CN101701039B - Variable regions of light chains and heavy chains of FMU-EPCAM-2A9 monoclonal antibodies - Google Patents
Variable regions of light chains and heavy chains of FMU-EPCAM-2A9 monoclonal antibodies Download PDFInfo
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Abstract
The invention discloses variable regions of light chains and heavy chains of FMU-EPCAM-2A9 monoclonal antibodies. The amino acid sequence of a monoclonal antibody light chain variable region is shown as SEQ ID NO.1, and the amino acid sequence of a monoclonal antibody heavy chain variable region is shown as SEQ ID NO.2. A set of mouse anti-human EPCAM monoclonal antibodies are prepared through recombining human EPCAM immune BALB/c mice, and hybridoma cell lines capable of stably secreting high-affinity anti-human EPCAM monoclonal antibodies are screened to prepare ascitic fluid so as to obtain the high-affinity anti-human EPCAM monoclonal antibodies. The uniqueness of the gene sequence and a corresponding protein sequence and a CDR sequence of the gene sequence are confirmed so as to provide supports for anti-human EPCAM chimeric or humanized gene engineering antibodies.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to a kind of monoclonal antibody, the light chain and the variable region of heavy chain of the FMU-EPCAM-2A9 monoclonal antibody of particularly a kind of anti-people EPCAM comprise its aminoacid sequence and nucleotide sequence.
Background technology
Epithelial cell adhesion molecule (EPCAM) is that the advantage epi-position as colorectal carcinoma is found at first, thinks that at that time its effect is cell adhesion and the treatment reliable connection site with antibody.Research at present thinks that EPCAM is glycosylated I type transmembrane glycoprotein, and molecular weight is 30 to 40kD, and its structure includes the after birth outskirt of an epidermal growth factor-like and a thyroglobulin sample, strides film district and one section 26 amino acid whose cytoplasmic domain for one section.EPCAM mainly is positioned at the tight junction in epithelial cell gap in normal cell.Intensity and the frequency expressed by EPCAM show, its basically high expression level in everyone adenocarcinoid, part squamous cell carcinoma, retinoblastoma and hepatocellular carcinoma.
Discovering the EPCAM function aspects: EPCAM has vital role before 2007 in cell proliferation, migration and signal transduction.For example: EPCAM expresses in the enhancing of people and rodent cell, has strengthened the propagation of cell grappling and the non-dependence of serum somatomedin and has increased as c-myc and cyclins in interior various target gene expression; At breast cancer cell line, the EPCAM signal is interfered through RNA, can cause the reduction of cell proliferation, migration and invasive ability.
Nearest studies confirm that: EPCAM can be used as one of surface marker of some noumenal tumour stem cells, also can be used as the integral part of normal stem cell surface marker.Simultaneously, result of study has also been explained the reason of EPCAM at the tumour cell high expression level well, and finds that itself and some tumour patient prognosis are relatively poor and survival rate is low relevant; And further explained the relation of EPCAM and tumour.In addition, mouse embryo stem cell experiment confirm, the EPCAM high expression level on tumor stem cell, normal stem cell and the rise of himself positive regeeration are to guaranteeing to keep these normal stem cells and extremely important with the propagation of pernicious stem cell.
The research that anti-EPCAM antibody is used for oncotherapy is the focus of Antybody therapy research always.As far back as the anti-EPCAM antibody 17-1A of mouse source property of exploitation in 1979, just be used for clinical treatment to tumour in nineteen eighty-two.But mouse endogenous antibody medicine is when human body uses, because it has immunogenicity, can cause the immune response of human body, thereby cause the removing or the immunocomplex mediated hypersensitivity of antagonist medicine.Can partly solve its immunogenicity problem to the genetic modification of mouse endogenous antibody, as making up chimeric antibody or humanized antibody etc.
