Abstract:
The present invention discloses antibodies that bind to mammalian occludin proteins which are transmembrane adhesion molecules of the tight junction. Nucleic acid and amino acid sequences encoding human, mammalian, and murine occludin are utilized to prepare and isolate occludin-specific antibodies. These antibodies may be used to elucidate and regulate the activities of tight junctions.

Description:
Application Ser. No. 08/945,826 is the national phase of PCT International Application No. PCT/JP97/00665 filed on Mar. 5, 1997 under 35 U.S.C. §371. The entire contents of each of the above identified applications are hereby incorporated by reference. 
     This application is a divisional application of U.S. application Ser. No. 08/945,826 filed Nov. 5, 1997. 
    
    
     The present invention relates to an amino acid sequence of membrane protein occludin in a tight junction (hereinafter referred to as “the TJ”) of a human, a dog and a mouse, and a DNA for encoding the amino acid sequence. Antibodies that bind to the occludin protein or portions thereof are also included in the present invention. 
     BACKGROUND OF THE INVENTION 
     In multicellular animals, the information on cellular adhesion between adjacent cells is deeply concerned with the regulation and maintenance of vital phenomena such as cellular proliferation and differentiation, inflammation and cancer metastasis. Intercellular adhesion molecules which take part in the adhesion of the cells frequently assemble together on the surfaces of these cells to form a specifically differentiated membrane region for the adhesion. Particularly, intercellular adhesion molecules such as cadherins are known to be firmly bound to cytoskeleton in the cytoplasmic domain of epithelial cells. Such membrane regions are called intercellular adhesion apparatus and chiefly classified into the following four structures: gap junction (GJ), adherens junction (AJ), desmosome and tight junction (TJ). 
     These adhesion apparatus has first been identified under an electron microscope, and as a result of the investigation and research of constituent proteins, the importance of their physiological and pathological significance has become the focus of increased interest. The proteins that are called the so-called adhesion molecules specifically exist in these adhesion apparatus, and the adhesion molecules of the AJ are cadherins and various kinds of cadherins such as N-cadherin and P-cadherin have been identified so far [Takeichi, M. et al., “Science”, Vol. 251, p. 1451-1455, (1991)]. As adhesion molecules of the desmosome, desmoglein and desmocollin are known, and according to recent studies, it has been elucidated that their structures are similar to those of the cadherins [Buxton, R. S. et al., “J. Cell Biol.”, Vol. 121, p. 481-484, (1993)]. The adhesion molecules of the GJ are called connexin, and it is known that connexin holds transmembrane domains at four different sites and both of its N-terminal and C-terminal protrude on the cytoplasmic side of the membrane. 
     The TJ is an intercellular adhesion apparatus peculiar to epithelial cells and endothelial cells, where the cell membranes of contiguous cells are seen completely tightly apposed. The TJ surrounds individual cells and functions as a barrier to block or regulate permeation of water-soluble molecules between the luminal and basement membrane sides of a cell layer. It has also been described to act as a fence partitioning the cell membrane into apical and basolateral sides in order to maintain the polar distribution of such membrane proteins as ion channels and pumps as well as lipids on the cell membrane [Schneeberger, E. E. et al., “Am. J. Physiol.”, Vol. 262, P. L647-L661, (1992)]. Owing to these functions of the TJ, milieus consisting of different fluid compositions are formed on the opposite sides of a cell layer, so that the polarity of the cell layer is maintained; hence the TJ can be said to be a fundamental structure of vital importance to multicellular organisms. 
     However, analysis of the molecular structure of the TJ has been less progressing, compared to other adhesion apparatus. In fact, it has constituted a serious drawback to the pursuit of molecular biological research on the TJ that the TJ adhesion molecule itself has not been identified yet. 
     The present inventors have established a method for isolation of AJ from rat liver, and have identified many proteins such as radixin and ZO-1 from this isolated AJ [Tsukita, Sh. et al., “Curr. Opin. Cell Biol.”, Vol. 4, P. 834-839, (1992)]. From researches on ZO-1 and histologic findings for the AJ and the TJ, it can be presumed that the proteins in the AJ also contain protein of the TJ. In view of this, the present inventors have isolated AJ from chick liver, prepared a monoclonal antibody against the AJ as the antigen, and carried out structural analysis of the TJ-constituting protein using the antibody specifically reacting with the TJ. As a result, the present inventor has been successful in the structural analysis of a novel constituent protein dissimilar to known proteins, and designated the protein as occludin [Furuse, M. et al., “J. Cell Biol.”, Vol. 123, p. 1777-1788, (1993)]. 
     This chick occludin is a 56 KDa protein composed of 504 amino acids, characterized conspicuously by transmembrane domains at four sites in the half of its N-terminus, with both the N- and C-terminals facing the cytoplasm and with two extracelluar loops. 
     From subsequent studies, occludin was inferred to be an important factor in the analysis of physiological function of the TJ at the cellular level as well as at the whole body level, and drew much attention of investigators. 
     No further study has progressed, nevertheless, since the said protein has its origin in the chicken species greatly remote from humans. Thus, structural analysis of occludin of human origin has. been expected for the sake of elucidation of the physiological function and medical analysis of the TJ. There is as yet no report of success in the elucidation of human occludin despite worldwide competition in research for this purpose in this field. 
     SUMMARY OF THE INVENTION 
     It is accordingly an object of the present invention to provide amino acid sequences of human, canine and mouse occludins and the DNAs encoding them. 
     In their report on the gene for human neuronal apoptosis inhibitory protein (NAIP), Roy et al. documented occurrence of a DNA fragment possessing a base sequence analogous to the C-terminal region of chick occludin in NAIP gene deletion mutants [Roy, N. et al., “Cell”, Vol. 80, p. 167-178, (1995)]. To ascertain whether the said sequence actually encoded a part of human analogue of occludin or not, the present inventor selected primers out of the base sequence analogous to that of chick occludin and made a scrupulous screening with a cDNA library of human intestinal epithelial cell strain T84 as a template for PCR. The present inventor has thus succeeded in the analysis of the whole structure of human occludin. Further, the inventor has completed analyses of mouse and canine occludins, prepared anti-occludin monoclonal antibodies, and verified with histologic staining that the occludins were transmembrane type TJ proteins. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS. 1A-B are photographs substituting for drawings showing the morphology of organisms. 
     FIG. 1A is a stained immunofluorescent photomicrograph of cultured human intestinal epithelial cell strain T84 with an anti-human occludin rat monoclonal antibody. 
     FIG. 1B is a stained immunofluorescent photomicrograph of cultured human intestinal epithelial cell strain T84 with a mouse monoclonal antibody against the TJ lining protein ZO-1. The same sites of T84 were photographed. 
    
