CN1281753C

CN1281753C - Monoclonal antibody CAb-1 heavy and light chain variable region gene of human carcinoma of large intestine, and its uses

Info

Publication number: CN1281753C
Application number: CN 200310118924
Authority: CN
Inventors: 陈志南; 邢金良; 杨向民; 张思河
Original assignee: 陈志南
Current assignee: Fourth Military Medical University FMMU
Priority date: 2003-12-08
Filing date: 2003-12-08
Publication date: 2006-10-25
Anticipated expiration: 2023-12-08
Also published as: CN1546664A

Abstract

The present invention relates to a gene of the variable regions of the heavy chain the light chain of monoclonal antibodies CAb-1 for resisting the large intestine cancer of a human body, a polypeptide coded by the gene, and the application of the gene and the polypeptide for preparing medicines used for diagnosing and treating the large intestine cancer (segmented intestine and rectum). In the present invention, the inventor uses a set of designed amplification primers to successfully clone corresponding antibody variable regions of the light chain and the heavy chain from the hybridoma cells of a cultured monoclonal antibodies CAb-1 for resisting the large intestine cancer; on the basis of the gene, the gene can be constructed and expressed into small molecule genetic engineering antibodies, such as Fab antibodies, chimeric antibodies, antibody fusion proteins, etc., in various types by the genetic engineering method and can be used for preparing medicines for diagnosing and treating the large intestine cancer (segmented intestine and rectum); the polypeptide coded on the basis of the gene can be crossly linked with various effect molecules for preparing medicines for diagnosing and treating large intestine cancer(segmented intestine and rectum).

Description

Anti-human large intestine cancer monoclonal antibody CAb-1 is light, heavy chain variable region gene and application thereof

Technical field

The present invention relates to light, heavy chain variable region gene and the encoded polypeptide thereof of the anti-human large intestine cancer monoclonal antibody of strain CAb-1, and described gene and polypeptide are used to diagnose and treat the application of the medicine of large bowel cancer in preparation.

Background technology

Large bowel cancer is harm crowd's a common cancer.Its sickness rate is in second the America and Europe, is number three in China, and along with the continuous adjustment of people's dietary structure, its sickness rate is still on the rise.Therefore diagnosis and the treatment of studying large bowel cancer have great importance.Though present nucleus magnetic resonance, endoscopic technique constantly develops, and the recall rate of early stage large bowel cancer tumour increases, and clinical practice detects and still has false positive, false negative phenomenon, and promptly susceptibility and specificity can not be satisfactory.Making a definite diagnosis of large bowel cancer is late period all clinically, even excised primary lesion, still about 50%, visible early stage discovery, diagnosis and treatment is crucial to the recurrence rate in 2 years.Since Kohler in 1975 and Milstein create the B lymphocyte hybridoma technology, various monoclonal antibodies (McAb) occur in succession, these monoclonal antibodies are not only being brought into play positive effect aspect the fundamental research of disease, also brought new hope for the diagnosis and treatment of multiple intractable tumour simultaneously.At present, the FdA approval is used for the existing 15 kinds of listings of tumor treatment antibody, and wherein the monoclonal antibody at large bowel cancer is the antigenic mouse of a target 17-1A source property monoclonal antibody.This series products enters the F that clinical trial all is the entirely anti-or antibody of the direct mark mouse of application of radiation isotropic substance source property (ab ') ₂Fragment.Up to now, the target antigen that is used for large bowel cancer diagnosis and treatment research both at home and abroad has 17-1A, TAG-72, CEA, A33 etc.Aspect the preparation of large bowel cancer monoclonal antibody, the methods of tradition immunity that adopt prepare antibody more, and promptly the colorectal cancer cells with soluble substance or cultivation is an immunogen.Because the variation or the partial loss of tumour antigen, thereby this method is difficult to obtain the large bowel cancer monoclonal antibody of high-affinity, high specific.We have obtained a strain large bowel cancer have been presented better reactivity and specific hybridoma cell strain CAb-1 through secular a large amount of colony screening, and it has higher using value and DEVELOPMENT PROSPECT.

