, cells were trypsinized, resuspended in 500 μl of PBS and analyzed by flow cytometry to count PRRSV infected cells via GFP expression. About 100,000 cells were analyzed from each sample. The results of this assay, shown in FIG. 16, indicate that the CD163 specific antibody was able to significantly reduce the number of cells infected when compared to cells incubated with normal goat IgG.

Example  29: pRSV - susCD163v2 Generation of Stable PRRSV-Proliferative Porcine Kidney Cell Lines Using

Porcine kidney cells (PK032495) were cultured as described in Example 21. For transfection, 24-well plates were seeded with 80% confluence and restored overnight. Transfection with the ligation pRSV-susCD163v2 DNA described in Example 5 was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Subsequent cloning and selection of PRRSV proliferative cells was performed essentially as described in Example 14. Initial cloning by limiting dilution did not yield a single cell derived clone, so five wells with PRRSV proliferatively acceptable cells were cloned again by limiting dilution to yield a clonal cell line. Ten clones were selected for further study, and one of these clones, PK-RSVScript-susCD163v2 # 9, showed the ability to support focus growth of PRRSV early after infection (see FIG. 18).

Example  30: pRSV - susCD163v2 Generation of Stable PRRSV-Proliferative Cat Renal Cell Line Using

NLFK cat kidney cells were cultured as described in Example 17. For transfection, 24 well plates were seeded at a maximum density of about 80%. After overnight incubation, the monolayer was transfected with RSV / susCD163v2 derived from ligation (see Example 5) using Lipofectamine according to the manufacturer's instructions. Cloning of the transfected cells and selection of PRRSV proliferative cell clones were performed essentially as described in Example 14. Of the 67 cell clones tested for PRRSV proliferation tolerance, 20 were found to be positive. An example of the staining observed is shown in FIG. 19.

Example  31: PK - RSVScript - susCD163v2  In the cell PRRSV Segregation P201 of Subculture

Amplification of the PRRSV clinical isolate was performed as follows. Using OptiMEM supplemented with 2% FBS, peripheral alveolar macrophages (PAM) cells were seeded in 6-well dishes at 5.4E6 cells / 10 cm 2. After 6 hours, the medium was aspirated and 2 ml aliquots of serum harvested from PRRSV infected pigs were added to the cells. After 90 minutes of adsorption, serum inoculum was removed and replaced with OptiMEM. After about 40 infection, the supernatants were harvested and clarified by centrifugation for 10 minutes. Supernatants were used directly to infect PK-RSVScript-susCD163v2 clone # 9 cells using 6 h adsorption. After removal of the inoculum, the cells were fed back with D-MEM. P201 virus was serially passaged on the PK-RSVScript-susCD163v2 # 9 cell line using alternating infected cells and acellular supernatant passages. For efficient spread of the virus, it was observed by the inventors that the flasks were seeded at 50-70% confluence the day before infection using a flask of cells maintained in sub-front growth. To follow the progress of infection, each passage was replicated in multiple wells of identically infected cells, one of the wells was fixed with acetone on each day and stained with FITC labeled monoclonal antibody SDOW17. If the percentage of infected cells was 50% or less and no significant progression of focus growth was seen compared to the previous day's observation, cells in equivalent wells were trypsinized and passaged into fresh multiple wells. This infected cell passage was typically 1: 4 divisions and sometimes included the addition of the same number of cells from uninfected cultures. Alternatively, if SDOW17 staining indicated that the infected cell focus had spread sufficiently to account for more than 50% of the total cells, the cell-free supernatants were harvested and used to infect multiple wells of freshly seeded cells (FIG. 20). After 11 passages, no intervening cell passage was necessary because the virus grew to sufficient titers to allow continuous cell-free supernatant passage of the virus.

Example  32: Various European and North American PRRSV On a separate note  For proliferation tolerance CD163  Screening of Cell Lines

 Various CD163 transgenic cell lines were evaluated for proliferative tolerance for low passage European and North American PRRSV isolates (see Table 7). Transgenic CD163 cell lines as described in the previous examples include NLFK-MARC CD163 D4, PK-RSVScript-susCD163v2 clone # 9 and PK-CMV-susCD163v1-A10. Each CD163 cell line along with the cell lines MARC-145, parental cat kidney, and parental pig kidney cell line (acting as controls) were seeded on 96 well tissue culture plates. Growth medium was removed from the monolayer and inoculated with 0.1 mL / well of each PRRSV isolate. Three days after infection, plates were fixed with 80% acetone and stained with FITC-conjugate monoclonal antibody SDOW17 (Rural Technologies Inc) specific for nucleocapsids. Fluorescent antibody (FA) assay results are provided in Table 7.

FA Results of Screening of Various CD163 Cell Lines on Proliferation Tolerance for European and North American PRRSV Isolates CD163 cell line PRRSV Isolation ^a EU98V226 P129 P201 1151 94-3 IND5 NLFK-MARC CD163 D4 ++ + +++ +++ ++ ++++ PK-RSVScript-susCD163v2 clone # 9 + + ++ + + ++ PK-CMV-susCD163v1-A10 + + ++ ++ ++ ++ MARC-145 ++ + +++ + ++++ +++ Pork Kidneys (Parent) - - - - - - Cat Kidneys (Parent) - - - - - - ^a All PRRSV isolates are North American except that EU98V226 is a European isolate.

Example  33: CD163 of Phorbol  12- Myristate  13-acetate ( PMA Induction of human U937 cells PRRSV  Allows proliferation for infection.

Human U937 cells obtained from ATCC (CRL-1593.2) were grown in RPMI medium containing serum and additives according to the ATCC instructions. These cells are known to express CD163 when activated by PMA treatment (Gronlund et al., 2000). U937 cells were seeded in duplicate into wells of 6 well plates. One set of wells was treated with 100 ng / ml PMA and the other was not. Three days after PMA stimulation, one well from each set was infected with P129 isolate of PRRSV. The other wells from each well were fixed and indirect immunofluorescent antibody assay for expression of CD163 using goat anti-human CD163 (R & D System) and donkey anti-goat IgG (BioDesign International) conjugated with FITC. Stained.

Untreated U937 cells continued to grow at high density after 3 days of initial seeding. PMA-treated U937 cells stopped proliferating, enlarged in size and adhered to the surface of the culture wells. A small fraction of untreated U937 was positive for CD163 staining, but most all PMA treated U937 was positive for CD163 staining. No PRRSV infected cells were observed in untreated U937. However, many PMA treated U937 cells were infected by PRRSV. This indicates that after chemical induction of CD163 expression, non-proliferative cells may be proliferable for PRRSV infection.

Additional aspects and variations of the invention will be apparent to those skilled in the art from the entire application, including the detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein may be recombined into additional embodiments that are also intended as aspects of the invention, whether or not combinations of features are expressly mentioned above as aspects or embodiments of the invention. have. In addition, only the limitations described herein as critical to the present invention should be considered as such: variants of the present invention that are not critical and not described herein as critical are intended as aspects of the present invention.

It will be apparent that the invention may be practiced otherwise than as specifically described in the foregoing description and examples.

Many variations and modifications of the invention are possible in light of the above teachings and are therefore within the scope of the invention.

The entire disclosures of all publications mentioned herein are incorporated herein by reference to the extent that they are inconsistent with the disclosures herein.

