Novel hemopoietin receptor protein, nr12

a technology of hemopoietin and receptor protein, applied in the field of new hemopoietin receptor protein, can solve the problems of difficult strict selection, unsatisfactory consensus sequence of yr motif, and difficult screening under normal hybridization experiment conditions, so as to increase durability and membrane permeability

Inactive Publication Date: 2009-04-23
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0131]An antisense oligonucleotide derivative is given to the patient by directly applying it onto the ailing site, by injecting it into the blood vessel and such, so that it will reach the ailing site. An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples are, liposome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives of them.

Problems solved by technology

However, it was extremely difficult to strictly select only those to which all 15 nucleotides that encode the motif would completely hybridize under the usual hybridization conditions, because the oligonucleotide “tggag(t / c)nnntggag(t / c)” (wherein “n” is an arbitrary nucleotide; SEQ ID NO:22) encoding the motif was short, having just 15 base pairs.
Therefore, performing screening under normal hybridization experiment conditions was extremely difficult.
However, this YR motif is not exactly a perfect consensus sequence, and the combination of the nucleotide sequences that encode the motif is very complicated.
Therefore, it is practically impossible to synthesize and provide oligonucleotides that encode all of the amino acid sequences as probes for hybridization, which is a practical method for screening, or as primers aimed for RT-PCR.

Method used

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  • Novel hemopoietin receptor protein, nr12
  • Novel hemopoietin receptor protein, nr12
  • Novel hemopoietin receptor protein, nr12

Examples

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example 1

Isolation of NR12 Gene

(1) Primary Screening by TblastN Search

[0177]Although sequencing of human genome is promoted extensively by human genome projects of institutes, the proportion of completely finished sequences to the whole human genome has not reached even 10%. However, information provided by above projects until today is counted as a good means for searching target genes, determining nucleotide sequences, and mapping genes. The informational basis of the above sequences consists of large information provided by the assembly of bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC), which aims to form a complete database in the future. The present inventors identified a human gene encoding a part of a novel hemopoietin receptor protein from a BAC clone sequence in one of public databases, “High Throughput Genomic Sequence (htgs)” of GenBank.

[0178]As mentioned above, the present inventors found motif sequences conserved in the hemopoietin receptor family, n...

example 2

Tissue Distribution Determination and Expression Pattern Analysis of NR12 Gene by RT-PCR

[0200]mRNA was detected using the RT-PCR method to analyze the expression distribution and the expression pattern of NR12.1 gene in different human organs. NR12-PPD primer with the sequence below was synthesized as a sense primer (downstream orientation) for the RT-PCR analysis. NR12-A1 primer synthesized in Example 1 (3) was used as the antisense primer (upstream orientation). The NR12-PPD primer was synthesized and purified as in Example 1 (3). It was expected that the common N-terminal region in all splice variants, NR12.1, NR12.2 and NR12.3, are amplified and detected using these primer sets (NR12-PPD and NR12-A1).

hNR12-PPD;(SEQ ID NO:17)5′-CCG CCA GAT ATT CCT GAT GAA GTA ACC-3′

[0201]The templates used were Human Multiple Tissue cDNA (MTC) Panel I (Clontech #K1420-1), Human MTC Panel II (Clontech #K1421-1), Human Immune System MTC Panel (Clontech #K1426-1), and Human Fetal MTC Panel (Clontech...

example 3

Verification of the Specificity of RT-PCR Product by Southern Blotting

[0206]In order to verify the specificity of amplification, the RT-PCR amplified target gene product in Example 2 was subjected to Southern blotting using NR12 specific cDNA fragment as a probe. At the same time, the amount of RT-PCR product was quantitatively detected by the strength of labeled signal to assess relative gene expression levels among different human organs. The RT-PCR product was electrophoresed on an agarose gel, blotted onto a charged nylon membrane, Hybond N (+) (Amersham, cat #RPN303B), and was subjected to hybridization. The 5′-RACE PCR product cDNA fragment corresponding to the N-terminus of the NR12 obtained in Example 1 (4) was used as a probe specific to NR12. Probes were prepared using the Mega Prime Kit (Amersham, cat #RPN1607), and labeled with radioisotope, [α-32P] dCTP (Amersham, cat #AA0005). Hybridization was performed using Express Hyb Hybridization Solution (Clontech #8015-2), and ...

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Abstract

A novel hemopoietin receptor gene (NR12) was successfully isolated by extracting motifs conserved among the amino acid sequences of known hemopoietin receptors and by using the predicted sequence. The NR12 gene encodes two forms of proteins, a transmembrane type and a soluble type. The expression of the NR12 gene was detected in tissues containing hematopoietic cells. NR12 is a novel hemopoietin receptor molecule involved in the regulation of immune system and hematopoiesis in vivo. Thus, NR12 is useful in the search for novel hematopoietic factors that functionally bind to the NR12 receptor, and in the development of therapeutic drugs for diseases associated with immunity or hematopoiesis.

Description

[0001]This application is a divisional of U.S. application Ser. Nos. 12 / 205,799 and 12 / 205,753, both filed Sep. 5, 2008, each being a divisional of U.S. application Ser. No. 11 / 595,320, filed Nov. 9, 2006, which is a continuation of U.S. application Ser. No. 11 / 274,375, filed Nov. 14, 2005, now abandoned, which is a divisional of U.S. application Ser. No. 10 / 105,930, filed Mar. 25, 2002, now U.S. Pat. No. 7,045,595, which is a continuation-in-part of International Patent Application No. PCT / JP00 / 06654, filed Sep. 27, 2000, which claims the benefit of Japanese Patent Application Nos. 11-273358, filed Sep. 27, 1999, and 2000-240397, filed Aug. 3, 2000. Each of the prior U.S. applications is herein incorporated by reference.TECHNICAL FIELD[0002]The present invention relates to novel hemopoietin receptor proteins, and genes encoding them, as well as methods for producing and using the same.BACKGROUND[0003]A large number of cytokines are known as humoral factors that regulate proliferati...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/00C07K14/715C07K16/28C12N1/21C12N15/12
CPCC07K16/2866C07K14/715A61P37/02
Inventor MAEDA, MASATSUGUYAGUCHI, NORIKO
Owner CHUGAI PHARMA CO LTD
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