    
     The present invention is concerned with amino acid sequences of a human, a canine and a mouse occludin, DNAs for encoding them, anti-occludin antibodies, and a genetic analysis method utilizing them. More specifically, the present invention is directed to the following aspects. 
     (1) A DNA for encoding a human occludin having an amino acid sequence described in Sequence No. 2. 
     (2) A DNA for encoding a human occludin as described in Sequence No. 7. 
     (3) A DNA described in Sequence No. 3 for encoding a canine occludin having an amino acid sequence described in Sequence No. 4. 
     (4) A DNA described in Sequence No. 5 for encoding a mouse occludin having an amino acid sequence described in Sequence No. 6. 
     (5) A human, a canine and a mouse occludin having the amino acid sequences described in Sequence Nos. 2, 4 and 6, respectively. 
     (6) An occludin variant having an amino acid sequence in which one or plural amino acids in the amino acid sequence of each occludin are added, deleted or substituted, and a DNA for encoding the variant. 
     (7) A vector which comprises any of DNAs for encoding a human, a canine or a mouse occludin or for encoding their variants. 
     (8) A transformant which holds the vector. 
     (9) A method for manufacturing an occludin protein which comprises the steps of cultivating the transformant, and collecting an expressed product. 
     (10) A DNA probe comprising containing the whole or part of the base sequence as defined in Sequence No. 1, 3 or 5. 
     (11) A DNA primer comprising containing part of the base sequence as defined in Sequence No. 1, 3 or 5. 
     (12) A polyclonal antibody or a monoclonal antibody specifically binding to a human, a canine or a mouse occludin protein. 
     (13) An assay method and an assay reagent for occludin in a biological specimen, wherein an anti-occludin antibody is used. 
     (14) An analysis method of an occludin gene in a biological specimen, wherein said DNA primer or said DNA probe is used. 
     (15) A screening method of a drug affecting the expression of occludin, wherein occludin-expressing cells and an analyte are allowed to coexist, and an expression quantity of an occludin gene of said cells is then determined by the use of a DNA primer or a DNA probe. 
     (16) An antisense DNA derived from a human occludin DNA. 
     (17) A laboratory animal whose occludin DNA is knocked out. 
     BRIEF DESCRIPTION OF DRAWINGS 
     FIG.  1 : Photographs of Immunofluorescence staining of cultured human intestinal epithelial T84 cells. 
     (a) immunofluorescence from rat anti-human occludin monoclonal antibody staining of cultured human intestinal epithelial cells (T84). 
     FIG. 1B; immunofluorescence of the same site from mouse monoclonal antibody against the TJ-lining protein ZO-1. 
     DETAILED DESCRIPTION OF THE INVENTION 
     With the success of the present inventor in identifying occludin analogues of mammals, it is now possible to structurally and functionally test the constitution and function of the TJ at the molecular level. The barrier and fencing functions of the TJ and the related regulatory mechanisms can be analyzed through experiments involving control of expression of the gene for occludin or inhibition of the occludin function with either an antisense probe or an antibody, using various types of cultured human, mouse and canine (MDCK) cells. For example, it is now possible to determine whether or not overexpression of occludin cDNA gives rise to an increase in number of TJ strands seen in freeze-fractured replicas and incidentally to an augmentation of the barrier function. Furthermore, the present invention has made it possible to establish a simple screening method for drugs affecting TJ function. For example, drugs affecting TJ function can be screened using various types of cells expressing occludin, by allowing the cell to react with a test article and subsequently by measuring the amount of cellular occludin gene or occludin protein expression. The gene analysis can be carried out by using a DNA probe or primer or other devices. It may be conducted by known methods, e.g. the Northern blot technique or Southern blot technique wherein RNA or DNA extracted from a test sample in the usual manner is pretreated when necessary, then electrophoresed on a membrane or gel, and hybridized with a labeled DNA probe, and the polymerase chain reaction (PCR) technique wherein the objective DNA is amplified using primers of about 20 bases corresponding to the relevant site and with a genomic DNA or cDNA as the template. The occludin protein can be quantitated, for example, by the use of an antibody. 
     Moreover, it is also made possible to ascertain how the TJ formation is involved in the morphogenesis of various organs and whether functional failure of the TJ has any relation to various pathologic states such as inflammation and tumor metastasis, by preparing various types of mutant mice and occludin gene-knocked out mice. The possibility of controlling a TJ function, especially its barrier function, is also of interest in connection with drug permeability. Thus, it would be feasible to control the blood-brain barrier via up- or down-regulation of occludin synthesis in epithelial cells of the brain. Control of the TJ function in the enteric epithelial cells is necessary to regulate drug absorption from the intestine. It will thus become possible to control drug absorption, particularly distribution to the brain tissue, by administering an effective substance screened out of drugs affecting the TJ function. Hence, the present invention is highly anticipated for elucidation of the physiological mechanisms primarily of the blood-brain barrier, as well as for analysis, diagnosis and treatment of disease states. 
     The DNA provided by the present invention can be utilized in the analysis of genes for occludin proteins and of gene expression thereof by using a part of it as a primer or a probe. The term a part here denotes that the oligonucleotide to be used as a primer or probe comprises containing at least a 10-relevant-base sequence, or preferably at least a 15-base sequence, or more preferably a corresponding polynucleotide comprising containing approximately 20- to 30-base sequence based on the DNA sequence of the present invention. As the probe, a higher macromolecular or even the whole DNA may be used. 
     There is a method utilizing antisense DNA or antisense RNA as a means to control the function of occludin. The method is intended to block the flow of gene expression by interfering with the reading of genetic information at any of the stages of gene expression such as DNA replication, transcription and translation, and the antisense technique employs nucleic acid or its analogue for the blockage (Wickstrome, E. ed., Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS. Wiley-Liss, New York, 1991). The elucidation of the occludin DNA according to the present invention has made possible the means for inhibiting occludin functions by the antisense method. The length of DNA oligomer has bearing on the double strand-forming capacity, membrane permeability and base sequence specificity; and at least 6 nucleotides, or preferably at least 10 mers, usually 15 to 30 mers, may be used. Appropriate sequences may be selected on the basis of the DNA sequence of the present invention, and verified by experimentation. Usually, the oligomer is chemically modified at its phosphate group, sugar moiety, and 3′ and 5′ tails in order to augment its stability (Cook, P. D., Anticancer Drig. Des., 5,585, 1991). Representative analogues are oligophosphorothioate where one of the oxygen atoms of the internucleoside phosphodiester group is replaced by a sulfur atom, and oligomethylsulfonate where the said oxygen is replaced by a methyl group; all such analogues are remarkably stable to nucleases. Besides, such oligomers as those with acridine or polylysine bonded to them and those containing N-methlythymidylate, to increase stability of the hybrid double strand, are also used. These oligomers can be synthesized by known chemical synthetic procedures. Antisense RNA derived from the DNA of the present invention may also be utilized. 
     The occludin protein of the present invention may be utilized for preparation of an antibody using the whole or a part of it as an epitope, and for use the antibody thus prepared as research and diagnostic reagents. The term epitope denotes an antigen determinant of polypeptide; epitope is usually comprised of at least 6 amino acids, and it is known that a polypeptide consisting of 6 amino acids combines with an antibody (JP-A-60-500684). The antigenic peptide of the subject protein signifies a polypeptide comprising a series of at least 6 amino acids, preferably a series of at least 8 amino acids, more preferably a series of at least 15 amino acids, or further preferably a series of at least 20 amino acids, based on the amino acid sequence of the present invention. Occludin provided by the present invention is a protein which, as inferred from its amino acid sequence in analogy with chick occludin, possesses transmembrane domains at four sites in a half of its N-terminal region, with the N- and C-terminus facing the cytoplasm, and which has two extracellular loops. In the case of human occludin of which amino acid 89-135 and 196-243 regions are presumed to be extracellularly apposed, various antibodies may be prepared by selecting antigenic sites appropriate for purposes and utilized as a means to elucidate the TJ function and as a means to suppression by the antibody of the TJ function. It is also possible to utilize the partial peptides as a means to screen compounds for those capable of binding to these peptides. 
     Proteins having an amino acid sequence of occludin of the present invention to which one or a plurality of amino acids are added or of which one or a plurality of amino acids are deleted or substituted are also encompassed by the present invention. 
     (1) Preparation of cDNA Library and Structural Analysis of Occludin 
     Preparation of RNA may be carried out using human or animal cells (cell strains) as the raw material, by for example extraction with a mixed solution of guanidine thiocyanate, a surfactant, a chelating agent and a reductant, followed by phenol extraction, fractionation in organic solvents (Chamezynski et al., Anal. Biochem., 162, 156, 1987) and subsequently density gradient ultracentrifugation procedure. Using the RNA thus obtained as a template, a double strand DNA is prepared in the usual manner such as by the cDNA synthesis technique (Gubler, U. et al., Gene, 25, 263, 1983) with the use of random primers, reverse transcriptase, DNA polymerase, etc. A DNA library can be prepared by insertion of the double strand DNA obtained into a bacteriophage such as λ zap or λ gt11 in the usual manner. Commercial cDNA libraries may also be used. 
     According to the report of Roy et al., it is possible to obtain a DNA fragment presumed to be of occludin DNA origin by properly selecting a primer region based on the base sequence analogous to the C-terminus of chick occludin, by amplification of the DNA with the PCR technique, and by its subclonation. Subsequent screening of the cDNA library with this DNA fragment as a probe and analysis of the base sequence of the clone isolated may yield a whole-length cDNA for occludin. The structure of the base sequence is determined by the Maxam-Gilbert method (Maxam, A. M. and Gilbert, W., Proc. Natl. Acad. Sci. USA, 74, 560, 1977) or by the dideoxynucleotide chain termination method (Sanger, F., Proc. Natl. Acad. Sci. USA, 74, 5463, 1977). The amino acid sequence is thus deduced on the basis of the base sequence. These gene manipulations can be performed by the known usual methods, for example in accordance with those described in Molecular Cloning. A Laboratory Manual, T. Maniatis et al. eds. (1989), Cold Spring Harbor Laboratory. 
     (2) Preparation of Antibodies 
     To prepare the monoclonal antibody according to the present invention, human, canine or mouse occludin is used as the antigen, its complex with a carrier protein is prepared if deemed necessary, and appropriate animals are immunized by inoculation with the antigen. Antibody-forming cells obtained from the spleen or lymphnodes of the above immunized animals are fused with myeloma cells, whereby hybridomas producing an antibody strongly specific to occludin are selected to prepare the monoclonal antibody. The preparation procedure may be in accordance with the known prior method. 
     As the immunogen, any of such products as purified natural products and products prepared by genetic recombination technique or chemical synthesis may be used. For preparation of occludin by the recombinant DNA technique, the cDNA encoding occludin can be religated to the promoter downstream of a vector appropriate for expression of occludin by the known method using restriction enzymes and DNA ligase to obtain a recombinant expression vector. The said vector is nonlimitative insofar as it can be replicated and amplified in a host. With regard to the promoter and terminator, there is also no particular limitation as long as they are concordant with the host used for expression of the base sequence encoding occludin; and appropriate combinations suited to the host may also be practicable. The recombinant expression vector thus obtained is introduced into the host by the competent cell technique (J. Mol. Biol., 53, 154, 1970) or the calcium phosphate procedure (Science, 221, 551, 1983) to prepare a transformant. Such organisms as  Escherichia coli  and animals are used as the host, and the transformant obtained is cultured in an appropriate medium suited to the host. The culture incubation is carried out usually at a temperature between 20° C. and 45° C. and at pH between 5 and 8, with aeration and/or stirring where required. Isolation and purification of occludin from the cultured microorganisms or cells may be performed by an appropriate combination of known methods of isolation and purification. These known methods include salting out, organic solvent method, dialysis, gel filtration, electrophoresis, ion exchange chromatography, affinity chromatography, and reverse-phase high performance liquid chromatography. 
