CN101712960A - Mac-2BP tumor antigen gene, protein, antibody and application thereof - Google Patents
Mac-2BP tumor antigen gene, protein, antibody and application thereof Download PDFInfo
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Abstract
The invention relates to a tumor antigen gene Mac-2BP containing fragments of SEQ ID NO:1 and SEQ ID NO:2, tumor cell secretory glycoprotein Mac-2BP coded by the gene, an antibody of the protein and application of the gene, the protein and the antibody in screening and/or preparing a medicament for diagnosing and/or treating tumors. The antigen protein Mac-2BP used as new tumor treating target protein is characterized by the high efficiency and the specificity of the antigen protein Mac-2BP used as the target protein in tumor diagnosis and/or treatment.
Description
Technical field
Albumen, this proteic antibody and the described gene, albumen, the antibody that the present invention relates to new tumor antigen gene, this genes encoding are used for diagnosing and/or treating the purposes of the medicine of tumour in screening and/or preparation.The invention still further relates to the test kit that is used for such use.
Background technology
Tumour is a normal cell that growing in the human body or sophisticated, under different factor long terms, hyperplasia occurs and becomes unusual differentiation and the true tumor of formation.In recent years, the sickness rate of tumour obviously increases, because the minimizing of transmissible disease, cardiovascular and cerebrovascular disease and tumour rise to first and second disease.
At present, the tumor treatment method mainly contains surgical operation, chemotherapy, radiotherapy, biotherapy and methods such as thermotherapy, optical dynamic therapy.Biotherapy is meant by biological response modifier (BRM) transfer host's natural defense mechanism or gives some material of body and obtain antineoplastic effect, reaches treatment tumour purpose.Biological response modifier mainly contains cytokine: as interleukin-(IL), Interferon, rabbit (IFN), tumour necrosis factor (TNF) etc.; Antineoplastic somatocyte and complementary hemopoietic stem cell: as LAK cell, til cell, TAK cell etc.; Antibody: comprise all kinds of anti-tumor monoclonal antibodies, anti-cell surface markers antibody etc.; Gene therapy; Tumor vaccine; Anti-angiogenic existence agent; Cell differentiation inducer or the like.
Gene therapy be exactly a kind of be the Human genome transfer techniques of purpose with prevention and treatment disease, be to treat based on the biomedicine of the genetic material that changes the people.Along with to the deepening continuously of tumor development Study on Molecular Mechanism, genetic treatment of tumor has become the new focus of current clinical tumor research.
Summary of the invention
The present inventor studies tumor antigen gene, has found the gene that works in tumour generation, evolution.The contriver carries out RNAi by implementation sequence and disturbs this expression of gene of proof reduction can suppress tumor growth and external invasion and attack behavior.The contriver proves that further the polypeptide of this genes encoding expresses in tumour cell, use the antibody of this polypeptide and can treat tumour.
Therefore one aspect of the present invention provides a kind of isolating polynucleotide, its have the polynucleotide passage of SEQ ID NO:1 and SEQ ID NO:2 or the fragment that can under rigorous condition, hybridize with the polynucleotide passage of SEQ ID NO:1 and SEQ ID NO:2 or with the fragment of above-mentioned fragment complementation.Described polynucleotide can be DNA or RNA.
The present invention relates to coding human tumor antigen gene M ac-2BP on the other hand, this gene contain the polynucleotide passage of SEQ ID NO:1 and SEQ ID NO:2 or the fragment that can under rigorous condition, hybridize with the polynucleotide passage of SEQ ID NO:1 and SEQ ID NO:2 or with the fragment of above-mentioned fragment complementation.Described polynucleotide can be DNA or RNA.
Another aspect of the invention relates to a peptide species, and it comprises with human tumor antigen gene M ac-2BP amino acid sequence coded having at least 50% identity and have identical or the aminoacid sequence of high biological activity more with the polypeptide with this aminoacid sequence.Preferably, this polypeptide has the aminoacid sequence shown in SEQ IDNO:8.
Another aspect of the invention relates to a kind of tumor marker, and described tumor marker is above-mentioned polypeptide.
Another aspect of the invention relates to the antibody that obtains as antigen with aforementioned polypeptides.This antibody can be monoclonal antibody.Preferably, described antibody is Mac-2BP monoclonal antibody 13H3.
Another aspect of the invention relates to above-mentioned polynucleotide and/or above-mentioned polypeptide and/or the above-mentioned purposes of antibody in the preparation anti-tumor drug.
Another aspect of the invention relates to above-mentioned polynucleotide and/or above-mentioned polypeptide and/or the above-mentioned purposes of antibody in preparation tumor marker preparation.
Tumour of the present invention can be lung cancer.
The invention still further relates to a kind of test kit, it comprises above-mentioned polynucleotide and/or above-mentioned polypeptide and/or above-mentioned antibody, and specification sheets.
Description of drawings
Figure 1A: the MALDI-TOF-PMF figure of monoclonal antibody 13H3 antigen protein.
Figure 1B: mass spectrum is identified 13H3 antigen protein Mac-2BP aminoacid sequence, and the sequence in the bracket is the aminoacid sequence of mass spectroscopy.
Fig. 2 A:siRNA is instantaneous to strike the influence of falling tumor cell proliferation.
Fig. 2 B:Mac-2BP is stable to strike the influence of falling tumor cell proliferation.
Instantaneous the striking of Fig. 3 A:siRNA fallen tumour cell and the adherent influence of matrix.
Stable the striking of Fig. 3 B:Mac-2BP fallen tumour cell and the adherent influence of matrix.
Fig. 4 A:Mac-2BP is instantaneous to strike the influence of falling tumor cell invasion.
Fig. 4 B:Mac-2BP is stable to strike the influence of falling tumor cell invasion.
Fig. 5: the fixed cell immunofluorescence detects the expression of Mac2-BP at lung carcinoma cell.
Fig. 6: cancerous lung tissue and the pairing immunohistochemical methods figure of cancer beside organism (* 100 times), 30# wherein, well-differentiated adenocarcinoma; 39#, middle differentiation phosphorus cancer; 92#, poorly differentiated adenocarcinoma; T: cancerous tissue; N: healthy tissues.
Fig. 7: Mac-2BP is steady to be changeed to strike and falls the cell experimentation on animals, Fig. 7 A wherein, growth of xenografted curve; Fig. 7 B, transplanted tumor weight; Fig. 7 C, transplanted tumor in nude mice figure; Fig. 7 D, inhibiting rate: p＜0.05.
Fig. 8: the experimentation on animals of Mac-2BP antibody interior therapeutic tumour, Fig. 8 A wherein, growth of xenografted curve; Fig. 8 B, the lung cancer transplanted tumor; Fig. 8 C, the transplanted tumor knurl is heavy; Fig. 8 D, 13H3 Antybody therapy inhibiting rate: P＜0.05.
Fig. 9: fluidic cell detects Mac-2BP and strikes and fall the back cell cycle, Fig. 9 A wherein, A549; Fig. 9 B, Ncontro1; Fig. 9 C, m2bpsiR1; Fig. 9 D, m2bpsiR2.
Figure 10: the Mac-2BP shRNA interference plasmid collection of illustrative plates (the SiRNA sequence is inserted between EcoRI and BamHI multiple clone site) of structure.
Embodiment
Except as otherwise noted, the present invention uses the routine techniques of immunology, molecular biology, microbiology, cytobiology and recombinant DNA, Sambrook for example, Fritsch and Maniatis, MOLECULAR CLONING:A LABORATORY MANUAL, the 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (people such as F.M.Ausubel compiles, (1987)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2:A PRACTICAL APPROACH (M.J.MacPherson, B.D.Hames and G.R.Taylor compile. and (1995)), Harlow and Lane compile, (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMALCELL CULTURE (R.I.Freshney compiles (1987)).
The invention provides isolating polynucleotide, its have the polynucleotide passage of SEQ ID NO:1 and SEQ ID NO:2 or the fragment that can under rigorous condition, hybridize with the polynucleotide passage of SEQ ID NO:1 and SEQ ID NO:2 or with the fragment of above-mentioned fragment complementation.Described polynucleotide can be DNA or RNA.
