JP4769009B2 - Vpr antigen for producing a hybridoma producing a Vpr-specific monoclonal antibody, an anti-Vpr-specific monoclonal antibody-producing hybridoma, an anti-Vpr-specific monoclonal antibody produced by the hybridoma, and immunological measurement of Vpr using the same - Google Patents

Description

  The present invention relates to a peptide for producing a hybridoma that produces an antibody that specifically recognizes the native type HIV accessory protein Vpr (hereinafter referred to as Vpr), an anti-Vpr-specific monoclonal antibody produced by the hybridoma, and the antibody. It relates to the immunoassay of Vpr used.

Immunodeficiency virus Immunodeficiency virus is known as a virus that causes immunodeficiency in various animals including humans. Known immunodeficiency viruses include, for example, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), etc., all of which are classified as retroviral lentiviruses. .

  Among these, human immunodeficiency virus (hereinafter referred to as HIV) has attracted particular attention as a pathogenic virus that causes acquired immunodeficiency syndrome (hereinafter referred to as AIDS) in humans. Currently, there are two types of HIV-1 and HIV-2. Are separated. Fig. 1 shows a schematic diagram of HIV virions, and Fig. 2 summarizes the genes identified in the HIV genome. The viral particle structure of HIV consists of a cylindrical core particle composed of a viral protein called p24 that wraps viral nucleic acids, reverse transcriptase, etc., and a p17 protein that lines the membrane on the outside, and on the outside It consists of an envelope with glycoproteins such as virus-specific gp120 and gp41. HIV genes are classified into structural genes (gag, pol, env), regulatory genes (Tat, Rev), and accessory genes (Nef, Vif, Vpr, Vpu, Vpx) according to the function and existence mode of the product. Structural genes are common to all retroviruses and play an important role in viral replication. These gene products, which are structural gene proteins (Gag, Pol, Env), are all synthesized as precursor proteins and cleaved by viral or host cell proteases to become mature proteins. Two types of regulatory proteins (Tat, Rev) are also called transactivators and function to control the expression of viral genes. Accessory proteins (Nef, Vif, Vpr, Vpu, Vpx) are specific to lentiviruses such as HIV / SIV, and their presence has not been confirmed in retroviruses other than lentiviruses such as HTLV. From this point of view, accessory proteins are thought to play an important role in the biological properties of lentivirus (Non-Patent Documents 1, 2, and 3). However, research on the mechanism of action of these proteins is delayed compared to structural proteins and regulatory proteins, and functional analysis of these proteins will become important in the future to elucidate the pathogenesis of AIDS, which is still unknown. It is considered.

  Like other retroviruses, the life cycle of HIV is adsorption to target cells, invasion and unshelling, synthesis of DNA complementary to viral RNA by retrovirus-specific reverse transcriptase, insertion into host cell chromosomal DNA, It takes the process of gene amplification and expression, virus particle construction and release. AIDS is generally associated with a long incubation period from HIV infection to onset. In clinical settings, it is very important to detect HIV infection as soon as possible, especially in order to prevent further infection to others due to blood transfusion. At present, a widely used practical method capable of detecting HIV infection at an earliest level is a method for detecting p24 protein by an immunochemical technique using an antibody against the p24 protein. Furthermore, there is a method for detecting by amplifying a part or all of the nucleic acid of HIV.

Meanwhile Vpr protein, Vpr (virus protein r) is immunodeficiency virus, in particular reported as an accessory protein having a molecular weight of about 15kDa with multiple functions in HIV-1. Vpr is thought to have cell cycle abnormalities, gene destabilization, G2 / M period arrest, apoptosis and HIV-1 replication promoting action (Non-Patent Documents 1, 2, and 3). Furthermore, it was pointed out that Vpr may be involved in HIV-1 infection and the onset of AIDS, and it has been considered that Vpr plays an important role. It is important to know the function of Vpr and control its function in order to elucidate the pathogenesis of AIDS, and thus to develop a new treatment method for AIDS.

Biological activity of Vpr protein In addition, with regard to the biological activity of Vpr protein, Vpr promotes HIV-1 replication in HIV-1 chronic persistently infected cells U1 cells, Vpr signaling through NFκB such as TNFα It is reported to be related to the system (Non-patent Documents 4 and 5). However, there is no report that Vpr has a specific receptor via a signal transduction system such as TNFα or IL-1 that induces inflammatory action.

  TNFα is known as a cytokine and is said to stimulate NFκB when bound to a specific receptor as a ligand. TNFα is considered to have a HIV-1 replication promoting action in HIV-1 chronic persistently infected cells U1 cells. Several monoclonal antibodies and polyclonal antibodies have already been prepared for TNFα, and some of them are known to neutralize the function of TNFα.

  In addition, Tat, which is an accessory protein like Vpr, is a 14 kDa protein and is considered to have multiple functions like TNFα. That is, it is said to have apoptosis and HIV-1 replication promoting action (Non-patent Document 6). Several monoclonal and polyclonal antibodies have already been prepared for Tat, and some of them are known to neutralize the function of Tat.

  Vpr is considered to have an HIV-1 replication promoting action, similar to TNFα and Tat. Therefore, the blood concentration rises early in the infection and promotes HIV-1 proliferation. Since the concentration in blood rises earlier than conventional p24 is detected in blood, it is possible to test HIV-1 infection early by measuring and detecting Vpr specific and highly sensitive This is presumed by using a polyclonal antibody against Vpr obtained by immunizing rabbits with 16 residues at the N-terminal side and 14 residues at the C-terminal side of Vpr (Non-patent Document 7).