The antibody monomer molecule is by two identical heavy chains (H chain) and two identical light chains (L chain), the tetrapeptide chain structure that is formed by connecting by interchain disulfide bond.H chain and L chain comprise amino (N) end and carboxyl (C) end, are made up of hypervariable region/complementary determining region (HVR/CDR) and skeleton district (FR) near the variable region (V district) of N end; Be constant region (C district) near the C end.The protein folding that variable region of heavy chain (VH) and variable region of light chain (VL) form is an antigen-binding site, and CDR/HVR wherein is antibody and the complementary bonded of epitope position, and the reaction after the antigen-antibody identification is caused in the C district.Antibody can divide for people source, mouse source etc. according to FR/C district difference, the mouse endogenous antibody has immunogenicity when using in human body, easily cause the immune response of human body, these immune responses can cause the removing of mouse endogenous antibody and immunocomplex mediated hypersensitivity.In order to overcome the defective of mouse endogenous antibody, need to make up specific chimeric antibody, single-chain antibody or the humanized antibody of high-affinity.
In transformation process, of paramount importance is at first to obtain to have good specificity and avidity mouse source property parental antibody, clones its light chain and heavy chain variable region gene, then these two genes is transformed, and makes up the recombinant antibodies gene.At these problems, the research and development of anti-EPCAM therapeutic antibodies constantly make progress, the rat of anti-EPCAM and mouse bi-specific antibody Catumaxomab, single-chain antibody Proxinium, humanized antibody ING-1, the research that humanized fusion antibody EMD and full-length human antibody Adecatumumab priorities such as (MT201) enter clinical treatment tumour.In clinical trial, obtained good effect at present by the antibody A decatumumab treatment mammary cancer of transforming.Therefore, filter out the anti-people EPCAM of high-affinity mouse source property monoclonal antibody, therefrom clone light, heavy chain variable region gene, to further improvement be with EPCAM target spot medicine preparation and tumor treatment had very important significance.
Summary of the invention
The problem that the present invention solves is to provide the light chain and the variable region of heavy chain of the FMU-EPCAM-2A9 monoclonal antibody of a kind of anti-people EPCAM, comprise its amino acid and nucleotide sequence, chimeric or humanized genetic engineering antibody provides support for the anti-EPCAM that makes up high-affinity.
The present invention is achieved through the following technical solutions:
The light chain of FMU-EPCAM-2A9 monoclonal antibody and variable region of heavy chain, 3 complementary determining regions (CDR) sequence of described variable region of light chain is respectively:
CDRl：Gln-Ser-Leu-Leu-Asp-Ser-Asp-Gly-Lys-Thr-Tyr；
CDR2：Val-Val-Ser-Lys-Leu-Asp-Ser；
CDR3：Trp-Gln-Gly-Thr-His-Phe-Pro-Trp-Thr；
3 complementary determining regions (CDR) sequence of described variable region of heavy chain is respectively:
CDR1：Gly-Tyr-Ser-Phe-Thr-Ser-Tyr-Trp；
CDR2：Ile-Tyr-Pro-Gly-Asn-Ser-Ala；
CDR3：Ile-Arg-Gly-Gly-Asn-Tyr。
The aminoacid sequence of described monoclonal antibody variable region of light chain such as SEQ ID NO.1, the aminoacid sequence of variable region of heavy chain is shown in SEQ ID NO.2.
The gene order of described coding monoclonal antibody variable region of light chain is shown in SEQ ID NO.3, and the gene order of coding monoclonal antibody variable region of heavy chain is shown in SEQ ID NO.4.
It is the preparation of the genetic engineering antibody or the diagnostic reagent of target spot that described monoclonal antibody is applied to make up with people EPCAM.
Compared with prior art, the present invention has following beneficial technical effects:
1, the FMU-EPCAM-2A9 monoclonal antibody provided by the invention monoclonal antibody that is the high-affinity of a kind of anti-people EPCAM, through indirect ELISA detect, flow cytometer detects and the pathological section immunohistochemistry detects and confirms that this monoclonal antibody can be specifically in conjunction with people EPCAM;
Clone this monoclonal antibody light chain, heavy chain variable region gene and aminoacid sequence, sequential analysis has confirmed the uniqueness of this antibody sequence.
