JP5397932B2 - Nuf2 protein antibody, its production method, antigen, and detection method - Google Patents

Description

  The present invention includes an antibody that specifically reacts with a Nuf2 protein localized in a cell nucleus, a Nuf2 protein immunoreagent containing the antibody, a method for producing a Nuf2 protein antibody, an antigen for producing a Nuf2 protein antibody, a method for detecting a Nuf2 protein, The present invention relates to a method for detecting another protein that interacts with Nuf2 protein.

Antibodies that specifically bind to specific proteins are frequently used as research tools in the life science field. Research on the cell nucleus has become active, and there is an increasing demand for antibodies that bind to proteins located in the cell nucleus.
It is already known that there is Nuf2 in the centromere protein constituting the cell nucleus (Non-patent Document 1, etc.).
Human NUF2 interacts with centromere-associated protein Eand is essential for a stable spindle microtubule-kinetochore attachment. Liu Det al., J Biol Chem 282: 21415-24 (2007)

In common across organisms, the transition from somatic cell division to meiosis dramatically changes the chromosomal arrangement from a centromeric structure to a telomeric structure in the nucleus.
The present inventors used fission yeast cells to search for molecules that retain the centromere in the SPB during somatic cell division and molecules that retain the telomere in the SPB during meiotic prophase. The protein Nuf2 and the telomeric protein Rap1 were identified. As a result of analyzing the function of the Nuf2 protein, it was found that it plays an essential role in chromosome separation.

There are commercially available antibodies that specifically bind to the Nuf2 protein (ABCAM product, http://www.abcam.co.jp/index.html?datasheet=17058), but the degree of specificity The reliability as a research reagent was not high enough.
There are Patent Documents 1 to 7 as conventional techniques related to antibodies and their production methods, but there is no disclosure regarding antibodies of Nuf2 protein.

JP 2006-31621 “Antibodies that specifically react with AGEs derived from 3,4-DGE” Japanese Patent Application Laid-Open No. 11-279200 “Dityrosine Polyclonal Antibody and Method for Producing the Same, and Antigen Used for the Same and Method for Producing the Same” Japanese Patent Application Laid-Open No. 10-218896 “Polyclonal Antibody and Method for Producing the Same, Antigen for Use in the Same and Method for Producing the Same” JP 10-72499 “Polyclonal antibodies and production methods” JP 2008-29353 “Method for producing anti-human soluble fibrin monoclonal antibody” JP2007-314493 “monoclonal antibody, production method and use thereof” JP 2007-244268 “Method for producing monoclonal antibody recognizing canine hematopoietic progenitor cells”

  Therefore, the present invention provides a highly specific Nuf2 protein antibody, a Nuf2 protein immunoreagent containing the same, a method for producing a Nuf2 protein antibody, an antigen for producing a Nuf2 protein antibody, a method for detecting the Nuf2 protein, and an interaction with the Nuf2 protein It is an object of the present invention to provide a method for detecting another protein.

In order to solve the above problems, the present invention has the following configuration.
That is, the Nuf2 protein antibody of the present invention is an antibody that specifically reacts with the Nuf2 protein, and uses a host animal using an antigen having CGGDYSAKIDEKTAELKRKMFMS as an epitope (antigenic determinant) among the amino acid sequences constituting the Nuf2 protein. After immunization, the product was obtained by isolation and purification from the body fluid of the host animal.

Here, the host animal may be a rabbit, and the body fluid of the host animal may be serum.

The antibody may be a polyclonal antibody.

The antibody may be a monoclonal antibody.

  The Nuf2 protein immunoreagent of the present invention is an immunoreagent containing the above Nuf2 protein antibody.

  A method for producing a Nuf2 protein antibody of the present invention is a method for producing an antibody that specifically reacts with a Nuf2 protein, wherein an antigen having CGGDYSAKIDEKTAELKRKMFMS as an epitope is used among amino acid sequences constituting the Nuf2 protein. When immunization is carried out and the production of antibodies by the host animal reaches a maximum, the body fluid of the host animal is extracted, and the antibody is isolated and purified from the body fluid.

  The antigen for producing a Nuf2 protein antibody of the present invention is an antigen used for the production of an antibody that specifically reacts with the Nuf2 protein, and is characterized by having CGGDYSAKIDEKTAELKRKMFMS as an epitope among amino acid sequences constituting the Nuf2 protein.

  The method of detecting the Nuf2 protein of the present invention is a method of detecting the Nuf2 protein, wherein the above-mentioned Nuf2 protein antibody and a sample having a protein fraction of a cell or tissue are used, and the antigen localization is microscopically determined by immunostaining. It is characterized by detecting below.

  Here, the presence of the antigen may be detected by Western blotting using a sample having the above Nuf2 protein antibody and a protein fraction of cells or tissues.

  The presence of the antigen may be detected by immunoprecipitation using a sample having the Nuf2 protein antibody and a protein fraction of cells or tissues.

  The method for detecting another protein that interacts with the Nuf2 protein of the present invention is a method for detecting another protein that interacts with the Nuf2 protein, the sample comprising the Nuf2 protein antibody and a protein fraction of a cell or tissue And another protein that specifically forms a complex with an antigen is detected by coimmunoprecipitation.

