JPH1180199A - Anti-nmt monoclonal antibody, hybridoma and detection of nmt - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a monoclonal antibody that specifically recognizes N- myristoyl transferase(NMT) and is useful in biochemical analysis and treatment and diagnosis for a variety of diseases. SOLUTION: This anti-NMT monoclonal antibody can specifically recognize the oligopeptide of the formula: Gly-Ile-Gly-Asp-Gly-Asn-Leu in the N-myristoyl transferase molecule. This anti-NMT monoclonal antibody is characterized by specifically recognizing N-myristoyl transferase of 88 kDa, 63 kDa and 57 kDa.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a monoclonal antibody specific to an enzyme N-myristoyltransferase that catalyzes N-myristoylation.

[0002]

2. Description of the Related Art For example, N-myristoyltransferase and a protein whose amino-terminal is myristoylated by the enzyme have been found in AIDS virus and oncoprotein products. The following substances are known as substances produced by involving N-myristoyltransferase.

For example, the protein from amino terminally myristoylated by N- myristoyl transferase cells, cAMP-including-dependent protein kinase, calcineurin B is phosphoprotein phosphatase, NADH-cytochrome b ₅ reductase, BC ₃ H ₁ myocytes system of intracellular proteins, pp
The p36K substrate protein of 60 ^v-src (src-gene gene product, tyrosine kinase), intracellular protein in macrophages, insulin receptor, vinculin and the like are known.

[0004] Concerning the constituent proteins of the virus whose amino terminal is myristoylated by N-myristoyltransferase, the Pr65 ^gag core protein of the mouse leukemia virus of Lausher and Moloney,
FeSV gag-onc protein, Moloney murine leukemia virus tyrosine protein kinase, Rous sarcoma virus (hereinafter RSV) pp60 ^V-src
Protein (AMSuhultz, et al. Science, 227, 427
(1985)], the p19 ^gag protein of the adult T-cell leukemia (ATL) virus (hereinafter referred to as HTLV-1), the virus that causes the acquired immunodeficiency syndrome (AIDS) (hereinafter,
HIV-1) p17 ^gag protein [S. Shoji,
J. Biochem., 103, 747-749 (1988)], polyoma virus and coat protein VP2 of SV40, core protein of Mason-Pfizer monkey virus, SI core of hepatitis B virus (hereinafter referred to as HBV). Protein (CHStreuli, et al. Nature (London), 326,
619 (1987)], Poliovirus core protein VP4
Etc. are known. In addition, the human oncogene (src-ge
ne) A myristoyl group was found in the product pp60 ^c-src , the α-subunit of guanosine triphosphate (GTP) binding protein, and the binding of the myristoyl group to immunoglobulins has also been suggested.

As described above, myristoylated proteins have been found in various intracellular proteins, virus constituent proteins, oncogene products, and the like. Especially,
As described above, the fact that many myristoyl groups are found in the proteins constituting the virus is presumed that the myristoylation of the protein is essential for the function of the virus.

Providing a monoclonal antibody that specifically recognizes the N-myristoyltransferase that performs myristoylation is useful for predicting and detecting the formation of each of the myristoyl compounds, and is particularly useful for viruses, particularly HI.
V-1, and analysis related to oncogene products, treatment,
It is expected to be useful for diagnosis, etc.

[0007] Conventionally, an antibody that specifically recognizes N-myristoyltransferase has a 63 kDa N-
An antiserum that recognizes myristoyltransferase (perimental cell research, 223, 348-356 (1996)),
And a monoclonal antibody that recognizes a 57-kDa N-myristoyltransferase [J. Biol. Chem., 270, 232].
26-23233 (1995)].

[0008]

The N-myristoyltransferase has been detected biochemically and immunochemically from various animal tissues and cultured cells, suggesting the existence of various isoforms. There was no discovery of the 88 kDa isoform.

Accordingly, an object of the present invention is to provide a novel monoclonal antibody that specifically recognizes N-myristoyltransferase, which is useful for biochemical analysis, treatment and diagnosis of various diseases, and the like.

