CN116987186A - Rabbit monoclonal antibody aiming at human ERG protein and application thereof - Google Patents
Rabbit monoclonal antibody aiming at human ERG protein and application thereof Download PDFInfo
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- CN116987186A CN116987186A CN202310995287.5A CN202310995287A CN116987186A CN 116987186 A CN116987186 A CN 116987186A CN 202310995287 A CN202310995287 A CN 202310995287A CN 116987186 A CN116987186 A CN 116987186A
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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Abstract
The invention belongs to the technical field of antibody preparation, and particularly relates to a rabbit monoclonal antibody aiming at human ERG protein and application thereof. The amino acid sequences of the light chain complementarity determining regions 1-3 of the rabbit monoclonal antibody aiming at the human ERG protein are respectively shown in SEQ ID NO. 3-5; the amino acid sequences of the heavy chain complementarity determining regions 1-3 are shown in SEQ ID NOS.8-10, respectively. The invention uses ERG protein 35-68AA segment coupled KLH protein as immunogen, and the prepared rabbit monoclonal antibody can specifically identify and combine ERG protein expressed by human cells or tissue samples, has the advantages of high sensitivity, high specificity and the like when being used for immunodetection of ERG protein, can effectively avoid false positive and false negative results, ensures the detection accuracy, and is suitable for high-accuracy and high-sensitivity immunodetection of multiple pathological samples, in particular for immunohistochemical detection.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a rabbit monoclonal antibody aiming at human ERG protein and application thereof.
Background
Erythrocyte transformation-specific related gene ERG is one of the gene family members of the E-26 transformation-specific (ETS) transcription factor that sequence-specific DNA-binding transcription factor. ERG gene plays an important and highly conserved role in vertebrate development, and in early embryo development, ERG is highly expressed in embryonic embryoid leaves and endothelial cells, and plays an important role in vascular system formation, genitourinary tract formation and bone development, and in late embryo development, ERG participates in physiological processes such as regulation of hematopoietic stem cell pluripotency, endothelial cell homeostasis and angiogenesis. ERG expression is not limited to development, but is normally observed in endothelial tissues (including adrenal, cartilage, heart, spleen, and lymph) and eosinophils in adult mice, and is primarily responsible for regulating and controlling endothelial apoptosis and angiogenesis and plays a sustained role in maintaining endothelial cell health.
ERG is expressed in both benign and malignant vascular tumors, and ERG antibodies are highly sensitive to vascular tumors, making them one of the highly sensitive and specific biomarkers of vascular-derived tumors. In addition, the gene is involved in chromosomal rearrangements that lead to deregulation of ERG activity and to different fusion gene products, associated with poor prognosis for a variety of different cancers. ERG has been found to be expressed in prostate cancer and high grade intraepithelial neoplasia (HGPIN), and ERG overexpression due to the TMPRSS2-ERG fusion gene formed by fusion of androgen regulated transmembrane serine protease 2 (TMPRSS 2) gene with ERG has been identified as a key driver for metastasis and poor prognosis in prostate cancer. But there is little positive expression of ERG in other epithelial tumors than prostate cancer, and negative expression is seen in all cancers of the breast, gastrointestinal, gynecological, kidney, lung, ovary, pancreas, salivary gland, skin, thyroid, and testes. ERG was also found to be expressed in a minority of EWS sarcoma (an invasive bone tumor and soft tissue tumor) due to chromosomal rearrangements resulting from fusion of ERG and EWSR1 genes, resulting in EWS/ERG chimeric proteins consisting of the amino-terminal transactivation domain (EWS) of EWS sarcoma breakpoint region 1 and the carboxy-terminal ETS domain of ERG. ERG also has expression staining in some meningiomas due to cross-reactivity with Fli-1. Chromosomal translocations between ERG and TLS/FUS or ERG and ELF4 are associated with acute myelogenous leukemia.
In view of the diagnostic and labeling value of ERG, ERG protein detection has important guiding significance in clinical diagnosis and prognosis evaluation of diseases such as angio-derived tumors, prostate cancer and the like. At present, an antibody-based immunological method such as an Immunohistochemical (IHC) is often used for detecting the expression level and the localization condition of ERG in pathological cells and tissues, however, the specificity of the currently marketed anti-human ERG protein antibody is not strong, and nonspecific staining is easily caused during IHC pathological diagnosis, so that the accuracy of a detection result is affected. Therefore, development of an anti-ERG antibody which has good specificity and can meet IHC detection requirements has important significance.
Disclosure of Invention
Aiming at the problems that the anti-human ERG protein antibody in the prior art has poor specificity and is difficult to be used for immunohistochemical detection, the invention provides a rabbit monoclonal antibody aiming at human ERG protein and provides application of the rabbit monoclonal antibody in preparation of reagents and/or kits for immunodetection of human ERG protein.
In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:
in a first aspect the invention provides a rabbit monoclonal antibody directed against human ERG protein comprising a light chain variable region and a heavy chain variable region, each comprising 3 complementarity determining regions, wherein: the amino acid sequences of the light chain complementarity determining region 1, the light chain complementarity determining region 2 and the light chain complementarity determining region 3 are shown in SEQ ID NO.3-5, respectively; the amino acid sequences of heavy chain complementarity determining region 1, heavy chain complementarity determining region 2, and heavy chain complementarity determining region 3 are shown in SEQ ID NOS.8-10, respectively.
Further, the light chain variable region and the heavy chain variable region each comprise 4 framework regions, and the 4 framework regions and the 3 complementarity determining regions are staggered in sequence to form a variable region; the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7.
Further, the amino acid sequence of the light chain of the rabbit monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6.
