CN111349171B

CN111349171B - Monoclonal antibody of peroxidase-6 and application thereof

Info

Publication number: CN111349171B
Application number: CN201811575067.2A
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: 2023-05-16
Anticipated expiration: 2038-12-21
Also published as: CN111349171A

Abstract

The invention provides a monoclonal antibody of peroxidase-6 and application thereof. Experimental results show that the antibody can be applied to antibody chip reaction, immunoprecipitation, western Blotting and detection of peroxidase-6 content in different cancer patients and Alzheimer disease patients aiming at the protein research.

Description

Monoclonal antibody of peroxidase-6 and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a peroxidase-6 monoclonal antibody and application thereof.

Background

Peroxidase-6 (PRDX 6) is a member of the family of thiol-specific antioxidant proteins. The protein is a bifunctional enzyme with two different active sites, which is involved in redox regulation of cells. The enzyme can reduce H ₂ O ₂ And short chain organics, fatty acids and phospholipid hydroperoxides, are thought to play a role in regulating phospholipid turnover and preventing oxidative damage.

It was found that there is a negative correlation between cancer incidence and Alzheimer's Disease (AD). The expression of the protein PRDX6 was significantly increased in human squamous lung carcinoma patients, but significantly decreased in AD patients comprising presenilin 2 (PS 2) mutants. Studies have shown that PS2 mutations inhibit lung tumor progression by inhibiting iPLA2 activity of PRDX6 through a gamma-secretase cleavage mechanism, and can explain the negative correlation between cancer and AD incidence.

There is currently no targeting antibody specifically directed against peroxidase-6 on the market, and there is a lack of effective antibody tools at the proteomics level for this field of research.

Disclosure of Invention

The antibody aims to provide a tool for researching targeted proteins so as to realize deeper research on protein expression changes and protein network patterns in different cancer patients and Alzheimer disease patients.

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 complementarity determining region 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 an antibody heavy chain having a heavy chain variable region and a heavy chain constant region according to 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 complementarity determining region 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 an antibody light chain having a light chain variable region and a light chain constant region according to 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 for a peroxidase-6 polypeptide.

In another preferred embodiment, the peroxidase-6 polypeptide is selected from the group consisting of: EADTTQGQIN (SEQ ID NO. 9) ', ' KLQDHVDWVN (SEQ ID NO. 10) ', ' GAFPYPIIAD (SEQ ID NO. 11) ', ' NVDEILRVID (SEQ ID NO. 12) ', or a combination 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 the heavy chain variable region of the first aspect of the invention, the sequence of the heavy chain of the second aspect of the invention, the sequence of the light chain variable region of the third aspect of the invention, the sequence of the light chain of the fourth aspect of the invention, or the sequence of the antibody of the fifth aspect of the invention; and

(ii) Optionally a tag sequence to assist expression and/or purification.

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

In another preferred embodiment, the recombinant protein comprises a bispecific antibody, 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) 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, or the antibody of the fifth aspect of the invention; or (b)

(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, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses and 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 incorporating a polynucleotide according to the seventh aspect of the invention.

In a tenth aspect of the present 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 attached 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 nanoparticle is selected from the group consisting of: gold nanoparticles, silver nanoparticles, 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 an inspection 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 detection preparation is used for detecting peroxidase-6.

In another preferred embodiment, the detection article comprises: test reagents, lateral flow sheets, chips, test strips, test plates, test strips.

In another preferred embodiment, the test strip includes: a detection zone to which another antibody against peroxidase-6 (antibody two) is immobilized, the antibody two being for capturing the peroxidase-6.

In another preferred embodiment, the test strip includes: a quality control region to which an antibody (secondary antibody) that binds to an antibody (primary antibody) directed against peroxidase-6 is immobilized, the secondary antibody being used to capture the antibody (primary antibody) directed against peroxidase-6.

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

In another preferred embodiment, the antibody (secondary antibody) that binds to an antibody (primary antibody) directed against peroxidase-6 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 the use of a heavy chain variable region according to the first aspect of the invention, a heavy chain variable region 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, 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 detection article according to the eleventh aspect of the invention, for the preparation of a detection article or kit for detecting the amount of peroxidase-6 in a subject.