2, analyze to obtain the CDR district of light chain, variable region of heavy chain, chimeric or humanized genetic engineering antibody provides support for the anti-people EPCAM of structure high-affinity on this basis.
Description of drawings
Fig. 1 is the FMU-EPCAM-2A9 monoclonal antibody of anti-people EPCAM and the EPCAM bonded flow cytometer detected result figure of COLO205 and SKBr-3 clone surface expression; Fig. 1 a combines detected result figure with COLO205 clone, and Fig. 1 b combines detected result figure with SKBr-3 clone; Wherein, FITC: fluorescein isothiocyanate, transverse axis are fluorescence intensity, and the longitudinal axis is relative cell counting, and M1 represents the positive cell scope;
Fig. 2 is the EPCAM bonded immunohistochemical staining detected result figure that expresses in the FMU-EPCAM-2A9 monoclonal antibody of anti-people EPCAM and the colorectal carcinoma sample; Fig. 2 a is 200 times of microscopy observations, and Fig. 2 b is 400 times of microscopy observations;
Fig. 3 is the FMU-EPCAM-2A9 monoclonal antibody chain variable region gene homology sequence detected result figure of anti-people EPCAM;
Fig. 4 is the FMU-EPCAM-2A9 monoclonal antibody heavy chain variable region gene homology sequence detected result figure of anti-people EPCAM;
Fig. 5 is the FMU-EPCAM-2A9 monoclonal antibody variable region of light chain amino acid identity sequential detection figure as a result of anti-people EPCAM;
Fig. 6 is the FMU-EPCAM-2A9 monoclonal antibody variable region of heavy chain amino acid identity sequential detection figure as a result of anti-people EPCAM.
Embodiment
The applicant uses recombinant human EPCAM immunity BALB/c mouse, prepared one group of mouse anti human EPCAM monoclonal antibody, filter out the hybridoma cell strain of the FMU-EPCAM-2A9 monoclonal antibody of the anti-people EPCAM of energy stably excreting high-affinity, preparation ascites obtains the anti-people EPCAM monoclonal antibody of high-affinity.Confirm the uniqueness and the CDR sequence thereof of this gene order and corresponding protein sequence; Chimeric or humanized genetic engineering antibody provides support for anti-people EPCAM.
Below in conjunction with accompanying drawing the present invention is elaborated, the explanation of the invention is not limited.
The present invention specifically implements according to the following steps:
The preparation of 1 mouse anti human EPCAM high-affinity antibody
1.1 MONOCLONAL ANTIBODIES SPECIFIC FOR, purifying
With reference to GENBANK NM_002354 sequences Design primer, and the proteic gene of amplification coding people EPCAM after birth outskirt; Then this gene clone is gone in the pGEX-4T-3 carrier, make up prokaryotic expression carrier and transfection host cell; Induce by IPTG, freeze thawing adds the soluble protein that the ultrasonic degradation method obtains recombinant human EPCAM after birth outskirt again.
(cell and molecular immunology experimental technique first version P9-P17), are used recombinant human EPCAM after birth outskirt protein immunization BALB/c mouse (available from The Fourth Military Medical University's Experimental Animal Center) to press method for preparing monoclonal antibody; Initial immunity uses the Fu Shi Freund's complete adjuvant, and follow-up immunization uses freund 's incomplete adjuvant, each 3 weeks at interval, be subcutaneous multi-point injection, and immunity is 4 times altogether.Last immunity after 7-10 days blood sampling survey it and tire, detect immune effect.Behind the At intervals of two to three weeks,, put to death animal after 3 days and get spleen and carry out cytogamy through intravenous injection antigen booster immunization again.