                         SEQUENCE LISTING <110> Calvert, Jay        Slade, David        Shields, shelly        Welch, Jenny   <120> Cellular Permissivity Factor for Viruses, and Uses Thereof <130> PC32282 <160> 48 <170> PatentIn version 3.2 <210> 1 <211> 2607 <212> DNA <213> Sus scrofa <400> 1 atggacaaac tcagaatggt gctacatgaa aactctggat ctgcagacct gaaactgaga 60 gtggtagatg gagtcactga atgttcagga agattggaag tgaaattcca aggagaatgg 120 ggaacaatct gtgatgatgg ctgggatagt gatgatgccg ctgtggcatg taagcaactg 180 ggatgtccaa ctgctgtcac tgccattggt cgagttaacg ccagtgaggg aactggacac 240 atttggcttg acagtgtttc ttgccatgga cacgagtctg ctctctggca gtgtagacac 300 catgaatggg gaaagcatta ttgcaatcat aatgaagatg ctggtgtgac atgttctgat 360 ggatcagatc tggaactgag acttaaaggt ggaggcagcc actgtgctgg gacagtggag 420 gtggaaattc agaaactggt aggaaaagtg tgtgatagaa gctggggact gaaagaagct 480 gatgtggttt gcaggcagct gggatgtgga tctgcactca aaacatcata tcaagtttat 540 tccaaaacca aggcaacaaa cacatggctg tttgtaagca gctgtaatgg aaatgaaact 600 tctctttggg actgcaagaa ttggcagtgg ggtggactta gttgtgatca ctatgacgaa 660 gccaaaatta cctgctcagc ccacaggaaa cccaggctgg ttggagggga cattccctgc 720 tctggtcgtg ttgaagtaca acatggagac acgtggggca ccgtctgtga ttctgacttc 780 tctctggagg cggccagcgt gctgtgcagg gaactacagt gcggcactgt ggtttccctc 840 ctggggggag ctcactttgg agaaggaagt ggacagatct gggctgaaga attccagtgt 900 gaggggcacg agtcccacct ttcactctgc ccagtagcac cccgccctga cgggacatgt 960 agccacagca gggacgtcgg cgtagtctgc tcaagataca cacaaatccg cttggtgaat 1020 ggcaagaccc catgtgaagg aagagtggag ctcaacattc ttgggtcctg ggggtccctc 1080 tgcaactctc actgggacat ggaagatgcc catgttttat gccagcagct taaatgtgga 1140 gttgcccttt ctatcccggg aggagcacct tttgggaaag gaagtgagca ggtctggagg 1200 cacatgtttc actgcactgg gactgagaag cacatgggag attgttccgt cactgctctg 1260 ggcgcatcac tctgttcttc agggcaagtg gcctctgtaa tctgctcagg gaaccagagt 1320 cagacactat ccccgtgcaa ttcatcatcc tcggacccat caagctctat tatttcagaa 1380 gaaaatggtg ttgcctgcat agggagtggt caacttcgcc tggtcgatgg aggtggtcgt 1440 tgtgctggga gagtagaggt ctatcatgag ggctcctggg gcaccatctg tgatgacagc 1500 tgggacctga atgatgccca tgtggtgtgc aaacagctga gctgtggatg ggccattaat 1560 gccactggtt ctgctcattt tggggaagga acagggccca tttggctgga tgagataaac 1620 tgtaatggaa aagaatctca tatttggcaa tgccactcac atggttgggg gcggcacaat 1680 tgcaggcata aggaggatgc aggagtcatc tgctcggagt tcatgtctct cagactgatc 1740 agtgaaaaca gcagagagac ctgtgcaggg cgcctggaag ttttttacaa cggagcttgg 1800 ggcagcgttg gcaagaatag catgtctcca gccacagtgg gggtggtatg caggcagctg 1860 ggctgtgcag acagagggga catcagccct gcatcttcag acaagacagt gtccaggcac 1920 atgtgggtgg acaatgttca gtgtcctaaa ggacctgaca ccctatggca gtgcccatca 1980 tctccatgga agaagagact ggccagcccc tcagaggaga catggatcac atgtgccaac 2040 aaaataagac ttcaagaagg aaacactaat tgttctggac gtgtggagat ctggtacgga 2100 ggttcctggg gcactgtgtg tgacgactcc tgggaccttg aagatgctca ggtggtgtgc 2160 cgacagctgg gctgtggctc agctttggag gcaggaaaag aggccgcatt tggccagggg 2220 actgggccca tatggctcaa tgaagtgaag tgcaagggga atgaaacctc cttgtgggat 2280 tgtcctgcca gatcctgggg ccacagtgac tgtggacaca aggaggatgc tgctgtgacg 2340 tgttcagaaa ttgcaaagag ccgagaatcc ctacatgcca caggtcgctc atcttttgtt 2400 gcacttgcaa tctttggggt cattctgttg gcctgtctca tcgcattcct catttggact 2460 cagaagcgaa gacagaggca gcggctctca gttttctcag gaggagagaa ttctgtccat 2520 caaattcaat accgggagat gaattcttgc ctgaaagcag atgaaacgga tatgctaaat 2580 ccctcaggag accactctga agtacaa 2607 <210> 2 <211> 869 <212> PRT <213> Sus scrofa <400> 2 Met Asp Lys Leu Arg Met Val Leu His Glu Asn Ser Gly Ser Ala Asp 1 5 10 15 Leu Lys Leu Arg Val Val Asp Gly Val Thr Glu Cys Ser Gly Arg Leu             20 25 30 Glu Val Lys Phe Gln Gly Glu Trp Gly Thr Ile Cys Asp Asp Gly Trp         35 40 45 Asp Ser Asp Asp Ala Ala Val Ala Cys Lys Gln Leu Gly Cys Pro Thr     50 55 60 Ala Val Thr Ala Ile Gly Arg Val Asn Ala Ser Glu Gly Thr Gly His 65 70 75 80 Ile Trp Leu Asp Ser Val Ser Cys His Gly His Glu Ser Ala Leu Trp                 85 90 95 Gln Cys Arg His His Glu Trp Gly Lys His Tyr Cys Asn His Asn Glu             100 105 110 Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Leu Arg Leu         115 120 125 Lys Gly Gly Gly Ser His Cys Ala Gly Thr Val Glu Val Glu Ile Gln     130 135 140 Lys Leu Val Gly Lys Val Cys Asp Arg Ser Trp Gly Leu Lys Glu Ala 145 150 155 160 Asp Val Val Cys Arg Gln Leu Gly Cys Gly Ser Ala Leu Lys Thr Ser                 165 170 175 Tyr Gln Val Tyr Ser Lys Thr Lys Ala Thr Asn Thr Trp Leu Phe Val             180 185 190 Ser Ser Cys Asn Gly Asn Glu Thr Ser Leu Trp Asp Cys Lys Asn Trp         195 200 205 Gln Trp Gly Gly Leu Ser Cys Asp His Tyr Asp Glu Ala Lys Ile Thr     210 215 220 Cys Ser Ala His Arg Lys Pro Arg Leu Val Gly Gly Asp Ile Pro Cys 225 230 235 240 Ser Gly Arg Val Glu Val Gln His Gly Asp Thr Trp Gly Thr Val Cys                 245 250 255 Asp Ser Asp Phe Ser Leu Glu Ala Ala Ser Val Leu Cys Arg Glu Leu             260 265 270 Gln Cys Gly Thr Val Val Ser Leu Leu Gly Gly Ala His Phe Gly Glu         275 280 285 Gly Ser Gly Gln Ile Trp Ala Glu Glu Phe Gln Cys Glu Gly His Glu     290 295 300 Ser His Leu Ser Leu Cys Pro Val Ala Pro Arg Pro Asp Gly Thr Cys 305 310 315 320 Ser His Ser Arg Asp Val Gly Val Val Cys Ser Arg Tyr Thr Gln Ile                 325 330 335 Arg Leu Val Asn Gly Lys Thr Pro Cys Glu Gly Arg Val Glu Leu Asn             340 345 350 Ile Leu Gly Ser Trp Gly Ser Leu Cys Asn Ser His Trp Asp Met Glu         355 360 365 Asp Ala His Val Leu Cys Gln Gln Leu Lys Cys Gly Val Ala Leu Ser     370 375 380 Ile Pro Gly Gly Ala Pro Phe Gly Lys Gly Ser Glu Gln Val Trp Arg 385 390 395 400 His Met Phe His Cys Thr Gly Thr Glu Lys His Met Gly Asp Cys Ser                 405 410 415 Val Thr Ala Leu Gly Ala Ser Leu Cys Ser Ser Gly Gln Val Ala Ser             420 425 430 Val Ile Cys Ser Gly Asn Gln Ser Gln Thr Leu Ser Pro Cys Asn Ser         435 440 445 Ser Ser Ser Asp Pro Ser Ser Ser Ile Ile Ser Glu Glu Asn Gly Val     450 455 460 Ala Cys Ile Gly Ser Gly Gln Leu Arg Leu Val Asp Gly Gly Gly Arg 465 470 475 480 Cys Ala Gly Arg Val Glu Val Tyr His Glu Gly Ser Trp Gly Thr Ile                 485 490 495 Cys Asp Asp Ser Trp Asp Leu Asn Asp Ala His Val Val Cys Lys Gln             500 505 510 Leu Ser Cys Gly Trp Ala Ile Asn Ala Thr Gly Ser Ala His Phe Gly         515 520 525 Glu Gly Thr Gly Pro Ile Trp Leu Asp Glu Ile Asn Cys Asn Gly Lys     530 535 540 Glu Ser His Ile Trp Gln Cys His Ser His Gly Trp Gly Arg His Asn 545 550 555 560 Cys Arg His Lys Glu Asp Ala Gly Val Ile Cys Ser Glu Phe Met Ser                 565 570 575 Leu Arg Leu Ile Ser Glu Asn Ser Arg Glu Thr Cys Ala Gly Arg Leu             580 585 590 Glu Val Phe Tyr Asn Gly Ala Trp Gly Ser Val Gly Lys Asn Ser Met         595 600 605 Ser Pro Ala Thr Val Gly Val Val Cys Arg Gln Leu Gly Cys Ala Asp     610 615 620 Arg Gly Asp Ile Ser Pro Ala Ser Ser Asp Lys Thr Val Ser Arg His 625 630 635 640 Met Trp Val Asp Asn Val Gln Cys Pro Lys Gly Pro Asp Thr Leu Trp                 645 650 655 Gln Cys Pro Ser Ser Pro Trp Lys Lys Arg Leu Ala Ser Pro Ser Glu             660 665 670 Glu Thr Trp Ile Thr Cys Ala Asn Lys Ile Arg Leu Gln Glu Gly Asn         675 680 685 Thr Asn Cys Ser Gly Arg Val Glu Ile Trp Tyr Gly Gly Ser Trp Gly     690 695 700 Thr Val Cys Asp Asp Ser Trp Asp Leu Glu Asp Ala Gln Val Val Cys 705 710 715 720 Arg Gln Leu Gly Cys Gly Ser Ala Leu Glu Ala Gly Lys Glu Ala Ala                 725 730 735 Phe Gly Gln Gly Thr Gly Pro Ile Trp Leu Asn Glu Val Lys Cys Lys             740 745 750 Gly Asn Glu Thr Ser Leu Trp Asp Cys Pro Ala Arg Ser Trp Gly His         755 760 765 Ser Asp Cys Gly His Lys Glu Asp Ala Ala Val Thr Cys Ser Glu Ile     770 775 780 Ala Lys Ser Arg Glu Ser Leu His Ala Thr Gly Arg Ser Ser Phe Val 785 790 795 800 Ala Leu Ala Ile Phe Gly Val Ile Leu Leu Ala Cys Leu Ile Ala Phe                 805 810 815 Leu Ile Trp Thr Gln Lys Arg Arg Gln Arg Gln Arg Leu Ser Val Phe             820 825 830 Ser Gly Gly Glu Asn Ser Val His Gln Ile Gln Tyr Arg Glu Met Asn         835 840 845 Ser Cys Leu Lys Ala Asp Glu Thr Asp Met Leu Asn Pro Ser Gly Asp     850 855 860 His Ser Glu Val Gln 865 <210> 3 <211> 3400 <212> DNA <213> Sus scrofa <400> 3 atggtgctac ttgaagactc tggatctgca gactttagaa gatgttctgc ccatttaagt 60 tccttcactt ttgctgtagt cgctgttctc agtgcctgct tggtcactag ttctcttgga 120 ggaaaagaca aggagctgag gctaacgggt ggtgaaaaca agtgctctgg aagagtggag 180 gtgaaagtgc aggaggagtg gggaactgtg tgtaataatg gctgggacat ggatgtggtc 240 tctgttgttt gtaggcagct gggatgtcca actgctatca aagccactgg atgggctaat 300 tttagtgcag gttctggacg catttggatg gatcatgttt cttgtcgagg gaatgagtca 360 gctctctggg actgcaaaca tgatggatgg ggaaagcata actgtactca ccaacaggat 420 gctggagtaa cctgctcaga tggatctgat ttagagatga ggctggtgaa tggaggaaac 480 cggtgcttag gaagaataga agtcaaattt caagagcggt ggggaacagt gtgtgatgat 540 aacttcaaca taaatcatgc ttctgtggtt tgtaaacaac ttgaatgtgg aagtgctgtc 600 agtttctctg gttcagctaa ttttggagaa ggttctggac caatctggtt tgatgatctt 660 gtatgcaatg gaaatgagtc agctctctgg aactgcaaac atgaaggatg gggaaagcac 720 aattgcgatc atgctgagga tgctggagtg atttgcttaa atggagcaga cctgaaactg 780 agagtggtag atggactcac tgaatgttca ggaagattgg aagtgaaatt ccaaggagaa 840 tggggaacaa tctgtgatga tggctgggat agtgatgatg ccgctgtggc atgtaagcaa 900 ctgggatgtc caactgctgt cactgccatt ggtcgagtta acgccagtga gggaactgga 960 cacatttggc ttgacagtgt ttcttgccat ggacacgagt ctgctctctg gcagtgtaga 1020 caccatgaat ggggaaagca ttattgcaat cataatgaag atgctggtgt gacatgttct 1080 gatggatcag atctggaact gagacttaaa ggtggaggca gccactgtgc tgggacagtg 1140 gaggtggaaa ttcagaaact ggtaggaaaa gtgtgtgata gaagctgggg actgaaagaa 1200 gctgatgtgg tttgcaggca gctgggatgt ggatctgcac tcaaaacatc atatcaagtt 1260 tattccaaaa ccaaggcaac aaacacatgg ctgtttgtaa gcagctgtaa tggaaatgaa 1320 acttctcttt gggactgcaa gaattggcag tggggtggac ttagttgtga tcactatgac 1380 gaagccaaaa ttacctgctc agcccacagg aaacccaggc tggttggagg ggacattccc 1440 tgctctggtc gtgttgaagt acaacatgga gacacgtggg gcaccgtctg tgattctgac 1500 ttctctctgg aggcggccag cgtgctgtgc agggaactac agtgcggcac tgtggtttcc 1560 ctcctggggg gagctcactt tggagaagga agtggacaga tctgggctga agaattccag 1620 tgtgaggggc acgagtccca cctttcactc tgcccagtag caccccgccc tgacgggaca 1680 tgtagccaca gcagggacgt cggcgtagtc tgctcaagat acacacaaat ccgcttggtg 1740 aatggcaaga ccccatgtga aggaagagtg gagctcaaca ttcttgggtc ctgggggtcc 1800 ctctgcaact ctcactggga catggaagat gcccatgttt tatgccagca gcttaaatgt 1860 ggagttgccc tttctatccc gggaggagca ccttttggga aaggaagtga gcaggtctgg 1920 aggcacatgt ttcactgcac tgggactgag aagcacatgg gagattgttc cgtcactgct 1980 ctgggcgcat cactctgttc ttcagggcaa gtggcctctg taatctgctc agggaaccag 2040 agtcagacac tatccccgtg caattcatca tcctcggacc catcaagctc tattatttca 2100 gaagaaagtg gtgttgcctg catagggagt ggtcaacttc gcctggtcga tggaggtggt 2160 cgttgtgctg ggagagtaga ggtctatcct ggggcatcct ggggcaccat ctgtgatgac 2220 agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg atgggccatt 2280 aatgccactg gttctgctca ttttggggaa ggaacagggc ccatttggct ggatgagata 2340 aactgtaatg gaaaagaatc tcatatttgg caatgccact cacatggttg ggggcggcac 2400 aattgcaggc ataaggagga tgcaggagtc atctgctcag agttcatgtc tctgagactg 2460 atcagtgaaa acagcagaga gacctgtgca gggcgcctgg aagtttttta caacggagct 2520 tggggcagcg ttggcaggaa tagcatgtct ccagccacag tgggggtggt atgcaggcag 2580 ctgggctgtg cagacagagg ggacatcagc cctgcatctt cagacaagac agtgtccagg 2640 cacatgtggg tggacaatgt tcagtgtcct aaaggacctg acacactatg gcagtgcccc 2700 tcatctccat ggaagaagag actggccagc ccctcagagg agacatggat cacatgtgcc 2760 aacaaaataa gacttcaaga aggaaacact aattgttctg gacgtgtgga gatctggtac 2820 ggaggttcct ggggcactgt gtgtgacgac tcctgggacc ttgaagatgc tcaggtggtg 2880 tgccgacagc tgggctgtgg ctcagctttg gaggcaggaa aagagcccgc atttggccag 2940 gggactgggc ccatatggct caatgaagtg aagtgcaagg ggaatgaacc ctccttgtgg 3000 gattgtcctg ccagatcctg gggccacagt gactgtggac acaaggagga tgctgctgtg 3060 acgtgctcag aaattgcaaa gagccgagaa tccctacatg ccacaggtcg ctcatctttt 3120 gttgcacttg caatctttgg ggtcattctg ttggcctgtc tcatcgcatt cctcatttgg 3180 actcagaagc gaagacagag gcagcggctc tcagttttct caggaggaga gaattctgtc 3240 catcaaattc aataccggga gatgaattct tgcctgaaag cagatgaaac ggatatgcta 3300 aatccctcag gagaccactc tgaagtacaa tgaaaaggaa aatgggaatt ataacctggt 3360 gagttcagcc tttaagatac cttgatgaag acctggacta 3400 <210> 4 <211> 1110 <212> PRT <213> Sus scrofa <400> 4 Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp Phe Arg Arg Cys Ser 1 5 10 15 Ala His Leu Ser Ser Phe Thr Phe Ala Val Val Ala Val Leu Ser Ala             20 25 30 Cys Leu Val Thr Ser Ser Leu Gly Lys Asp Lys Glu Leu Arg Leu         35 40 45 Thr Gly Gly Glu Asn Lys Cys Ser Gly Arg Val Glu Val Lys Val Gln     50 55 60 Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp Asp Met Asp Val Val 65 70 75 80 Ser Val Val Cys Arg Gln Leu Gly Cys Pro Thr Ala Ile Lys Ala Thr                 85 90 95 Gly Trp Ala Asn Phe Ser Ala Gly Ser Gly Arg Ile Trp Met Asp His             100 105 110 Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp Asp Cys Lys His Asp         115 120 125 Gly Trp Gly Lys His Asn Cys Thr His Gln Gln Asp Ala Gly Val Thr     130 135 140 Cys Ser Asp Gly Ser Asp Leu Glu Met Arg Leu Val Asn Gly Gly Asn 145 150 155 160 Arg Cys Leu Gly Arg Ile Glu Val Lys Phe Gln Glu Arg Trp Gly Thr                 165 170 175 Val Cys Asp Asp Asn Phe Asn Ile Asn His Ala Ser Val Val Cys Lys             180 185 190 Gln Leu Glu Cys Gly Ser Ala Val Ser Phe Ser Gly Ser Ala Asn Phe         195 200 205 Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp Asp Leu Val Cys Asn Gly     210 215 220 Asn Glu Ser Ala Leu Trp Asn Cys Lys His Glu Gly Trp Gly Lys His 225 230 235 240 Asn Cys Asp His Ala Glu Asp Ala Gly Val Ile Cys Leu Asn Gly Ala                 245 250 255 Asp Leu Lys Leu Arg Val Val Asp Gly Leu Thr Glu Cys Ser Gly Arg             260 265 270 Leu Glu Val Lys Phe Gln Gly Glu Trp Gly Thr Ile Cys Asp Asp Gly         275 280 285 Trp Asp Ser Asp Asp Ala Ala Val Ala Cys Lys Gln Leu Gly Cys Pro     290 295 300 Thr Ala Val Thr Ala Ile Gly Arg Val Asn Ala Ser Glu Gly Thr Gly 305 310 315 320 His Ile Trp Leu Asp Ser Val Ser Cys His Gly His Glu Ser Ala Leu                 325 330 335 Trp Gln Cys Arg His His Glu Trp Gly Lys His Tyr Cys Asn His Asn             340 345 350 Glu Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Leu Arg         355 360 365 Leu Lys Gly Gly Gly Ser His Cys Ala Gly Thr Val Glu Val Glu Ile     370 375 380 Gln Lys Leu Val Gly Lys Val Cys Asp Arg Ser Trp Gly Leu Lys Glu 385 390 395 400 Ala Asp Val Val Cys Arg Gln Leu Gly Cys Gly Ser Ala Leu Lys Thr                 405 410 415 Ser Tyr Gln Val Tyr Ser Lys Thr Lys Ala Thr Asn Thr Trp Leu Phe             420 425 430 Val Ser Ser Cys Asn Gly Asn Glu Thr Ser Leu Trp Asp Cys Lys Asn         435 440 445 Trp Gln Trp Gly Gly Leu Ser Cys Asp His Tyr Asp Glu Ala Lys Ile     450 455 460 Thr Cys Ser Ala His Arg Lys Pro Arg Leu Val Gly Gly Asp Ile Pro 465 470 475 480 Cys Ser Gly Arg Val Glu Val Gln His Gly Asp Thr Trp Gly Thr Val                 485 490 495 Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala Ser Val Leu Cys Arg Glu             500 505 510 Leu Gln Cys Gly Thr Val Val Ser Leu Leu Gly Gly Ala His Phe Gly         515 520 525 Glu Gly Ser Gly Gln Ile Trp Ala Glu Glu Phe Gln Cys Glu Gly His     530 535 540 Glu Ser His Leu Ser Leu Cys Pro Val Ala Pro Arg Pro Asp Gly Thr 545 550 555 560 Cys Ser His Ser Arg Asp Val Gly Val Val Cys Ser Arg Tyr Thr Gln                 565 570 575 Ile Arg Leu Val Asn Gly Lys Thr Pro Cys Glu Gly Arg Val Glu Leu             580 585 590 Asn Ile Leu Gly Ser Trp Gly Ser Leu Cys Asn Ser His Trp Asp Met         595 600 605 Glu Asp Ala His Val Leu Cys Gln Gln Leu Lys Cys Gly Val Ala Leu     610 615 620 Ser Ile Pro Gly Gly Ala Pro Phe Gly Lys Gly Ser Glu Gln Val Trp 625 630 635 640 Arg His Met Phe His Cys Thr Gly Thr Glu Lys His Met Gly Asp Cys                 645 650 655 Ser Val Thr Ala Leu Gly Ala Ser Leu Cys Ser Ser Gly Gln Val Ala             660 665 670 Ser Val Ile Cys Ser Gly Asn Gln Ser Gln Thr Leu Ser Pro Cys Asn         675 680 685 Ser Ser Ser Ser Asp Pro Ser Ser Ser Ile Ile Ser Glu Glu Ser Gly     690 695 700 Val Ala Cys Ile Gly Ser Gly Gln Leu Arg Leu Val Asp Gly Gly Gly 705 710 715 720 Arg Cys Ala Gly Arg Val Glu Val Tyr Pro Gly Ala Ser Trp Gly Thr                 725 730 735 Ile Cys Asp Asp Ser Trp Asp Leu Asn Asp Ala His Val Val Cys Lys             740 745 750 Gln Leu Ser Cys Gly Trp Ala Ile Asn Ala Thr Gly Ser Ala His Phe         755 760 765 Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp Glu Ile Asn Cys Asn Gly     770 775 780 Lys Glu Ser His Ile Trp Gln Cys His Ser His Gly Trp Gly Arg His 785 790 795 800 Asn Cys Arg His Lys Glu Asp Ala Gly Val Ile Cys Ser Glu Phe Met                 805 810 815 Ser Leu Arg Leu Ile Ser Glu Asn Ser Arg Glu Thr Cys Ala Gly Arg             820 825 830 Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly Ser Val Gly Arg Asn Ser         835 840 845 Met Ser Pro Ala Thr Val Gly Val Val Cys Arg Gln Leu Gly Cys Ala     850 855 860 Asp Arg Gly Asp Ile Ser Pro Ala Ser Ser Asp Lys Thr Val Ser Arg 865 870 875 880 His Met Trp Val Asp Asn Val Gln Cys Pro Lys Gly Pro Asp Thr Leu                 885 890 895 Trp Gln Cys Pro Ser Ser Pro Trp Lys Lys Arg Leu Ala Ser Pro Ser             900 905 910 Glu Glu Thr Trp Ile Thr Cys Ala Asn Lys Ile Arg Leu Gln Glu Gly         915 920 925 Asn Thr Asn Cys Ser Gly Arg Val Glu Ile Trp Tyr Gly Gly Ser Trp     930 935 940 Gly Thr Val Cys Asp Asp Ser Trp Asp Leu Glu Asp Ala Gln Val Val 945 950 955 960 Cys Arg Gln Leu Gly Cys Gly Ser Ala Leu Glu Ala Gly Lys Glu Pro                 965 970 975 Ala Phe Gly Gln Gly Thr Gly Pro Ile Trp Leu Asn Glu Val Lys Cys             980 985 990 Lys Gly Asn Glu Pro Ser Leu Trp Asp Cys Pro Ala Arg Ser Trp Gly         995 1000 1005 His Ser Asp Cys Gly His Lys Glu Asp Ala Ala Val Thr Cys Ser     1010 1015 1020 Glu Ile Ala Lys Ser Arg Glu Ser Leu His Ala Thr Gly Arg Ser     1025 1030 1035 Ser Phe Val Ala Leu Ala Ile Phe Gly Val Ile Leu Leu Ala Cys     1040 1045 1050 Leu Ile Ala Phe Leu Ile Trp Thr Gln Lys Arg Arg Gln Arg Gln     1055 1060 1065 Arg Leu Ser Val Phe Ser Gly Gly Glu Asn Ser Val His Gln Ile     1070 1075 1080 Gln Tyr Arg Glu Met Asn Ser Cys Leu Lys Ala Asp Glu Thr Asp     1085 1090 1095 Met Leu Asn Pro Ser Gly Asp His Ser Glu Val Gln     1100 1105 1110 <210> 5 <211> 2930 <212> DNA <213> Sus scrofa <400> 5 gtaataatac aagaagattt aaatgggcat aaaaccttgg aatggacaaa ctcagaatgg 60 tgctacatga aaactctgga tctgcagacc tgaaactgag agtggtagat ggagtcactg 120 aatgttcagg aagattggaa gtgaaattcc aaggagaatg gggaacaatc tgtgatgatg 180 gctgggatag tgatgatgcc gctgtggcat gtaagcaact gggatgtcca actgctgtca 240 ctgccattgg tcgagttaac gccagtgagg gaactggaca catttggctt gacagtgttt 300 cttgccatgg acacgagtct gctctctggc agtgtagaca ccatgaatgg ggaaagcatt 360 attgcaatca taatgaagat gctggtgtga catgttctga tggatcagat ctggaactga 420 gacttaaagg tggaggcagc cactgtgctg ggacagtgga ggtggaaatt cagaaactgg 480 taggaaaagt gtgtgataga agctggggac tgaaagaagc tgatgtggtt tgcaggcagc 540 tgggatgtgg atctgcactc aaaacatcat atcaagttta ttccaaaacc aaggcaacaa 600 acacatggct gtttgtaagc agctgtaatg gaaatgaaac ttctctttgg gactgcaaga 660 attggcagtg gggtggactt agttgtgatc actatgacga agccaaaatt acctgctcag 720 cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 780 aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 840 tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggga gctcactttg 900 gagaaggaag tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc 960 tttcactctg cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg 1020 gcgtagtctg ctcaagatac acacaaatcc gcttggtgaa tggcaagacc ccatgtgaag 1080 gaagagtgga gctcaacatt cttgggtcct gggggtccct ctgcaactct cactgggaca 1140 tggaagatgc ccatgtttta tgccagcagc ttaaatgtgg agttgccctt tctatcccgg 1200 gaggagcacc ttttgggaaa ggaagtgagc aggtctggag gcacatgttt cactgcactg 1260 ggactgagaa gcacatggga gattgttccg tcactgctct gggcgcatca ctctgttctt 1320 cagggcaagt ggcctctgta atctgctcag ggaaccagag tcagacacta tccccgtgca 1380 attcatcatc ctcggaccca tcaagctcta ttatttcaga agaaaatggt gttgcctgca 1440 tagggagtgg tcaacttcgc ctggtcgatg gaggtggtcg ttgtgctggg agagtagagg 1500 tctatcatga gggctcctgg ggcaccatct gtgatgacag ctgggacctg aatgatgccc 1560 atgtggtgtg caaacagctg agctgtggat gggccattaa tgccactggt tctgctcatt 1620 ttggggaagg aacagggccc atttggctgg atgagataaa ctgtaatgga aaagaatctc 1680 atatttggca atgccactca catggttggg ggcggcacaa ttgcaggcat aaggaggatg 1740 caggagtcat ctgctcggag ttcatgtctc tcagactgat cagtgaaaac agcagagaga 1800 cctgtgcagg gcgcctggaa gttttttaca acggagcttg gggcagcgtt ggcaagaata 1860 gcatgtctcc agccacagtg ggggtggtat gcaggcagct gggctgtgca gacagagggg 1920 acatcagccc tgcatcttca gacaagacag tgtccaggca catgtgggtg gacaatgttc 1980 agtgtcctaa aggacctgac accctatggc agtgcccatc atctccatgg aagaagagac 2040 tggccagccc ctcagaggag acatggatca catgtgccaa caaaataaga cttcaagaag 2100 gaaacactaa ttgttctgga cgtgtggaga tctggtacgg aggttcctgg ggcactgtgt 2160 gtgacgactc ctgggacctt gaagatgctc aggtggtgtg ccgacagctg ggctgtggct 2220 cagctttgga ggcaggaaaa gaggccgcat ttggccaggg gactgggccc atatggctca 2280 atgaagtgaa gtgcaagggg aatgaaacct ccttgtggga ttgtcctgcc agatcctggg 2340 gccacagtga ctgtggacac aaggaggatg ctgctgtgac gtgttcagaa attgcaaaga 2400 gccgagaatc cctacatgcc acaggtcgct catcttttgt tgcacttgca atctttgggg 2460 tcattctgtt ggcctgtctc atcgcattcc tcatttggac tcagaagcga agacagaggc 2520 agcggctctc agttttctca ggaggagaga attctgtcca tcaaattcaa taccgggaga 2580 tgaattcttg cctgaaagca gatgaaacgg atatgctaaa tccctcagga gaccactctg 2640 aagtacaatg aaaaggaaaa tgggaattat aacctggtga gttcagcctt taagatacct 2700 tgatgaagac ctggactatt gaatgagcaa gaatctgcct cttacactga agattacaat 2760 acagtcctct gtctcctggt attccaaaga ctgctgttga atttctaaaa aatagattgg 2820 tgaatgtgac tactcaaagt tgtatgtaag actttcaagg gcattaaata aaaaagaata 2880 ttgctgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2930 <210> 6 <211> 32 <212> DNA <213> Artificial <220> PCR primers <400> 6 cggaattccg cggatgtaat aatacaagaa ga 32 <210> 7 <211> 36 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 7 ccgctcgagt agtccaggtc ttcatcaagg tatctt 36 <210> 8 <211> 38 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 8 acactcgaca tgtcgatgta cgggccagat atacgcgt 38 <210> 9 <211> 38 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 9 ttccttacag agctcgaggt gcacaccaat gtggtgaa 38 <210> 10 <211> 46 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 10 cggtccggag cggccgcgat gtaataatac aagaagattt aaatgg 46 <210> 11 <211> 38 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 11 cggttggtac ccagcaatat tcttttttat ttaatgcc 38 <210> 12 <211> 3623 <212> DNA <213> Sus scrofa <400> 12 gtaataatac aagaagattt aaatggcata aaaccttgga atggacaaac tcagaatggt 60 gctacatgaa aactctggat ctgcagactt tagaagatgt tctgcccatt taagttcctt 120 cacttttgct gtagtcgctg ttctcagtgc ctgcttggtc actagttctc ttggaggaaa 180 agacaaggag ctgaggctaa cgggtggtga aaacaagtgc tctggaagag tggaggtgaa 240 agtgcaggag gagtggggaa ctgtgtgtaa taatggctgg gacatggatg tggtctctgt 300 tgtttgtagg cagctgggat gtccaactgc tatcaaagcc actggatggg ctaattttag 360 tgcaggttct ggacgcattt ggatggatca tgtttcttgt cgagggaatg agtcagctct 420 ctgggactgc aaacatgatg gatggggaaa gcataactgt actcaccaac aggatgctgg 480 agtaacctgc tcagatggat ctgatttaga gatggggctg gtgaatggag gaaaccggtg 540 cttaggaaga atagaagtca aatttcaagg acggtgggga acagtgtgtg atgataactt 600 caacataaat catgcttctg tggtttgtaa acaacttgaa tgtggaagtg ctgtcagttt 660 ctctggttca gctaattttg gagaaggttc tggaccaatc tggtttgatg atcttgtatg 720 caatggaaat gagtcagctc tctggaactg caaacatgaa ggatggggaa agcacaattg 780 cgatcatgct gaggatgctg gagtgatttg cttaaatgga gcagacctga aactgagagt 840 ggtagatgga gtcactgaat gttcaggaag attggaagtg aaattccaag gagaatgggg 900 aacaatctgt gatgatggct gggatagtga tgatgccgct gtggcatgta agcaactggg 960 atgtccaact gctgtcactg ccattggtcg agttaacgcc agtgagggaa ctggacacat 1020 ttggcttgac agtgtttctt gccatggaca cgagtctgct ctctggcagt gtagacacca 1080 tgaatgggga aagcattatt gcaatcatga tgaagatgct ggtgtgacat gttctgatgg 1140 atcagatctg gaactgagac ttaaaggtgg aggcagccac tgtgctggga cagtggaggt 1200 ggaaattcag aaactggtag gaaaagtgtg tgatagaagc tggggactga aagaagctga 1260 tgtggtttgc aggcagctgg gatgtggatc tgcactcaaa acatcatatc aagtttattc 1320 caaaaccaag gcaacaaaca catggctgtt tgtaagcagc tgtaatggaa atgaaacttc 1380 tctttgggac tgcaagaatt