JP6614431B2 - Anti-zebrafish CD4 monoclonal antibody or antibody fragment, anti-zebrafish CD8α monoclonal antibody or antibody fragment and nucleic acids encoding them - Google Patents

Description

  The present invention relates to anti-zebrafish CD4 monoclonal antibody or antibody fragment, anti-zebrafish CD8α monoclonal antibody or antibody fragment and nucleic acids encoding them.

  Conventionally, mice and rats have been mainly used as experimental animals. However, it seems that it will become increasingly difficult to use these experimental animals in large quantities due to cost and ethical issues.

  On the other hand, small fish such as zebrafish are easy to breed, are prolific, have a short generation cycle, generate in vitro, and have a basic structure as a vertebrate. It has organs and tissues. In addition, since the embryo is transparent and the development proceeds quickly, for example, using an embryo about 24 hours after fertilization, an organ abnormality or the like when a critical gene is lost can be easily observed from the outside.

  Because of these characteristics, small fish such as zebrafish have recently attracted attention as models for human diseases. The background is that the genome is almost deciphered and the gene homology between humans and fish is high.

  As a fish model for human diseases, many mutants related to nervous system, immune system, urinary system, hematopoietic system and circulatory system disease have been prepared so far. In addition, mutants of disease models called lifestyle-related diseases such as transgenic fish that cause acute T-cell leukemia and obesity models have also been produced (see Non-Patent Document 1, for example).

  In recent years, small fish have attracted attention in screening large-scale new drug compounds for drug discovery. Since zebrafish and the like are about 3 cm in length, the breeding space and cost are reduced, and the number of eggs is large. For example, a large number of eggs and embryos can be screened using a 96-well microplate. it can. In addition, since the generation speed is high, the result can be obtained quickly (see, for example, Non-Patent Document 1).

Teruyuki Nakanishi, Fish as a Human Disease Model and Prospects of Drug Discovery Research Using Fish, Applied Pharmacology, Vol. 80, No. 5/6, p. 99, 2011

  Thus, in recent years, zebrafish have attracted attention as experimental animals. However, antibodies against CD4 and CD8α expressed on the surface of zebrafish immune system cells are not known. These antibodies are essential for analyzing the zebrafish immune system. Accordingly, an object of the present invention is to provide a monoclonal antibody or antibody fragment against the zebrafish CD4 antigen and a monoclonal antibody or antibody fragment against the zebrafish CD8α antigen. The present invention also aims to provide nucleic acids encoding these antibodies or antibody fragments.

The present invention is as follows.
(1) The heavy chain variable region comprising CDRs 1 to 3 each consisting of the amino acid sequence of SEQ ID NO: 1 to 3 or the amino acid sequence of SEQ ID NO: 1 to 3, wherein one or several amino acids are deleted, substituted or added , And CDR1 to CDR3 are amino acid sequences of SEQ ID NOs: 4 to 6 or amino acid sequences of SEQ ID NOs: 4 to 6, respectively, wherein one or several amino acids are deleted, substituted, or added, and the light chain variable region An anti-zebrafish CD4 monoclonal antibody or antibody fragment.
(2) CDRs 1 to 3 each have a heavy chain variable region consisting of the amino acid sequence of SEQ ID NOs: 1 to 3, and CDRs 1 to 3 each have a light chain variable region consisting of the amino acid sequence of SEQ ID NOs: 4 to 6, The anti-zebrafish CD4 monoclonal antibody or antibody fragment according to (1).
(3) the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 7, and the amino acid sequence of SEQ ID NO: 8, or The anti-zebrafish CD4 monoclonal antibody according to (1) or (2), which has a light chain variable region consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 8 Or an antibody fragment.
(4) The anti-zebrafish CD4 monoclonal antibody or antibody fragment according to (3), which has a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.
(5) A nucleic acid encoding the anti-zebrafish CD4 monoclonal antibody or antibody fragment according to any one of (1) to (4).
(6) The heavy chain variable region comprising CDR 1 to 3 each having the amino acid sequence of SEQ ID NOs: 11 to 13 or the amino acid sequence of SEQ ID NOs: 11 to 13, wherein one or several amino acids are deleted, substituted, or added. And CDR1 to CDR3 are amino acid sequences of SEQ ID NOs: 14 to 16 or amino acid sequences of SEQ ID NOs: 14 to 16, respectively, wherein one or several amino acids are deleted, substituted, or added, and the light chain variable region An anti-zebrafish CD8α monoclonal antibody or antibody fragment.
(7) CDRs 1 to 3 each have a heavy chain variable region consisting of the amino acid sequence of SEQ ID NOs: 11 to 13, and CDRs 1 to 3 each have a light chain variable region consisting of the amino acid sequence of SEQ ID NOs: 14 to 16, respectively. The anti-zebrafish CD8α monoclonal antibody or antibody fragment described in (6).
(8) the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 17, and the amino acid sequence of SEQ ID NO: 18, or The anti-zebrafish CD8α monoclonal antibody according to (6) or (7), which has a light chain variable region comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 18 Or an antibody fragment.
(9) The anti-zebrafish CD8α monoclonal antibody or antibody fragment according to (8), which has a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.
(10) A nucleic acid encoding the anti-zebrafish CD8α monoclonal antibody or antibody fragment according to any one of (6) to (9).