" hybridize under the rigorous condition " that nucleic acid hybrids is stable under this condition.The stability of crossbred is reflected in the melting temperature(Tm) (Tm) of crossbred.Usually, the stability of crossbred is the function of Na ion concentration and temperature.Typically, described hybridization carries out under rigorous condition." rigorous condition " is meant some conditions, this conditions permit herbicide-tolerant polynucleotide and the identity (identity) that has with this herbicide-tolerant polynucleotide about 60%, preferred about 75% identity, the complementary nucleic acid combination of more preferably about 85% identity; The identity that has greater than about 90% with herbicide-tolerant polynucleotide is particularly preferred.Rigorous condition is to be equivalent under 42 ℃, hybridization under 50% methane amide, 5 * Denhart ' s solution, 5 * SSPE, 0.2%SDS, and then under 65 ℃, at 0.2 * SSPE, 0.2%SDS is the condition of washing down.
The present invention also provides a peptide species, and it comprises with human tumor antigen gene M ac-2BP amino acid sequence coded having at least 50% identity and have identical or the aminoacid sequence of high biological activity more with the polypeptide with this aminoacid sequence.Preferably, this polypeptide has the aminoacid sequence shown in SEQ IDNO:8.
Among the present invention " polypeptide " and " protein " but the use of general mutual alternative is meant single polypeptide chain, it may be modified by adding non-amino acid group, also may not modify." protein " among the present invention and " polypeptide " also comprise varient, mutant, modifier, stand-in and/or the derivative of described polypeptide.Amino acid sequence of polypeptide mutant of the present invention can prepare by the Nucleotide of introducing suitable change in nucleic acid of the present invention, or by external synthetic required polypeptide.Described mutant is included in deletion, insertion or the replacement of carrying out residue in the aminoacid sequence, as long as final polypeptide product has required character.
The % identity (identity) of polypeptide is by GAP (Needleman and Wunsch, 1970) analyzing (GCG program) determines, its breach produces point penalty (gap creation penalty)=5, and breach extends point penalty (gap extension penalty)=0.3.At least 15 amino acid longs of search sequence, GAP are analyzed at least 15 amino acid whose zone comparison two sequences.Preferably, at least 50 amino acid longs of search sequence, GAP are analyzed at least 50 amino acid whose zone comparison two sequences.More preferably, GAP analyzes the total length of two sequences is compared.
" biological activity " used herein fragment is meant the part of polypeptide of the present invention, and it has kept the activity of determining of this full-length polypeptide.Bioactive fragment can be random length, as long as they have kept described definite activity.
The present invention also provides polypeptide of the present invention or its segmental mono-clonal or polyclonal antibody.Preferably, described antibody is Mac-2BP monoclonal antibody 13H3.Polyclonal antibody uses the selected Mammals (mouse, rabbit, goat, horse etc.) of immunogenic polypeptide immunity of the present invention if desired.Collect and handle the serum of immune animal according to known program.For making described antibody, the present invention also provides polypeptide of the present invention or its fragment to be connected to other polypeptide as the immunogen in the animal as haptens.
Those skilled in the art also can easily produce the monoclonal antibody of anti-polypeptide of the present invention.The general method of making monoclonal antibody by hybridoma is that people know.The clone that produces immortal antibody can produce by cytogamy, also can transform bone-marrow-derived lymphocyte by other technologies such as carcinogenic DNA, or Epstein-Barr virus (Epstein-Barr virus) transfection.Preferably, antibody of the present invention has detectable mark.
The present invention also provides above-mentioned polynucleotide and/or above-mentioned polypeptide and/or the above-mentioned purposes of antibody in the preparation anti-tumor drug.Described tumour can be lung cancer.
The present invention also provides above-mentioned polynucleotide and/or above-mentioned polypeptide and/or the above-mentioned purposes of antibody in preparation tumor marker preparation.Tumor marker preparation according to the present invention can be used to identify related neoplasms, described tumor marker preparation can also comprise the preparation known in the art that this polypeptide is detected for having the polypeptide of human tumor antigen gene M ac-2BP amino acid sequence coded.Described tumour can be lung cancer.
The present invention also provides a kind of tumor marker, and described tumor marker can be above-mentioned polypeptide.Described tumour can be lung cancer.
The present invention also provides a kind of test kit that is used for oncotherapy or tumor marker, and it comprises above-mentioned polynucleotide and/or above-mentioned polypeptide and/or above-mentioned antibody, and specification sheets.Described tumour can be lung cancer.
Anti-tumor drug produced according to the present invention, tumor marker preparation and test kit relate to aspects such as goal gene, carrier and recipient cell, described goal gene can be polynucleotide of the present invention, its have the polynucleotide passage of SEQ ID NO:1 and SEQ ID NO:2 or the fragment that can under rigorous condition, hybridize with the polynucleotide passage of SEQ IDNO:1 and SEQ ID NO:2 or with the fragment of above-mentioned fragment complementation.Described polynucleotide can be DNA or RNA.Described goal gene also can be the human tumor antigen gene M ac-2BP of total length.
Polynucleotide in anti-tumor drug produced according to the present invention, tumor marker preparation and the test kit can import target cell as goal gene with gene transfer technique by methods known in the art, this gene transfer technique can shift or directly these polynucleotide is injected in the body for ex vivo, these polynucleotide can be injected merely, also can inject with subsidiary such as liposome.
Can comprise a kind of recombinant vectors in anti-tumor drug produced according to the present invention, tumor marker preparation and the test kit, this recombinant vectors comprises at least a separation polynucleotide of the present invention that are inserted in any carrier that polynucleotide of the present invention can be delivered to host cell.One type recombinant vectors comprises the exercisable polynucleotide molecule of the present invention that is connected to expression vector.Exercisable connection is meant that the mode that polynucleotide molecule this molecule when being transformed in the host cell can be expressed is inserted in the expression vector.Expression vector used herein is meant can transformed host cell and can effective expression specify the DNA or the RNA carrier of polynucleotide molecule.
Can comprise a kind of reconstitution cell in anti-tumor drug produced according to the present invention, tumor marker preparation and the test kit, this reconstitution cell comprises one or more polynucleotide molecule transformed host cells of the present invention, or its daughter cell.Can polynucleotide molecule be transformed by any method that polynucleotide molecule can be inserted in the cell and enter cell.Transformation technology includes but not limited to that transfection, electroporation, microinjection, lipofection, absorption and protoplasma merge.
Anti-tumor drug produced according to the present invention is meant the antibody that comprises active substance such as anti-Mac-2BP polypeptide and the combination of pharmaceutical carrier or other material, and other materials can be assistant agent, thinner, wedding agent, stablizer, buffer reagent, salt, lipophilic solvent, sanitas, assistant agent etc.Pharmaceutical carrier can comprise pharmaceutical excipient and additive, protein, peptide, amino acid, lipid and carbohydrate, and it can exist alone or in combination, comprises the 1-99.99% weight or meausurement of form alone or in combination.
" pharmaceutical carrier " comprises the pharmaceutical carrier of any standard among the present invention, such as phosphate buffered salt solution, water and milk sap, such as oil/water or water/oil emulsion oil emulsion and various types of wetting agent.Comprise composition and medicine with SEQ ID NO:1 and SEQ ID NO:2 polynucleotide and/or Mac-2BP polypeptide and/or Mac-2BP monoclonal antibody 13H3 produced according to the present invention and/or that use can comprise stablizer and sanitas and any above-mentioned carrier.The example of carrier, stablizer and assistant agent is seen Martin REMINGTON ' S PHARM.SCI, the 15th edition (Mack Publ.Co., Easton (1975) and Williams ﹠amp; Williams, (1995) and " PHYSICIAN ' S DESK REFERENCE ", the 52nd edition, Medical Economics, Montvale, N.J. (1998).
Anti-tumor drug produced according to the present invention can give the patient with the various formulations that are suitable for selected route of administration, selected route of administration is oral administration or passes through intravenously, intramuscular, part or subcutaneous route parenterai administration, for example by perfusion or injection intravenously or intraperitoneal administration.
In some cases, anti-tumor drug produced according to the present invention also can with for example inert diluent or the administration of assimilable edible carrier Combined with Oral of pharmaceutical excipient.These pharmaceutical compositions and preparation should contain at least 0.1% active compound.Certainly, the per-cent of said composition and reagent can change to some extent, for example can between set unit dosage form weight about 2 to about 60% to about 90% between.