Further, the rabbit monoclonal antibody is a full length antibody or an antibody fragment selected from the group consisting of a Fab fragment, F (ab) 2 Fragments, (Fv) fragments 2 Fragments, scFv fragments and sc (Fv) 2 At least one of the fragments.
In a second aspect the invention provides a nucleic acid molecule for encoding a rabbit monoclonal antibody against a human ERG protein as described above.
Further, the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody is shown as SEQ ID NO.12, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 14.
Further, the nucleotide sequence of the light chain of the rabbit monoclonal antibody is shown as SEQ ID NO.11, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 13.
In a third aspect the present invention provides a recombinant vector comprising a nucleic acid molecule as described above.
In a fourth aspect, the invention provides the use of a rabbit monoclonal antibody directed against human ERG protein as described above, a nucleic acid molecule as described above, a recombinant vector as described above in the preparation of a reagent and/or kit for immunodetection of human ERG protein.
Further, the immunodetection method is an immunohistochemical method, and the immunodetection pathological sample comprises one or more of human tonsil, human extranodal NK/T cell lymphoma, human placenta and human appendix.
The invention has the advantages and positive effects that:
according to the invention, the ERG protein 35-68AA fragment is coupled with KLH protein as an immunogen to immunize a white rabbit, then a monoclonal antibody development technology based on single B lymphocyte screening and culturing is adopted to obtain the anti-ERG protein rabbit monoclonal antibody, the anti-ERG protein rabbit monoclonal antibody with the complementarity determining region can specifically identify and bind ERG protein expressed by human cells or tissue samples, when immunohistochemistry is adopted to detect positive tissue samples expressing ERG protein and negative tissue samples not expressing ERG protein, all positive samples and negative samples can be accurately detected, positive staining is clear, nonspecific staining is avoided, the background is clean, the specificity of the antibody is proved to be high, the sensitivity is high, the specificity is high when the anti-ERG protein monoclonal antibody is used for immunodetection of ERG protein, false positive and false negative results can be effectively avoided, the detection accuracy is ensured, and the anti-ERG protein monoclonal antibody is suitable for high-accuracy and high-sensitivity immunodetection of multiple pathological samples, and especially immunohistochemical detection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of a vector used for constructing a rabbit monoclonal antibody 1G8 expression vector according to example 1 of the present invention, from left to right, respectively pRB322 vector map carrying light chain constant regions and heavy chain constant regions in advance;
FIG. 2 shows the specific localization and expression of ERG protein in human appendiceal tissue samples detected by immunohistochemistry using rabbit monoclonal antibody 1G8 in accordance with example 2 of the present invention;
FIG. 3 shows the specific localization and expression of ERG protein in human tonsil tissue samples detected by immunohistochemistry using rabbit monoclonal antibody 1G8 in example 2 of the present invention;
FIG. 4 shows the detection of specific localization and expression of ERG protein in human prostate tissue samples by immunohistochemistry using rabbit monoclonal antibody 1G8 according to example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In addition, the terms "comprising," "including," "containing," "having," and the like are intended to be non-limiting, as other steps and other ingredients may be added that do not affect the result.
In addition, it is noted that unless otherwise defined, in the context of the present invention, scientific and technical terms used should have meanings commonly understood by one of ordinary skill in the art.
The terms "comprising," "including," "having," and the like are intended to be non-limiting, as other steps and other ingredients not affecting the result may be added. The term "and/or" should be taken to refer to a specific disclosure of each of the two specified features or components with or without the other. For example, "a and/or B" will be considered to encompass the following: (i) A, (ii) B, and (iii) A and B.
In the context of the present invention, the terms "rabbit monoclonal antibody", "antibody" and the like have the same meaning and are used interchangeably to refer to antibodies that specifically bind to Human (Human) ERG protein. The modifier "rabbit" means that the Complementarity Determining Regions (CDRs) of the antibody are derived from a rabbit immunoglobulin sequence.
An antibody is an immunoglobulin molecule that specifically binds to an antigen or epitope of interest through at least one antigen recognition site located in the variable region. In the present invention, the term "antibody" is to be interpreted in the broadest sense and includes different antibody structures, including but not limited to so-called full length antibodies, antibody fragments, and genetic or chemical modifications thereof, as long as they exhibit the desired antigen binding activity. Where "antibody fragment" refers to one or more portions or fragments of a full-length antibody, in typical examples, the antibody fragment comprises: fab, fab', F (ab) 2 、F(ab’) 2 、Fv、(Fv) 2 、scFv、sc(Fv) 2 。
A typical antibody molecule (full length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types, kappa and lambda chains, respectively; heavy chains can be categorized into five, μ, δ, γ, α and ε chains, respectively, and antibodies are defined as IgM, igD, igG, igA and IgE, respectively. The amino acid sequences of the heavy and light chains near the N-terminus vary greatly, the other portions of the amino acid sequences are relatively constant, the region of the light and heavy chains near the N-terminus, where the amino acid sequences vary greatly, is referred to as the variable region (V), and the region near the C-terminus, where the amino acid sequences are relatively stable, is referred to as the constant region (C). Heavy chain variable regions (VH) and light chain variable regions (VL) are typically the most variable parts of antibodies and contain antigen recognition sites. The VH and VL regions can be further subdivided into hypervariable regions (hypervariable region, HVR) also known as Complementarity Determining Regions (CDRs) which are circular structures, and Framework Regions (FR) where the heavy and light chain CDRs are held closely together and cooperate to form a surface complementary to the three-dimensional structure of the antigen or epitope of interest, which determines the specificity of the antibody, and are the sites for antibody recognition and binding to the antigen. The FR region is the more conserved part of VH and VL, which are generally in the β -sheet configuration, joined by three CDRs forming a connecting loop. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
CDRs may be identified according to Kabat definitions, chothia definitions, a combination of both Kabat and Chothia definitions, abM definitions, contact definitions, IMGT unique numbering definitions and/or conformational definitions, or any CDR determination method known in the art. As used herein, CDRs are defined by the Kabat numbering system.
