CN109593131A - A kind of anti-14-3-3 η protein monoclonal antibody and application thereof - Google Patents

Abstract

The present invention provides a kind of anti-14-3-3 η protein monoclonal antibodies and application thereof, specifically the present inventor successfully obtains the monoclonal antibody of the anti-14-3-3 η albumen of one plant of specificity in the course of the research, the experimental results showed that, the monoclonal antibody can specifically combine 14-3-3 η albumen, and affinity is strong, high sensitivity.The antibody is used not only for detection blood plasma, the 14-3-3 η albumen in flesh tissue, it may also be used for the detection of the clinical tissue sample slice of paraffin embedding.It is significant for being studied in laboratory research and clinic.

Description

A kind of anti-14-3-3 η protein monoclonal antibody and application thereof

Technical field

The invention belongs to fields of biomedicine, specifically, the present invention relates to a kind of anti-14-3-3 η protein monoclonal antibodies And application thereof.

Background technique

14-3-3 is a kind of phosphoserine/threonine binding protein family, including α/β, γ, ε, σ, ξ, θ/τ and η seven Hypotype.Existing research shows: 5 kinds of 14-3-3 protein subunits (α/β, γ, ε, σ and ξ) take part in tumor progression.

Tumour (tumor) refers to that cell loses the normal regulation grown to it under the effect of tumorigenesis factor, leads to abnormal increasing Raw and generation disease.Tumour can be divided into benign and malignant tumour two major classes.The former slow growth is clear with surrounding tissue boundary Chu does not shift, little to human health damage.The latter's growth rapidly, can be transferred to other body parts, can also generate Evil substance, destroys normal organ structure, so that body function is lacked of proper care, life-threatening.

It is to endanger a kind of disease of human health most serious at present that malignant tumour, which is also cancer (cancer),.In the U.S., dislike The death rate of property tumour is only second to cardiovascular disease and occupies second.The early diagnosis of cancer has huge in the treatment of cancer Meaning.In addition, needing by being detected to tumor-marker albumen, and then in the treatment of cancer to the molecule of cancer Type is assessed, to formulate more reasonable therapeutic scheme.

Therefore, those skilled in the art be dedicated to develop detection effect it is more accurate, sensitivity is higher, and use is more square Just the detection technique of tumor-marker albumen.

Summary of the invention

The purpose of the present invention is to provide a kind of anti-14-3-3 η protein monoclonal antibodies and application thereof.

The first aspect of the present invention provides a kind of anti-14-3-3 η protein monoclonal antibody,

The first aspect of the present invention, provides a kind of antibody, and the antibody includes

(1) heavy chain variable region；And/or

(2) light chain variable region；

Wherein, the heavy chain variable region includes following three complementary determining region CDR:

CDR1 shown in SEQ ID NO.:1,

CDR2 shown in SEQ ID NO.:2, and

CDR3 shown in SEQ ID NO.:3；

The light chain variable region includes following three complementary determining region CDR:

CDR1' shown in SEQ ID NO.:5,

CDR2' shown in SEQ ID NO.:6, and

CDR3' shown in SEQ ID NO.:7.

In another preferred example, the heavy chain variable region has amino acid sequence shown in SEQ ID NO.:4.

In another preferred example, the light chain variable region has amino acid sequence shown in SEQ ID NO.:8.

In another preferred example, the antibody further includes heavy chain constant region.

In another preferred example, the heavy chain constant region is source of people, source of mouse or rabbit source.

In another preferred example, the antibody further includes constant region of light chain.

In another preferred example, the constant region of light chain is source of people, source of mouse or rabbit source.

In another preferred example, the antibody is the antibody of the anti-14-3-3 η albumen of specificity.

In another preferred example, the antibody includes: single-chain antibody, double-chain antibody, monoclonal antibody, chimeric antibody (such as human mouse chimeric antibody), source of mouse antibody, rabbit source antibody or humanized antibody.

The second aspect of the present invention provides a kind of polynucleotides, it encodes antibody as described in the first aspect of the invention.

The third aspect of the present invention provides a kind of carrier, it contains polynucleotides described in second aspect of the present invention.

In another preferred example, the carrier include: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, Mammalian cell virus such as adenovirus, retrovirus or other carriers.

The fourth aspect of the present invention provides a kind of genetically engineered host cell, it contains third aspect present invention Polynucleotides described in second aspect of the present invention are integrated in the carrier or genome.

The fifth aspect of the present invention provides a kind of immune conjugate, which contains:

(a) antibody as described in the first aspect of the invention；With

(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor, radionuclide or Enzyme.

In another preferred example, the conjugate is selected from: (magnetic is total by fluorescence or luminous marker, radioactively labelled substance, MRI Vibration imaging) CT (x-ray tomography of electronic computer) contrast agent or can generate detectable product enzyme, radiation Property nucleic, biotoxin, cell factor (such as IL-2), antibody, antibody Fc fragment, antibody scFv fragment, gold nano grain/receive Rice stick, virion, liposome, magnetic nanosphere, pro-drug activation enzymes are (for example, DT- diaphorase (DTD) or biphenyl base hydrolase- Sample protein (BPHL)), chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..

The sixth aspect of the present invention provides a kind of pharmaceutical composition, which is characterized in that it contains:

(i) antibody as described in the first aspect of the invention, the immune conjugate as described in fifth aspect present invention；And

(ii) pharmaceutically acceptable carrier.

In another preferred example, the pharmaceutical composition is injection type.

In another preferred example, the pharmaceutical composition is used to prepare the drug for the treatment of tumour, and the tumour is selected from The following group: liver cancer, gastric cancer, leukaemia, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, large intestine Cancer, cervical carcinoma, carcinoma of endometrium, carcinoma of penis, adrenal tumor or tumor of bladder.

The seventh aspect of the present invention provides antibody as described in the first aspect of the invention, such as fifth aspect present invention institute The purposes for the immune conjugate stated is used to prepare medicament, reagent, detection plate or kit；

The reagent, detection plate or kit are used for:

(1) 14-3-3 η albumen in test sample；And/or

(2) endogenic 14-3-3 η albumen in tumour cell is detected；And/or

(3) tumour cell of detection expression 14-3-3 η albumen；

The medicament is used to treat or prevent the tumour of expression 14-3-3 η albumen.