Nature Med. Vol 8 pp673-680 2002 Molecular Cell Biology Dictionary, Tokyo Chemical Co., Ltd., p345, 654 and 808, 1997 Dictionary of immunology terms (third edition), latest medical company, p275, 1993 Proc. Natl. Acad. Sci. USA, Vol.92, pp.3621-3625, April 1995, Yosef Refaeliet al. NATURE MEDICINE, Vol.3, No.10, October 1997, p.1117-1123, Velpandi Ayyavooet al. J. Biol. Chem., Vol.274, No.41, October 8, 1999, pp.28837-28840, Kuan-Teh Jeanget al. Virus Research 90, 263-268, 2002 FASEB J., 13, 621-637, 1999 Rev. Med. Virol. 9: p.39-49 (1999) Cell, Vol.40, p.9-17, 1985, Simon WH et al. AIDS Research and human retroviruses, vol.3, p.33-39, 1987, Flossie WS et al. J. Virology, Vol.63, p.4110-4114, 1989, K. Ogawa et al. J. AIDS, vol.3, p.11-18, 1990, Eric AC et al. Nucl. Acids. Res. 25., p.3389-3402, 1997

  However, the establishment of a method for measuring and detecting Vpr protein and its related peptides simply and specifically with high sensitivity has not been established. In addition, a monoclonal antibody obtained using a full-length Vpr protein expressed using a yeast expression system as an immunogen has been reported (Non-patent Document 8), but the specificity of the monoclonal antibody has not been sufficiently confirmed. In other words, it has not been used in methods for measuring and detecting Vpr protein and related peptides.

  The present invention has been made in view of the above problems, and provides a new monoclonal antibody that specifically recognizes the Vpr protein and its related peptide with high affinity, as well as a cell that produces this monoclonal antibody, An object of the present invention is to provide a method for specifically and highly sensitively measuring Vpr protein and related peptides using an antibody.

  A sandwich ELISA is preferable as a method for simply and highly sensitively and specifically detecting Vpr in blood. In order to supply a sandwich ELISA stably, it is necessary to be able to supply the antibody to be used stably. When there is no detailed information on the presence state of the target protein, generally, in order to construct a sandwich ELISA, examination is performed using antibodies on the N-terminal side and C-terminal side of the target protein. However, in many cases, antibodies obtained by preparing polyclonal antibodies such as rabbits using a synthetic peptide as an immunogen do not have the performance used for sandwich ELISA, that is, high affinity. Moreover, even if a high-affinity antibody can be purified from the prepared polyclonal antibody, it is very difficult to secure an antibody amount that can stably supply a sandwich ELISA. Therefore, the present inventor first prepared a recombinant Vpr protein for the purpose of using it as a standard for immunogen and sandwich ELISA. When the Vpr protein is expressed in a solubilized state, it is known that it is difficult to obtain a large amount of protein due to its toxicity because the cells into which the gene has been introduced are killed. Therefore, it is necessary to select a vector that can recover the expressed protein in the insoluble fraction. Although pET vectors can be expressed in an IPTG-inducible manner, the expression level of proteins is high, but the disadvantage is that they often form inclusion bodies and become insoluble. However, since this point is effective in the expression of the Vpr protein this time, this vector was selected.

  In order to stably supply a sandwich ELISA, it must be possible to stably supply antibodies. As a result of intensive studies aimed at solving the above problems, the present invention has revealed that when a polyclonal antibody is produced using a recombinant Vpr protein as an immunogen, most of the amino acid sequence of the N-terminal side, MEQAPEDQGPQREPYN: amino acid residue of SEQ ID NO: 1 An antibody against the C-terminal region, which is found to be an antibody that recognizes a region containing groups 1 to 16 and is difficult to obtain stably with a polyclonal antibody, is an amino acid sequence of 14 residues on the C-terminal side (1) VTRQRRARNGASRS It has been found that a specific and high affinity monoclonal antibody can be obtained against the C-terminal side of the Vpr protein by immunizing with a specific peptide having the same amino acid sequence as a part of the amino acid sequence as an antigen. Further, an immunoassay is performed using a monoclonal antibody obtained using the peptide as an antigen and an antibody recognizing the N-terminal side of the Vpr protein. And by, we found that it is possible to measure and detect the Vpr and specifically and with high sensitivity related compounds, and completed the present invention.

The present invention is as follows.
(1) An antigen characterized by the following a) or b):
a) An antigen having a peptide consisting of 14 amino acids from the C-terminal end of the Vpr protein (amino acids 83 to 96 in SEQ ID NO: 1 in the sequence listing: VTRQRRARNGASRS) and exhibiting antigenic activity
b) Amino acid sequence in which one to several amino acids have been deleted, substituted or added from the peptide consisting of 14 amino acids from the C-terminal of the Vpr protein (amino acids 83 to 96 in SEQ ID NO: 1 in the sequence listing: VTRQRRARNGASRS) An antigen having a peptide comprising and exhibiting antigenic activity.
(2) A fused cell obtained by fusing an antibody-producing cell obtained from a mammal immunized with the antigen described in (1) above and a myeloma cell line from a mammal.
(3) The fused cell according to (2) above, wherein the fused cell is clone 2C7 (accession number: FERM AP-20367) or clone 4D2 (accession number: FERM AP-20366).
(4) A monoclonal antibody that binds to an HIV-1-derived Vpr protein produced by the fused cell according to (2) or (3).
(5) A method for immunological measurement of Vpr protein using the monoclonal antibody according to (4) as a solid phase antibody, comprising the following steps a) to c) and d) to f):
a) After reacting a standard substance with an immobilized antibody obtained by binding the monoclonal antibody described in (4) to an insoluble support,
b) reacting a labeled antibody obtained by labeling an antibody that recognizes Vpr having a recognition site different from the monoclonal antibody described in (4) with an enzyme;
c) creating a calibration curve by measuring the amount of reaction product of the enzyme; and
d) After reacting the sample with the immobilized antibody,
e) reacting a labeled antibody obtained by labeling an antibody that recognizes Vpr having a recognition site different from the monoclonal antibody described in (4) with an enzyme;
f) measuring the amount of the reaction product of the enzyme and measuring the Vpr protein contained in the specimen from the calibration curve (6) comprising the following steps a) to c) and d) to f) Immunological measurement method of Vpr protein using the monoclonal antibody according to (4) as a detection antibody:
a) After reacting a standard substance with an immobilized antibody in which an antibody recognizing Vpr having a recognition site different from the monoclonal antibody described in (4) is bound to an insoluble support,
b) reacting a labeled antibody obtained by labeling the monoclonal antibody according to (4) with an enzyme,
c) creating a calibration curve by measuring the amount of reaction product of the enzyme; and
d) After reacting the sample with the immobilized antibody,
e) reacting a labeled antibody obtained by labeling the monoclonal antibody described in (4) with an enzyme,
f) measuring the amount of the reaction product of the enzyme and measuring the Vpr protein contained in the specimen from the calibration curve (7) An antibody that recognizes Vpr having a recognition site different from the monoclonal antibody described in (4) The immunological measurement method according to (5) or (6) above, which is any of the following a) to c):
a) From a mammal having a peptide consisting of 16 amino acids from the N-terminus of the Vpr protein (1st to 16th amino acids in SEQ ID NO: 1 in the sequence listing: MEQAPEDQGPQREPYN) and immunized with an antigen exhibiting antigenic activity A monoclonal antibody produced from a fused cell obtained by fusing an obtained antibody-producing cell and a mammalian myeloma cell.
b) One to several amino acids of the peptide consisting of 16 amino acids from the N-terminal of the Vpr protein (1st to 16th amino acids in SEQ ID NO: 1 in the sequence listing: MEQAPEDQGPQREPYN) were deleted, substituted or added Monoclonal produced from a fusion cell obtained by fusing an antibody-producing cell obtained from a mammal having a peptide comprising an amino acid sequence and immunized with an antigen exhibiting antigenic activity, and a mammalian myeloma cell line antibody.
c) Polyclonal antibody purified from antiserum prepared by immunizing a mammal using recombinant Vpr as an immunogen (8) The standard substance is recombinant Vpr (5) to (7) above The immunological measurement method according to any one of the above.
(9) The immunological measurement method according to any one of (5) to (8), wherein the labeling substance is an enzyme, a fluorescent substance, or biotin.