Summary of the invention

One object of the present invention is to provide heavy chain variable region gene and chain variable region gene and the encoded polypeptides thereof of the monoclonal antibody CAb-1 of Chinese People's Anti-Japanese Military and Political College's intestinal cancer, can give expression to specific recognition after its reorganization and in conjunction with the antibody activity fragment of large bowel cancer related antigen.

Another object of the present invention is to the gene of described antibody CAb-1 or polypeptide is used for diagnosing and treating the large bowel cancer medicine in preparation application.

According to an aspect of the present invention, the present invention relates to heavy chain and the chain variable region gene of the monoclonal antibody CAb-1 of a kind of Chinese People's Anti-Japanese Military and Political College intestinal cancer.The Chinese People's Anti-Japanese Military and Political College of the present invention intestinal cancer monoclonal antibody CAb-1 heavy chain and chain variable region gene are to clone to obtain from the hybridoma cell strain CAb-1 of the mouse source property CAb-1 monoclonal antibody that can secrete greater activity.The inventor utilizes a cover primer amplification of design to go out full-length light chains and the heavy chain Fd gene of large bowel cancer monoclonal antibody CAb-1, through sequencing with in NCBI after the BLAST comparative analysis, confirm that described CAb-1 heavy chain variable region gene total length is 351bp, its nucleotide sequence as in the sequence table＜400〉1 shown in, its amino acid sequence coded as in the sequence table＜400〉2 shown in.Described CAb-1 chain variable region gene total length is 393bp, its nucleotide sequence as in the sequence table＜400〉3 shown in, its amino acid sequence coded as in the sequence table＜400〉4 shown in, above-mentioned two genes can give expression to specific recognition and in conjunction with two strain colorectal cancer cells SW480,8693 monoclonal antibody active fragments after reorganization.

Light, the heavy chain variable region gene of the large bowel cancer specific monoclonal antibody CAb-1 antibody of successfully cloning for the inventor, use known antibodies geneseq database (IMGT) professional on the Internet respectively and (NCBI) institute's calling sequence is carried out the homology comparison and show with its germline gene source analysis, the gene order that is obtained is all not quite identical from the various antibody gene sequences of mouse germline gene and existing report really.The mouse antibodies variable region that gained VH and VL gene codified are correct.

According to another aspect of the present invention, the invention still further relates to the gene of described monoclonal antibody CAb-1 and encoded polypeptides is used for diagnosing and treating the large bowel cancer tumour medicine in preparation application thereof.

The amplimer that the inventor uses an Analysis of Nested Design has cloned from the hybridoma of the intestinal cancer specific monoclonal antibody CAb-1 of the strain Chinese People's Anti-Japanese Military and Political College that cultivates successfully that its corresponding antibody is light, heavy chain variable region gene.Light, heavy chain variable region gene based on above-mentioned monoclonal antibody CAb-1, can adopt gene engineering method, make up and be expressed as the small molecules genetic engineering antibody of various ways, as Fab antibody, chimeric antibody, antibody fusion protein etc., preparation is used to diagnose and treat the medicine of large bowel cancer tumour.Based on light, the heavy chain variable region gene encoded polypeptide product of above-mentioned CAb-1, can crosslinkedly go up wide variety of effector molecules, preparation is used to diagnose and treat the medicine of large bowel cancer tumour.

Description of drawings

Fig. 1 is light for the anti-human large intestine cancer monoclonal antibody CAb-1 of RT-PCR amplification, the agarose gel electrophoresis figure of heavy chain variable region gene.

Fig. 2 is the order-checking collection of illustrative plates of anti-human large intestine cancer monoclonal antibody CAb-1 chain variable region gene.

