CN117467003B - Anti-human MSH6 protein rabbit monoclonal antibody and application thereof - Google Patents
Anti-human MSH6 protein rabbit monoclonal antibody and application thereof Download PDFInfo
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- CN117467003B CN117467003B CN202311298896.1A CN202311298896A CN117467003B CN 117467003 B CN117467003 B CN 117467003B CN 202311298896 A CN202311298896 A CN 202311298896A CN 117467003 B CN117467003 B CN 117467003B
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Classifications
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/565—Complementarity determining region [CDR]
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of antibody preparation, and particularly relates to a rabbit monoclonal antibody for resisting human MSH6 protein and application thereof. 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 of the rabbit monoclonal antibody of the anti-human MSH6 protein are respectively shown in SEQ ID NO. 3-5; 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. The antibody provided by the invention can specifically identify human MSH6 protein expressed by cells and tissues, has good binding affinity with the human MSH6 protein, is suitable for the field of immunodetection of MSH6 protein, and particularly has the advantages of high accuracy, high sensitivity, high specificity, excellent anti-interference capability and the like when being used for immunodetection, and is particularly suitable for immunohistochemical detection.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a rabbit monoclonal antibody for resisting human MSH6 protein and application thereof.
Background
The MutS homolog 6 (MSH 6) gene is a member of the MutS family of mismatch repair (MISMATCH REPAIR, MMR) genes, located on chromosome 2, spanning the 24kb genomic sequence, consisting of 10 exons, encoding 1360 amino acid proteins, encoding MSH6 proteins that are primarily involved in recognition, repair of base substitutions and single base insertion/deletion mismatches. MSH6 protein is defective in most microsatellite highly unstable (MSI-H) patients; in addition, MSH6 proteins are also defective in certain malignancies, such as hereditary non-polyposis colorectal cancer (HNPCC, also known as Lynch syndrome) and endometrial cancer. HNPCC is an autosomal dominant genetic disease associated with a significant increase in susceptibility to cancer and is characterized by familial susceptibility to early-onset colorectal cancer (CRC) and extra-colonic cancers of the gastrointestinal tract, urinary system and female genital tract. Cancers in HNPCC originate from benign neoplastic polyps called adenomas. MSH6 mutations have been reported to be associated with atypical HNPCC, in particular with the development of endometrial cancer or atypical endometrial hyperplasia (presumed to be a precursor to endometrial cancer). MSH6 defects are also found in familial colorectal cancers that do not meet HNPCC criteria (suspected or incomplete HNPCC). MSH6 deficiency is also a cause of susceptibility to endometrial cancer (ENDMC). Clinically, MSH6 is usually detected in combination with MLH1, MSH2 and PMS2 for screening of HNPCC.
The expression of MSH6 protein in pathological samples is detected by immunoblotting (WB), immunohistochemistry (IHC) and other immunological methods in clinic, the technical core is monoclonal antibody which specifically binds the protein, and the reliability and accuracy of the detection result are directly determined by the quality of the antibody performance. However, there are few antibodies against MSH6 protein with high specificity on the market, and fewer antibodies can be applied to immunological detection, so developing a monoclonal antibody with high binding specificity and good MSH6 protein binding capacity has important significance and broad market prospect for realizing immunological detection of MSH6 protein expression level.
Disclosure of Invention
Aiming at the problems that an anti-human MSH6 protein antibody is difficult to be used for immunological detection and the like in the prior art, the invention provides a rabbit monoclonal antibody for resisting human MSH6 protein, and provides application of the rabbit monoclonal antibody in preparation of human MSH6 protein immunodetection reagents and/or kits, and the antibody has the advantages of high accuracy, high sensitivity, high specificity, excellent anti-interference capability and the like when being used for immunodetection.
In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:
The first aspect of the invention provides a rabbit monoclonal antibody against human MSH6 protein, comprising 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, and 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 respectively shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the amino acid sequences of the heavy chain complementarity determining region 1, the heavy chain complementarity determining region 2, and the heavy chain complementarity determining region 3 are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.
Further, 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, a light chain constant region and a heavy chain constant region are included, the light chain constant region and the light chain variable region comprising a light chain, the heavy chain constant region and the heavy chain variable region comprising a heavy chain; the amino acid sequence of the light chain is shown as SEQ ID NO.1, the amino acid sequence of the heavy chain is shown as SEQ ID NO.6, and the heavy chain is of the IgG type.