According to the present invention, two types of monoclonal antibodies having reactivity specific to Vpr are provided. By using this monoclonal antibody, an immunological measurement method for Vpr was provided. This measurement method made it possible to detect Vpr in a liquid phase such as blood. As a result, it becomes possible to determine HIV infection by a new method. This measurement method is effective for primary screening of a large amount of samples at the time of blood donation because it can easily measure a large number of samples at a time with a small amount of samples, and prevents secondary infection due to blood transfusion. Can greatly contribute to doing.

  In addition, the antibody for Vpr and the method for measuring / detecting Vpr of the present invention are not only useful for measuring the concentration of Vpr in blood, but include, for example, the identification of the location of Vpr in cells and tissues and the involvement of Vpr. As a means to conduct research that could not be done by conventional analysis methods such as elucidation of intracellular signal transduction mechanisms that are expected to occur, this is considered as an abnormality in intracellular signal transduction mechanisms caused by the involvement of Vpr A means of elucidating the cause of AIDS pathogenesis from HIV-1 infection is provided. Moreover, the monoclonal antibody used for these is provided and the useful fusion cell which produces this monoclonal antibody is further provided.

Peptide derived from Vpr protein of amino acid sequence (1) VTRQRRARNGASRS which is the first of the present invention In the present invention, Vpr protein means an accessory protein having a multifunctional function in immunodeficiency virus of about 12 kDa to about 18 kDa, preferably about 15 kDa. Vpr is generally known for biological functions such as cell cycle abnormalities, gene destabilization, G2 / M period arrest, apoptosis and immunodeficiency virus replication promoting action including HIV-1. Vpr is particularly well studied in HIV-1, but its presence has been confirmed in viruses such as HIV-2 and SIV, and it shows the same amino acid sequence as HIV-1. In addition to Vpr, HIV-2 has a protein called Vpx having a high base sequence and amino acid sequence homology with Vpr (Non-patent Document 9).

  The amino acid sequence of the Vpr protein is known, and is described in, for example, documents such as Non-Patent Document 10, Non-Patent Document 11, Non-Patent Document 12, and Non-Patent Document 13. However, one or more of the natural proteins may be due to differences in the varieties of the species that produce them, gene mutations due to differences in ecotypes, or the presence of similar isozymes. It is well known that mutant proteins having amino acid mutations exist. Therefore, the Vpr-derived amino acid sequence (1) and amino acid sequence (2) of interest in the present invention are not limited to their full length, but one or more amino acid residues are substituted, or one to several amino acid residues are missing. Including lost mutants.

  In particular, Vpr is typically associated with an immunodeficiency virus, which is a retrovirus, and is generally more susceptible to mutations than normal proteins. As used herein, the term “amino acid mutation” means substitution, deletion, insertion and / or addition of one or more amino acids.

  As mentioned above, Vpr is intended to include all homologous proteins as long as they have the properties described herein. The homology is at least 70% or more, preferably 80% or more, more preferably 90% or more. The three-dimensional structure of amino acid residues constituting the peptide can be D, L, or DL, but the peptide is usually L-form.

  In the present specification, the percent homology can be determined by comparing with the sequence information using, for example, the BLAST program described in Altschul et al. The program can be used on the Internet from the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ) website. Various conditions (parameters) for homology search using the BLAST program are described in detail on the same site, and some settings can be changed as appropriate. The search is performed using default values.

  In general, substitution between amino acids having similar properties (for example, substitution from one hydrophobic amino acid to another hydrophobic amino acid, substitution from one hydrophilic amino acid to another hydrophilic amino acid, substitution from one acidic amino acid to another When a substitution to an acidic amino acid or a substitution from one basic amino acid to another basic amino acid is introduced, the resulting mutant protein often has the same properties as the original protein. Techniques for modifying recombinant proteins with desired mutations using genetic recombination techniques are well known to those skilled in the art, and such mutant proteins are also included within the scope of the present invention. The present invention relates to a peptide having the following amino acid sequence (1) VTRQRRARNGASRS from the C-terminus of the Vpr protein according to claim 1 (amino acid residues 83 to 96 of SEQ ID NO: 1), or an amino acid sequence (1) The present invention relates to an antigen consisting of an amino acid sequence in which one to several amino acids are deleted, substituted or added, and consisting of a peptide exhibiting an antigenic activity equivalent to that of amino acid sequence (1).