ggcagtgggg tggacttagt tgtgatcact atgacgaagc 1440 caaaattacc tgctcagccc acaggaaacc caggctggtt ggaggggaca ttccctgctc 1500 tggtcgtgtt gaagtacaac atggagacac gtggggcacc gtctgtgatt ctgacttctc 1560 tctggaggcg gccagcgtgc tgtgcaggga actacagtgc ggcactgtgg tttccctcct 1620 ggggggagct cactttggag aaggaagtgg acagatctgg gctgaagaat tccagtgtga 1680 ggggcacgag tcccaccttt cactctgccc agtagcaccc cgccctgacg ggacatgtag 1740 ccacagcagg gacgtcggcg tagtctgctc aagatacaca caaatccgct tggtgaatgg 1800 caagacccca tgtgaaggaa gagtggagct caacattctt gggtcctggg ggtccctctg 1860 caactctcac tgggacatgg aagatgccca tgttttatgc cagcagctta aatgtggagt 1920 tgccctttct atcccgggag gagcaccttt tgggaaagga agtgagcagg tctggaggca 1980 catgtttcac tgcactggga ctgagaagca catgggagat tgttccgtca ctgctctggg 2040 cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc tgctcaggga accagagtca 2100 gacactatct ccgtgcaatt catcatcctc ggacccatca agctctatta tttcagaaga 2160 aaatggtgtt gcctgcatag ggagtggtca acttcgcctg gtcgatggag gtggtcgttg 2220 tgctgggaga gtagaggtct atcatgaggg ctcctggggc accatctgtg atgacagctg 2280 ggacctgaat gatgcccatg tggtgtgcaa acagctgagc tgtggatggg ccattaatgc 2340 cactggttct gctcattttg gggaaggaac agggcccatt tggctggatg agataaactg 2400 taatggaaaa gaatctcata tttggcaatg ccactcacat ggttgggggc ggcacaattg 2460 caggcataag gaggatgcag gagtcatctg ctcagagttc atgtctctga gactgatcag 2520 tgaaaacagc agagagacct gtgcagggcg cctggaagtt ttttacaacg gagcttgggg 2580 cagcgttggc aggaatagca tgtctccagc cacagtgggg gtggtatgca ggcagctggg 2640 ctgtgcagac agaggggaca tcagccctgc atcttcagac aagacagtgt ccaggcacat 2700 gtgggtggac aatgttcagt gtcctaaagg acctgacaca ctatggcagt gcccatcatc 2760 tccatggaag aagagactgg ccagcccctc agaggagaca tggatcacat gtgccaacaa 2820 aataagactt caagaaggaa acactaattg ttctggacgt gtggagatct ggtacggagg 2880 ttcctggggc actgtgtgtg acgactcctg ggaccttgaa gatgctcagg tggtgtgccg 2940 acagctgggc tgtggctcag ctttggaggc aggaaaagag gccgcatttg gccaggggac 3000 tgggcccata tggctcaatg aagtgaagtg caaggggaat gaaacctcct tgtgggattg 3060 tcctgccaga tcctggggcc acagtgactg tggacacaag gaggatgctg ctgtgacgtg 3120 ctcagaaatt gcaaagagcc gagaatccct acatgccaca ggtcgctcat cttttgttgc 3180 acttgcaatc tttggggtca ttctgttggc ctgtctcatc gcattcctca tttggactca 3240 gaagcgaaga cagaggcagc ggctctcagt tttctcagga ggagagaatt ctgtccatca 3300 aattcaatac cgggagatga attcttgcct gaaagcagat gaaacggata tgctaaatcc 3360 ctcaggagac cactctgaag tacaatgaaa aggaaaatgg gaattataac ctggtgagtt 3420 cagcctttaa gataccttga tgaagacctg gactattgaa tgagcaagaa tctgcctctt 3480 acactgaaga ttacaataca gtcctctgtc tcctggtatt ccaaagactg ctgctgaatt 3540 tctaaagaat agattggtga atgtgactac tcaaagttgt atgtaagact ttcaagggca 3600 ttaaataaaa aagaatattg ctg 3623 <210> 13 <211> 3345 <212> DNA <213> Sus scrofa <400> 13 atggacaaac tcagaatggt gctacatgaa aactctggat ctgcagactt tagaagatgt 60 tctgcccatt taagttcctt cacttttgct gtagtcgctg ttctcagtgc ctgcttggtc 120 actagttctc ttggaggaaa agacaaggag ctgaggctaa cgggtggtga aaacaagtgc 180 tctggaagag tggaggtgaa agtgcaggag gagtggggaa ctgtgtgtaa taatggctgg 240 gacatggatg tggtctctgt tgtttgtagg cagctgggat gtccaactgc tatcaaagcc 300 actggatggg ctaattttag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgagggaatg agtcagctct ctgggactgc aaacatgatg gatggggaaa gcataactgt 420 actcaccaac aggatgctgg agtaacctgc tcagatggat ctgatttaga gatggggctg 480 gtgaatggag gaaaccggtg cttaggaaga atagaagtca aatttcaagg acggtgggga 540 acagtgtgtg atgataactt caacataaat catgcttctg tggtttgtaa acaacttgaa 600 tgtggaagtg ctgtcagttt ctctggttca gctaattttg gagaaggttc tggaccaatc 660 tggtttgatg atcttgtatg caatggaaat gagtcagctc tctggaactg caaacatgaa 720 ggatggggaa agcacaattg cgatcatgct gaggatgctg gagtgatttg cttaaatgga 780 gcagacctga aactgagagt ggtagatgga gtcactgaat gttcaggaag attggaagtg 840 aaattccaag gagaatgggg aacaatctgt gatgatggct gggatagtga tgatgccgct 900 gtggcatgta agcaactggg atgtccaact gctgtcactg ccattggtcg agttaacgcc 960 agtgagggaa ctggacacat ttggcttgac agtgtttctt gccatggaca cgagtctgct 1020 ctctggcagt gtagacacca tgaatgggga aagcattatt gcaatcatga tgaagatgct 1080 ggtgtgacat gttctgatgg atcagatctg gaactgagac ttaaaggtgg aggcagccac 1140 tgtgctggga cagtggaggt ggaaattcag aaactggtag gaaaagtgtg tgatagaagc 1200 tggggactga aagaagctga tgtggtttgc aggcagctgg gatgtggatc tgcactcaaa 1260 acatcatatc aagtttattc caaaaccaag gcaacaaaca catggctgtt tgtaagcagc 1320 tgtaatggaa atgaaacttc tctttgggac tgcaagaatt ggcagtgggg tggacttagt 1380 tgtgatcact atgacgaagc caaaattacc tgctcagccc acaggaaacc caggctggtt 1440 ggaggggaca ttccctgctc tggtcgtgtt gaagtacaac atggagacac gtggggcacc 1500 gtctgtgatt ctgacttctc tctggaggcg gccagcgtgc tgtgcaggga actacagtgc 1560 ggcactgtgg tttccctcct ggggggagct cactttggag aaggaagtgg acagatctgg 1620 gctgaagaat tccagtgtga ggggcacgag tcccaccttt cactctgccc agtagcaccc 1680 cgccctgacg ggacatgtag ccacagcagg gacgtcggcg tagtctgctc aagatacaca 1740 caaatccgct tggtgaatgg caagacccca tgtgaaggaa gagtggagct caacattctt 1800 gggtcctggg ggtccctctg caactctcac tgggacatgg aagatgccca tgttttatgc 1860 cagcagctta aatgtggagt tgccctttct atcccgggag gagcaccttt tgggaaagga 1920 agtgagcagg tctggaggca catgtttcac tgcactggga ctgagaagca catgggagat 1980 tgttccgtca ctgctctggg cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc 2040 tgctcaggga accagagtca gacactatct ccgtgcaatt catcatcctc ggacccatca 2100 agctctatta tttcagaaga aaatggtgtt gcctgcatag ggagtggtca acttcgcctg 2160 gtcgatggag gtggtcgttg tgctgggaga gtagaggtct atcatgaggg ctcctggggc 2220 accatctgtg atgacagctg ggacctgaat gatgcccatg tggtgtgcaa acagctgagc 2280 tgtggatggg ccattaatgc cactggttct gctcattttg gggaaggaac agggcccatt 2340 tggctggatg agataaactg taatggaaaa gaatctcata tttggcaatg ccactcacat 2400 ggttgggggc ggcacaattg caggcataag gaggatgcag gagtcatctg ctcagagttc 2460 atgtctctga gactgatcag tgaaaacagc agagagacct gtgcagggcg cctggaagtt 2520 ttttacaacg gagcttgggg cagcgttggc aggaatagca tgtctccagc cacagtgggg 2580 gtggtatgca ggcagctggg ctgtgcagac agaggggaca tcagccctgc atcttcagac 2640 aagacagtgt ccaggcacat gtgggtggac aatgttcagt gtcctaaagg acctgacaca 2700 ctatggcagt gcccatcatc tccatggaag aagagactgg ccagcccctc agaggagaca 2760 tggatcacat gtgccaacaa aataagactt caagaaggaa acactaattg ttctggacgt 2820 gtggagatct ggtacggagg ttcctggggc actgtgtgtg acgactcctg ggaccttgaa 2880 gatgctcagg tggtgtgccg acagctgggc tgtggctcag ctttggaggc aggaaaagag 2940 gccgcatttg gccaggggac tgggcccata tggctcaatg aagtgaagtg caaggggaat 3000 gaaacctcct tgtgggattg tcctgccaga tcctggggcc acagtgactg tggacacaag 3060 gaggatgctg ctgtgacgtg ctcagaaatt gcaaagagcc gagaatccct acatgccaca 3120 ggtcgctcat cttttgttgc acttgcaatc tttggggtca ttctgttggc ctgtctcatc 3180 gcattcctca tttggactca gaagcgaaga cagaggcagc ggctctcagt tttctcagga 3240 ggagagaatt ctgtccatca aattcaatac cgggagatga attcttgcct gaaagcagat 3300 gaaacggata tgctaaatcc ctcaggagac cactctgaag tacaa 3345 <210> 14 <211> 1115 <212> PRT <213> Sus scrofa <400> 14 Met Asp Lys Leu Arg Met Val Leu His Glu Asn Ser Gly Ser Ala Asp 1 5 10 15 Phe Arg Arg Cys Ser Ala His Leu Ser Ser Phe Thr Phe Ala Val Val             20 25 30 Ala Val Leu Ser Ala Cys Leu Val Thr Ser Ser Leu Gly Gly Lys Asp         35 40 45 Lys Glu Leu Arg Leu Thr Gly Gly Glu Asn Lys Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Asp Met Asp Val Val Ser Val Val Cys Arg Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Thr Gly Trp Ala Asn Phe Ser Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Asn Cys Thr His Gln Gln     130 135 140 Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Met Gly Leu 145 150 155 160 Val Asn Gly Gly Asn Arg Cys Leu Gly Arg Ile Glu Val Lys Phe Gln                 165 170 175 Gly Arg Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asn His Ala             180 185 190 Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe Ser         195 200 205 Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp Asp     210 215 220 Leu Val Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His Glu 225 230 235 240 Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val Ile                 245 250 255 Cys Leu Asn Gly Ala Asp Leu Lys Leu Arg Val Val Asp Gly Val Thr             260 265 270 Glu Cys Ser Gly Arg Leu Glu Val Lys Phe Gln Gly Glu Trp Gly Thr         275 280 285 Ile Cys Asp Asp Gly Trp Asp Ser Asp Asp Ala Ala Val Ala Cys Lys     290 295 300 Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn Ala 305 310 315 320 Ser Glu Gly Thr Gly His Ile Trp Leu Asp Ser Val Ser Cys His Gly                 325 330 335 His Glu Ser Ala Leu Trp Gln Cys Arg His His Glu Trp Gly Lys His             340 345 350 Tyr Cys Asn His Asp Glu Asp Ala Gly Val Thr Cys Ser Asp Gly Ser         355 360 365 Asp Leu Glu Leu Arg Leu Lys Gly Gly Gly Ser His Cys Ala Gly Thr     370 375 380 Val Glu Val Glu Ile Gln Lys Leu Val Gly Lys Val Cys Asp Arg Ser 385 390 395 400 Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys Gly                 405 410 415 Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Thr Lys Ala Thr             420 425 430 Asn Thr Trp Leu Phe Val Ser Ser Cys Asn Gly Asn Glu Thr Ser Leu         435 440 445 Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Ser Cys Asp His Tyr     450 455 460 Asp Glu Ala Lys Ile Thr Cys Ser Ala His Arg Lys Pro Arg Leu Val 465 470 475 480 Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Gln His Gly Asp                 485 490 495 Thr Trp Gly Thr Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala Ser             500 505 510 Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Leu Leu Gly         515 520 525 Gly Ala His Phe Gly Glu Gly Ser Gly Gln Ile Trp Ala Glu Glu Phe     530 535 540 Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala Pro 545 550 555 560 Arg Pro Asp Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val Cys                 565 570 575 Ser Arg Tyr Thr Gln Ile Arg Leu Val Asn Gly Lys Thr Pro Cys Glu             580 585 590 Gly Arg Val Glu Leu Asn Ile Leu Gly Ser Trp Gly Ser Leu Cys Asn         595 600 605 Ser His Trp Asp Met Glu Asp Ala His Val Leu Cys Gln Gln Leu Lys     610 615 620 Cys Gly Val Ala Leu Ser Ile Pro Gly Gly Ala Pro Phe Gly Lys Gly 625 630 635 640 Ser Glu Gln Val Trp Arg His Met Phe His Cys Thr Gly Thr Glu Lys                 645 650 655 His Met Gly Asp Cys Ser Val Thr Ala Leu Gly Ala Ser Leu Cys Ser             660 665 670 Ser Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln Thr         675 680 685 Leu Ser Pro Cys Asn Ser Ser Ser Ser Asp Pro Ser Ser Ser Ile Ile     690 695 700 Ser Glu Glu Asn Gly Val Ala Cys Ile Gly Ser Gly Gln Leu Arg Leu 705 710 715 720 Val Asp Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Val Tyr His Glu                 725 730 735 Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Asn Asp Ala             740 745 750 His Val Val Cys Lys Gln Leu Ser Cys Gly Trp Ala Ile Asn Ala Thr         755 760 765 Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp Glu     770 775 780 Ile Asn Cys Asn Gly Lys Glu Ser His Ile Trp Gln Cys His Ser His 785 790 795 800 Gly Trp Gly Arg His Asn Cys Arg His Lys Glu Asp Ala Gly Val Ile                 805 810 815 Cys Ser Glu Phe Met Ser Leu Arg Leu Ile Ser Glu Asn Ser Arg Glu             820 825 830 Thr Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly Ser         835 840 845 Val Gly Arg Asn Ser Met Ser Pro Ala Thr Val Gly Val Val Cys Arg     850 855 860 Gln Leu Gly Cys Ala Asp Arg Gly Asp Ile Ser Pro Ala Ser Ser Asp 865 870 875 880 Lys Thr Val Ser Arg His Met Trp Val Asp Asn Val Gln Cys Pro Lys                 885 890 895 Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Lys Lys Arg             900 905 910 Leu Ala Ser Pro Ser Glu Glu Thr Trp Ile Thr Cys Ala Asn Lys Ile         915 920 925 Arg Leu Gln Glu Gly Asn Thr Asn Cys Ser Gly Arg Val Glu Ile Trp     930 935 940 Tyr Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu Glu 945 950 955 960 Asp Ala Gln Val Val Cys Arg Gln Leu Gly Cys Gly Ser Ala Leu Glu                 965 970 975 Ala Gly Lys Glu Ala Ala Phe Gly Gln Gly Thr Gly Pro Ile Trp Leu             980 985 990 Asn Glu Val Lys Cys Lys Gly Asn Glu Thr Ser Leu Trp Asp Cys Pro         995 1000 1005 Ala Arg Ser Trp Gly His Ser Asp Cys Gly His Lys Glu Asp Ala     1010 1015 1020 Ala Val Thr Cys Ser Glu Ile Ala Lys Ser Arg Glu Ser Leu His     1025 1030 1035 Ala Thr Gly Arg Ser Ser Phe Val Ala Leu Ala Ile Phe Gly Val     1040 1045 1050 Ile Leu Leu Ala Cys Leu Ile Ala Phe Leu Ile Trp Thr Gln Lys     1055 1060 1065 Arg Arg Gln Arg Gln Arg Leu Ser Val Phe Ser Gly Gly Glu Asn     1070 1075 1080 Ser Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu Lys     1085 1090 1095 Ala Asp Glu Thr Asp Met Leu Asn Pro Ser Gly Asp His Ser Glu     1100 1105 1110 Val Gln     1115 <210> 15 <211> 50 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 15 caccgcggcc gcgaagttat aaatcgccac catgagcaaa ctcagaatgg 50 <210> 16 <211> 40 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 16 tgctccggta cctagtccag gtcttcatca aggtatctta 40 <210> 17 <211> 3434 <212> DNA <213> Homo sapiens <400> 17 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgactt cagaagacat 60 tttgtcaacc tgagtccctt caccattact gtggtcttac ttctcagtgc ctgttttgtc 120 accagttctc ttggaggaac agacaaggag ctgaggctag tggatggtga aaacaagtgt 180 agcgggagag tggaagtgaa agtccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cggtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 cctggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggattgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgacc tgctcagatg gatccaattt ggaaatgagg 480 ctgacgcgtg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacggtgg 540 ggaacagtgt gtgatgataa cttcaacata gatcatgcat ctgtcatttg tagacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcatctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgaccat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggggaatg ggggacaata tgtgatgacg gctgggacag ttacgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgccgtca cagccattgg tcgagttaac 960 gccagtaagg gatttggaca catctggctt gacagcgttt cttgccaggg acatgaacct 1020 gctgtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtga catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatctt atcaagtgta ctccaaaatc caggcaacaa acacatggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggagggg acattccctg ttctggacgt gttgaagtga agcatggtga cacgtggggc 1500 tccatctgtg attcggactt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag ttgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccgtgtgagg gcagagtgga gctcaaaacg 1800 cttggtgcct ggggatccct ctgtaactct cactgggaca tagaagatgc ccatgttctt 1860 tgccagcagc ttaaatgtgg agttgccctt tctaccccag gaggagcacg ttttggaaaa 1920 ggaaatggtc agatctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct aggtgcttca ttatgtcctt cagagcaagt ggcctctgta 2040 atctgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcgtc tttgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ctggtaaatg gaggaggtcg ctgtgctggg agagtagaga tctatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agtgatgccc acgtggtttg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggggaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca 2400 cacggctggg ggcagcaaaa ttgcaggcac aaggaggatg cgggagttat ctgctcagaa 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctggaa 2520 gttttttaca atggagcttg gggcactgtt ggcaagagta gcatgtctga aaccactgtg 2580 ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaaccc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccagccc ctcggaggag 2760 acctggatca catgtgacaa caagataaga cttcaggaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcatgg aggttcctgg gggacagtgt gtgatgactc ttgggacttg 2880 gacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccg atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccatagtga gtgtgggcac 3060 aaggaagacg ctgcagtgaa ttgcacagat atttcagtgc agaaaacccc acaaaaagcc 3120 acaacaggtc gctcatcccg tcagtcatcc tttattgcag tcgggatcct tggggttgtt 3180 ctgttggcca ttttcgtcgc attattcttc ttgactaaaa agcgaagaca gagacagcgg 3240 cttgcagttt cctcaagagg agagaactta gtccaccaaa ttcaataccg ggagatgaat 3300 tcttgcctga atgcagatga tctggaccta atgaattcct caggaggcca ttctgagcca 3360 cactgaaaag gaaaatggga atttataacc cagtgagttc agcctttaag ataccttgat 3420 gaagacctgg acta 3434 <210> 18 <211> 3363 <212> DNA <213> Homo sapiens <400> 18 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgactt cagaagacat 60 tttgtcaacc tgagtccctt caccattact gtggtcttac ttctcagtgc ctgttttgtc 120 accagttctc ttggaggaac agacaaggag ctgaggctag tggatggtga aaacaagtgt 180 agcgggagag tggaagtgaa agtccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cggtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 cctggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggattgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgacc tgctcagatg gatccaattt ggaaatgagg 480 ctgacgcgtg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacggtgg 540 ggaacagtgt gtgatgataa cttcaacata gatcatgcat ctgtcatttg tagacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcatctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgaccat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggggaatg ggggacaata tgtgatgacg gctgggacag ttacgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgccgtca cagccattgg tcgagttaac 960 gccagtaagg gatttggaca catctggctt gacagcgttt cttgccaggg acatgaacct 1020 gctgtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtga catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatctt atcaagtgta ctccaaaatc caggcaacaa acacatggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggagggg acattccctg ttctggacgt gttgaagtga agcatggtga cacgtggggc 1500 tccatctgtg attcggactt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag ttgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccgtgtgagg gcagagtgga gctcaaaacg 1800 cttggtgcct ggggatccct ctgtaactct cactgggaca tagaagatgc ccatgttctt 1860 tgccagcagc ttaaatgtgg agttgccctt tctaccccag gaggagcacg ttttggaaaa 1920 ggaaatggtc agatctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct aggtgcttca ttatgtcctt cagagcaagt ggcctctgta 2040 atctgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcgtc tttgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ctggtaaatg gaggaggtcg ctgtgctggg agagtagaga tctatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agtgatgccc acgtggtttg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggggaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca 2400 cacggctggg ggcagcaaaa ttgcaggcac aaggaggatg cgggagttat ctgctcagaa 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctggaa 2520 gttttttaca atggagcttg gggcactgtt ggcaagagta gcatgtctga aaccactgtg 2580 ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaaccc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccagccc ctcggaggag 2760 acctggatca catgtgacaa caagataaga cttcaggaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcatgg aggttcctgg gggacagtgt gtgatgactc ttgggacttg 2880 gacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccg atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccatagtga gtgtgggcac 3060 aaggaagacg ctgcagtgaa ttgcacagat atttcagtgc agaaaacccc acaaaaagcc 3120 acaacaggtc gctcatcccg tcagtcatcc tttattgcag tcgggatcct tggggttgtt 3180 ctgttggcca ttttcgtcgc attattcttc ttgactaaaa agcgaagaca gagacagcgg 3240 cttgcagttt cctcaagagg agagaactta gtccaccaaa ttcaataccg ggagatgaat 3300 tcttgcctga atgcagatga tctggaccta atgaattcct caggaggcca ttctgagcca 3360 cac 3363 <210> 19 <211> 1121 <212> PRT <213> Homo sapiens <400> 19 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Phe Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Thr Val Val             20 25 30 Leu Leu Leu Ser Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Asp         35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Pro Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln     130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asn Leu Glu Met Arg 145 150 155 160 Leu Thr Arg Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe                 165 170 175 Gln Gly Arg Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asp His             180 185 190 Ala Ser Val Ile Cys Arg Gln Leu Glu Cys Gly Ser Ala Val Ser Phe         195 200 205 Ser Gly Ser Ser Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp     210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val                 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val             260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly         275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser Tyr Asp Ala Ala Val Ala Cys     290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Lys Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln                 325 330 335 Gly His Glu Pro Ala Val Trp Gln Cys Lys His His Glu Trp Gly Lys             340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly         355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly     370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys                 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala             420 425 430 Thr Asn Thr Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser         435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His     450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly                 485 490 495 Asp Thr Trp Gly Ser Ile Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala             500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu         515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu     530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val                 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys             580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Gly Ala Trp Gly Ser Leu Cys         595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu     610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala Arg Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Ile Trp Arg His Met Phe His Cys Thr Gly Thr Glu                 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys             660 665 670 Pro Ser Glu Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln         675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr     690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp             740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp     770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg             820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly         835 840 845 Thr Val Gly Lys Ser Ser Met Ser Glu Thr Thr Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Pro Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro                 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys             900 905 910 Arg Leu Ala Ser Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys         915 920 925 Ile Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile     930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asp Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu                 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp             980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys         995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp     1010 1015 1020 Ala Ala Val Asn Cys Thr Asp Ile Ser Val Gln Lys Thr Pro Gln     1025 1030 1035 Lys Ala Thr Thr Gly Arg Ser Ser Arg Gln Ser Ser Phe Ile Ala     1040 1045 1050 Val Gly Ile Leu Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu     1055 1060 1065 Phe Phe Leu Thr Lys Lys Arg Arg Gln Arg Gln Arg Leu Ala Val     1070 1075 1080 Ser Ser Arg Gly Glu Asn Leu Val His Gln Ile Gln Tyr Arg Glu     1085 1090 1095 Met Asn Ser Cys Leu Asn Ala Asp Asp Leu Asp Leu Met Asn Ser     1100 1105 1110 Ser Gly Gly His Ser Glu Pro His     1115 1120 <210> 20 <211> 37 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 20 caccgcggcc gccacacgga gccatcaaaa tcatcaa 37 <210> 21 <211> 46 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 21 ggtaccgcga acaagcaaac caatagcaat attgtttaat tccctc 46 <210> 22 <211> 3556 <212> DNA <213> Mus musculus <400> 22 gctttggaat gggtggacac agaatggttc ttcttggagg tgctggatct cctggttgta 60 aaaggtttgt ccatctaggt ttctttgttg tggctgtgag ctcacttctc agtgcctctg 120 ctgtcactaa cgctcctgga gaaatgaaga aggaactgag actggcgggt ggtgaaaaca 180 actgtagtgg