The light chain constant region (CL) and the heavy chain constant region (CH) are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of an antibody. CL lengths of different classes of igs (κ or λ) are substantially identical, but CH lengths of different classes of igs are different, e.g. IgG, igA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4. The amino acid sequences of the antibody heavy and light chain constant regions are well known in the art.
Full length antibodies are the most complete antibody molecular structure, having a typical Y-type molecular structure, and thus, "full length antibodies", "complete antibodies" and "Y-type antibodies" are used interchangeably in the context of the present invention.
The term "Antigen-binding fragment (Fab)" is used in the context of an antibody moleculeThe antigen-binding region, which consists of the complete light chain (variable and constant regions) and a portion of the heavy chain (variable and one constant region fragment), can be cleaved by protease to give fragments such as Fab, F (ab ') 2, fab' and the like. For example, igG can be degraded into two Fab fragments and one Fc fragment by papain; igG can be degraded into a F (ab') under the action of pepsin 2 Fragments and a pFC' fragment. F (ab') 2 The fragment was further reduced to form two Fab' fragments. Because the Fab has an antigen binding region and a partial constant region, the Fab not only has antibody-antigen affinity like a single chain antibody (scFv), excellent tissue penetrating power and the like, but also has a more stable structure, and is widely applied to clinical diagnosis and treatment.
The term "variable fragment (Fv)" is located at the N-terminus of an antibody Fab fragment, contains only the variable region, and consists of one variable region of one light chain and one heavy chain, is a dimer of one VH and one VL that are non-covalently bound (VH-VL dimer), and the 3 CDRs of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer, with the ability to recognize and bind antigen, although with less avidity than an intact antibody.
The term "Single-chain antibody (scFv)" refers to a minimum antibody fragment in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked by a flexible linker (linker, typically consisting of 10 to 25 amino acids), which retains the binding specificity of the original antibody to an antigen, and the linker in the present invention is not particularly limited as long as it does not interfere with the expression of the antibody variable regions linked at both ends thereof. Compared with full-length antibodies, scFv has the characteristic of small molecular weight, thus having higher penetrability and lower immune side reaction.
The full length sequences of the antibodies or antibody fragments of the invention may be from a single species, such as rabbit, or may be chimeric or humanized antibodies to reduce body rejection while maintaining the desired specificity, affinity. The term "chimeric antibody" antibody refers to an antibody in which a portion is derived from a particular source or species, while the remainder is derived from a different source or species. The term "humanized antibody" is a chimeric antibody in which the CDR regions of a non-human antibody, such as a rabbit antibody, and the FR regions derived from a human, in some cases, the variable regions of a non-human antibody bind to the constant regions of a human antibody, e.g., a human rabbit chimeric antibody; in other cases, the CDR regions of a non-human antibody bind to FR regions and constant regions derived from human antibody sequences, i.e., the CDR regions of a non-human antibody are grafted onto human antibody Framework (FR) sequences derived from single or multiple other human antibody variable region framework sequences. In the present invention, the CDR regions in the chimeric or humanized antibody are derived from rabbit-derived CDR regions.
The term "monoclonal antibody" refers to a homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen or epitope. "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as limiting the structure, source, or manner of preparation of the antibody.
The term "specific binding" is a term well known in the art that exhibits "specific binding," "specific binding," or is referred to as "preferential binding" if a molecule reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular antigen or epitope of interest than to other antigens or epitopes of interest, and does not necessarily require (although may include) exclusive binding.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
The embodiment of the invention provides a rabbit monoclonal antibody aiming at human ERG protein, which comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise 3 Complementarity Determining Regions (CDRs), and the amino acid sequences of the light chain CDR1, the light chain CDR2 and the light chain CDR3 are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.
According to the invention, the ERG protein 35-68AA amino acid fragment is coupled with KLH protein as an immunogen, the protein specificity of the 35-68AA expression region is high, the protein is not cross-identified with Fli-1, the immunogenicity of an epitope is strong, the hydrophilicity is good, and the immunogen is easily soluble and is convenient for immunizing animals, so that a white rabbit is immunized by adopting the protein fragment, and then a monoclonal antibody development technology based on single B lymphocyte screening and culturing is adopted to obtain an anti-ERG protein rabbit monoclonal antibody, and heavy chain and light chain amino acid and nucleotide sequences.
The biological function of an antibody is that the CDR regions of the antibody are complementary to the spatial conformation between the antigenic determinants, and therefore the length and amino acid composition of the CDR sequences largely determine the specificity and affinity of the antibody for binding to an antigen. The rabbit monoclonal antibody with the CDR can specifically identify and combine ERG proteins expressed by human cells or tissue samples, has good affinity and anti-interference capability, is used as a detection antibody, adopts an immunohistochemical method to specifically dye a plurality of positive tissue samples expressing ERG proteins and negative tissue samples not expressing ERG proteins, can accurately detect all positive samples, has clear positive dyeing and no nonspecific dyeing, has clean background, has no specific dyeing on all negative samples, confirms that the specificity of the antibody is high, has the advantages of high sensitivity, high specificity and the like when being used for immunodetection of ERG proteins, can effectively avoid false positive and false negative results, ensures the detection accuracy, is suitable for the immunodetection of high accuracy and high sensitivity of multiple pathological samples, and provides reliable antibody raw materials for immunodetection of diagnosis of diseases with abnormal ERG protein expression, determination of primary parts and pathological typing, and particularly immunohistochemical detection.