     The immunogen, occludin, is preferably to retain its whole structure but may be in the form of a fragment or peptide having its partial structure; it may be appropriately selected from the whole amino acid sequence of occludin. For preparation of the fragment or peptide, a method such as chemical synthesis, the above mentioned gene recombination procedure or degradation of a naturally occurring article is employed. 
     Various condensing agents may be used for preparation of the immunogen-carrier protein complex; such reagents as glutaraldehyde, carbodiimide, and maleimide activated ester may be used. 
     The carrier protein may be any of those commonly used such as bovine serum albumin, thyroglobulin and hemocyanin, and usually the method wherein a 1- to 5-fold quantity of a carrier protein is coupled to antigen is used. 
     Animals employed for immunization include the mouse, rat, rabbit, and guinea pigs, and inoculation is made by subcutaneous, intramuscular or intraperitoneal injection. The administration of immunogen may be carried out in the form of a mixture with complete Freund adjuvant or with incomplete adjuvant, and is usually made once every 2 to 5 weeks. Antibody-producing cells obtained from the spleen or lymphnodes of the immunized animals are fused with myeloma cells and isolated as hybrydomas. The myeloma cells used are those of mouse, rat or human origin, preferably allogeneic to the antigen-producing cells used but, in some instances, can be xenogeneic. 
     The manipulation of cell fusion can be conducted in accordance, for example, with the method of Milstein and Köhler (Nature, 256, 495, 1975). Fusogens used include such agents as polyethylene glycol and Sendai virus, and the cell fusion can be made by incubating antibody-producing cells with myeloma cells in an approximate population ratio of 1:1 to 10:1 at a temperature between 20 and 40° C., preferably between 30 and 37° C., for about 1 to 10 minutes, using polyethylene glycol (mean molecular weight: 1,000 to 4,000) usually at a concentration of about 20 to 50%. 
     Various immunochemical methods can be used for screening hybridomas producing an anti-occludin antibody. The methods include enzyme-linked immunosorbent assay (ELISA) using microplates coated with occludin, enzyme immunoassay (EIA) using micoplates coated with an anti-immunoglobulin antibody, and Western blotting technique in which samples containing occludin are electrophoresed with the subsequent use of nitrocellulose transfer membranes. 
     Clones are obtained from these wells further by, for example, limiting dilution. Screening and breeding of hybridomas are performed in a culture medium for animal cells (e.g. RPMI 1640) containing 10-20% fetal bovine serum usually with added HAT (hypoxanthine, aminopterin and thymidine). The clone thus obtained is intraperitoneally transplanted into BALB/c mice previously dosed with bristan, and ascites containing a high concentration of a monoclonal antibody is collected 10-14 days later so that the ascites can be used as a source for purification of the antibody. Furthermore, the cloned hybridoma cells are cultured so that the cultured cells can be used as the source for purification of the antibody. Known methods for purification of immunoglobulin may be used for recovering the monoclonal antibody; the recovery can be readily accomplished for example by such means as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, utilization of anion exchangers, and affinity chromatography. 
     With immunological methods using the anti-occludin monoclonal antibody obtained in accordance with the present invention, it is possible to make qualitative and quantitative determination of occludin in biological specimens. As the immunological methods, conventional methods such as immunohistologic staining, enzyme immunoassay, agglutination test, competitive assay, and sandwich technique may be applied to samples from biological specimens that have been appropriately processed, where required, e.g. isolation of cells and extraction. The immunohistologic staining can be performed for example by the direct method using a labeled antibody or the indirect method using a labeled antibody directed to the antibody bound to target antigen. Any of such known labeling substances as fluorescent agents, radioactive substances, enzymes, metals and dyes may be used as labeling agents. 
     The monoclonal antibody of the present invention may be used in the form of Fab′ or Fab fragment after removal of its Fc′ or Fc region, or in the form of polymer of either fragment. Furthermore, it may also be in the form of a chimeric antibody, a humanized antibody, or a human antibody. 
     EXAMPLE 
     The present invention will now be illustrated in detail with specific embodiments by the following examples. Of course, the present invention shall not be limited to the following examples. 
     Example 1 
     Structural Analysis of Human Occludin 
     Based on a base sequence of a part of human NAIP-deficient gene analogous to the C-terminus of chick occludin, PCR was performed using as primers the oligonucleotides of Sequence Nos. 7 and 8. λgt11 cDNA library was prepared by purifying poly(A)+RNA from a source consisting of human intestinal epithelial cell strain T84 and by using TimeSaver cDNA Synthesis Kit (trade name; Pharmacia LKB Biotechnology Inc.)and GIGAPACK II Packaging Extract (Stratagene Inc.). The PCR was carried out with this library as the template and with said two primers yielded a cDNA fragment of 363 base pairs. 
     This DNA fragment was DIG-labeled using DIG Labeling Kit (trade name; Boehringer Mannheim), and said library was screened with it used as the probe. As a result, three cDNA clones were isolated, their insertion sites were cut, and they were subcloned to pBluescript SK(−). Of these, the two clones phOc6 and phOc16 were presumed to contain a total ORF, and these two cloned strands were analyzed for their base sequences, with the results demonstrating that said base sequences encoded the whole structure of human occludin. The coding sequence was determined using a 7-deaza Sequenase Version Deoxy Terminator Cycle Sequencing Kit (trade name; Applied Biosystems). The base sequence is shown in Sequence No. 1, and the amino acid sequence deduced therefrom in Sequence No. 2. 
     Structure of canine and mouse occludins were determined in the same manner as described above using λgt11 and λgt10 cDNA libraries, respectively, prepared from dog kidney (MDCK) cells and mouse lung cells. The base sequence and amino acid sequence of canine occludin are shown in Sequence Nos. 3 and 4, and the base sequence and amino acid sequence of mouse occludin in Sequence Nos. 5 and 6 . Escherichia coli  JM 109 containing the human occludin cDNA has been deposited (Deposition No. FERM BP-5477) with the National Institute of Bioscience and Human Technology, Ministry of International Trade and Industry, Japan (address: 1-1-3, Higashi, Tsukuba, Ibaraki, 305 Japan) as of Mar. 15, 1996. 
     Example 2 
     Preparation of an Anti-human Occludin Monoclonal Antibody 
     The cDNA fragment encoding the cytoplasmic region on the C-terminal side of human occludin was obtained by cutting the pBluescript SK(−) vector containing Sequence No. 1 described in Example 1 with restriction enzymes Ssp I and EcoR I (both being products of Takara Shuzo Co., Ltd.). This fragment was introduced into pGEX-3X vector, and  Escherichia coli  transformed with this vector was cultured to prepare a GST fused protein. Rats were immunized with this fused protein as an antigen, so that a monoclonal antibody was prepared. 
     The rat immunization was performed by injecting the antigen in doses of 300 μg/injection into the hindlimb paw, first as an emulsion with complete Freund adjuvant and the antigen alone twice thereafter (days 3 and 7 after the first). On the day following the last injection, inguinal lymphnodes were excised from the immunized animals and used for cellular fusion. 
     The rat lymphocytes and mouse myeloma P3 cells were combined in a ratio of 2.5:1, and the mixture was incubated in RPMI medium containing 1 g of polyethylene glycol (mean MW 4,000) dissolved in it, for 2 minutes according to a modified method of Köhler et al., to permit fusion of the cells. Fused cells were seeded in 24-well plates with HAT medium containing 10% HCF (Bokusui-Braun) for 9 days, followed by incubation in HT medium and subsequently in flasks with RPMI medium. Hybridomas were cloned by assaying supernatants of wells showing cellular growth for antibody titer using immunoblot technique and fluorescent antibody staining in cultures of human intestinal epithelial cell strain T84, and by limiting dilution from proper wells. The hybridoma cells were seeded at calculated concentration of 7 cells/well in microtiter plates, and screened by immunoblotting technique to verify and isolate clonal hybridoma cell strains. Antibodies were purified from culture supernatants of said hybridoma. 
     Example 3 
     Cell Staining 
     Human intestinal epithelial cells were fixed in 3% formalin in phosphate buffered saline (PBS) at room temperature for 15 minutes, and further treated with 0.2% Triton X-100 in PBS at room temperature for 15 minutes. After blocking the cells with 1% bovine serum albumin (BSA), the test substance was added and incubated for 30 minutes at room temperature. After subsequent washing, FITC-labeled anti-rat immunoglobulin antibody was added and incubated for 30 minutes at room temperature, followed by washing off unreacted antibody and examination with a fluorescent microscope. 
     Results of double immunofluorescent staining with monoclonal antibody to the TJ-related protein ZO-1 and the anti-human occludin monoclonal antibody of the present invention are shown in the figure. As totally the same staining pattern as that of ZO-1 (reported in the literature) was observed, the human protein of the present invention has proven to be a human homologue of the TJ adhesion molecule occludin. 
     Example 4 
     Expression of Occludin in Cerebrovascular Cells 
     Since cerebral vascular endothelial cells are thought to have a high electroresistant TJ, which form the brain-blood barrier unlike peripheral vascular endothelial cells, I examined the distribution and expression of occludin in cultured porcine brain vascular endothelial cells (PBEC) possessing the high electroresistant TJ and cultured porcine aortic endothelial cells (PAEC). 
     As porcine occludin cDNA fragment, a 363 base fragment was prepared by amplification with PCR technique using as primers 1359-1391 sense strand (Sequence No. 7) and 1692-1721 antisense strand (Sequence No. 8) from the human occludin DNA sequence (Sequence No. 1). The amino acid sequence based on analysis of the coding base sequence of said fragment showed a high degree of homology with amino acid sequences of human, mouse and canine occludins, thus verifying the fragment to be a cDNA for porcine occludin.  32 P-labeled said fragment was used as a probe. 
     To prepare mRNA from the cultured cells, an agarose gel electrophoresed sample was transferred onto nitrocellulose membrane and hybridized with the c DNA probe under highly stringent conditions, using an RNA isolation kit (Stratagene). As a result, the occludin mRNA showed a strong band at about 2.4 kb in PBEC, whereas in PAEC, only a very weak band was noted at that position. 
     Expression of occludin in these cells was compared using anti-mouse occludin antibody as a monoclonal antibody specifically recognizing mammalian occludins and an antibody against the TJ-related protein ZO-1. 
     Anti-mouse occludin rat antibody was prepared using mouse occludin:glutathione-S-transferase fused protein as a antigen, and FITC-labeled anti-rat IgG sheep antibody was used for detection of said antibody. When equal protein quantities of extracts from disrupted cultured cells were analyzed by immunoblotting after one-dimensional gel electrophoresis, a strong band of occludin was detected at about 58 KD in PBEC while a considerably weaker band was noted at that position in PAEC. On the other hand, there was no appreciable difference in expression of ZO-1 between the two types of cells. With immunostaining, PBEC exhibited a marked occludin expression with the same continuous intercellular localization as ZO-1, as seen in the immunoblotting study. In PAEC, in contrast, occludin was scarcely detected and ZO-1 showed a discontinuous intercellular localization. These results suggest that the relatively marked expression of occludin in PBEC is required for the formation of the highly electroresistant TJ, and provide evidence that occludin is the constituent protein of the TJ. 
     