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

In another preferred embodiment, the subject is a cancer patient and/or an Alzheimer's disease patient.

In another preferred embodiment, the detection article comprises a chip, an immune microparticle coated with an antibody.

In a thirteenth aspect of the invention, there is provided a detection 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 detection article according to the eleventh aspect of the invention.

In a fourteenth aspect of the present invention, there is provided a method of detecting peroxidase-6 in a subject, the method comprising 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-peroxidase-6 complex" in said mixture, wherein the presence of said complex, if any, indicates the presence of peroxidase-6 in said sample; if the complex is not present, it is an indication that peroxidase-6 is not present in the sample.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

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

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

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

Detailed Description

Through extensive and intensive studies, the present inventors have unexpectedly obtained a high affinity mab (murine mab) that recognizes and binds to peroxidase-6, which can be used in antibody chip reactions, immunoprecipitation, western Blotting, and detection of peroxidase-6 levels in different subjects (e.g., cancer patients and Alzheimer's disease patients) for which the protein is being studied. On this basis, the present invention has been completed.

Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods 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, as 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, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (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 described herein.

Antibodies to

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 the heavy chain by a covalent disulfide bond. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; 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. Specific amino acid residues form an 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 results in 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 fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

As used herein, the term "monoclonal antibody" 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 highly specific for a single antigenic site. 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 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 any particular method for producing the antibody.

The invention also includes monoclonal antibodies having the corresponding amino acid sequences of the anti-peroxidase-6 (PRDX 6) monoclonal antibodies, monoclonal antibodies having the variable region chains of the anti-peroxidase-6 (PRDX 6) monoclonal antibodies, and other proteins or protein conjugates and fusion expression products having these chains. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain comprising a hypervariable region (complementarity determining region, CDR), provided that the hypervariable region is identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light chain and heavy chain 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 to the anti-peroxidase-6 (PRDX 6) monoclonal antibody or fragment thereof. The invention also includes cell surface markers or antigens that bind to the anti-peroxidase-6 (PRDX 6) monoclonal antibodies, or fragments thereof.

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

As used herein, the terms "heavy chain variable region" and "V _H "interchangeably enableIs used.

As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determiningregion, CDR)".

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

VH CDR1 with amino acid sequence GSTFNNYW (SEQ ID No. 1);

VH CDR2 with amino acid sequence ISPSDNET (SEQ ID No. 2);

VH CDR3 with amino acid sequence ARYYRSFAF (SEQ ID No. 3);

in another preferred embodiment, the amino acid sequence of the heavy chain variable region (94 aa) is:

QVQSGPELRPASVKSCKASGSTFNNYWLHWKQRPGQLEWIGMISPSDNETRLNFKKAGKSSTAMQTSEDAVYFCARYYRSFAFGPKGTQVTVSA(SEQID NO.4)；

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

As used herein, the terms "light chain variable region" and "V _L "interchangeably used.

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, whose amino acid sequence is ESVDTYGKSL (SEQ ID No. 5);

VL CDR2, the amino acid sequence of which is LASTRUMT (SEQ ID NO. 6);

VL CDR3, the amino acid sequence of which is QQNNEI (SEQ ID NO. 7);

in another preferred embodiment, the amino acid sequence of the light chain variable region (100 aa) is:

YIVLTQSPASLAVSLGQRATISCRASESVDTYGKSLMHWYQQKPGQPPKLLIFLASVRMTNSVARFSGSRTFLIDPEFDAAYYCQQNNEIFGGGTKLEIK(SEQ ID NO.8)；

in a preferred embodiment of the present invention, the light chain of the antibody comprises the light chain variable region described above and a 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 peroxidase-6 (PRDX 6), 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 an initiating methionine.

The invention also provides other proteins or fusion expression products having the antibodies 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 regions of the heavy and light chains of the antibodies of the invention.

In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.

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

The invention includes not only intact monoclonal antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as 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 one skilled in the art and would be well known in light of the teachings herein.