Fig. 3 is the order-checking collection of illustrative plates of anti-human large intestine cancer monoclonal antibody CAb-1 heavy chain variable region gene.

Fig. 4 utilizes the gene constructed Fab antibody expression vector of the CAb-1 of acquisition synoptic diagram.

Fig. 5 is by cell (SW480) ELISA of the gene constructed Fab antibody of CAb-1.

Fig. 6 is by cell (SW480) immunofluorescence of the gene constructed Fab antibody of CAb-1.

Embodiment

(1) clone of light, the heavy chain variable region gene of anti-human large intestine cancer monoclonal antibody CAb-1

Used cell strain be the inventor with fresh Colorectal Carcinoma homogenate immune mouse, adopt traditional fusion method and a plant height avidity that obtains, the anti-people's colorectal cell of the mouse source property cancer monoclonal antibody CAb-1 hybridoma cell strain of high specific.Its excretory antibody not with CEA, 17-1A, TAG-72, CEA, tumor associated antigen combinations such as A33 are to be about new tumor-associated glycoprotein antigen about 200KD at a kind of molecular weight.Its excretory antibody molecule hypotype is IgG1 κ.

Get the CAb-1 hybridoma (5 * 10 that is in logarithmic phase ⁶), adopt the guanidinium isothiocyanate single stage method to extract total RNA, take a morsel and carry out the quantitative and 1% denaturing formaldehyde agarose gel electrophoresis detection of ultraviolet spectrophotometer.Subsequently with Oligo (dT) ₁₅(Promega company) is random primer, and cDNA first chain is synthesized in reverse transcription.Then, utilize one of design to overlap full-length light chains and the heavy chain Fd gene that primer amplifies this strain large bowel cancer monoclonal antibody CAb-1 respectively.(VH, amplified production VL) cut glue and separate the target fragment behind 1% LMP agarose gel electrophoresis will to contain variable region gene.Behind gel-purified test kit (promegaInc) recovery purifying, gel electrophoresis is identified, referring to accompanying drawing 1.Need to prove, the heavy chain FD gene of the large bowel cancer monoclonal antibody CAb-1 of shown amplification is to obtain from the skeleton district FR1 of antibody among Fig. 1, thereby its size is littler than the light chain gene that contains signal peptide. the chain variable region gene of monoclonal antibody CAb-1 increases from the signal peptide position.Afterwards, to the T carrier, sequencing analysis is referring to accompanying drawing 2,3 with the purpose fragment cloning.

Oligo (dT) ₁₅(Promega company) is as follows for the reverse transcription scheme of random primer: add the total RNA of 1 μ g (2 μ L) in the 20 μ L reaction systems successively, 0.5ug random primer Oligo (dT) ₁₅(1 μ L), 4ulMgCL2 (25mM) 2 μ L 5 * dNTPs, 2 μ L, 10 * damping fluid, 0.5 μ L RNase inhibitor, add ThermoScript II AMV 15U (0.75 μ L), water is mended to 20 μ L, mixing, 42 ℃ of water-bath 1h boil 3min, reaction product place-20 ℃ standby.

Pcr amplification reaction carries out according to a conventional method: with above-mentioned product is template, uses full-length light chains, the heavy chain Fd gene of 5 counterweight strand primers and 6 couples of light chain primer amplification large bowel cancer monoclonal antibody CAb-1 respectively.Reaction system is: template cDNA 2.5ul, dNTP (each 0.4mM), 10 * Buffer 5ul, Ex Taq archaeal dna polymerase 1.25u, 5 ' end and 3 ' end each 5ul of primer (about 30pmol) add water to 50ul, mixing, instantaneous centrifugal after, add 1-2 drop of liquid paraffin, put on the PCR instrument and react.Reaction conditions: 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 1min, 35 circulations, last 72 ℃ are extended 10min.