In another preferred example, the tumour includes: gastric cancer, liver cancer, leukaemia, tumor of kidney, lung cancer, carcinoma of small intestine, bone Cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical carcinoma, carcinoma of endometrium, carcinoma of penis, Small Cell Lung Cancer, melanin Tumor or H/N tumors or adrenal tumor.

In another preferred example, the reagent includes the immune particle of chip, coated antibody.

The eighth aspect of the present invention provides a kind of method of 14-3-3 η albumen in test sample, and the method includes steps It is rapid:

(1) sample is contacted with antibody described in first aspect present invention；

(2) it detects whether to form antigen-antibody complex, wherein forming compound means that there are 14-3-3 η in sample Albumen.

In another preferred example, it is detected in step (2) by ELISA method.

In another preferred example, the sample includes: human or animal tissues sample, tumor resection sample, cast-off cells sample Product.

In another preferred example, the method is used for nondiagnostic purpose.

The ninth aspect of the present invention provides a kind of kit, includes: in the kit

The first container contains antibody described in first aspect present invention in the first container.

In another preferred example, in the kit further include:

Second container contains cell cracking agent in the second container.

In another preferred example, the antibody in the first container has detectable label.

The tenth aspect of the present invention provides a kind of preparation method of preparation and reorganization polypeptide, and this method includes:

(a) under conditions suitable for the expression, host cell described in fourth aspect present invention is cultivated；

(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is antibody described in first aspect present invention.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Detailed description of the invention

Fig. 1 shows coloration result of the 108A5H11 monoclonal antibody in liver cancer tissue cell.

Fig. 2 shows coloration result of the 108A5H11 monoclonal antibody in normal liver cell.

Fig. 3 shows coloration result of the 108A5H11 monoclonal antibody in normal liver cell.

Fig. 4 shows coloration result of the 108A5H11 monoclonal antibody in normal liver cell.

Fig. 5 shows the electrophoresis result of WB detection hepatocarcinoma patient liver samples 14-3-3 η.

Fig. 6 shows the electrophoresis result of WB detection hepatocarcinoma patient liver samples 14-3-3 η.

Specific embodiment

The present inventor successfully obtains the anti-14-3-3 η albumen of one plant of specificity by largely screening by in-depth study Monoclonal antibody, the experimental results showed that, which can specifically combine 14-3-3 η albumen, and affinity is strong, sensitivity It is high.The antibody is used not only for the 14-3-3 η albumen in detection flesh tissue, it may also be used for formalin fixes, paraffin packet The detection of the clinical tissue sample slice buried.On this basis, the present invention is completed.

14-3-3 is a kind of phosphoserine/threonine binding protein family, including α/β, γ, ε, σ, ξ, θ/τ and η seven Hypotype.Existing research shows: 5 kinds of 14-3-3 protein subunits (α/β, γ, ε, σ and ξ) take part in liver cancer process.The present inventor's research It was found that: compared to hypotype has been reported, 14-3-3 η exists in the tumour cell and vascular endothelial cell in liver cancer tissue to be divided Cloth, and its expression gradually rises with liver cancer progress.Be further discovered that: 14-3-3 η is equal in liver cancer and vascular endothelial cell It can be by forming regenerative feedback loop with p-ERK1/2, and then promote the process of liver cancer, and the high table of liver cancer patient tissue 14-3-3 η Up to Sorafenib resist and prognosis mala it is related.

The amino acid sequence of 14-3-3 η is as follows:

The 14-3-3 η albumen that the present inventor is surprised to find that in the course of the research in liver cancer patient blood serum significantly increases, because This can be used as the significant albumen of liver cancer.The blood plasma detection of 14-3-3 η is pre- as potential liver cancer early detection, tumor prognosis Measuring tool has important value.

But the 14-3-3 η detection in liver cancer patient tumor tissues is extremely inconvenient, the present inventor attempts to examine in serum/slurry The level of 14-3-3 η is surveyed, but since 14-3-3 η molecular weight is smaller, about 28KD, close to the light chain size of albumen, therefore The difference of 14-3-3 η level in Serum of Cancer Patients/slurry sample and normal human serum/slurry sample can not be observed in testing result It is different.

The present inventor further removes high-abundance proteins in plasma/serum (such as IgG, IgM, IgA, albumin, α 1- acid Property glycoprotein etc.) after, 14-3-3 η is detected in serum/slurry sample by the immunoblotting analysis detection method success of improvement, and And have been surprisingly found that 14-3-3 η level has significant difference in Serum of Cancer Patients/slurry sample and normal human serum/slurry sample.Into One step, it is compared and analyzed by large sample hepatocarcinoma patient plasma sample and human normal plasma's sample, finds 14-3- in blood plasma 3 η can be used as hepatocarcinoma mark object, can be used for potential liver cancer early detection, tumor prognosis prediction etc..

Specifically, the present invention is using recombination 14-3-3 η protein immune balb/c mice, and uses 14-3-3 η albumen Recombinant protein obtains positive monoclonal hybridoma cell strain as screening antigen, screening.Final 7 plants of acquisition can be specifically bound The cell strain of 14-3-3 η albumen.

7 plants of monoclonal cell strains are subjected to mouse hydroperitoneum antibody of RGDV preparation, formalin are fixed, the liver cancer tissue of paraffin embedding Slice is detected, final to obtain one plant and specific recognition paraffin tissue sections 14-3-3 η albumen and can use serum The monoclonal antibody of middle destination protein detection.

As used herein, term " antibody " or " immunoglobulin " are about 150000 dalton for having identical structure feature Different four glycan albumen is made of two identical light chains (L) and two identical heavy chains (H).Every light chain is total by one Valence disulfide bond is connected with heavy chain, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different.Each heavy chain and The intrachain disulfide bond at light chain also regular interval.One end of each heavy chain has variable region (VH), is followed by multiple constant regions.Every One end of light chain has variable region (VL), and the other end has constant region；The constant region of light chain is opposite with first constant region of heavy chain, gently The variable region of chain and the variable region of heavy chain are opposite.Special amino acid residue forms boundary between the variable regions of the light chain and the heavy chain Face.