       
         
           
             8 
           
           
             
               2379 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               cDNA 
             
             
               Homo sapiens 
               Cell strain T84
           
               Epithelial, intestinal 
             
             
               CDS 
                168..1733
 
             
             1
CTCCCGCGTC CACCTCTCCC TCCCTGCTTC CTCTGGCGGA GGCGGCAGGA ACCGAGAGCC     60
AGGTCCAGAG CGCCGAGGAG CCGGTCTAGG ACGCAGCAGA TTGGTTTATC TTGGAAGCTA    120
AAGGGCATTG CTCATCCTGA AGATCAGCTG ACCATTGACA ATCAGCC ATG TCA TCC      176
                                                    Met Ser Ser
                                                      1
AGG CCT CTT GAA AGT CCA CCT CCT TAC AGG CCT GAT GAA TTC AAA CCG      224
Arg Pro Leu Glu Ser Pro Pro Pro Tyr Arg Pro Asp Glu Phe Lys Pro
      5                  10                  15
AAT CAT TAT GCA CCA AGC AAT GAC ATA TAT GGT GGA GAG ATG CAT GTT      272
Asn His Tyr Ala Pro Ser Asn Asp Ile Tyr Gly Gly Glu Met His Val
 20                  25                  30                  35
CGA CCA ATG CTC TCT CAG CCA GCC TAC TCT TTT TAC CCA GAA GAT GAA      320
Arg Pro Met Leu Ser Gln Pro Ala Tyr Ser Phe Tyr Pro Glu Asp Glu
                 40                  45                  50
ATT CTT CAC TTC TAC AAA TGG ACC TCT CCT CCA GGA GTG ATT CGG ATC      368
Ile Leu His Phe Tyr Lys Trp Thr Ser Pro Pro Gly Val Ile Arg Ile
             55                  60                  65
CTG TCT ATG CTC ATT ATT GTG ATG TGC ATT GCC ATC TTT GCC TGT GTG      416
Leu Ser Met Leu Ile Ile Val Met Cys Ile Ala Ile Phe Ala Cys Val
         70                  75                  80
GCC TCC ACG CTT GCC TGG GAC AGA GGC TAT GGA ACT TCC CTT TTA GGA      464
Ala Ser Thr Leu Ala Trp Asp Arg Gly Tyr Gly Thr Ser Leu Leu Gly
     85                  90                  95
GGT AGT GTA GGC TAC CCT TAT GGA GGA AGT GGC TTT GGT AGC TAC GGA      512
Gly Ser Val Gly Tyr Pro Tyr Gly Gly Ser Gly Phe Gly Ser Tyr Gly
100                 105                 110                 115
AGT GGC TAT GGC TAT GGC TAT GGT TAT GGC TAT GGC TAC GGA GGC TAT      560
Ser Gly Tyr Gly Tyr Gly Tyr Gly Tyr Gly Tyr Gly Tyr Gly Gly Tyr
                120                 125                 130
ACA GAC CCA AGA GCA GCA AAG GGC TTC ATG TTG GCC ATG GCT GCC TTT      608
Thr Asp Pro Arg Ala Ala Lys Gly Phe Met Leu Ala Met Ala Ala Phe
            135                 140                 145
TGT TTC ATT GCC GCG TTG GTG ATC TTT GTT ACC AGT GTT ATA AGA TCT      656
Cys Phe Ile Ala Ala Leu Val Ile Phe Val Thr Ser Val Ile Arg Ser
        150                 155                 160
GAA ATG TCC AGA ACA AGA AGA TAC TAC TTA AGT GTG ATA ATA GTG AGT      704
Glu Met Ser Arg Thr Arg Arg Tyr Tyr Leu Ser Val Ile Ile Val Ser
    165                 170                 175
GCT ATC CTG GGC ATC ATG GTG TTT ATT GCC ACA ATT GTC TAT ATA ATG      752
Ala Ile Leu Gly Ile Met Val Phe Ile Ala Thr Ile Val Tyr Ile Met
180                 185                 190                 195
GGA GTG AAC CCA ACT GCT CAG TCT TCT GGA TCT CTA TAT GGT TCA CAA      800
Gly Val Asn Pro Thr Ala Gln Ser Ser Gly Ser Leu Tyr Gly Ser Gln
                200                 205                 210
ATA TAT GCC CTC TGC AAC CAA TTT TAT ACA CCT GCA GCT ACT GGA CTC      848
Ile Tyr Ala Leu Cys Asn Gln Phe Tyr Thr Pro Ala Ala Thr Gly Leu
            215                 220                 225
TAC GTG GAT CAG TAT TTG TAT CAC TAC TGT GTT GTG GAT CCC CAG GAG      896
Tyr Val Asp Gln Tyr Leu Tyr His Tyr Cys Val Val Asp Pro Gln Glu
        230                 235                 240
GCC ATT GCC ATT GTA CTG GGG TTC ATG ATT ATT GTG GCT TTT GCT TTA      944
Ala Ile Ala Ile Val Leu Gly Phe Met Ile Ile Val Ala Phe Ala Leu
    245                 250                 255
ATA ATT TTC TTT GCT GTG AAA ACT CGA AGA AAG ATG GAC AGG TAT GAC      992
Ile Ile Phe Phe Ala Val Lys Thr Arg Arg Lys Met Asp Arg Tyr Asp
260                 265                 270                 275
AAG TCC AAT ATT TTG TGG GAC AAG GAA CAC ATT TAT GAT GAG CAG CCC     1040
Lys Ser Asn Ile Leu Trp Asp Lys Glu His Ile Tyr Asp Glu Gln Pro
                280                 285                 290
CCC AAT GTC GAG GAG TGG GTT AAA AAT GTG TCT GCA GGC ACA CAG GAC     1088
Pro Asn Val Glu Glu Trp Val Lys Asn Val Ser Ala Gly Thr Gln Asp
            295                 300                 305
GTG CCT TCA CCC CCA TCT GAC TAT GTG GAA AGA GTT GAC AGT CCC ATG     1136
Val Pro Ser Pro Pro Ser Asp Tyr Val Glu Arg Val Asp Ser Pro Met
        310                 315                 320
GCA TAC TCT TCC AAT GGC AAA GTG AAT GAC AAG CGG TTT TAT CCA GAG     1184
Ala Tyr Ser Ser Asn Gly Lys Val Asn Asp Lys Arg Phe Tyr Pro Glu
    325                 330                 335
TCT TCC TAT AAA TCC ACG CCG GTT CCT GAA GTG GTT CAG GAG CTT CCA     1232
Ser Ser Tyr Lys Ser Thr Pro Val Pro Glu Val Val Gln Glu Leu Pro
340                 345                 350                 355
TTA ACT TCG CCT GTG GAT GAC TTC AGG CAG CCT CGT TAC AGC AGC GGT     1280
Leu Thr Ser Pro Val Asp Asp Phe Arg Gln Pro Arg Tyr Ser Ser Gly
                360                 365                 370
GGT AAC TTT GAG ACA CCT TCA AAA AGA GCA CCT GCA AAG GGA AGA GCA     1328
Gly Asn Phe Glu Thr Pro Ser Lys Arg Ala Pro Ala Lys Gly Arg Ala
            375                 380                 385
GGA AGG TCA AAG AGA ACA GAG CAA GAT CAC TAT GAG ACA GAC TAC ACA     1376
Gly Arg Ser Lys Arg Thr Glu Gln Asp His Tyr Glu Thr Asp Tyr Thr
        390                 395                 400
ACT GGC GGC GAG TCC TGT GAT GAG CTG GAG GAG GAC TGG ATC AGG GAA     1424
Thr Gly Gly Glu Ser Cys Asp Glu Leu Glu Glu Asp Trp Ile Arg Glu
    405                 410                 415
TAT CCA CCT ATC ACT TCA GAT CAA CAA AGA CAA CTG TAC AAG AGG AAT     1472
Tyr Pro Pro Ile Thr Ser Asp Gln Gln Arg Gln Leu Tyr Lys Arg Asn
420                 425                 430                 435
TTT GAC ACT GGC CTA CAG GAA TAC AAG AGC TTA CAA TCA GAA CTT GAT     1520
Phe Asp Thr Gly Leu Gln Glu Tyr Lys Ser Leu Gln Ser Glu Leu Asp
                440                 445                 450
GAG ATC AAT AAA GAA CTC TCC CGT TTG GAT AAA GAA TTG GAT GAC TAT     1568
Glu Ile Asn Lys Glu Leu Ser Arg Leu Asp Lys Glu Leu Asp Asp Tyr
            455                 460                 465
AGA GAA GAA AGT GAA GAG TAC ATG GCT GCT GCT GAT GAA TAC AAT AGA     1616
Arg Glu Glu Ser Glu Glu Tyr Met Ala Ala Ala Asp Glu Tyr Asn Arg
        470                 475                 480
CTG AAG CAA GTG AAG GGA TCT GCA GAT TAC AAA AGT AAG AAG AAT CAT     1664
Leu Lys Gln Val Lys Gly Ser Ala Asp Tyr Lys Ser Lys Lys Asn His
    485                 490                 495
TGC AAG CAG TTA AAG AGC AAA TTG TCA CAC ATC AAG AAG ATG GTT GGA     1712
Cys Lys Gln Leu Lys Ser Lys Leu Ser His Ile Lys Lys Met Val Gly
500                 505                 510                 515
GAC TAT GAT AGA CAG AAA ACA TAGAAGGCTG ATGCCAAGTT GTTTGAGAAA        1763
Asp Tyr Asp Arg Gln Lys Thr
                520
TTAAGTATCT GACATCTCTG CAATCTTCTC AGAAGGCAAA TGACTTTGGA CCATAACCCC   1823
GGAAGCCAAA CCTCTGTGAG CATCACAAAG TTTTGGTTGC TTTAACATCA TCAGTATTGA   1883
AGCATTTTAT AAATCGCTTT TGATAATCAA CTGGGCTGAA CACTCCAATT AAGGATTTTA   1943
TGCTTTAAAC ATTGGTTCTT GTATTAAGAA TGAAATACTG TTTGAGGTTT TTAAGCCTTA   2003
AAGGAAGGTT CTGGTGTGAA CTAAACTTTC ACACCCCAGA CGATGTCTTC ATACCTACAT   2063
GTATTTGTTT GCATAGGTGA TCTCATTTAA TCCTCTCAAC CACCTTTCAG ATAACTGTTA   2123
TTTATAATCA CTTTTTTCCA CATAAGGAAA CTGGGTTCCT GCAATGAAGT CTCTGAAGTG   2183
AAACTGCTTG TTTCCTAGCA CACACTTTTG GTTAAGTCTG TTTTATGACT TCATTAATAA   2243
TAAATTCCCT GGCCTTTCAT ATTTTAGCTA CTATATATGT GATGATCTAC CAGCCTCCCT   2303
ATTTTTTTTC TGTTATATAA ATGGTTAAAA GAGGTTTTTC TTAAATAATA AAGATCATGT   2363
AAAAGTAAAA AAAAAA                                                   2379
 