[0010]

The present inventors have found the presence of 88 kDa for the enzyme N-myristoyltransferase (abbreviation: NMT) that catalyzes N-myristoylation. That is, the present inventors have found that NMT of 88 kDa is well expressed in cancer cells,
It was noted that the creation of a monoclonal antibody against NMT of Da could be used for cancer diagnosis and the like and was useful. Furthermore, the present inventors have found that the expression of 88 kDa NMT is significantly reduced in HIV-1 infected cells, and if a monoclonal antibody against 88 kDa NMT is created, it can be used for diagnosis of HIV-1 infection and the like. We focused on the potential and usefulness.

The present inventors have prepared Chou-Fausm based on the amino acid sequence of human NMT as an antigen in order to prepare a monoclonal antibody specific to human NMT.
The secondary structure was estimated by the an method, and an amino acid sequence considered to be suitable as an antigen was determined as follows.

The present inventor has proposed an oligo at a position which is located at residues 388-394 from the amino terminus of human NMT and which is presumed to have a bent structure at the joint of α-helix structure and β-sheet structure. Focusing on peptides, the following formula (1) (SEQ ID NO: 1), which is well conserved among species, Gly-Ile-Gly-Asp-Gly-Asn-Leu: an oligopeptide represented by formula (1) The present invention was completed using it as an antigen.

That is, the anti-NMT monoclonal antibody of the present invention is an anti-NMT monoclonal antibody capable of specifically recognizing the oligopeptide represented by the above formula (1) in the N-myristoyltransferase molecule. I do.

Further, the anti-NMT monoclonal antibody of the present invention is characterized in that it can specifically recognize 88 kDa, 63 kDa and 57 kDa N-myristoyltransferase.

[0015] The hybridoma of the present invention comprises Gly-Ile-Gly- in N-myristoyltransferase.
Asp-Gly-Asn-Leu molecules can be specifically recognized, or 88 kDa, 63 kDa and 5 kDa.
It is a hybridoma having a monoclonal antibody-producing ability capable of specifically recognizing a 7 kDa N-myristoyltransferase.

The method for immunologically detecting N-myristoyltransferase of the present invention comprises the steps of 88 kDa and 63 kDa.
A human or animal tissue, body fluid suspected of containing a Da or 57 kDa N-myristoyltransferase;
The monoclonal antibody is brought into contact with a sample containing cells or cell lines to detect 88-kDa, 63-kDa or 57-kDa N-myristoyltransferase.

The immunochemical detection methods applicable to the present invention include, for example, an agglutination reaction method, an agglutination inhibition method, an immunodiffusion method,
Immunonephelometry, immunoturbidimetry, latex nephelometry, competitive reaction (solid phase method, second antibody method, etc.), sandwich method, immunometric method, liposome immunolysis assay (L
ILA) and the like, and a detection method using a labeling substance includes a lazyme immunoassay (RIA), an enzyme immunoassay (EIA), and a fluorescence immunoassay (FI
A), chemiluminescence immunoassay, metal immunoassay,
Spin immunoassay and the like.

[0018]

【Example】

[Example 1] Establishment of hybridoma : The following formula (1) Gly-Ile-Gly-Asp-Gly-Asn-Leu: An oligopeptide represented by the formula (1) is an antigen at the tip of a branched structure of poly-L-lysine. Peptides are linked, and the following formula (2) Gly-Ile-Gly-Asp-Gly-Asn-Leu-poly-L-lysine (8): Multiple antigen peptide (8) represented by formula (2) [abbreviation: MA
P (8)] to enhance immunogenicity. The structure of the multiple antigen peptide (8) used in Example 1 is represented by the chemical structural formula in FIG. 1 in which amino acids are represented by the one-letter notation method. The multiple antigen peptide (8) was bound to a solid phase to obtain an immunizing antigen. 200 μg of the immunizing antigen per mouse was intraperitoneally administered four times per week, then 3 weeks later, 40 μg of the immunizing antigen was intravenously injected per mouse, and 3 days later, the mice were killed by slaughter. Cells were obtained.