As used herein, " variable " the certain parts for indicating variable region in antibody of term are different in sequence, its shape Combination and specificity at various specific antibodies to its specific antigen.However, changeability and being unevenly distributed over entire anti- In body variable region.It concentrates in light chain and heavy chain variable region three segments being known as in complementary determining region (CDR) or hypervariable region In.More conservative part is known as framework region (FR) in variable region.Four FR are respectively contained in the variable region of native heavy and light chain Area, they are generally in beta sheet configuration, are connected by three CDR of formation connection ring, can form part β folding in some cases Stack structure.CDR in every chain is by the area FR firmly against the antigen for together forming antibody together and with the CDR of another chain Binding site (referring to Kabat etc., NIH Publ.No.91-3242, rolls up I, 647-669 pages (1991)).Constant region is not joined directly With the combination of antibody and antigen, but they show different effector functions, such as participate in antibody dependent on the thin of antibody Cellular toxicity.

" light chain " of vertebrate antibodies (immunoglobulin) can be classified as obviously not according to the amino acid sequence of its constant region One kind in same two classes (referred to as κ and λ).According to the amino acid sequence of its heavy chain constant region, immunoglobulin can be divided into not Same type.Mainly there are 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them can also be further separated into subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.Light chain constant corresponding to different immunoglobulin like protein is distinguished It is also known as α, δ, ε, γ and μ.The subunit structure and 3-d modelling of different immunoglobulin like protein are known to those skilled in the art 's.

As used herein, term " monoclonal antibody (monoclonal antibody) " refers to the antibody obtained from a kind of substantially uniform group, i.e., The single antibody for including in the group be it is identical, in addition to a small number of mutation that may be present naturally occurred.Monoclonal antibody is high Specifically it is directed to single antigen site.Moreover, (usually having for different determinants from conventional polyclonal antibody preparation Different antibodies) it is different, each monoclonal antibody is for the single determinant on antigen.Other than their specificity, herein Rabbit monoclonal antibodies be that overall length rabbit monoclonal antibodies base is constructed by the method for molecular biosciences after screening by phage library Because of expression vector, which is transferred to eukaryotic expression system, cell conditioned medium is collected after culture and obtains, it will not be by other immune balls Protein contamination.Modifier " monoclonal " illustrates the characteristic of antibody, is obtained from substantially uniform antibody population, this should not be by It is construed to need to produce antibody with any specific process.

The invention also includes the monoclonals of the corresponding amino acid sequence with the anti-14-3-3 η protein monoclonal antibody Antibody, the monoclonal antibody with the anti-14-3-3 η protein monoclonal antibody variable region chain, and its with these chains His protein or protein conjugate and fusion expressed product.Specifically, the present invention includes having containing hypervariable region (complementation decision Area, CDR) light chain and any protein or protein conjugate and fusion expressed product of heavy chain (immune conjugate and melt Close expression product), as long as the hypervariable region is identical as the hypervariable region of light chain of the invention and heavy chain or at least 90% homology, preferably Ground at least 95% homology.

As it is known by the man skilled in the art, immune conjugate and fusion expressed product include: drug, toxin, cell factor (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and the 14-3-3 η protein monoclonal antibody or its Segment in conjunction with and the conjugate of formation.The invention also includes with the anti-14-3-3 η protein monoclonal antibody or its segment In conjunction with cell surface marker object or antigen.

The present invention not only includes complete monoclonal antibody, further includes having immunocompetent antibody fragment, such as Fab or (Fab')₂Segment；Heavy chain of antibody；Antibody light chain.

As used herein, term " heavy chain variable region " and " V_H" be used interchangeably.

As used herein, term " variable region " and " complementary determining region (complementarity determining Region, CDR) " it is used interchangeably.

In a preferred embodiment of the invention, the heavy chain variable region of the antibody includes that following three complementations are determined Determine area CDR:

CDR1, amino acid sequence are GYTFTSYIS (SEQ ID NO.:1)；

CDR2, amino acid sequence are QRYDA (SEQ ID NO.:2)；

CDR3, amino acid sequence are YYPAFYDKAMVNL (SEQ ID NO.:3).

In another preferred example, the amino acid sequence of the heavy chain variable region are as follows:

EVQLQQSGPGSVKPGASLKLSCKASGYTFTSYISAISKQRQTPEKSLEWIGSIQRYDATAYMDSVGKR GTISYDRARLINYLDMSSLRSEDTAMYFCARYYPAFYDKAMVNLDWGQGTSVTVSS(SEQ ID NO.:4)。

In a preferred embodiment of the invention, the heavy chain of the antibody includes above-mentioned heavy chain variable region and heavy chain Constant region, the heavy chain constant region can be source of mouse, source of people or rabbit source.

As used herein, term " light chain variable region " and " V_L" be used interchangeably.

In a preferred embodiment of the invention, the light chain variable region of antibody according to the present invention has and is selected from The complementary determining region CDR of the following group:

CDR1', amino acid sequence are WKPVGTGKFTPHEVI (SEQ ID NO.:5)；

CDR2', amino acid sequence are PVRETLS (SEQ ID NO.:6)；

CDR3', amino acid sequence are QDNTPYT (SEQ ID NO.:7)；

In another preferred example, the amino acid sequence of the light chain variable region are as follows:

DIVLTQSPSSLAVQTGQTMICSYWKPVGTGKFTPHEVIWYQQKPGQPPRLLIYPVRETLSGVPDRFSG SGSGTDFTLTIHMVEAEDAAVYYCQDNTPYTFGGTKEEGGR(SEQ ID NO.:8)。

In a preferred embodiment of the invention, the light chain of the antibody includes above-mentioned light chain variable region and light chain Constant region, the constant region of light chain can be source of mouse, source of people or rabbit source.

In the present invention, term " antibody of the present invention ", " albumen of the present invention " or " polypeptide of the present invention " is used interchangeably, all Refer to the antibody of specific binding 14-3-3 η albumen.They can be with or without initial methionine.