Alternatively, the light chain variable region and the heavy chain variable region each comprise 4 Framework Regions (FR), 4 FR and 3 CDRs sequentially staggered to form the variable region. The amino acid sequence of the light chain variable region (VL) of the rabbit monoclonal antibody is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region (VH) is shown as SEQ ID NO. 7.
Optionally, the rabbit monoclonal antibodies of the invention further comprise a light chain constant region (CH) and a heavy chain constant region (VH), CL and VL comprising the complete light chain, CH and VH comprising the complete heavy chain. The constant regions of antibodies are typically obtained by public interrogation, such as: through IMGT online database (www.imgt.org), rabbit source IgG gamma C reign is searched for CH and rabbit source IgG Kappa C reign is searched for CL.
Alternatively, the amino acid sequence of the light chain is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown as SEQ ID NO.6, and correspondingly, the heavy chain is of the IgG type.
Alternatively, the rabbit monoclonal antibodies of the invention may be full length antibodies or antibody fragments including, but not limited to: (i) A Fab fragment, a monovalent fragment consisting of the variable region and the first constant region of each heavy and light chain; (ii) F (ab) 2 A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fv fragment consisting of one heavy chain variable region and one light chain variable region of an antibody; (iv) (Fv) 2 Fragments consisting of two Fv fragments covalently linked together; (v) An scFv fragment, an Fv fragment consisting of a single polypeptide chain, a heavy chain variable region and a light chain variable region joined by a linker; (vi) sc (Fv) 2 A fragment is a fragment in which two heavy chain variable regions and two light chain variable regions are linked by a linker or the like. These antibody fragments can be obtained by conventional techniques known to those skilled in the art.
The antibody fragment of the present invention has CDR regions having amino acid sequences shown in SEQ ID NO.3-5 and SEQ ID NO.8-10, more preferably has variable regions having amino acid sequences shown in SEQ ID NO.2 and 7, and is capable of retaining intact antigen recognition and binding sites, thereby binding to the same antigen, particularly to the same epitope, as a full-length antibody.
Yet another embodiment of the invention provides a nucleic acid molecule for encoding a rabbit monoclonal antibody directed against a human ERG protein as described above.
The nucleic acid molecule may be in the form of DNA (e.g., cDNA or genomic DNA or synthetic DNA) or RNA (e.g., mRNA or synthetic RNA). The DNA may be single-stranded or double-stranded, or may be a coding strand or a non-coding strand. The sequence of the nucleic acid molecule is deduced by conventional means such as codon encoding rules according to the amino acid sequence of the antibody.
Illustratively, the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody of the invention is shown as SEQ ID NO.12, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 14. Further, the nucleotide sequence of the light chain is shown as SEQ ID NO.11, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 13. It will be appreciated by those skilled in the art that nucleic acid molecules other than those exemplified above may likewise be encoded to provide rabbit monoclonal antibodies of the invention due to the degeneracy of the genetic code, and therefore the nucleic acid molecules of the examples should not be taken as limiting the scope of the invention.
Another embodiment of the invention provides a recombinant vector comprising a nucleic acid molecule as described above.
The starting vector from which the recombinant vector is constructed is a variety of vectors conventional in the art, as long as it is capable of harboring the nucleic acid molecule. Typical vectors include plasmids, viral vectors, phages, cosmids and minichromosomes. Plasmids are the most common form of vector, and thus, in the context of the present invention, vectors are used interchangeably with plasmids. The vector may be a cloning vector (i.e., for transferring the nucleic acid molecule into a host and for mass propagation in a host cell) or an expression vector (i.e., comprising the necessary genetic elements to allow expression of the nucleic acid molecule inserted into the vector in a host cell). Thus, a cloning vector may contain a selectable marker, and an origin of replication that matches the cell type specified by the cloning vector, while an expression vector contains regulatory elements (e.g., promoters, enhancers) for expression in a specified host cell. The nucleic acid molecules of the invention may be inserted into a suitable vector to form a cloning vector or an expression vector carrying the nucleic acid molecules of the invention. This is well known in the art and will not be described in detail herein.
Nucleic acid molecules encoding the heavy and light chains of an antibody of the invention may be cloned into a vector, each nucleic acid sequence being linked downstream of a suitable promoter; for example, each nucleic acid sequence encoding a heavy chain and a light chain may be operably linked to a different promoter, or alternatively, the nucleic acid sequences encoding the heavy chain and the light chain may be operably linked to a single promoter such that both the heavy chain and the light chain are expressed from the same promoter. In other embodiments, nucleic acid molecules encoding the heavy and light chains of an antibody of the invention may also be constructed separately on two vectors, which may be introduced into the same or different host cells. When the heavy and light chains are expressed in different host cells, each chain may be isolated from the host cell in which it is expressed and the isolated heavy and light chains mixed and incubated under appropriate conditions to form the antibody. The choice of the aforementioned expression vector/promoter depends on the type of host cell used to produce the antibody.
Transformation of host cells with recombinant vectors can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl 2 By a method or MgCl 2 The method may also be performed by microinjection, electroporation, or liposome encapsulation, and the procedures used are well known in the art. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, microinjection, electroporation, liposome packaging, and the like.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomycete, salmonella typhimurium, fungal cells such as yeast, insect cells of drosophila S2 or Sf9, CHO, COS7, 293 series cells, and the like. After obtaining a host cell transformed with the expression vector as described above, the cell is cultured under appropriate conditions to express the monoclonal antibody, and then isolated to obtain a purified antibody.
In a preferred embodiment, the recombinant vector is a mammalian expression vector pBR322 and the host cell is a human kidney epithelial cell (293F cell).