  According to the present invention, a monoclonal antibody or antibody fragment against zebrafish CD4 antigen and a monoclonal antibody or antibody fragment against zebrafish CD8α antigen can be provided. In addition, nucleic acids encoding these antibodies or antibody fragments can be provided.

(A) And (b) is a graph which shows the result of Experimental example 3. FIG. (A) And (b) is a graph which shows the result of Experimental example 4. FIG. (A) And (b) is a graph which shows the result of Experimental example 5. FIG. (A) And (b) is a graph which shows the result of Experimental example 6. FIG. (A) And (b) is a graph which shows the result of Experimental example 7. FIG. 10 is a photograph showing the results of Experimental Example 8. 10 is a photograph showing the results of Experimental Example 9.

[Anti-zebrafish CD4 monoclonal antibody or antibody fragment]
In one embodiment, the present invention provides that the complementarity determining regions (CDRs) 1 to 3 are deleted of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 to 3 or the amino acid sequence of SEQ ID NO: 1 to 3, respectively. A heavy chain variable region consisting of a substituted or added amino acid sequence, and CDRs 1-3 are deleted in the amino acid sequence of SEQ ID NOs: 4-6 or the amino acid sequences of SEQ ID NOs: 4-6, respectively, An anti-zebrafish CD4 monoclonal antibody or antibody fragment having a light chain variable region comprising a substituted or added amino acid sequence is provided. Here, several in the CDR 1 to 3 of the heavy chain variable region or the light chain variable region mean 5, 4, 3 or 2.

Examples of the antibody fragment include Fab, F (ab ′) ₂ , single-chain Fv (scFv) in which a heavy chain variable region and a light chain variable region are linked with an appropriate linker. Examples of the scFv linker include peptides such as (GGGGS) ₃ (SEQ ID NO: 21).

  Since the anti-zebrafish CD4 monoclonal antibody or antibody fragment of this embodiment can bind well to the CD4 antigen on the surface of lymphocytes in zebrafish, it can be used for analysis of the immune mechanism of zebrafish by FACS analysis, fluorescence microscopy, etc. Can be used.

  In the anti-zebrafish CD4 monoclonal antibody or antibody fragment, CDRs 1 to 3 are each a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 to 3, and CDRs 1 to 3 are respectively consisting of the amino acid sequence of SEQ ID NOs: 4 to 6. It may have a light chain variable region.

  Further, the anti-zebrafish CD4 monoclonal antibody or antibody fragment may have a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

  Further, as long as the anti-zebrafish CD4 monoclonal antibody or antibody fragment has reactivity to the CD4 antigen of zebrafish, one or several amino acids in the amino acid sequence of SEQ ID NO: 7 have been deleted, substituted or added. It may have a heavy chain variable region comprising an amino acid sequence, and a light chain variable region comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 8. . Here, several in the heavy chain variable region or the light variable region are 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, It means 4, 3 or 2.

  The anti-zebrafish CD4 monoclonal antibody or antibody fragment may be labeled with a labeling substance. Examples of labeling substances include peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, lactate dehydrogenase, amylase, and the like; fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), Cy3, Cy5, R- Examples include fluorescent substances such as phycoerythrin, B-phycoerythrin, Alexafluoro488, and Alexafluoro647; radionuclides such as tritium, iodine125, and iodine131; biotin; streptavidin and the like.