In another preferred example, the antibody is mouse or the people's mouse chimeric mAb of anti-14-3-3 η albumen, it Heavy chain constant region and/or constant region of light chain can be the heavy chain constant region or constant region of light chain of humanization.It is highly preferred that described The heavy chain constant region or constant region of light chain of humanization are the heavy chain constant region or constant region of light chain of human IgG1, IgG2 etc..

The present invention also provides other protein or fusion expressed product with antibody of the present invention.Specifically, of the invention It is (i.e. immune even including any protein or protein conjugate and fusion expressed product with heavy chain and light chain containing variable region Join object and fusion expressed product), as long as the variable region is identical as the variable region of the heavy chain of antibody of the present invention and light chain or at least 90% homology, preferably at least 95% homology.

Generally, the antigenic binding property of antibody can be described by being located at 3 specific regions of heavy chain and light chain variable region, The section is partitioned into 4 frame areas (FR) by referred to as Variable Area (CDR), and the amino acid sequence of 4 FR is relatively conservative, Association reaction is not participated in directly.These CDR form cyclic structure, the β-pleated sheet formed by FR therebetween phase on space structure Mutually close, the CDR on CDR and corresponding light chain on heavy chain constitutes the antigen binding site of antibody.It can be by comparing similar The amino acid sequence of the antibody of type determines be which Amino acid profile FR or CDR region domain.

The heavy chain of antibody of the present invention and/or the variable region of light chain are particularly interesting, because at least partly relating in them And combine antigen.Therefore, the present invention includes those molecules with the monoclonal antibody light chain with CDR and heavy chain variable region, only Want its CDR and CDR that identifies herein have 90% or more (preferably 95% or more, most preferably 98% or more) homology.

The present invention not only includes complete monoclonal antibody, further include have immunocompetent antibody segment or antibody with The fusion protein that other sequences are formed.Therefore, the invention also includes the segments of the antibody, derivative and analogue.

As used herein, term " segment ", " derivative " refer to that be kept substantially antibody of the present invention identical with " analog " Biological function or active polypeptide.Polypeptide fragment of the invention, derivative or the like, which can be (i), one or more Conservative or non-conservative amino acid residue (preferably conservative amino acid) substituted polypeptide, and such substituted amino Sour residue, which can be, may not be by genetic code encoding, or (ii) has substitution in one or more amino acid residues The polypeptide of group, or (iii) mature polypeptide and another compound (for example extend the compound of polypeptide half-life period, such as poly- second Glycol) fusion is formed by polypeptide, or (iv) additional amino acid sequence is fused to this polypeptide sequence and the polypeptide that is formed is (as before Lead sequence or secretion sequence or for purifying the sequence of this polypeptide or proprotein sequence, or merge egg with what 6His label was formed It is white).According to the teaching of this article, these segments, derivative and analogue belong to scope known to those skilled in the art.

Antibody of the present invention refers to polypeptide with 14-3-3 η protein binding activity, including above-mentioned CDR region.The term also wraps Include the variant form with polypeptide with antibody identical function of the present invention, comprising above-mentioned CDR region.These variant forms include (but being not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) Missing, insertion and/or the substitution of amino acid, and C-terminal and/or N-terminal addition it is one or several (usually 20 with It is interior, be more preferably within 5 within preferably 10) amino acid.For example, in the art, use is similar in performance When amino acid is replaced, the function of protein is not usually changed.For another example, C-terminal and/or N-terminal add one or Several amino acid will not generally also change the function of protein.The term further includes that the active fragment of antibody of the present invention and activity are spread out Biology.

The variant form of the polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation, induction Mutant, the encoded albumen of DNA that can hybridize with the coding DNA of antibody of the present invention under the conditions of high or low stringency, with And more peptide or proteins of the antiserum acquisition using anti-antibody of the present invention.

The present invention also provides other polypeptides, such as the fusion protein comprising human antibody or its segment.In addition to almost overall length Outside polypeptide, the invention also includes the segments of antibody of the present invention.In general, the segment has at least about 50 companies of antibody of the present invention Continue amino acid, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably at least about 100 continuous amino acids.

In the present invention, " conservative variant of antibody of the present invention " refers to compared with the amino acid sequence of antibody of the present invention, There are at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are with similar or analogous properties Amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on Table A and generate.

Table A

The polynucleotides for encoding mature polypeptide of the invention include: the coded sequence of an encoding mature polypeptide；Mature polypeptide Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and non-volume Code sequence.

The term polynucleotides of polypeptide " coding " can be the polynucleotides including encoding this polypeptide, be also possible to further include The polynucleotides of additional code and/or non-coding sequence.

The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least 70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with it is of the present invention more The interfertile polynucleotides of nucleotide.In the present invention, " stringent condition " refers to: (1) compared with low ionic strength and higher temperature Under hybridization and elution, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant, such as 50% (v/v) formyl when (2) hybridization Amine, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) only the phase same sex between two sequences at least 90% with On, more preferably 95% or more when, just hybridizes.

The nucleotide full length sequence of antibody of the invention or its segment can usually use PCR amplification method, recombination method or artificial Synthetic method obtains.A kind of feasible method is that manually synthetic method synthesizes related sequence, especially fragment length When shorter.In general, being then attached the very long segment of available sequence again by first synthesizing multiple small fragments.In addition, may be used also The coded sequence of heavy chain and expression label (such as 6His) are fused together, fusion protein is formed.

Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method. Biomolecule (nucleic acid, albumen etc.) according to the present invention includes existing biomolecule in a separate form.

At present, it is already possible to obtain encoding albumen of the present invention (or its segment or its derivative by chemical synthesis completely Object) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or such as carrier) and In cell.In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.

The invention further relates to the carriers comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence.This A little carriers can be used for converting host cell appropriate, allow it to expression protein.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high Equal eukaryocytes, such as mammalian cell.Representative example has: Escherichia coli, streptomyces；The bacterium of salmonella typhimurium Cell；Fungal cell's such as yeast；The insect cell of drosophila S2 or Sf9；CHO, COS7, zooblast of 293 cells etc..

It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is using MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection method can be selected: calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.

Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as Fruit needs, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process is used Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.

Antibody of the invention can be used alone, can also be with detectable marker (for diagnostic purpose), therapeutic agent, PK (egg White kinases) combination of modified part or any the above substance combines or coupling.