  The amino acid sequence (1) which is the antigen of the present invention was found as follows. The basic HIV-1-derived Vpr consists of 96 amino acids (SEQ ID NO: 1). Based on the amino acid sequence of Vpr, the secondary structure was compared by epitope analysis software according to a conventional method. (1) Sites with high hydrophilicity by hydrophilicity prediction, (2) Sites with β-turns by secondary structure prediction (3) Sites where flexible sites exist by Vpredity prediction and can be epitopes on the surface of Vpr The amino acid sequence at the C-terminal (1) VTRQRRARNGASRS and the amino acid sequence at the N-terminal (2) MEQAPEDQGPQREPYN were selected (amino acid residues 1 to 16 in SEQ ID NO: 1). As described above, the Vpr protein is known to be a protein that is very susceptible to mutation like other HIV-related proteins. Therefore, amino acid sequences (1) and (2) are almost mutated in the Vpr protein sequence compared to the Vpr protein amino acid sequence predicted from the Vpr gene isolated from individual AIDS patients that have already been reported in large numbers. It was confirmed that there was no site. The comparison results are shown in FIG. However, it is difficult to infer that an antibody obtained using a peptide predicted from epitope analysis software as an antigen can recognize the target protein and be used in the target measurement method. Therefore, in order to confirm whether the peptide predicted by the epitope analysis software could be an antibody recognition site, rabbits were immunized with recombinant Vpr to produce a polyclonal antibody, and the antibody recognition site was verified. Table 1 shows a summary of the reactivity of the polyclonal antibody prepared with the recombinant Vpr performed by the present inventors and the result of verification of the recognition site performed using the synthetic peptide.

ウサギ２羽(Rab #1, Rab. #2)に大腸菌で発現させたN末端側に6Hisタグを導入したリコンビナントVpr(His-Vpr、実施例２に示す)を免疫し、得られた抗血清中に含まれる抗体の種類をHis-Vpr、N末端側に6Hisタグを導入したVprとは異なるリコンビナント蛋白質(His-NC)、Vprのアミノ酸番号1から16番目（アミノ酸配列：MEQAPEDQGPQREPYN）の合成ペプチド(N-ter.)、Vprのアミノ酸番号83から96番目（アミノ酸配列：VTRQRRARNGASRS）の合成ペプチド(C-ter.)を固相用抗原として作製した、それぞれのマイクロタイタープレートを用いて行った、ELISAの結果を示す。それぞれのウサギから得られた抗血清中に含まれる抗体の殆どは、Vprのアミノ酸番号1から16番目（アミノ酸配列：MEQAPEDQGPQREPYN）に対する抗体であった。Rab. #2抗血清中には、Vprのアミノ酸番号83から96番目（アミノ酸配列：VTRQRRARNGASRS）に対する抗体も含まれていることから、アミノ酸番号1から16番目よりは低いが、抗原性があることも確認できた。Vprのアミノ酸番号1から16番目（アミノ酸配列：MEQAPEDQGPQREPYN）に対する抗体を作製する場合は今回用いたリコンビナント蛋白或いは、Vprのアミノ酸番号1から16番目（アミノ酸配列：MEQAPEDQGPQREPYN）の合成ペプチドでも作製できることが推測される。Vprのアミノ酸番号83から96番目（アミノ酸配列：VTRQRRARNGASRS）に対する抗体を作製する場合は合成ペプチドが望ましいことが予測される。

Antiserum obtained by immunizing two rabbits (Rab # 1, Rab. # 2) with recombinant Vpr (His-Vpr, shown in Example 2) introduced with 6His tag at the N-terminal side expressed in E. coli Recombinant protein (His-NC), which is different from Vpr with the His-Vpr and 6His tag introduced on the N-terminal side, and the synthetic peptide of amino acids 1 to 16 of Vpr (amino acid sequence: MEQAPEDQGPQREPYN) (N-ter.), Vpr amino acid number 83 to 96 (amino acid sequence: VTRQRRARNGASRS) synthetic peptide (C-ter.) Prepared as a solid phase antigen, each microtiter plate was used, The result of ELISA is shown. Most of the antibodies contained in the antiserum obtained from each rabbit were antibodies against amino acid numbers 1 to 16 of Vpr (amino acid sequence: MEQAPEDQGPQREPYN). Rab. # 2 antiserum contains antibodies against amino acids 83 to 96 of Vpr (amino acid sequence: VTRQRRARNGASRS), so it is lower than amino acids 1 to 16 but has antigenicity. Was also confirmed. When producing an antibody against amino acid number 1 to 16 of Vpr (amino acid sequence: MEQAPEDQGPQREPYN), it is speculated that the recombinant protein used this time or a synthetic peptide of amino acid number 1 to 16 (amino acid sequence: MEQAPEDQGPQREPYN) of Vpr can also be prepared. Is done. It is predicted that a synthetic peptide is desirable when producing an antibody against amino acid numbers 83 to 96 of Vpr (amino acid sequence: VTRQRRARNGASRS).

  In order to develop a sandwich ELISA, two types of antibodies with different recognition sites are required. As is well known, polyclonal antibodies have different reactivity to antigenic proteins among immunized individuals, that is, the proportion of antibodies required to construct a measurement system contained in antibodies against the target protein. It is not preferable to supply a measurement system. However, when recombinant Vpr is used as an antigen, a high-affinity antibody that recognizes the N-terminal region can be stably and easily produced using a rabbit. A large amount of antibody that specifically recognizes the other C-terminal region necessary to construct the Vpr ELISA is required. Since it is difficult to obtain a polyclonal antibody in this region, a monoclonal antibody is required. To obtain a monoclonal antibody, an antibody-producing cell obtained from a mammal immunized with an antigen is fused with a mammalian myeloma cell, and then a fused cell that produces the target antibody is selected. The advantage is that the same monoclonal antibody can be easily obtained in a large amount by culturing the obtained fused cells. FIG. 4 shows the verification results of the reactivity of the polyclonal antibody and the monoclonal antibody conducted by the present inventors.