The embodiment of the invention also provides application of the rabbit monoclonal antibody aiming at the human ERG protein, the nucleic acid molecule and the recombinant vector in preparation of reagents and/or kits for immunodetection of the human ERG protein.
The advantages of the rabbit monoclonal antibody, the nucleic acid molecule and the recombinant vector for human ERG protein in the preparation of the reagent and/or the kit for immunodetection of human ERG protein are the same as those of the rabbit monoclonal antibody for human ERG protein in the prior art, and are not described in detail herein.
The antibodies of the invention may be used alone or may be conjugated or coupled to a detectable label (for detection purposes). Detectable labels for detection purposes include, but are not limited to: biotin, fluorescein, chemiluminescent groups, fluorescent proteins, enzymes (e.g., horseradish peroxidase, acid phosphatase), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies, or combinations thereof.
In immunodetection, the antibody of the present invention is used as an antigen-binding antibody (such as a capture antibody or primary antibody) which specifically recognizes and binds to the ERG protein in the sample to be detected, and then a recognizable signal change is generated by a detectable label attached thereto, or a detection antibody (or secondary antibody) to which a detectable label is coupled specifically binds to the antibody of the present invention to generate a recognizable signal change, thereby achieving qualitative or quantitative detection of the human ERG protein.
In a preferred embodiment, the immunodetection method is immunohistochemistry, and suitable immunodetection pathological samples include, but are not limited to: human tonsils, human extranodal NK/T cell lymphomas, human placenta and human appendix.
The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.
EXAMPLE 1 preparation of Rabbit monoclonal antibodies against human ERG protein
1. Antigen preparation
In this example, the 35-68AA fragment of human ERG protein (NCBI Gene ID:2078, swissProt: P11308) was selected as antigen, and the expressed region protein had high specificity, no cross recognition with Fli-1, strong immunogenicity of antigen epitope, and good hydrophilicity, so that the fragment was selected as antigen. Coupled via KLH or BSA as an immunogen. Wherein the amino acid sequence of the ERG protein 35-68AA fragment is as follows:
ASSSSDYGQTSKMSPRVPQQDWLSQPPARVTIKM (see SEQ ID NO. 15).
The preparation method of the KLH coupling ERG protein 35-68AA antigen comprises the following steps:
(1) Preparing an amine-sulfhydryl crosslinking agent SMCC solution: (1) 2mL of 1 XPBS was added to a SMCC bottle having a mass of 50mg, and the mixture was transferred to a 10mL EP tube after mixing; (2) Removing the gun head of the liquid transfer device, placing one side, installing a new gun head, adding 2mL of 1 XPBS into the bottle, cleaning residual SMCC in the bottle, changing the gun head into the original gun head, and transferring the liquid in the bottle into the EP tube, wherein the process is repeated for 3 times; (3) Turning over, mixing, and heating in a water bath at 55deg.C for 5-6min; (4) Cooled to room temperature, 2mL of 1 XPBS was added to the EP tube to a volume of 10mL to give a SMCC concentration of 5mg/mL; (5) After being turned over and mixed evenly, the mixture is packaged into an EP tube with the volume of 1.5mL and preserved at the temperature of minus 20 ℃ for standby.
(2) KLH coupled with thiol: (1) The total mass of the coupled polypeptide is proportionally Peptide according to the requirement: klh=2: 1 weighing proper KLH on an analytical balance, dissolving the KLH by using 1 XPBS, keeping the concentration at 3mg/mL, and shaking uniformly; (2) A suitable volume of SMCC is taken according to the mass of KLH, and the proportion of KLH is: smcc=10: 1.5, peptides: BSA: smcc=4: 4:1, adding the mixture into dissolved KLH, and uniformly shaking the mixture on a rotary incubator for 1 hour at room temperature to obtain white floccules; (3) 1 XPBS with pH 7.4 was used in the volume ratio of PBS: klh=15: 1 dialyzing for 1h, removing free SMCC; (4) Testing and dissolving polypeptide, firstly adding 100 mu L of 1 XPBS into a small tube for containing polypeptide, testing and dissolving to see whether the polypeptide can be completely dissolved, otherwise, using 8M urea; (5) The polypeptide solution (10 mg/mL) was slowly added to activated KLH (dialysis completed) in multiple portions. Placing into a small shaking table or rotary incubator, observing at minimum rotation speed for one half hour, and if precipitate is generated, adding urea continuously at room temperature for 4h or overnight at 4deg.C.
(3) KLH coupled with amino group: (1) KLH dissolution is the same as thiol-coupled KLH dissolution; (2) Filling dissolved KLH into dialysis bag, placing into 300mL 1 XPBS, adding 6mL glutaraldehyde (for activating KLH) into the dialysate, placing on magnetic stirrer, adjusting rotation speed to 1/3 of rotation speed scale, and dialyzing for 90min; (3) Changing the dialysate into 300mL of 1 XPBS, changing the dialysate for 2 times, 40min each time, and rotating at the same speed; (4) The polypeptide dissolution mode is the same as the polypeptide dissolution mode in the sulfhydryl coupling; (5) The polypeptide solution is slowly added into activated KLH for a plurality of times, and then is put into a small shaking table or a rotary incubator, the rotation speed is the lowest, the observation is carried out once for half an hour, if precipitation is generated, urea is continuously added, and the KLH coupling ERG protein 35-68AA antigen is obtained after 4 hours or overnight at the room temperature.