The treatment significant quantity of medicine of the present invention needs to change with patient and disease or physiological problem to be treated.For example, the therapeutic dose between 30 to 112,000 μ g/kg body weight is effective to intravenous administration.This medicine can unit dosage form form administration; For example contain 1 to 1000mg, preferably 10 to 750mg, more preferably is 20 to 500mg polypeptide (antibody)/unit dosage forms.Polynucleotide/polypeptide of the present invention/antibody drug can pass through intraperitoneal injection, for example by abdominal injection antibody drug of the present invention, its dosage can be for 1-100mg/kg/ time, preferably can be for 10-80mg/kg/ time, more preferably can be for 12.5-50mg/kg/ time, its number of times can as 4-7 time, be treated several weeks even longer time continuously for weekly for several times.Be familiar with as those skilled in the art, this dosage can change to some extent according to medication.Treatment also changes with route of administration to some extent with required content of medicines of the present invention, but also changes to some extent with institute's sanatory character and patient age and situation, and finally physician or the clinicist by nursing determines.
Embodiment
The authentic monoclonal antibody 13H3 that uses the preparation of inventor herein's Immune Fusion carries out the immunoaffinity purification experiment and (sees Hu Hai, Ran Yu-Liang, Chen Li-Zhao, Yu Long, Sun Li-Xing, Yang Zhi-Hua.Identification of functional antibodies of lungcancer by screening of functional antibody library.Cancer Prevention (Chinese) .2007; 34 (6): 395-398.), separation and purification 13H3 antigen protein from the tumor tissues total protein that the RIPA method is extracted, utilize the purified 13H3 antigen protein (Figure 1A) of MALDI-TOF-PMF technical evaluation, the Mascot database analysis shows that the 13H3 antigen protein is people galectin-3binding protein (having another name called Mac-2BP).Figure 1B is a Mac-2BP Argine Monohydrochloride sequence, and the sequence in the bracket is the aminoacid sequence of mass spectroscopy.
Use the RNAi perturbation technique, Mac-2BP can grow fast at external promotion lung carcinoma cell from gene level proof antigen gene.According to GeneBank database analysis Mac-2BP gene order, design the siRNA sequence (the sharp rich biological company limited in Guangzhou) of two target Mac-2BP genes:
Sequence name: m2bpsiR1
Target sequence SEQ ID NO:1:CCATCAGCGTGAATGTGCA
Positive-sense strand (5 '-3 ') SEQ ID NO:3:CCAUCAGCGUGAAUGUGCA dTdT
Antisense strand (3 '-5 ') SEQ ID NO:4:dTdT GGUAGUCGCACUUACACGU;
Sequence name: m2bpsiR2
Target sequence SEQ ID NO:2:GGACCTGTATGCCTATGCA
Positive-sense strand (5 '-3 ') SEQ ID NO:5:GGACCUGUAUGCCUAUGCA dTdT
Antisense strand (3 '-5 ') SEQ ID NO:6:dTdT CCUGGACAUACGGAUACGU.
The operation instructions that provides according to sharp rich company is carried out siRNA transient transfection clpp gene and is fallen experiment, transient transfection strike fall 48 hours after digestion tumour cell A549 and accurate counting, the adjustment cell density is 25000/ml, and 200 μ l/ holes are inoculated in the 96 porocyte culture plates, inoculates 5 96 orifice plates.Got plate in per 24 hours and measure, every hole adds 20 μ l CCK-8 reagent.CO
2Incubator continues to cultivate 1 hour, measures the CCK-8 light absorption value with the microplate reader dual wavelength.Draw the growth of tumour cell curve (Fig. 2 A) of parent's group, Ncontrol group and siRNA experimental group, Mac-2BPsiRNA is to A549 cell proliferation inhibition rate=(Ncontrol cell mean-siRNA experimental group mean value)/Ncontrol cell mean * 100%.
Make up two shRNA interference plasmids that are used for the stable transfection tumour cell.With above-mentioned 2 siRNA sequences serves as that the basis adds EcoRI restriction enzyme site and terminal protection sequence at its 5 ' end, adds BamHI restriction enzyme site and terminal protection sequence at its 3 ' end, and it is as follows to form new sequence:
Sequence name: m2bpsiR1 '
Target sequence SEQ ID NO:1:CCATCAGCGTGAATGTGCA
Positive-sense strand (5 '-3 ') SEQ ID NO:10:5 '-GATCCGCCATCAGCGTGAATGTGCACAAGAGACATGCACATTCACGCTGATGGTTT TTTGGAAA-3 '
Antisense strand (3 '-5 ') SEQ ID NO:11:3 '-CTAGGCGGTAGTCGCACTTACACGTGTTCTCTGTACGTGTAAGTGCGACTACCAAA AAACCTTT-5 '
Sequence name: m2bpsiR2 '
Target sequence SEQ ID NO:2:GGACCTGTATGCCTATGCA
Positive-sense strand (5 '-3 ') SEQ ID NO:12:5 '-GATCCGGGACCUGUAUGCCUAUGCACAAGAGACATGCATAGGCATACAGGTCCTTT TTTGGAAA-3 '
Antisense strand (3 '-5 ') SEQ ID NO:13:3 '-CTAGGCCCTGGACATACGGATACGTGTTCTCTGTACGTATCCGTATGTCCAGGAAA AAACCTTT-5 '.
Dissolve SiRNA sequence and complementary single stranded oligonucleotide thereof respectively to final concentration 1mg/ml.Get above-mentioned nucleotide acid fragment solution 2 μ l, add 46 μ l Annealing buffer (pH 7.4 for 100mMKAc, 30mM HEPES-KOH), fully mixing.With 90 ℃ of sex change of mixed solution 3 minutes, slowly be cooled to 37 ℃ through 1 hour then, make above-mentioned complementary single stranded oligonucleotide annealing form double chain oligonucleotide, carry out double digestion with DNA restriction enzyme EcoRI and BamHI.Simultaneously the pSilencer3.1-H1neo plasmid vector is carried out double digestion and recovery with DNA restriction enzyme EcoRI and BamHI.Double chain oligonucleotide chain behind the double digestion is connected with pSilencer3.1-H1neo plasmid vector behind the double digestion, finally prepares and make up two shRNA interference plasmids that are used for the stable transfection tumour cell.Plasmid promptly is used for this experiment after order-checking is accredited as correctly.The Mac-2BP shRNA interference plasmid collection of illustrative plates that makes up is seen accompanying drawing 10 (the SiRNA sequence is inserted between EcoRI and BamHI multiple clone site).
The Mac-2BP shRNA interference plasmid that utilize to make up (the lucky triumphant biological company limited in Shanghai) transfection human tumor cells A549, and obtain to stablize by the G418 screening and strike two cell strain C13 and the C36 that falls the Mac-2BP gene.With parental cell A549, unloaded control cells Vector and two steady change to strike fall after cell C13 and C36 be cultured to logarithmic phase, peptic cell is also accurately counted, the adjustment cell density is 25000/ml, and 200 μ l/ holes are inoculated in the 96 porocyte culture plates, inoculates 5 96 orifice plates.Got plate in per 24 hours and measure, every hole adds 20 μ l CCK-8 reagent.CO
2Incubator continues to cultivate 1 hour, measures the CCK-8 light absorption value with the microplate reader dual wavelength.Draw growth of tumour cell curve (Fig. 2 B).
Transient transfection and the steady proliferate curve that changes cell show that after the Mac-2BP clpp gene fell, tumour cell reduced by 38.5% and 50% respectively in the proliferate ability of vitro culture.Illustrate that antigen gene Mac-2BP can promote tumour cell in external growing multiplication function, reduce Mac-2BP genetic expression and can obviously reduce growth of tumour cell speed.
Embodiment 3 antigen gene Mac-2BP promote the adhesion of tumour cell and extracellular matrix
Mac-2BP is a kind of tumor cell secretion adhesion molecule, in the external adhesive capacity that can promote tumour cell and extracellular matrix, in the present embodiment, utilizes the RNAi perturbation technique to fall and steady transgenosis is struck and fallen cell two aspects and carry out from the transient transfection clpp gene.The digestion tumour cell is also accurately counted, the adjustment cell density is 4 * 105/ml, wrap respectively by 96 orifice plates (collagen I 20 μ g/ml with different substrates the day before yesterday in experiment, matrigel 30 μ g/ml, FN 10 μ g/ml), 2%BSA seals non-specific site, before adhering to experiment with the PBS inferior plank of giving a baby a bath on the third day after its birth.Every hole adds 100 μ l cell suspensions, CO
237 ℃ of 5%CO of incubator
2Cultivated 1 hour under the condition, siphon away all liquid in the hole, with serum-free medium vibration washing 3-5 time, exhaust all liquid in the hole then, every hole adds 100 μ l serum-free mediums and 10 μ l CCK-8 reagent.CO
2Incubator continues to cultivate 2 hours, measures the CCK-8 light absorption value with the microplate reader dual wavelength.The light absorption value of compare group and experimental group, cell adhesion inhibiting rate=(control group mean value-empirical average value)/control group mean value * 100%.
