CN111349170A

CN111349170A - Monoclonal antibody of immune related GTP enzyme family M (IRGM) and application thereof

Info

Publication number: CN111349170A
Application number: CN201811574995.7A
Authority: CN
Inventors: 翁炜宁
Original assignee: Abmart Pharmaceutical Technology Shanghai Co ltd
Current assignee: Abmart Pharmaceutical Technology Shanghai Co ltd
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2020-06-30
Anticipated expiration: 2038-12-21
Also published as: CN111349170B

Abstract

The invention provides a monoclonal antibody of an immune related GTP enzyme family M (IRGM) and application thereof. The experimental result shows that the antibody can be applied to antibody chip reaction, immunoprecipitation and Western Blotting for researching the protein and detecting the content of the marker protein IRGM in different occurrence stages of gastric cancer.

Description

Monoclonal antibody of immune related GTP enzyme family M (IRGM) and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an immune-related GTP enzyme family M (IRGM) monoclonal antibody and application thereof.

Background

Immune-related gtpase family M is a human protein encoded by the IRGM gene and the encoded protein may play a role in innate immune responses by modulating autophagy formation of intracellular pathogens, which contributes significantly to autophagy by inflammation and infection.

Recent studies have shown that autophagy may play a key role in tumorigenesis. The data show that the IRGM mRNA levels in Peripheral Blood Mononuclear Cells (PBMCs) from patients with gastric cancer are significantly higher than PBMCs from healthy controls, and furthermore, the mRNA and protein levels in cancer tissues are significantly higher compared to adjacent non-cancerous stomach tissues, while the correlation between IRGM expression and cancer stage shows that the mRNA and protein levels of IRGM in cancer tissues from stage IV patients are higher than those from stage I patients. This suggests that IRGM expression is deregulated in gastric cancer and that this molecule may affect disease progression.

Currently, no targeting antibody of immune related GTP enzyme family M specially aiming at IRGM in cancer tissues of gastric cancer patients exists in the market, and an effective antibody tool is lacked in the aspect of proteomics for the research field.

Disclosure of Invention

The antibody aims to provide a tool for targeted protein research so as to realize deeper research on protein expression changes and protein network maps of different gastric cancer patients and different stages of cancers.

In a first aspect of the invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region having one or more of the following complementarity determining regions CDRs:

VH CDR1 shown in SEQ ID NO.1,

VH CDR2 shown in SEQ ID NO.2, and

VH CDR3 shown in SEQ ID NO. 3;

preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 4.

In a second aspect of the invention there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region and a heavy chain constant region as described in the first aspect of the invention.

In another preferred embodiment, the heavy chain constant region is murine.

In a third aspect of the invention, there is provided a light chain variable region of an antibody, said light chain variable region having one or more of the following complementarity determining regions CDRs:

VL CDR1 shown in SEQ ID NO.5,

VL CDR2 shown in SEQ ID NO.6, and

VL CDR3 shown in SEQ ID NO. 7;

preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 8.

In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region and a light chain constant region as described in the third aspect of the invention.

In another preferred embodiment, the constant region of the light chain is murine.

In a fifth aspect of the invention, there is provided an antibody having:

(1) a heavy chain variable region according to the first aspect of the invention; and/or

(2) A light chain variable region according to the third aspect of the invention;

preferably, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.

In another preferred embodiment, the antibody is an antibody specific against an immune-related gtpase family m (irgm) polypeptide.

In another preferred embodiment, said immune-related gtpase family m (irgm) polypeptide is selected from the group consisting of: 'ADGNLPEVIS (SEQ ID NO. 9)' 'KASPPTELVK (SEQ ID NO. 10)' 'SHFSNVVLWD (SEQ ID NO. 11)' 'QLLQIRENVL (SEQ ID NO. 12)', or combinations thereof.

In another preferred embodiment, the antibody comprises: single chain antibodies (scFv), diabodies, monoclonal antibodies, chimeric antibodies, murine antibodies.

In a sixth aspect of the present invention, there is provided a recombinant protein having:

(i) the sequence of a heavy chain variable region according to the first aspect of the invention, the sequence of a heavy chain according to the second aspect of the invention, the sequence of a light chain variable region according to the third aspect of the invention, the sequence of a light chain according to the fourth aspect of the invention, or the sequence of an antibody according to the fifth aspect of the invention; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the tag sequence is selected from the group consisting of 6 × His tag, GGGS sequence, FLAG tag.

In another preferred embodiment, the recombinant protein comprises a bispecific antibody or a chimeric antibody.