The present invention has the following effects by providing the above configuration.
That is, by generating an antibody using an antigen having CGGDYSAKIDEKTAELKRKMFMS as an epitope among the amino acid sequences constituting the Nuf2 protein, a highly specific Nuf2 protein antibody can be obtained and used to detect the Nuf2 protein. Detection of other proteins that interact with the Nuf2 protein.

Hereinafter, embodiments of the present invention will be described based on examples shown in the drawings. The embodiment is not limited to the following examples, and the design can be changed as appropriate using a conventionally known technique such as the above-mentioned patent document without departing from the gist of the present invention.
Previously, there was no effective antibody that specifically binds Nuf2 protein, but the present inventors immunized rabbits with an antigen having CGGDYSAKIDEKTAELKRKMFMS as an epitope of the antigen among the amino acid sequences constituting Nuf2 protein. When the production of the antibody reached a maximum, serum was extracted and the antibody was successfully isolated and purified.

As a method for preparing an antibody, a conventionally known method for producing an antibody by immunizing a host animal can be appropriately used.
The type of host animal to be immunized is not particularly limited, and for example, mammals such as rabbits, rats, mice, goats, sheep, horses, pigs, guinea pigs, and birds such as chickens, pigeons, ducks, quails can be used. .
The administration method of the antigen is not particularly limited, and intradermal administration, subcutaneous administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be appropriately used.

When preparing a polyclonal antibody, body fluid such as serum or ascites fluid of the immunized host animal may be collected and the antibody isolated and purified.
When preparing a monoclonal antibody, for example, a hybridoma is prepared by fusing myeloma cells with antibody-producing cells such as spleen cells and lymphoid cells in the immunized host animal, and the hybridoma is proliferated. What is necessary is just to isolate and purify the hybridoma cell which produces the antibody with specificity.

The method for purifying the polyclonal antibody and the monoclonal antibody is not particularly limited, and for example, salting out, dialysis, ion exchange chromatography, affinity chromatography, electrophoresis and the like can be used as appropriate.

The method for screening antibody production is not particularly limited, and for example, a radioimmunoassay method, an enzyme immunoassay method and the like can be used as appropriate.

The antibody thus obtained may be used as an antibody per se, or may be used as an active fragment of an antibody obtained by performing an enzyme treatment or the like, or may be mixed with other drugs etc. as a reagent. Etc. may be used.

Example:
FIG. 1 is a mass spectrum confirming the purification of the antigen.
The antigen was prepared by a conventional method, and the peptide was expressed in the process of adding a His tag to express and purify the protein, and cutting off and purifying the His tag. The target molecular ion was shown by the peak of the graph, and the quality of the obtained antigen peptide was confirmed.

FIG. 2 is a table showing the immunization schedule for rabbits, and FIG. 3 is a table showing changes in antibody titer due to the immunization.
The antigen solution was mixed with an equal volume of Freund's complete adjuvant to be emulsified and administered to 20 rabbits on the back of the rabbit. The dose was 2 mg in terms of protein mass.
It was administered every other week, and after the second immunization, an emulsion mixed with an equal amount of Freund's incomplete adjuvant was used, and the dose was 1 mg in terms of protein mass.

Blood was collected over time from the start of the first immunization, and the antibody titer was measured by ELISA.
Blood was collected from the ear vein and the serum volume was 2 ml. Seven weeks later, whole blood was collected from the carotid artery to obtain 78 ml of serum. The antibody titer was confirmed to be sufficiently increased by 4 immunizations.

FIG. 4 is a photograph in which the localization of the antigen was detected under a microscope by immunostaining using the Nuf2 protein antibody of the present invention.
The localization of Nuf2 protein (green) was shown, along with DAPI staining (pink) that stains chromosomes in the cell nucleus of cultured cells. 4A shows the cell cycle of prometaphase, and FIG. 4B shows the cell cycle of metaphase.

FIG. 5 is a photograph in which the presence of an antigen was detected by Western blotting using the Nuf2 protein antibody of the present invention.
In the illustrated example, those diluted 500 times, 1000 times, and 5000 times are shown. Even when diluted 5000 times, a specific band at 50 KDa indicating the molecular weight of the Nuf2 protein was confirmed.

In the present invention, the Nuf2 protein can be detected by appropriately utilizing a conventionally known immunostaining method, Western blotting method or immunoprecipitation method.
The immunostaining method is a histochemical method for detecting an antigen in a sample using an antibody, and involves a color development operation in order to visualize an immune reaction that is originally invisible. Western blotting is a method in which electrophoretic protein molecules are transferred to a specific membrane and immunostained with an antibody.

  For the immune reaction, the primary antibody that directly reacts with the antigen is labeled and the direct method in which the immune reaction is performed only once is performed, or the first immune reaction is performed using an unlabeled primary antibody, and the primary antibody is combined with the antigen. An indirect method can be used in which another secondary antibody is labeled and further immunoreacted. In general, the amplification is repeated as the immune reaction is repeated, so that the detection sensitivity can be increased, but the specificity is lowered.