The murine myeloma cell SP2/0 counting of taking the logarithm and growing prepares the immune spleen cell suspension simultaneously.Myeloma cell and splenocyte are carried out cytogamy by 1: 10 mixed.Merge the back cell suspension and add 96 orifice plates that contain feeder cell, 37 ℃, 5%CO
2Incubator is cultivated.After treating that the clone occurs, indirect ELISA detects, and selects positive colony.Adopt limiting dilution assay to carry out cloning to the cell that contains the positive colony hole, until obtain can stably excreting antibody hybridoma cell line (in-vitro cultivation is above 6 months), and it is carried out conventional caryogram is identified and Ig subclass mensuration (result is the IgG2a subclass).
Obtain can the hybridoma cell strain of stably excreting antibody after, by mouse ascites preparation method preparation comprise monoclonal antibody ascites (cell and molecular immunology experimental technique first version, P9-P17).Ascites adopts QFF anion exchange chromatography purifying behind 40% saturated ammonium sulphate.With the purity of SDS-PAGE purification Identification antibody, the monoclonal antibody FMU-EPCAM-2A9 purity of purifying reaches 95%.
1.2 anti-people EPCAM monoclonal antibody titration
Relative affinity with mAb behind ascites and the purifying before the indirect ELISA method mensuration purifying.Wherein envelope antigen is the soluble protein of recombinant human EPCAM after birth outskirt, and testing sample is mAb behind the ascites of serial dilution and the purifying, and detecting antibody is sheep anti mouse-HRP enzymic-labelled antibody, and substrate uses ABTS.The high-affinity FMU-EPCAM-2A9mAb that is filtered out, it is 1 * 10 that ascites is tired
-7, tiring behind the purifying is 0.5ng/ml, reaches 1 * 10 and generally adopt indirect ELISA detection ascites to tire
-5Above antibody can use.
1.3 the flow cytometry fluorescent dye detects anti-people EPCAM monoclonal antibody FMU-EPCAM-2A9 and cell surface EPCAM bonded activity
As negative antibody control, the EPCAM of Flow cytometry FMU-EPCAM-2A9mAb and COLO205 and SKBr-3 clone surface expression combines with SEDmAb;
To logarithmic phase, adjusting cell concn is 5 * 10 with the 10%FCS-RPMI1640 culture medium culturing for COLO205 and SKBr-3 clone
6～1 * 10
7/ ml; Get 50 μ l cell suspensions and add specificity FMU-EPCAM-2A9mAb ascites 1 μ l, add 1 μ l deactivation normal rabbit serum again, hatch 30min for 4 ℃; Same method prepares negative control group.
With washings (5%FCS-PBS-4%NaN
3) washed cell 2 times, add washings 2ml at every turn, abandon supernatant after 1000rpm * 5min is centrifugal; The sheep anti mouse fluorescent-labeled antibody that adds 100 μ l working concentrations, shake well is hatched 30min for 4 ℃;
With washings washing 2 times, each liquid feeding 2ml abandons supernatant after 1000rpm * 5min is centrifugal; Add a certain amount of stationary liquid, carry out flow cytometry FMU-EPCAM-2A9mAb then and combine situation with the EPCAM of cell surface;
The result as shown in Figure 1, in COLO205 clone: compare with negative control, combine FMU-EPCAM-2A9mAb and the COLO205 cell that falls into M1 positive cell scope reaches 46%;
In SKBr-3 clone: compare with negative control, combine FMU-EPCAM-2A9mAb and the SKBr-3 cell that falls into M1 positive cell scope reaches 24%;
Above flow cytometry fluorescent dye detects and shows that FMU-EPCAM-2A9mAb can discern and in conjunction with the natural EPCAM of COLO205 and SKBr-3 cell surface expression.