The antibody of the present invention refers to a polypeptide having peroxidase-6 (PRDX 6) binding activity, which includes the above-described CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions 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 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of 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 a human antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, 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 of the antibody of the invention.

In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to Table 1.

TABLE 1

Initial residues	Representative substitution	Preferred substitution
			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 antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

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

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

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in 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 generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. 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 that exist in an isolated form.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of 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: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The transformant obtained 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 culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such 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 (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

The antibodies of the invention may be used alone or may be conjugated or coupled to a detectable label (for detection purposes).

Detectable labels for detection purposes include, but are not limited to: biotin, fluorescein, chemiluminescent groups, fluorescent proteins, enzymes, colloidal gold, colored magnetic beads, latex particles, biotin labels, radionuclides, antibodies, ligands, antigens, receptors, nanoparticles, or combinations thereof.

Typically, the nanoparticle is selected from the group consisting of: gold nanoparticles, silver nanoparticles, 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 hybridoma cell strains capable of producing the peroxidase-6 monoclonal antibodies of the invention; preferably, the present invention provides hybridoma cell lines with high titers against peroxidase-6 monoclonal antibodies.

After obtaining the hybridoma producing the peroxidase-6 monoclonal antibody of the invention, the hybridoma cell line can be used to prepare the antibody by a person skilled in the art with ease. Furthermore, the structure of the antibodies of the invention (e.g., the heavy and light chain variable regions of the antibodies) can be readily known to those skilled in the art, and then the monoclonal antibodies of the invention can be prepared by recombinant methods.

Preparation of monoclonal antibodies

Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, the antigens of the invention may be administered to animals to induce monoclonal antibody production. For monoclonal antibodies, hybridoma technology can be used to prepare (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 Hybridomas, 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 levels of antibody production by the antibody-producing cell of choice, and are sensitive to the medium (HAT medium matrix), including myeloma cell lines, e.g., 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 TypeCulture Collection, rocyveromyces, 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); techniques and applications for the production of monoclonal antibodies by Brodeur et al (Monoclonal AntibodiesProduction Techniques and Applications), pages 51-63 (Marcel Dekker, inc., new York, 1987) ].

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

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

The present invention provides a monoclonal antibody directed against peroxidase-6. In a preferred embodiment of the invention, the monoclonal antibodies are prepared by culturing hybridoma cells. Taking supernatant of hybridoma cell culture, roughly extracting IgG by a saturated ammonium sulfate precipitation method, and purifying the roughly extracted antibody by an affinity chromatography column (Protein G-Sepharose).

In a preferred embodiment of the present invention, the monoclonal antibody is prepared by the method of producing a monoclonal antibody by using Balb/C mouse ascites. Approximately hybridoma cells were inoculated into the abdominal cavity of sensitized mice, and obvious abdominal distension was seen around 10 days. Extracting ascites, coarse extracting with saturated ammonium sulfate precipitation, and purifying the coarse extracted antibody with affinity chromatographic column (Protein G-Sepharose).

The invention has the main advantages that:

(1) The antibody of the present invention is the first antibody known to be applicable to peroxidase-6;

(2) The antibodies of the invention are the first antibodies known to be useful in antibiotic screening;

(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 is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratory, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Materials and methods

1 antibody production:

the production route of the antibody adopts the patent technology SEAL of Aibi-Mary company ^TM . The specific process route is as follows (figure 1):

(1) antigen preparation: peroxidase-6 polypeptide 'EADTTQGQIN (SEQ ID NO. 9)' 'KLQDHVDWVN (SEQ ID NO. 10)' 'GAFPYPIIAD (SEQ ID NO. 11)' 'NVDEILRVID (SEQ ID NO. 12)' was synthesized as an immunogen to which was coupled an immunogenicity enhancing factor coupled VLP as well as conventional KLH systems.