Production of the monoclonal antibody, which is the second monoclonal antibody of the present invention, and the fused cell, which is the third of the present invention, will be described below.
(A. Antigen)
A peptide having the amino acid sequence (1) was synthesized by a solid phase method or a liquid phase method, and then the obtained crude product was purified by reverse phase chromatography to a purity of 95% or more and used as an immunogen. Since the peptide is a relatively small molecule, it is difficult to produce an antibody even if this peptide is immunized as it is. Therefore, (1) introduces a cysteine residue artificially on the N-terminal side, and uses m-maleimide-benzoyl-N-hydoroxysuccinimide (MBS) via the SH group present in the cysteine residue as a carrier protein. Used as an immunogen by binding to The N-terminal amino group can be used as an immunogen by binding to a carrier protein using 1-ethyl-3- (3-dimethylaminoprppyl) carbodiimide hydrochloride (EDC). When EDC is used, care must be taken when an amino acid having an amino group, such as a lysine residue, is present in the antigenic peptide. The carrier protein used was Keyhole Limpet Hemocyanin (KLH), but albumin and thyroglobulin can also be used.

(B. Immunization with the above antigen)
As immunized animals, mice, mammals, rats, hamsters, and the like can be used. Usually, mice are most commonly used, and BALB / c mice and other strain mice (BDF1, C3H, etc.) can be used. At this time, the immunization plan and the concentration of the antigen should be selected so that a sufficient amount of antigen-stimulated lymphocytes are formed. For example, the antigen is mixed with Freund's complete adjuvant or Freund's incomplete adjuvant to form an emulsion. One mouse is immunized subcutaneously, intradermally or peritoneally with 25 μg of antigen as a peptide. After collecting blood of the mouse and confirming that antibody is produced in the blood according to a conventional method, 25 μg of antigen dissolved in PBS or the like is further administered intravenously. Spleen cells for fusion are removed several days after the final immunization.

(C. Cell fusion)
The spleen is aseptically removed from the immunized mammalian individual as described above, and a single cell suspension is prepared therefrom. The spleen cells (antibody producing cells) are fused with a myeloma cell established as an appropriate cell line by using an appropriate cell fusion promoter. As myeloma cells, those derived from mammals of the same species as the immunized animal are desirable, but spleen cells such as rats and hamsters can be fused with mouse myeloma cells. As the antibody-producing cells, lymph node cells that have been antigen-stimulated by immunizing a foot pad can also be used. A preferred fusion ratio of antibody producing cells and myeloma cells is in the range of about 20: 1 to about 2: 1. The use of 0.5-1.5 mL of fusion medium is appropriate for about 10 ⁸ antibody producing cells. As a preferred fusion promoter, for example, polyethylene glycol having an average molecular weight of 1000 to 6000 can be advantageously used, but other fusion promoters known in this field (for example, Sendai virus) can also be used. In addition to the method using these fusion promoters, cell fusion may be performed by a method using electric shock.

(D. Selection of fusion cells producing the desired monoclonal antibody)
Cells that have undergone cell fusion treatment are unfused antibody-producing cells, unfused myeloma cells, and fused cells (fused cells of antibody-producing cells and myeloma cells, fused cells of antibody-producing cells and antibody-producing cells, myeloma Cells and myeloma cells). The mixed cells are diluted in a separate container (eg, a microtiter plate) with selective medium that does not support the myeloma cells and cultured for a time sufficient to kill the myeloma cells (about 2 weeks). As the selective medium, for example, HAT medium can be used. In this selective medium, the myeloma cells die. Antibody-producing cells are non-neoplastic and are not immortalized. Therefore, it will die after a certain period of time (about 1 week). In contrast, fusion cells of antibody-producing cells and myeloma cells have the characteristics of immortality of the parent myeloma cells and the viability of the parent antibody-producing cells in the selective medium, and therefore can survive in the selective medium. Thus, after the fusion cell is detected, the antibody against the peptide having the amino acid sequence (1) is screened by an enzyme linked immunosorbent assay to produce a monoclonal antibody that specifically binds to the immunogen. Select only fused cells. If the target monoclonal antibody is a protein and the immunogen is a substance consisting of a part of the target protein such as a synthetic peptide, a monoclonal antibody that specifically binds to the target protein is further produced. Select fused cells. Examples of such fused cells include fused cell clone 2C7 (accession number: FERM AP-20367) or fused cell clone 4D2 (accession number: FERM AP-20366).

(E. Acquisition of the desired monoclonal antibody)
After the fusion cell producing the target monoclonal antibody is cloned by an appropriate method (for example, limiting dilution method), the antibody can be produced by two different methods. According to the first method, a monoclonal antibody produced by the fusion cell can be obtained from the culture supernatant by culturing the fusion cell in an appropriate medium for a certain period. According to the second method, the fused cells can be administered and proliferated into the peritoneal cavity of an immunized animal having an isogenic or semi-isogenic gene. A monoclonal antibody produced by the fusion cell can be obtained from the ascites fluid of the host for a certain period after the administration of the fusion cell intraperitoneally. Usually, in order to efficiently obtain ascites, prior to administering the fused cells into the peritoneal cavity, pristene or the like is administered to the peritoneal cavity in advance.

The fourth immunological measurement method of the present invention The immunological measurement method of the present invention can be performed by a known immunological measurement method (for example, sandwich method) for the antibody. In the method, a monoclonal antibody can be used as a solid phase antibody, or a monoclonal antibody can be used as a detection antibody.

  Either monoclonal or polyclonal antibody is immobilized on a suitable insoluble carrier. Then, in order to avoid non-specific binding between the insoluble carrier and the test sample to be measured, the surface of the insoluble carrier is coated with a suitable substance (for example, bovine serum albumin). The insoluble carrier on which the first antibody thus obtained is immobilized is contacted with the test sample at a constant time and at a constant temperature. Thus, the Vpr protein binds to the first antibody. Next, after washing with an appropriate detergent, the solution of the second antibody labeled with an appropriate labeling substance (eg, enzyme) is contacted with the free Vpr protein bound to the first antibody in an insoluble carrier at a constant time and at a constant temperature. And react. This is washed with an appropriate detergent, and then the amount of the labeled substance of the second antibody (labeled antibody) bound on the insoluble carrier is measured.

