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Assay method for human orotate phosphoribosyltransferase protein

A technology of orotate phosphoribosyl and determination method, applied in anti-enzyme immunoglobulin, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of complicated operation, unable to fully reflect protein quality and enzyme activity, etc.

Inactive Publication Date: 2008-02-27
TAIHO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to post-transcriptional regulatory mechanisms, etc., it is possible that the measurement of the amount of mRNA may not fully reflect the amount of protein and enzyme activity.
In addition, quantification by measuring enzyme activity is very cumbersome due to the use of mainly radiolabeled substrates

Method used

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  • Assay method for human orotate phosphoribosyltransferase protein
  • Assay method for human orotate phosphoribosyltransferase protein

Examples

Experimental program
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Effect test

Embodiment

[0035] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Embodiment 1

[0055] Embodiment 1 (preparation of antibody)

[0056] According to the following operation, anti-OPRT polyclonal antibody was obtained.

[0057] (1) Peptide synthesis

[0058] Synthesize a peptide having the amino acid sequence of 86th to 108th from the N-terminus of human OPRT, a peptide having the sequence of amino acids 428th to 446th from the N-terminus of human OPRT, and a peptide having an amino acid sequence of 428th to 446th from the N-terminus of human OPRT The peptides of the 454th to 474th amino acid sequences were obtained as artificial peptides consisting of the following amino acid sequences.

[0059] Peptide having the amino acid sequence at position 86-108 from the N-terminal of human OPRT (antigen name: OPRT-A)

[0060] Cys-Ser-Thr-Asn-Gln-Ile-Pro-Met-Leu-Ile-Arg-Arg-Lys-Glu-Thr-Lys-Asp-Tyr-Gly-Thr-Lys-Arg-Leu (SEQ ID NO. 1)

[0061] Peptide having an amino acid sequence from 428th to 446th from the N-terminus of human OPRT (antigen name: OPRT-B)

[0062]...

Embodiment 2

[0075] Example 2 (quantification of human OPRT by sandwich ELISA)

[0076] (1) rhOPRT obtained by culturing Escherichia coli was used as a standard. The preparation method is as follows: use PCR to clone the full-length cDNA of human OPRT, prepare a plasmid expressing the fusion protein of glutathione-S-transferase (GST) and rhOPRT, introduce the plasmid into E. In the presence of penicillin, culture was shaken overnight at 37° C. in 100 mL of LB medium (manufactured by Wako Pure Chemical Industries, Ltd.). 10 mL of this culture solution was transferred to an Erlenmeyer flask containing 1 L of LB medium, and further cultured at 37° C. for 4 hours. After collecting bacteria by centrifugation, suspend the bacteria in 100mL of bacteria collection buffer (50mM Tris, 8M Urea, 1mM PMSF, 5mM EDTA, 5mMDTT, pH 7.4), and slowly stir at 4°C for 30 minutes. After the suspension was sonicated at 4°C, it was centrifuged at 15,000×g and 4°C for 30 minutes. Then, the concentration of urea ...

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Abstract

It is intended to establish an immunoassay method for human OPRT. An assay method for human orotate phosphoribosyltransferase protein using an anti-human orotate phosphoribosyltransferase antibody that recognizes an epitope present in an amino acid region between positions 86 and 108 from the N-terminus of human orotate phosphoribosyltransferase and an anti-human orotate phosphoribosyltransferase antibody that recognizes an epitope present in an amino acid region between positions 454 and 474 from the N-terminus of human orotate phosphoribosyltransferase in combination.

Description

technical field [0001] The present invention relates to a novel antibody against human orotate phosphoribosyltransferase and a method for detecting human orotate phosphoribosyltransferase protein using the antibody. Background technique [0002] Orotate phosphoribosyl transferase (orotate phosphoribosyl transferase; EC2.4.2.10, hereinafter referred to as "OPRT") is an enzyme that catalyzes the reaction of uridine monophosphate (UMP) from orotic acid, and has the function of supplying nucleic acid synthesis Functions as an essential pyrimidine nucleotide and is one of the main rate-limiting enzymes in a nucleic acid precursor supply pathway. Therefore, it is known that its activity is high in tumor tissues or digestive tract epithelium where cell proliferation is vigorous. [0003] In addition, it is known that 5-fluorouracil-based anticancer agents exert an antitumor effect by activating OPRT as a rate-limiting enzyme. The effect is relatively large for patients with a larg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40G01N33/537
CPCC12Q1/48C07K16/40G01N2333/91142G01N33/537
Inventor 坂本一树杉本芳一
Owner TAIHO PHARMA CO LTD
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