CN112538117B - Anti-human carcinoembryonic antigen antibody and coding gene and application thereof - Google Patents

Abstract

The invention discloses an anti-human carcino-embryonic antigen antibody, a coding gene and an application thereof, the anti-human carcino-embryonic antigen antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of three hypervariable regions of the heavy chain variable region, namely CDRH1, CDRH 2and CDRH3, are respectively as follows:GDSISGD、HHSGS、AARAAMDRSFDF(ii) a The amino acid sequences of three hypervariable regions of a light chain variable region of the light chain variable region are respectively CDRL1, CDRL 2and CDRL3TGSGSNIGAGYDVH、LNNNRPS、QSYDKSLRGGL. The antibody is a fully human anti-human carcinoembryonic antigen antibody, has high affinity with human carcinoembryonic antigen, and can be used for detecting and treating human cancer diseases.

Description

Anti-human carcinoembryonic antigen antibody and coding gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an anti-human carcino-embryonic antigen antibody and a coding gene and application thereof.

Background

Tumor markers (Tumor markers) are chemical species that reflect the presence of tumors. They are either absent from normal adult tissues and present only in embryonic tissues, or present in tumor tissues in amounts substantially exceeding those in normal tissues, and their presence or amount may be indicative of the nature of the tumor, thereby providing insight into tumorigenesis,CellsDifferentiation, cell function to aid in the diagnosis, classification, prognosis and treatment guidance of tumors.

In the fight against malignant tumors, doctors, patients and family members often pay the most attention to how to discover the generation, development and metastasis of tumors as early as possible. The development of diagnostic instruments and technologies such as an X-ray diagnostic machine, a CT machine, an MRI machine and the like enables tumors with the diameter larger than 5mm to be found at present, and provides powerful support for early detection of tumors, evaluation of treatment effect and assistance of doctors in modification of treatment schemes. However, some "residual tillering" of the tumor is still difficult to be identified because of too small volume or special position in the body, and these "fish with net leakage" grow quietly in dark places, so that the tumor recurs and metastasizes, and finally the life of many patients is lost. Then, there is no other way to help identify these potential threats? The answer is an important index, namely a tumor marker widely applied to clinical diagnosis and treatment at present. They are substances that can be detected by assaying blood, urine or tumor tissue and reflect the condition of the tumor in vivo, and are called "invisible appearance" as compared with the above-mentioned imaging morphological examination method, and are a powerful complement to morphological diagnosis.

In a broad sense, tumor markers are substances that are present on certain tumor cells or secreted and excreted into body fluids, and can be roughly classified into two types, tumor cell secretions and tumor cell expression products. The former isTumor cellsThe more the tumor grows and the more the substance is produced in the occurrence and development, and conversely, the tumor growth is suppressed and the production amount is reduced. These substances are often glycoproteins, which can be detected and monitored by testing body fluids such as blood. Currently, lung cancer tumor markers (CEA, Cyfra21-1, NSE, etc.), digestive tract tumor markers (CEA, CA199, CA242, CA724, etc.), CA153 (breast cancer, etc.), CA125 (ovarian cancer, etc.), AFP (liver cancer, etc.), PSA (prostate cancer, etc.), HCG (choriocarcinoma, etc.) [ Tannous BA, Teng J (2011) "segmented blood reporters: instruments and applications" Biotechnol adv.29(6): 997-; khunger M, Kumar U, Roy HK, Tiwari AK. (2014) "Dysplasia and cancer screening in 21st century." APMIS 122(8): 674-; grunnet M, Mau-

M.(2014)“Serum tumor markers in bile duct cancer—a review.”Biomarkers 19(6):437-443；Napier KJ,Scheerer M,Misra S.(2014)“Esophageal cancer:A Review of epidemiology,pathogenesis,staging workup and treatment modalities.”World J Gastrointest Oncol 6(5):112-120.]. They are often detected when the tumor is small, which helps to find the focus early; it suggests that the therapeutic effect may be poor; if the marker is progressively increased after a period of surgical removal of the tumor, it is often indicated that there may have been tumor cell proliferation and growth in the body, and if the marker is significantly decreased after treatment, it is indicated that the treatment is effective, otherwise, it needs to be closely monitored.

Therefore, an experienced oncologist will often perform an image examination of the patient, a blood drawing examination while assessing the size of the tumor body, and a dynamic observation of the corresponding markers to see if the tumor growth is still active, if its activity is inhibited after treatment, etc. They are characterized by sensitive reflection, and can often judge the growth state of the tumor earlier than means such as CT, MRI and the like, and remind doctors whether to change the treatment scheme; the weakness is that they are less accurate and less reliable than imaging diagnostics, and therefore, it is often necessary to test several markers together, dynamically observe their changes, and make decisions in conjunction with other clinical characterizations. Tumor marker detection is commonly used clinically: (1) early warning of tumor occurrence and development; (2) dynamic monitoring to reflect treatment effects; (3) when the tumor specimen can not be obtained, some prompts are made for the tumor property when the pathological diagnosis is definite, and a reference basis is provided for experimental treatment.