           
           
             
               522 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             2
Met Ser Ser Arg Pro Leu Glu Ser Pro Pro Pro Tyr Arg Pro Asp Glu
  1               5                  10                  15
Phe Lys Pro Asn His Tyr Ala Pro Ser Asn Asp Ile Tyr Gly Gly Glu
             20                  25                  30
Met His Val Arg Pro Met Leu Ser Gln Pro Ala Tyr Ser Phe Tyr Pro
         35                  40                  45
Glu Asp Glu Ile Leu His Phe Tyr Lys Trp Thr Ser Pro Pro Gly Val
     50                  55                  60
Ile Arg Ile Leu Ser Met Leu Ile Ile Val Met Cys Ile Ala Ile Phe
 65                  70                  75                  80
Ala Cys Val Ala Ser Thr Leu Ala Trp Asp Arg Gly Tyr Gly Thr Ser
                 85                  90                  95
Leu Leu Gly Gly Ser Val Gly Tyr Pro Tyr Gly Gly Ser Gly Phe Gly
            100                 105                 110
Ser Tyr Gly Ser Gly Tyr Gly Tyr Gly Tyr Gly Tyr Gly Tyr Gly Tyr
        115                 120                 125
Gly Gly Tyr Thr Asp Pro Arg Ala Ala Lys Gly Phe Met Leu Ala Met
    130                 135                 140
Ala Ala Phe Cys Phe Ile Ala Ala Leu Val Ile Phe Val Thr Ser Val
145                 150                 155                 160
Ile Arg Ser Glu Met Ser Arg Thr Arg Arg Tyr Tyr Leu Ser Val Ile
                165                 170                 175
Ile Val Ser Ala Ile Leu Gly Ile Met Val Phe Ile Ala Thr Ile Val
            180                 185                 190
Tyr Ile Met Gly Val Asn Pro Thr Ala Gln Ser Ser Gly Ser Leu Tyr
        195                 200                 205
Gly Ser Gln Ile Tyr Ala Leu Cys Asn Gln Phe Tyr Thr Pro Ala Ala
    210                 215                 220
Thr Gly Leu Tyr Val Asp Gln Tyr Leu Tyr His Tyr Cys Val Val Asp
225                 230                 235                 240
Pro Gln Glu Ala Ile Ala Ile Val Leu Gly Phe Met Ile Ile Val Ala
                245                 250                 255
Phe Ala Leu Ile Ile Phe Phe Ala Val Lys Thr Arg Arg Lys Met Asp
            260                 265                 270
Arg Tyr Asp Lys Ser Asn Ile Leu Trp Asp Lys Glu His Ile Tyr Asp
        275                 280                 285
Glu Gln Pro Pro Asn Val Glu Glu Trp Val Lys Asn Val Ser Ala Gly
    290                 295                 300
Thr Gln Asp Val Pro Ser Pro Pro Ser Asp Tyr Val Glu Arg Val Asp
305                 310                 315                 320
Ser Pro Met Ala Tyr Ser Ser Asn Gly Lys Val Asn Asp Lys Arg Phe
                325                 330                 335
Tyr Pro Glu Ser Ser Tyr Lys Ser Thr Pro Val Pro Glu Val Val Gln
            340                 345                 350
Glu Leu Pro Leu Thr Ser Pro Val Asp Asp Phe Arg Gln Pro Arg Tyr
        355                 360                 365
Ser Ser Gly Gly Asn Phe Glu Thr Pro Ser Lys Arg Ala Pro Ala Lys
    370                 375                 380
Gly Arg Ala Gly Arg Ser Lys Arg Thr Glu Gln Asp His Tyr Glu Thr
385                 390                 395                 400
Asp Tyr Thr Thr Gly Gly Glu Ser Cys Asp Glu Leu Glu Glu Asp Trp
                405                 410                 415
Ile Arg Glu Tyr Pro Pro Ile Thr Ser Asp Gln Gln Arg Gln Leu Tyr
            420                 425                 430
Lys Arg Asn Phe Asp Thr Gly Leu Gln Glu Tyr Lys Ser Leu Gln Ser
        435                 440                 445
Glu Leu Asp Glu Ile Asn Lys Glu Leu Ser Arg Leu Asp Lys Glu Leu
    450                 455                 460
Asp Asp Tyr Arg Glu Glu Ser Glu Glu Tyr Met Ala Ala Ala Asp Glu
465                 470                 475                 480
Tyr Asn Arg Leu Lys Gln Val Lys Gly Ser Ala Asp Tyr Lys Ser Lys
                485                 490                 495
Lys Asn His Cys Lys Gln Leu Lys Ser Lys Leu Ser His Ile Lys Lys
            500                 505                 510
Met Val Gly Asp Tyr Asp Arg Gln Lys Thr
        515                 520
 
           
           