2. Immunization of animals
4 New Zealand white rabbits are immunized by using a KLH coupled ERG protein 35-68AA polypeptide sequence, each white rabbit is immunized by 300 mug, and the immunogen is mixed with an equivalent amount of complete Freund's adjuvant to prepare an emulsifying agent before the first immunization, and the emulsifying agent is subcutaneously injected into the abdomen and the back of the rabbits at multiple points. 150 μg of immunogen is mixed with an equal amount of incomplete Freund's adjuvant every 3 weeks after the primary immunization to prepare an emulsifier, and the emulsifier is subcutaneously injected into the abdomen and the back of a rabbit for two times of boosting. After three immunizations, rabbit serum samples were collected, titers against ERG proteins were determined by enzyme-linked immunosorbent assay (ELISA), and the last immunized serum was purified to antibodies, and the specificity of the antibodies was detected using IHC tissue chips (including tonsil, extranodal NK/T cell lymphoma, prostate cancer, glioblastoma and brain (neuronal) tissue samples). Rabbits with high serum titer and best endogenous detection specificity were then boosted once with 150 μg of immunogen by subcutaneous multipoint injection, and three days later spleens were sacrificed.
3. Sorting and culturing of B lymphocytes
Spleen cells were first isolated: taking out a culture dish in a safe cabinet in a sterile operation mode, adding 30-40mL of basic culture medium, placing a cell screen, taking out spleen, placing the spleen in the cell screen, shearing superfluous connective tissue and fat on rabbit spleen tissue, shearing spleen tissue, placing the spleen tissue into the cell screen for grinding, taking a clean grinding rod, and grinding the tissue by rolling the tail end of the pressed part. The cells in the membrane slowly come out and are suspended in the culture dish solution after passing through a cell sieve; the washed cell screen was washed with 10mL of basal medium and the basal medium outside the cell screen was collected. Centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 13mL of RBC erythrocyte lysate at room temperature (purchased from BioGems company), gently blowing off cell clusters by using a pipettor, timing for 1min, performing erythrocyte lysis, adding 37mL of basal medium, uniformly mixing, stopping erythrocyte lysis, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 40mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, resuspending cells, completing the first cleaning, centrifuging at room temperature for 5min by using a centrifugal force of 400g, removing supernatant, reserving cells, adding 20mL of basal medium placed at room temperature, gently blowing off cell clusters by using a pipettor, and resuspending cells; the resuspended cells were filtered again through a cell screen to remove agglomerated cells, after which the cells were counted.
Then the method of separating single antigen specific B lymphocyte from spleen cell (publication No. CN 110016462A) and the method of separating and culturing B lymphocyte in vitro culture system and application of B lymphocyte (publication No. CN 111518765A) are adopted.
4. Cloning of genes encoding Rabbit monoclonal antibodies
The cultured B lymphocyte supernatant was subjected to antigen-coated ELISA to identify B lymphocyte positive clones capable of recognizing and binding to human ERG protein. Positive clones were collected and lysed and then treated with Quick-RNA TM MicroPrep kit instructions (available from ZYMO, cat# R1051) extract RNA and reverse transcribe into cDNA. And then, using the cDNA as a template, adopting a light chain variable region primer pair shown in SEQ ID NO.16-17 and a heavy chain variable region primer pair shown in SEQ ID NO.18-19 to carry out PCR amplification, amplifying the VH and VL genes of the naturally paired rabbit monoclonal antibodies, and determining the sequence by sequencing. The PCR reaction system comprises: 4. Mu.L of cDNA, 1. Mu.L of forward primer (10 mM), 1. Mu.L of reverse primer (10 mM), 12.5. Mu.L of 2 XGloriaHiFi (Wuhan Aibolag Biotechnology Co., ltd., cat# RK 20717) and 6.5. Mu. L H 2 O. The PCR amplification procedure included: predenaturation at 98℃for 30s followed by 10s at 98℃and 30s at 64℃for 72 s And (3) carrying out 40 times of circulation at the temperature of 30s, and finally keeping the temperature at 72 ℃ for 5min, and preserving the obtained reaction liquid at 4 ℃.
The nucleotide sequences (5 '-3') of the aforementioned amplified VL and VH primer pairs are shown below, F representing the forward primer, and R representing the reverse primer:
VL-F: tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC (see SEQ ID NO. 16);
VL-R: cacacacacgatggtgactgTTCCAGTTGCCACCTGATCAG (see SEQ ID NO. 17);
VH-F: tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG (see SEQ ID NO. 18);
VH-R: gtagcctttgaccaggcagcCCAGGGTCACCGTGGAGCTG (see SEQ ID NO. 19).
5. Production and purification of monoclonal antibodies
In order to obtain a plurality of rabbit monoclonal antibodies for recognizing ERG proteins, heavy chain genes and light chain genes of the rabbit monoclonal antibodies are respectively loaded on expression vectors, specifically, variable region genes (upstream carrying signal peptide coding genes) of the heavy chain and the light chain obtained by amplification are purified and then are respectively loaded on a mammal expression vector pBR322 carrying CL and CH sequences in advance by adopting a homologous recombination mode, and insertion positions are between NheI and XbaI restriction enzymes. The expression pattern of the mammalian expression vector pBR322 carrying the CL and CH sequences in advance is shown in FIG. 1, in which pRB322origin and f1origin are replication promoters in E.coli (E.Coli), ampcillin is a plasmid resistance gene, CMV immearly promotor is a promoter in eukaryotes, SV40 PA terminator is a tailing signal, light chain constant is the nucleic acid sequence of the light chain constant region (CL) (left panel), heavy chain constant is the nucleic acid sequence of the heavy chain constant region (CH) (right panel).