gagagtggaa cttaagatcc atgacaagtg gggcacagtg tgcagtaacg 240 gctggagcat gaatgaagtg tccgtggttt gccagcagct gggatgccca acttctatta 300 aagcccttgg atgggctaac tccagcgccg gctctggata tatctggatg gacaaagttt 360 cttgtacagg gaatgagtca gctctttggg actgcaaaca tgatgggtgg ggaaagcata 420 actgtaccca tgaaaaagat gctggagtga cctgctcaga tggatctaat ttggagatga 480 gactggtgaa cagtgcgggc caccgatgct taggaagagt agaaataaag ttccagggaa 540 agtgggggac ggtgtgtgac gacaacttca gcaaagatca cgcttctgtg atttgtaaac 600 agcttggatg tggaagtgcc attagtttct ctggctcagc taaattggga gctggttctg 660 gaccaatctg gctcgatgac ctggcatgca atggaaatga gtcagctctc tgggactgca 720 aacaccgggg atggggcaag cataactgtg accatgctga ggatgtcggt gtgatttgct 780 tagagggagc agatctgagc ctgagactag tggatggagt gtccagatgt tcaggaagat 840 tggaagtgag attccaagga gaatggggga ccgtgtgtga tgataactgg gatctccggg 900 atgcttctgt ggtgtgcaag caactgggat gtccaactgc catcagtgcc attggtcgag 960 ttaatgccag tgagggatct ggacagattt ggcttgacaa catttcatgc gaaggacatg 1020 aggcaactct ttgggagtgt aaacaccaag agtggggaaa gcattactgt catcatagag 1080 aagacgctgg cgtgacatgt tctgatggag cagatctgga acttagactt gtaggtggag 1140 gcagtcgctg tgctggcatt gtggaggtgg agattcagaa gctgactggg aagatgtgta 1200 gccgaggctg gacactggca gatgcggatg tggtttgcag acagcttgga tgtggatctg 1260 cgcttcaaac ccaggctaag atctactcta aaactggggc aacaaatacg tggctctttc 1320 ctggatcttg taatggaaat gaaactactt tttggcaatg caaaaactgg cagtggggcg 1380 gcctttcctg tgataatttc gaagaagcca aagttacctg ctcaggccac agggaaccca 1440 gactggttgg aggagaaatc ccatgctctg gtcgtgtgga agtgaaacac ggagacgtgt 1500 ggggctccgt ctgtgatttt gacttgtctc tggaagctgc cagtgtggtg tgcagggaat 1560 tacaatgtgg aacagtcgtc tctatcctag ggggagcaca ttttggagaa ggaagtggac 1620 agatctgggg tgaagaattc cagtgtagtg gggatgagtc ccatctttca ctatgctcag 1680 tggcgccccc gctagacaga acttgtaccc acagcaggga tgtcagcgta gtctgctcac 1740 gatacataga tattcgtctg gcaggcggcg agtcctcctg tgagggaaga gtggagctca 1800 agacactcgg agcctggggt cccctctgca gttctcattg ggacatggaa gatgctcatg 1860 tcttatgtca gcagctgaag tgtggggttg cccaatctat tccagaagga gcacattttg 1920 ggaaaggagc tggtcaggtc tggagtcaca tgttccactg cactggaact gaggaacata 1980 taggagattg cctcatgact gctctgggtg cgccgacgtg ttccgaagga caggtggcct 2040 ctgtcatctg ctcaggaaac caatcccaga cactattgcc atgtagttca ttgtctccag 2100 tccaaacaac aagctctaca attccaaagg agagtgaagt tccctgcata gcaagtggcc 2160 agcttcgctt ggtaggtgga ggtggtcgct gcgctggaag agtggaggtc taccacgagg 2220 gctcttgggg caccgtctgt gatgacaatt gggatatgac tgatgccaat gtggtgtgca 2280 agcagctgga ctgtggcgtg gcaattaacg ccactggctc tgcttacttc ggggaaggag 2340 caggagctat ctggctagac gaagtcatct gcactgggaa agagtctcat atttggcagt 2400 gccattcaca tggctgggga cgccataact gcaggcacaa agaagatgca ggtgttatct 2460 gctccgagtt catgtctctg aggctgacca acgaagccca caaagaaaac tgcacaggtc 2520 gccttgaagt gttttacaat ggtacatggg gcagtattgg cagtagcaat atgtctccaa 2580 ccactgtggg ggtggtgtgc cgtcagctgg gctgtgcaga caacgggact gtgaaaccca 2640 taccttcaga caagacacca tccaggccca tgtgggtaga tcgtgtgcag tgtccaaaag 2700 gagttgacac tttgtggcag tgcccctcgt caccttggaa acagagacag gccagcccct 2760 cctcccagga gtcctggatc atctgtgaca acaaaataag actccaggaa gggcatacag 2820 actgttctgg acgtgtggag atctggcaca aaggttcctg gggaacagtg tgtgatgact 2880 cctgggatct taatgatgct aaggttgtat gtaagcagtt gggctgtggc caagctgtga 2940 aggcactaaa agaagcagca tttggtccag gaactgggcc catatggctc aatgaaatta 3000 agtgtagagg gaatgagtct tccctgtggg attgtcctgc caaaccgtgg agtcacagcg 3060 actgtgggca caaagaagat gcttccatcc agtgcctccc aaaaatgact tcagaatcac 3120 atcatggcac aggtcacccc accctcacgg cactcttggt ttgtggagcc attctattgg 3180 tcctcctcat tgtcttcctc ctgtggactc tgaagcgacg acagattcag cgacttacag 3240 tttcctcaag aggagaggtc ttgatacatc aagttcagta ccaagagatg gattcaaagg 3300 cggatgatct ggacttgctg aaatcctcgg gggtcattca gaggcacact gagaaggaaa 3360 atgataattt ataatccact gaggttggag tttaagaagc cttgacagga cagccagcta 3420 aatggaacaa gagcccaggc aacgcacgga tgaccacagc tgcatcttca tgcagtcctt 3480 tgtttcctgg aactctgctg aacctgcaaa aaccatattt gtgaatgtga ccacttaata 3540 gagatgggag actttt 3556 <210> 23 <211> 3363 <212> DNA <213> Mus musculus <400> 23 atgggtggac acagaatggt tcttcttgga ggtgctggat ctcctggttg taaaaggttt 60 gtccatctag gtttctttgt tgtggctgtg agctcacttc tcagtgcctc tgctgtcact 120 aacgctcctg gagaaatgaa gaaggaactg agactggcgg gtggtgaaaa caactgtagt 180 gggagagtgg aacttaagat ccatgacaag tggggcacag tgtgcagtaa cggctggagc 240 atgaatgaag tgtccgtggt ttgccagcag ctgggatgcc caacttctat taaagccctt 300 ggatgggcta actccagcgc cggctctgga tatatctgga tggacaaagt ttcttgtaca 360 gggaatgagt cagctctttg ggactgcaaa catgatgggt ggggaaagca taactgtacc 420 catgaaaaag atgctggagt gacctgctca gatggatcta atttggagat gagactggtg 480 aacagtgcgg gccaccgatg cttaggaaga gtagaaataa agttccaggg aaagtggggg 540 acggtgtgtg acgacaactt cagcaaagat cacgcttctg tgatttgtaa acagcttgga 600 tgtggaagtg ccattagttt ctctggctca gctaaattgg gagctggttc tggaccaatc 660 tggctcgatg acctggcatg caatggaaat gagtcagctc tctgggactg caaacaccgg 720 ggatggggca agcataactg tgaccatgct gaggatgtcg gtgtgatttg cttagaggga 780 gcagatctga gcctgagact agtggatgga gtgtccagat gttcaggaag attggaagtg 840 agattccaag gagaatgggg gaccgtgtgt gatgataact gggatctccg ggatgcttct 900 gtggtgtgca agcaactggg atgtccaact gccatcagtg ccattggtcg agttaatgcc 960 agtgagggat ctggacagat ttggcttgac aacatttcat gcgaaggaca tgaggcaact 1020 ctttgggagt gtaaacacca agagtgggga aagcattact gtcatcatag agaagacgct 1080 ggcgtgacat gttctgatgg agcagatctg gaacttagac ttgtaggtgg aggcagtcgc 1140 tgtgctggca ttgtggaggt ggagattcag aagctgactg ggaagatgtg tagccgaggc 1200 tggacactgg cagatgcgga tgtggtttgc agacagcttg gatgtggatc tgcgcttcaa 1260 acccaggcta agatctactc taaaactggg gcaacaaata cgtggctctt tcctggatct 1320 tgtaatggaa atgaaactac tttttggcaa tgcaaaaact ggcagtgggg cggcctttcc 1380 tgtgataatt tcgaagaagc caaagttacc tgctcaggcc acagggaacc cagactggtt 1440 ggaggagaaa tcccatgctc tggtcgtgtg gaagtgaaac acggagacgt gtggggctcc 1500 gtctgtgatt ttgacttgtc tctggaagct gccagtgtgg tgtgcaggga attacaatgt 1560 ggaacagtcg tctctatcct agggggagca cattttggag aaggaagtgg acagatctgg 1620 ggtgaagaat tccagtgtag tggggatgag tcccatcttt cactatgctc agtggcgccc 1680 ccgctagaca gaacttgtac ccacagcagg gatgtcagcg tagtctgctc acgatacata 1740 gatattcgtc tggcaggcgg cgagtcctcc tgtgagggaa gagtggagct caagacactc 1800 ggagcctggg gtcccctctg cagttctcat tgggacatgg aagatgctca tgtcttatgt 1860 cagcagctga agtgtggggt tgcccaatct attccagaag gagcacattt tgggaaagga 1920 gctggtcagg tctggagtca catgttccac tgcactggaa ctgaggaaca tataggagat 1980 tgcctcatga ctgctctggg tgcgccgacg tgttccgaag gacaggtggc ctctgtcatc 2040 tgctcaggaa accaatccca gacactattg ccatgtagtt cattgtctcc agtccaaaca 2100 acaagctcta caattccaaa ggagagtgaa gttccctgca tagcaagtgg ccagcttcgc 2160 ttggtaggtg gaggtggtcg ctgcgctgga agagtggagg tctaccacga gggctcttgg 2220 ggcaccgtct gtgatgacaa ttgggatatg actgatgcca atgtggtgtg caagcagctg 2280 gactgtggcg tggcaattaa cgccactggc tctgcttact tcggggaagg agcaggagct 2340 atctggctag acgaagtcat ctgcactggg aaagagtctc atatttggca gtgccattca 2400 catggctggg gacgccataa ctgcaggcac aaagaagatg caggtgttat ctgctccgag 2460 ttcatgtctc tgaggctgac caacgaagcc cacaaagaaa actgcacagg tcgccttgaa 2520 gtgttttaca atggtacatg gggcagtatt ggcagtagca atatgtctcc aaccactgtg 2580 ggggtggtgt gccgtcagct gggctgtgca gacaacggga ctgtgaaacc cataccttca 2640 gacaagacac catccaggcc catgtgggta gatcgtgtgc agtgtccaaa aggagttgac 2700 actttgtggc agtgcccctc gtcaccttgg aaacagagac aggccagccc ctcctcccag 2760 gagtcctgga tcatctgtga caacaaaata agactccagg aagggcatac agactgttct 2820 ggacgtgtgg agatctggca caaaggttcc tggggaacag tgtgtgatga ctcctgggat 2880 cttaatgatg ctaaggttgt atgtaagcag ttgggctgtg gccaagctgt gaaggcacta 2940 aaagaagcag catttggtcc aggaactggg cccatatggc tcaatgaaat taagtgtaga 3000 gggaatgagt cttccctgtg ggattgtcct gccaaaccgt ggagtcacag cgactgtggg 3060 cacaaagaag atgcttccat ccagtgcctc ccaaaaatga cttcagaatc acatcatggc 3120 acaggtcacc ccaccctcac ggcactcttg gtttgtggag ccattctatt ggtcctcctc 3180 attgtcttcc tcctgtggac tctgaagcga cgacagattc agcgacttac agtttcctca 3240 agaggagagg tcttgataca tcaagttcag taccaagaga tggattcaaa ggcggatgat 3300 ctggacttgc tgaaatcctc gggggtcatt cagaggcaca ctgagaagga aaatgataat 3360 tta 3363 <210> 24 <211> 1121 <212> PRT <213> Mus musculus <400> 24 Met Gly Gly His Arg Met Val Leu Leu Gly Gly Ala Gly Ser Pro Gly 1 5 10 15 Cys Lys Arg Phe Val His Leu Gly Phe Phe Val Val Ala Val Ser Ser             20 25 30 Leu Leu Ser Ala Ser Ala Val Thr Asn Ala Pro Gly Glu Met Lys Lys         35 40 45 Glu Leu Arg Leu Ala Gly Gly Glu Asn Asn Cys Ser Gly Arg Val Glu     50 55 60 Leu Lys Ile His Asp Lys Trp Gly Thr Val Cys Ser Asn Gly Trp Ser 65 70 75 80 Met Asn Glu Val Ser Val Val Cys Gln Gln Leu Gly Cys Pro Thr Ser                 85 90 95 Ile Lys Ala Leu Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Tyr Ile             100 105 110 Trp Met Asp Lys Val Ser Cys Thr Gly Asn Glu Ser Ala Leu Trp Asp         115 120 125 Cys Lys His Asp Gly Trp Gly Lys His Asn Cys Thr His Glu Lys Asp     130 135 140 Ala Gly Val Thr Cys Ser Asp Gly Ser Asn Leu Glu Met Arg Leu Val 145 150 155 160 Asn Ser Ala Gly His Arg Cys Leu Gly Arg Val Glu Ile Lys Phe Gln                 165 170 175 Gly Lys Trp Gly Thr Val Cys Asp Asp Asn Phe Ser Lys Asp His Ala             180 185 190 Ser Val Ile Cys Lys Gln Leu Gly Cys Gly Ser Ala Ile Ser Phe Ser         195 200 205 Gly Ser Ala Lys Leu Gly Ala Gly Ser Gly Pro Ile Trp Leu Asp Asp     210 215 220 Leu Ala Cys Asn Gly Asn Glu Ser Ala Leu Trp Asp Cys Lys His Arg 225 230 235 240 Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Val Gly Val Ile                 245 250 255 Cys Leu Glu Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val Ser             260 265 270 Arg Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly Thr         275 280 285 Val Cys Asp Asp Asn Trp Asp Leu Arg Asp Ala Ser Val Val Cys Lys     290 295 300 Gln Leu Gly Cys Pro Thr Ala Ile Ser Ala Ile Gly Arg Val Asn Ala 305 310 315 320 Ser Glu Gly Ser Gly Gln Ile Trp Leu Asp Asn Ile Ser Cys Glu Gly                 325 330 335 His Glu Ala Thr Leu Trp Glu Cys Lys His Gln Glu Trp Gly Lys His             340 345 350 Tyr Cys His His Arg Glu Asp Ala Gly Val Thr Cys Ser Asp Gly Ala         355 360 365 Asp Leu Glu Leu Arg Leu Val Gly Gly Gly Ser Arg Cys Ala Gly Ile     370 375 380 Val Glu Val Glu Ile Gln Lys Leu Thr Gly Lys Met Cys Ser Arg Gly 385 390 395 400 Trp Thr Leu Ala Asp Ala Asp Val Val Cys Arg Gln Leu Gly Cys Gly                 405 410 415 Ser Ala Leu Gln Thr Gln Ala Lys Ile Tyr Ser Lys Thr Gly Ala Thr             420 425 430 Asn Thr Trp Leu Phe Pro Gly Ser Cys Asn Gly Asn Glu Thr Thr Phe         435 440 445 Trp Gln Cys Lys Asn Trp Gln Trp Gly Gly Leu Ser Cys Asp Asn Phe     450 455 460 Glu Glu Ala Lys Val Thr Cys Ser Gly His Arg Glu Pro Arg Leu Val 465 470 475 480 Gly Gly Glu Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly Asp                 485 490 495 Val Trp Gly Ser Val Cys Asp Phe Asp Leu Ser Leu Glu Ala Ala Ser             500 505 510 Val Val Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu Gly         515 520 525 Gly Ala His Phe Gly Glu Gly Ser Gly Gln Ile Trp Gly Glu Glu Phe     530 535 540 Gln Cys Ser Gly Asp Glu Ser His Leu Ser Leu Cys Ser Val Ala Pro 545 550 555 560 Pro Leu Asp Arg Thr Cys Thr His Ser Arg Asp Val Ser Val Val Cys                 565 570 575 Ser Arg Tyr Ile Asp Ile Arg Leu Ala Gly Gly Glu Ser Ser Cys Glu             580 585 590 Gly Arg Val Glu Leu Lys Thr Leu Gly Ala Trp Gly Pro Leu Cys Ser         595 600 605 Ser His Trp Asp Met Glu Asp Ala His Val Leu Cys Gln Gln Leu Lys     610 615 620 Cys Gly Val Ala Gln Ser Ile Pro Glu Gly Ala His Phe Gly Lys Gly 625 630 635 640 Ala Gly Gln Val Trp Ser His Met Phe His Cys Thr Gly Thr Glu Glu                 645 650 655 His Ile Gly Asp Cys Leu Met Thr Ala Leu Gly Ala Pro Thr Cys Ser             660 665 670 Glu Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln Thr         675 680 685 Leu Leu Pro Cys Ser Ser Leu Ser Pro Val Gln Thr Thr Ser Ser Thr     690 695 700 Ile Pro Lys Glu Ser Glu Val Pro Cys Ile Ala Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Gly Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Val Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Val Cys Asp Asp Asn Trp Asp Met Thr Asp             740 745 750 Ala Asn Val Val Cys Lys Gln Leu Asp Cys Gly Val Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala Tyr Phe Gly Glu Gly Ala Gly Ala Ile Trp Leu Asp     770 775 780 Glu Val Ile Cys Thr Gly Lys Glu Ser His Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Arg His Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Asn Glu Ala His Lys             820 825 830 Glu Asn Cys Thr Gly Arg Leu Glu Val Phe Tyr Asn Gly Thr Trp Gly         835 840 845 Ser Ile Gly Ser Ser Asn Met Ser Pro Thr Thr Val Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Asn Gly Thr Val Lys Pro Ile Pro Ser 865 870 875 880 Asp Lys Thr Pro Ser Arg Pro Met Trp Val Asp Arg Val Gln Cys Pro                 885 890 895 Lys Gly Val Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Lys Gln             900 905 910 Arg Gln Ala Ser Pro Ser Ser Gln Glu Ser Trp Ile Ile Cys Asp Asn         915 920 925 Lys Ile Arg Leu Gln Glu Gly His Thr Asp Cys Ser Gly Arg Val Glu     930 935 940 Ile Trp His Lys Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp 945 950 955 960 Leu Asn Asp Ala Lys Val Val Cys Lys Gln Leu Gly Cys Gly Gln Ala                 965 970 975 Val Lys Ala Leu Lys Glu Ala Ala Phe Gly Pro Gly Thr Gly Pro Ile             980 985 990 Trp Leu Asn Glu Ile Lys Cys Arg Gly Asn Glu Ser Ser Leu Trp Asp         995 1000 1005 Cys Pro Ala Lys Pro Trp Ser His Ser Asp Cys Gly His Lys Glu     1010 1015 1020 Asp Ala Ser Ile Gln Cys Leu Pro Lys Met Thr Ser Glu Ser His     1025 1030 1035 His Gly Thr Gly His Pro Thr Leu Thr Ala Leu Leu Val Cys Gly     1040 1045 1050 Ala Ile Leu Leu Val Leu Leu Ile Val Phe Leu Leu Trp Thr Leu     1055 1060 1065 Lys Arg Arg Gln Ile Gln Arg Leu Thr Val Ser Ser Arg Gly Glu     1070 1075 1080 Val Leu Ile His Gln Val Gln Tyr Gln Glu Met Asp Ser Lys Ala     1085 1090 1095 Asp Asp Leu Asp Leu Leu Lys Ser Ser Gly Val Ile Gln Arg His     1100 1105 1110 Thr Glu Lys Glu Asn Asp Asn Leu     1115 1120 <210> 25 <211> 3639 <212> DNA <213> Mus musculus <400> 25 gctttggaat gggtggacac agaatggttc ttcttggagg tgctggatct cctggttgta 60 aaaggtttgt ccatctaggt ttctttgttg tggctgtgag ctcacttctc agtgcctctg 120 ctgtcactaa cgctcctgga gaaatgaaga aggaactgag actggcgggt ggtgaaaaca 180 actgtagtgg gagagtggaa cttaagatcc atgacaagtg gggcacagtg tgcagtaacg 240 gctggagcat gaatgaagtg tccgtggttt gccagcagct gggatgccca acttctatta 300 aagcccttgg atgggctaac tccagcgccg gctctggata tatctggatg gacaaagttt 360 cttgtacagg gaatgagtca gctctttggg actgcaaaca tgatgggtgg ggaaagcata 420 actgtaccca tgaaaaagat gctggagtga cctgctcaga tggatctaat ttggagatga 480 gactggtgaa cagtgcgggc caccgatgct taggaagagt agaaataaag ttccagggaa 540 agtgggggac ggtgtgtgac gacaacttca gcaaagatca cgcttctgtg atttgtaaac 600 agcttggatg tggaagtgcc attagtttct ctggctcagc taaattggga gctggttctg 660 gaccaatctg gctcgatgac ctggcatgca atggaaatga gtcagctctc tgggactgca 720 aacaccgggg atggggcaag cataactgtg accatgctga ggatgtcggt gtgatttgct 780 tagagggagc agatctgagc ctgagactag tggatggagt gtccagatgt tcaggaagat 840 tggaagtgag attccaagga gaatggggga ccgtgtgtga tgataactgg gatctccggg 900 atgcttctgt ggtgtgcaag caactgggat gtccaactgc catcagtgcc attggtcgag 960 ttaatgccag tgagggatct ggacagattt ggcttgacaa catttcatgc gaaggacatg 1020 aggcaactct ttgggagtgt aaacaccaag agtggggaaa gcattactgt catcatagag 1080 aagacgctgg cgtgacatgt tctgatggag cagatctgga acttagactt gtaggtggag 1140 gcagtcgctg tgctggcatt gtggaggtgg agattcagaa gctgactggg aagatgtgta 1200 gccgaggctg gacactggca gatgcggatg tggtttgcag acagcttgga tgtggatctg 1260 cgcttcaaac ccaggctaag atctactcta aaactggggc aacaaatacg tggctctttc 1320 ctggatcttg taatggaaat gaaactactt tttggcaatg caaaaactgg cagtggggcg 1380 gcctttcctg tgataatttc gaagaagcca aagttacctg ctcaggccac agggaaccca 1440 gactggttgg aggagaaatc ccatgctctg gtcgtgtgga aatgaaacac ggagacgtgt 1500 ggggctccgt ctgtgatttt gacttgtctc tggaagctgc cagtgtggtg tgcagggaat 1560 tacaatgtgg aacagtcgtc tctatcctag ggggagcaca ttttggagaa ggaagtggac 1620 agatctgggg tgaagaattc cagtgtagtg gggatgagtc ccatctttca ctatgctcag 1680 tggcgccccc gctagacaga acttgtaccc acagcaggga tgtcagcgta gtctgctcac 1740 gatacataga tattcgtctg gcaggcggcg agtcctcctg tgagggaaga gtggagctca 1800 agacactcgg agcctggggt cccctctgca gttctcattg ggacatggaa gatgctcatg 1860 tcttatgtca gcagctgaag tgtggggttg cccaatctat tccagaagga gcacattttg 1920 ggaaaggagc tggtcaggtc tggagtcaca tgttccactg cactggaact gaggaacata 1980 taggagattg cctcatgact gctctgggtg cgccgacgtg ttccgaagga caggtggcct 2040 ctgtcatctg ctcaggaaac caatcccaga cactattgcc atgtagttca ttgtctccag 2100 tccaaacaac aagctctaca attccaaagg agagtgaagt tccctgcata gcaagtggcc 2160 agcttcgctt ggtaggtgga ggtggtcgct gcgctggaag agtggaggtc taccacgagg 2220 gctcttgggg caccgtctgt gatgacaatt gggatatgac tgatgccaat gtggtgtgca 2280 agcagctgga ctgtggcgtg gcaattaacg ccactggctc tgcttacttc ggggaaggag 2340 caggagctat ctggctagac gaagtcatct gcactgggaa agagtctcat atttggcagt 2400 gccattcaca tggctgggga cgccataact gcaggcacaa agaagatgca ggtgttatct 2460 gctccgagtt catgtctctg aggctgacca acgaagccca caaagaaaac tgcacaggtc 2520 gccttgaagt gttttacaat ggtacatggg gcagtattgg cagtagcaat atgtctccaa 2580 ccactgtggg ggtggtgtgc cgtcagctgg gctgtgcaga caacgggact gtgaaaccca 2640 taccttcaga caagacacca tccaggccca tgtgggtaga tcgtgtgcag tgtccaaaag 2700 gagttgacac tttgtggcag tgcccctcgt caccttggaa acagagacag gccagcccct 2760 cctcccagga gtcctggatc atctgtgaca acaaaataag actccaggaa gggcatacag 2820 actgttctgg acgtgtggag atctggcaca aaggttcctg gggaacagtg tgtgatgact 2880 cctgggatct taatgatgct aaggttgtat gtaagcagtt gggctgtggc caagctgtga 2940 aggcactaaa agaagcagca tttggtccag gaactgggcc catatggctc aatgaaatta 3000 agtgtagagg gaatgagtct tccctgtggg attgtcctgc caaaccgtgg agtcacagcg 3060 actgtgggca caaagaagat gcttccatcc agtgcctccc caaaatgact tcagaatcac 3120 atcatggcac aggtcacccc accctcacgg cactcttggt ttgtggagcc attctattgg 3180 tcctcctcat tgtcttcctc ctgtggactc tgaagcgacg acagattcag cgacttacag 3240 tttcctcaag aggagaggtc ttgatacatc aagttcagta ccaagagatg gattcaaagg 3300 cggatgatct ggacttgctg aaatcctcgg aaaattccaa caattcatat gattttaatg 3360 atgatggact gacatctttg tctaaatatc ttcctatttc tggaattaaa aaggggtcat 3420 tcagaggcac actgagaagg aaaatgataa tttataatcc actgaggttg gagtttaaga 3480 agccttgaca ggacagccag ctaaatggaa caagagccca ggcaacgcac ggatgaccac 3540 agctgcatct tcatgcagtc ctttgtttcc tggaactctg ctgaacctgc aaaaaccata 3600 tttgtgaatg tgaccactta atagagatgg gagactttt 3639 <210> 26 <211> 3477 <212> DNA <213> Mus musculus <400> 26 atgggtggac acagaatggt tcttcttgga ggtgctggat ctcctggttg taaaaggttt 60 gtccatctag gtttctttgt tgtggctgtg agctcacttc tcagtgcctc tgctgtcact 120 aacgctcctg gagaaatgaa gaaggaactg agactggcgg gtggtgaaaa caactgtagt 180 gggagagtgg aacttaagat ccatgacaag tggggcacag tgtgcagtaa cggctggagc 240 atgaatgaag tgtccgtggt ttgccagcag ctgggatgcc caacttctat taaagccctt 300 ggatgggcta actccagcgc cggctctgga tatatctgga tggacaaagt ttcttgtaca 360 gggaatgagt cagctctttg ggactgcaaa catgatgggt ggggaaagca taactgtacc 420 catgaaaaag atgctggagt gacctgctca gatggatcta atttggagat gagactggtg 480 aacagtgcgg gccaccgatg cttaggaaga gtagaaataa agttccaggg aaagtggggg 540 acggtgtgtg acgacaactt cagcaaagat cacgcttctg tgatttgtaa acagcttgga 600 tgtggaagtg ccattagttt ctctggctca gctaaattgg gagctggttc tggaccaatc 660 tggctcgatg acctggcatg caatggaaat gagtcagctc tctgggactg caaacaccgg 720 ggatggggca agcataactg tgaccatgct gaggatgtcg gtgtgatttg cttagaggga 780 gcagatctga gcctgagact agtggatgga gtgtccagat gttcaggaag attggaagtg 840 agattccaag gagaatgggg gaccgtgtgt gatgataact gggatctccg ggatgcttct 900 gtggtgtgca agcaactggg atgtccaact gccatcagtg ccattggtcg agttaatgcc 960 agtgagggat ctggacagat ttggcttgac aacatttcat gcgaaggaca tgaggcaact 1020 ctttgggagt gtaaacacca agagtgggga aagcattact gtcatcatag agaagacgct 1080 ggcgtgacat gttctgatgg agcagatctg gaacttagac ttgtaggtgg aggcagtcgc 1140 tgtgctggca ttgtggaggt ggagattcag aagctgactg ggaagatgtg tagccgaggc 1200 tggacactgg cagatgcgga tgtggtttgc agacagcttg gatgtggatc tgcgcttcaa 1260 acccaggcta agatctactc taaaactggg gcaacaaata cgtggctctt tcctggatct 1320 tgtaatggaa atgaaactac tttttggcaa tgcaaaaact ggcagtgggg cggcctttcc 1380 tgtgataatt tcgaagaagc caaagttacc tgctcaggcc acagggaacc cagactggtt 1440 ggaggagaaa tcccatgctc tggtcgtgtg gaaatgaaac acggagacgt gtggggctcc 1500 gtctgtgatt ttgacttgtc tctggaagct gccagtgtgg tgtgcaggga attacaatgt 1560 ggaacagtcg tctctatcct agggggagca cattttggag aaggaagtgg acagatctgg 1620 ggtgaagaat tccagtgtag tggggatgag tcccatcttt cactatgctc agtggcgccc 1680 ccgctagaca gaacttgtac ccacagcagg gatgtcagcg tagtctgctc acgatacata 1740 gatattcgtc tggcaggcgg cgagtcctcc tgtgagggaa gagtggagct caagacactc 1800 ggagcctggg gtcccctctg cagttctcat tgggacatgg aagatgctca tgtcttatgt 1860 cagcagctga agtgtggggt tgcccaatct attccagaag gagcacattt tgggaaagga 1920 gctggtcagg tctggagtca catgttccac tgcactggaa ctgaggaaca tataggagat 1980 tgcctcatga ctgctctggg tgcgccgacg tgttccgaag gacaggtggc ctctgtcatc 2040 tgctcaggaa accaatccca gacactattg ccatgtagtt cattgtctcc agtccaaaca 2100 acaagctcta caattccaaa ggagagtgaa gttccctgca tagcaagtgg ccagcttcgc 2160 ttggtaggtg gaggtggtcg ctgcgctgga agagtggagg tctaccacga gggctcttgg 2220 ggcaccgtct gtgatgacaa ttgggatatg actgatgcca atgtggtgtg caagcagctg 2280 gactgtggcg tggcaattaa cgccactggc tctgcttact tcggggaagg agcaggagct 2340 atctggctag acgaagtcat ctgcactggg aaagagtctc atatttggca gtgccattca 2400 catggctggg gacgccataa ctgcaggcac aaagaagatg caggtgttat ctgctccgag 2460 ttcatgtctc tgaggctgac caacgaagcc cacaaagaaa actgcacagg tcgccttgaa 2520 gtgttttaca atggtacatg gggcagtatt ggcagtagca atatgtctcc aaccactgtg 2580 ggggtggtgt gccgtcagct gggctgtgca gacaacggga ctgtgaaacc cataccttca 2640 gacaagacac catccaggcc catgtgggta gatcgtgtgc agtgtccaaa aggagttgac 2700 actttgtggc agtgcccctc gtcaccttgg aaacagagac aggccagccc ctcctcccag 2760 gagtcctgga tcatctgtga caacaaaata agactccagg aagggcatac agactgttct 2820 ggacgtgtgg agatctggca caaaggttcc tggggaacag tgtgtgatga ctcctgggat 2880 cttaatgatg ctaaggttgt atgtaagcag ttgggctgtg gccaagctgt gaaggcacta 2940 aaagaagcag catttggtcc aggaactggg cccatatggc tcaatgaaat taagtgtaga 3000 gggaatgagt cttccctgtg ggattgtcct gccaaaccgt ggagtcacag cgactgtggg 3060 cacaaagaag atgcttccat ccagtgcctc cccaaaatga cttcagaatc acatcatggc 3120 acaggtcacc ccaccctcac ggcactcttg gtttgtggag ccattctatt ggtcctcctc 3180 attgtcttcc tcctgtggac tctgaagcga cgacagattc agcgacttac agtttcctca 3240 agaggagagg tcttgataca tcaagttcag taccaagaga tggattcaaa ggcggatgat 3300 ctggacttgc tgaaatcctc ggaaaattcc aacaattcat atgattttaa tgatgatgga 3360 ctgacatctt tgtctaaata tcttcctatt tctggaatta aaaaggggtc attcagaggc 3420 acactgagaa ggaaaatgat aatttataat ccactgaggt tggagtttaa gaagcct 3477 <210> 27 <211> 1159 <212> PRT <213> Mus musculus <400> 27 Met Gly Gly His Arg Met Val Leu Leu Gly Gly Ala Gly Ser Pro Gly 1 5 10 15 Cys Lys Arg Phe Val His Leu Gly Phe Phe Val Val Ala Val Ser Ser             20 25 30 Leu Leu Ser Ala Ser Ala Val Thr Asn Ala Pro Gly Glu Met Lys Lys         35 40 45 Glu Leu Arg Leu Ala Gly Gly Glu Asn Asn Cys Ser Gly Arg Val Glu     50 55 60 Leu Lys Ile His Asp Lys Trp Gly Thr Val Cys Ser Asn Gly Trp Ser 65 70 75 80 Met Asn Glu Val Ser Val Val Cys Gln Gln Leu Gly Cys Pro Thr Ser                 85 90 95 Ile Lys Ala Leu Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Tyr Ile             100 105 110 Trp Met Asp Lys Val Ser Cys Thr Gly Asn Glu Ser Ala Leu Trp Asp         115 120 125 Cys Lys His Asp Gly Trp Gly Lys His Asn Cys Thr His Glu Lys Asp     130 135 140 Ala Gly Val Thr Cys Ser Asp Gly Ser Asn Leu Glu Met Arg Leu Val 145 150 155 160 Asn Ser Ala Gly His Arg Cys Leu Gly Arg Val Glu Ile Lys Phe Gln                 165 170 175 Gly Lys Trp Gly Thr Val Cys Asp Asp Asn Phe Ser Lys Asp His Ala             180 185 190 Ser Val Ile Cys Lys Gln Leu Gly Cys Gly Ser Ala Ile Ser Phe Ser         195 200 205 Gly Ser Ala Lys Leu Gly Ala Gly Ser Gly Pro Ile Trp Leu Asp Asp     210 215 220 Leu Ala Cys Asn Gly Asn Glu Ser Ala Leu Trp Asp Cys Lys His Arg 225 230 235 240 Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Val Gly Val Ile                 245 250 255 Cys Leu Glu Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val Ser             260 