According to a conventional method, cells were fused with P3U1 using polyethylene glycol, and hybridomas were selected using HAT medium. Antibody producing hybridomas
Using an antigen peptide synthesized by the multi-pin method, a multi-pin-enzyme-linked immunosorbent assay (Multi
-Pin-ELISA method), and then a hybridoma producing anti-NMT monoclonal antibody was established by limiting dilution.

This hybridoma has been accorded an accession number FE from August 8, 1997 to the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.
Deposited as RM P-16377.

Example 2 Purification of Anti-NMT Monoclonal Antibody: Large-scale preparation and purification of anti-NMT monoclonal antibody were carried out according to a conventional method. Pristane 2,6,10,14-tetramethylpentadecane was administered to adult Balb / c mice intraperitoneally at a dose of 500 μl / mouse, and hybridomas were transplanted 7 to 10 days later. Monoclonal antibody-producing hybridomas
A suspension of cells in HBSS buffer at 1.0 × 10 ⁷ cells / 200 μl / mouse was transplanted into the peritoneal cavity of the mouse. After 10 to 14 days, the abdomen of the hypertrophied mouse was incised by hybridoma growth, and ascites was collected. The collected ascites is separated into cells and serum by centrifugation,
Serum alone was collected, saturated ammonium sulfate was added so as to be 50% saturated, left at 4 ° C., and a globulin fraction was obtained by centrifugation. This is dissolved in an appropriate amount of PBS (-), and PBS (-)
After dialysis, the antibody was partially purified. After dialysis, further purification was carried out using a protein A-Sepharose CL-4B column, and the obtained IgG fraction was washed with PBS.
The purified antibody was obtained by dialysis against (−).

Example 3 Competition assay : GIGDGGN was used as various competitor peptides.
L-poly L-lysine, GIGDG, GIGD, GNL
Q, GDGNLQ, and these various competitor peptides were adjusted to a concentration of 16 mg / ml with a competitor lysis buffer (50
mM Tris-HCl (pH 8.0), 10 mM M
gCl ₂ , 0.1% polyoxyethylene (20) sorbitan monooleate) dissolved in a series of 1- to 512-fold dilutions using the same buffer solution 50
μl was prepared, and protein A-Sepharose CL was added thereto.
50 μl of anti-human NMT monoclonal antibody purified by a −4B column was added, and preincubation was performed at 37 ° C. for 1 hour. Using this mixed solution as a primary antibody, 50 μl was added to an ELISA plate on which the antigen peptide represented by the formula (2) had been previously adsorbed as an immobilized antigen, and the antibody titer was measured by ELISA.

In the ELISA method, the primary antibody is
After reacting at 7 ° C. for 1.5 hours, peroxidase (abbreviation: POD) -labeled anti-mouse IgG was used as a secondary antibody at 37 ° C. for 4 hours.
After reacting for 5 minutes, TMBZ (3, 3 ',
5,5'-tetramethylbenzidine), and using 0.3 N H ₂ SO ₄ as a coloring stop solution, dominant wavelength 450n
m, the absorbance at a reference wavelength of 630 nm was measured. The results are shown in the graph of FIG. 2 in which the horizontal axis represents the concentration of the competitor and the vertical axis represents the% inhibition.

The multiple antigen peptide represented by the formula (2) has an IC ₅₀ (50% inhibitory concentration) of 33.3 nmol.
Met. In addition, oligopeptides derived from other immunizing antigens also exhibited an IC ₅₀ comparable to that of the multiple antigen peptides represented by the formula (2). From the above, it is clear that the present antibody recognizes the peptide represented by the formula (1).