The signal peptide of this example can be expressed by using an antibody commonly used in the art, such as the "high affinity Human IL-5 rabbit monoclonal antibody" and its application (publication No. CN115819578A, publication No. 2023-03-21), the light chain variable region has the signal peptide "MDTRAPTQLLGLLLLWLPGARC" upstream, the coding gene "ATGGAC ACGAGGGCCCCCACTCAGCTGCTGGGTCTCCTGTTGTTGTGGCTGCCTGGCGCAAGGTG T", the heavy chain variable region has the signal peptide "METGLRWLLLVAVLKGVQC" upstream, and the coding gene "ATGGAGA CTGGGCTGCGCTGGCTTCTCCTGGTGGCAGTGCTGAAGGGAGTACAGTGC".
The constructed pBR322 plasmid is transfected into 293F cells for 72 to 96 hours to obtain the recombinant antibody which can recognize ERG protein in cell culture supernatant. Recombinant rabbit monoclonal antibodies recognizing ERG Protein were purified from the culture supernatant following transfection using Protein A affinity gel resin (available from Tiandi Kong, cat. SA 023100) at an antibody concentration of 0.13mg/mL, as specified in the specification. And (5) after the antibody is qualified, subpackaging, and preserving at a low temperature of-20 ℃ for standby.
The anti-human ERG protein rabbit monoclonal antibody obtained in this example was designated 1G8, and the amino acid sequence and the nucleotide sequence of the encoding gene are shown in Table 1. For convenience of description, light chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by LCDR1, LCDR2 and LCDR3, respectively, heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by HCDR1, HCDR2 and HCDR3, respectively, AA represents the amino terminal sequence, and DNA represents the nucleotide sequence.
TABLE 1 summary of sequence information related to Rabbit monoclonal antibody 1G8 of the invention
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EXAMPLE 2 immunohistochemical analysis of Rabbit monoclonal antibodies directed against human ERG protein
1. Pathological chip selection
ERG proteins are expressed in human samples mainly in lymphocyte and endothelial cell nuclei, and TMA000-24-HGenaral pathological chips are adopted, and the ERG proteins comprise human positive samples expressing human ERG proteins and human negative samples not expressing human ERG proteins. Positive samples (4 total) were human tonsils, extranodal NK/T cell lymphomas, placenta and appendix. Negative samples (20 cases total) were human prostate, glioblastoma, small cell lung cancer, colon invasive adenocarcinoma, high-grade urothelial carcinoma, breast invasive ductal carcinoma, ovary, endometrium-like carcinoma, ovarian serous carcinoma, cervix, cervical squamous cell carcinoma, endometrial stromal sarcoma, hepatocellular carcinoma, hepatobiliary carcinoma, lung squamous carcinoma, lung adenocarcinoma, human lung, and human brain.
2. Immunohistochemical (IHC) staining and analysis
1) Sample preparation and baking: placing paraffin slices on a slice rack in the same direction, and placing the paraffin slices into a constant temperature box at 56 ℃ to bake the slices for 30min; simultaneously placing the dewaxing liquid 1 cylinders into an incubator at 56 ℃; dewaxing to water: placing paraffin slices and a slice frame into a dewaxing liquid 1 jar, taking out the paraffin slices and the slice frame from an incubator and placing the paraffin slices and the slice frame into a normal temperature dewaxing liquid 2 jar, taking out the paraffin slices and immersing the paraffin slices into the dewaxing liquid 2 jar, and sequentially placing paraffin slices into the dewaxing liquid 2, the dewaxing liquid 3, the absolute ethyl alcohol 1, the absolute ethyl alcohol 2 and the absolute ethyl alcohol 3 according to the sequence of the dewaxing liquid 2, the dewaxing liquid reagent jar for 5min each, and the absolute ethyl alcohol reagent jar for 3min each; the sections were washed with running water for 3min. The dewaxing liquid 1-3 is purchased from the original industry and industry limited company of the Wuxi city river;
2) Antigen retrieval: high-pressure heat repair of 0.01M Tris-EDTA repair liquid (pH 9.0);
3) Inactivation of endogenous peroxidases: immersing and washing for 3 times by using PBS buffer solution for 1min each time, and removing the buffer solution on the slice; immersing the slices into 3% hydrogen peroxide solution completely, and incubating for 10min at room temperature;
4) Closing: immersing and washing for 3 times by using PBS buffer solution for 3min each time, and removing the buffer solution on the slice; an immunohistochemical water pen is used for delineating a tissue region to be detected on a slide, and PBS blocking solution is dripped into the delineating region; horizontally placing the slices in an incubation wet box with water at the bottom, incubating for 30min at normal temperature, and starting timing from dripping the sealing liquid;
5) Incubation resistance: removing the blocking solution, dripping 200 mu L of rabbit monoclonal antibody 1G8 (the primary antibody dilution ratio is 1:2000) diluted by antibody diluent-PBS working solution on the tissue slice, horizontally placing in an incubation wet box, and incubating for 60min at normal temperature; removing antibody working solution, quickly rinsing with PBS buffer solution for 1 time, soaking and washing with the buffer solution PBS for 3 times, and 3 minutes each time; repeatedly lifting up and down for multiple times during soaking and washing;
6) Secondary antibody incubation: dripping a ready-to-use secondary antibody working solution (purchased from Dako company, product number K5007) on a tissue slice, horizontally placing the tissue slice in an incubation wet box, and incubating for 25min at normal temperature; removing reagents on the sections, quickly rinsing the sections for 1 time by using buffer PBS, soaking and washing the sections for 3 times by using the buffer PBS for 3 minutes each time; repeatedly lifting up and down for multiple times during soaking and washing;
7) Color development: dropwise adding a color development liquid working solution on a tissue slice, closely observing the color change condition under a microscope, and obtaining proper dyeing intensity; immersing the slices in a large amount of distilled water to terminate the color development; after the color development is stopped, the slices are washed in running water for 10min;
8) Counterstaining: immersing the slightly drained tissue slice into Mayer's hematoxylin for counterstaining for 1min, and then washing with running water for 3min;
9) Returning blue: the slightly drained slices were immersed in a saturated aqueous solution of lithium carbonate for bluing for 3s and washed with running water for 3min.