[Nucleic acid encoding anti-zebrafish CD4 monoclonal antibody or antibody fragment]
In one embodiment, the present invention provides a nucleic acid encoding the anti-zebrafish CD4 monoclonal antibody or antibody fragment described above.

  Examples of such nucleic acids include the heavy chain variable region gene of the above-described anti-zebrafish CD4 monoclonal antibody, the light chain variable region gene of the above-described anti-zebrafish CD4 monoclonal antibody, and the heavy chain of the above-described anti-zebrafish CD4 monoclonal antibody. A gene encoding a part of a variable region and a constant region, a gene encoding a light chain variable region and a part of a constant region of the anti-zebrafish CD4 monoclonal antibody described above, and a heavy chain full length of the anti-zebrafish CD4 monoclonal antibody described above. Encoding gene, gene encoding the full-length light chain of the anti-zebrafish CD4 monoclonal antibody described above, and scFv obtained by linking the heavy chain variable region and the light chain variable region of the anti-zebrafish CD4 monoclonal antibody described above with an appropriate linker Do Gene, and the like.

  The heavy chain variable region gene may be a heavy chain variable region gene consisting of residues 58 to 414 of the nucleotide sequence set forth in SEQ ID NO: 9. The light chain variable region gene may be a light chain variable region gene consisting of residues 58 to 378 of the base sequence shown in SEQ ID NO: 10.

  In the above heavy chain variable region gene, CDRs 1 to 3 are each composed of the amino acid sequences set forth in SEQ ID NOs: 1 to 3, and a framework region other than CDRs encodes a heavy chain variable region derived from a non-rat antibody. It may be a gene. In the above light chain variable region gene, CDRs 1 to 3 are each composed of the amino acid sequences set forth in SEQ ID NOs: 4 to 6, and a framework region other than CDRs encodes a light chain variable region derived from a non-rat antibody. It may be a gene. Here, examples of the non-rat antibody include antibodies such as mouse, guinea pig, hamster, rabbit, dog, cat, goat, donkey, cow, horse, pig, monkey, and human.

  The nucleic acid of the present embodiment is a combination of the above-described heavy chain variable region gene of anti-zebrafish CD4 monoclonal antibody or a gene derived therefrom with the light chain variable region gene of anti-zebrafish CD4 monoclonal antibody or a gene derived therefrom. It is preferable that Here, as a gene derived from the heavy chain variable region gene, for example, CDRs 1 to 3 are each composed of the amino acid sequences set forth in SEQ ID NOs: 1 to 3, and a framework region other than the CDR is derived from a non-rat antibody. And a gene encoding the chain variable region. Similarly, as a gene derived from the light chain variable region gene, for example, CDR1 to CDR3 are each composed of the amino acid sequences set forth in SEQ ID NOs: 4 to 6, and framework regions other than CDR are derived from non-rat antibodies. Examples include a gene encoding a light chain variable region.

  The nucleic acid of this embodiment may be a vector that holds the above-described nucleic acid encoding the anti-zebrafish CD4 monoclonal antibody or antibody fragment in an expressible manner. With such a vector, the nucleic acid of this embodiment can be expressed, and the above-described anti-zebrafish CD4 monoclonal antibody or antibody fragment can be produced. A vector suitable for each expression system can be used. For example, pET for expression of E. coli, pAUR for expression of yeast, pIEx for expression of insect cells, pCMV for expression of animal cells, adenovirus vector, retrovirus A viral vector, a lentiviral vector, etc. are mentioned.

[Anti-zebrafish CD8α monoclonal antibody or antibody fragment]
In one embodiment, the present invention relates to an amino acid sequence in which CDRs 1 to 3 are each deleted, substituted, or added in the amino acid sequence of SEQ ID NOs: 11 to 13 or the amino acid sequence of SEQ ID NOs: 11 to 13, respectively. A heavy chain variable region consisting of the amino acid sequences of SEQ ID NOs: 14-16, or amino acid sequences in which one or several amino acids are deleted, substituted, or added in the amino acid sequences of SEQ ID NOs: 14-16, respectively. An anti-zebrafish CD8α monoclonal antibody or antibody fragment having a light chain variable region consisting of: Here, several in the CDR 1 to 3 of the heavy chain variable region or the light chain variable region mean 5, 4, 3 or 2.