Further, the rabbit monoclonal antibody is a full-length antibody or an antibody fragment having immunological activity; the antibody fragment is selected from at least one of Fab fragment, F (ab) 2 fragment, fv fragment, (Fv) 2 fragment, scFv fragment and sc (Fv) 2 fragment.
Further, the immunogen for preparing the rabbit monoclonal antibody is a hemocyanin coupled human MSH6 protein 2-35AA amino acid sequence, wherein the human MSH6 protein 2-35AA amino acid sequence is shown as SEQ ID NO. 11.
In a second aspect the invention provides a nucleic acid molecule encoding a rabbit monoclonal antibody as described above against human MSH6 protein.
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 against human MSH6 protein as described above, a nucleic acid molecule as described above, a recombinant vector as described above in the preparation of a human MSH6 protein immunoassay reagent and/or kit.
Further, the immunodetection method is immunoblotting or immunohistochemistry.
Further, the immunodetection sample comprises a serum, cell or tissue sample, wherein the cell comprises a human embryonic kidney cell and the tissue comprises one or more of human tonsil, human appendix, human pancreas and human colon cancer.
The invention has the advantages and positive effects that:
the antibody provided by the invention can specifically identify human MSH6 protein expressed by cells and tissues, has good binding affinity with the human MSH6 protein, is suitable for the field of immunodetection of MSH6 protein, and particularly has the advantages of high accuracy, high sensitivity, high specificity, excellent anti-interference capability and the like when being used for immunodetection, and is particularly suitable for 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 mammalian vector pBR322 used for constructing a rabbit monoclonal antibody expression vector against human MSH6 protein according to example 1 of the present invention, from left to right, respectively, a pRB322 vector map carrying light chain constant regions and heavy chain constant regions in advance;
FIG. 2 is a diagram showing the result of immunoblotting detection of the binding of the rabbit monoclonal antibody against human MSH6 protein of example 2 to 293F cells before and after MSH6 gene knockout;
FIG. 3 is a graph showing the results of immunohistochemical detection of the binding of the rabbit monoclonal antibody against human MSH6 protein of example 2 of the present invention to human appendiceal tissue;
FIG. 4 is a graph showing the results of immunohistochemical detection of the binding of the rabbit monoclonal antibody against human MSH6 protein of example 2 of the present invention to human colon cancer tissue;
FIG. 5 is a comparison chart of immunohistochemical detection results of the binding of the rabbit monoclonal antibody against human MSH6 protein of example 2 of the present invention to normal human colon cancer tissue and human colon cancer tissue lacking MSH6 gene.
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, 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) MSH6 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 capable of specifically binding to an antigen or epitope of interest through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. 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 with one another by the FR regions to form surfaces complementary to the three-dimensional structure of the antigen or epitope of interest, determining 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 a region of an antibody molecule that binds Antigen and consists of the complete light chain (variable and constant regions) and part of the heavy chain (variable and one constant region fragment), and fragments such as Fab, F (ab ') 2, fab' can be obtained by proteolytic cleavage of the full-length antibody. For example, igG can be degraded into two Fab fragments and one Fc fragment by papain; igG can be degraded into a F (ab ') 2 fragment and a pFC' fragment by pepsin. The F (ab ') 2 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. In some embodiments, the monoclonal antibodies are prepared by a hybridoma method, phage display method, yeast display method, recombinant DNA method, single cell screening, or single cell sequencing method.
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 for resisting human MSH6 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 complementarity determining region 1 (CDR 1), the light chain complementarity determining region 2 (CDR 2) and the light chain complementarity determining region 3 (CDR 3) are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the amino acid sequences of heavy chain complementarity determining region 1 (CDR 1), heavy chain complementarity determining region 2 (CDR 2) and heavy chain complementarity determining region 3 (CDR 3) are shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.