             
               1961 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               cDNA 
             
             
               Canine 
               MDCK
           
               Kidney cell 
             
             
               CDS 
                72..1634
 
             
             3
CAGGTTGGCT TATTTTGGGG AGCTCTGGGA TCCTGCTCGT CCTGAAGATC GGGTGATCAT     60
TGACATCAGC C ATG TCA TCG AGG CCT TTT GAG AGT CCA CCT CCG TAT AGA     110
             Met Ser Ser Arg Pro Phe Glu Ser Pro Pro Pro Tyr Arg
               1               5                  10
CCT GAT GAA TTC AAA CCC AAT CAT TAT GCA CCG AGC AAT GAT GTG TAC      158
Pro Asp Glu Phe Lys Pro Asn His Tyr Ala Pro Ser Asn Asp Val Tyr
     15                  20                  25
GGT GGG GAC ATG CAC GTC CGA CCC ATG CTC TCT CAG CCG GCG TAT TCT      206
Gly Gly Asp Met His Val Arg Pro Met Leu Ser Gln Pro Ala Tyr Ser
 30                  35                  40                  45
TTC TAC CCA GAA GAT GAA ATT CTT CAC TTC TAC AAA TGG ACC TCT CCT      254
Phe Tyr Pro Glu Asp Glu Ile Leu His Phe Tyr Lys Trp Thr Ser Pro
                 50                  55                  60
CCA GGA GTA ATT CGG ATT CTG TCC ATG CTT GTC ATT GTG ATG TGC ATC      302
Pro Gly Val Ile Arg Ile Leu Ser Met Leu Val Ile Val Met Cys Ile
             65                  70                  75
GCC ATA TTT GGC TGT GTC GCG TCC ACG CTC GCC TGG GAT AGA GGC TAT      350
Ala Ile Phe Gly Cys Val Ala Ser Thr Leu Ala Trp Asp Arg Gly Tyr
         80                  85                  90
GGA ACT GGC TTA ATG GGT GGT AGC ATA GGC TAC CCT TAC GGA AGT GGC      398
Gly Thr Gly Leu Met Gly Gly Ser Ile Gly Tyr Pro Tyr Gly Ser Gly
     95                 100                 105
TTC GGG AGC TAC GGG ACT GGC TAC GGC TAC GGG TTT GGC TAC GGC TAC      446
Phe Gly Ser Tyr Gly Thr Gly Tyr Gly Tyr Gly Phe Gly Tyr Gly Tyr
110                 115                 120                 125
GGC TAC GGC GGC TAC ACG GAT CCC AGA GCA GCA AAG GGC TTC CTC CTG      494
Gly Tyr Gly Gly Tyr Thr Asp Pro Arg Ala Ala Lys Gly Phe Leu Leu
                130                 135                 140
GCC ATG GTG GCC TTT TGT TTT ATC GCT GCA TTG GTG ATA TTT GTT ACC      542
Ala Met Val Ala Phe Cys Phe Ile Ala Ala Leu Val Ile Phe Val Thr
            145                 150                 155
AGC GTT ATA AGG TCT GAC ATA TCC AGA ACC AGA AGG TAC TAC TTG ACT      590
Ser Val Ile Arg Ser Asp Ile Ser Arg Thr Arg Arg Tyr Tyr Leu Thr
        160                 165                 170
GTA ATA ATA CTG AGT GCC TTC CTG GGC GTC ATG ATG TTC ATT GCT ACA      638
Val Ile Ile Leu Ser Ala Phe Leu Gly Val Met Met Phe Ile Ala Thr
    175                 180                 185
ATT GTC TAT ATA ATG GGA GTC AAT CCA ACT GCC CAG GCT TCT GGG TCT      686
Ile Val Tyr Ile Met Gly Val Asn Pro Thr Ala Gln Ala Ser Gly Ser
190                 195                 200                 205
TTA TAC AGT TCA CAG ATA TAT GCC ATG TGC AAC CAG TTC TAT GCA TCT      734
Leu Tyr Ser Ser Gln Ile Tyr Ala Met Cys Asn Gln Phe Tyr Ala Ser
                210                 215                 220
ACA GCT ACC GGA CTC TAC ATG GAT CAG TAT TTG TAT CAC TAC TGT GTG      782
Thr Ala Thr Gly Leu Tyr Met Asp Gln Tyr Leu Tyr His Tyr Cys Val
            225                 230                 235
GTG GAT CCC CAA GAG GCA ATT GCC ATT GTC CTG GGA TTC ATG GTG ATT      830
Val Asp Pro Gln Glu Ala Ile Ala Ile Val Leu Gly Phe Met Val Ile
        240                 245                 250
GTG GCT TTT GCT TTA ATA ATT TTC TTT GCT GTG AAA ACT CGA AGA AAG      878
Val Ala Phe Ala Leu Ile Ile Phe Phe Ala Val Lys Thr Arg Arg Lys
    255                 260                 265
ATG GAC CGG TAT GAC AAG TCG AAT ATA TTG TGG GAC AAG GAA CAT ATT      926
Met Asp Arg Tyr Asp Lys Ser Asn Ile Leu Trp Asp Lys Glu His Ile
270                 275                 280                 285
TAT GAT GAA CAA CCC CCC AAT GTT GAA GAG TGG GTT AAA AAC GTT TCT      974
Tyr Asp Glu Gln Pro Pro Asn Val Glu Glu Trp Val Lys Asn Val Ser
                290                 295                 300
GCA GGC ACA CAA GAC ATG CCT CCT CCC CCT TCT GAC TAT GTG GAG AGA     1022
Ala Gly Thr Gln Asp Met Pro Pro Pro Pro Ser Asp Tyr Val Glu Arg
            305                 310                 315
GTG GAC AGT CCC ATG GCG TAC TCT TCC AAT GGT AAA GTG AAT GAC AAG     1070
Val Asp Ser Pro Met Ala Tyr Ser Ser Asn Gly Lys Val Asn Asp Lys
        320                 325                 330
CGG TTG TAT CCA GAG TCT TCC TAT AAA TCA ACA CCG GTC CCC GAA GTG     1118
Arg Leu Tyr Pro Glu Ser Ser Tyr Lys Ser Thr Pro Val Pro Glu Val
    335                 340                 345
GTG CAG GAG CTG CCC GCC ACC TCC CCT GCG GAT GAC TTC AGG CAG CCT     1166
Val Gln Glu Leu Pro Ala Thr Ser Pro Ala Asp Asp Phe Arg Gln Pro
350                 355                 360                 365
CGC TAC AGC AGC AGC GGG CAC TTG GAG CCA CCT TCG AAG AGG GCC CCC     1214
Arg Tyr Ser Ser Ser Gly His Leu Glu Pro Pro Ser Lys Arg Ala Pro
                370                 375                 380
TCG AAA GGA AGA ACG GGA AGG CCC AAG AGG CTG GAG CAG GAC CAC TAT     1262
Ser Lys Gly Arg Thr Gly Arg Pro Lys Arg Leu Glu Gln Asp His Tyr
            385                 390                 395
GAG ACA GAC TAC ACG ACG GGC GGC GAG TCG TGT GAC GAG CTG GAG GAG     1310
Glu Thr Asp Tyr Thr Thr Gly Gly Glu Ser Cys Asp Glu Leu Glu Glu
        400                 405                 410
GAC TGG ATC AGG GAA TAT CCA CCT ATC ACT TCA GAT CAA CAA AGA CAA     1358
Asp Trp Ile Arg Glu Tyr Pro Pro Ile Thr Ser Asp Gln Gln Arg Gln
    415                 420                 425
CTC TAC AAG AGA AAT TTT GAC ACT GGC CTG CAG GAA TAC AAG AGC TTA     1406
Leu Tyr Lys Arg Asn Phe Asp Thr Gly Leu Gln Glu Tyr Lys Ser Leu
430                 435                 440                 445
CAA GCA GAA CTT GAT GAG ATC AAT AAA GAA CTC TCT CGC CTG GAT AAA     1454
Gln Ala Glu Leu Asp Glu Ile Asn Lys Glu Leu Ser Arg Leu Asp Lys
                450                 455                 460
GAA TTG GAT GAC TAT AGA GAA GAA AGT GAA GAG TAC ATG GCT GCT GCT     1502
Glu Leu Asp Asp Tyr Arg Glu Glu Ser Glu Glu Tyr Met Ala Ala Ala
            465                 470                 475
GAT GAG TAC AAT AGA CTG AAG CAA GTT AAG GGA TCT CCA GAT TAC AAA     1550
Asp Glu Tyr Asn Arg Leu Lys Gln Val Lys Gly Ser Pro Asp Tyr Lys
        480                 485                 490
AAT AAG AGG AAT TAT TGC AAG CAG TTG AAG AGC AAA TTG TCC CAC ATC     1598
Asn Lys Arg Asn Tyr Cys Lys Gln Leu Lys Ser Lys Leu Ser His Ile
    495                 500                 505
AAG AAG ATG GTT GGA GAC TAT GAT AGA CAG AAA ACA TAGAAGGCAG          1644
Lys Lys Met Val Gly Asp Tyr Asp Arg Gln Lys Thr
510                 515                 520
ATGCCACACA GTTTGAGAGA TTGTGAAGTA TTTGACATAT CTGCAACGTT GTCAGAAGGC   1704
AGAATGACTT TGGATTTCGA ACCCAGGAGG CCAGATCTTT GTGATCATTA CAAAGTTTTG   1764
GTAGCTTTAA TATCATCAGT ATTGAAGCAT TTTACACATA GCTTTTGATA ATCAACTGGG   1824
CTGAACACTC CCGATTAAGG ATTCTGTGCT TTAGACTTTG GCTGTTGTGC TAAAGGACTG   1884
AGTATAGGTG GAGGTTTTCA GACCTTGGAA GAAGGTCCCA CGGTGAACTT GTGCTGTGAA   1944
CTTGCACACT TGGGGCA                                                  1961
 
           
           
             
               521 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             4
Met Ser Ser Arg Pro Phe Glu Ser Pro Pro Pro Tyr Arg Pro Asp Glu
  1               5                  10                  15
Phe Lys Pro Asn His Tyr Ala Pro Ser Asn Asp Val Tyr Gly Gly Asp
             20                  25                  30
Met His Val Arg Pro Met Leu Ser Gln Pro Ala Tyr Ser Phe Tyr Pro
         35                  40                  45
Glu Asp Glu Ile Leu His Phe Tyr Lys Trp Thr Ser Pro Pro Gly Val
     50                  55                  60
Ile Arg Ile Leu Ser Met Leu Val Ile Val Met Cys Ile Ala Ile Phe
 65                  70                  75                  80
Gly Cys Val Ala Ser Thr Leu Ala Trp Asp Arg Gly Tyr Gly Thr Gly
                 85                  90                  95
Leu Met Gly Gly Ser Ile Gly Tyr Pro Tyr Gly Ser Gly Phe Gly Ser
            100                 105                 110
Tyr Gly Thr Gly Tyr Gly Tyr Gly Phe Gly Tyr Gly Tyr Gly Tyr Gly
        115                 120                 125
Gly Tyr Thr Asp Pro Arg Ala Ala Lys Gly Phe Leu Leu Ala Met Val
    130                 135                 140
Ala Phe Cys Phe Ile Ala Ala Leu Val Ile Phe Val Thr Ser Val Ile
145                 150                 155                 160
Arg Ser Asp Ile Ser Arg Thr Arg Arg Tyr Tyr Leu Thr Val Ile Ile
                165                 170                 175
Leu Ser Ala Phe Leu Gly Val Met Met Phe Ile Ala Thr Ile Val Tyr
            180                 185                 190
Ile Met Gly Val Asn Pro Thr Ala Gln Ala Ser Gly Ser Leu Tyr Ser
        195                 200                 205
Ser Gln Ile Tyr Ala Met Cys Asn Gln Phe Tyr Ala Ser Thr Ala Thr
    210                 215                 220
Gly Leu Tyr Met Asp Gln Tyr Leu Tyr His Tyr Cys Val Val Asp Pro
225                 230                 235                 240
Gln Glu Ala Ile Ala Ile Val Leu Gly Phe Met Val Ile Val Ala Phe
                245                 250                 255
Ala Leu Ile Ile Phe Phe Ala Val Lys Thr Arg Arg Lys Met Asp Arg
            260                 265                 270
Tyr Asp Lys Ser Asn Ile Leu Trp Asp Lys Glu His Ile Tyr Asp Glu
        275                 280                 285
Gln Pro Pro Asn Val Glu Glu Trp Val Lys Asn Val Ser Ala Gly Thr
    290                 295                 300
Gln Asp Met Pro Pro Pro Pro Ser Asp Tyr Val Glu Arg Val Asp Ser
305                 310                 315                 320
Pro Met Ala Tyr Ser Ser Asn Gly Lys Val Asn Asp Lys Arg Leu Tyr
                325                 330                 335
Pro Glu Ser Ser Tyr Lys Ser Thr Pro Val Pro Glu Val Val Gln Glu
            340                 345                 350
Leu Pro Ala Thr Ser Pro Ala Asp Asp Phe Arg Gln Pro Arg Tyr Ser
        355                 360                 365
Ser Ser Gly His Leu Glu Pro Pro Ser Lys Arg Ala Pro Ser Lys Gly
    370                 375                 380
Arg Thr Gly Arg Pro Lys Arg Leu Glu Gln Asp His Tyr Glu Thr Asp
385                 390                 395                 400
Tyr Thr Thr Gly Gly Glu Ser Cys Asp Glu Leu Glu Glu Asp Trp Ile
                405                 410                 415
Arg Glu Tyr Pro Pro Ile Thr Ser Asp Gln Gln Arg Gln Leu Tyr Lys
            420                 425                 430
Arg Asn Phe Asp Thr Gly Leu Gln Glu Tyr Lys Ser Leu Gln Ala Glu
        435                 440                 445
Leu Asp Glu Ile Asn Lys Glu Leu Ser Arg Leu Asp Lys Glu Leu Asp
    450                 455                 460
Asp Tyr Arg Glu Glu Ser Glu Glu Tyr Met Ala Ala Ala Asp Glu Tyr
465                 470                 475                 480
Asn Arg Leu Lys Gln Val Lys Gly Ser Pro Asp Tyr Lys Asn Lys Arg
                485                 490                 495
Asn Tyr Cys Lys Gln Leu Lys Ser Lys Leu Ser His Ile Lys Lys Met
            500                 505                 510
Val Gly Asp Tyr Asp Arg Gln Lys Thr
        515                 520
 