1.4 immunohistochemical staining detects combining of EPCAM in FMU-EPCAM-2A9mAb and the colorectal carcinoma sample
Colorectal carcinoma paraffin section (The Fourth Military Medical University's Pathological Staff Room provides), respectively by dimethylbenzene I, II, 100% alcohol I, II, 95%, 90%, 80% and 70% each 10min of alcohol makes to dewax to water; 0.3% methyl alcohol-H
2O
230min is with deactivating endogenous peroxydase in the effect section; 0.01M PBS (pH7.3) rinsing 10min, rinsing 3 times; 95 ℃ of antigen retrieval liquid (available from Fuzhou Maixin biotechnology Development Co., Ltd) effect 20min; Sealing 30min (confining liquid is 1% normal rabbit serum or 5% normal sheep serum);
Add one anti-(the FMU-EPCAM-2A9mAb ascites of dilution in 1: 100), 4 ℃ of wet boxes are hatched more than the 18h; 0.01M PBS (pH7.3) rinsing 10min * 3 times; The sheep anti mouse two that adds the HRP mark is anti-, and the wet box of room temperature is hatched 2h～3h;
0.01M PBS (pH7.3) rinsing 10min * 3 times; PBS stops behind DAB colour developing 7～8min.
Light microscopic is observed down, haematoxylin redyeing 10～20s; The result as shown in Figure 2, the position that combines FMU-EPCAM-2A9mAb on the colorectal carcinoma sample presents brown, explanation can be discerned the EPCAM that the colorectal carcinoma sample is expressed.
By above-mentioned 3 kinds of FMU-EPCAM-2A9mAb in conjunction with experiment, qualification result at different levels shows, FMU-EPCAM-2A9mAb both can discern recombinant expressed EPCAM (indirect ELISA detection), again can recognizing cells the EPCAM (immunohistochemical staining) of system and the natural expression of tissue surface, illustrate that FMU-EPCAM-2A9mAb combines with corresponding antigens EPCAM and has good specificity and avidity.
The FMU-EPCAM-2A9 monoclonal antibody light chain of 2 anti-people EPCAM and the clone of heavy chain variable region gene
2.1FMU-EPCAM-2A9 the cultivation of hybridoma
(P88) FMU-EPCAM-2A9 hybridoma is cultivated based on 37 ℃ 5%CO with the RPMI 1640 that contains 10% calf serum for cell cultures, first version in recovery according to a conventional method
2Be cultured to logarithmic phase in the incubator.
2.2 the extraction of total RNA and cDNA first chain is synthetic
With TRIZOL Reagent (available from U.S. GIBCO company), by specification extracts total RNA.
2.3RT-PCR VL and the VH gene of method amplification FMU-EPCAM-2A9mb
Single stage method RT-PCR amplification kit is pressed the VL of test kit specification sheets amplification FMU-EPCAM-2A9mAb and the VH gene of FMU-EPCAM-2A9mAb available from TakaRa company;
Utilizing VL F (upstream primer) and VL B (downstream primer) and VH F and two pairs of primers of VH B, is that template is carried out RT-PCR with total RNA;
Reaction volume 50 μ l, reaction conditions is: 94 ℃ of 5min; 95 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 1min circulate 35 times; 72 ℃ of 7min; Primer sequence is:
VL?F：gttagatctc?cagcttggtc?cc 22；
VL?B：gacattcagc?tgacccagtc?tcca 24；
VH?F：tgaggagacg?gtgaccgtgg?tcccttggcc?ccag?34；
VH?B：aggtsmarct?gcagsagtcw?gg 22；
(the IUB standard annexs base code: s:c/g; M:a/c; R:a/g; W:a/t)
2.4PCR the clone of amplified production and screening
The PCR product is through 1.5% agarose gel electrophoresis; reclaim test kit (can take charge of) with a small amount of glue and reclaim pcr amplified fragment available from Shanghai China Shun biotechnology is limited; with dna ligation kit (available from TakaRa company) with this fragment by specification; utilization adds the A tail and inserts in the pMD-T18 carrier (available from TakaRa company); connector Transformed E .coli is (available from Chinese common micro-organisms DSMZ; CGMCC, Beijing), 37 ℃ of overnight incubation in Amp resistance LB agar culture dish.