The invention takes human MSH6 protein (Uniprot number: P52701) 2-35AA amino acid sequence (see SEQ ID No. 11) coupled with hemocyanin (KLH) as immunogen, and 2-35AA amino acid fragment is positioned at N end, has good specificity, high homology with big and small mice and good antigen epitope, so that the protein fragment is adopted to immunize rabbits, and then monoclonal antibody development technology based on single B lymphocyte screening and culture is adopted to obtain the rabbit monoclonal antibody of the anti-human MSH6 protein. The obtained antibody can specifically identify the human MSH6 protein expressed by cells and tissues, has good binding affinity with the human MSH6 protein, high specificity and good anti-interference capability. Taking the antibody as a detection antibody, detecting a positive cell sample (293F cell) which highly expresses MSH6 protein and a negative cell sample (MSH 6 gene knockout 293F cell) which does not express human MSH6 protein by adopting an immunoblotting method, and detecting a single band in the positive cell only, wherein the protein is correct in size; the method further adopts an immunohistochemical method to dye a plurality of positive tissue samples expressing MSH6 protein, and the result shows that the antibody has accurate dyeing positioning, clear dyeing and no nonspecific dyeing, the identification sensitivity of the positive tissue samples reaches 100%, the accuracy of detection results is ensured, and false positive and false negative results are effectively avoided, so that the antibody is suitable for the field of immunodetection of MSH6 protein, particularly immunohistochemical detection, and has the advantages of high accuracy, high sensitivity, high specificity, excellent anti-interference capability and the like.
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: the rabbit source IGG GAMMA C REIGN was searched for CH and the rabbit source IGG KAPPA C REIGN was searched for CL via IMGT online database (www.imgt.org).
Specifically, the amino acid sequence of the light chain comprising CL is shown as SEQ ID NO.1, the amino acid sequence of the heavy chain comprising CH is shown as SEQ ID NO.6, and correspondingly, the heavy chain is of the IgG type.
The rabbit monoclonal antibody of the invention may be a full length antibody or an antibody fragment, which refers to a polypeptide that retains substantially the same biological function or activity as the full length form of the rabbit monoclonal antibody, in particular, an antibody fragment comprising the CDR regions (SEQ ID No.3-5 and SEQ ID No. 8-10) as described above, more preferably having the variable regions (SEQ ID No.2 and SEQ ID No. 7) as described above, thereby retaining intact antigen recognition and binding sites capable of binding to the same antigen, in particular to the same epitope, as the full length antibody. Such antibody fragments include, but are 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) A F (ab) 2 fragment, a bivalent fragment comprising 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 fragment 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) The sc (Fv) 2 fragment is obtained by ligating two heavy chain variable regions and two light chain variable regions via a linker or the like. These antibody fragments can be obtained by conventional techniques known in the art.
Yet another embodiment of the invention provides a nucleic acid molecule encoding a rabbit monoclonal antibody as described above against human MSH6 protein.
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.
The full-length sequence of the nucleic acid molecule or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis.
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). The cloning vector may contain a selectable marker and an origin of replication that matches the cell type specified by the cloning vector, while the expression vector contains regulatory elements (e.g., promoters, enhancers) for expression in the 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 molecule. 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 the antibodies of the invention may 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. In other embodiments, nucleic acid molecules encoding the heavy and light chains of a rabbit monoclonal antibody of the invention may also 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 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. The choice of 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 are capable of absorbing DNA, can be obtained after the exponential growth phase and treated with CaCl 2 or MgCl 2. Microinjection, electroporation, or liposome encapsulation may also be used if desired. 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: bacterial cells of E.coli, streptomyces, salmonella typhimurium, fungal cells such as insect cells of yeast, drosophila S2 or Sf9, CHO, COS7, 293 series cells, etc. 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 of the anti-human MSH6 protein, the nucleic acid molecule or the recombinant vector in preparation of human MSH6 protein immunodetection reagent and/or kit.
The application advantages of the anti-human MSH6 protein rabbit monoclonal antibody, nucleic acid molecule or recombinant vector in preparing human MSH6 protein immunodetection reagent and/or kit are the same as those of the anti-human MSH6 protein rabbit monoclonal antibody described above with respect to the prior art, and are not described in detail herein.
The rabbit monoclonal antibodies of the invention can be used in the field of conventional immunodetection, including but not limited to: immunoblotting (Western blot, WB), enzyme immunoassay (Enzyme immunoassay, EIA), enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), enzyme-linked immunospot (Enzyme-linked Immunospot, ELISPOT), immunofluorescence (IF), immunohistochemistry (Immunohistochemistry, IHC), flow Cytometry (FC). In a preferred embodiment, the immunodetection method is immunoblotting or immunohistochemistry.
The test sample includes a serum, cell or tissue sample, wherein the cells include, but are not limited to: human embryonic kidney cells 293F, tissues including but not limited to: human tonsils, human appendices, human pancreas, and human colon cancers.