  The antibody fragment is the same as described above. Since the anti-zebrafish CD8α monoclonal antibody or antibody fragment of this embodiment can bind well to the CD8α antigen on the surface of lymphocytes in zebrafish, it can be used for analysis of the immune mechanism of zebrafish by FACS analysis, fluorescence microscopy, etc. Can be used.

  In the anti-zebrafish CD8α monoclonal antibody or antibody fragment, CDR1 to CDR3 consist of the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 11-13, respectively, and CDR1 to CDR3 consist of the amino acid sequence of SEQ ID NO: 14 to 16, respectively. It may have a light chain variable region.

  Further, the anti-zebrafish CD8α monoclonal antibody or antibody fragment may have a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18.

  In addition, as long as the anti-zebrafish CD8α monoclonal antibody or antibody fragment has reactivity with the zebrafish CD8α antigen, one or several amino acids in the amino acid sequence of SEQ ID NO: 17 are deleted, substituted or added. It may have a heavy chain variable region comprising an amino acid sequence and a light chain variable region comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 18. . Here, several in the heavy chain variable region or the light variable region are 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, It means 4, 3 or 2.

  The anti-zebrafish CD8α monoclonal antibody or antibody fragment may be labeled with a labeling substance. The labeling substance is the same as described above.

[Nucleic acid encoding anti-zebrafish CD8α monoclonal antibody or antibody fragment]
In one embodiment, the present invention provides a nucleic acid encoding the anti-zebrafish CD8α monoclonal antibody or antibody fragment described above.

  Examples of such nucleic acids include the heavy chain variable region gene of the above-described anti-zebrafish CD8α monoclonal antibody, the light chain variable region gene of the above-described anti-zebrafish CD8α monoclonal antibody, and the heavy chain of the above-described anti-zebrafish CD8α monoclonal antibody. A gene encoding a part of a variable region and a constant region, a gene encoding a light chain variable region and a part of a constant region of the above-described anti-zebrafish CD8α monoclonal antibody, and a heavy chain full length of the above-described anti-zebrafish CD8α monoclonal antibody. A gene encoding the above, a gene encoding the full-length light chain of the anti-zebrafish CD8α monoclonal antibody described above, and an scFv in which the heavy chain variable region and the light chain variable region of the above-described anti-zebrafish CD8α monoclonal antibody are connected by an appropriate linker. Such as genes over de like.

  The heavy chain variable region gene may be a heavy chain variable region gene consisting of residues 58 to 402 in the nucleotide sequence set forth in SEQ ID NO: 19. The light chain variable region gene may be a light chain variable region gene consisting of the 61st to 381st residues of the nucleotide sequence set forth in SEQ ID NO: 20.

  In the above heavy chain variable region gene, CDRs 1 to 3 each have the amino acid sequence set forth in SEQ ID NOs: 11 to 13, and a framework region other than CDRs encodes a heavy chain variable region derived from a non-rat antibody. It may be a gene. In the above light chain variable region gene, CDRs 1 to 3 are each composed of the amino acid sequences set forth in SEQ ID NOs: 14 to 16, and a framework region other than CDRs encodes a light chain variable region derived from a non-rat antibody. It may be a gene. Here, examples of the non-rat antibody include the same ones as described above.

  The nucleic acid of this embodiment is a combination of the heavy chain variable region gene of the above-described anti-zebrafish CD8α monoclonal antibody or a gene derived therefrom with the light chain variable region gene of the anti-zebrafish CD8α monoclonal antibody or a gene derived therefrom. It is preferable that Here, as a gene derived from the heavy chain variable region gene, for example, CDRs 1 to 3 are each composed of the amino acid sequences set forth in SEQ ID NOs: 11 to 13, and framework regions other than the CDRs are derived from non-rat antibodies. And a gene encoding the chain variable region. Similarly, as a gene derived from the light chain variable region gene, for example, CDR1 to CDR3 are each composed of the amino acid sequences set forth in SEQ ID NOs: 14 to 16, and framework regions other than CDR are derived from non-rat antibodies. Examples include a gene encoding a light chain variable region.

  The nucleic acid of this embodiment may be a vector that holds the above-described nucleic acid encoding the anti-zebrafish CD8α monoclonal antibody or antibody fragment in an expressible manner. With such a vector, the nucleic acid of this embodiment can be expressed, and the above-described anti-zebrafish CD8α monoclonal antibody or antibody fragment can be produced. Examples of the vector include the same ones as described above.

  Next, although an experiment example is shown and this invention is demonstrated in detail, this invention is not limited to the following experiment examples.