The CAb-1 monoclonal antibody variable region gene pcr amplification primer sequence of design is as follows:

Murine heavy chain V district 5 ' end primer

VH1：5’-GGG?GAT?ATC?CAC?CAT?GG?(AG)?ATG?(CG)?AG?CTG?(TG)?GT?(CA)AT?(CG)?CT?CTT-3’

VH2：5’-GGG?GAT?ATC?CAC?CAT?G(AG)A?CTT?CGG?G(TC)T?GAG?CT(TG)GGT?TTT-3’

VH3：5’-GGG?GAT?ATC?CAC?CAT?GGC?TGT?CTT?GGG?GCT?GCT?CTT?CT-3’

VH4：5’-GGG?GAT?ATC?CAC?CAT?GAT(AG)GT?GTT(AG)AG?TCT?T(CT)T?GT(AG)CCT?G3’

H-FR1：5′-AGG?T(GC)(AC)?A(GA)C?T(GT)C?TCG?AGT?C(AT)GG-3′；

Murine heavy chain V district 3 ' end primer

Fd?3′：5′-AGG?CTT?ACT?AGT?ACA?ATC?CCT?GGG?CAC?AAT-3′；

Mouse light chain V district 5 ' end primer

VL1：5’-GGG?GAT?ATC?CAC?CAT?GGA?GAC?AGA?CAC?ACT?CCT?GCT?AT-3’

VL2：5’-GGG?GAT?ATC?CAC?CAT?GGA?TTT?TCA?AGT?GCA?GAT?TTT?CAG-3’

VL3：5’-GGG?GAT?ATC?CAC?CAT?GGA?G(AT)C?ACA(GT)(AT)C?TCG?GGTCTT?T(GA)T?A-3’

VL4：5’-GGG?GAT?ATC?CAC?CAT?G(GT)C?CCC(AT)(AG)C?TCA?G(CT)T?C(CT)C?T(TG)G?T-3’

VL5：5’-GGG?GAT?ATC?CAC?CAT?GAA?GTT?GCC?TGT?TAG?GCT?GTT?G-3’

L-FR1：5′-GAT?GTG?AGC?TCG?TGA?TGA?CCC?AGA?CTC?C-3′；

Mouse light chain light chain 3 ' end primer:

Lc3′：5′-GCG?CCG?TCT?AGA?ATT?AAC?ACT?CAT?TCC?TGT?TGA?A-3′；

Pseudogene is rejected primer

Pseudogene 5 ' end primer: 5 '-TCT GGC TAT AGT TAT ATG CAC-3 ';

Pseudogene 3 ' end primer: 5 '-TGT AAG CTC CCT AAT GTG CTG-3 '.

Purpose fragment T carrier cloning order-checking scheme is as follows: with reclaiming after the PCR product gel electrophoretic separation, be connected into the pMD18-T carrier.The ligation system is: pMD18-T carrier 1ul, PCR product gel purifying heavy chain (or light chain) 3ul, deionized water 1ul, connect damping fluid 5ul, 4 ℃ are spent the night behind the mixing, transformed into escherichia coli JM109, three of screening recombinant clones, adopt the universal sequencing primer thing to carry out two-way sequencing reaction then, sequencing result is seen Fig. 2 and Fig. 3.

Further as follows to the anti-human large intestine cancer monoclonal antibody CAb-1 that cloned is light, heavy chain variable region gene carries out sequential analysis:

Use following two databases respectively;

http：//imgt.cines.fr：8104/cgi-bin/IMGTdnap.jv

http：//www.ncbi.nlm.nih.gov/blast/Blast.cgi，

Institute's calling sequence and existing other various antibody genes of having reported are carried out homology relatively, and analyze its germline gene source, the result is as follows:

(1) CAb-1 heavy chain＜400〉1 germline gene source:

V-GENE：L14363?IGHV9S3*02。

D-GENE：M23243?IGHD-ST4*01。

J-GENE：V00770，IGHJ2*01。

Analyze demonstration by FR-IMGT and CDR-IMGT:

CDR1：GGG?TAT?ACC?TTC?ACA?GCC?TAT?GGA；

CDR2：ATA?AAC?ACC?TAC?ACT?GGA?GAA?CCA；

CDR3：GCA?AGA?CGG?GGG?TAC?GGC?TAC?TAC?TTT?GAC?TAC

The homology comparative result shows among the NCBI:

Request?ID1067677802-5302-660581.BLASTQ3

Query＝Databa?se：All?GenBank+EMBL+DDBJ+PDB?sequences(but?noEST，STS，GSS，or?pha?se?0，1?or?2?HTGS?sequences)

Sequences?producing?significant?alignments：(bits)

Value

gi|195813|gb|M60239.1|MUSIGHP139?Mouse?rearranged?anti-NPa...452?e-124

gi|31322148|gb|AY169677.1|Mus?musculus?clone?VG2J2.8immun...446?e-122

gi|18307783|gb|AF393386.1|AF393386?Mus?musculus?clone?C7an...446?e-122

(2) CAb-1 light chain＜400〉3 germline genes source:

V-GENE：D00080?IGKV1-110*01。

J-GENE：V00777?IGKJ5*01。

Analyze demonstration by FR-IMGT and CDR-IMGT:

CDR1：CAG?AGC?CTT?GTA?CAC?AGT?GAT?GGA?GAC?ACC?TAT

CDR2：AAA?GTT?TCC

CDR3：TCG?CAA?AGT?GCA?CAT?GTT?TCT?CCC?ACG

The homology comparative result shows among the NCBI:

Sequences?producing?significant?alignments：(bits)Valuegi|639654|gb|M32381.1|MUSIGK1025?Mus?musculus(clone10-25)...676 0.0

gi|18044163|gb|BC019760.1|Mus?musculus?immunoglobulinkapp...662 0.0

gi|639660|gb|M32382.1|MUSIGK324A?Mus?musculus(clone3-24)...638 e-180

Above-mentioned analytical results shows: the large bowel cancer monoclonal antibody CAb-1 gene order that is obtained is all not quite identical from various other known antibodies genes of mouse germline gene and existing report really, is to belong to a kind of new antibody gene at large bowel cancer that function is arranged.

(2) structure and the expression of the FAb form antibody of anti-human large intestine cancer monoclonal antibody CAb-1

GIII albumen among the phasmid expression vector pCOMB3 is excised, light, the heavy chain variable region gene of the large bowel cancer monoclonal antibody CAb-1 that is cloned with the present invention reassemble into solubility FAb expression vector and carry out the prokaryotic expression (see figure 4). with the negative contrast of the normal colorectal cell of people, detect the active (see figure 5) of expression product-solubility FAb with ELISA, found that expressed solubility FAb small molecular antibody, a specificity combines and does not combine with normal colorectal cell with colorectal cancer cells.In addition, with the immunofluorescence experiment (see figure 6) of this solubility FAb small molecular antibody of expressing, but show that expressed solubility FAb small molecular antibody specificity is combined on the cytolemma of colorectal cancer cells in conjunction with above-mentioned two kinds of cells.

(3) light, heavy chain variable region gene of anti-human large intestine cancer monoclonal antibody CAb-1 and encoded polypeptides product thereof are used for the application of diagnosis or treatment large intestine (colon, rectum) cancer drug in preparation.