[Example 4] Detection of NMT using anti-NMT monoclonal antibody : Using the anti-NMT monoclonal antibody obtained in Example 2 above, human colon adenocarcinoma cells (COLO 320DM) and leukemia human T cells ( Western immuno-blotting assay was performed on each cell solubilized supernatant of CEM). E. coli as a control. coli (JM101),
The E.C. Recombinant human NMT expressed in E. coli was used. The results are shown in FIGS. 3 (control) and 4 (COLO 320
DM; CEM). According to FIG. 3 and FIG. 4, the anti-NMT monoclonal antibody of the present invention was used for human colon adenocarcinoma cells (COLO 320DM) and leukemia human T cells in 8
It can be seen that N-myristoyltransferase of 8 kDa, 63 kDa and 57 kDa is specifically recognized.

[Example 5] Continuous feeling of HIV-1 using anti-NMT monoclonal antibody
Detection against stained cell line: anti-NM obtained in Example 2 above
Using the T monoclonal antibody, HIV-1 uninfected human T-cell line H9, human T-cell line Molt-4
And human macrophage cell line U937, and HIV-1 persistently infected human T-
Western immuno-blotting of cell solubilized supernatants of cell line H9 / MN, HIV-1 persistently infected human T-cell line Molt-4 / IIIB and HIV-1 persistently infected human macrophage cell line U937 / IIIB, respectively -The assay was performed. The result is shown in FIG. As shown in FIG. 5, in HIV-1 infected cells, 88 kDa
It can be seen that the expression of NMT was significantly reduced.

[0027]

The anti-NMT monoclonal antibody of the present invention specifically recognizes 88-kDa, 63-kDa and 57-kDa N-myristoyltransferases and has Gly-Ile-Gly-Asp-Gly-A.
N-myristoyltransferase containing a peptide consisting of sn-Leu is specifically recognized. This is expected to be useful for prediction, detection, etc. of the formation of myristoyl compounds, and particularly for analysis, therapy, diagnosis, etc. relating to viruses, especially HIV-1, and oncogene products.

[0028]

[Sequence list]

SEQ ID NO: 1 Sequence length: 7 Sequence type: amino acid Topology: Linear Sequence type: Peptide

[Brief description of the drawings]

FIG. 1 shows the chemical structure of the multiple antigen peptide (8) used in Example 1 by the one-letter notation method of amino acids.

FIG. 2 is a graph showing the results of a competition assay using the anti-NMT monoclonal antibody of the present invention, in which the horizontal axis represents the concentration of the competitor and the vertical axis represents the inhibition%.

FIG. 3 shows a control obtained when a western immuno-blotting assay was performed using the anti-NMT monoclonal antibody of the present invention [E. coli (JM101)]
And E. recombinant human NMT expressed in E. coli
4 is a photograph showing a result of electrophoresis of FIG.

FIG. 4 shows a human colon adenocarcinoma cell (COLO 320D) when a western immuno-blotting assay was performed using the anti-NMT monoclonal antibody of the present invention.
M) is a photograph showing the result of electrophoresis of leukemia human T cells (CEM).

FIG. 5 shows the results of Western immuno-blotting assay performed on each cell line not infected with HIV-1 and each cell line infected with HIV-1 using the anti-NMT monoclonal antibody of the present invention. It is a photograph which shows the result of electrophoresis.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/577 C12N 15/00 ZNAC // (C12N 5/10 C12R 1:91)

Claims (4)

[Claims] 1. The following formula in an N-myristoyltransferase molecule: Gly-Ile-Gly-Asp-Gly-Asn-L
An anti-NMT monoclonal antibody capable of specifically recognizing the oligopeptide represented by eu. 2. 88 kDa, 63 kDa and 57 kD
An anti-NMT monoclonal antibody capable of specifically recognizing N-myristoyltransferase a. 3. A hybridoma having an anti-NMT monoclonal antibody-producing ability capable of specifically recognizing the N-myristoyltransferase according to claim 1 or 2. 4. The anti-NMT monoclonal antibody according to claim 1 against a sample containing a human or animal tissue, body fluid, cell or cell line suspected of containing the N-myristoyltransferase according to claim 1 or 2. A method for detecting 88 kDa, 63 kDa or 57 kDa N-myristoyltransferase immunologically by contacting.