Polypeptides having phospholipase a2 activity

a polypeptide and activity technology, applied in the field of phospholipase a2 polypeptides, can solve the problems of difficult purification and isolation from tissues or cells, undesirable use of non-specific chemicals, and specific cytoplasmic phospholipas

Inactive Publication Date: 2008-02-21
KYOWA HAKKO KIRIN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0229] The expression-regulating compound can be identified by searching for a test sample increasing or decreasing the content of the mRNA encoding the polypeptide of the present invention as compared with the system without the addition of the test sample.
[0240] The expression-regulating compound can be identified by searching for a test sample increasing or decreasing the content of the polypeptide encoded by the reporter gene as compared with the system without the addition of the test sample.
[0262] (10) Administration of the antibody inhibiting the function of the polypeptide of the present invention (phospholipase A2 activity) is effective for the treatment or prevention of diseases such as asthma, ischemic diseases, arthritis, rheumatism, sepsis, dermatitis, arteriosclerosis, pain, Parkinson disease, Alzheimer disease, malignant tumor, nephritis, diabetes and ischemic reperfusion injury.

Problems solved by technology

In either case of inhibiting or enhancing phospholipase A2 activity, use of nonspecific chemicals is undesirable because of effect on the phospholipid metabolism in tissues and cells other than target tissues and cells.
However, the expression of cytoplasmic phospholipase A2α, β and γ is ubiquitous, and no tissue- or cell-specific cytoplasmic phospholipase A2 has so far been known.
In the case of cytoplasmic phospholipase A2, purification and isolation from tissues or cells is not easy because it exists only in extremely small amounts.
The limitation of currently employed purification methods and the difficulty in confirming that a single purified enzyme preparation has been obtained hinder the isolation of a novel subtype using conventional enzymological techniques.

Method used

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  • Polypeptides having phospholipase a2 activity
  • Polypeptides having phospholipase a2 activity
  • Polypeptides having phospholipase a2 activity

Examples

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Effect test

example 1

Cloning of cDNA Encoding the Human-Derived Polypeptide of the Present Invention

[0294] Unless otherwise noted, the genetic engineering techniques in the following examples were carried out according to the known methods described in Molecular Cloning, Second Edition.

(1) Preparation of a cDNA Library Derived from Human Small Intestine

[0295] Total RNA was extracted from human small intestine using an RNA extraction kit (#27-9270-01) produced by Pharmacia. Thereafter, mRNA was extracted and purified in accordance with the polyA(+)RNA purification method described in literature [J. Sambrook, E. F. Fritsch & T. Maniatis, Molecular Cloning Second Edition, Cold Spring Harbor Laboratory Press (1989)].

[0296] A cDNA library was prepared from each of polyA(+)RNA according to the oligo-cap method [Gene, 138, 171 (1994)]. BAP (bacterial alkaline phosphatase) treatment, TAP (tobacco acid pyrophosphatase) treatment, RNA ligation, single-stranded cDNA synthesis and RNA removal were carried out ...

example 2

Analysis of Expression Using RT-PCR Method

[0315] A 5′-end DNA primer having the nucleotide sequence shown in SEQ ID NO: 13 and a 3′-end DNA primer having the nucleotide sequence shown in SEQ ID NO. 14 were designed and synthesized based on the information on the nucleotide sequence determined in Example 1.

[0316] PCR was carried out using 20 μl of a reaction solution containing 1.0 μmol / l each of the two primers (SEQ ID NOS: 13 and 14), 2 μl of a cDNA library prepared from each of the mRNAs of various human organs, a mixed solution of dNTPs (dATP, dGTP, dCTP and dTTP) containing 200 μmol / l each of the components, 200 μmol / l each of dNTPs (dATP, dGTP, dCTP and dTTP), 2.5 units of Taq Gold polymerase (Perkin Elmer) and 1× Taq Gold (Mg plus) buffer (Perkin Elmer) under the following conditions.