(1) set out with light, heavy chain variable region gene of the present invention, can reconstruct and be expressed as the protein drug of various ways, can be directly used in the diagnosis and the treatment of large intestine (colon, rectum) cancer.For example: (1-1) chimeric antibody.Be that C district with the V district of mouse MAb and people Ig is formed by connecting and is people-mouse chimeric antibody.Because it has intactly kept specificity and the avidity of mouse MAb, has reduced untoward reactions such as HAMA simultaneously, so demonstrate good effect in the treatment of large bowel cancer.(1-2) humanized antibody.Humanization modified at the variable region gene structure, comprise that CDR transplants, surface amino groups acid residue frosting, the exchange of skeleton district, the location keeps and the epi-position guiding is selected etc., thereby the mouse source property that has not only reduced the variable region has kept specificity and the avidity of mouse MAb simultaneously again.(1-3) small molecular antibody.Mainly contain by VH-CH1 and VL-C1 form Fab antibody (seeing Fig. 4-6), with a polypeptide (GLy4Ser) ₃The single domain antibody that joint connects single-chain antibody that VH gene and VL gene form, be made up of with non covalent bond be combined into Fv fragment antibody, by VH or functional domain of VL VH and VL, the atom that constitutes by single CDR etc.(1-4) multivalence miniantibody.Mainly contain double-stranded antibody, (ScFv) ₂, Flex miniantibody, LD miniantibody, F (ab ') ₂, F (ab ') ₃, (ScFv) ₄Deng.Because the polyvalent antigen binding site is arranged, the avidity height, molecular size is moderate, can penetrate Colorectal Carcinoma, also slower characteristics of the clearance rate in kidney and have high clinical value.(1-5) bi-specific antibody.Be the antibody that a class has dual specificity and dual-use function, claim bifunctional antibody again.(1-6) recombinant antibody fusion proteins.Gene fragments such as Fab or Fv, with having that other protein genes such as the toxin of non-antibody or enzyme are connected to form a kind of recombinant protein of specific biological activity guiding target site.(1-7) recombinant immunotoxin.The gene recombination of encoding antibody and toxin is produced, and characteristics are efficient, and non-special toxicity is low, and the body internal stability is good, easily penetrate use in large bowel cancer tumour and the body safer.(1-8) phage antibody.The V district gene of Ig is connected after transfecting host bacterium with last gene III of filobactivirus DNA or gene VIII, makes its fusion protein product at film surface outer casing protein expression Fab or ScFv.By this product is taken turns the affine absorption of related antigen more, therefrom wash in a pan and sift out required more high-affinity and more specific new antibodies.

(2) set out with light, heavy chain variable region gene encoded polypeptides of the present invention and derivative thereof, can crosslinkedly go up cytotoxic drug, toxin, radionuclide, enzyme and biological response modifier etc., also can be used for the diagnosis and the treatment of large intestine (colon, rectum) cancer as targeted drug.(2-1) antibody-nucleic conjugate.This conjugate nucleic can be led effectively large bowel cancer and intestinal inflammation tissue local, thus the normal tissue injury that causes because of radiotherapy external radiation exposure etc. and relevant untoward reaction reduced, and can position diagnosis and targeted therapy to large bowel neoplasm.This method is radio-immuno-image and radioimmunotherapy.Have with monoclonal antibody link coupled nucleic commonly used ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tcm, ¹⁸⁸Re and ¹⁸⁶Re etc.(2-2) antibody-chemotherapeutics conjugate.This conjugate large bowel cancer tumour that can lead specifically can lower the damage to healthy tissues, reduces the toxic side effect of chemotherapeutics.Normal link coupled chemotherapeutics is as the phosphoamide in the alkylating agent; Methotrexate in the antimetabolic, 5 FU 5 fluorouracil; Antibiotics Zorubicin, pidorubicin, daunorubicin; Plant class vincristine(VCR), mitomycin etc.(2-3) antibody-toxin conjugated thing.This conjugate claims immunotoxin again.Its killer cell effect is strong and do not rely on biological auxiliary mechanism.It kills and wounds large intestine (colon, rectum) tumor mechanism and radiotherapy, and the chemotherapy difference so the tumour of general radiotherapy, chemotherapy effect difference can be used, has a extensive future.Toxin commonly used has ricin, diphtheria toxin, abrin, Saponaria officinalis toxin, pseudomonas extracellular toxin, streptolysin, pore-forming protein etc.(2-4) antibody-biological response modifier (BRM) conjugate.BRM regulates the immunological competence and the killing tumor cell of body separately and has obtained curative effect preferably.But owing to only have part to arrive large intestine (colon, rectum) tumor target site after in its input body, its lethal effect is not in full use and toxic side effect.With BRM and monoclonal antibody coupling, carry its arrival target site by monoclonal antibody and bring into play the effect that kills and wounds large bowel cancer, make the effect of these factors stronger, and more single-minded.BRM commonly used has INF and IL-2 etc.(2-5) antibody targeted enzymolysis prodrug.The conjugate of antibody and drug specificity activating enzymes is injected in the body, inject prodrug behind the certain hour at interval, make it change into high density antitumour activity medicine with killing tumor cell at the large bowel cancer tumor locus.At present, can be used as the glutamine derivative that phenylformic acid hydrogen mustard is arranged, phosphoric acid Podophyllum emodi var chinense ethylidene ring, phosphoric acid mitomycin, glucuronide daunorubicin, Zorubicin, 5-flurocytosine and the cynnematin mustargen etc. of prodrug.Activating enzymes have carboxypeptidase G 2, alkaline phosphatase, penicillin amidase, β-Nei Xiananmei, Isocytosine deaminase, beta-glucosidase enzyme and aminopeptidase etc.(2-6) immunoliposome.The surface energy that MAb is coupled to liposome fully combines the characteristic of MAb and antigen-specific bonded guidance quality and lipid physical efficiency parcel high amount of drug together, and relevant medicine in the embedding then can improve drug targeting and curative effect again.