Transient gene strikes the attached experimental result of viscosity reduction (Fig. 3 A) and shows, the adhesive capacity of lung cell A549 and three kinds of extracellular matrix collagen I (collagen I), matrigel (matrigel) and FN has descended 40%, 50% and 35% respectively behind the Mac-2BP gene knockout; And steady transgenosis is struck the adhesion experimental result (Fig. 3 B) of falling cell and is shown, steady change to strike cell falls and collagen I (collagen I), matrigel (matrigel) and three kinds of matrix adhesive capacities of FN reduce respectively more than 50%, 38% and 37%.Clpp gene falls and experimental results show that Mac-2BP antigen can promote the adhesion function of tumour cell and extracellular matrix.
Embodiment 4 antigen gene Mac-2BP promote the external invasion and attack behavior of tumour cell
In the present embodiment, utilize the RNAi perturbation technique to fall with steady transgenosis and strike the influence of falling cell two aspects evaluation Mac-2BP gene pairs tumor cell invasion function from the transient transfection clpp gene.The digestion tumour cell is also accurately counted, and it is 4 * 10 that serum-free medium is adjusted the A549 cell density
5/ ml tests the freeze thawing Matrigel that spends the night on ice the day before yesterday, adds the Matrigel of 60 μ l 1mg/ml on the Transwell upper strata in the chamber, and 37 ℃ of bags were by 1 hour, and PBS washs 2 times.The good every hole 100ul of tumour cell adds upper strata Transwell cell with digesting counting, and washing is generally 40,000/hole to cell through serum-free, and Transwell lower floor cell adds the complete cell culture fluid of 600 μ l.37 ℃ of 5%CO2 continue to cultivate 24 hours in the CO2 incubator, scrape off cell in the Transwell cell of upper strata with cotton swab, and 50% methyl alcohol/50% acetone fixed is washed 3 times with PBS after 15 minutes again, the DAPI mounting, and the fluorescence microscopy is taken pictures, the IPP6.0 counting cells.The invasion and attack cell count of compare group and experimental group, cell invasion inhibiting rate=(control group mean value-empirical average value)/control group mean value * 100%.
Instantaneous striking fallen invasion and attack experimental result (Fig. 4 A) and steady commentaries on classics and struck and fall cell invasion experimental result (Fig. 4 B) and show that after the Mac-2BP clpp gene fell, the invasion by tumor cells cell count had reduced by 29% and 32.9% respectively.Proof Mac-2BP can promote the invasion by tumor cells function.
Embodiment 5 cellular immunofluorescences detect the expression of Mac-2BP in multiple lung carcinoma cell
Use the inventor herein and merge the Mac-2BP monoclonal antibody 13H3 (IgM hypotype) of preparation, detect the expression of Mac-2BP at multiple lung carcinoma cell by the cellular immunofluorescence method.4000/hole of inoculated tumour cell in 96 orifice plates, CO
237 ℃ of 5%CO of incubator
2Cultivated 48 hours under the condition.This moment, cell was in logarithmic phase, removed nutrient solution, PBS washed cell 1 time.For the viable cell immunofluorescence, cell need not fixing and penetrating processing, the direct and antibody incubation of cell.For the fixed cell immunofluorescence, cell is fixed earlier, promptly adds cell fixation liquid (50% methyl alcohol/50% acetone, fresh preparation), get rid of stationary liquid behind the incubated at room 10min gently, with PBS (PBST) washing that contains 0.05%Tween-20 5 times, each 5min; Add an anti-solution (13H antibody is 10 μ g/ml, and mouse-anti α-tublin is 1: 1000), incubated at room 1h, PBST wash plate 5 times, each 5min; Add two anti-solution (the sheep anti mouse IgM1 of biotin mark: 300, the anti-mouse IgG of the horse of biotin mark 1: 300), incubated at room 30min, PBST wash plate 5 times, each 5min; Add three anti-solution (Avidin of Cy3 mark 1: 300), incubated at room 30min, PBST wash plate 5 times, each 5min; Add 60 μ l, 1 μ g/ml DAPI (being dissolved in 50%PBS/50% glycerine), under fluorescent microscope, observe and take pictures.
The result is negative for the viable cell immunofluorescence dyeing, illustrates that Mac-2BP does not express or the location at the tumour cell surface of cell membrane; Fixed cell immunofluorescence dyeing result (Fig. 5) shows that there is expression in Mac-2BP in multiple lung carcinoma cell, and different tumor cells expressions is strong and weak different, and Mac-2BP is expressed in tumour cell cytoplasm and nucleus.
Embodiment 6Mac-2BP is at the express spectra of people's lung cancer and pairing normal lung tissue thereof
Use the expression that Mac-2BP monoclonal antibody 13H3 that the inventor herein merges preparation has detected antigen gene Mac-2BP in 105 routine lung cancer patient cancerous lung tissues and the pairing normal lung tissue thereof.Paraffin-embedded tissue (tissue sample is collected in Cancer Hospital of Chinese Academy of Medical Sciences) carries out the routine paraffin wax section, through dimethylbenzene dewaxing, gradient alcohol aquation.3% hydrogen peroxide at room temperature 10min, the activity of blocking-up endogenous peroxydase.(0.01M carries out antigen retrieval with microwave heating method in pH6.0) at citrate buffer in section.With 10% normal goats serum room temperature sealing 10min.Abandon confining liquid, add the anti-Mac-2BP protein monoclonal antibody 13H3 of 10 μ g/ml, 4 ℃ of overnight incubation.Wash 3 times with PBS then, every all over 3min.Add biotin labeled sheep anti mouse two anti-(available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing) the incubated at room 10min of proper concn.PBS washes 3 times then, and is every all over 3min, adds the strepto-avidin of HRP mark, incubated at room 10min.DAB dyeing, Hematorylin is redyed, hydrochloric acid-alcohol color separation, ammoniacal liquor returns indigo plant, mounting.If the negative contrast of PBS.Microscopically observations (Fig. 6).As seen Mac-2BP is expressed in cytoplasm and the nucleus, and is higher than cytoplasm at nuclear expression level, and Mac-2BP is significantly higher than pairing normal lung tissue at the expression level of cancerous lung tissue.The statistic analysis result of immunohistochemical methods (table 1A, 1B) show, Mac-2BP is 91.4% (96/105) in the The positive expression rate of cancerous lung tissue, cancer is compared Mac-2BP genetic expression and is had significant difference (p＜0.01) with healthy tissues, the differentiation degree of Mac-2BP genetic expression and cancerous lung tissue has obvious dependency (p＜0.05).The patient of lung cancer had better differentiation, the Mac-2BP expression level is lower, and the differentiation degree of cancerous tissue is poor more, the Mac-2BP expression level is high more.
In addition, the organization chip of using 44 routine lung squamous cancers and 32 routine adenocarcinoma of lung patient's cancerous lung tissues and pairing normal lung tissue preparation carries out the immunohistochemical methods of 13H3 antibody, further prove the Mac-2BP gene the expression of cancerous lung tissue (table 2A, 2B).In 76 routine lung cancer pairing organization chips, cancerous lung tissue Mac-2BP is positive, and the example is 85.5% (65/76), the Mac-2BP of normal lung tissue feminine gender is not expressed and is accounted for 89.5% (68/76), and there is significant difference (P＜0.0001) in the Mac-2BP expression level between cancer and healthy tissues; The differentiation degree significant correlation of Mac-2BP genetic expression and lung cancer patient (P＜0.01) does not have dependency with sex, age and tumor type.Immunohistochemical experiment prompting Mac-2BP is a kind of new tumor markers, might be that a kind of new tumour patient prognosis detects index.
The situation of table 1A Mac-2BP in 105 routine lung cancer and related normal tissue
Table 1B is according to the situation of patient's clinical pathologic characteristic Mac-2BP in 105 routine lung cancer and related normal tissue
Annotate: SCC, squamous cell carcinoma; AD, gland cancer; LCC, large cell carcinoma; SCLC, small cell lung cancer.