In a seventh aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or

(2) The recombinant protein according to the sixth aspect of the present invention.

In an eighth aspect of the invention, there is provided a vector comprising a polynucleotide according to the seventh aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses.

In a ninth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the eighth aspect of the invention into which has been integrated a polynucleotide according to the seventh aspect of the invention.

In a tenth aspect of the invention, there is provided a conjugate comprising:

(a) an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention; and

(b) a detectable label linked to the antibody or recombinant protein of (a).

In another preferred embodiment, the detectable label is selected from the group consisting of: biotin, fluorescein, chemiluminescent groups, fluorescent proteins, enzymes, colloidal gold, colored magnetic beads, latex particles, biotin labels, radionuclides, antibodies, ligands, antigens, receptors, nanoparticles, or combinations thereof.

In another preferred embodiment, the nanoparticles are selected from the group consisting of: nanogold, nanosilver, quantum dots, or combinations thereof.

In another preferred embodiment, the enzyme is selected from the group consisting of: horseradish peroxidase, acid phosphatase, or a combination thereof.

In an eleventh aspect of the present invention, there is provided a detection article comprising:

(1) an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or a conjugate according to the tenth aspect of the invention; and

(2) optionally a buffer solution or buffer.

In another preferred embodiment, the assay preparation is for the detection of immune-related gtpase family m (irgm).

In another preferred embodiment, the detection article comprises: detection reagent, lateral flow sheet, chip, test strip, detection plate and test sheet.

In another preferred embodiment, the test strip comprises: a detection zone to which is immobilized another antibody (antibody two) against immune-related gtpase family m (irgm) for capturing said immune-related gtpase family m (irgm).

In another preferred embodiment, the test strip comprises: a quality control region having immobilized thereto an antibody (secondary antibody) that binds to an antibody (primary antibody) directed against an immune-related gtpase family m (irgm) for capturing said antibody (primary antibody) directed against an immune-related gtpase family m (irgm).

In another preferred embodiment, the test strip is selected from the group consisting of: multiwell plates, preferably 96-well plates, PVDF membranes.

In another preferred embodiment, said antibody (secondary antibody) that binds to an antibody (primary antibody) directed against the immune-related gtpase family m (irgm) is selected from the group consisting of: HRP-labeled goat anti-mouse IgG secondary antibody.

In a twelfth aspect of the invention, there is provided a use of the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, the antibody of the fifth aspect of the invention, the recombinant protein of the sixth aspect of the invention, or the conjugate of the tenth aspect of the invention, or the detection article of the eleventh aspect of the invention, in the preparation of a detection article or kit for detecting the content of immune-related gtpase family m (irgm) in a subject.

In another preferred embodiment, the subject is a human or non-human other mammal.

In another preferred embodiment, the subject is a cancer patient.

In another preferred example, the subject is a gastric cancer patient or a suspected gastric cancer patient.

In another preferred embodiment, the detection article comprises a chip and immune microparticles coated with antibodies.

In a thirteenth aspect of the invention, there is provided a test kit comprising an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or a conjugate according to the tenth aspect of the invention or a test article according to the eleventh aspect of the invention.

In a fourteenth aspect of the present invention, there is provided a method of detecting immune-related gtpase family m (irgm) in a subject, characterized in that said method comprises the steps of:

(a) providing a sample to be detected;

(b) mixing the sample with an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, a conjugate according to the tenth aspect of the invention, or a detection article according to the eleventh aspect of the invention to form a mixture;

(c) detecting the presence or absence of an "antibody-immune related gtpase family m (irgm) complex" in said mixture, wherein the presence of said complex, if present, indicates the presence of immune related gtpase family m (irgm) in said sample; the absence of said complex indicates the absence of immune-related GTPase family M (IRGM) in said sample.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a specific process route for antibody production.

FIG. 2 is a graph of antibody endogenous protein immunoblot results.

FIG. 3 is a graph showing the results of the fluorescence reaction of the antibody chip.

FIG. 4 is a graph of the standard test for the IRGM content.

Detailed Description

The present inventors have made extensive and intensive studies to unexpectedly obtain a high affinity monoclonal antibody (murine monoclonal antibody) that recognizes and binds to immune-related gtpase family m (IRGM), which can be applied to antibody chip reaction, immunoprecipitation, Western Blotting and detection of IRGM content, which is a marker protein for different stages of gastric cancer development, studied against the protein. On the basis of this, the present invention has been completed.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

Antibodies

As used herein, the term "antibody" consists of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which form the binding and specificity of each particular antibody for its particular antigen, however, the variability is not evenly distributed throughout the antibody variable region it is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions the more conserved portions of the variable regions are called Framework Regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, roughly in a β -fold configuration, connected by three CDRs forming a connecting loop, and in some cases may form a partial β fold structure.