           
           
             
               2839 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               cDNA 
             
             
               Mouse 
               Lung cell 
             
             
               CDS 
                223..1785
 
             
             5
GGAGTTTCAG GTGAATGGGT CACCGAGGGA GGAGGCTGGC CACGCCACAC CTCGTCGCTA     60
GTGCCCACCT CCCGGCCCCT CTTTCCTTAG GCGACAGCGG TGGAGTTGCG GGAGAGCGGT    120
CCAGCGCACG GAGCAACCGG CTAGGGGCTC GGCAGGTTCG CTTATCTTGG GAGCCTGGAC    180
ATTTTGCTCA TCATAAAGAT TAGGTGACCA GTGACATCAG CC ATG TCC GTG AGG       234
                                               Met Ser Val Arg
                                                 1
CCT TTT GAA AGT CCA CCT CCT TAC AGA CCT GAT GAA TTC AAA CCC AAT      282
Pro Phe Glu Ser Pro Pro Pro Tyr Arg Pro Asp Glu Phe Lys Pro Asn
  5                  10                  15                  20
CAT TAT GCA CCA AGC AAT GAC ATG TAT GGC GGA GAG ATG CAT GTC CGG      330
His Tyr Ala Pro Ser Asn Asp Met Tyr Gly Gly Glu Met His Val Arg
                 25                  30                  35
CCG ATG CTC TCT CAG CCA GCG TAC TCT TTT TAT CCG GAA GAT GAA ATT      378
Pro Met Leu Ser Gln Pro Ala Tyr Ser Phe Tyr Pro Glu Asp Glu Ile
             40                  45                  50
CTT CAC TTC TAC AAA TGG ACG TCG CCC CCA GGG GTG ATC CGG ATC CTG      426
Leu His Phe Tyr Lys Trp Thr Ser Pro Pro Gly Val Ile Arg Ile Leu
         55                  60                  65
TCT ATG CTC ATT ATT GTG ATG TGC ATC GCC ATA TTT GCC TGT GTG GCT      474
Ser Met Leu Ile Ile Val Met Cys Ile Ala Ile Phe Ala Cys Val Ala
     70                  75                  80
TCC ACA CTT GCT TGG GAC AGA GGC TAT GGG ACA GGG CTC TTT GGA GGA      522
Ser Thr Leu Ala Trp Asp Arg Gly Tyr Gly Thr Gly Leu Phe Gly Gly
 85                  90                  95                 100
AGC CTA AAC TAC CCT TAT AGT GGC TTT GGC TAC GGA GGT GGC TAT GGA      570
Ser Leu Asn Tyr Pro Tyr Ser Gly Phe Gly Tyr Gly Gly Gly Tyr Gly
                105                 110                 115
GGC GGC TAT GGA GGC TAT GGC TAT GGC TAT GGC GGA TAT ACA GAC CCA      618
Gly Gly Tyr Gly Gly Tyr Gly Tyr Gly Tyr Gly Gly Tyr Thr Asp Pro
            120                 125                 130
AGA GCA GCC AAA GGC TTC CTG TTG GCC ATG GCA GCC TTC TGC TTC ATC      666
Arg Ala Ala Lys Gly Phe Leu Leu Ala Met Ala Ala Phe Cys Phe Ile
        135                 140                 145
GCT TCC TTA GTA ATA TTT GTG ACC AGT GTT ATA AGA TCT GGA ATG TCC      714
Ala Ser Leu Val Ile Phe Val Thr Ser Val Ile Arg Ser Gly Met Ser
    150                 155                 160
AGG ACA AGA AGA TAT TAC TTG ATC GTG ATC ATA GTC AGC GCT ATC CTG      762
Arg Thr Arg Arg Tyr Tyr Leu Ile Val Ile Ile Val Ser Ala Ile Leu
165                 170                 175                 180
GGC ATC ATG GTG TTT ATT GCC ACG ATC GTG TAC ATA ATG GGA GTG AAC      810
Gly Ile Met Val Phe Ile Ala Thr Ile Val Tyr Ile Met Gly Val Asn
                185                 190                 195
CCG ACG GCC CAG GCT TCT GGA TCT ATG TAC GGC TCA CAG ATA TAT ATG      858
Pro Thr Ala Gln Ala Ser Gly Ser Met Tyr Gly Ser Gln Ile Tyr Met
            200                 205                 210
ATC TGC AAC CAG TTT TAT ACT CCT GGA GGT ACT GGT CTC TAC GTG GAT      906
Ile Cys Asn Gln Phe Tyr Thr Pro Gly Gly Thr Gly Leu Tyr Val Asp
        215                 220                 225
CAA TAT TTG TAT CAC TAC TGT GTG GTT GAT CCC CAG GAG GCT ATA GCC      954
Gln Tyr Leu Tyr His Tyr Cys Val Val Asp Pro Gln Glu Ala Ile Ala
    230                 235                 240
ATT GTC CTG GGG TTC ATG ATT ATC GTG GCT TTT GCT TTA ATC ATC TTT     1002
Ile Val Leu Gly Phe Met Ile Ile Val Ala Phe Ala Leu Ile Ile Phe
245                 250                 255                 260
TTT GCT GTG AAA ACC CGA AGA AAG ATG GAT CGG TAT GAT AAG TCC AAT     1050
Phe Ala Val Lys Thr Arg Arg Lys Met Asp Arg Tyr Asp Lys Ser Asn
                265                 270                 275
ATT TTG TGG GAT AAG GAA CAC ATT TAT GAT GAA CAG CCC CCC AAT GTT     1098
Ile Leu Trp Asp Lys Glu His Ile Tyr Asp Glu Gln Pro Pro Asn Val
            280                 285                 290
GAA GAG TGG GTT AAA AAT GTG TCT GCA GGC ACA CAG GAC ATG CCT CCA     1146
Glu Glu Trp Val Lys Asn Val Ser Ala Gly Thr Gln Asp Met Pro Pro
        295                 300                 305
CCC CCA TCT GAC TAT GCG GAA AGA GTT GAC AGT CCA ATG GCC TAC TCC     1194
Pro Pro Ser Asp Tyr Ala Glu Arg Val Asp Ser Pro Met Ala Tyr Ser
    310                 315                 320
TCC AAT GGC AAA GTG AAT GGC AAG CGA TCA TAC CCA GAG TCT TTC TAT     1242
Ser Asn Gly Lys Val Asn Gly Lys Arg Ser Tyr Pro Glu Ser Phe Tyr
325                 330                 335                 340
AAG TCA ACA CCT CTG GTG CCT GAA GTG GCC CAG GAG ATT CCT CTG ACC     1290
Lys Ser Thr Pro Leu Val Pro Glu Val Ala Gln Glu Ile Pro Leu Thr
                345                 350                 355
TTG AGT GTG GAT GAC TTC AGG CAG CCT CGG TAC AGC AGC AAT GGT AAC     1338
Leu Ser Val Asp Asp Phe Arg Gln Pro Arg Tyr Ser Ser Asn Gly Asn
            360                 365                 370
CTA GAG ACA CCT TCT AAA AGG GCT CCC ACG AAG GGG AAA GCA GGA AAG     1386
Leu Glu Thr Pro Ser Lys Arg Ala Pro Thr Lys Gly Lys Ala Gly Lys
        375                 380                 385
GGC AAG AGG ACG GAC CCT GAC CAC TAT GAA ACA GAC TAC ACG ACA GGT     1434
Gly Lys Arg Thr Asp Pro Asp His Tyr Glu Thr Asp Tyr Thr Thr Gly
    390                 395                 400
GGG GAG TCC TGC GAG GAG CTG GAG GAG GAC TGG GTC AGG GAA TAT CCA     1482
Gly Glu Ser Cys Glu Glu Leu Glu Glu Asp Trp Val Arg Glu Tyr Pro
405                 410                 415                 420
CCT ATC ACT TCA GAT CAA CAA AGA CAA CTC TAC AAG AGA AAT TTT GAT     1530
Pro Ile Thr Ser Asp Gln Gln Arg Gln Leu Tyr Lys Arg Asn Phe Asp
                425                 430                 435
GCA GGT CTG CAG GAG TAT AAG AGC TTA CAG GCA GAA CTA GAC GAC GTC     1578
Ala Gly Leu Gln Glu Tyr Lys Ser Leu Gln Ala Glu Leu Asp Asp Val
            440                 445                 450
AAT AAA GAG CTC TCT CGT CTA GAT AAA GAG CTG GAT GAC TAC AGA GAG     1626
Asn Lys Glu Leu Ser Arg Leu Asp Lys Glu Leu Asp Asp Tyr Arg Glu
        455                 460                 465
GAG AGT GAA GAG TAC ATG GCT GCT GCT GAT GAA TAT AAT AGA CTA AAG     1674
Glu Ser Glu Glu Tyr Met Ala Ala Ala Asp Glu Tyr Asn Arg Leu Lys
    470                 475                 480
CAA GTT AAG GGA TCT GCA GAT TAT AAA AGT AAG AGG AAT TAC TGC AAG     1722
Gln Val Lys Gly Ser Ala Asp Tyr Lys Ser Lys Arg Asn Tyr Cys Lys
485                 490                 495                 500
CAG TTG AAG AGC AAA TTA TCG CAC ATC AAG AGG ATG GTG GGA GAC TAT     1770
Gln Leu Lys Ser Lys Leu Ser His Ile Lys Arg Met Val Gly Asp Tyr
                505                 510                 515
GAC AGA CGG AAA CCT TAGAGAGATG CCAGTTGCGG GAGAAGGGAG AGGTGCATCT     1825
Asp Arg Arg Lys Pro
            520
GCCTGCACGA TGTCTCTGCA ATTCTCTCCA GAGGCAAACT GACTTTGGAC TCTAATCTGG   1885
GAAGTTAAAA CTTTGTGATC ATTACAAAGT TTCCATGGCT TTAATTCCAT CAGTTTCCTA   1945
TCTCCAGTAT TGAAGCATTT TATAAATGGC TTTTGATAAT TGACTGGGCT GAACACTCCA   2005
ATTAAGGATT TTACAGTTTC AACATTGATT CTTGTATTAA GAATTAAAAT GTTGCTTGAG   2065
GTTTTAAATG TCAAGAAAGG TCCTGGTGTG AGCTGTGATG TGTGTGAGCT GTGATGTGAA   2125
GGTTCACACG CCAGGCAGCG TGTTCCTCCA GGTAGACCGT CTAATCAATC TTTGCAGCAG   2185
CCCTCAGGTG ACTGTTATTT AGAATCAGGT TGTTTTTGGT TTTCCAGACA GGGTTTCTCT   2245
GTGTAGCCCT GGCTGACCTA GAACTTACGC TGTAGACCAG GCTGGCCTTG AACTCACACA   2305
GCTCCTCTGA GTGCTGGTGC AGGAGTTAAC GTCGTGGACC GGTATCATCA CTTTTCCTGC   2365
GGTGACTTCT CCAAACTGAA ACTGCTAAGG CAGTTTTGGC TAAGTCTGTT TTATGACTGC   2425
AAATGACAGC ATTCCTGCCT TTGTATTTCA GGGGAAATAC GATACATTAT ATCGGCCATG   2485
TTCCCCACCA CTGTTTTTCT TATATTGACT TTTAACAAAT GAATAGGATT ATTTTTGGCT   2545
TTACATTTTT TCCTAACACT TAAGATCATA TAAAATTAAC AAATATGTGA AATTTAAGAA   2605
TTGTAAATAT ATATTTACGT TTGAAAGATG ATTTTAAATC CAGGGTTAAA GTGCTTTTTA   2665
TCTTGTATAG TTTACATGCT TTTTTTTTTT TTTGATAACC CACTAGACCT TTCCATTGTA   2725
TCAGAGTATC CAATTACATT TACAATTATG ACTTGAATTG TATTTCACAG GAATGCTCAA   2785
GTTTTGTACA TATTTTATAA GGTATTAAAC CTGATGTTCT CTTTCTAAAA AAAA         2839
 