Detectable marker for diagnostic purposes includes but is not limited to: fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product Enzyme.

Can in conjunction with antibody of the present invention or coupling therapeutic agent include but is not limited to: 1. radionuclides (Koppe etc., 2005, (Cancer metastasis reviews) 24,539 is commented in metastasis of cancer)；2. biology poison (Chaudhary etc., 1989, Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gillies etc., 1992, National Academy of Sciences proceeding (PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. gold nano (Lapotko etc., 2005, cancer communicates (Cancer letters) 239,36 to particle/nanometer rods；Huang etc., 2006, it is Americanized Association magazine (Journal of the American Chemical Society) 128,2115)；5. virion (Peng Deng 2004, gene therapy (Gene therapy) 11,1234)；6. liposome (Mamot etc., 2005, cancer research (Cancer Research) 65,11631)；7. magnetic nanosphere；8. pro-drug activation enzymes are (for example, DT- diaphorase (DTD) or xenyl hydrolysis Enzyme-sample protein (BPHL))；10. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..

The present invention also provides a kind of compositions.In preference, the composition is pharmaceutical composition, it contains The antibody stated or its active fragment or its fusion protein and pharmaceutically acceptable carrier.In general, these substances can be prepared In nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is usually about 5-8, and preferably pH is about 6-8, although pH value can be varied with the property and illness to be treated for being formulated substance.Prepared pharmaceutical composition It can be administered by conventional route, including (but being not limited to): tumor is interior, peritonaeum is interior, intravenous or local administration.

Pharmaceutical composition of the invention can be directly used for combining 14-3-3 η protein molecular, thus can be used for preventing and treating Tumour.In addition, also can be used simultaneously other therapeutic agents.

Pharmaceutical composition of the invention contain safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more Good ground 0.1-80wt%) the above-mentioned monoclonal antibody (or its conjugate) and pharmaceutically acceptable carrier or tax of the present invention Shape agent.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.Drug system Agent should match with administration mode.Pharmaceutical composition of the invention can be made into injection form, such as with physiological saline or contain There are glucose and the aqueous solution of other adjuvants to be prepared by conventional method.Pharmaceutical composition such as injection, solution are preferably sterile Under the conditions of manufacture.The dosage of active constituent is therapeutically effective amount, such as about 5 mg/kg of about 1 microgram/kg body weight-daily Weight.In addition, polypeptide of the invention can be also used together with other therapeutic agents.

It is that the immune conjugate of safe and effective amount is applied to mammal when using pharmaceutical composition, the wherein safety Effective quantity typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg weight, preferably The ground dosage is about 1 mg/kg weight of about 10 micrograms/kg body weight-.Certainly, specific dosage is also contemplated that administration route, disease The factors such as people's health status, within the scope of these are all skilled practitioners technical ability.

The preparation of monoclonal antibody

Antibody of the invention can be prepared by various technologies known to those skilled in the art.For example, this hair Bright antigen can be applied to animal to induce the generation of monoclonal antibody.For monoclonal antibody, can using hybridoma technology come Preparation is (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), display technique of bacteriophage or the recombinant DNA method (U.S. can be used The patent No. 4,816,567) preparation.

Representative myeloma cell is effective integration, the stabilization Gao Shui for supporting by the antibody produced cell of selection antibody It shows no increases in output and gives birth to and to those of culture medium (HAT medium matrix) sensitivity myeloma cell, including myeloma cell strain, such as mouse The myeloma cell strain of class (is purchased from Salk including the myeloma cell strain derived from MOPC-21 and MPC-11 mouse tumor Institute Cell Distribution Center, Santiago, California, the U.S.) and SP-2, NZ0 or X63-Ag8-653 cell (is purchased from American Type Culture Collection, Luo Keweier, Maryland, the U.S.). Human myeloma and mouse-human heteromyeloma's cell strain be also described for generate human monoclonal antibodies [Kozbor, J.Immunol., (1984) 133:3001；Brodeur etc., the production technology and application (Monoclonal of monoclonal antibody Antibodies Production Techniques and Applications), 51-63 pages (Marcel Dekker, Inc., New York, 1987)].

Growth of Hybridoma Cell is analyzed in culture medium therein to detect and have the monoclonal of required specificity anti- The generation of body, e.g., by external binding analysis for example, Enzyme Linked Immunoadsorbent Assay (ELISA) or radiommunoassay (RIA). The position for expressing the cell of antibody can be detected with FACS.Then, hybridoma clone can be formed by limiting dilution procedures It is subcloned (subcloned), and grows (Goding, monoclonal antibody (Monoclonal by standard method Antibodies): principle and practicing (Principles and Practice), Academic Press (1986) 59-103 Page).The suitable culture medium used to reach this purpose includes, for example, DMEM or RPMI-1640 culture medium.In addition, Hybridoma can be grown as ascites tumor in animal body.

Pass through conventional immunoglobulin purification from culture medium, ascites or serum by the monoclonal antibody of subclone secretion Technique is suitably separated, these purifying process are for example, Protein A-agarose method (protein A-Sepharose), hydroxyl Base apatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Display technique of bacteriophage is a screening technique, and allogenic polypeptide or albumen are merged table with the capsid protein of bacteriophage It reaches, fusion protein is shown on the surface of virion, and the DNA for encoding the fusant is then located in virion, to make big It establishes between amount polypeptide and its DNA encoding sequence and directly contacts, make various target molecules (antibody, enzyme, cell surface receptor etc.) Polypeptide ligand Rapid identification is able to by elutriation.

In of the invention one preferred scheme, monoclonal antibody is using culture hybridoma method preparation.It takes miscellaneous The supernatant for handing over oncocyte culture, is purified through affinity column (Protein A/G-Sephrose).

In the preferred scheme of of the invention one, monoclonal antibody is using Balb/C mouse ascites production monoclonal antibody Method preparation.Hybridoma is inoculated into the mouse peritoneal of sensitization, ascites is extracted, through affinity column (Protein A/ G-Sephrose it) is purified.