Embodiment 7Mac-2BP gene promotes tumour in the intravital fast breeding growth of nude mice
In order to prove the effect in the tumor development in vivo of Mac2BP gene, use steady the commentaries on classics and strike the lung cancer A549 cell that falls the Mac-2BP gene and carry out interior animal experiment.With two steady change to strike clone C13 falls, C36, empty plasmid vehicle Control cell Vector and parental cell A549 after external enlarged culturing grows to logarithmic phase, peptic cell, subcutaneous with every 4,000,000 cell inoculation in nude mice.After observing nude mice one-tenth knurl, measured the nude mice diameter of tumor every 2 days with vernier callipers, gross tumor volume=π/6 major diameter minor axis 2, calculate gross tumor volume, draw tumor growth curve (Fig. 7 A), gather in the crops tumour (Fig. 7 B) in good time, claim knurl heavy, calculate every group of average knurl heavy (Fig. 7 C) and tumour inhibiting rate (Fig. 7 D).Tumour inhibiting rate=(control group tumor weight-treatment group tumor weight)/control group tumor weight * 100%.The t check is adopted in test of significance.
The situation of table 2A Mac-2BP in 76 routine lung cancer and related normal tissue
Table 2B is according to the situation of patient's clinical pathologic characteristic Mac-2BP in 76 routine lung cancer and related normal tissue
Tumor-bearing mice was raised after 50 days, put to death mouse, claimed tumor weight and assessed Mac-2BP genetic expression role in vivo.Experimental group is compared with control group, and after the Mac-2BP clpp gene fell, the tumor growth rate of clone cell C13 and C36 was very slow; Seriously lag behind control group Vector cell and parent A549 cell.And the tumor weight of experimental group is far smaller than control group, the inhibitory rate to 80% of experimental group.Zooperal result proves absolutely that the Mac-2BP gene can significantly promote the fast breeding and the growth of tumour in vivo.
Embodiment 8 adopts Mac-2BP antibody that tumour is treated
Use Mac-2BP monoclonal antibody 13H3 and carry out transplanted tumor inhibition experiment in the nude mouse.Identify that 13H3 antibody is in vivo to the tumor treatment effect.It is subcutaneous that lung carcinoma cell ANIP-973 is inoculated in nude mice with every 3,000,000, set up height (50mg/kg/ time), in (25mg/kg/ time), low (12.5mg/kg/ time) three dosage experiments groups and PBS control group and ANIP-973 do not treat group.Begin abdominal injection treatment, every day 1 time, continuous 2 weeks next day behind the inoculated tumour cell; After this with same dose, 4 times weekly, treated for 4 weeks continuously.Cell inoculation begins to measure the knurl volume after 8 days, draw tumor growth curve (Fig. 8 A).Put to death nude mice after 35 days, strip tumour (Fig. 8 B), calculate every group of average knurl heavy (Fig. 8 C) and tumour inhibiting rate (Fig. 8 D).Tumour inhibiting rate=(control group tumor weight-treatment group tumor weight)/control group tumor weight * 100%.The t check is adopted in test of significance.
Antibody 13H3 treatment embodies the experimental result of people's tumor growth, PBS group knurl weighs 0.97 ± 0.29g, parental cell is not treated the group knurl and is weighed 0.88 ± 0.24g, treatment group high dose group knurl weighs 0.39 ± 0.19g, middle dosage group knurl weighs 0.44 ± 0.14g, the low dose group knurl weighs 0.54 ± 0.34g, these presentation of results 13H3 antibody can obviously suppress growth of tumor in nude mouse, the tumour inhibiting rate of high, medium and low three dosage groups is respectively 60.14%, 54.86% and 43.84%, and tumour inhibiting rate and antibody are dose-dependence.Experimental group is compared with control group, and all there were significant differences for gross tumor volume and tumor weight (P＜0.05).13H3 antibody has important use and is worth in the immunotherapy tumour.
The influence of embodiment 9Mac-2BP gene pairs tumour cell cycle
Use Mac-2BP gene siRNA sequence (m2bpsiR-1, m2bpsiR-2) and negative control NControl siRNA sequence transient transfection human tumor cells A549.Transfection digested tumour cell after 48 hours, and PBS washes twice, and the centrifugal 5min collecting cell of 1200rpm is resuspended in 300 μ l PBS and adds the dehydrated alcohol 700 μ l of-20 ℃ of precoolings.Mixing place 4 ℃ 24 hours, the PBS washed cell twice then, adds 200 μ l RNase A solution (1mg/ml) re-suspended cells, 37 ℃ of water-bath 30min add 800 μ l iodate, third ingot (propidium iodide, PI) PI staining fluid (50 μ g/ml) again, mixing, 4 ℃ of lucifuge dyeing 30min.The cells were tested by flow cytometry cell cycle.
The RNAi technology is instantaneous strike fall carry out the cell cycle behind the tumour cell Mac-2BP gene measurement result as shown in Figure 9, table 3 is percentage of cells of cell cycle G1, G2 and S phase.Behind the Mac-2BP gene knockout, the experimental group cell is compared with cellular control unit as can be seen, and the massive tumor cell is trapped in the cell cycle G1 phase, thereby the cell poor growth, and this is consistent with our observed in vitro and in vivo phenotype.The Mac-2BP gene is described by the regulate tumor cell cycle progression, thereby influences the growing multiplication ability of tumour cell.