[0317] That is, using a thermal cycler, PTC-200 (MJ Research), PCR was carried out, after heating at 95° C. for 10 minutes, by 35 cycles, one cycle consisting of reaction at 94° C. for one minu...

example 3

Analysis of Expression of mRNA by Northern Hybridization

[0319] PCR was carried out using 50 μl of a reaction solution containing 0.2 μmol / l each of the two primers (SEQ ID NOS: 13 and 14), a mixed solution of dNTPs (dATP, dGTP, dCTP and dTTP) containing 200 μmol / l each of the components, 200 μmol / l each of dNTPs (dATP, dGTP, dCTP and dTTP), 2 μl of Human Kidney Marathon-Ready cDNA, 2.5 units of Ampli Taq Gold polymerase (Perkin Elmer) and 1× Taq Gold buffer under the following conditions.

[0320] That is, using a thermal cycler, PTC-200, PCR was carried out, after heating at 95° C. for 10 minutes, by 35 cycles, one cycle consisting of reaction at 94° C. for one minute and reaction at 60° C. for one minute, followed by heating at 72° C. for 8 minutes.

[0321] A 5 μl aliquot of the resulting PCR reaction mixture was subjected to agarose gel electrophoresis to confirm that an about 0.6 kb DNA fragment was amplified, The DNA fragment was then purified using QIAEX II Gel Extraction Kit (Q...

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Abstract

The present invention relates to a novel phospholipase A2 polypeptide, DNA encoding the polypeptide, a vector comprising the DNA, a transformant transformed with the vector, and a process for producing the phospholipase A2 polypeptide. The present invention also relates to a method of utilizing the polypeptide, e.g., a method of screening for a compound having agonist or antagonist activity by using the polypeptide or an antibody to the polypeptide, and a pharmaceutical comprising the polypeptide or an antibody to the polypeptide. The present invention further relates to a polypeptide inhibiting the phospholipase A2 activity of a phospholipase A2 polypeptide (hereinafter referred to as inhibitor polypeptide), DNA encoding the inhibitor polypeptide, a vector comprising the DNA encoding the inhibitor polypeptide, a transformant transformed with the vector, a pharmaceutical comprising the inhibitor polypeptide, and a process for producing the inhibitor polypeptide.

Description

[0001] This application is a division of application Ser. No. 10 / 380,873 filed on Mar. 19, 2003, which in turn is a national phase of PCT Application No. PCT / JP01 / 08138 filed Sep. 19, 2001.TECHNICAL FIELD [0002] The present invention relates to a novel phospholipase A2 polypeptide, DNA encoding the polypeptide, a vector comprising the DNA, a transformant transformed with the vector, and a process for producing the phospholipase A2 polypeptide. The present invention also relates to a method of utilizing the polypeptide, e.g., a method of screening for a compound having agonist or antagonist activity by using the polypeptide or an antibody to the polypeptide, and a pharmaceutical comprising the polypeptide or an antibody to the polypeptide. The present invention further relates to a polypeptide inhibiting the phospholipase A2 activity of a phospholipase A2 polypeptide (hereinafter sometimes referred to as inhibitor polypeptide), DNA encoding the inhibitor polypeptide, a vector compris...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395A61P1/00A61P11/06A61P17/00A61P19/02A61P25/16A61P25/28A61P3/10A61P35/00C07H21/04C07K14/435C07K16/40C12N1/00C12N1/21C12N5/04C12N5/06C12P21/06G01N33/53A61K38/00A61P5/38A61P7/00A61P9/10A61P13/12A61P39/00C12N9/16C12N9/20C12N15/55
CPCA61K38/00A61K2039/505C12N9/20C12N9/16C07K16/40A61P1/00A61P11/06A61P13/12A61P17/00A61P19/00A61P19/02A61P25/04A61P25/16A61P25/28A61P29/00A61P29/02A61P3/10A61P31/04A61P35/00A61P39/00A61P43/00A61P5/38A61P7/00A61P9/10
Inventor MIYAJI, HIROMASAHARUOKA, MOTOKONAGATA, HIROYUKIOTA, TOSHIOKAWABATA, AYAKOSUGANO, SUMIONAKAMURA, YUSUKE
Owner KYOWA HAKKO KIRIN CO LTD
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