[Experimental Example 1]
[Preparation of anti-zebrafish CD4 monoclonal antibody]
(Establishment of Nymbuna CD4 stably expressing NRK cell line)
The inventors have succeeded in cloning the CD4 gene of Carbius auratus langsdorfii in the past. The nucleotide sequence of Gimbuna CD4 gene is shown in SEQ ID NO: 22. The guinea beech CD4 gene was incorporated into an expression vector, introduced into an NRK cell line, which is a rat kidney-derived cell line, and cultured in the presence of neomycin to establish a guinea beech CD4 stably expressing NRK cell line.

(Rat immunity and production of hybridoma)
Conventionally, with respect to an antigen prepared based on genetic information, a recombinant protein prepared using E. coli or the like has been mainly used as an immunizing antigen. However, most antibodies obtained by immunization with recombinant proteins cannot recognize the primary structure of the target molecule, but cannot recognize the three-dimensional structure when expressed on the cell surface.

  Therefore, the present inventors immunized rats by the rat iliac lymph node method. More specifically, the above-mentioned Gimbuna CD4 stably expressing NRK cell line was immunized to rats syngeneic with the NRK cell line. Thereby, it was considered that an antibody recognizing only non-self guimbuna CD4 expressed on the cell surface of the NRK cell line was obtained.

  Gimbuna CD4 stably expressing NRK cell line was administered to the footpad of Wistar rat (Japan SLC Co., Ltd.) for immunization. Subsequently, inguinal lymph nodes were collected and lymphocytes were collected. Subsequently, the recovered lymphocytes and the mouse myeloma cell line P3X63-Ag8.653 cells were fused. The obtained cells were cultured in a HAT medium to prepare hybridomas in which lymphocytes and mouse myeloma cells were fused. From the obtained hybridoma, a clone producing a monoclonal antibody that reacts with guinea beech CD4 was selected, and an anti-guinea bean CD4 monoclonal antibody was established.

  Subsequently, the established anti-guimbuna CD4 monoclonal antibody gene was cloned by a conventional method, and its nucleotide sequence was determined. The nucleotide sequence of the heavy chain variable region of the anti-guimbuna CD4 monoclonal antibody is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 7. The base sequence of the light chain variable region of the anti-guimbuna CD4 monoclonal antibody is shown in SEQ ID NO: 10, and the amino acid sequence is shown in SEQ ID NO: 8. As will be described later, the inventors have clarified that this antibody reacts with the zebrafish CD4 antigen.

[Experimental example 2]
[Preparation of anti-zebrafish CD8α monoclonal antibody]
(Establishment of Gimbuna CD8α stable expression NRK cell line)
The inventors have succeeded in cloning the CD8α gene of Gimbuna (Carassius auratus langsdorfii) in the past. The nucleotide sequence of Gimbuna CD8α gene is shown in SEQ ID NO: 23. The guimbuna CD8α gene was incorporated into an expression vector, introduced into an NRK cell line, which is a rat kidney-derived cell line, and cultured in the presence of neomycin to establish a guimbuna CD8α stable expression NRK cell line.

  Subsequently, in the same manner as in Experimental Example 1, a hybridoma was prepared by immunizing a rat similar to the NRK cell line with the above-described Nymbuna CD8α stably expressing NRK cell line, and a clone producing a monoclonal antibody that reacts with Ginna CD8α. Select and established anti-guimbuna CD8α monoclonal antibody.

  Subsequently, the established anti-guimbuna CD8α monoclonal antibody gene was cloned by a conventional method, and its base sequence was determined. The nucleotide sequence of the heavy chain variable region of the anti-guimbuna CD8α monoclonal antibody is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 17. Further, the base sequence of the light chain variable region of the anti-guimbuna CD8α monoclonal antibody is shown in SEQ ID NO: 20, and the amino acid sequence is shown in SEQ ID NO: 18. As will be described later, the inventors have clarified that this antibody reacts with the zebrafish CD8α antigen.

[Experimental Example 3]
[Analysis of antibody reactivity to zebrafish lymphocyte population (1)]
Zebrafish lymphocyte populations were stained with the monoclonal antibodies prepared in Experimental Examples 1 and 2 and analyzed by FACS. It is known that fish are hematopoietic in the kidney. Therefore, first, a cell population containing lymphocytes was collected from zebrafish kidney. More specifically, first, leukocytes and erythrocytes were separated from a zebrafish kidney to prepare a cell suspension. Since the obtained cell suspension contains a large amount of red blood cells, the red blood cells were removed by hemolysis, and the obtained whole leukocyte population was stained with the monoclonal antibody described above and analyzed by FACS.