Table 3Mac-2BP strikes and falls back tumour cell cycle G1, G2 and the percentage of cells of S phase
Sequence table
＜110〉Tumour Inst., Chinese Medical Academy
＜120〉Mac-2BP tumor antigen gene, albumen, antibody and uses thereof
<130>880248CG
<160>13
<170>PatentIn?version?3.4
<210>1
<211>19
<212>DNA
＜213〉human (Homo sapiens)
<400>1
ccatcagcgt?gaatgtgca 19
<210>2
<211>19
<212>DNA
＜213〉human (Homo sapiens)
<400>2
ggacctgtat?gcctatgca 19
<210>3
<211>19
<212>RNA
＜213〉artificial sequence
<220>
<221>misc_RNA
<222>(1)..(19)
＜223〉the double-stranded siRNA positive-sense strand of m2bpsiR1,3 ' end has two outstanding base dTdT
<400>3
ccaucagcgu?gaaugugca 19
<210>4
<211>19
<212>RNA
＜213〉artificial sequence
<220>
<221>misc_RNA
<222>(1)..(19)
＜223〉the double-stranded siRNA antisense strand of m2bpsiR1,3 ' end has two outstanding base dTdT
<400>4
gguagucgca?cuuacacgu 19
<210>5
<211>19
<212>RNA
＜213〉artificial sequence
<220>
<221>misc_RNA
<222>(1)..(19)
<220>
<221>misc_RNA
<222>(1)..(19)
＜223〉the double-stranded siRNA positive-sense strand of m2bpsiR2,3 ' end has two outstanding base dTdT
<400>5
ggaccuguau?gccuaugca 19
<210>6
<211>19
<212>RNA
＜213〉artificial sequence
<220>
<221>misc_RNA
<222>(1)..(19)
＜223〉the double-stranded siRNA antisense strand of m2bpsiR2,3 ' end has two outstanding base dTdT
<400>6
ccuggacaua?cggauacgu 19
<210>7
<211>1758
<212>DNA
＜213〉human (Homo sapiens)
<220>
<221>CDS
<222>(1)..(1758)
＜223〉Mac-2BP encoding sequence
<400>7
atg?acc?cct?ccg?agg?ctc?ttc?tgg?gtg?tgg?ctg?ctg?gtt?gca?gga?acc 48
Met?Thr?Pro?Pro?Arg?Leu?Phe?Trp?Val?Trp?Leu?Leu?Val?Ala?Gly?Thr
1 5 10 15
caa?ggc?gtg?aac?gat?ggt?gac?atg?cgg?ctg?gcc?gat?ggg?ggc?gcc?acc 96
Gln?Gly?Val?Asn?Asp?Gly?Asp?Met?Arg?Leu?Ala?Asp?Gly?Gly?Ala?Thr
20 25 30
aac?cag?ggc?cgc?gtg?gag?atc?ttc?tac?aga?ggc?cag?tgg?ggc?act?gtg 144
Asn?Gln?Gly?Arg?Val?Glu?Ile?Phe?Tyr?Arg?Gly?Gln?Trp?Gly?Thr?Val
35 40 45
tgt?gac?aac?ctg?tgg?gac?ctg?act?gat?gcc?agc?gtc?gtc?tgc?cgg?gcc 192
Cys?Asp?Asn?Leu?Trp?Asp?Leu?Thr?Asp?Ala?Ser?Val?Val?Cys?Arg?Ala
50 55 60
ctg?ggc?ttc?gag?aac?gcc?acc?cag?gct?ctg?ggc?aga?gct?gcc?ttc?ggg 240
Leu?Gly?Phe?Glu?Asn?Ala?Thr?Gln?Ala?Leu?Gly?Arg?Ala?Ala?Phe?Gly
65 70 75 80
caa?gga?tca?ggc?ccc?atc?atg?ctg?gac?gag?gtc?cag?tgc?acg?gga?acc 288
Gln?Gly?Ser?Gly?Pro?Ile?Met?Leu?Asp?Glu?Val?Gln?Cys?Thr?Gly?Thr
85 90 95
gag?gcc?tca?ctg?gcc?gac?tgc?aag?tcc?ctg?ggc?tgg?ctg?aag?agc?aac 336
Glu?Ala?Ser?Leu?Ala?Asp?Cys?Lys?Ser?Leu?Gly?Trp?Leu?Lys?Ser?Asn
100 105 110
tgc?agg?cac?gag?aga?gac?gct?ggt?gtg?gtc?tgc?acc?aat?gaa?acc?agg 384
Cys?Arg?His?Glu?Arg?Asp?Ala?Gly?Val?Val?Cys?Thr?Asn?Glu?Thr?Arg
115 120 125
agc?acc?cac?acc?ctg?gac?ctc?tcc?agg?gag?ctc?tcg?gag?gcc?ctt?ggc 432
Ser?Thr?His?Thr?Leu?Asp?Leu?Ser?Arg?Glu?Leu?Ser?Glu?Ala?Leu?Gly
130 135 140
cag?atc?ttt?gac?agc?cag?cgg?ggc?tgc?gac?ctg?tcc?atc?agc?gtg?aat 480
Gln?Ile?Phe?Asp?Ser?Gln?Arg?Gly?Cys?Asp?Leu?Ser?Ile?Ser?Val?Asn
145 150 155 160
gtg?cag?ggc?gag?gac?gcc?ctg?ggc?ttc?tgt?ggc?cac?acg?gtc?atc?ctg 528
Val?Gln?Gly?Glu?Asp?Ala?Leu?Gly?Phe?Cys?Gly?His?Thr?Val?Ile?Leu
165 170 175
act?gcc?aac?ctg?gag?gcc?cag?gcc?ctg?tgg?aag?gag?ccg?ggc?agc?aat 576
Thr?Ala?Asn?Leu?Glu?Ala?Gln?Ala?Leu?Trp?Lys?Glu?Pro?Gly?Ser?Asn
180 185 190
gtc?acc?atg?agt?gtg?gat?gct?gag?tgt?gtg?ccc?atg?gtc?agg?gac?ctt 624
Val?Thr?Met?Ser?Val?Asp?Ala?Glu?Cys?Val?Pro?Met?Val?Arg?Asp?Leu
195 200 205
ctc?agg?tac?ttc?tac?tcc?cga?agg?att?gac?atc?acc?ctg?tcg?tca?gtc 672
Leu?Arg?Tyr?Phe?Tyr?Ser?Arg?Arg?Ile?Asp?Ile?Thr?Leu?Ser?Ser?Val
210 215 220
aag?tgc?ttc?cac?aag?ctg?gcc?tct?gcc?tat?ggg?gcc?agg?cag?ctg?cag 720
Lys?Cys?Phe?His?Lys?Leu?Ala?Ser?Ala?Tyr?Gly?Ala?Arg?Gln?Leu?Gln
225 230 235 240
ggc?tac?tgc?gca?agc?ctc?ttt?gcc?atc?ctc?ctc?ccc?cag?gac?ccc?tcg 768
Gly?Tyr?Cys?Ala?Ser?Leu?Phe?Ala?Ile?Leu?Leu?Pro?Gln?Asp?Pro?Ser
245 250 255
ttc?cag?atg?ccc?ctg?gac?ctg?tat?gcc?tat?gca?gtg?gcc?aca?ggg?gac 816
Phe?Gln?Met?Pro?Leu?Asp?Leu?Tyr?Ala?Tyr?Ala?Val?Ala?Thr?Gly?Asp
260 265 270
gcc?ctg?ctg?gag?aag?ctc?tgc?cta?cag?ttc?ctg?gcc?tgg?aac?ttc?gag 864
Ala?Leu?Leu?Glu?Lys?Leu?Cys?Leu?Gln?Phe?Leu?Ala?Trp?Asn?Phe?Glu
275 280 285
gcc?ttg?acg?cag?gcc?gag?gcc?tgg?ccc?agt?gtc?ccc?aca?gac?ctg?ctc 912
Ala?Leu?Thr?Gln?Ala?Glu?Ala?Trp?Pro?Ser?Val?Pro?Thr?Asp?Leu?Leu
290 295 300
caa?ctg?ctg?ctg?ccc?agg?agc?gac?ctg?gcg?gtg?ccc?agc?gag?ctg?gcc 960
Gln?Leu?Leu?Leu?Pro?Arg?Ser?Asp?Leu?Ala?Val?Pro?Ser?Glu?Leu?Ala
305 310 315 320
cta?ctg?aag?gcc?gtg?gac?acc?tgg?agc?tgg?ggg?gag?cgt?gcc?tcc?cat 1008
Leu?Leu?Lys?Ala?Val?Asp?Thr?Trp?Ser?Trp?Gly?Glu?Arg?Ala?Ser?His
325 330 335
gag?gag?gtg?gag?ggc?ttg?gtg?gag?aag?atc?cgc?ttc?ccc?atg?atg?ctc 1056
Glu?Glu?Val?Glu?Gly?Leu?Val?Glu?Lys?Ile?Arg?Phe?Pro?Met?Met?Leu
340 345 350
cct?gag?gag?ctc?ttt?gag?ctg?cag?ttc?aac?ctg?tcc?ctg?tac?tgg?agc 1104
Pro?Glu?Glu?Leu?Phe?Glu?Leu?Gln?Phe?Asn?Leu?Ser?Leu?Tyr?Trp?Ser
355 360 365
cac?gag?gcc?ctg?ttc?cag?aag?aag?act?ctg?cag?gcc?ctg?gaa?ttc?cac 1152
His?Glu?Ala?Leu?Phe?Gln?Lys?Lys?Thr?Leu?Gln?Ala?Leu?Glu?Phe?His
370 375 380
act?gtg?ccc?ttc?cag?ttg?ctg?gcc?cgg?tac?aaa?ggc?ctg?aac?ctc?acc 1200
Thr?Val?Pro?Phe?Gln?Leu?Leu?Ala?Arg?Tyr?Lys?Gly?Leu?Asn?Leu?Thr
385 390 395 400
gag?gat?acc?tac?aag?ccc?cgg?att?tac?acc?tcg?ccc?acc?tgg?agt?gcc 1248
Glu?Asp?Thr?Tyr?Lys?Pro?Arg?Ile?Tyr?Thr?Ser?Pro?Thr?Trp?Ser?Ala
405 410 415
ttt?gtg?aca?gac?agt?tcc?tgg?agt?gca?cgg?aag?tca?caa?ctg?gtc?tat 1296
Phe?Val?Thr?Asp?Ser?Ser?Trp?Ser?Ala?Arg?Lys?Ser?Gln?Leu?Val?Tyr
420 425 430
cag?tcc?aga?cgg?ggg?cct?ttg?gtc?aaa?tat?tct?tct?gat?tac?ttc?caa 1344