In the immunoassay, a sample to be examined is contacted with an anti-human MSH6 protein monoclonal antibody, and then the monoclonal antibody is detected. In some embodiments, anti-human MSH6 protein monoclonal antibodies may be conjugated to a detectable label to effect qualitative or quantitative detection of MSH6 protein by detecting a change in the identifiable signal produced by the detectable label. In other embodiments, the anti-human MSH6 protein monoclonal antibody (primary or capture antibody) is not labeled, and the detectable label is conjugated to a secondary antibody (detection antibody) or other molecule that binds to the monoclonal antibody, e.g., if the anti-human MSH6 protein antibody is a rabbit IgG antibody, the secondary antibody may be an anti-rabbit IgG antibody, whereby the secondary antibody conjugated with the detectable label specifically binds to the primary antibody to produce a change in the recognizable signal.
Such detectable labels for producing identifiable signal changes 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, or combinations thereof.
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 MSH6 protein
1. Antigen preparation
In the embodiment, a human MSH6 (Uniprot number: P52701) protein 2-35AA amino acid fragment is selected as a hapten, the region is positioned at the N end of the protein, the specificity is good, the homology with a rat is high, the epitope is good, the hapten is coupled through hemocyanin (KLH) and then used as an immunogen, wherein the amino acid fragments from the 2 nd position to the 35 th position of the human MSH6 protein are shown as follows:
SRQSTLYSFFPKSPALSDANKASARASREGGRAA (see SEQ ID NO. 11).
The preparation method of the KLH coupled human MSH6 protein 2-35AA immunogen comprises the following steps:
Dissolving KLH protein to 3mg/mL by using PBS solution for standby; putting dissolved KLH into dialysis bag, putting into 300mL 1 XPBS, adding 6mL glutaraldehyde (activated KLH) into the dialysate, placing on magnetic stirrer, adjusting rotation speed to 1/3 of rotation speed scale, and dialyzing for 90min; changing the dialysate into 300mL of 1 XPBS, changing the dialysate for 2 times, 40min each time, and rotating at the same speed; dissolving the polypeptide to a concentration of 10mg/mL; 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, and the reaction is carried out for 4 hours at room temperature at the lowest rotating speed. Packaging the prepared immunogen solution, and preserving at-20deg.C for use.
2. Immunization of animals
4 New Zealand white rabbits are immunized by taking KLH protein coupled human MSH6 protein 2-35AA polypeptide sequence as immunogen; each Japanese white rabbit was immunized 300. Mu.g, and the immunogen was mixed with an equivalent amount of complete Freund's adjuvant to prepare an emulsion before the first immunization, and the emulsion was subcutaneously injected at the abdomen and back of the rabbit 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 MSH6 protein were determined by enzyme-linked immunosorbent assay (ELISA), and the last immunized serum was purified to antibodies, and endogenous samples were detected by immunoprecipitation (WB) and Immunohistochemistry (IHC). The endogenous samples comprise 293T cell lysate (293T-WT) which is a positive cell sample expressing MSH6 protein and 293T-MSH6 -/- knockout cell lysate which is a negative cell sample, and the tissue samples comprise 3 cases of human tonsils, 3 cases of human appendices, 3 cases of human pancreas, 3 cases of human colon cancer and 1 case of MSH6 deleted human colon cancer.
Rabbits with high serum titer and best endogenous detection were boosted once with 150 μg immunogen by subcutaneous multipoint injection and spleens were taken three days later.
3. Isolation and culture of B lymphocytes in spleen
Spleen cells were 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 normal temperature (purchased from BioGems company, product number 64010-00-100), gently blowing off cell clusters by using a pipette, counting 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 normal temperature, gently blowing off cell clusters by using a pipette, 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 normal temperature, gently blowing off cell clusters by using a pipette, and resuspending cells; the resuspended cells were filtered again through a cell screen to remove agglomerated cells, after which the cells were counted.
Sorting and culturing of B lymphocytes: see patent "method for efficiently isolating single antigen-specific B lymphocytes from spleen cells (publication No. CN110016462A, publication No. 2019-07-16)" and patent "an in vitro B lymphocyte culture system and application (publication No. CN111518765A, publication No. 2020-08-11)".