  In FACS analysis, lymphocyte populations were identified and gated from forward scatter (FSC) and side scatter (SSC) patterns, and the reactivity of monoclonal antibodies against lymphocyte populations was analyzed.

  As the primary antibody, the anti-CD4 monoclonal antibody prepared in Experimental Example 1 or the anti-CD8α monoclonal antibody prepared in Experimental Example 2 was used, and Alexa Fluoro488 (Life Technologies) labeled anti-rat IgG antibody was used as the secondary antibody. .

  FIG. 1A is a graph showing the results of staining with an anti-CD4 monoclonal antibody. As a result, about 9.2% of the zebrafish lymphocyte population was stained. In addition, the broken line of Fig.1 (a) shows the negative control dye | stained with the isotype control antibody.

  FIG. 1 (b) is a graph showing the results of staining with an anti-CD8α monoclonal antibody. As a result, about 9.1% of the zebrafish lymphocyte population was stained. In addition, the broken line of FIG.1 (b) shows the negative control dye | stained with the isotype control antibody.

[Experimental Example 4]
[Analysis of antibody reactivity to zebrafish lymphocyte population (2)]
In the same manner as in Experimental Example 3, antibody reactivity to zebrafish lymphocyte populations was analyzed. Specifically, the zebrafish lymphocyte population was double-stained using the monoclonal antibody prepared in Experimental Example 1 or 2 and the anti-ZAP-70 antibody.

  ZAP-70 is a T cell specific marker. Further, as the anti-ZAP-70 antibody, an anti-human ZAP-70 antibody was used. ZAP-70, a T cell-specific transcription factor, is conserved in vertebrates, and antibodies against human ZAP-70 have been reported to cross-react in fish. The present inventors have now confirmed that anti-human ZAP-70 antibody reacts with zebrafish and guinea bean T cells.

  FIG. 2 (a) is a graph showing the results of double staining of a zebrafish lymphocyte population with the anti-CD4 monoclonal antibody and anti-ZAP-70 antibody prepared in Experimental Example 1. FIG. As a result, it was revealed that the cells recognized by the anti-CD4 monoclonal antibody are T cells.

  FIG. 2 (b) is a graph showing the results of double staining of a zebrafish lymphocyte population with the anti-CD8α monoclonal antibody and anti-ZAP-70 antibody prepared in Experimental Example 2. As a result, it was revealed that the cells recognized by the anti-CD8α monoclonal antibody are T cells.

[Experimental Example 5]
[Analysis of antibody reactivity to HEK293T cells forcibly expressing zebrafish CD4]
Whether or not the anti-CD4 monoclonal antibody prepared in Experimental Example 1 recognizes the zebrafish CD4 antigen was examined. Specifically, first, the zebrafish CD4 gene was forcibly expressed in HEK293T cells, a human embryonic kidney cell line. The base sequence of the zebrafish CD4 gene is shown in SEQ ID NO: 24.

  Here, the HA tag was linked to the zebrafish CD4 gene expressed in HEK293T cells. Therefore, HEK293T cells expressing the introduced zebrafish CD4 gene can be stained with an anti-HA antibody.

  Subsequently, the HEK293T cells were stained with anti-HA antibody or the anti-CD4 monoclonal antibody prepared in Experimental Example 1 and analyzed by FACS.

  FIG. 3 (a) is a graph showing the result of staining HEK293T cells in which the zebrafish CD4 gene was forcibly expressed with an anti-HA antibody. This result can be said to indicate gene transfer efficiency. As a result, about 94.8% of HEK293T cells were stained. In addition, the broken line of Fig.3 (a) shows the negative control dye | stained with the isotype control antibody.

  FIG. 3B is a graph showing the results of staining HEK293T cells in which the zebrafish CD4 gene was forcibly expressed with the anti-CD4 monoclonal antibody prepared in Experimental Example 1. As a result, about 81.4% of HEK293T cells were stained. In addition, the broken line of FIG.3 (b) shows the negative control dye | stained with the isotype control antibody.