           
           
             
               521 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             6
Met Ser Val Arg Pro Phe Glu Ser Pro Pro Pro Tyr Arg Pro Asp Glu
  1               5                  10                  15
Phe Lys Pro Asn His Tyr Ala Pro Ser Asn Asp Met Tyr Gly Gly Glu
             20                  25                  30
Met His Val Arg Pro Met Leu Ser Gln Pro Ala Tyr Ser Phe Tyr Pro
         35                  40                  45
Glu Asp Glu Ile Leu His Phe Tyr Lys Trp Thr Ser Pro Pro Gly Val
     50                  55                  60
Ile Arg Ile Leu Ser Met Leu Ile Ile Val Met Cys Ile Ala Ile Phe
 65                  70                  75                  80
Ala Cys Val Ala Ser Thr Leu Ala Trp Asp Arg Gly Tyr Gly Thr Gly
                 85                  90                  95
Leu Phe Gly Gly Ser Leu Asn Tyr Pro Tyr Ser Gly Phe Gly Tyr Gly
            100                 105                 110
Gly Gly Tyr Gly Gly Gly Tyr Gly Gly Tyr Gly Tyr Gly Tyr Gly Gly
        115                 120                 125
Tyr Thr Asp Pro Arg Ala Ala Lys Gly Phe Leu Leu Ala Met Ala Ala
    130                 135                 140
Phe Cys Phe Ile Ala Ser Leu Val Ile Phe Val Thr Ser Val Ile Arg
145                 150                 155                 160
Ser Gly Met Ser Arg Thr Arg Arg Tyr Tyr Leu Ile Val Ile Ile Val
                165                 170                 175
Ser Ala Ile Leu Gly Ile Met Val Phe Ile Ala Thr Ile Val Tyr Ile
            180                 185                 190
Met Gly Val Asn Pro Thr Ala Gln Ala Ser Gly Ser Met Tyr Gly Ser
        195                 200                 205
Gln Ile Tyr Met Ile Cys Asn Gln Phe Tyr Thr Pro Gly Gly Thr Gly
    210                 215                 220
Leu Tyr Val Asp Gln Tyr Leu Tyr His Tyr Cys Val Val Asp Pro Gln
225                 230                 235                 240
Glu Ala Ile Ala Ile Val Leu Gly Phe Met Ile Ile Val Ala Phe Ala
                245                 250                 255
Leu Ile Ile Phe Phe Ala Val Lys Thr Arg Arg Lys Met Asp Arg Tyr
            260                 265                 270
Asp Lys Ser Asn Ile Leu Trp Asp Lys Glu His Ile Tyr Asp Glu Gln
        275                 280                 285
Pro Pro Asn Val Glu Glu Trp Val Lys Asn Val Ser Ala Gly Thr Gln
    290                 295                 300
Asp Met Pro Pro Pro Pro Ser Asp Tyr Ala Glu Arg Val Asp Ser Pro
305                 310                 315                 320
Met Ala Tyr Ser Ser Asn Gly Lys Val Asn Gly Lys Arg Ser Tyr Pro
                325                 330                 335
Glu Ser Phe Tyr Lys Ser Thr Pro Leu Val Pro Glu Val Ala Gln Glu
            340                 345                 350
Ile Pro Leu Thr Leu Ser Val Asp Asp Phe Arg Gln Pro Arg Tyr Ser
        355                 360                 365
Ser Asn Gly Asn Leu Glu Thr Pro Ser Lys Arg Ala Pro Thr Lys Gly
    370                 375                 380
Lys Ala Gly Lys Gly Lys Arg Thr Asp Pro Asp His Tyr Glu Thr Asp
385                 390                 395                 400
Tyr Thr Thr Gly Gly Glu Ser Cys Glu Glu Leu Glu Glu Asp Trp Val
                405                 410                 415
Arg Glu Tyr Pro Pro Ile Thr Ser Asp Gln Gln Arg Gln Leu Tyr Lys
            420                 425                 430
Arg Asn Phe Asp Ala Gly Leu Gln Glu Tyr Lys Ser Leu Gln Ala Glu
        435                 440                 445
Leu Asp Asp Val Asn Lys Glu Leu Ser Arg Leu Asp Lys Glu Leu Asp
    450                 455                 460
Asp Tyr Arg Glu Glu Ser Glu Glu Tyr Met Ala Ala Ala Asp Glu Tyr
465                 470                 475                 480
Asn Arg Leu Lys Gln Val Lys Gly Ser Ala Asp Tyr Lys Ser Lys Arg
                485                 490                 495
Asn Tyr Cys Lys Gln Leu Lys Ser Lys Leu Ser His Ile Lys Arg Met
            500                 505                 510
Val Gly Asp Tyr Asp Arg Arg Lys Pro
515 520
 
           
           
             
               33 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               other nucleic acid 
               /desc = “Synthesis DNA” 
             
             7
TATGAGACAG ACTACACAAC TGGCGGCGAG TCC                                  33
 
           
           
             
               30 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               other nucleic acid 
               /desc = “Synthesis DNA” 
             
             8
ATCATAGTCT CCAACCATCT TCTTGATGTG                                      30