  Further, the immunological measurement method of the present invention can also be performed by a one-step method. That is, the first antibody immobilized on the insoluble carrier, the labeled second antibody, and the test sample are mixed at the same time, reacted at a constant time and at a constant temperature, and the amount of the labeling substance is measured. be able to.

  The kit based on the immunoassay method of the present invention is configured based on a combination of reagents in a normal sandwich method. That is, when (1) the first antibody immobilized on an insoluble carrier, (2) the labeled second antibody, (3) the solubilizer, (4) the detergent, and (5) the labeling substance is an enzyme A substrate for measuring enzyme activity and a reaction terminator.

  As the insoluble carrier, for example, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, cross-linked dextran, polysaccharide, and other paper, glass, metal, agarose, and combinations thereof can be used. As the shape of the insoluble carrier, various shapes such as a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a cell, and a test tube are applicable.

  As the labeling substance for the labeled antibody, it is advantageous to use enzymes, fluorescent substances, luminescent substances, radioactive substances, and the like. Enzymes include peroxidase, alkaline phosphatase, β-D-galactosidase, fluorescent materials such as fluorescein isothiocyanate and phycobiliprotein, luminescent materials such as isolucinol and lucigenin, and radioactive materials such as 125I, 131I and 14C. , 3H, etc. can be used, but these are not limited to those exemplified, and other materials can be used as long as they can be used for immunological measurement.

  When the labeling substance is an enzyme, a substrate and, if necessary, a color former are used for measuring the activity. When peroxidase is used as an enzyme, H2O2 is used as a substrate, 2,2'-azinodi- [3-ethylbenzthiazolinesulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, o-phenylenediamine, When using 4-aminoantivillin, 3,3 ', 5,5'-tetramethylbenzidine, etc., when alkaline phosphatase is used as an enzyme, when using o-nitrophenyl phosphate as a substrate, when using β-D-galactosidase as an enzyme As the substrate, fluorescein-di- (β-D-galactopyranoside), 4-methylumbelliferyl-β-D-galactopyranoside, and the like can be used.

  In the immunological measurement kit, (3) the solubilizing agent may be any one that is usually used for immunological measurement. For example, pH containing phosphate buffer, Tris-HCl buffer, acetate buffer, etc. Is in the range of 6.0 to 8.0 as a suitable example. Furthermore, as (4) the detergent, those generally used for immunological measurement are used as they are. Examples thereof include physiological saline, phosphate buffer, Tris-HCl buffer, and mixtures thereof. These detergents may further contain a nonionic surfactant such as Triton X-100, Tween 20 or Brig 35, and an ionic surfactant such as sodium dodecyl sulfate.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples. However, these are not intended to limit the technical scope of the present invention, and various forms can be adopted as long as they belong to the technical scope of the present invention. Not too long. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are also included in the technical scope of the present invention. In the following examples, the steric structure of the amino acid residue is L form unless otherwise specified.

(Example 1) Preparation of immunogen A peptide having the following amino acid sequence was synthesized and used as an immunogen and an antigen for antibody titer confirmation.
(1) H-VTRQRRARNGASRS-OH
(2) H-MEQAPEDQGPQREPYNC-OH
Since the peptide is a relatively small molecule, it is difficult to produce an antibody even if this peptide is immunized as it is. Therefore, (1) was used as an immunogen by binding the amino group on the N-terminal side to a carrier protein using 1-ethyl-3- (3-dimethylaminoprppyl) carbodiimide hydrochloride (EDC). (2) was used as an immunogen by binding a cysteine residue artificially introduced to the C-terminal side with a carrier protein using m-maleimide-benzoyl-N-hydoroxysuccinimide (MBS). In this example, Keyhole Limpet Hemocyanin (KLH) was used as the carrier protein, but albumin and thyroglobulin can also be used.

Example 2 Acquisition of Recombinant Vpr As a standard substance for quantifying Vpr in a living body, cDNA synthesis was performed based on an amino acid sequence encoding the full length of Vpr, and Vpr was prepared using an E. coli expression system. The Vpr gene (cDNA) was co-fitted with E. coli using the base sequence for expression, and the entire sequence was synthesized and introduced into the vector. Since the expressed protein must be recovered in the insolubilized fraction and then solubilized and purified with urea or the like, pET28a capable of introducing His-tag was selected. As the vector, if the Vpr protein can be recovered in the insoluble fraction first, the purpose is achieved. Therefore, a vector other than pET28a can be used. After expression as a fusion protein with His-tag, purification was performed using Ni-NTA agarose (Qiagen).

(Example 3) BALB / c mice were used as mice for immunization. At this time, the antigen described in Example 1 was mixed with Freund's complete adjuvant or Freund's incomplete adjuvant to form an emulsion, and one mouse was immunized subcutaneously, intradermally or intraperitoneally with 25 μg of the antigen as a peptide. After collecting the blood of the mouse and confirming that antibodies are produced in the blood according to a conventional method, 25 μg of antigen dissolved in PBS or the like is further administered intravenously, and for fusion several days after the final immunization. Spleen cells were removed.

(Example 4) Cell fusion and selection and acquisition of fusion cells producing the target monoclonal antibody As described above, a spleen cell suspension is prepared, and mouse myeloma cells are mixed with polyethylene glycol having an average molecular weight of 1000 to 6000. Fused. The fusion ratio of antibody-producing cells and myeloma cells was in the range of about 20: 1 to about 2: 1. The mixed cells were diluted with a selective medium HAT medium that does not support myeloma cells in a microtiter plate, and cultured for a sufficient time (about 2 weeks) to kill the myeloma cells to obtain fused cells. In the obtained fused cells, antibodies against the peptide having the amino acid sequence (1) are screened by an enzyme immunoassay (Enzyme Linked Imunosorbent Assay), and only those fused cells that produce monoclonal antibodies that specifically bind to the immunogen. Selected. Thus, clone 2C7 (accession number: FERM AP-20367), clone 4D2 (accession number: FERM AP-20366), clone 1H1, and clone 4H1 were obtained as fusion cells. After cloning the fusion cells producing the desired monoclonal antibody by the limiting dilution method, the fusion cells are administered into the peritoneal cavity of the mouse, and the monoclonal antibody produced by the fusion cell is obtained from the host ascites for a certain period of time. It was. In order to obtain ascites efficiently, pristene was administered to the abdominal cavity in advance before administering the fused cells into the abdominal cavity.