[Experimental Example 6]
[Analysis of antibody reactivity to HEK293T cells in which zebrafish CD4L2 is forcibly expressed]
In fish, in addition to the CD4 antigen homologous to the mammalian CD4 antigen, there is a fish-specific CD4 antigen (referred to as “CD4L2” or “CD4rel”). In this experimental example, it was examined whether or not the anti-CD4 monoclonal antibody prepared in Experimental Example 1 recognizes the zebrafish CD4L2 antigen.

  Specifically, first, the zebrafish CD4L2 gene was forcibly expressed in HEK293T cells. The base sequence of the zebrafish CD4L2 gene is shown in SEQ ID NO: 25.

  Here, the HA tag was linked to the zebrafish CD4L2 gene expressed in HEK293T cells. Therefore, HEK293T cells expressing the introduced zebrafish CD4L2 gene can be stained with an anti-HA antibody.

  Subsequently, the HEK293T cells were stained with anti-HA antibody or the anti-CD4 monoclonal antibody prepared in Experimental Example 1 and analyzed by FACS.

  FIG. 4 (a) is a graph showing the result of staining HEK293T cells in which the zebrafish CD4L2 gene is forcibly expressed with an anti-HA antibody. This result can be said to indicate gene transfer efficiency. As a result, about 97.2% of HEK293T cells were stained. In addition, the broken line of Fig.4 (a) shows the negative control dye | stained with the isotype control antibody.

  FIG. 4B is a graph showing the result of staining HEK293T cells in which the zebrafish CD4L2 gene was forcibly expressed with the anti-CD4 monoclonal antibody prepared in Experimental Example 1. As a result, HEK293T cells were hardly stained. In addition, the broken line of FIG.4 (b) shows the negative control dye | stained with the isotype control antibody.

[Experimental Example 7]
[Analysis of antibody reactivity to HEK293T cells forcibly expressing zebrafish CD8α]
Whether or not the anti-CD8α monoclonal antibody prepared in Experimental Example 2 recognizes the zebrafish CD8α antigen was examined.

  Specifically, first, the zebrafish CD8α gene was forcibly expressed in HEK293T cells. The base sequence of the zebrafish CD8α gene is shown in SEQ ID NO: 26.

  Here, the HA tag was linked to the zebrafish CD8α gene expressed in HEK293T cells. Therefore, HEK293T cells expressing the introduced zebrafish CD8α gene can be stained with an anti-HA antibody.

  Subsequently, the HEK293T cells were stained with an anti-HA antibody or the anti-CD8α monoclonal antibody prepared in Experimental Example 2 and analyzed by FACS.

  FIG. 5 (a) is a graph showing the result of staining HEK293T cells in which the zebrafish CD8α gene was forcibly expressed with an anti-HA antibody. This result can be said to indicate gene transfer efficiency. As a result, about 97.2% of HEK293T cells were stained. In addition, the broken line of Fig.5 (a) shows the negative control dye | stained with the isotype control antibody.

  FIG. 5 (b) is a graph showing the results of staining HEK293T cells in which the zebrafish CD8α gene was forcibly expressed with the anti-CD8α monoclonal antibody prepared in Experimental Example 2. As a result, about 69.6% of HEK293T cells were stained. In addition, the broken line of FIG.5 (b) shows the negative control dye | stained with the isotype control antibody.

[Experimental Example 8]
[Gene expression analysis in fractionated cell population (1)]
The cell population stained with the anti-CD4 monoclonal antibody prepared in Experimental Example 1 was collected and examined for gene expression.

  Specifically, first, a whole leukocyte population of zebrafish was collected in the same manner as in Experimental Example 3, and stained with the anti-CD4 monoclonal antibody prepared in Experimental Example 1. Subsequently, a total lymphocyte population, a CD4 negative cell population, and a CD4 positive cell population were sorted using a cell sorter (model “FACS Aria II”, BD Bioscience).

  Subsequently, total RNA was extracted from each sorted cell population and subjected to reverse transcription to synthesize cDNA. Perform PCR reaction using each synthesized cDNA as a template to express zebrafish CD4 gene, zebrafish CD8α gene, zebrafish T cell receptor constant site (TCRAC) gene, zebrafish immunoglobulin light chain constant site (IgLC) gene It was investigated. In addition, each β cDNA was standardized using the β-actin gene as an internal standard gene. Table 1 shows the primer names of each gene amplification primer and the sequence number of the base sequence.