(3) in addition,, can also be applied to the fundamental research of large bowel cancer related antigen, or be used to find the new epi-position of new large bowel cancer tumor associated antigen or known large bowel cancer tumour antigen with large bowel cancer CAb-1 monoclonal antibody of the present invention.These antigens all can be further used for clinical Serological testing, have certain clinical diagnosis meaning.

Sequence table

＜110〉Chen Zhinan

＜120〉light, heavy chain variable region gene of anti-human large intestine cancer monoclonal antibody CAb-1 and application thereof

<160>4

<210>1

<211>353

<212>DNA

＜213〉mouse

<220>

<221>V_region

<222>(3)...(353)

<400>1

ag?gtg?aaa?ctg?cag?cag?tct?gga?cct?gag?ctg?aag?aag?cct?gga?gag?aca?gtc?aag?atc 59

tcc?tgc?aag?gtt?tct?ggg?tat?acc?ttc?aca?gcc?tat?gga?atg?aac?tgg?gtg?aag?cag?gct 119

cca?gga?aag?ggt?ttg?aag?tgg?atg?ggc?tgg?ata?aac?acc?tac?act?gga?gaa?cca?aca?tat 179

ggt?gat?gac?ttc?aag?gga?cga?ttt?gcc?ttc?tct?ttg?gaa?acc?tct?gtc?agc?act?gcc?cat 239

ttg?cag?atc?agt?aac?gtc?aga?aat?gcg?gac?acg?gct?aca?tat?ttc?tgt?gca?aga?cgg?ggg 299

tac?ggc?tac?tac?ttt?gac?tac?tgg?ggc?caa?ggc?acc?act?ctc?aca?gtc?tcc?tca 353

<210>2

<211>117

<212>PRT

＜213〉mouse

<220>

<221>V_segment

<400>2

Val?Lys?Leu?Gln?Gln?Ser?Gly?Pro?Glu?Leu?Lys?Lys?Pro?Gly?Glu?Thr?Val?Lys 18

Ile?Ser?Cys?Lys?Val?Ser?Gly?Tyr?Thr?Phe?Thr?Ala?Tyr?Gly?Met?Asn?Trp?Val 36

Lys?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Lys?Trp?Met?Gly?Trp?Ile?Asn?Thr?Tyr?Thr 54