(Example 5) Confirmation of specificity of monoclonal antibody The monoclonal antibody produced from the fused cell clone obtained in Example 4 is a monoclonal antibody obtained using a synthetic peptide as a source. Then, the reactivity with respect to Vpr protein was confirmed by the three types of methods shown below.
(5-1) Confirmation of Reactivity to Recombinant Vpr by ELISA Fusion Cell Clone 2C7 (Accession Number: FERM AP-20367), Clone 4D2 (Accession Number: FERM AP-20366), Clone 1H1, obtained in Example 4 FIG. 3 shows the results of confirming the reactivity to recombinant Vpr by ELISA for the four types of monoclonal antibodies produced from clone 4H1. In FIG. 4, “2C7” is a monoclonal antibody produced from clone 2C7, “4D2” is a monoclonal antibody produced from clone 4D2, “1H1” is a monoclonal antibody produced from clone 1H1, and “4H1” is a clone. Monoclonal antibody produced from 4H1, “N-term” is a rabbit polyclonal antibody against amino acids 1 to 16 of Vpr (amino acid sequence: MEQAPEDQGPQREPYN), “C-term” is amino acids 83 to 96 of Vpr (amino acid sequence) : Rabbit polyclonal antibody against VTRQRRARNGASRS). ELISA was performed according to a standard method, and a plate obtained by immobilizing recombinant Vpr at a concentration of 10 mg / mL on a microtiter plate (Nunk) was used. Mouse monoclonal antibody was detected using HRP-labeled anti-mouse IgG (H; L) (Bio-Rad), and rabbit polyclonal antibody was detected using HRP-labeled anti-rabbit IgG (H; L) (Bio-Rad). It was. Both monoclonal antibodies were antibodies that recognize recombinant Vpr immobilized on a microplate, but it was revealed that monoclonal antibodies 2C7 and 4D2 reacted more strongly than monoclonal antibodies 1H1 and 4H1.

(5-2) Confirmation of reactivity to recombinant Vpr by Western blot (WB) method Next, the result of confirmation of reactivity to recombinant Vpr by WB is shown in FIG. In FIG. 5, “2C7” is a monoclonal antibody produced from clone 2C7, “4D2” is a monoclonal antibody produced from clone 4D2, “1H1” is a monoclonal antibody produced from clone 1H1, and “4H1” is a clone. Monoclonal antibody produced from 4H1, “N-term” is a rabbit polyclonal antibody against amino acids 1 to 16 of Vpr (amino acid sequence: MEQAPEDQGPQREPYN), “C-term” is amino acids 83 to 96 of Vpr (amino acid sequence) : Rabbit polyclonal antibody against VTRQRRARNGASRS). The WB was performed after treating the Escherichia coli extract containing the recombinant Vpr with the sample treatment solution for SDS-PAGE, performing SDS-PAGE at 2 mg per lane, and transferring the protein to the PVDF membrane according to a conventional method. CBB in the figure is a Coomassie-stained membrane after transfer. Each antibody against Vpr was reacted at a concentration of 1 mg / mL. Mouse monoclonal antibody was detected using HRP-labeled anti-mouse IgG (H; L) (manufactured by Bio-Rad), and rabbit polyclonal antibody was detected using HRP-labeled anti-rabbit IgG (H; L) (manufactured by Bio-Rad). All monoclonal antibodies recognized recombinant Vpr, but 4H1 monoclonal antibody was less reactive than the other three monoclonal antibodies. In addition, there was no significant difference in the reactivity to Vpr, which seems to be a monomer, for three monoclonal antibodies other than the 4H1 antibody (lower arrow in the figure), but the reactivity to Vpr, which seems to be a dimer. The results were slightly different for each of the three monoclonal antibodies (upper arrow in the figure).

(5-3) Confirmation of Reactivity to Recombinant Vpr by Immunoprecipitation Method This monoclonal antibody is intended to be applied to the detection of Vpr in human serum. Therefore, the reactivity of Vpr in the liquid phase was confirmed using serum with human recombinant serum supplemented with recombinant Vpr (shown as + in the figure) or unadded serum (shown with-in the figure). The results are shown in FIG. In the method, 50 mg of purified monoclonal antibody was adsorbed on 20 mL of commercially available Protein A gel (manufactured by Amersham Pharmacia). The serum after the addition of purified recombinant Vpr to 100 mg / mL or human serum without addition was diluted 10-fold with a buffer solution, and then 100 mL was added to the gel for mixing and reaction. The gel after the reaction with the serum was washed well and then treated with a sample treatment solution for SDS-PAGE to perform SDS-PAGE. The gel after electrophoresis was confirmed by WB using biotin-labeled rabbit polyclonal antibody against amino acids 83 to 96 of Vpr (amino acid sequence: VTRQRRARNGASRS) and HRP-labeled streptavidin. The left figure shows the result of Coomassie staining (CBB) of the gel after electrophoresis, and the right figure shows the result of WB. Both monoclonal antibodies were antibodies that recognized recombinant Vpr in the liquid phase, but monoclonal antibody 4D2 showed stronger reactivity than monoclonal antibodies 2C7 and 1H1, and monoclonal antibody 4H1 was weaker than monoclonal antibodies 2C7 and 1H1. The result showed sex. Monoclonal antibodies 2C7 and 4D2 were selected for the detection of Vpr in human serum by combining the results of immunoprecipitation with the results of ELISA and Western blot shown above.