4. Cloning of genes encoding Rabbit monoclonal antibodies
The cultured B cell supernatants were subjected to antigen-coated ELISA to identify B lymphocyte positive clones capable of recognizing and binding to human MSH6 protein. RNA was extracted using the Quick-RNA TM MicroPrep kit (available from ZYMO company under the trade designation R1100-250) and reverse transcribed into cDNA. The cDNA is used as a template, a PCR method is adopted to amplify the light chain variable region (VL) and heavy chain variable region (VH) genes of the naturally paired rabbit monoclonal antibodies from the cDNA of the corresponding positive clone, and the sequence is determined by sequencing, and the sequencing work is completed by Jin Kairui biotechnology company. Wherein, 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 2X GloriaHiFi (from Wuhan Aibotac Biotechnology Co., ltd., product No. RK 20717) and 6.5. Mu. L H 2 O. The PCR amplification procedure included: the reaction mixture was subjected to preliminary denaturation at 98℃for 30s, followed by 40 cycles at 98℃for 10s,64℃for 30s, and 72℃for 30s, and finally kept at 72℃for 5min, and the resulting reaction mixture was kept at 4 ℃. Wherein the nucleic acid sequences of the primer pairs for amplifying the VL and VH genes are shown below, F represents the forward primer, and R represents the reverse primer:
VL-F：5'-tgaattcgagctcggtacccatggacacgagggcccccac-3'(SEQ ID NO.12)；
VL-R：5'-cacacacacgatggtgactgttccagttgccacctgatcag-3'(SEQ ID NO.13)；
VH-F：5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3'(SEQ ID NO.14)；
VH-R：5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3'(SEQ ID NO.15)。
5. Production and purification of Rabbit monoclonal antibodies
In order to obtain a plurality of rabbit monoclonal antibodies recognizing human MSH6 protein, VL and VH genes of the selected rabbit monoclonal antibodies are respectively loaded on a mammalian expression vector pBR322 which carries a light chain constant region (CL) and a heavy chain constant region gene (CH) in advance, the expression patterns of which are shown in FIG. 1, pRB322origin and f1origin are replication promoters in E.coli (E.Coli), AMPCILLIN is a plasmid resistance gene, CMV IMMEARLY promotor is a promoter in eukaryote, SV40 PA terminator is an tailing signal, LIGHT CHAIN constant is a nucleic acid sequence of a light chain constant region (left panel), HEAVY CHAIN constant is a nucleic acid sequence of a heavy chain constant region (right panel), and the constant region sequences are obtained by querying an IMGT online database (www.imgt.org).
The construction process of the rabbit monoclonal antibody expression vector carrying the light chain and heavy chain genes is as follows: carrying out conventional linearization treatment on a mammalian cell expression vector pBR322 containing rabbit monoclonal antibodies CH and CL by using NheI and XbaI restriction endonucleases respectively, purifying the amplified PCR product, and respectively constructing a VL gene and a VH gene with signal peptide coding genes at the upstream into the expression vector pBR322 by adopting a homologous recombination mode; after sequencing verification, the expression vectors containing the light chain genes and the heavy chain genes of the corresponding rabbit monoclonal antibodies are transfected into 293F cells together; culturing for 72-96h after transfection, and culturing to obtain supernatant containing recombinant rabbit monoclonal antibody recognizing human MSH6 protein. The rabbit monoclonal antibody recognizing human MSH6 protein was then purified from the culture supernatant using protein A affinity gel resin (available from Tiandi Kong, cat. SA 023100), and the purification protocol was followed according to the protein A affinity gel resin instructions and will not be described again. Purifying to obtain antibody with concentration of 0.65mg/mL, verifying antibody, packaging, and storing at-20deg.C.
The signal peptide of this example may be expressed by using an antibody commonly used in the art, such as the "high affinity Human IL-5 rabbit monoclonal antibody and its use (publication No. CN115819578A, publication No. 2023-03-21)", the light chain variable region has the signal peptide "MDTRAPTQLLGLLLLWLPGARC" upstream and the heavy chain variable region has the signal peptide "METGLRWLLLVAVLKGVQC" upstream.
The amino acid sequences of the rabbit monoclonal antibodies against human MSH6 protein obtained in this example are shown in Table 1, and for convenience of description, the light chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by LCDR1, LCDR2 and LCDR3, respectively, and the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are denoted by HCDR1, HCDR2 and HCDR3, respectively.