Human carcinoembryonic antigen (CEA) is an acidic glycoprotein that is widely present in cancers of the digestive system of the endoderm origin and also in the tissue of the digestive tract of normal embryos, and was originally found in colon cancer and fetal intestinal tissue, and is known as carcinoembryonic antigen. Carcinoembryonic antigen is a protein synthesized in the gastrointestinal tract, liver, pancreas at the time of embryo. There is also a small amount of synthesis in the adult gastrointestinal tract, but not into the blood system but rather is excreted through the gastrointestinal tract, so that there is only a trace of carcinoembryonic antigen in normal adult serum.

Carcinoembryonic antigen is a broad-spectrum tumor marker which can reflect the existence of a plurality of tumors to people, CEA elevation is commonly found in colorectal Cancer, pancreatic Cancer, gastric Cancer, Lung Cancer, liver Cancer, bile duct Cancer, breast Cancer, ovary, medullary thyroid Cancer and urinary tumors [ Grunnet M, Sorensen JB (2012)' Carcinoembryonic antigen (CEA) as tumor marker in Lung Cancer76(2): 138-43; nagai Y, Beppu T, Sakamoto Y, et al (2014) "Carbonic antibacterial Half-life Is an Early Predictor of Therapeutic Effects in indication Chemotherapy for Liver metals from colour cancer" Anticancer Res.34(10): 5529-35; chao YJ, Sy ED, Hsu HP, Shann YS (2014) "dividers for removability and survival in localization advanced scientific cancer after transistor gel-based neo ado therapy" BMC Surg.14:72 ]. In addition, CEA is a good tumor marker for the curative effect judgment, disease development, monitoring and pre-estimation of colorectal cancer, breast cancer and lung cancer.

The paracarcinoma normal mucosa has little or negative CEA content. For example, the CEA positive rate of gastric cancer is 85.58%; the CEA positivity of gastric mucinous adenocarcinoma and signet ring cell carcinoma (also called mucinous cell carcinoma) is 100% [ Chung JK, Lee MC, Chung HK, Lim SM, Jang JJ, Koh CS (1995) "Concentration and distribution of tumor associated antigens TAG-72and CEA in stomach cancer." an Nucl Med.9(1):7-13 ]. The reason for the high CEA content in the cytoplasm of cancer is related to the increase of CEA synthesis and the block of CEA excretion of cancer cells. When cancer cells are degenerated and killed, the structure of the inner membrane of the cell is damaged and broken, and CEA can be present in the matrix of the cytoplasm. The mucous cell carcinoma CEA is distributed in the whole cell membrane and cytoplasmic membrane structure, CEA epitope is glycoprotein, and the infiltration and metastasis of tumor cells are related to the glycosylation change of cell membrane glycoprotein. In addition, the mucus cell cancer can secrete and release a large amount of proteolytic enzyme, destroy cancer cell calcium bridges, and dissolve soft tissues around cancer nests. Therefore, the gastric mucus cell carcinoma has strong invasiveness and high metastasis rate.

Heretofore, no CEA-based imaging diagnosis has been reported and applied. Therefore, the antibody capable of detecting CEA protein is very significant for the detection, imaging diagnosis and treatment of various tumors. Chinese patent application publication No. CN104628859 discloses an anti-human carcinoembryonic antigen antibody, and a preliminary study on tumor image detection using the antibody.

Disclosure of Invention

Compared with the existing anti-human carcino-embryonic antigen antibody, the anti-human carcino-embryonic antigen antibody provided by the invention has the advantages that the affinity coefficient Kd with human CEA protein is remarkably improved, and meanwhile, the anti-human carcino-embryonic antigen antibody has a remarkable inhibiting effect on tumor cells, and can be used for preparing anti-tumor drugs.

An anti-human carcinoembryonic antigen antibody (anti-human CEA protein antibody for short) comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of three hypervariable regions of the heavy chain variable region, namely CDRH1, CDRH 2and CDRH3, are respectively as follows:GDSISGD(SEQ ID No.5)、HHSGS(SEQ ID No.6)、AARAAMDRSFDF(SEQ ID No.7)(ii) a The amino acid sequences of three hypervariable regions of a light chain variable region of the light chain variable region are respectively CDRL1, CDRL 2and CDRL3TGSGSNIGAGYDVH(SEQ ID No.8)、LNNNRPS(SEQ ID No.9)、QSYDKSLRGGL(SEQ ID No.10)。

Wherein, three hypervariable regions CDRH1, CDRH 2and CDRH3 of the heavy chain variable region are respectively positioned at the 26 th to 32 th, 52 th to 56 th and 98 th to 114 th positions of the heavy chain variable region; the three hypervariable regions CDRL1, CDRL 2and CDRL3 of the light chain variable region are respectively located at positions 24-34, 50-56 and 89-96 of the light chain variable region.