(Example 6) Construction of measurement method of Vpr protein using monoclonal antibody as solid phase antibody (FIG. 7)
FIG. 7 shows the results of a Vpr sandwich ELISA performed using the monoclonal antibody 2C7 or 4D2 produced from the fused cell clone selected in Example 5 as the solid phase antibody. The immobilization on the microtiter plate was performed according to a conventional method. The solid phase was prepared by adding 0.1 mL of antibody solution prepared to 10 mg / mL with PBS per well. As a comparative control, a rabbit polyclonal antibody against amino acids 83 to 96 of Vpr (amino acid sequence: VTRQRRARNGASRS) (in the figure)
The solid phase of cAb) was also performed at the same time. Each antibody-immobilized plate was blocked with BSA and then used for ELISA. The specimen used was a purified recombinant Vpr prepared using a buffer solution so as to be 50, 5, 0.5, 0 (ng / mL). As a sample, 0.1 mL per well of a microtiter plate on which an antibody was immobilized was reacted, and Vpr in the liquid phase was captured. Captured Vpr was performed using biotin-labeled rabbit polyclonal antibody against amino acid numbers 1 to 16 of Vpr (amino acid sequence: MEQAPEDQGPQREPYN) and HRP-labeled streptavidin. Both monoclonal antibodies were confirmed to be usable as solid phase antibodies for Vpr sandwich ELISA.

(Example 7) Construction of a method for measuring Vpr protein using a monoclonal antibody as a detection antibody A Vpr protein was detected using the monoclonal antibody 2C7 or 4D2 produced from the fused cell clone selected in Example 5 as a detection antibody. The result of sandwich ELISA is shown in FIG. In the same manner as in Example 6, the solid phase was prepared by adding 0.1 mL per well of a rabbit polyclonal antibody against amino acid numbers 1 to 16 of Vpr (amino acid sequence: MEQAPEDQGPQREPYN) prepared in PBS to 10 mg / mL. went. As a comparative control, a rabbit polyclonal antibody against the sandwich ELISA (amino acid sequence 83 to 96 of Vpr (amino acid sequence: VTRQRRARNGASRS) performed in Example 6 was used for solid phase, and amino acids 1 to 16 of biotin-labeled Vpr (amino acids ELISA using a rabbit polyclonal antibody against the sequence (MEQAPEDQGPQREPYN) as a detection antibody, indicated by N-biotin in the figure). Both monoclonal antibodies were confirmed to be usable antibodies as detection antibodies for Vpr sandwich ELISA.

The genes identified in the HIV genome are indicated. A schematic diagram of HIV virus particles is shown. The amino acid site to be mutated in the Vpr protein and the mutated amino acid are shown. The graph showing the relationship of the light absorbency with respect to the antibody (purified IgG) density | concentration when using recombinant Vpr protein is shown. The figure which confirmed the binding property of the antibody (purified IgG) with respect to Vpr protein by Western blotting is shown. The figure which confirmed the binding property of the antibody (purified IgG) with respect to Vpr protein which exists in a liquid phase by the immunoprecipitation method is shown. The graph showing the result of ELISA implemented using the monoclonal antibody with respect to Vpr as an antibody for solid phases is shown. The graph showing the result of ELISA implemented using the monoclonal antibody with respect to Vpr as a detection antibody is shown.

Claims (9)

An antigen characterized by the following a):
a) An antigen having a peptide consisting of only 14 from the C-terminus of the Vpr protein (the 83rd to 96th amino acids in sequence number 1 of the sequence listing: VTRQRRARNGASRS) and exhibiting antigenic activity.   A fused cell obtained by fusing an antibody-producing cell obtained from a mammal immunized with the antigen of claim 1 and a mammalian myeloma cell. The fused cell according to claim 2, wherein the fused cell is clone 2C7 (accession number: FERM P- 20367) or clone 4D2 (accession number: FERM P- 20366).   A monoclonal antibody that binds to an HIV-1-derived Vpr protein produced by the fused cell according to claim 2 or 3. A method for immunological measurement of Vpr protein using the monoclonal antibody according to claim 4 as a solid phase antibody, comprising the following steps a) to c) and d) to f).
a) after reacting a standard substance with an immobilized antibody obtained by binding the monoclonal antibody according to claim 4 to an insoluble support,
b) reacting a labeled antibody obtained by labeling an antibody recognizing Vpr having a recognition site different from the monoclonal antibody according to claim 4 with an enzyme;
c) creating a calibration curve by measuring the amount of reaction product of the enzyme; and
d) After reacting the sample with the immobilized antibody,
e) reacting a labeled antibody obtained by labeling an antibody that recognizes Vpr having a recognition site different from the monoclonal antibody according to claim 4 with an enzyme;
f) measuring the amount of the reaction product of the enzyme, and measuring the Vpr protein contained in the sample from the calibration curve The method for immunological measurement of Vpr protein using the monoclonal antibody according to claim 4 as a detection antibody, comprising the following steps a) to c) and d) to f).
a) After reacting a standard substance with an immobilized antibody obtained by binding an antibody recognizing Vpr having a recognition site different from the monoclonal antibody of claim 4 to an insoluble support,
b) reacting a labeled antibody obtained by labeling the monoclonal antibody according to claim 4 with an enzyme;
c) creating a calibration curve by measuring the amount of reaction product of the enzyme; and
d) After reacting the sample with the immobilized antibody,
e) reacting a labeled antibody obtained by labeling the monoclonal antibody according to claim 4 with an enzyme,
f) measuring the amount of the reaction product of the enzyme, and measuring the Vpr protein contained in the sample from the calibration curve The immunological method according to claim 5 or 6, wherein the antibody recognizing Vpr having a recognition site different from that of the monoclonal antibody according to claim 4 is one of the following a) or b): Measuring method.
a) A mammal having a peptide consisting only of 16 amino acids from the N-terminus of the Vpr protein (amino acids 1 to 16 in SEQ ID NO: 1 in the sequence listing: MEQAPEDQGPQREPYN) and immunized with an antigen exhibiting antigenic activity A monoclonal antibody produced from a fusion cell obtained by fusing an antibody-producing cell obtained from the above and a mammalian myeloma cell line, or b) an anti-antibody produced by immunizing a mammal with a recombinant Vpr as an immunogen Polyclonal antibody purified from serum   The immunoassay method according to claim 5, wherein the standard substance is recombinant Vpr.   The immunoassay method according to any one of claims 5 to 8, wherein the labeling substance is an enzyme, a fluorescent substance or biotin.

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