As used herein, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The invention also comprises a monoclonal antibody with the corresponding amino acid sequence of the anti-immune related GTP enzyme family M (IRGM) monoclonal antibody, a monoclonal antibody with the variable region chain of the anti-immune related GTP enzyme family M (IRGM) monoclonal antibody, and other proteins or protein conjugates and fusion expression products with the chains. Specifically, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having light and heavy chains with hypervariable regions (complementarity determining regions, CDRs) so long as the hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.

As known to those skilled in the art, conjugates include: a detectable label such as a colloidal gold label, a colored magnetic bead or latex particle, a biotin label, a horseradish peroxidase label, a radionuclide label, a fluorescein label, a nanoparticle label, or a conjugate formed by binding the anti-immune related gtpase family m (irgm) monoclonal antibody or a fragment thereof. The invention also includes cell surface markers or antigens that bind to the anti-immune related gtpase family m (irgm) monoclonal antibodies or fragments thereof.

The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab')₂A fragment; an antibody heavy chain; the light chain of the antibody.

As used herein, the terms "heavy chain variable region" and "V_H"may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

In a preferred embodiment of the invention, the heavy chain variable region (VH) of the antibody has complementarity determining regions CDRs selected from the group consisting of:

VH CDR1, the amino acid sequence of which is GYSFTGYT (SEQ ID NO. 1);

VH CDR2 having the amino acid sequence INPYNGGT (SEQ ID NO. 2);

VH CDR3 having amino acid sequence ARGNSDGEAWFVY (SEQ ID NO. 3);

in another preferred embodiment, the amino acid sequence of said heavy chain variable region (85aa) is: VHLQQGELKPGSMKCKASGYSFTGYTNWKQHGLEWILINPYNGGTIYNQKFTGKATLTVDKARGNSDGEAWFVYWGQGTLITVSA(SEQ IDNO.4)；

In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and a heavy chain constant region, which may be murine.

As used herein, the terms "light chain variable region" and "V_L"may be used interchangeably.

In a preferred embodiment of the invention, the light chain variable region (VL) of the antibody according to the invention has complementarity determining regions CDRs selected from the group consisting of:

VL CDR1 having amino acid sequence KSVSTSGYSY (SEQ ID NO. 5);

VL CDR2 having amino acid sequence PTQLYFFRI (SEQ ID NO. 6);

VL CDR3 having the amino acid sequence QHIRELT (SEQ ID NO. 7);

in another preferred embodiment, the amino acid sequence of said light chain variable region (96aa) is: DVTQSP ALAVLGQRATISYKSVSTSGYSYMWNQQPGQPPRLLIPTQLYFFRINLEGVARSGGSGTTLNHPVEEEDAAQHIRELTTQQANTFLKFRI(SEQ ID NO.8)；

In a preferred embodiment of the present invention, the light chain of the antibody comprises the light chain variable region and the light chain constant region, which may be murine.

In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and refer to an antibody that specifically binds to the immune-related gtpase family m (irgm), such as a protein or polypeptide having a heavy chain variable region (e.g., the amino acid sequence of SEQ ID No. 4) and/or a light chain variable region (e.g., the amino acid sequence of SEQ ID No. 8). They may or may not contain the initial methionine.

The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., conjugate and fusion expression product) having heavy and light chains comprising a variable region, provided that the variable region is identical or at least about 90% homologous, preferably at least about 95% homologous, to the variable region of the heavy and light chains of the antibody of the invention.

In general, the antigen binding properties of an antibody can be described by 3 specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs), which are separated by 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

The invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having binding activity of immune-related GTP enzyme family M (IRGM) comprising the CDR regions described above. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences, the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention, where "stringent conditions" are defined as (1) hybridization and elution at lower ionic strength and higher temperatures, e.g., 0.2 × SSC, 0.1% SDS,60 ℃, or (2) hybridization with denaturing agents, e.g., 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃, etc., or (3) hybridization only if the identity between the two sequences is at least 90% or more, more preferably 95% or more, and where the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides of SEQ ID No.4 and/or SEQ ID No. 8.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

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: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated 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, colloidal gold, colored magnetic beads, latex particles, biotin labels, radionuclides, antibodies, ligands, antigens, receptors, nanoparticles, or combinations thereof.