In the preferred scheme of of the invention one, monoclonal antibody sets up eukaryotic expression system, wink using recombinant DNA method Turn HEK293 cell to express antibody, then by antibody of the secretion in culture medium through affinity column (Protein A/G- Sephrose it) is purified.

Method and sample

The present invention relates to for the method in tissue samples detection cancer.This method step approximately as: obtain tissue sample This；Detect the level of 14-3-3 η albumen in the sample.Sample used in the method for the present invention is serum sample or clinic The commonly used formalin of pathology is fixed, the histotomy of paraffin embedding.

Sample (sample) employed in the present invention includes tissue samples and biopsy specimen.Terminology used in the present invention is " living Inspection " should include the biopsy of all kinds well known by persons skilled in the art.Therefore biopsy used in the present invention may include example As tumour excision sample, pass through tissue samples prepared by the puncture of endoscopic procedures or organ or needle puncture biopsy.

Sample used in the present invention may include fixed or preservation tissue samples.Tissue samples can be protected for example There are can commercially obtain in the sample collection of standard, storage or conveying medium, such as known to those skilled in the art It saves medium (formalin, Cytyc " PreservCyt " or Tripath Imaging " Cytorich " etc.).It is suitable to save Medium may include it is one or more selected from alcohol, aldehyde, ketone, acid, metal ion or mercury, ether etc. for saving the mixed of cellular component Close object.Alcohol includes methanol, ethyl alcohol, (just or different) propyl alcohol, (just, exclusive or uncle) butanol or Gao Zhilian or unbranched alcohol.Aldehyde includes Formaldehyde, acetaldehyde, glutaraldehyde etc..Also the ketone of such as acetone can be used.The acid used in the sample medium of standard includes The inorganic acid of machine acid (acetic acid, trichloroacetic acid, salicylic acid and picric acid) or such as chromic acid.May include in the sample solution of standard Metal such as silver, copper, chromium, mercury, osmium and uranium.The salting liquid of uranyl acetate, two potassium chromates, ammonium sulfate etc. can be preservation and be situated between The component of matter.

Kit

The present invention also provides a kind of kits for containing only antibody of the invention (or its segment), at of the invention one In preference, the kit further includes container, operation instructions, buffer etc..

The present invention is further designed for the detection kit of detection 14-3-3 η albumen, which includes identification 14-3- The antibody of 3 η albumen detects required common reagent and buffer, such as various buffers, enzyme-linked tag secondary antibody, detection label, Detection substrate etc..The detection kit can be in-vitro diagnosis device.

Main advantages of the present invention are:

(1) antibody provided by the invention for 14-3-3 η albumen, specificity is high, and affinity is strong, and can largely make Standby, monoclonal antibody quality is easy to control.

(2) the antibody test sensitivity provided by the invention for 14-3-3 η albumen is high, can be used in serum sample The detection of destination protein.

(3) antibody provided by the invention is used in the method for detection 14-3-3 η albumen provided by the invention, stability is good.

(4) monoclonal antibody and detection method provided by the invention, suitable for the early diagnosis of associated cancer, and can be used In the prognosis of comment cancer.

Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002) Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.

The preparation of the anti-14-3-3 η protein monoclonal antibody of embodiment 1

1, mouse immune

Immunogene is recombination 14-3-3 η albumen (SEQ ID NO.:17), and 4 week old female Balb/C healthy mices is taken (to be purchased from This Leco Corp. of Shanghai), it is immunized.It is immune for the first time: 200 μ g (250 μ l) antigen and 250 μ l Freund's complete adjuvants to be mixed, note It penetrates in the subcutaneous multiple spot of Balb/c mouse and vola.It is immune with second of dosage that first time immunization interval carries out same method after three weeks.The Behind secondary immunity interval two weeks, third time is carried out with dosage with method and is immunized.After third time immunization interval two weeks, with method with dosage into Row the 4th time immune.

Mouse docking blood sampling is carried out before being immunized every time, is used as detection anti-using 14-3-3 η protein recombinant protein (5 μ g/ml) Original, ELISA method detection mice serum potency are greater than 1:10 to immune serum potency titre, and extracting spleen cell is melted when 000 It closes.

2, cell fusion and culture

By the myeloma cell SP2/0 (being purchased from ATCC) of well-grown logarithmic growth phase, and collect according to a conventional method The splenocyte fusion of immune mouse, splenocyte: SP2/0 ratio is 10:1, with 2 × 10⁴The density of a cell is by fused cell kind Enter 96 well culture plates, 37 DEG C, 5%CO₂It is fostered in constant incubator.After culture 14 days, ELISA detection screening 14-3-3 η is carried out Protein antibodies positive colony.

Positive hybridoma screening is carried out using indirect ELISA, antigen is immunogen protein mixed liquor, after wrapper sheet 4 DEG C overnight, 5% skimmed milk power is closed, 37 DEG C of effect 2h, after washing pats dry, hybridoma supematant is added and positive (P), negative (N) and sky is arranged White control, 37 DEG C of reaction 1h, washing pat dry after add 37 DEG C of sheep anti mouse-HRP secondary antibody (Sigma A2554) reaction 45~ 60min is developed the color using TMB, 2M H₂SO₄It terminates.Amplification is positive clone by continuous detection twice.

3, hybridoma is subcloned

It is subcloned using limiting dilution assay.Cell suspension is diluted to 60/ml, every hole adds 100 μ in 96 orifice plates L (about 6 cells/wells).2 rows are inoculated with, remaining cell suspension culture solution makees doubling dilution, inoculates 2 rows.It is repeated once.It sets 37 DEG C, 5%CO₂It is incubated in cell incubator.Every 2~3 days, 1/2 culture solution is replaced.After cultivating about 10d, single clone is selected The positive hole of growth carries out programmed screening and subclone.After being continuously subcloned three times, it is through ELISA method detection antibody positive rate It is determined as stable cell line when 100%.Altogether obtain seven plants combine immunogen protein stabilization monoclonal antibody strain 105A5H18, 0795、0823、0826、108A5H19、109A5H15、0931。

4, the preparation of monoclonal antibody

The Balb/c female mice of 6 week old is shifted to an earlier date 1 week and injects sensitization with atoleine in advance, then intraperitoneal injection hybridization Oncocyte, 1~2x10⁶Hybridoma/only, every kind of hybridoma injects 3.Ascites is extracted after 7-10 days.It is detected by ELISA Titer of ascites reaches purifying with Protein A/G affinity column chromatography method for 1:10,000 or more.