Clone in the picking LB agar culture dish, 37 ℃ are shaken bacterium and spend the night in Amp resistance LB substratum, are template with 1 μ l bacterium liquid, by above-mentioned primer at light chain, variable region of heavy chain design, with the positive E.coli clone of PCR method screening reorganization;
Institute is obtained the positive E.coli clone of reorganization shake bacterium, bacterium liquid is delivered Shanghai biotechnology Services Co., Ltd and is finished gene sequencing, the gene order of variable region of light chain is shown in SEQ ID NO.3, and the gene order of variable region of heavy chain is shown in SEQ ID NO.4.
The nucleotide sequence of 3FMU-EPCAM-2A9mAb light chain and variable region of heavy chain and homology analysis
3.1 after determining that order-checking is errorless, in the GenBank+EMBL+DDBJ+PDB database, carry out nucleotide sequence homology analysis (Blastn).
The FMU-EPCAM-2A9mAb chain variable region gene is the highest with the mouse Ig chain variable region gene homology that is numbered GeneID:243420, reaches 292/299 (97%), as shown in Figure 3;
The FMU-EPCAM-2A9mAb heavy chain variable region gene is the highest with the mouse Ig heavy chain variable region gene homology that is numbered GeneID:668469, is 262/288 (90%), as shown in Figure 4.
Homology analysis shows, the nucleotide sequence of light, the variable region of heavy chain of coding FMU-EPCAM-2A9mAb, although certain homology is arranged with other gene order, do not find and the identical gene order of the present invention, show that the present invention has uniqueness on gene order.
3.2 variable region gene is translated into aminoacid sequence, carries out amino acid sequence analysis
The aminoacid sequence of monoclonal antibody variable region of light chain such as SEQ ID NO.1, the aminoacid sequence of variable region of heavy chain is shown in SEQ ID NO.2.
In non-redundant Genbank CDS translations+PDB+SwissProt+PIR+PRF Protein Data Bank, carry out amino acid sequence homology analysis (Blastp).
Analytical results shows that the FMU-EPCAM-2A9mAb light-chain amino acid sequence is the highest with the mouse Ig κ chain homology that is numbered ABR32168.1GI:149799217, reaches 106/111 (95%), as shown in Figure 5;
The FMU-EPCAM-2A9mAb heavy chain amino acid sequence is the highest with the homology of the mouse Ig heavy chain protein that is numbered S55541 GI:1363159, is 98/117 (83%), as shown in Figure 6.
Homology analysis shows, FMU-EPCAM-2A9mAb is light, the aminoacid sequence of variable region of heavy chain, although certain homology is arranged with other Argine Monohydrochloride sequence, do not find and the identical aminoacid sequence of the present invention, show that the present invention also has uniqueness on aminoacid sequence.
3.3 utilize IMGT/V-QUEST to analyze the variable region structure, determine the CDR district.
To check order gained FMU-EPCAM-2A9mAb light chain of antibody and weight chain variabl area sequence, (http://imgt.cines.fr/IMGT_vquest/vquest) analyzes in the IMGT/V-QUEST website, draws its CDR district.
3 complementary determining regions (CDR) sequence of variable region of light chain shown in SEQ ID NO.1 line part, is specially:
CDR1：Gln-Ser-Leu-Leu-Asp-Ser-Asp-Gly-Lys-Thr-Tyr；
CDR2：Val-Val-Ser-Lys-Leu-Asp-Ser；
CDR3：Trp-Gln-Gly-Thr-His-Phe-Pro-Trp-Thr；
3 complementary determining regions (CDR) sequence of described variable region of heavy chain is specially shown in SEQ ID NO.2 line part:
CDR1：Gly-Tyr-Ser-Phe-Thr-Ser-Tyr-Trp；
CDR2：Ile-Tyr-Pro-Gly-Asn-Ser-Ala；
CDR3：Ile-Arg-Gly-Gly-Asn-Tyr。
4. genetic engineering antibody design
Based on expression, purifying and the sequential analysis of anti-people EPCAM monoclonal antibody FMU-EPCAM-2A9, the following biological products of design construction
1) structure of single-chain antibody: can FMU-EPCAM-2A9mAb of the present invention is light, heavy chain variable region gene connects by linker, insert protokaryon or carrier for expression of eukaryon, transform host bacterium or transfecting eukaryotic cells, being used for preparation can be to medicative single-chain antibodies of tumour such as mammary cancer, colorectal carcinoma and carcinoma of the pancreas.