Typically, the nanoparticles are selected from the group consisting of: nanogold, nanosilver, quantum dots, or combinations thereof.

Typically, the enzyme is selected from the group consisting of: horseradish peroxidase, acid phosphatase, or a combination thereof.

Hybridoma cell strain

The invention also provides a hybridoma cell strain capable of producing the monoclonal antibody against the immune related GTP enzyme family M (IRGM) of the invention; preferably, the invention provides a hybridoma cell strain with high titer aiming at the immune related GTP enzyme family M (IRGM) monoclonal antibody.

After obtaining the hybridoma producing the monoclonal antibody of the immune-related gtpase family m (irgm) of the present invention, one skilled in the art can conveniently prepare the antibody using the hybridoma cell line. In addition, the structure of the antibody of the present invention (e.g., the heavy chain variable region and the light chain variable region of the antibody) can be easily known by those skilled in the art, and then the monoclonal antibody of the present invention can be prepared by recombinant methods.

Preparation of monoclonal antibodies

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the antigens of the invention can be administered to an animal to induce the production of monoclonal antibodies. For Monoclonal antibodies, they can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol.6:511,1976; Kohler et al, Eur. J. Immunol.6:292,1976; Hammerling et al, In Monoclonal antibodies and T Cell hybrids, Elsevier, N.Y.,1981) or can be prepared using recombinant DNA methods (U.S. Pat. No.4,816,567).

Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to medium (HAT medium matrix), including myeloma Cell lines, such as murine myeloma Cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (available from salk institute Cell Distribution Center, san diego, california, usa), and SP-2, NZ0, or X63-Ag8-653 cells (available from American Type Culture Collection, rockwell, maryland, usa). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [ Kozbor, j.immunol., 133: 3001 (1984); brodeur et al, Techniques for the Production and use of monoclonal antibodies (monoclonal antibodies Production Techniques and Applications), pp 51-63 (Marcel Dekker, Inc., New York, 1987).

The medium in which the hybridoma cells are grown is assayed to detect the production of monoclonal antibodies of the desired specificity, e.g., by in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or Radioimmunoassay (RIA). The location of the antibody-expressing cells can be detected by FACS. The hybridoma clones can then be subcloned by limiting dilution procedures (subcloned) and grown by standard methods (Goding, monoclonal antibodies): Principles and Practice (Principles and Practice), Academic Press (1986) pp 59-103). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow in animals as ascites tumors.

The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The invention provides a monoclonal antibody aiming at immune related GTP enzyme family M (IRGM). In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. Taking supernatant fluid of hybridoma cell culture, carrying out saturated ammonium sulfate precipitation to obtain IgG, and purifying the antibody obtained by crude extraction through an affinity chromatography column (Protein G-Sepharose).

In a preferred embodiment of the invention, the monoclonal antibody is prepared by a method for producing the monoclonal antibody by Balb/C mouse ascites. The hybridoma cells are inoculated into the abdominal cavity of the sensitized mouse, and the abdomen is obviously swelled after about 10 days. Ascites is extracted, and after the crude extraction by saturated ammonium sulfate precipitation, the antibody of the crude extraction is purified by an affinity chromatography column (Protein G-Sepharose).

The main advantages of the invention are:

(1) the antibody of the invention is the first antibody known to be applicable to the detection of immune-related gtpase family M;

(2) the antibody has the characteristics of high affinity, high specificity, multiple application scenes and the like.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Materials and methods

1 antibody production:

the production route of the antibody adopts the patent technology SEAL of Eibomatt company^TM. The specific process route is as follows (fig. 1):

① antigen preparation an immune-related GTPase family M polypeptide 'ADGNLPEVIS (SEQ ID NO. 9)' 'KASPPTELVK (SEQ ID NO. 10)' 'SHFSNVVLWD (SEQ ID NO. 11)' 'QLLQIRENVL (SEQ ID NO. 12)' coupled to VLPs and immunogenic enhancers of the conventional KLH system was synthesized as an immunogen.

② mice were immunized, each group of antigens was used to immunize 6 Balb/c mice (8-12 weeks old) and their serum titers were monitored to determine the optimal number of immunizationsThe individual immunogenic polypeptides are combined into a group. The optimized adjuvant and immunization method can produce high affinity antibodies (IgG subtype) against most antigenic polypeptides. The initial immunization is strengthened for 3 to 4 times, and the mouse serum is taken to detect the titer after the strengthening (the invention uses recombinant protein as antigen coating, the recombinant protein adopts Escherichia coli prokaryotic expression, and the specific preparation method is shown in Advanced genetic constructs for recombinant protein expression in Escherichia coli.