Gln?Ser?Arg?Arg?Gly?Pro?Leu?Val?Lys?Tyr?Ser?Ser?Asp?Tyr?Phe?Gln
435 440 445
gcc?ccc?tct?gac?tac?aga?tac?tac?ccc?tac?cag?tcc?ttc?cag?act?cca 1392
Ala?Pro?Ser?Asp?Tyr?Arg?Tyr?Tyr?Pro?Tyr?Gln?Ser?Phe?Gln?Thr?Pro
450 455 460
caa?cac?ccc?agc?ttc?ctc?ttc?cag?gac?aag?agg?gtg?tcc?tgg?tcc?ctg 1440
Gln?His?Pro?Ser?Phe?Leu?Phe?Gln?Asp?Lys?Arg?Val?Ser?Trp?Ser?Leu
465 470 475 480
gtc?tac?ctc?ccc?acc?atc?cag?agc?tgc?tgg?aac?tac?ggc?ttc?tcc?tgc 1488
Val?Tyr?Leu?Pro?Thr?Ile?Gln?Ser?Cys?Trp?Asn?Tyr?Gly?Phe?Ser?Cys
485 490 495
tcc?tcg?gac?gag?ctc?cct?gtc?ctg?ggc?ctc?acc?aag?tct?ggc?ggc?tca 1536
Ser?Ser?Asp?Glu?Leu?Pro?Val?Leu?Gly?Leu?Thr?Lys?Ser?Gly?Gly?Ser
500 505 510
gat?cgc?acc?att?gcc?tac?gaa?aac?aaa?gcc?ctg?atg?ctc?tgc?gaa?ggg 1584
Asp?Arg?Thr?Ile?Ala?Tyr?Glu?Asn?Lys?Ala?Leu?Met?Leu?Cys?Glu?Gly
515 520 525
ctc?ttc?gtg?gca?gac?gtc?acc?gat?ttc?gag?ggc?tgg?aag?gct?gcg?att 1632
Leu?Phe?Val?Ala?Asp?Val?Thr?Asp?Phe?Glu?Gly?Trp?Lys?Ala?Ala?Ile
530 535 540
ccc?agt?gcc?ctg?gac?acc?aac?agc?tcg?aag?agc?acc?tcc?tcc?ttc?ccc 1680
Pro?Ser?Ala?Leu?Asp?Thr?Asn?Ser?Ser?Lys?Ser?Thr?Ser?Ser?Phe?Pro
545 550 555 560
tgc?ccg?gca?ggg?cac?ttc?aac?ggc?ttc?cgc?acg?gtc?atc?cgc?ccc?ttc 1728
Cys?Pro?Ala?Gly?His?Phe?Asn?Gly?Phe?Arg?Thr?Val?Ile?Arg?Pro?Phe
565 570 575
tac?ctg?acc?aac?tcc?tca?ggt?gtg?gac?tag 1758
Tyr?Leu?Thr?Asn?Ser?Ser?Gly?Val?Asp
580 585
<210>8
<211>585
<212>PRT
＜213〉human (Homo sapiens)
<400>8
Met?Thr?Pro?Pro?Arg?Leu?Phe?Trp?Val?Trp?Leu?Leu?Val?Ala?Gly?Thr
1 5 10 15
Gln?Gly?Val?Asn?Asp?Gly?Asp?Met?Arg?Leu?Ala?Asp?Gly?Gly?Ala?Thr
20 25 30
Asn?Gln?Gly?Arg?Val?Glu?Ile?Phe?Tyr?Arg?Gly?Gln?Trp?Gly?Thr?Val
35 40 45
Cys?Asp?Asn?Leu?Trp?Asp?Leu?Thr?Asp?Ala?Ser?Val?Val?Cys?Arg?Ala
50 55 60
Leu?Gly?Phe?Glu?Asn?Ala?Thr?Gln?Ala?Leu?Gly?Arg?Ala?Ala?Phe?Gly
65 70 75 80
Gln?Gly?Ser?Gly?Pro?Ile?Met?Leu?Asp?Glu?Val?Gln?Cys?Thr?Gly?Thr
85 90 95
Glu?Ala?Ser?Leu?Ala?Asp?Cys?Lys?Ser?Leu?Gly?Trp?Leu?Lys?Ser?Asn
100 105 110
Cys?Arg?His?Glu?Arg?Asp?Ala?Gly?Val?Val?Cys?Thr?Asn?Glu?Thr?Arg
115 120 125
Ser?Thr?His?Thr?Leu?Asp?Leu?Ser?Arg?Glu?Leu?Ser?Glu?Ala?Leu?Gly
130 135 140
Gln?Ile?Phe?Asp?Ser?Gln?Arg?Gly?Cys?Asp?Leu?Ser?Ile?Ser?Val?Asn
145 150 155 160
Val?Gln?Gly?Glu?Asp?Ala?Leu?Gly?Phe?Cys?Gly?His?Thr?Val?Ile?Leu
165 170 175
Thr?Ala?Asn?Leu?Glu?Ala?Gln?Ala?Leu?Trp?Lys?Glu?Pro?Gly?Ser?Asn
180 185 190
Val?Thr?Met?Ser?Val?Asp?Ala?Glu?Cys?Val?Pro?Met?Val?Arg?Asp?Leu
195 200 205
Leu?Arg?Tyr?Phe?Tyr?Ser?Arg?Arg?Ile?Asp?Ile?Thr?Leu?Ser?Ser?Val
210 215 220
Lys?Cys?Phe?His?Lys?Leu?Ala?Ser?Ala?Tyr?Gly?Ala?Arg?Gln?Leu?Gln
225 230 235 240
Gly?Tyr?Cys?Ala?Ser?Leu?Phe?Ala?Ile?Leu?Leu?Pro?Gln?Asp?Pro?Ser
245 250 255
Phe?Gln?Met?Pro?Leu?Asp?Leu?Tyr?Ala?Tyr?Ala?Val?Ala?Thr?Gly?Asp
260 265 270
Ala?Leu?Leu?Glu?Lys?Leu?Cys?Leu?Gln?Phe?Leu?Ala?Trp?Asn?Phe?Glu
275 280 285
Ala?Leu?Thr?Gln?Ala?Glu?Ala?Trp?Pro?Ser?Val?Pro?Thr?Asp?Leu?Leu
290 295 300
Gln?Leu?Leu?Leu?Pro?Arg?Ser?Asp?Leu?Ala?Val?Pro?Ser?Glu?Leu?Ala
305 310 315 320
Leu?Leu?Lys?Ala?Val?Asp?Thr?Trp?Ser?Trp?Gly?Glu?Arg?Ala?Ser?His
325 330 335
Glu?Glu?Val?Glu?Gly?Leu?Val?Glu?Lys?Ile?Arg?Phe?Pro?Met?Met?Leu
340 345 350
Pro?Glu?Glu?Leu?Phe?Glu?Leu?Gln?Phe?Asn?Leu?Ser?Leu?Tyr?Trp?Ser
355 360 365
His?Glu?Ala?Leu?Phe?Gln?Lys?Lys?Thr?Leu?Gln?Ala?Leu?Glu?Phe?His
370 375 380
Thr?Val?Pro?Phe?Gln?Leu?Leu?Ala?Arg?Tyr?Lys?Gly?Leu?Asn?Leu?Thr
385 390 395 400
Glu?Asp?Thr?Tyr?Lys?Pro?Arg?Ile?Tyr?Thr?Ser?Pro?Thr?Trp?Ser?Ala
405 410 415
Phe?Val?Thr?Asp?Ser?Ser?Trp?Ser?Ala?Arg?Lys?Ser?Gln?Leu?Val?Tyr
420 425 430
Gln?Ser?Arg?Arg?Gly?Pro?Leu?Val?Lys?Tyr?Ser?Ser?Asp?Tyr?Phe?Gln
435 440 445
Ala?Pro?Ser?Asp?Tyr?Arg?Tyr?Tyr?Pro?Tyr?Gln?Ser?Phe?Gln?Thr?Pro
450 455 460
Gln?His?Pro?Ser?Phe?Leu?Phe?Gln?Asp?Lys?Arg?Val?Ser?Trp?Ser?Leu
465 470 475 480
Val?Tyr?Leu?Pro?Thr?Ile?Gln?Ser?Cys?Trp?Asn?Tyr?Gly?Phe?Ser?Cys
485 490 495
Ser?Ser?Asp?Glu?Leu?Pro?Val?Leu?Gly?Leu?Thr?Lys?Ser?Gly?Gly?Ser
500 505 510
Asp?Arg?Thr?Ile?Ala?Tyr?Glu?Asn?Lys?Ala?Leu?Met?Leu?Cys?Glu?Gly
515 520 525
Leu?Phe?Val?Ala?Asp?Val?Thr?Asp?Phe?Glu?Gly?Trp?Lys?Ala?Ala?Ile
530 535 540
Pro?Ser?Ala?Leu?Asp?Thr?Asn?Ser?Ser?Lys?Ser?Thr?Ser?Ser?Phe?Pro
545 550 555 560
Cys?Pro?Ala?Gly?His?Phe?Asn?Gly?Phe?Arg?Thr?Val?Ile?Arg?Pro?Phe
565 570 575
Tyr?Leu?Thr?Asn?Ser?Ser?Gly?Val?Asp
580 585
<210>9
<211>2254
<212>DNA
＜213〉human (Homo sapiens)
<220>
<221>gene
<222>(1)..(2254)
＜223〉Mac-2BP gene complete sequence
<400>9
aatcgaaagt?agactctttt?ctgaagcatt?tcctgggatc?agcctgacca?cgctccatac 60
tgggagaggc?ttctgggtca?aaggaccagt?ctgcagaggg?atcctgtggc?tggaagcgag 120
gaggctccac?acggccgttg?cagctaccgc?agccaggatc?tgggcatcca?ggcacggcca 180
tgacccctcc?gaggctcttc?tgggtgtggc?tgctggttgc?aggaacccaa?ggcgtgaacg 240
atggtgacat?gcggctggcc?gatgggggcg?ccaccaacca?gggccgcgtg?gagatcttct 300
acagaggcca?gtggggcact?gtgtgtgaca?acctgtggga?cctgactgat?gccagcgtcg 360
tctgccgggc?cctgggcttc?gagaacgcca?cccaggctct?gggcagagct?gccttcgggc 420
aaggatcagg?ccccatcatg?ctggacgagg?tccagtgcac?gggaaccgag?gcctcactgg 480
ccgactgcaa?gtccctgggc?tggctgaaga?gcaactgcag?gcacgagaga?gacgctggtg 540
tggtctgcac?caatgaaacc?aggagcaccc?acaccctgga?cctctccagg?gagctctcgg 600
aggcccttgg?ccagatcttt?gacagccagc?ggggctgcga?cctgtccatc?agcgtgaatg 660
tgcagggcga?ggacgccctg?ggcttctgtg?gccacacggt?catcctgact?gccaacctgg 720
aggcccaggc?cctgtggaag?gagccgggca?gcaatgtcac?catgagtgtg?gatgctgagt 780
gtgtgcccat?ggtcagggac?cttctcaggt?acttctactc?ccgaaggatt?gacatcaccc 840
tgtcgtcagt?caagtgcttc?cacaagctgg?cctctgccta?tggggccagg?cagctgcagg 900
gctactgcgc?aagcctcttt?gccatcctcc?tcccccagga?cccctcgttc?cagatgcccc 960