(2) Immunized mice

Each group of antigens will be used to immunize 6 Balb/c mice (8-12 weeks old) and their serum titers monitored to determine the optimal number of immunizations. Each immunogenic polypeptide is mixed into a set. Optimized adjuvants and immunization methods are capable of producing high affinity antibodies (IgG subtypes) against most antigenic polypeptides. The mice serum is taken to detect titer after 3 to 4 boosts after primary immunization (the recombinant protein is used as antigen coating in the invention, the recombinant protein is expressed by escherichia coli procaryon, and the specific preparation method is shown in Advanced genetic strategies for recombinant protein expression in Escherichia coli.

HP, mortensen kk.j biotechnol.2005jan 26;115 (2):113-28.). Mice with acceptable titers will be impacted once and used for fusion, and failed mice will continue to be boosted one to two times to fusion after the titer is highest.

(3) Serum detection and screening: the immunized mice were bled from the orbit and serum titers were detected by ELISA (recombinant proteins as antigen coating). Serum titers were greater than 10K, otherwise boost was continued.

(4) Fusion and screening: whole spleen and 1/2 lymph nodes were taken and fused with myeloma SP2/0 cell line. The process is optimized PEG fusion. The fused cells were plated onto 4 384-well plates (102 to 104 per well) and cultured. The supernatants from all wells were collected, screened for polypeptide detecting precursors by ELISA, and cultured in a positive Kong Zhuaidao 96-well plate with cells visualized. After several days of growth, the supernatants from all wells were collected and assayed for reaction with soluble fragment detector by ELISA. The positive wells further detect soluble fragment detection pro-binding at different dilutions for affinity sequencing. The 20 parent clones with highest affinity for each polypeptide immunogen enter subclones. The 60 parental clones with highest affinity for each soluble fragment immunogen enter subclones.

(5) Subcloning and screening: subcloning is performed by limiting dilution and ELISA screening to obtain monoclonal hybridoma cells. Cells were plated in 96-well plates and cultured to cover about 1/6 of the bottom. ELISA detects the reaction of the supernatant of each hole on the soluble fragment detecting antigen and the corresponding polypeptide detecting antigen, and two holes with high OD value and good cell state are taken to enter the subcloning of the next round. The above procedure was repeated until the cell line positive rate in the wells was 100%. At this time, the inventors obtained a monoclonal cell line. After the last round of subcloning, all positive cells were immediately expanded, one part was frozen for later use and the other part was prepared as supernatant or ascites.

(6) Antibody supernatant preparation: finally, the inventors obtained 8 monoclonal cell lines and injected F1 mice through the abdomen for antibody production. The ascites produced was purified with Protein A/G and used for subsequent detection.

2 antibody validation

ELISA, western blotting, co-immunoprecipitation, mass spectrometry, antibody chip and the like are carried out on the obtained 8 monoclonal antibody cell strains, and the most effective antibody is determined.

Example 1 Elisa (immune enzyme linked) pairing verification of antibodies and antigen polypeptides

And (3) coating the ascites antibody to be paired on a 96-well ELISA plate, incubating, washing, blocking with skimmed milk overnight, washing with PBS, and preserving at 4 ℃ for later use. Antigen polypeptide incubation, PBS wash, and control were set. HRP-labeled detection antibody was added to the ELISA plate incubated with the above. TMB color reaction, and reading by an enzyme label instrument. Titers of 8 cell lines obtained by screening are shown in table 2 below:

table 28 titers of cell lines

Antibody name	Clone number	3.125K	6.25K	12.5K	25K	50K	100K	200K	400K	800K	1600K	PC	NC	Whether or not to pass	Valency of
																anti-PRDX6	3F12	3.075	3.093	3.161	2.688	1.888	1.519	1.161	0.973	0.652	0.396	3.016	0.238	Qualified product	1600K
anti-PRDX6	4B7	3.089	2.849	2.709	2.406	1.593	1.813	0.757	0.591	0.242	0.205	3.188	0.151	Qualified product	400K
																anti-PRDX6	4F11	3.062	2.993	2.916	2.702	1.966	1.734	1.008	0.775	0.556	0.206	3.08	0.229	Qualified product	800K
anti-PRDX6	8D2	3.049	2.942	2.717	1.788	0.91	0.644	0.418	0.279	0.251	0.191	2.985	0.184	Qualified product	800K
																anti-PRDX6	2H9	3.086	2.84	2.713	2.567	2.287	2.372	1.634	1.271	0.793	0.464	3.017	0.154	Qualified product	1600K
anti-PRDX6	2C6	3.001	2.958	3.055	2.637	1.462	1.439	0.541	0.249	0.234	0.172	2.899	0.145	Qualified product	200K
																anti-PRDX6	7C10	3.097	2.814	2.681	1.539	1.039	0.832	0.345	0.242	0.218	0.189	3.015	0.225	Qualified product	200K
anti-PRDX6	5A8	2.935	2.924	2.56	2.157	1.821	1.627	0.962	0.427	0.231	0.202	2.997	0.153	Qualified product	400K