265 270 Arg Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly Thr         275 280 285 Val Cys Asp Asp Asn Trp Asp Leu Arg Asp Ala Ser Val Val Cys Lys     290 295 300 Gln Leu Gly Cys Pro Thr Ala Ile Ser Ala Ile Gly Arg Val Asn Ala 305 310 315 320 Ser Glu Gly Ser Gly Gln Ile Trp Leu Asp Asn Ile Ser Cys Glu Gly                 325 330 335 His Glu Ala Thr Leu Trp Glu Cys Lys His Gln Glu Trp Gly Lys His             340 345 350 Tyr Cys His His Arg Glu Asp Ala Gly Val Thr Cys Ser Asp Gly Ala         355 360 365 Asp Leu Glu Leu Arg Leu Val Gly Gly Gly Ser Arg Cys Ala Gly Ile     370 375 380 Val Glu Val Glu Ile Gln Lys Leu Thr Gly Lys Met Cys Ser Arg Gly 385 390 395 400 Trp Thr Leu Ala Asp Ala Asp Val Val Cys Arg Gln Leu Gly Cys Gly                 405 410 415 Ser Ala Leu Gln Thr Gln Ala Lys Ile Tyr Ser Lys Thr Gly Ala Thr             420 425 430 Asn Thr Trp Leu Phe Pro Gly Ser Cys Asn Gly Asn Glu Thr Thr Phe         435 440 445 Trp Gln Cys Lys Asn Trp Gln Trp Gly Gly Leu Ser Cys Asp Asn Phe     450 455 460 Glu Glu Ala Lys Val Thr Cys Ser Gly His Arg Glu Pro Arg Leu Val 465 470 475 480 Gly Gly Glu Ile Pro Cys Ser Gly Arg Val Glu Met Lys His Gly Asp                 485 490 495 Val Trp Gly Ser Val Cys Asp Phe Asp Leu Ser Leu Glu Ala Ala Ser             500 505 510 Val Val Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu Gly         515 520 525 Gly Ala His Phe Gly Glu Gly Ser Gly Gln Ile Trp Gly Glu Glu Phe     530 535 540 Gln Cys Ser Gly Asp Glu Ser His Leu Ser Leu Cys Ser Val Ala Pro 545 550 555 560 Pro Leu Asp Arg Thr Cys Thr His Ser Arg Asp Val Ser Val Val Cys                 565 570 575 Ser Arg Tyr Ile Asp Ile Arg Leu Ala Gly Gly Glu Ser Ser Cys Glu             580 585 590 Gly Arg Val Glu Leu Lys Thr Leu Gly Ala Trp Gly Pro Leu Cys Ser         595 600 605 Ser His Trp Asp Met Glu Asp Ala His Val Leu Cys Gln Gln Leu Lys     610 615 620 Cys Gly Val Ala Gln Ser Ile Pro Glu Gly Ala His Phe Gly Lys Gly 625 630 635 640 Ala Gly Gln Val Trp Ser His Met Phe His Cys Thr Gly Thr Glu Glu                 645 650 655 His Ile Gly Asp Cys Leu Met Thr Ala Leu Gly Ala Pro Thr Cys Ser             660 665 670 Glu Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln Thr         675 680 685 Leu Leu Pro Cys Ser Ser Leu Ser Pro Val Gln Thr Thr Ser Ser Thr     690 695 700 Ile Pro Lys Glu Ser Glu Val Pro Cys Ile Ala Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Gly Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Val Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Val Cys Asp Asp Asn Trp Asp Met Thr Asp             740 745 750 Ala Asn Val Val Cys Lys Gln Leu Asp Cys Gly Val Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala Tyr Phe Gly Glu Gly Ala Gly Ala Ile Trp Leu Asp     770 775 780 Glu Val Ile Cys Thr Gly Lys Glu Ser His Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Arg His Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Asn Glu Ala His Lys             820 825 830 Glu Asn Cys Thr Gly Arg Leu Glu Val Phe Tyr Asn Gly Thr Trp Gly         835 840 845 Ser Ile Gly Ser Ser Asn Met Ser Pro Thr Thr Val Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Asn Gly Thr Val Lys Pro Ile Pro Ser 865 870 875 880 Asp Lys Thr Pro Ser Arg Pro Met Trp Val Asp Arg Val Gln Cys Pro                 885 890 895 Lys Gly Val Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Lys Gln             900 905 910 Arg Gln Ala Ser Pro Ser Ser Gln Glu Ser Trp Ile Ile Cys Asp Asn         915 920 925 Lys Ile Arg Leu Gln Glu Gly His Thr Asp Cys Ser Gly Arg Val Glu     930 935 940 Ile Trp His Lys Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp 945 950 955 960 Leu Asn Asp Ala Lys Val Val Cys Lys Gln Leu Gly Cys Gly Gln Ala                 965 970 975 Val Lys Ala Leu Lys Glu Ala Ala Phe Gly Pro Gly Thr Gly Pro Ile             980 985 990 Trp Leu Asn Glu Ile Lys Cys Arg Gly Asn Glu Ser Ser Leu Trp Asp         995 1000 1005 Cys Pro Ala Lys Pro Trp Ser His Ser Asp Cys Gly His Lys Glu     1010 1015 1020 Asp Ala Ser Ile Gln Cys Leu Pro Lys Met Thr Ser Glu Ser His     1025 1030 1035 His Gly Thr Gly His Pro Thr Leu Thr Ala Leu Leu Val Cys Gly     1040 1045 1050 Ala Ile Leu Leu Val Leu Leu Ile Val Phe Leu Leu Trp Thr Leu     1055 1060 1065 Lys Arg Arg Gln Ile Gln Arg Leu Thr Val Ser Ser Arg Gly Glu     1070 1075 1080 Val Leu Ile His Gln Val Gln Tyr Gln Glu Met Asp Ser Lys Ala     1085 1090 1095 Asp Asp Leu Asp Leu Leu Lys Ser Ser Glu Asn Ser Asn Asn Ser     1100 1105 1110 Tyr Asp Phe Asn Asp Asp Gly Leu Thr Ser Leu Ser Lys Tyr Leu     1115 1120 1125 Pro Ile Ser Gly Ile Lys Lys Gly Ser Phe Arg Gly Thr Leu Arg     1130 1135 1140 Arg Lys Met Ile Ile Tyr Asn Pro Leu Arg Leu Glu Phe Lys Lys     1145 1150 1155 Pro      <210> 28 <211> 26 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 28 caccggaatg agcaaactca gaatgg 26 <210> 29 <211> 40 <212> DNA <213> Artificial <220> <223> PCR Primer <400> 29 tgctccggta cctagtccag gtcttcatca aggtatctta 40 <210> 30 <211> 3414 <212> DNA <213> African green monkey <400> 30 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60 tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc 120 accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180 agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgact tgctcagatg gatccgattt ggaaatgagg 480 ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540 ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac 960 gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020 gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtaa catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500 tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800 cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860 tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920 ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040 atttgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcatc tctgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca 2400 catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520 gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580 ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaaccc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760 acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880 aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060 aaggaagacg ctgcagtgaa ttgcacagat atttcaacga acaaaacccc acaaaaagcc 3120 acaacaggtc agtcatccct tattgcagtc ggaatccttg gagttgttct cttggtcatt 3180 ttcgtcgcat tattcttgac tcaaaagcga agacagagac agcggcttac agtttcctca 3240 agaggagaga acttagtcca ccaaattcaa taccgggaga tgaattcttg cctgaatgca 3300 gatgatctgg acctaatgaa ttcctcagga ggccattctg aggcacactg aaaaggaaaa 3360 tgggaattta taacccagtg agccttgaag ataccttgat gaagacctgg acta 3414 <210> 31 <211> 3348 <212> DNA <213> African green monkey <400> 31 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60 tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc 120 accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180 agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgact tgctcagatg gatccgattt ggaaatgagg 480 ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540 ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac 960 gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020 gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtaa catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500 tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800 cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860 tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920 ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040 atttgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcatc tctgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca 2400 catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520 gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580 ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaaccc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760 acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880 aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060 aaggaagacg ctgcagtgaa ttgcacagat atttcaacga acaaaacccc acaaaaagcc 3120 acaacaggtc agtcatccct tattgcagtc ggaatccttg gagttgttct cttggtcatt 3180 ttcgtcgcat tattcttgac tcaaaagcga agacagagac agcggcttac agtttcctca 3240 agaggagaga acttagtcca ccaaattcaa taccgggaga tgaattcttg cctgaatgca 3300 gatgatctgg acctaatgaa ttcctcagga ggccattctg aggcacac 3348 <210> 32 <211> 1116 <212> PRT <213> African green monkey <400> 32 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Val Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Ala Val Val             20 25 30 Leu Leu Leu Arg Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Thr         35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Ile Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Thr Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln     130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Met Arg 145 150 155 160 Leu Thr Asn Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe                 165 170 175 Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asn His             180 185 190 Ala Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe         195 200 205 Ser Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp     210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val                 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val             260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly         275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser His Asp Ala Ala Val Ala Cys     290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Ile Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Glu Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln                 325 330 335 Gly His Glu Pro Ala Val Trp Gln Cys Lys His His Glu Trp Gly Lys             340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly         355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly     370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys                 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala             420 425 430 Thr Asn Met Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser         435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His     450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly                 485 490 495 Asp Thr Trp Gly Ser Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala             500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu         515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu     530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val                 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys             580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Asn Ala Trp Gly Ser Leu Cys         595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu     610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala His Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Val Trp Arg His Met Phe His Cys Thr Gly Thr Glu                 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys             660 665 670 Pro Ser Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln         675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr     690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp             740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp     770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg             820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly         835 840 845 Ser Val Gly Arg Ser Asn Met Ser Glu Thr Thr Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Pro Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro                 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys             900 905 910 Arg Leu Ala Arg Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys         915 920 925 Met Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile     930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asn Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu                 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp             980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys         995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp     1010 1015 1020 Ala Ala Val Asn Cys Thr Asp Ile Ser Thr Asn Lys Thr Pro Gln     1025 1030 1035 Lys Ala Thr Thr Gly Gln Ser Ser Leu Ile Ala Val Gly Ile Leu     1040 1045 1050 Gly Val Val Leu Leu Val Ile Phe Val Ala Leu Phe Leu Thr Gln     1055 1060 1065 Lys Arg Arg Gln Arg Gln Arg Leu Thr Val Ser Ser Arg Gly Glu     1070 1075 1080 Asn Leu Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu     1085 1090 1095 Asn Ala Asp Asp Leu Asp Leu Met Asn Ser Ser Gly Gly His Ser     1100 1105 1110 Glu ala his     1115 <210> 33 <211> 3348 <212> DNA <213> African green monkey <400> 33 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60 tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc 120 accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180 agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtaact tgctcagatg gatccgattt ggaaatgagg 480 ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540 ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac 960 gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020 gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtaa catgttctga tggatcaggt ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500 tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tggactgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800 cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860 tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920 ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040 atttgctcag gaaaccagtc ccaaacactg tcctcgcgca attcatcatc tctgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca 2400 catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520 gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580 ggtgtagtgt gcaggcagct gggctgtgca gacaaaggga aaatcaactc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760 acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880 aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcgaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060 aaggaagacg ctgcagtgaa ttgcacagat atttcaacgc gcaaaacccc acaaaaagcc 3120 acaacaggtc agtcatccct tattgcagtc ggaatccttg gagttgttct cttggccatt 3180 ttcgtcgcat tattcttgac tcaaaagcga agacagagac agcggcttac agtttcctca 3240 agaggagaga acttagtcca ccaaattcaa taccgggaga tgaattcttg cctgaatgca 3300 gatgatctgg acctaatgaa ttcctcagga ggccattctg aggcacac 3348 <210> 34 <211> 1116 <212> PRT <213> African Green Monkey <400> 34 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Val Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Ala Val Val             20 25 30 Leu Leu Leu Arg Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Thr         35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Ile Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Thr Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln     130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Met Arg 145 150 155 160 Leu Thr Asn Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe                 165 170 175 Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asn His             180 185 190 Ala Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe         195 200 205 Ser Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp     210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val                 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val             260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly         275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser His Asp Ala Ala Val Ala Cys     290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Ile Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Glu Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln                 325 330 335 Gly His Glu Pro Ala Val Trp Gln Cys Lys His His Glu Trp Gly Lys             340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly         355 360 365 Ser Gly Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly     370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys                 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala             420 425 430 Thr Asn Met Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser         435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His     450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly                 485 490 495 Asp Thr Trp Gly Ser Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala             500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu         515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Thr Glu Glu     530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val                 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys             580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Asn Ala Trp Gly Ser Leu Cys         595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu     610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala His Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Val Trp Arg His Met Phe His Cys Thr Gly Thr Glu                 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys             660 665 670 Pro Ser Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln         675 680 685 Thr Leu Ser Ser Arg Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr     690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp             740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp     770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg             820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly         835 840 845 Ser Val Gly Arg Ser Asn Met Ser Glu Thr Thr Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Ser Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro                 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys             900 905 910 Arg Leu Ala Arg Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys         915 920 925 Met Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile     930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asn Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu                 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp             980 985 990 Leu Asn Glu Val Lys Cys Glu Gly Asn Glu Ser Ser Leu Trp Asp Cys         995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp     1010 1015 1020 Ala Ala Val Asn Cys Thr Asp Ile Ser Thr Arg Lys Thr Pro Gln     1025 1030 1035 Lys Ala Thr Thr Gly Gln Ser Ser Leu Ile Ala Val Gly Ile Leu     1040 1045 1050 Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu Phe Leu Thr Gln     1055 1060 1065 Lys Arg Arg Gln Arg Gln Arg Leu Thr Val Ser Ser Arg Gly Glu     1070 1075 1080 Asn Leu Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu     1085 1090 1095 Asn Ala Asp Asp Leu Asp Leu Met Asn Ser Ser Gly Gly His Ser     1100 1105 1110 Glu ala his     1115 <210> 35 <211> 3453 <212> DNA <213> African Green Monkey <400> 35 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60 tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc 120 accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180 agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtaact tgctcagatg gatccgattt ggaaatgagg 480 ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540 ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac 960 gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020 gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtaa catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500 tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tggactgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800 cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860 tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920 ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040 atttgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcatc tctgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca 2400 catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520 gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580 ggtgtagtgt gcaggcagct gggctgtgca gacaaaggga aaatcaactc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760 acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880 aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060 aaggaagacg ctgcagtgaa ttgcacagat atttcaacgc gcaaaacccc acaaaaagcc 3120 acaacaggtc agtcatccct tattgcagtc ggaatccttg gagttgttct cttggccatt 3180 ttcgtcgcat tattcttgac tcaaaagcga agacagagac agcggcttac agtttcctca 3240 agaggagaga acttagtcca ccaaattcaa taccgggaga tgaattcttg cctgaatgca 3300 gatgatctgg acctaatgaa ttcctcagaa aattccaatg agtcagctga tttcaatgct 3360 gctgaactaa tttctgtgtc taaatttctt cctatttctg gaatggaaaa ggaggccatt 3420 ctgaggcaca ctgaaaagga aaatgggaat tta 3453 <210> 36 <211> 1151 <212> PRT <213> African Green Monkey <400> 36 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Val Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Ala Val Val             20 25 30 Leu Leu Leu Arg Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Thr         35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Ile Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Thr Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln     130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Met Arg 145 150 155 160 Leu Thr Asn Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe                 165 170 175 Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asn His             180 185 190 Ala Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe         195 200 205 Ser Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp     210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val                 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val             260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly         275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser His Asp Ala Ala Val Ala Cys     290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Ile Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Glu Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln                 325 330 335 Gly His Glu Pro Ala Val Trp Gln Cys Lys His His Glu Trp Gly Lys             340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly         355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly     370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys                 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala             420 425 430 Thr Asn Met Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser         435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His     450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly                 485 490 495 Asp Thr Trp Gly Ser Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala             500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu         515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Thr Glu Glu     530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val                 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys             580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Asn Ala Trp Gly Ser Leu Cys         595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu     610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala His Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Val Trp Arg His Met Phe His Cys Thr Gly Thr Glu                 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys             660 665 670 Pro Ser Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln         675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr     690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp             740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp     770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg             820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly         835 840 845 Ser Val Gly Arg Ser Asn Met Ser Glu Thr Thr Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Ser Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro                 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys             900 905 910 Arg Leu Ala Arg Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys         915 920 925 Met Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile     930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asn Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu                 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp             980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys         995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp     1010 1015 1020 Ala Ala Val Asn Cys Thr Asp Ile Ser Thr Arg Lys Thr Pro Gln     1025 1030 1035 Lys Ala Thr Thr Gly Gln Ser Ser Leu Ile Ala Val Gly Ile Leu     1040 1045 1050 Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu Phe Leu Thr Gln     1055 1060 1065 Lys Arg Arg Gln Arg Gln Arg Leu Thr Val Ser Ser Arg Gly Glu     1070 1075 1080 Asn Leu Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu     1085 1090 1095 Asn Ala Asp Asp Leu Asp Leu Met Asn Ser Ser Glu Asn Ser Asn     1100 1105 1110 Glu Ser Ala Asp Phe Asn Ala Ala Glu Leu Ile Ser Val Ser Lys     1115 1120 1125 Phe Leu Pro Ile Ser Gly Met Glu Lys Glu Ala Ile Leu Arg His     1130 1135 1140 Thr Glu Lys Glu Asn Gly Asn Leu     1145 1150 <210> 37 <211> 3273 <212> DNA <213> African Green Monkey <400> 37 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60 tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc 120 accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180 agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtaact tgctcagatg gatccgattt ggaaatgagg 480 ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540 ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac 960 gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020 gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtaa catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500 tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tggactgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800 cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860 tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920 ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040 atttgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcatc tctgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca 2400 catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520 gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580 ggtgtagtgt gcaggcagct gggctgtgca gacaaaggga aaatcaactc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760 acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880 aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060 aaggaagacg ctgcggtgaa ttgcacagat atttcaacgc gcaaaacccc acaaaaagcc 3120 acaacggttt cctcaagagg agagaactta gtccaccaaa ttcaataccg ggagatgaat 3180 tcttgcctga atgcagatga tctgaaccta atgaattcct caggaggcca ttctgaggca 3240 cactgaaaag gaaaatggga atttataacc cag 3273 <210> 38 <211> 1081 <212> PRT <213> African Green Monkey <400> 38 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Val Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Ala Val Val             20 25 30 Leu Leu Leu Arg Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Thr         35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Ile Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Thr Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln     130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Met Arg 145 150 155 160 Leu Thr Asn Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe                 165 170 175 Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asn His             180 185 190 Ala Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe         195 200 205 Ser Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp     210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val                 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val             260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly         275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser His Asp Ala Ala Val Ala Cys     290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Ile Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Glu Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln                 325 330 335 Gly His Glu Pro Ala Val Trp Gln Cys Lys His His Glu Trp Gly Lys             340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly         355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly     370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys                 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala             420 425 430 Thr Asn Met Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser         435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His     450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly                 485 490 495 Asp Thr Trp Gly Ser Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala             500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu         515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Thr Glu Glu     530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val                 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys             580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Asn Ala Trp Gly Ser Leu Cys         595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu     610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala His Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Val Trp Arg His Met Phe His Cys Thr Gly Thr Glu                 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys             660 665 670 Pro Ser Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln         675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr     690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp             740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp     770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg             820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly         835 840 845 Ser Val Gly Arg Ser Asn Met Ser Glu Thr Thr Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Ser Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro                 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys             900 905 910 Arg Leu Ala Arg Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys         915 920 925 Met Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile     930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asn Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu                 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp             980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys         995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp     1010 1015 1020 Ala Ala Val Asn Cys Thr Asp Ile Ser Thr Arg Lys Thr Pro Gln     1025 1030 1035 Lys Ala Thr Thr Val Ser Ser Arg Gly Glu Asn Leu Val His Gln     1040 1045 1050 Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu Asn Ala Asp Asp Leu     1055 1060 1065 Asn Leu Met Asn Ser Ser Gly Gly His Ser Glu Ala His     1070 1075 1080 <210> 39 <211> 3279 <212> DNA <213> African Green Monkey <400> 39 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60 tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc 120 accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180 agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgact tgctcagatg gatccgattt ggaaatgagg 480 ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540 ggaacagtgt gtgatgataa cttcaacgtc aatcatgcat ctgtggtttg taaacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac 960 gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020 gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtaa catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500 tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800 cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860 tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920 ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040 atttgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcatc tctgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gtatttggca gtgccattca 2400 catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520 gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580 ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaactc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760 acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880 aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060 aaggaagacg ctgcagtgaa ttgcacagca caaaaaattt caacgcacaa aaccccacaa 3120 aaagccacaa cagtttcctc aagaggagag aacttagtcc accaaattca ataccgggag 3180 atgaattctt gcctgaatgc agatgatctg gacctaatga attcctcagg aggccattct 3240 gaggcacact gaaaaggaaa atgggaattt ataacccag 3279 <210> 40 <211> 1083 <212> PRT <213> African Green Monkey <400> 40 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Val Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Ala Val Val             20 25 30 Leu Leu Leu Arg Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Thr         35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Ile Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Thr Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln     130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Met Arg 145 150 155 160 Leu Thr Asn Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe                 165 170 175 Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Val Asn His             180 185 190 Ala Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe         195 200 205 Ser Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp     210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val                 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val             260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly         275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser His Asp Ala Ala Val Ala Cys     290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Ile Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Glu Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln                 325 330 335 Gly His Glu Pro Ala Val Trp Gln Cys Lys His His Glu Trp Gly Lys             340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly         355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly     370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys                 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala             420 425 430 Thr Asn Met Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser         435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His     450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly                 485 490 495 Asp Thr Trp Gly Ser Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala             500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu         515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu     530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val                 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys             580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Asn Ala Trp Gly Ser Leu Cys         595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu     610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala His Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Val Trp Arg His Met Phe His Cys Thr Gly Thr Glu                 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys             660 665 670 Pro Ser Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln         675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr     690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp             740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp     770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg             820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly         835 840 845 Ser Val Gly Arg Ser Asn Met Ser Glu Thr Thr Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Ser Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro                 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys             900 905 910 Arg Leu Ala Arg Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys         915 920 925 Met Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile     930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asn Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu                 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp             980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys         995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp     1010 1015 1020 Ala Ala Val Asn Cys Thr Ala Gln Lys Ile Ser Thr His Lys Thr     1025 1030 1035 Pro Gln Lys Ala Thr Thr Val Ser Ser Arg Gly Glu Asn Leu Val     1040 1045 1050 His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu Asn Ala Asp     1055 1060 1065 Asp Leu Asp Leu Met Asn Ser Ser Gly Gly His Ser Glu Ala His     1070 1075 1080 <210> 41 <211> 3354 <212> DNA <213> African Green Monkey <400> 41 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60 tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc 120 accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180 agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgact tgctcagatg gatccgattt ggaaatgagg 480 ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540 ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac 960 gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020 gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtaa catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500 tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800 cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860 tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920 ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040 atttgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcatc tctgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gtatttggca gtgccattca 2400 catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520 gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580 ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaactc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760 acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880 aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060 aaggaagacg ctgcagtgaa ttgcacagca caaaaaattt caacgcacaa aaccccacaa 3120 aaagccacaa caggtcggtc attccttatt gcattcggaa tccttggagt tgttctcttg 3180 gccattttcg tcgcattatt cttgactcaa aagcgaagac agagacagcg gcttacagtt 3240 tcctcaagag gagagaactt agtccaccaa attcaatacc gggagatgaa ttcttgcctg 3300 aatgcagatg atctggacct aatgaattcc tcaggaggcc attctgaggc acac 3354 <210> 42 <211> 1118 <212> PRT <213> African Green Monkey <400> 42 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Val Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Ala Val Val             20 25 30 Leu Leu Leu Arg Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Thr         35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Ile Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Thr Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln     130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Met Arg 145 150 155 160 Leu Thr Asn Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe                 165 170 175 Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asn His             180 185 190 Ala Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe         195 200 205 Ser Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp     210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val                 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val             260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly         275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser His Asp Ala Ala Val Ala Cys     290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Ile Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Glu Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln                 325 330 335 Gly His Glu Pro Ala Val Trp Gln Cys Lys His His Glu Trp Gly Lys             340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly         355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly     370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys                 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala             420 425 430 Thr Asn Met Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser         435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His     450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly                 485 490 495 Asp Thr Trp Gly Ser Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala             500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu         515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu     530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val                 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys             580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Asn Ala Trp Gly Ser Leu Cys         595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu     610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala His Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Val Trp Arg His Met Phe His Cys Thr Gly Thr Glu                 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys             660 665 670 Pro Ser Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln         675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr     690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp             740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp     770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg             820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly         835 840 845 Ser Val Gly Arg Ser Asn Met Ser Glu Thr Thr Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Ser Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro                 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys             900 905 910 Arg Leu Ala Arg Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys         915 920 925 Met Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile     930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asn Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu                 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp             980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys         995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp     1010 1015 1020 Ala Ala Val Asn Cys Thr Ala Gln Lys Ile Ser Thr His Lys Thr     1025 1030 1035 Pro Gln Lys Ala Thr Thr Gly Arg Ser Phe Leu Ile Ala Phe Gly     1040 1045 1050 Ile Leu Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu Phe Leu     1055 1060 1065 Thr Gln Lys Arg Arg Gln Arg Gln Arg Leu Thr Val Ser Ser Arg     1070 1075 1080 Gly Glu Asn Leu Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser     1085 1090 1095 Cys Leu Asn Ala Asp Asp Leu Asp Leu Met Asn Ser Ser Gly Gly     1100 1105 1110 His Ser Glu Ala His     1115 <210> 43 <211> 3459 <212> DNA <213> African Green Monkey <400> 43 atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60 tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc 120 accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180 agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc 300 actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt 360 cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgact tgctcagatg gatccgattt ggaaatgagg 480 ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540 ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt 600 gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac 960 gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020 gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtaa catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260 aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440 gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500 tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560 tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620 tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800 cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860 tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920 ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040 atttgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcatc tctgggccca 2100 acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160 ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220 ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280 ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gtatttggca gtgccattca 2400 catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460 ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520 gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580 ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaactc tgcatcttta 2640 gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760 acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880 aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060 aaggaagacg ctgcagtgaa ttgcacagca caaaaaattt caacgcacaa aaccccacaa 3120 aaagccacaa caggtcagtc attccttatt gcattcggaa tccttggagt tgttctcttg 3180 gccattttcg tcgcattatt cttgactcaa aagcgaagac agagacagcg gcttacagtt 3240 tcctcaagag gagagaactt agtccaccaa attcaatacc gggagatgaa ttcttgcctg 3300 aatgcagatg atctggacct aatgaattcc tcagaaaatt ccaatgagtc agctgatttc 3360 aatgctgctg aactaatttc tgtgtctaaa tttcttccta tttctggaat ggaaaaggag 3420 gccattctga ggcacactga aaaggaaaat gggaattta 3459 <210> 44 <211> 1153 <212> PRT <213> African Green Monkey <400> 44 Met Ser Lys Leu Arg Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp 1 5 10 15 Val Arg Arg His Phe Val Asn Leu Ser Pro Phe Thr Ile Ala Val Val             20 25 30 Leu Leu Leu Arg Ala Cys Phe Val Thr Ser Ser Leu Gly Gly Thr Thr         35 40 45 Lys Glu Leu Arg Leu Val Asp Gly Glu Asn Lys Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Ile Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Ser Met Glu Ala Val Ser Val Ile Cys Asn Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Thr Gly Trp Ala Asn Ser Ser Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Ser Asn Cys Thr His Gln     130 135 140 Gln Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Met Arg 145 150 155 160 Leu Thr Asn Gly Gly Asn Met Cys Ser Gly Arg Ile Glu Ile Lys Phe                 165 170 175 Gln Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asn His             180 185 190 Ala Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe         195 200 205 Ser Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp     210 215 220 Asp Leu Ile Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His 225 230 235 240 Gln Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val                 245 250 255 Ile Cys Ser Lys Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val             260 265 270 Thr Glu Cys Ser Gly Arg Leu Glu Val Arg Phe Gln Gly Glu Trp Gly         275 280 285 Thr Ile Cys Asp Asp Gly Trp Asp Ser His Asp Ala Ala Val Ala Cys     290 295 300 Lys Gln Leu Gly Cys Pro Thr Ala Ile Thr Ala Ile Gly Arg Val Asn 305 310 315 320 Ala Ser Glu Gly Phe Gly His Ile Trp Leu Asp Ser Val Ser Cys Gln                 325 330 335 Gly His Glu Pro Ala Val Trp Gln Cys Lys His His Glu Trp Gly Lys             340 345 350 His Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly         355 360 365 Ser Asp Leu Glu Leu Arg Leu Arg Gly Gly Gly Ser Arg Cys Ala Gly     370 375 380 Thr Val Glu Val Glu Ile Gln Arg Leu Leu Gly Lys Val Cys Asp Arg 385 390 395 400 Gly Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys                 