2) structure of people-mouse-anti EPCAM chimeric antibody: can FMU-EPCAM-2A9mAb of the present invention is light, heavy chain variable region gene inserts in the universal chimeric antibody expression vector, acquisition contains the carrier transfecting eukaryotic cells of mosaic gene, is used for preparation and is expected medicative chimeric antibodies of tumour such as mammary cancer, colorectal carcinoma and carcinoma of the pancreas.
3) structure of humanized antibody: can be transplanted in the skeleton district (FR) of human antibody variable region in CDR district FMU-EPCAM-2A9mAb of the present invention is light, heavy chain variable region gene, form complementary determining region (CDR) grafted antibody (CDR-grafted antibody), also weigh structure antibody (reshapingantibody) or humanized antibody (humanized antibody).
Utilize CDR implantation technique engineered antibody, can obtain both to have kept mouse source property parent mAb specificity, again more near the novel antibody of people's antibody, being used for preparation can be to medicative humanized antibodies of tumour such as mammary cancer, colorectal carcinoma and carcinoma of the pancreas.
4) can be according to gene order of the present invention and amino acid sequence coded thereof, preparation is at other biological products of people EPCAM functional epitope.
5) can utilize the FMU-EPCAM-2A9 specific monoclonal antibody of anti-people EPCAM of the present invention as the instrument of distinguishing epithelium and non-epithelial origin tissue, to make a definite diagnosis the disease that some are easy to obscure.By adopting the FMU-EPCAM-2A9 specific monoclonal antibody dyeing of anti-people EPCAM of the present invention, the expression intensity of EPCAM in the tissues observed may be as the judgement symbol of some epithelial origin tumor prognosis and differentiation degree.
6) can use the proteic ELISA test kit of EPCAM after birth outskirt in the high-affinity FMU-EPCAM-2A9mAb formation determination body fluid of the present invention, be expected in the clinical diagnosis of tumour generation, development and prognosis, to be with a wide range of applications.
Amino acid and nucleotides sequence tabulation
＜110〉The Fourth Military Medical University of P.L.A
＜120〉light chain of FMU-EPCAM-2A9 monoclonal antibody and variable region of heavy chain
<160>4
<210>1
<211>111
<212>PRT
＜213〉synthetic
<400>1
Asp?Val?Val?Leu?Thr?Gln?Ser?Pro?Leu?Thr?Leu?Ser?Val?Thr?Ile?Gly?Gln?Pro?Ala?Ser
1 5 10 15 20
Ile?Ser?Cys?Lys?Ser?Ser?
Gln?Ser?Leu?Leu?Asp?Ser?Asp?Gly?Lys?Thr?Tyr?Leu?Asn?Trp
25 30 35 40
Leu?Phe?Gln?Arg?Pro?Gly?Gln?Ser?Pro?Lys?Arg?Leu?Ile?Tyr?
Val?Val?Ser?Lys?Leu?Asp
45 50 55 60
Ser?Gly?Val?Pro?Asp?Arg?Phe?Tyr?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile
65 70 75 80
Ser?Arg?Val?Glu?Ala?Glu?Asp?Leu?Gly?Val?Tyr?Tyr?Cys?
Trp?Gln?Gly?Thr?His?Phe?Pro
85 90 95 100
Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile
105 110
<210>2
<211>113
<212>PRT
＜213〉synthetic
<400>2
Glu?Val?Lys?Leu?Gln?Glu?Ser?Gly?Thr?Val?Leu?Ala?Arg?Pro?Gly?Thr?Ser?Val?Lys?Met
1 5 10 15 20
Ser?Cys?Arg?Ala?Ser?