HP, Mortensen kk.jbiotechnol.2005jan 26; 115(2):113-28.). Mice that are eligible for titer will be bumped once and used for fusion, and ineligible mice will continue to be fused after one to two boosts to the highest titer.

③ serum detection and screening, the orbit of the immunized mouse is bled, and ELISA is used for detecting the serum titer (recombinant protein is used as antigen coating), the serum titer needs to be more than 10K, otherwise, the boosting is continued.

④ fusion and selection, taking whole spleen and 1/2 lymph nodes, fusing with myeloma SP2/0 cell line, fusing with optimized PEG, spreading the fused cells onto 4 384-well plates (102 to 104 cells per well), culturing, collecting supernatant of all wells, screening polypeptide detecting source by ELISA, transferring positive wells with microscopic cells to 96-well plates for further culturing, collecting supernatant of all wells after several days of growth, detecting the reaction of soluble fragment detecting source by ELISA, further detecting the combination of soluble fragment detecting source with different dilution in the positive wells for affinity sorting, entering 20 parent clones with highest immunogenic affinity for each polypeptide into subclones, and entering 60 parent clones with highest immunogenic affinity for each soluble fragment into subclones.

⑤ subcloning and screening, namely, carrying out subcloning by a limiting dilution method and ELISA screening to obtain monoclonal hybridoma cells, laying cells on a 96-well plate, and culturing until the cell covers the bottom of about 1/6, detecting the reaction of supernatant of each well to a soluble fragment detection source and a corresponding polypeptide detection source by ELISA, taking two holes with high OD values and good cell states, entering the next round of subcloning, repeating the steps until the positive rate of cell strains in the holes is 100 percent, obtaining the monoclonal cell strains by the inventor at the moment, immediately carrying out expanded culture on all positive cells after the last round of subcloning, freezing one part of the positive cells for later use, and preparing supernatant or ascites by the other part of the positive cells.

⑥ preparation of antibody supernatant:

finally, the inventors obtained 8 monoclonal cell strains and injected the strains into F1 mice by abdomen for antibody production. The ascites fluid produced was purified with Protein A/G and used for subsequent detection.

2 antibody validation

And (3) carrying out ELISA, Western blotting, co-immunoprecipitation and mass spectrometry, antibody chip verification and the like on the obtained 8 monoclonal antibody cell strains to determine the most effective antibody.

Example 1 Elisa (immunoenzyme-linked) pairing validation of antibodies and antigenic polypeptides

And (3) coating a 96-hole ELISA plate with the ascites antibody to be paired, incubating, washing, sealing the degreased milk overnight, washing with PBS, and storing at 4 ℃ for later use. Antigen polypeptide incubation, PBS wash, with controls. HRP-labeled detection antibody was added to the ELISA plate incubated with the aforementioned. TMB color reaction, reading by a microplate reader. The titers of the 8 cell lines obtained by the screening are shown in the following table 2:

TABLE 28 Titers of cell lines

Example 2 endogenous protein imprinting (WB) validation of antibodies

WB validation was performed using whole protein lysates of duck meat with antibody dilution concentrations of 1:1000, 1:2000 and 1: 5000. The experimental results (FIG. 2) show that anti-IRGM (clone 2B11) specifically recognized the 47kd band in WB validation, consistent with the expected size.

Example 3 Immunoprecipitation (IP) additive Spectroscopy validation of antibodies

1mg of the whole protein of the duck meat is extracted, immunoprecipitation is carried out by using anti-IRGM (clone 2B11), and 47kd size bands are cut out from an IP product for mass spectrum detection. Mass spectrometry results (table 3 below) show that IRGM is enriched in IP samples in large amounts, indicating that the antibodies are highly specific for IRGM recognition. Furthermore, the antibody can be applied to IP experiments.

TABLE 3 Mass spectrometric detection results of 47kd size bands of IP products

Example 4 antibody chip assay

An anti-IRGM (clone 2B11) antibody and a control antibody were spotted on a glass plate with an NC membrane as a substrate using a chip spotting machine to form antibody spots having a diameter of 100 μm. The whole protein of the duck meat is labeled by biotin, incubated on an antibody chip at the concentration of 2ug/ml, and incubated at room temperature for half an hour. The cells were gently washed three times with PBS, incubated with CY3-SA fluorescent secondary antibody, washed three times with PBS, and the chip was scanned 523nm using a GenePix fluorescent chip scanner.

