Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-11-05
2004-01-20
Kunz, Gary (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S827000, C530S835000
Reexamination Certificate
active
06680370
ABSTRACT:
TECHNICAL FIELD
The present invention belongs to the field of genetic engineering and specifically relates to isolation of a gene of renal cells.
BACKGROUND ART
Sixty trillion various cells in vivo essentially comprise identical genomic DNA. For the normal physiological functions, the expression of these genes is strictly controlled by signals received by cell lines and cells. Therefore, elucidation of genes expressed in each cell type is very important.
A mesangial cell plays a pivotal role in maintaining the structure and function of a glomerulus and also has a central meaning of pathophysiology for each type of nephritis. For example, proliferation of mesangial cells and accumulation of extracellular mesangial matrix are thought to be important pathological finding of glomerulosclerosis in a patient suffering from various glomerular diseases such as chronic nephritis and diabetic nephropathy.
Therefore, identification of genes expressed specifically in mesangial cells and elucidation of its function are helpful for understanding biological characteristics of mesangial cells and the causes of diseases relating to mesangial cells, and in turn, treating or diagnosing diseases relating to mesangial cells.
Thy1 antigen is known as a marker for mesangial cells in rats. However, this gene is not specific to mesangial cells and is not expressed in human mesangial cells (Miyata T. et al., Immunology, 1989,67: 531-533; and Miyata T. et al., Immunology, 1990, 69: 391-395). Mesangial cells are known to express &agr; smooth muscle actin when activated, but this gene is also not specific to mesangial cells. Any genes characteristically in mesangial cells have not been reported.
The present inventor has previously reported MEGSIN as a protein that is expressed specifically in the mesangial cells (J. Clin. Invest, Aug. 15, 1998 102: 4, 828-36). The present invention relates to a novel protein having a structure that is distinctly different from the MEGSIN.
DISCLOSURE OF THE INVENTION
An objective of the present invention is to isolate a gene highly expressed in mesangial cells.
The current inventor isolated mRNA from in vitro cultures of human mesangial cells to construct a cDNA library of 3′ side. Sequences of numerous clones were randomly determined from the cDNA library and compared with the known nucleotide sequences of cDNA clones of 3′ side obtained from various organs and cells to determine the clones expressed in mesangial cells. One of the clones, which appeared with high frequency in the mesangial cells, was selected. Furthermore, the &lgr;ZIPLox cDNA library prepared from mesangial cells using the insert of this clone as a probe was screened to determine the nucleotide sequence of the positive clone. Based on the determined cDNA nucleotide sequence, the amino acid sequence of the longest open reading frame was elucidated. Several characteristic motifs were found in this amino acid sequence. Since homology was confirmed with mouse ATP dependent metalloprotease (ATP-MP), a known protein, this amino acid sequence was presumed to be the amino acid sequence of the protein encoded by the cDNA of this invention. The protein of this invention having this amino acid sequence was named Meg-4by the present inventor. The nucleotide sequence of human Meg-4 cDNA and the deduced amino acid sequence for human Meg-4 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
An amino acid sequence homology search performed on this amino acid sequence using the SwissProt database confirmed that Meg-4 is a novel protein containing an AAA motif (Walker, J. E. et al., EMBO J. 1982, 8: 945-951; Swaffield, J. C. et al., Nature, 374: 88-91; Fry, D. C. et al., Proc. Natl. Acad. Sci. USA. 1997, 83: 907-911; Frohlich, K. U. et al., J. Cell. Biol. 1991, 114:443-453) characteristic of AAA protein family (ATPases associated with different cellular activities protein family). Furthermore, when the topography of Meg-4 was observed by Northern blotting, expression of Meg-4 was hardly observed in the human lung and liver, and expression was observed in the kidney as well as in other tissues, such as the heart, brain, placenta, skeletal muscle, and pancreas. At the cellular level, especially high levels of expression in the mesangial cells was characteristic. Expression was also observed in fibroblasts and epithelial cells. This invention was completed based on these findings.
This invention specifically includes the following:
(1) A protein comprising the amino acid sequence of SEQ ID NO: 2, or a protein comprising the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are replaced, deleted, added, and/or inserted, and being functionally equivalent to the protein comprising the amino acid sequence of SEQ ID NO: 2.
(2) The protein of (1), wherein the protein comprises an amino acid sequence that has not less than 90% homology to the amino acid sequence of SEQ ID NO: 2.
(3) The protein of (1), wherein the protein comprises the amino acid sequence of SEQ ID NO: 2.
(4) A DNA encoding the protein of (1).
(5) The DNA of (4), wherein the DNA comprises a nucleotide sequence that has not less than 85% homology to the nucleotide sequence of SEQ ID NO: 1.
(6) The DNA of (5), wherein the DNA comprises a protein coding region in the nucleotide sequence of SEQ ID NO: 1.
(7) A DNA encoding the protein of (1), the DNA hybridizing under stringent conditions with DNA comprising the nucleotide sequence of SEQ ID NO: 1.
(8) A DNA hybridizing specifically with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or with the complementary strand thereof, the DNA having a chain length of at least 15 nucleotides.
(9) An antisense DNA against the DNA of (6) or a portion thereof.
(10) A vector comprising the DNA of any one of (4), (5), (6), and (7).
(11) A transformant expressively carrying the DNA of any one of (4) (5), (6), and (7).
(12) A method for producing the protein of (1), the method comprising culturing the transformant of (11) and collecting an expression product of the DNA of any one of (4), (5), (6), and (7).
(13) An antibody binding to the protein of (1).
(14) The antibody of (13), wherein the antibody recognizes a protein comprising an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 2.
(15) The antibody of (14), wherein the antibody is a monoclonal antibody.
(16) An immunoassay method for measuring the protein of (3) or a fragment thereof based on immunological binding of the antibody of (14) or (15) to the protein of (2) or a fragment thereof.
(17) A reagent for an immunoassay for the protein of (3) or a fragment thereof, the reagent comprising the antibody of (14) or (15).
(18) A method for detecting mesangial proliferative nephropathy, the method comprising measuring the protein of (3) or a fragment thereof contained in a biological sample and comparing the measured value with that obtained from a normal sample.
(19) A transgenic nonhuman vertebrate in which the expression level of a gene encoding Meg-4 is modified.
(20) The transgenic nonhuman vertebrate of (19), wherein the nonhuman vertebrate is a mouse.
(21) The transgenic nonhuman vertebrate of (20), wherein the nonhuman vertebrate is a knockout mouse in which the expression of a gene encoding Meg-4 is inhibited.
To fulfill the issues mentioned above, the present inventor used a 3′-directed CDNA library. This method avoids the effect of the size of cDNA on cloning efficiency. The sequence at the 3′ region characterizes each of the genes, and the sequence data of approximately 200 to 300 bp are large enough to demonstrate the characteristics of the gene.
The DNA encoding human Meg-4 of the present invention can be obtained by preparing mRNA from mesangial cells and converting them to the double stranded cDNA by the known methods. mRNA can be prepared by, for example, the guanidine isothiocyanate-cesium chloride method (Chirwin, et al., Biochemistry 18, 5294, 1979), and the treatment with a surfactant and phenol in the presence of deoxyribonuclease (Berger & Birkenmeier, Biochemistry 18, 5143, 1979)
Bozicevic Karl
Bozicevic Field & Francis LLP
Kunz Gary
Miyata Toshio
Turner Sharon
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