405 410 415 Gly Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Ile Gln Ala             420 425 430 Thr Asn Met Trp Leu Phe Leu Ser Ser Cys Asn Gly Asn Glu Thr Ser         435 440 445 Leu Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Thr Cys Asp His     450 455 460 Tyr Glu Glu Ala Lys Ile Thr Cys Ser Ala His Arg Glu Pro Arg Leu 465 470 475 480 Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly                 485 490 495 Asp Thr Trp Gly Ser Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala             500 505 510 Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Ile Leu         515 520 525 Gly Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu     530 535 540 Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala 545 550 555 560 Pro Arg Pro Glu Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val                 565 570 575 Cys Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Lys Thr Pro Cys             580 585 590 Glu Gly Arg Val Glu Leu Lys Thr Leu Asn Ala Trp Gly Ser Leu Cys         595 600 605 Asn Ser His Trp Asp Ile Glu Asp Ala His Val Leu Cys Gln Gln Leu     610 615 620 Lys Cys Gly Val Ala Leu Ser Thr Pro Gly Gly Ala His Phe Gly Lys 625 630 635 640 Gly Asn Gly Gln Val Trp Arg His Met Phe His Cys Thr Gly Thr Glu                 645 650 655 Gln His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Ser Leu Cys             660 665 670 Pro Ser Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln         675 680 685 Thr Leu Ser Ser Cys Asn Ser Ser Ser Leu Gly Pro Thr Arg Pro Thr     690 695 700 Ile Pro Glu Glu Ser Ala Val Ala Cys Ile Glu Ser Gly Gln Leu Arg 705 710 715 720 Leu Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Ile Tyr His                 725 730 735 Glu Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp             740 745 750 Ala His Val Val Cys Arg Gln Leu Gly Cys Gly Glu Ala Ile Asn Ala         755 760 765 Thr Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp     770 775 780 Glu Met Lys Cys Asn Gly Lys Glu Ser Arg Ile Trp Gln Cys His Ser 785 790 795 800 His Gly Trp Gly Gln Gln Asn Cys Arg His Lys Glu Asp Ala Gly Val                 805 810 815 Ile Cys Ser Glu Phe Met Ser Leu Arg Leu Thr Ser Glu Ala Ser Arg             820 825 830 Glu Ala Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly         835 840 845 Ser Val Gly Arg Ser Asn Met Ser Glu Thr Thr Val Gly Val Val Cys     850 855 860 Arg Gln Leu Gly Cys Ala Asp Lys Gly Lys Ile Asn Ser Ala Ser Leu 865 870 875 880 Asp Lys Ala Met Ser Ile Pro Met Trp Val Asp Asn Val Gln Cys Pro                 885 890 895 Lys Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Glu Lys             900 905 910 Arg Leu Ala Arg Pro Ser Glu Glu Thr Trp Ile Thr Cys Asp Asn Lys         915 920 925 Met Arg Leu Gln Glu Gly Pro Thr Ser Cys Ser Gly Arg Val Glu Ile     930 935 940 Trp His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu 945 950 955 960 Asn Asp Ala Gln Val Val Cys Gln Gln Leu Gly Cys Gly Pro Ala Leu                 965 970 975 Lys Ala Phe Lys Glu Ala Glu Phe Gly Gln Gly Thr Gly Pro Ile Trp             980 985 990 Leu Asn Glu Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys         995 1000 1005 Pro Ala Arg Arg Trp Gly His Ser Glu Cys Gly His Lys Glu Asp     1010 1015 1020 Ala Ala Val Asn Cys Thr Ala Gln Lys Ile Ser Thr His Lys Thr     1025 1030 1035 Pro Gln Lys Ala Thr Thr Gly Gln Ser Phe Leu Ile Ala Phe Gly     1040 1045 1050 Ile Leu Gly Val Val Leu Leu Ala Ile Phe Val Ala Leu Phe Leu     1055 1060 1065 Thr Gln Lys Arg Arg Gln Arg Gln Arg Leu Thr Val Ser Ser Arg     1070 1075 1080 Gly Glu Asn Leu Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser     1085 1090 1095 Cys Leu Asn Ala Asp Asp Leu Asp Leu Met Asn Ser Ser Glu Asn     1100 1105 1110 Ser Asn Glu Ser Ala Asp Phe Asn Ala Ala Glu Leu Ile Ser Val     1115 1120 1125 Ser Lys Phe Leu Pro Ile Ser Gly Met Glu Lys Glu Ala Ile Leu     1130 1135 1140 Arg His Thr Glu Lys Glu Asn Gly Asn Leu     1145 1150 <210> 45 <211> 3345 <212> DNA (213) Canis familiaris <400> 45 atgagcaaac tcagaatggt cccacatgga aactctggat ctgctgactt tagaagatgt 60 tttgccctct tgtgtccctc tgctgtggct gtggtctcca ttctcagtac ctgtttgatg 120 accaattctc ttgggagagc agataaagag atgaggctaa cggatggtga agacaattgc 180 tccgggagag tggaagtgaa agtccaggag gagtggggaa cggtgtgtaa taatggctgg 240 ggcatggatg aagtctctgt gatttgcagg cagctgggat gtcccactgc tatcaaagcc 300 gctggatggg ccaattccag ggcaggctct ggacgaatct ggatggatca tgtttcttgt 360 cgagggaatg aatctgctct ctgggactgc aaacatgatg gatggggaaa gcacaactgc 420 agtcatcaac aggatgctgg agtaacctgt tcagatggat ccagtttgga gatgaggttg 480 atgaacggcg gaaaccagtg ttctggcaga atagaagtca agttccaggg acagtgggga 540 acagtgtgtg atgacaactt caacatagat catgcttctg tggtttgtaa acagctcgaa 600 tgtggaagtg ctgtcagttt ctctggttca gctaattttg gagaaggttc tgggccaatc 660 tggtttgatg atcttgtgtg cagtggaaat gagtcagctc tctggaactg caagcatgaa 720 ggatggggaa agcataactg tgatcacgct gaggatgttg gagtgatttg cttggatgga 780 gcagatctga gcctgagact ggtagatgga gtcactgaat gttcaggaag attagaagta 840 aaattccaag gggaatgggg gacagtgtgt gatgatggct gggatagtaa tgatgctgct 900 gtggtatgta aacaactggg atgcccaact gctgtcaccg ccattggtcg agttaacgcc 960 agtgagggaa gtggacacat ttggcttgac aatctttcct gccaaggaga cgaatctgct 1020 ctctggcagt gtagacacca tgaatgggga aagcattatt gcaatcataa tgaagatgct 1080 ggtgtgacat gttctgatgg atcagacctg gagctgagac ttgtcggtgg aggcagccgc 1140 tgtgctggga cagtggaggt tgaaattcag aaactgctag ggaaagtatg tgatagaggc 1200 tggggactga aagaagccga tgtggtttgc aagcagttgg gatgtggatc tgctctcaaa 1260 acgtcctatc agcgttattc caaagttaag gcaacaaaca catggctgtt tttaagccgc 1320 tgtagtggca atgaaacttc cctttgggac tgcaagaact ggcagtgggg tggactgagc 1380 tgtgatcact atgaagaagc taaagttacc tgctcagccc acagggaacc cagactagtt 1440 ggaggagata ttccctgctc tggtcgtgtt gaagtgaaac atggtgacac atggggcacc 1500 gtctgtgatt ccgacttctc tttggaagct gccagtgtgc tgtgcagaga gttacagtgt 1560 ggcacagtca tctccatcct agggggagct cactttggag aaggaaatgg acagatctgg 1620 gctgaagaat tccagtgtga ggggcaggag tcccatcttt cactctgttc agtagcctct 1680 cgcccagatg ggacctgtag ccacagcagg gatgttggag tcgtctgctc aagatacacg 1740 gaaatccgct tggtgaatgg ccagtccccg tgtgaaggaa gagtggagct caagatactt 1800 gggaactggg gatccctctg caactctcac tgggacatag aagatgccca tgttttctgt 1860 cagcagctca aatgtggagt tgccctttct attccgggag gagcacattt tgggaaagga 1920 agtggtcaga tctggaggca catgtttcac tgcactggga ctgagcagca catgggagat 1980 tgccctgtaa ctgctctggg cgcgacgctg tgttctgctg ggcaagtggc ctctgtaatc 2040 tgctcaggaa atcagagcca gacgctatcc ccatgcaatt caacatctct ggacccaaca 2100 agatctacca cttcggaaga aagtgctgtt gcttgtattg cgagtgggca acttcgcctg 2160 gtaaatggag gcggtcgctg tgctgggaga atagaggtct accatgaggg ctcctggggc 2220 accatctgtg atgacagctg ggacctgagt gatgcccatg tggtgtgcag acagctgggc 2280 tgtggagtgg ccattaatgc cactggctct gctcattttg gggaaggaac agggcccatc 2340 tggctggacg aggtgaactg taatggaaag gaatctcata tctggcaatg ccgctcacac 2400 ggctgggggc aacacaactg cagacataag gaggatgcag gagttatctg ctcagagttc 2460 atgtctctca gactgattga tgaaaccagc agagacatct gtgcagggcg tcttgaagtt 2520 ttttacaatg gagcttgggg cagcgttggc aagagtaata tgtctgcaac cactgtggag 2580 gtggtatgca ggcaactggg ttgtgcagac aaggggagca tcaaccctgc atcttcagac 2640 aagcccatgt ccaggcacat gtgggtggac aatgtccagt gtccaaaagg acctgacacc 2700 ttatggcagt gcccatcttc tccatggaaa cagagagtgg ccagttcttc agaggagacc 2760 tggatcacat gtgccaacaa gataagactt caagaaggaa cctctaattg ttctggacgt 2820 gtggagctct ggcacggagg ttcctggggg acagtgtgcg atgactcctg ggaccttgaa 2880 gatgcacaag tggtgtgtcg acagctgggc tgtggcccag cattagaagc actaaaagag 2940 gcagcatttg gtcaggggac tgggcctata tggctcaatg acgtgaagtg caaagggaat 3000 gagtcttcct tgtgggattg tcctgctaga ccctgggggc acagtgactg tggccacaag 3060 gaagatgctg ctgtgaggtg ctcagaaatt gcaatggccc aaagatcatc aaatcctaga 3120 ggtcactcat cccttgttgc attggggatc tttggtgtca ttcttctggc ctttctcatc 3180 gctctcctct tgtggactca aaggcgaaga cagcaacagc ggcttacagt ttccttgaga 3240 ggagagaatt ctgtccacca aattcaatac cgggaaatga attcttccct gaaagcagat 3300 gatctggacg tgctgacttc ctcagaagac cattttgagg tacac 3345 <210> 46 <211> 1115 <212> PRT (213) Canis familiaris <400> 46 Met Ser Lys Leu Arg Met Val Pro His Gly Asn Ser Gly Ser Ala Asp 1 5 10 15 Phe Arg Arg Cys Phe Ala Leu Leu Cys Pro Ser Ala Val Ala Val Val             20 25 30 Ser Ile Leu Ser Thr Cys Leu Met Thr Asn Ser Leu Gly Arg Ala Asp         35 40 45 Lys Glu Met Arg Leu Thr Asp Gly Glu Asp Asn Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Gly Met Asp Glu Val Ser Val Ile Cys Arg Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Ala Gly Trp Ala Asn Ser Arg Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Asn Cys Ser His Gln Gln     130 135 140 Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Ser Leu Glu Met Arg Leu 145 150 155 160 Met Asn Gly Gly Asn Gln Cys Ser Gly Arg Ile Glu Val Lys Phe Gln                 165 170 175 Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asp His Ala             180 185 190 Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe Ser         195 200 205 Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp Asp     210 215 220 Leu Val Cys Ser Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His Glu 225 230 235 240 Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Val Gly Val Ile                 245 250 255 Cys Leu Asp Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val Thr             260 265 270 Glu Cys Ser Gly Arg Leu Glu Val Lys Phe Gln Gly Glu Trp Gly Thr         275 280 285 Val Cys Asp Asp Gly Trp Asp Ser Asn Asp Ala Ala Val Val Cys Lys     290 295 300 Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn Ala 305 310 315 320 Ser Glu Gly Ser Gly His Ile Trp Leu Asp Asn Leu Ser Cys Gln Gly                 325 330 335 Asp Glu Ser Ala Leu Trp Gln Cys Arg His His Glu Trp Gly Lys His             340 345 350 Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly Ser         355 360 365 Asp Leu Glu Leu Arg Leu Val Gly Gly Gly Ser Arg Cys Ala Gly Thr     370 375 380 Val Glu Val Glu Ile Gln Lys Leu Leu Gly Lys Val Cys Asp Arg Gly 385 390 395 400 Trp Gly Leu Lys Glu Ala Asp Val Val Cys Lys Gln Leu Gly Cys Gly                 405 410 415 Ser Ala Leu Lys Thr Ser Tyr Gln Arg Tyr Ser Lys Val Lys Ala Thr             420 425 430 Asn Thr Trp Leu Phe Leu Ser Arg Cys Ser Gly Asn Glu Thr Ser Leu         435 440 445 Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Ser Cys Asp His Tyr     450 455 460 Glu Glu Ala Lys Val Thr Cys Ser Ala His Arg Glu Pro Arg Leu Val 465 470 475 480 Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly Asp                 485 490 495 Thr Trp Gly Thr Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala Ser             500 505 510 Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Ile Ser Ile Leu Gly         515 520 525 Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu Phe     530 535 540 Gln Cys Glu Gly Gln Glu Ser His Leu Ser Leu Cys Ser Val Ala Ser 545 550 555 560 Arg Pro Asp Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val Cys                 565 570 575 Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Gln Ser Pro Cys Glu             580 585 590 Gly Arg Val Glu Leu Lys Ile Leu Gly Asn Trp Gly Ser Leu Cys Asn         595 600 605 Ser His Trp Asp Ile Glu Asp Ala His Val Phe Cys Gln Gln Leu Lys     610 615 620 Cys Gly Val Ala Leu Ser Ile Pro Gly Gly Ala His Phe Gly Lys Gly 625 630 635 640 Ser Gly Gln Ile Trp Arg His Met Phe His Cys Thr Gly Thr Glu Gln                 645 650 655 His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Thr Leu Cys Ser             660 665 670 Ala Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln Thr         675 680 685 Leu Ser Pro Cys Asn Ser Thr Ser Leu Asp Pro Thr Arg Ser Thr Thr     690 695 700 Ser Glu Glu Ser Ala Val Ala Cys Ile Ala Ser Gly Gln Leu Arg Leu 705 710 715 720 Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Ile Glu Val Tyr His Glu                 725 730 735 Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp Ala             740 745 750 His Val Val Cys Arg Gln Leu Gly Cys Gly Val Ala Ile Asn Ala Thr         755 760 765 Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp Glu     770 775 780 Val Asn Cys Asn Gly Lys Glu Ser His Ile Trp Gln Cys Arg Ser His 785 790 795 800 Gly Trp Gly Gln His Asn Cys Arg His Lys Glu Asp Ala Gly Val Ile                 805 810 815 Cys Ser Glu Phe Met Ser Leu Arg Leu Ile Asp Glu Thr Ser Arg Asp             820 825 830 Ile Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly Ser         835 840 845 Val Gly Lys Ser Asn Met Ser Ala Thr Thr Val Val Glu Val Val Cys Arg     850 855 860 Gln Leu Gly Cys Ala Asp Lys Gly Ser Ile Asn Pro Ala Ser Ser Asp 865 870 875 880 Lys Pro Met Ser Arg His Met Trp Val Asp Asn Val Gln Cys Pro Lys                 885 890 895 Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Lys Gln Arg             900 905 910 Val Ala Ser Ser Ser Glu Glu Thr Trp Ile Thr Cys Ala Asn Lys Ile         915 920 925 Arg Leu Gln Glu Gly Thr Ser Asn Cys Ser Gly Arg Val Glu Leu Trp     930 935 940 His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu Glu 945 950 955 960 Asp Ala Gln Val Val Cys Arg Gln Leu Gly Cys Gly Pro Ala Leu Glu                 965 970 975 Ala Leu Lys Glu Ala Ala Phe Gly Gln Gly Thr Gly Pro Ile Trp Leu             980 985 990 Asn Asp Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys Pro         995 1000 1005 Ala Arg Pro Trp Gly His Ser Asp Cys Gly His Lys Glu Asp Ala     1010 1015 1020 Ala Val Arg Cys Ser Glu Ile Ala Met Ala Gln Arg Ser Ser Asn     1025 1030 1035 Pro Arg Gly His Ser Ser Leu Val Ala Leu Gly Ile Phe Gly Val     1040 1045 1050 Ile Leu Leu Ala Phe Leu Ile Ala Leu Leu Leu Trp Thr Gln Arg     1055 1060 1065 Arg Arg Gln Gln Gln Arg Leu Thr Val Ser Leu Arg Gly Glu Asn     1070 1075 1080 Ser Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Ser Leu Lys     1085 1090 1095 Ala Asp Asp Leu Asp Val Leu Thr Ser Ser Glu Asp His Phe Glu     1100 1105 1110 Val his     1115 <210> 47 <211> 3399 <212> DNA (213) Canis familiaris <400> 47 atgagcaaac tcagaatggt cccacatgga aactctggat ctgctgactt tagaagatgt 60 tttgccctct tgtgtccctc tgctgtggct gtggtctcca ttctcagtac ctgtttgatg 120 accaattctc ttgggagagc agataaagag atgaggctaa cggatggtga agacaattgc 180 tccgggagag tggaagtgaa agtccaggag gagtggggaa cggtgtgtaa taatggctgg 240 ggcatggatg aagtctctgt gatttgcagg cagctgggat gtcccactgc tatcaaagcc 300 gctggatggg ccaattccag ggcaggctct ggacgaatct ggatggatca tgtttcttgt 360 cgagggaatg aatctgctct ctgggactgc aaacatgatg gatggggaaa gcacaactgc 420 agtcatcaac aggatgctgg agtaacctgt tcagatggat ccagtttgga gatgaggttg 480 atgaacggcg gaaaccagtg ttctggcaga atagaagtca agttccaggg acagtgggga 540 acagtgtgtg atgacaactt caacatagat catgcttctg tggtttgtaa acagctcgaa 600 tgtggaagtg ctgtcagttt ctctggttca gctaattttg gagaaggttc tgggccaatc 660 tggtttgatg atcttgtgtg cagtggaaat gagtcagctc tctggaactg caagcatgaa 720 ggatggggaa agcataactg tgatcacgct gaggatgttg gagtgatttg cttggatgga 780 gcagatctga gcctgagact ggtagatgga gtcactgaat gttcaggaag attagaagta 840 aaattccaag gggaatgggg gacagtgtgt gatgatggct gggatagtaa tgatgctgct 900 gtggtatgta aacaactggg atgcccaact gctgtcaccg ccattggtcg agttaacgcc 960 agtgagggaa gtggacacat ttggcttgac aatctttcct gccaaggaga cgaatctgct 1020 ctctggcagt gtagacacca tgaatgggga aagcattatt gcaatcataa tgaagatgct 1080 ggtgtgacat gttctgatgg atcagacctg gagctgagac ttgtcggtgg aggcagccgc 1140 tgtgctggga cagtggaggt tgaaattcag aaactgctag ggaaagtatg tgatagaggc 1200 tggggactga aagaagccga tgtggtttgc aagcagttgg gatgtggatc tgctctcaaa 1260 acgtcctatc agcgttattc caaagttaag gcaacaaaca catggctgtt tttaagccgc 1320 tgtagtggca atgaaacttc cctttgggac tgcaagaact ggcagtgggg tggactgagc 1380 tgtgatcact atgaagaagc taaagttacc tgctcagccc acagggaacc cagactagtt 1440 ggaggagata ttccctgctc tggtcgtgtt gaagtgaaac atggtgacac atggggcacc 1500 gtctgtgatt ccgacttctc tttggaagct gccagtgtgc tgtgcagaga gttacagtgt 1560 ggcacagtca tctccatcct agggggagct cactttggag aaggaaatgg acagatctgg 1620 gctgaagaat tccagtgtga ggggcaggag tcccatcttt cactctgttc agtagcctct 1680 cgcccagatg ggacctgtag ccacagcagg gatgttggag tcgtctgctc aagatacacg 1740 gaaatccgct tggtgaatgg ccagtccccg tgtgaaggaa gagtggagct caagatactt 1800 gggaactggg gatccctctg caactctcac tgggacatag aagatgccca tgttttctgt 1860 cagcagctca aatgtggagt tgccctttct attccgggag gagcacattt tgggaaagga 1920 agtggtcaga tctggaggca catgtttcac tgcactggga ctgagcagca catgggagat 1980 tgccctgtaa ctgctctggg cgcgacgctg tgttctgctg ggcaagtggc ctctgtaatc 2040 tgctcaggaa atcagagcca gacgctatcc ccatgcaatt caacatctct ggacccaaca 2100 agatctacca cttcggaaga aagtgctgtt gcttgtattg cgagtgggca acttcgcctg 2160 gtaaatggag gcggtcgctg tgctgggaga atagaggtct accatgaggg ctcctggggc 2220 accatctgtg atgacagctg ggacctgagt gatgcccatg tggtgtgcag acagctgggc 2280 tgtggagtgg ccattaatgc cactggctct gctcattttg gggaaggaac agggcccatc 2340 tggctggacg aggtgaactg taatggaaag gaatctcata tctggcaatg ccgctcacac 2400 ggctgggggc aacacaactg cagacataag gaggatgcag gagttatctg ctcagagttc 2460 atgtctctca gactgattga tgaaaccagc agagacatct gtgcagggcg tcttgaagtt 2520 ttttacaatg gagcttgggg cagcgttggc aagagtaata tgtctgcaac cactgtggag 2580 gtggtatgca ggcaactggg ttgtgcagac aaggggagca tcaaccctgc atcttcagac 2640 aagcccatgt ccaggcacat gtgggtggac aatgtccagt gtccaaaagg acctgacacc 2700 ttatggcagt gcccatcttc tccatggaaa cagagagtgg ccagttcttc agaggagacc 2760 tggatcacat gtgccaacaa gataagactt caagaaggaa cctctaattg ttctggacgt 2820 gtggagctct ggcacggagg ttcctggggg acagtgtgcg atgactcctg ggaccttgaa 2880 gatgcacaag tggtgtgtcg acagctgggc tgtggcccag cattagaagc actaaaagag 2940 gcagcatttg gtcaggggac tgggcctata tggctcaatg acgtgaagtg caaagggaat 3000 gagtcttcct tgtgggattg tcctgctaga ccctgggggc acagtgactg tggccacaag 3060 gaagatgctg ctgtgaggtg ctcagaaatt gcaatggccc aaagatcatc aaatcctaga 3120 ggtcactcat cccttgttgc attggggatc tttggtgtca ttcttctggc ctttctcatc 3180 gctctcctct tgtggactca aaggcgaaga cagcaacagc ggcttacagt ttccttgaga 3240 ggagagaatt ctgtccacca aattcaatac cgggaaatga attcttccct gaaagcagat 3300 gatctggacg tgctgacttc ctcagaatat cccaatgagt cagatgattt taatgatgct 3360 gggctaattt ctgtgtctaa atctcttcct atttctgga 3399 <210> 48 <211> 1133 <212> PRT (213) Canis familiaris <400> 48 Met Ser Lys Leu Arg Met Val Pro His Gly Asn Ser Gly Ser Ala Asp 1 5 10 15 Phe Arg Arg Cys Phe Ala Leu Leu Cys Pro Ser Ala Val Ala Val Val             20 25 30 Ser Ile Leu Ser Thr Cys Leu Met Thr Asn Ser Leu Gly Arg Ala Asp         35 40 45 Lys Glu Met Arg Leu Thr Asp Gly Glu Asp Asn Cys Ser Gly Arg Val     50 55 60 Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Gly Met Asp Glu Val Ser Val Ile Cys Arg Gln Leu Gly Cys Pro Thr                 85 90 95 Ala Ile Lys Ala Ala Gly Trp Ala Asn Ser Arg Ala Gly Ser Gly Arg             100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp         115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Asn Cys Ser His Gln Gln     130 135 140 Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Ser Leu Glu Met Arg Leu 145 150 155 160 Met Asn Gly Gly Asn Gln Cys Ser Gly Arg Ile Glu Val Lys Phe Gln                 165 170 175 Gly Gln Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asp His Ala             180 185 190 Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe Ser         195 200 205 Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp Asp     210 215 220 Leu Val Cys Ser Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His Glu 225 230 235 240 Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Val Gly Val Ile                 245 250 255 Cys Leu Asp Gly Ala Asp Leu Ser Leu Arg Leu Val Asp Gly Val Thr             260 265 270 Glu Cys Ser Gly Arg Leu Glu Val Lys Phe Gln Gly Glu Trp Gly Thr         275 280 285 Val Cys Asp Asp Gly Trp Asp Ser Asn Asp Ala Ala Val Val Cys Lys     290 295 300 Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn Ala 305 310 315 320 Ser Glu Gly Ser Gly His Ile Trp Leu Asp Asn Leu Ser Cys Gln Gly                 325 330 335 Asp Glu Ser Ala Leu Trp Gln Cys Arg His His Glu Trp Gly Lys His             340 345 350 Tyr Cys Asn His Asn Glu Asp Ala Gly Val Thr Cys Ser Asp Gly Ser         355 360 365 Asp Leu Glu Leu Arg Leu Val Gly Gly Gly Ser Arg Cys Ala Gly Thr     370 375 380 Val Glu Val Glu Ile Gln Lys Leu Leu Gly Lys Val Cys Asp Arg Gly 385 390 395 400 Trp Gly Leu Lys Glu Ala Asp Val Val Cys Lys Gln Leu Gly Cys Gly                 405 410 415 Ser Ala Leu Lys Thr Ser Tyr Gln Arg Tyr Ser Lys Val Lys Ala Thr             420 425 430 Asn Thr Trp Leu Phe Leu Ser Arg Cys Ser Gly Asn Glu Thr Ser Leu         435 440 445 Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Ser Cys Asp His Tyr     450 455 460 Glu Glu Ala Lys Val Thr Cys Ser Ala His Arg Glu Pro Arg Leu Val 465 470 475 480 Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Lys His Gly Asp                 485 490 495 Thr Trp Gly Thr Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala Ser             500 505 510 Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Ile Ser Ile Leu Gly         515 520 525 Gly Ala His Phe Gly Glu Gly Asn Gly Gln Ile Trp Ala Glu Glu Phe     530 535 540 Gln Cys Glu Gly Gln Glu Ser His Leu Ser Leu Cys Ser Val Ala Ser 545 550 555 560 Arg Pro Asp Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val Cys                 565 570 575 Ser Arg Tyr Thr Glu Ile Arg Leu Val Asn Gly Gln Ser Pro Cys Glu             580 585 590 Gly Arg Val Glu Leu Lys Ile Leu Gly Asn Trp Gly Ser Leu Cys Asn         595 600 605 Ser His Trp Asp Ile Glu Asp Ala His Val Phe Cys Gln Gln Leu Lys     610 615 620 Cys Gly Val Ala Leu Ser Ile Pro Gly Gly Ala His Phe Gly Lys Gly 625 630 635 640 Ser Gly Gln Ile Trp Arg His Met Phe His Cys Thr Gly Thr Glu Gln                 645 650 655 His Met Gly Asp Cys Pro Val Thr Ala Leu Gly Ala Thr Leu Cys Ser             660 665 670 Ala Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln Thr         675 680 685 Leu Ser Pro Cys Asn Ser Thr Ser Leu Asp Pro Thr Arg Ser Thr Thr     690 695 700 Ser Glu Glu Ser Ala Val Ala Cys Ile Ala Ser Gly Gln Leu Arg Leu 705 710 715 720 Val Asn Gly Gly Gly Arg Cys Ala Gly Arg Ile Glu Val Tyr His Glu                 725 730 735 Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Ser Asp Ala             740 745 750 His Val Val Cys Arg Gln Leu Gly Cys Gly Val Ala Ile Asn Ala Thr         755 760 765 Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp Glu     770 775 780 Val Asn Cys Asn Gly Lys Glu Ser His Ile Trp Gln Cys Arg Ser His 785 790 795 800 Gly Trp Gly Gln His Asn Cys Arg His Lys Glu Asp Ala Gly Val Ile                 805 810 815 Cys Ser Glu Phe Met Ser Leu Arg Leu Ile Asp Glu Thr Ser Arg Asp             820 825 830 Ile Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly Ser         835 840 845 Val Gly Lys Ser Asn Met Ser Ala Thr Thr Val Val Glu Val Val Cys Arg     850 855 860 Gln Leu Gly Cys Ala Asp Lys Gly Ser Ile Asn Pro Ala Ser Ser Asp 865 870 875 880 Lys Pro Met Ser Arg His Met Trp Val Asp Asn Val Gln Cys Pro Lys                 885 890 895 Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Lys Gln Arg             900 905 910 Val Ala Ser Ser Ser Glu Glu Thr Trp Ile Thr Cys Ala Asn Lys Ile         915 920 925 Arg Leu Gln Glu Gly Thr Ser Asn Cys Ser Gly Arg Val Glu Leu Trp     930 935 940 His Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu Glu 945 950 955 960 Asp Ala Gln Val Val Cys Arg Gln Leu Gly Cys Gly Pro Ala Leu Glu                 965 970 975 Ala Leu Lys Glu Ala Ala Phe Gly Gln Gly Thr Gly Pro Ile Trp Leu             980 985 990 Asn Asp Val Lys Cys Lys Gly Asn Glu Ser Ser Leu Trp Asp Cys Pro         995 1000 1005 Ala Arg Pro Trp Gly His Ser Asp Cys Gly His Lys Glu Asp Ala     1010 1015 1020 Ala Val Arg Cys Ser Glu Ile Ala Met Ala Gln Arg Ser Ser Asn     1025 1030 1035 Pro Arg Gly His Ser Ser Leu Val Ala Leu Gly Ile Phe Gly Val     1040 1045 1050 Ile Leu Leu Ala Phe Leu Ile Ala Leu Leu Leu Trp Thr Gln Arg     1055 1060 1065 Arg Arg Gln Gln Gln Arg Leu Thr Val Ser Leu Arg Gly Glu Asn     1070 1075 1080 Ser Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Ser Leu Lys     1085 1090 1095 Ala Asp Asp Leu Asp Val Leu Thr Ser Ser Glu Tyr Pro Asn Glu     1100 1105 1110 Ser Asp Asp Phe Asn Asp Ala Gly Leu Ile Ser Val Ser Lys Ser     1115 1120 1125 Leu Pro Ile Ser Gly     1130