Specificity and the affinity detection of 2 monoclonal antibody of embodiment

ELISA detects monoclonal antibody cross reaction, and antigen selects the α/β of 14-3-3, γ, ε, σ, ξ, θ/τ and η seven respectively A hypotype recombinant protein, 2 μ g/ml wrapper sheets, 4 DEG C overnight, and after PBST washing pats dry, the closing of 5% skimmed milk power, 37 DEG C of effects are added 2h or 4 DEG C overnight, after PBST washing pats dry, 1 μ g/ml of monoclonal antibody is added, 37 DEG C of reaction 1h, PBST washings add goat-anti after patting dry 37 DEG C of reaction 45~60min, PBST washings of mouse-HRP secondary antibody (Sigma A2554) (1:10000) pat dry rear TMB colour developing and 2M H₂SO₄It terminates, is read at OD450nm.The result shows that: 105A5H18,108A5H11,108A5H19,109A5H15 monoclonal antibody can be special Property combination 14-3-3 η albumen, with other subtype proteins without apparent cross reaction.

Testing result shows that the affinity constant of 105A5H18 monoclonal antibody is 2.76E^-9, 108A5H11 monoclonal antibody Affinity constant be 8.76E^-9, the affinity constant of 108A5H19 monoclonal antibody is 1.68E^-10, 109A5H15 monoclonal is anti- The affinity constant of body is 5.23E^-9。

Antibody cloning sequencing is carried out after extracting the total serum IgE reverse transcription of 108A5H11 monoclonal antibody hybridoma cell:

Weight chain variabl area sequence information is as follows:

Light-chain variable sequence information is as follows:

The 14-3-3 η albumen in liver cancer tissue detects in 3 immunohistochemistry staining method of embodiment

To be primary antibody to liver cancer tissue and liver using 105A5H18,108A5H11,108A5H19,109A5H15 monoclonal antibody Normal tissue carries out immunohistochemical staining.After paraffin tissue sections are sequentially placed into dimethylbenzene, are impregnated in ethyl alcohol, it is put into In 0.01M sodium citrate buffer solution, boil；It is closed 20 minutes after washing with the TBST room temperature containing 10% calf serum；It is added Anti-Mouse IgG-HRP is added dropwise in test antibodies, antibody concentration 15ug/ml after incubation at room temperature, be incubated at room temperature 30 minutes and show Color observation.

108A5H11 monoclonal antibody has apparent dyeing (Fig. 1, Fig. 2) in liver cancer tissue cell, in normal liver cell Middle dyeing weaker (Fig. 3, Fig. 4), dyeing background is clean, distinguishes significantly, therefore, the experimental results showed that 108A5H11 monoclonal antibody Identification tumor cells of hepatocellular carcinoma that can be special.108A5H19 monoclonal antibody has apparent dyeing in liver cancer tissue cell, but It is that dyeing background is deeper, there are obvious background coloration, is difficult to distinguish background and tumour cell；And 105A5H18, 109A5H15 monoclonal antibody dye-free in liver cancer tissue.Therefore, the experimental results showed that only 108A5H11 monoclonal antibody energy Enough special identification tumor cells of hepatocellular carcinoma.

Embodiment 4WB detection

The 14-3-3 η albumen in tissue samples is detected using 108A5H11 monoclonal antibody in the present embodiment.

1, the preparation and concentration mensuration of albumen supernatant in organizing

1) tissue is cut into tiny fragment；

2) melt RIPA lysate, mix.The lysate for taking appropriate amount is using addition enzyme inhibitor three in first several minutes Multi pack.

3) organize the ratio that 100 μ l lysates are added that lysate is added according to every 20mg.

4) it is ground in refiner, until tissue block disappears in lysate.

5) sufficiently after cracking, 12000rpm is centrifuged 5 minutes, takes supernatant.

6) part supernatant protein BCA determination of protein concentration kit measurement protein concentration is taken, as follows:

1. A liquid and B liquid are configured to working solution with the ratio of 50:1 in kit specification；

2. taking the BSA standard items of 2000 μ g/ml, 2000 μ g/ml, 1500 μ g/ml, 1000 μ are diluted to PBS (pH 7.4) G/ml, 750 μ g/ml, 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 25 μ g/ml, 0 μ g/ml totally 9 concentration gradients；

3. taking 10 μ l of protein extract, 10 times are diluted with PBS；

4. the histone extracting solution after every 100 μ l protein standard substance of Guan Zhongjia or dilution, along with stating working solution 2ml, Absorbance is surveyed at 37 DEG C of incubations 30min, 562nm, records OD value；

5. drawing standard curve according to OD value and protein standard substance concentration, sample total protein concentration is calculated.

2, WB (Western blotting) detects 14-3-3 η

1) encapsulating

1. distilled water cleans encapsulating glass plate, dry vertically.

2. preparing 15% separation gel 10ml according to the above method: water 2.3ml, 30% acrylamide 5.0ml, 1.5MTris buffering Encapsulating immediately after 4 μ l of liquid 2.5ml, 10%SDS 0.1ml, urea 6.4g, TEMED, 10% (w/v) AP100 μ l are mixed fills extremely 2~3mm of stripping fork lower edge (marking in advance) is stored at room temperature 45 with isopropanol closing liquid level to remove bubble removing and completely cut off air Minute polymerize completely to glue.

3. after glue to be separated polymerize completely, the deionized water inclined at the top of it is blotted water with filter paper.

4. preparing 5% concentration glue 6ml: water 4ml, 30% acrylamide 1ml, 1.0M Tris buffer 1ml, 10%SDS 80ul, 10%AP 60ul, TEMED 8ul mix encapsulating immediately, fill to top, and be inserted perpendicularly into Teflon stripping fork, room temperature is quiet It sets 20 minutes and polymerize to glue.

5. extracting comb after glue polymerize completely, gel is placed in electrophoresis tank, electrophoretic buffer is added, uses running buffer Liquid rinses loading hole to remove bubble removing.