TABLE 1 summary of amino acid sequence information of Rabbit monoclonal antibodies of the invention
EXAMPLE 2 reaction specificity and application Studies of Rabbit monoclonal antibodies
1. Immunoblot analysis of Rabbit monoclonal antibody (Western blot, WB)
Taking a 293T cell sample (293T-WT, positive) with high expression of MSH6 protein and a 293T cell sample (293T-MSH 6 -/-, negative) with MSH6 gene deleted, detecting the identification specificity of the monoclonal antibody by using an immunoblotting method, wherein the reference gene is beta-actin, and the method comprises the following steps: respectively lysing the cell samples to obtain protein extract, performing 10% polyacrylamide gel electrophoresis, and transferring gel protein bands onto PVDF membrane in an electrotransfer system according to a conventional method; placing the membrane in TBST blocking solution containing 5% skimmed milk powder, incubating for 1h at room temperature, adding rabbit monoclonal antibody (primary antibody dilution ratio 1:6500, primary antibody working concentration after dilution 0.1 μg/mL) against MSH6 protein, and incubating at 4deg.C overnight; then, washing the membrane by TBST, adding goat anti-rabbit secondary antibody (purchased from ABclonal, cat# AS014, secondary antibody dilution ratio 1:10000), and incubating for 1h at room temperature; the membrane was washed again with TBST, and ECL hypersensitivity developing solution was added, and developed, and the WB detection result was shown in FIG. 2, with lane WT representing 293T-WT cells and lane MSH6 KO representing 293T-MSH6 -/- cells.
The western blot staining result shows that under the 3S exposure time, compared with the negative cell sample 293T-MSH6 -/-, the positive cell sample 293T-WT has obvious single target band at 180KD, and the protein size is correct, which indicates that the rabbit monoclonal antibody resisting the human MSH6 protein has good specificity, can uniquely identify the MSH6 protein in the cell sample lysate, and has good binding affinity with the MSH6 protein.
2. Immunohistochemical analysis of Rabbit monoclonal antibodies (Immunohistochemistry, IHC)
Human MSH6 protein is mainly expressed in colon cancer and rectal cancer cells in human samples, protein subcells are positioned in cell nuclei, and positive tissue samples for expressing the MSH6 protein are selected as follows: human appendix, human tonsil, human pancreas and human colon cancer, each tissue included 3 parallel samples, negative tissue samples were: 1 case of MSH6 gene deleted human colon cancer.
The IHC staining and analysis operations are referred to the pathological chip instruction book, and specifically comprise the following steps:
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 cylinder together into a constant temperature box at 56 ℃, placing paraffin slices and a slice frame together into the dewaxing liquid 1 cylinder, taking out the paraffin slices from the constant temperature box together and placing the paraffin slices at normal temperature, taking out the paraffin slices after 5min, immersing the paraffin slices into a normal temperature dewaxing liquid 2 cylinder, and sequentially placing the 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 3, the absolute ethyl alcohol 2 and the absolute ethyl alcohol 3, wherein each dewaxing liquid reagent cylinder is 5 min; washing the slices 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;
Antigen retrieval: high-pressure heat repair of 0.01M Tris-EDTA repair liquid (pH 9.0);
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;
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;
Incubation resistance: removing the blocking solution, dripping rabbit monoclonal antibody diluted by antibody diluent-PBS working solution (primary antibody dilution ratio is 1:200, primary antibody working concentration after dilution is 3.25 mug/mL) on a 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;
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 the secondary antibody working solution on the slice, quickly rinsing the slice for 1 time by using PBS buffer solution, soaking and washing the slice for 3 times by using the PBS buffer solution for 3 minutes each time; repeatedly lifting up and down for multiple times during soaking and washing;
Color development: dropwise adding a color development liquid working solution on the slice, closely observing the color change condition under a microscope to obtain proper dyeing intensity, immersing the slice in a large amount of distilled water to terminate color development, and washing the slice in running water for 10min after the color development is terminated;
Counterstaining: immersing the slightly drained tissue slice into Mayer's hematoxylin for counterstaining for 1min, and then washing with running water for 3min;
Returning blue: immersing the slightly drained slice into a saturated aqueous solution of lithium carbonate to blu for 3s, and cleaning the slice with running water for 3min;
Dehydrating: 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; completely drying at high temperature (54-58 deg.C) in constant temperature blast drying oven;
Sealing piece: 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;
And finally, slice scanning is carried out.