Gly?Glu?Pro?Thr?Tyr?Gly?Asp?Asp?Phe?Lys?Gly?Arg?Phe?Ala?Phe?Ser?Leu?Glu 72

Thr?Ser?Val?Ser?Thr?Ala?His?Leu?Gln?Ile?Ser?Asn?Val?Arg?Asn?Ala?Asp?Thr 90

Ala?Thr?Tyr?Phe?Cys?Ala?Arg?Arg?Gly?Tyr?Gly?Tyr?Tyr?Phe?Asp?Tyr?Trp?Gly 108

Gln?Gly?Thr?Thr?Leu?Thr?Val?Ser?Ser 117

<210>3

<211>406

<212>DNA

＜213〉mouse

<220>

<221>V_region

<222>(14)...(406)

<400>3

ggggatatccacc?atg?aag?ttg?cct?gtt?agg?ctg?ttg?gtg?ctg?atg?ttc?tgg?att?cct?gct?tcc 64

acc?agt?gat?att?gtg?atg?acc?cag?act?cca?ctc?tcc?ctg?cct?gtc?agt?ctt?gga?gat?caa 124

gcc?tcc?atc?tct?tgc?aga?tct?agt?cag?agc?ctt?gta?cac?agt?gat?gga?gac?acc?tat?tta 184

cat?tgg?tac?ctg?cag?aag?cca?ggc?cag?tct?cca?aag?ctc?ctg?atc?tac?aaa?gtt?tcc?aac 244

cga?ttt?tct?ggg?gtc?cca?gac?agg?ttc?agt?ggc?agt?gga?tca?ggg?aca?gat?ttc?aca?ctc 304

aag?atc?agc?aga?gtg?gag?gct?gag?gat?ctg?gga?gtt?tat?ttt?tgc?tcg?caa?agt?gca?cat 364

gtt?tct?ccc?acg?ttc?ggt?gct?ggg?acc?aag?ctg?gag?ctg?aaa 406

<210>4

<211>140

<212>PRT

＜213〉mouse

<220>

<221>V_segment

<400>4

Met?Gly?Cys?Ser?Trp?Val?Met?Leu?Phe?Leu?Val?Ala?Thr?Ala?Thr?Gly?Val?His 18

Ser?Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?Glu?Leu?Val?Arg?Pro?Gly?Val?Ser 36

Val?Lys?Ile?Ser?Cys?Lys?Gly?Ser?Gly?Tyr?Thr?Phe?Thr?Asp?Tyr?Thr?Met?His 54

Trp?Val?Lys?Gln?Ser?His?Ala?Lys?Ser?Leu?Glu?Trp?Ile?Gly?Val?Ile?Asn?Thr 72

Tyr?Ser?Gly?Asn?Thr?Lys?Tyr?Asn?Gln?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Met?Thr 90

Val?Asp?Lys?Ser?Ser?Arg?Thr?Ala?Tyr?Met?Glu?Leu?Val?Arg?Leu?Thr?Ser?Glu 108

Asp?Ser?Ala?Ile?Tyr?Tyr?Cys?Val?Arg?Gly?Asn?Ser?Val?Arg?Asn?Tyr?Tyr?Val 126

Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Ser?Val?Thr?Val?Ser?Ser 140

Claims

1, the gene of the anti-human large intestine cancer monoclonal antibody of a kind of separated coding CAb-1, this gene comprises heavy chain variable region gene and chain variable region gene, described heavy chain variable region gene has sequence table＜400〉1 sequence, chain variable region gene has sequence table＜400〉3 sequence.

2, by the polypeptide of the described genes encoding of claim 1, it has sequence table＜400〉2 and＜400〉4 sequence.

3, the described gene of claim 1 is used for diagnosing or treating the application of large bowel cancer medicine in preparation.

4, the described polypeptide of claim 2 is used for diagnosing or treating the application of large bowel cancer medicine in preparation.