2) electrophoresis

1. taking each processing histone extracting solution, protein concentration and the mixing of isometric 5 × sample-loading buffer are adjusted, as Sample solution.

2. sample solution is boiled 5min in 100 DEG C of boiling water, makes albuminous degeneration, be quenched on ice, 3000 turns/min centrifugation 1min。

3. every hole adds sample liquid 10ul, a hole is stayed to add the Marker of 10 μ l pre-dyed.Electrophoretic buffer is filled it up with, slot cover is covered, Powering on, first uses 70v constant pressure electrophoresis, about 30min uses 90v constant pressure electrophoresis instead after indicator bromine Finland enters separation gel, when Stop electrophoresis when its electrophoretic band of 25Kd Marker is to separation gel middle position, close power supply, takes out offset plate.

3) albumen and immune detection are transferred

1. pvdf membrane is impregnated 15s in methyl alcohol in advance, then uses ddH before electrophoresis closes to an end₂O rinses 2min, leaching It steeps and starts subsequent operation after 5min in transfering buffering liquid.

2. prizing glue in water, repairs glue to be soaked in transfering buffering liquid after glue and balance 15min.

3. pressing black flour (cathode) → sponge → filter paper → glue → pvdf membrane (0.2um) → filter paper → sponge → red face (anode) Sequence prepare transferring film " sandwich ", every layer complete after first drive bubble away and repave another layer.Sanming City is prepared in transfering buffering liquid Control the generation of avoidable bubble.

4. connecting positive and negative anodes, transfer box is put into electroporation to the direction of anode by film, transferring film buffer is added.

5. electroporation is placed in ice water, 200mA constant current transferring film 70min.

6. after transferring film, quickly removing pvdf membrane, it is put into 5%BSA room temperature closing 2h.

7. film is taken out, in washing film 5min × 3 time with TBST on shaking table.

8. be added in incubation bags the diluted anti-14-3-3 η antibody of TBST (105A5H18,108A5H11,108A5H19, 109A5H15 monoclonal antibody), 4 DEG C of overnight incubations.

9. TBST washes film 5min × 3 time, the sheep anti mouse secondary antibody of horseradish peroxidase (HRP) label is incubated at room temperature 2h.

10. TBST washes film 10min × 3 time.Film reacts 2min in chemiluminescence detection reagent (reagent A: reagent B=1:1), Film is taken out, extra liquid is got rid of, pvdf membrane is wrapped with preservative film, uses X light reaching the film, development, fixing in darkroom.

Experimental result is as shown in Figure 5 and Figure 6, and each swimming lane is respectively 108A5H11 (swimming lane 1,2,3), 105A5H18 in Fig. 5 Immunoblot results (three repetitions of the respectively same hepatocarcinoma patient operation on liver sample of (swimming lane 4,5,6) monoclonal antibody Sample, albumen applied sample amount 40ug, antibody dilution 1:2500), wherein 1-3 swimming lane has obvious clear band, 4-6 at 28kd Purpose band can be observed at 28kd, but purpose band is unintelligible.

Each swimming lane is respectively 108A5H19 (swimming lane 1,2,3), 109A5H15 (swimming lane 4,5,6) monoclonal antibody in Fig. 6 Immunoblot results (three repeated samples of the respectively same hepatocarcinoma patient operation on liver sample, albumen applied sample amount 40ug, antibody Dilution 1:2500), wherein 1-6 swimming lane has obvious clear band at 28kd, but antibody miscellaneous band is more, and specificity is not strong.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

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Shanghai is stood upright Biotechnology Co., Ltd

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Claims (10)

1. a kind of anti-14-3-3 η protein monoclonal antibody, which is characterized in that the antibody includes

(1) heavy chain variable region；And/or

(2) light chain variable region；

Wherein, the heavy chain variable region includes following three complementary determining region CDR:

CDR1 shown in SEQ ID NO.:1,

CDR2 shown in SEQ ID NO.:2, and

CDR3 shown in SEQ ID NO.:3；

The light chain variable region includes following three complementary determining region CDR:

CDR1' shown in SEQ ID NO.:5,

CDR2' shown in SEQ ID NO.:6, and

CDR3' shown in SEQ ID NO.:7.

2. a kind of polynucleotides, which is characterized in that it encodes antibody as described in claim 1.

3. a kind of carrier, which is characterized in that it contains polynucleotides as claimed in claim 2.

4. a kind of genetically engineered host cell, which is characterized in that it contains in carrier or genome as claimed in claim 3 It is integrated with polynucleotides as claimed in claim 2.

5. a kind of immune conjugate, which is characterized in that the immune conjugate contains:

(a) antibody as described in claim 1；With

(b) coupling moiety selected from the group below: detectable marker, drug, toxin, cell factor, radionuclide or enzyme.

6. a kind of pharmaceutical composition, which is characterized in that it contains:

(i) antibody, immune conjugate as claimed in claim 5 as described in claim 1；And

(ii) pharmaceutically acceptable carrier.

7. the purposes of antibody as described in claim 1, immune conjugate as claimed in claim 5 is used to prepare medicament, examination Agent, detection plate or kit；

The reagent, detection plate or kit are used for:

(1) 14-3-3 η albumen in test sample；And/or

(2) endogenic 14-3-3 η albumen in tumour cell is detected；And/or

(3) tumour cell of detection expression 14-3-3 η albumen；

The medicament is used to treat or prevent the tumour of expression 14-3-3 η albumen.

8. a kind of method of 14-3-3 η albumen in test sample, which is characterized in that the method includes the steps:

(1) sample is contacted with antibody described in claim 1；

(2) it detects whether to form antigen-antibody complex, wherein forming compound means that there are 14-3-3 η albumen in sample.

9. a kind of kit, which is characterized in that include: in the kit

The first container contains antibody described in claim 1 in the first container.

10. a kind of preparation method of preparation and reorganization polypeptide, which is characterized in that this method includes:

(a) under conditions suitable for the expression, host cell as claimed in claim 4 is cultivated；

(b) recombinant polypeptide is isolated from culture, the recombinant polypeptide is antibody described in claim 1.