Example 2 endogenous protein imprinting (WB) validation of antibodies

WB validation was performed using whole protein lysates of plutella xylostella with antibody dilution concentrations of 1:1000, 1:2000 and 1:5000. The experimental results (FIG. 2) show that anti-PRDX6 (clone 2H 9) can specifically recognize 24kd bands in WB validation, consistent with the expected size.

EXAMPLE 3 Immunoprecipitation (IP) of antibodies plus mass spectrometry validation

1mg of plutella xylostella total protein is extracted, immunoprecipitation is carried out by using anti-PRDX6 (clone 2H 9), and an IP product is cut into 24kd size bands for mass spectrum detection. The mass spectrum results (shown in table 3) show that PRDX6 is enriched in a large amount in the IP sample, which indicates that the specificity of the antibody to PRDX6 recognition is high. Furthermore, the antibody can be applied to IP experiments.

TABLE 3 detection results of 24kd size band mass spectrum for IP products

Example 4 antibody chip detection experiments

anti-PRDX6 (clone 2H 9) antibody and control antibody were spotted on NC membrane-based glass plates using a chip spotter to form antibody spots with a diameter of 100. Mu.m. The whole protein of plutella xylostella is subjected to biotin labeling, and incubated on an antibody chip at a concentration of 2ug/ml for half an hour at room temperature. The PBS was gently washed three times, incubated with CY3-SA fluorescent secondary antibody, washed three times with PBS, and the chip was scanned using a GenePix fluorescent chip scanner at 523 nm.

The experimental results (figure 3) show that the anti-PRDX6 antibody has obvious enrichment binding effect on target protein, the fluorescence intensity is strong, and the antigen-antibody binding reaction of the control antibody does not occur.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

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Claims

1. An anti-peroxidase-6 antibody, wherein the heavy chain variable region of said antibody has the following 3 complementarity determining region 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; and is also provided with

The light chain variable region of the antibody has the following 3 complementarity determining region 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.

2. The antibody of claim 1, wherein the heavy chain variable region amino acid sequence of the antibody is set forth in SEQ ID No. 4.

3. The antibody of claim 1, wherein the light chain variable region amino acid sequence of the antibody is set forth in SEQ ID No. 8.

4. A recombinant protein, said recombinant protein comprising:

(i) The sequence of the antibody of claim 1; and

(ii) Optionally a tag sequence to assist expression and/or purification.

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

(1) The antibody of claim 1; or (b)

(2) The recombinant protein according to claim 4.

6. A vector comprising the polynucleotide of claim 5.

7. A genetically engineered host cell comprising the vector or genome of claim 6 integrated with the polynucleotide of claim 5.

8. A conjugate, the conjugate comprising:

(a) The antibody of claim 1 or the recombinant protein of claim 4; and

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

9. An inspection article, the inspection article comprising:

(1) The antibody of claim 1, the recombinant protein of claim 4, or the conjugate of claim 8; and

(2) Optionally a buffer solution or buffer.

10. Use of the antibody of claim 1, the recombinant protein of claim 4, or the conjugate of claim 8, or the detection article of claim 9 for the preparation of a detection article or kit for detecting the amount of peroxidase-6 in a subject.

11. A test kit comprising the antibody of claim 1, the recombinant protein of claim 4, or the conjugate of claim 8 or the test article of claim 9.