Immunohistochemical staining results were divided into: positive (tan staining in specific tissues and cells) and negative (no tan staining in specific tissues and cells). Positive expression must be tan colored and low background in antigen expressing cells of a particular tissue to be considered positive, blue staining being nuclear localization. For example, MSH6 protein is highly expressed in the cell nucleus of the positive sample human appendiceal cell and the rest of the cell nucleus is not expressed, MSH6 protein is highly expressed in the cell nucleus of the positive sample human colon cancer cell and the rest of the cell nucleus is not expressed, so the cell nucleus of the MSH6 protein expression cell is brown and the rest of the cell nucleus is not brown. The staining results of a part of the tissue samples are shown in fig. 3-5, wherein fig. 3 is a positive sample human appendiceal tissue staining result, fig. 4 is a positive sample human colon cancer staining result, and fig. 5 is a comparison graph of normal human colon cancer (left graph) and MSH6 gene deleted human colon cancer (right graph) staining results.
The immunohistochemical staining result shows that the anti-human MSH6 rabbit monoclonal antibody is used for accurately positioning, has clear staining, no nonspecific staining and clean background, further proves that the antibody has high specificity and strong anti-cell component interference capability, and is beneficial to improving the detection accuracy. Counting the staining results of the tissue chip, wherein in the positive samples, 3/3 cases of human tonsils, 3/3 cases of human appendices, 3/3 cases of human pancreas and 3/3 cases of human colon cancers are positive in nuclear staining; in 1 case of human colon cancer cell negative samples with MSH6 gene deletion, 1/1 case of human colon cancer cells with MSH6 deletion have no brown coloration, and the detection result is negative; the whole immunohistochemical detection sensitivity is 12/12=100%, the specificity reaches 1/1=100%, the accuracy of the visible detection result is high, the reliability is good, and no false positive and false negative result exists.
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 against human MSH6 protein, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region each comprise 3 complementarity determining regions, wherein the amino acid sequences of light chain complementarity determining region 1, light chain complementarity determining region 2, and light chain complementarity determining region 3 are shown in SEQ ID No.3, SEQ ID No.4, and SEQ ID No.5, respectively; the amino acid sequences of the heavy chain complementarity determining region 1, the heavy chain complementarity determining region 2, and the heavy chain complementarity determining region 3 are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.
2. The rabbit monoclonal antibody against human MSH6 protein according to claim 1, characterized in that the amino acid sequence of said light chain variable region is shown in SEQ ID NO.2 and the amino acid sequence of said heavy chain variable region is shown in SEQ ID NO. 7.
3. The rabbit monoclonal antibody against human MSH6 protein according to claim 2, further comprising a light chain constant region and a heavy chain constant region, the light chain constant region and the light chain variable region comprising a light chain, the heavy chain constant region and the heavy chain variable region comprising a heavy chain; the amino acid sequence of the light chain is shown as SEQ ID NO.1, the amino acid sequence of the heavy chain is shown as SEQ ID NO.6, and the heavy chain is of the IgG type.
4. The rabbit monoclonal antibody against human MSH6 protein according to claim 1 or 2, wherein the rabbit monoclonal antibody is a full length antibody or an immunologically active antibody fragment; the antibody fragment is selected from at least one of Fab fragment, F (ab) 2 fragment, fv fragment, (Fv) 2 fragment, scFv fragment and sc (Fv) 2 fragment.
5. The rabbit monoclonal antibody against human MSH6 protein according to claim 1, characterized in that the immunogen for preparing said rabbit monoclonal antibody is hemocyanin coupled human MSH6 protein 2-35AA amino acid sequence, wherein said human MSH6 protein 2-35AA amino acid sequence is shown as SEQ ID NO. 11.
6. A nucleic acid molecule encoding a rabbit monoclonal antibody against a human MSH6 protein according to any one of claims 1-5.
7. A recombinant vector comprising the nucleic acid molecule of claim 6.
8. Use of a rabbit monoclonal antibody against human MSH6 protein according to any one of claims 1-5 for the preparation of a human MSH6 protein immunoassay reagent and/or kit.
9. The use of a rabbit monoclonal antibody against human MSH6 protein according to claim 8 for the preparation of a reagent and/or kit for immunodetection of human MSH6 protein, wherein the immunodetection method is immunoblotting or immunohistochemistry.
10. The use of a rabbit monoclonal antibody against human MSH6 protein according to claim 8 for the preparation of a human MSH6 protein immunoassay reagent and/or kit, wherein the immunoassay sample comprises a serum, cell or tissue sample, wherein the cell comprises human embryonic kidney cells and the tissue comprises one or more of human tonsils, human appendices, human pancreas and human colon cancer.
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