10 Dewatering: soaking the cleaned slice in absolute ethyl alcohol for 1 time, lifting up and down for several times during the soaking period, and taking out after timing for 10 seconds; and (3) placing the mixture in a constant-temperature blast drying oven to be completely dried at a high temperature (54-58 ℃).
11 Sealing plate): dripping a proper amount of neutral gum at the center of the slice, covering a cover glass, wherein the glue adding amount is proper, and the cover glass is required to cover tissues completely and cannot overflow the glue;
12 Slice scan).
Immunohistochemical staining results were divided into: positive (brown coloration in specific tissues and cells) and negative (no brown coloration in specific tissues and cells), wherein positive expression requires brown coloration in specific antigenic sites of tissues and cells and no background color of antigenic sites (cells and tissues which should not be colored theoretically), i.e. no non-specific coloration, such as high expression of EGR protein in nuclei of positive samples human tonsil endothelial cells, corresponding sites should be brown colored, and the rest should be no brown coloration. The nuclei were blue after hematoxylin counterstain and showed a background profile of cells stained with specific immunity for determining subcellular localization of the target protein ERG.
The scan showed that there was specific staining in the nuclei of endothelial cells expressed by ERG protein in 4 positive tissue samples, while cells without antigen expression were brown-brown, and the detection results were positive for 4 positive samples. Of the 20 negative tissue samples, only human brain endothelial cells were positive, the remaining cells were negative, and consistent with the theoretical localization, were judged to be negative, and the remaining 19 samples were 1/1 prostate, 1/1 glioblastoma, 1/1 small cell lung cancer, 1/1 colon invasive adenocarcinoma, 1/1 high-grade urothelial carcinoma, 1/1 breast invasive ductal carcinoma, 1/1 ovary, 1/1 endometrium-like carcinoma, 1/1 ovary serous carcinoma, 1/1 cervix, 1/1 cervical squamous cell carcinoma, 1/1 endometrium sarcoma, 1/1 hepatocellular carcinoma, 1/1 hepatobiliary duct carcinoma, 1/1 lung adenocarcinoma, and 1/1 human lung were not brown colored, and the detection results were negative. The results are shown in FIGS. 2-4, wherein FIGS. 2-4 show the results of detection of human appendix, human tonsils and human prostate tissue samples using rabbit monoclonal antibody 1G8, respectively. The graph shows that the color development position of the antibody 1G8 is consistent with the theoretical positioning, the dyeing positioning is accurate, the positive dyeing is clear, the non-specific dyeing is avoided, the background is clean, the specificity of the antibody is good, the detection accuracy is improved, the analysis sensitivity and the specificity are high, the sensitivity reaches 4/4=100%, the specificity reaches 20/20=100%, false positive and false negative results can be effectively avoided, and the antibody 1G8 disclosed by the invention can meet the requirements of high-accuracy and high-accuracy detection of multiple pathological samples.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A rabbit monoclonal antibody directed against human ERG protein comprising a light chain variable region and a heavy chain variable region, each comprising 3 complementarity determining regions, wherein:
the amino acid sequences of the light chain complementarity determining region 1, the light chain complementarity determining region 2 and the light chain complementarity determining region 3 are shown in SEQ ID NO.3-5, respectively;
the amino acid sequences of heavy chain complementarity determining region 1, heavy chain complementarity determining region 2, and heavy chain complementarity determining region 3 are shown in SEQ ID NOS.8-10, respectively.
2. The rabbit monoclonal antibody directed against human ERG protein of claim 1, wherein said light chain variable region and said heavy chain variable region each comprise 4 framework regions, 4 of said framework regions being staggered in sequence with 3 of said complementarity determining regions, constituting a variable region;
the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7.
3. The rabbit monoclonal antibody against human ERG protein of claim 1, wherein the amino acid sequence of the light chain of said rabbit monoclonal antibody is shown in SEQ ID No.1 and the amino acid sequence of the heavy chain is shown in SEQ ID No. 6.
4. The rabbit monoclonal antibody directed against human ERG protein of claim 1, wherein said rabbit monoclonal antibody is a full length antibody or an antibody fragment selected from the group consisting of Fab fragments, F (ab) 2 Fragments, (Fv) fragments 2 Fragments, scFv fragments and sc (Fv) 2 At least one of the fragments.
5. A nucleic acid molecule encoding a rabbit monoclonal antibody directed against a human ERG protein according to any one of claims 1-4.
6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence of the light chain variable region of the rabbit monoclonal antibody is shown in SEQ ID NO.12 and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 14.
7. The nucleic acid molecule of claim 6, wherein the light chain of the rabbit monoclonal antibody has a nucleotide sequence shown in SEQ ID NO.11 and the heavy chain has a nucleotide sequence shown in SEQ ID NO. 13.
8. A recombinant vector comprising the nucleic acid molecule of any one of claims 5-7.
9. Use of a rabbit monoclonal antibody directed against human ERG protein according to any of claims 1-4 for the preparation of a reagent and/or kit for immunodetection of human ERG protein.
10. The use of a rabbit monoclonal antibody directed against human ERG protein according to claim 9 for the preparation of reagents and/or kits for the immunodetection of human ERG protein, wherein the immunodetection method is immunohistochemistry, and the immunodetection of pathological samples comprises one or more of human tonsils, human extranodal NK/T cell lymphomas, human placenta and human appendix.
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