FIG. 6 is a photograph of agarose gel electrophoresis showing the results of the PCR reaction. In FIG. 6, “total leukocytes” are the results using the cDNA of the total leukocyte population as a template, “total lymphocytes” are the results of using the cDNA of the entire lymphocyte population as a template, and “CD4 ⁻ ” is the CD4 negative cell. The result is the result of using the cDNA of the population as a template, “CD4 ⁺ ” is the result of using the cDNA of the CD4 positive cell population as a template, and the result of the “negative control” is the result of the control without the template. The number on the right side of the photograph indicates the number of PCR reaction cycles.

  As a result, expression of the zebrafish CD4 gene was observed only in the CD4 positive cell population. Moreover, expression of the TCRAC gene was observed in all fractions other than the whole leukocyte population. The reason why no expression of TCRAC gene was observed in the whole leukocyte population was considered to be due to the low proportion of T cells in the total leukocyte population. In addition, IgLC gene expression was observed only in the whole lymphocyte population and the CD4 negative cell population.

[Experimental Example 9]
[Gene expression analysis in fractionated cell population (2)]
The cell population stained with the anti-CD8α monoclonal antibody prepared in Experimental Example 2 was collected and examined for gene expression.

  Specifically, first, a whole leukocyte population of zebrafish was collected in the same manner as in Experimental Example 3, and stained with the anti-CD8α monoclonal antibody prepared in Experimental Example 2. Subsequently, a total lymphocyte population, a CD8α-negative cell population, and a CD8α-positive cell population were sorted using a cell sorter (model “FACS Aria II”, BD Bioscience).

  Subsequently, total RNA was extracted from each sorted cell population and subjected to reverse transcription to synthesize cDNA. PCR reaction was performed using each synthesized cDNA as a template, and the expression of zebrafish CD4 gene, zebrafish CD8α gene, zebrafish TCRAC gene, and zebrafish IgLC gene was examined. In addition, each β cDNA was standardized using the β-actin gene as an internal standard gene. As the primers for gene amplification, the same primers as in Experimental Example 8 were used.

FIG. 7 is a photograph of agarose gel electrophoresis showing the results of the PCR reaction. In FIG. 7, “total leukocytes”, “total lymphocytes”, and “negative control” have the same meaning as in Experimental Example 8. “CD8α ⁻ ” is the result of using the cDNA of the CD8α negative cell population as a template, and “CD8α ⁺ ” is the result of using the cDNA of the CD8α positive cell population as a template. The number on the right side of the photograph indicates the number of PCR reaction cycles.

  As a result, expression of the zebrafish CD8α gene was observed only in the CD8α-positive cell population. Moreover, expression of the TCRAC gene was observed in all fractions other than the whole leukocyte population. The reason why no expression of TCRAC gene was observed in the whole leukocyte population was considered to be due to the low proportion of T cells in the total leukocyte population. In addition, IgLC gene expression was observed only in the whole lymphocyte population and the CD8α-negative cell population.

  The results of Experimental Examples 3 to 9 above show that the anti-CD4 monoclonal antibody prepared in Experimental Example 1 reacts with zebrafish CD4 antigen, and the anti-CD8α monoclonal antibody prepared in Experimental Example 2 reacts with zebrafish CD8α antigen. Indicates to do.

  According to the present invention, a monoclonal antibody or antibody fragment against zebrafish CD4 antigen and a monoclonal antibody or antibody fragment against zebrafish CD8α antigen can be provided. In addition, nucleic acids encoding these antibodies or antibody fragments can be provided.

Claims (6)

CDRs 1 to 3 are a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1-3, and, CDRs 1 to 3 are a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4-6, that having a anti Zebrafish CD4 monoclonal antibody or antibody fragment. Heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region, comprising the amino acid sequence of SEQ ID NO: 8, anti zebrafish CD4 monoclonal antibody or antibody fragment of claim 1. A nucleic acid encoding the anti-zebrafish CD4 monoclonal antibody or antibody fragment according to claim 1 or 2 . CDRs 1 to 3 are a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11-13, and, CDRs 1 to 3 are a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14-16, that having a anti Zebrafish CD8α monoclonal antibody or antibody fragment. The anti-zebrafish CD8α monoclonal antibody or antibody fragment according to claim 4 , which has a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18. A nucleic acid encoding the anti-zebrafish CD8α monoclonal antibody or antibody fragment according to claim 4 or 5 .