tggacctgta?tgcctatgca?gtggccacag?gggacgccct?gctggagaag?ctctgcctac 1020
agttcctggc?ctggaacttc?gaggccttga?cgcaggccga?ggcctggccc?agtgtcccca 1080
cagacctgct?ccaactgctg?ctgcccagga?gcgacctggc?ggtgcccagc?gagctggccc 1140
tactgaaggc?cgtggacacc?tggagctggg?gggagcgtgc?ctcccatgag?gaggtggagg 1200
gcttggtgga?gaagatccgc?ttccccatga?tgctccctga?ggagctcttt?gagctgcagt 1260
tcaacctgtc?cctgtactgg?agccacgagg?ccctgttcca?gaagaagact?ctgcaggccc 1320
tggaattcca?cactgtgccc?ttccagttgc?tggcccggta?caaaggcctg?aacctcaccg 1380
aggataccta?caagccccgg?atttacacct?cgcccacctg?gagtgccttt?gtgacagaca 1440
gttcctggag?tgcacggaag?tcacaactgg?tctatcagtc?cagacggggg?cctttggtca 1500
aatattcttc?tgattacttc?caagccccct?ctgactacag?atactacccc?taccagtcct 1560
tccagactcc?acaacacccc?agcttcctct?tccaggacaa?gagggtgtcc?tggtccctgg 1620
tctacctccc?caccatccag?agctgctgga?actacggctt?ctcctgctcc?tcggacgagc 1680
tccctgtcct?gggcctcacc?aagtctggcg?gctcagatcg?caccattgcc?tacgaaaaca 1740
aagccctgat?gctctgcgaa?gggctcttcg?tggcagacgt?caccgatttc?gagggctgga 1800
aggctgcgat?tcccagtgcc?ctggacacca?acagctcgaa?gagcacctcc?tccttcccct 1860
gcccggcagg?gcacttcaac?ggcttccgca?cggtcatccg?ccccttctac?ctgaccaact 1920
cctcaggtgt?ggactagacg?cgtggccaag?ggtggtgaga?accggagaac?cccaggacgc 1980
cctcactgca?ggctcccctc?ctcggcttcc?ttcctctctg?caatgacctt?caacaaccgg 2040
ccaccagatg?tcgccctact?cacctgaggc?tcagcttcaa?gaaattactg?gaaggcttcc 2100
actagggtcc?accaggagtt?ctcccaccac?ctcaccagtt?tccaggtggt?aagcaccagg 2160
aggccctcga?ggttgctctg?gatcccccca?cagcccctgg?tcagtctgcc?cttgtcactg 2220
gtctgaggtc?attaaaatta?cattgaggtt?ccta 2254
<210>10
<211>64
<212>DNA
＜213〉artificial sequence
<400>10
gatccgccat?cagcgtgaat?gtgcacaaga?gacatgcaca?ttcacgctga?tggttttttg 60
<210>11
<211>64
<212>DNA
＜213〉artificial sequence
<400>11
ctaggcggta?gtcgcactta?cacgtgttct?ctgtacgtgt?aagtgcgact?accaaaaaac 60
<210>12
<211>64
<212>DNA
＜213〉artificial sequence
<400>12
gatccgggac?ctgtatgcct?atgcacaaga?gacatgcata?ggcatacagg?tccttttttg 60
<210>13
<211>64
<212>DNA
＜213〉artificial sequence
<400>13
ctaggccctg?gacatacgga?tacgtgttct?ctgtacgtat?ccgtatgtcc?aggaaaaaac 60
Claims (13)
1. isolating polynucleotide, it is selected from:
1) as SEQ ID NO:1 and the described polynucleotide passage of SEQ ID NO:2;
2) fragment that can under rigorous condition, hybridize with SEQ ID NO:1 and the described polynucleotide passage of SEQ ID NO:2; Or
3) with the fragment of above-mentioned fragment complementation.
2. polynucleotide according to claim 1 are DNA or RNA.
3. coding human tumor antigen gene M ac-2BP is characterized in that it contains polynucleotide as claimed in claim 1 or 2.
4. a peptide species, it has with aminoacid sequence at least 50% identity of polynucleotide encoding as claimed in claim 3 and has identical or the aminoacid sequence of high biological activity more with the polypeptide with this aminoacid sequence.
5. polypeptide according to claim 4, it has the aminoacid sequence shown in SEQ ID NO:8.
6. antibody that polypeptide as claimed in claim 4 obtains as antigen.
7. antibody according to claim 6 is characterized in that described antibody is monoclonal antibody.
8. antibody according to claim 7 is characterized in that described antibody is 13H3 antibody.
9. polynucleotide as claimed in claim 1 and/or the described polypeptide of claim 4 and/or the described antibody of claim 6 purposes in the preparation anti-tumor drug.
10. polynucleotide as claimed in claim 1 and/or the described polypeptide of claim 4 and/or the described antibody of claim 6 purposes in preparation tumor marker preparation.
11. according to claim 9 or 10 described purposes, wherein said tumour is a lung cancer.
12. a tumor marker is characterized in that described tumor marker is a polypeptide as claimed in claim 4.
13. a test kit, it comprises polynucleotide as claimed in claim 1 and/or described polypeptide of claim 4 and/or the described antibody of claim 6, and specification sheets.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810167779A CN101712960A (en) | 2008-10-07 | 2008-10-07 | Mac-2BP tumor antigen gene, protein, antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374072A (en) * | 2012-04-13 | 2013-10-30 | 中国医学科学院肿瘤研究所 | Preparation of anti-Mac-2BP humanized antibody |
| CN106065397A (en) * | 2010-06-07 | 2016-11-02 | 广州呼吸疾病研究所 | Human lung adenocarcinoma derived cell strain A1015 |
| CN108283013A (en) * | 2015-07-24 | 2018-07-13 | 公立大学法人名古屋市立大学 | The marker and device for assisting method, the method for prediction for assisting prognosis and the monitoring method of therapeutic effect of the diagnosis of the state of myleo disease and using in these methods |
-
2008
- 2008-10-07 CN CN200810167779A patent/CN101712960A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106065397A (en) * | 2010-06-07 | 2016-11-02 | 广州呼吸疾病研究所 | Human lung adenocarcinoma derived cell strain A1015 |
| CN103374072A (en) * | 2012-04-13 | 2013-10-30 | 中国医学科学院肿瘤研究所 | Preparation of anti-Mac-2BP humanized antibody |
| CN103374072B (en) * | 2012-04-13 | 2017-10-31 | 中国医学科学院肿瘤医院 | A kind of preparation of anti-Mac 2BP humanized antibodies |
| CN108283013A (en) * | 2015-07-24 | 2018-07-13 | 公立大学法人名古屋市立大学 | The marker and device for assisting method, the method for prediction for assisting prognosis and the monitoring method of therapeutic effect of the diagnosis of the state of myleo disease and using in these methods |
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