Gly?Tyr?Ser?Phe?Tyr?Ser?Tyr?Trp?Leu?His?Trp?Ile?Lys?Gln?Arg
25 30 35 40
Pro?Gly?Gln?Gly?Leu?Glu?Trp?Val?Gly?Gly?
Ile?Tyr?Pro?Gly?Asn?Ser?Ala?Thr?Ser?Tyr
45 50 55 60
Lys?Gln?Lys?Phe?Lys?Asp?Lys?Ala?Thr?Leu?Thr?Ala?Val?Thr?Ser?Ala?Ser?Thr?Ala?Tyr
65 70 75 80
Met?Glu?Leu?Ser?Ser?Leu?Thr?Asn?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys?
Ile?Arg?Gly?Gly
85 90 95 100
Asn?Tyr?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser
105 110
<210>3
<211>333
<212>DNA
＜213〉synthetic
<400>3
gatgttgtgc?tgacccagtc?tccactcact?ttgtcggtta?ccattggaca?accagcctcc 60
atctcttgca?agtcaagtca?gagcctctta?gatagtgatg?gaaagacata?tttgaattgg 120
ttgttccaga?ggccaggcca?gtctccaaag?cgcctgatct?atgtggtgtc?taaactggac 180
tctggagtcc?ctgacaggtt?cactggcagt?ggatcaggga?cagatttcac?actgaaaatc 240
agtagagtgg?aggctgagga?tttgggagtt?tattattgct?ggcaaggtac?acactttccg 300
tggacgttcg?gtggagggac?caagctggag?atc 333
<210>4
<211>339
<212>DNA
＜213〉synthetic
<400>4
gaggtgaagc?tgcaggagtc?tgggactgtg?ctggcaaggc?ctgggacttc?cgtgaagatg 60
tcctgcaggg?cttctggcta?cagttttacc?agctactggt?tgcactggat?aaaacagagg 120
cctggacagg?gtctagaatg?ggttggtggt?atctatcctg?gaaatagtgc?tactagttac 180
aaacagaagt?ttaaggacaa?ggccacactg?actgcagtca?catccgccag?tactgcctac 240
atggaactca?gtagcctgac?aaatgaggac?tctgcggtct?attactgcat?aagagggggg 300
aactactggg?gccaagggac?cacggtcacc?gtctcctca 339
Claims (4)
1.FMU-EPCAM-2A9 monoclonal antibody comprises light chain and heavy chain, it is characterized in that, 3 complementary determining regions (CDR) sequence of the variable region of described light chain is:
CDR1：Gln-Ser-Leu-Leu-Asp-Ser-Asp-Gly-Lys-Thr-Tyr；
CDR2：Val-Val-Ser-Lys-Leu-Asp-Ser；
CDR3：Trp-Gln-Gly-Thr-His-Phe-Pro-Trp-Thr；
3 complementary determining regions (CDR) sequence of the variable region of described heavy chain is:
CDR1：Gly-Tyr-Ser-Phe-Thr-Ser-Tyr-Trp；
CDR2：Ile-Tyr-Pro-Gly-Asn-Ser-Ala；
CDR3：Ile-Arg-Gly-Gly-Asn-Tyr。
2. FMU-EPCAM-2A9 monoclonal antibody as claimed in claim 1 is characterized in that, the aminoacid sequence of described monoclonal antibody variable region of light chain such as SEQ ID NO.1, and the aminoacid sequence of variable region of heavy chain is shown in SEQ ID NO.2.
3. FMU-EPCAM-2A9 monoclonal antibody as claimed in claim 1 is characterized in that, the gene order of coding monoclonal antibody variable region of light chain is shown in SEQ ID NO.3, and the gene order of coding monoclonal antibody variable region of heavy chain is shown in SEQ ID NO.4.
4. to be applied to make up with people EPCAM be the preparation of the genetic engineering antibody or the diagnostic reagent of target spot to the described FMU-EPCAM-2A9 monoclonal antibody of claim 1.
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