The experimental results (fig. 3) show that the anti-IRGM antibody has significant enrichment binding effect on the target protein, with strong fluorescence intensity, while the control antibody does not undergo antigen-antibody binding reaction.

Example 5IRGM content assay

The antibody can be used as a detection antibody for specially detecting the content of IRGM protein due to the unique specificity of the antibody to the immunity-related GTP enzyme family M, or further developed by a kit. The invention has already made some preliminary exploratory verification experiments.

And (3) standard substance: recombinant proteins of the immune related gtpase family M were quantified by BCA protein and run gel checked for concentration and diluted at 10ug/ml,5ug/ml,2.5ug/ml,1.25ug/ml fold.

Detection antibodies were subjected to an ELISA assay for the standard at a fixed concentration (1mg/ml) and the data (see Table 4) were obtained to plot a standard curve (FIG. 4) for reference in concentration calculations for subsequent detection of the test sample.

TABLE 4ELISA Rapid detection method

Concentration of standard substance mg/ml	10	5	2.5	1.25
					OD value of standard	3.045	2.373	1.734	0.986
Concentration of standard substance	10	5	2.5	1.25

Tissue fluid samples of different animals were taken for ELISA experiments, the same immobilized antibody concentration (1mg/ml) was used for detection, and the obtained OD values were substituted into a standard curve to calculate the actual protein content of different animal tissues, with the results shown in Table 5 below.

TABLE 5 results of ELISA experiments performed on different animal tissue samples

	Detecting the OD value	Conversion of enzyme concentration to mg/ml	Expression level
				Stomach cancer tissue	3.267	7.62	Height of
Duck meat	1.768	1.70	Is low in
				Mouse	1.355	1.13	Is low in
Peripheral blood of human	1.259	1.02	Is low in

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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<120> monoclonal antibody of immune related GTP enzyme family M (IRGM) and application thereof

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Claims

1. An antibody heavy chain variable region having one or more of the following Complementarity Determining Regions (CDRs):

VH CDR1 shown in SEQ ID NO.1,

VH CDR2 shown in SEQ ID NO.2, and

VH CDR3 shown in SEQ ID NO. 3;

2. An antibody heavy chain having the heavy chain variable region and the heavy chain constant region of claim 1.

3. An antibody light chain variable region having one or more of the following Complementarity Determining Regions (CDRs):

VL CDR1 shown in SEQ ID NO.5,

VL CDR2 shown in SEQ ID NO.6, and

VL CDR3 shown in SEQ ID NO. 7;

4. An antibody light chain comprising the light chain variable region and the light chain constant region of claim 3.

5. An antibody, wherein said antibody has:

(1) the heavy chain variable region of claim 1; and/or

(2) The light chain variable region of claim 3;

preferably, the antibody has: the heavy chain of claim 2; and/or the light chain of claim 4.

6. A recombinant protein, said recombinant protein having:

(i) the sequence of the heavy chain variable region of claim 1, the sequence of the heavy chain of claim 2, the sequence of the light chain variable region of claim 3, the sequence of the light chain of claim 4, or the sequence of the antibody of claim 5; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

7. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or the antibody of claim 5; or

(2) The recombinant protein of claim 6.

8. A vector comprising the polynucleotide of claim 7.

9. A genetically engineered host cell comprising the vector or genome of claim 8 having the polynucleotide of claim 7 integrated therein.

10. A conjugate, wherein the conjugate comprises:

(a) the antibody of claim 5 or the recombinant protein of claim 6; and

(b) a detectable label linked to the antibody or recombinant protein of (a).

11. An detection article, comprising:

(1) the antibody of claim 5, the recombinant protein of claim 6, or the conjugate of claim 10; and

(2) optionally a buffer solution or buffer.

12. Use of the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, the antibody of claim 5, the recombinant protein of claim 6, or the conjugate of claim 10, or the assay preparation of claim 11, in the preparation of an assay preparation or kit for detecting the level of immune-related gtpase family m (irgm) in a subject.

13. A test kit comprising the antibody of claim 5, the recombinant protein of claim 6, or the conjugate of claim 10 or the test article of claim 11.

14. A method of detecting immune-related gtpase family m (irgm) in a subject, comprising the steps of:

(a) providing a sample to be detected;

(b) mixing the sample with the antibody of claim 5, the recombinant protein of claim 6, the conjugate of claim 10, or the detection article of claim 11 to form a mixture;