  For staining, in addition to the method of binding a dye or fluorescent dye to the antibody, autoradiography in which a radioisotope is bound as a label and exposed to photographic paper, or gold and silver particles are bound and observed with an electron microscope or the like A gold colloid method, a gold colloid silver sensitization method, an enzyme antibody method in which a specific enzyme is bound, and the coloration of the dye product is observed with an optical microscope, an immunoperoxidase method, and the like can be used as appropriate.

  Further, the antigen may be detected by specifically reacting and precipitating the antigen and the antibody by immunoprecipitation. Usually, the antibody is bound to a carrier such as Sepharose beads to facilitate precipitation. Polyclonal antibodies are more suitable than monoclonal antibodies because they recognize multiple sites, but nonspecific adsorption is also likely to occur.

In the present invention, another protein that specifically forms a complex with the Nuf2 protein can be detected by co-immunoprecipitation to obtain knowledge about the interaction between the proteins.
The co-immunoprecipitation method is a method of recovering a complex with another protein that specifically forms a complex with a target protein by immunoprecipitation. This may be combined with mass spectrometry and used to identify an unknown protein that interacts with a known protein.

  According to the present invention, an antibody having high specificity for the Nuf2 protein localized in the cell nucleus can be obtained, which contributes to research in the life science field. For example, it is useful for investigating the mechanism of various diseases such as dysplasia of germ cells, which is a problem in reproductive medicine research, and for finding out the mechanism of such diseases. Is expensive.

Mass spectrum of antigen Table showing immunization schedule for rabbits Table showing changes in antibody titer due to immunization The photograph (a) in which the localization of the antigen was detected under a microscope using the Nuf2 protein antibody of the present invention by immunostaining, the cell cycle is prometaphase, and (b) the cell cycle is metaphase. Photograph of detecting the presence of an antigen by Western blotting using the Nuf2 protein antibody of the present invention

Claims (10)

A polyclonal antibody that specifically reacts with the epitope CGGDYSAKIDEKTAELKRKMFKMS of the Nuf2 protein,
After immunizing a host animal (excluding humans) with an antigen that has CGGDYSAKIDEKTAELKRKMFKMS as the only epitope among the amino acid sequences that make up the Nuf2 protein, it is isolated and purified from the body fluid of the host animal (excluding humans) A Nuf2 protein antibody obtained by A monoclonal antibody that specifically reacts with the epitope CGGDYSAKIDEKTAELKRKMFKMS of the Nuf2 protein,
After immunizing a host animal (excluding humans) with an antigen having CGGDYSAKIDEKTAELKRKMFKMS as the only epitope among the amino acid sequences that make up the Nuf2 protein, antibody producing cells and myeloma of the host animal (excluding humans) A Nuf2 protein antibody obtained by preparing a hybridoma by fusing with a cell and isolating and purifying a hybridoma cell producing the antibody. The Nuf2 protein antibody according to claim 1 or 2, wherein the host animal (excluding human) is a rabbit, and the body fluid of the host animal (excluding human) is serum. An immunoreagent containing an antibody comprising:
A Nuf2 protein immunoreagent, wherein the antibody is the Nuf2 protein antibody according to any one of claims 1 to 3. A method for producing an antibody that specifically reacts with the epitope CGGDYSAKIDEKTAELKRKMFKMS of the Nuf2 protein,
Among the amino acid sequences that make up the Nuf2 protein, immunize host animals (except humans) with antigens that have CGGDYSAKIDEKTAELKRKMFKMS as the only epitope,
When antibody production by a host animal (excluding humans) is almost maximized, the body fluid of the host animal (excluding humans) is extracted,
A method for producing a Nuf2 protein antibody, comprising isolating and purifying an antibody from the body fluid. An antigen used to produce an antibody that specifically reacts with Nuf2 protein,
An antigen for producing a Nuf2 protein antibody, characterized by having CGGDYSAKIDEKTAELKRKMFKMS as a unique epitope among the amino acid sequences constituting Nuf2 protein. A method for detecting Nuf2 protein,
A sample having the Nuf2 protein antibody according to claim 1 and a protein fraction of a cell or tissue,
A method for detecting a Nuf2 protein, wherein the localization of an antigen is detected under a microscope by immunostaining. A method for detecting Nuf2 protein,
A sample having the Nuf2 protein antibody according to claim 1 and a protein fraction of a cell or tissue,
A method for detecting Nuf2 protein, characterized by detecting the presence of an antigen by Western blotting. A method for detecting Nuf2 protein,
A sample having the Nuf2 protein antibody according to claim 1 and a protein fraction of a cell or tissue,
A method for detecting Nuf2 protein, characterized by detecting the presence of an antigen by immunoprecipitation. A method for detecting another protein that interacts with Nuf2 protein,
A sample having the Nuf2 protein antibody according to claim 1 and a protein fraction of a cell or tissue,
A method for detecting another protein that interacts with a Nuf2 protein, comprising detecting another protein that specifically forms a complex with an antigen by co-immunoprecipitation.