Claims (15)

(a) causing increased CD163 polypeptide expression in vertebrate cells A method of promoting infection of vertebrate cells by porcine genital respiratory syndrome virus (PRRSV), comprising ex vivo or in vitro. A method of infecting vertebrate cells with PRRSV in vitro or in vitro, comprising causing an increase in CD163 polypeptide expression in vertebrate cells and infecting the cells with PRRSV. Producing a culture of PRRSV in vitro or in vitro, comprising causing an increase in CD163 polypeptide expression in vertebrate cells, infecting the cells with PRRSV and producing a culture of PRRSV. Way. The method of claim 1, wherein said cell is a mammalian cell. The method of claim 1, wherein said CD163 polypeptide comprises a transmembrane domain. The method of claim 1, wherein said increase in expression is achieved by the introduction of an exogenous nucleic acid. The method of claim 1, wherein said increase in expression is achieved by chemical treatment. The method of claim 1, wherein the cells were PRRSV proliferative incapable and become PRRSV proliferative by step (a). The method of claim 2, wherein the PRRSV is a European phenotype. The method of claim 2, wherein the PRRSV is a North American phenotype. A method of producing a PRRSV vaccine comprising the method of any one of claims 2 to 10. The method of claim 11, wherein the vaccine is a killing vaccine. The method of claim 11, wherein the vaccine is an attenuated live vaccine. a) providing a sample containing nucleic acid from a test cell line; And b) determining the amount of the polynucleotide encoding the CD163 polypeptide or complement thereof in the sample Wherein the increased amount of polynucleotide encoding the CD163 polypeptide relative to a control sample derived from a control cell line known to not support the proliferation of PRRSV indicates the propensity of the test cell line to support replication of PRRSV, A method of measuring the propensity of a test cell line to allow infection by PRRSV. (a) providing a sample containing a polypeptide from a test cell line; And (b) determining the amount of CD163 polypeptide in the sample Infection with PRRSV, wherein the increased amount of CD163 polypeptide is indicative of the propensity of the test cell line to support replication of PRRSV compared to a control sample derived from a control cell line known to not support the proliferation of PRRSV. How to measure the propensity of test cell lines to allow.

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