The amino acid sequences of the three hypervariable regions vary greatly over the variable regions of the antibody molecule, while the amino acid sequences of the regions between the hypervariable regions vary less. These three hypervariable regions are sterically complementary to antigenic determinants, and are therefore also referred to as Complementarity Determining Regions (CDRs), with the CDRs of the different heavy and light chains determining the specificity of the antibody for the antigen.

Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2.

The anti-human carcinoembryonic antigen antibody can be a whole antibody or an antigen-binding portion of a whole antibody. The whole antibody is preferably IgG1 type; the antigen binding portion is preferably a Fab fragment, Fab 'fragment, F (ab')₂Fragments or single chain antibodies, more preferably single chain antibodies.

The antigen binding part not only reserves the area capable of being specifically bound with the antigen, but also avoids the side effect caused by the antigenicity of the Fc fragment; the single-chain antibody has the advantages of easy penetration into tumor tissue, increased medicine concentration, small immunogenicity, short half-life period in vivo circulation, easy elimination, easy connection with toxin or enzyme gene to directly obtain immunotoxin or enzyme-labeled antibody, etc.

Antigen binding portions can be prepared by recombinant DNA techniques or by enzymatic/chemical cleavage of whole antibodies. In the specific embodiment of the present invention, the preparation method of the single-chain antibody against human carcinoembryonic antigen (CEA) is as follows: a human single-chain antibody library is constructed by adopting a method disclosed by Chinese patent document with publication number CN 1444651A, and an anti-human CEA protein antibody is screened from the human single-chain antibody library.

The invention also provides the coding gene of the anti-human carcino-embryonic antigen antibody, which comprises a heavy chain variable region coding gene and a light chain variable region coding gene; specifically, the nucleotide sequence of the heavy chain variable region encoding gene is shown in SEQ ID No.3, and the light chain variable region encoding gene is shown in SEQ ID No. 4.

The invention also provides a recombinant vector or an expression system containing the coding gene. The original vector of the recombinant vector is pACT2 or pET27 b.

The invention also provides a host cell comprising the expression system.

The invention also provides the application of the anti-human carcinoembryonic antigen antibody in preparing in vitro diagnostic reagents or tumor image diagnostic reagents. The anti-human carcinoembryonic antigen antibody can be specifically combined with human carcinoembryonic antigen and can be used for detecting a CEA tumor marker in a serum sample of a tumor patient; the anti-human carcinoembryonic antigen antibody can also be injected into a human body to carry out image detection on tumors through image diagnosis.

The invention also provides the application of the anti-human carcinoembryonic antigen antibody in preparing a medicament for inhibiting or treating tumors. Furthermore, the anti-human carcinoembryonic antigen antibody is an IgG1 type full antibody, and the sequence of the variable region of the anti-human carcinoembryonic antigen antibody is the same as that of the variable region of the single-chain antibody hCEA-1. After 40 days of treatment with the anti-CEA-1 full length antibody IgG-CEA-1, the tumor size was only about 50% of the control group.

Among the three applications, the human carcinoembryonic antigen is a broad-spectrum tumor marker, and can be used for detecting and preventing various tumor diseases, such as: carcinoma of large intestine, pancreatic cancer, gastric cancer, lung cancer, hepatocarcinoma, cholangiocarcinoma, breast cancer, ovary cancer, medullary thyroid cancer, and urinary system tumor.

The invention also provides a pharmaceutical composition, which comprises a pharmaceutically effective amount of the anti-human carcinoembryonic antigen antibody. The anti-human carcino-embryonic antigen antibody is mixed with a medicinal carrier. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal use (e.g., by injection or infusion).

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present application can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response without toxicity to the patient, composition, and mode of administration. The selected dosage level will depend upon a variety of factors including the activity of the particular composition or ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds or/and substances used in combination with the pharmaceutical composition, the age, sex, body weight, condition, general health and previous medical history of the patient being treated, and like factors well known in the medical arts.

In some embodiments, the pharmaceutical composition needs to be sterile and fluid to the extent that it can be delivered by syringe, and apart from water, may be isotonic with buffered saline solution, ethanol, polyols, and the like, or suitable mixtures.

In some embodiments, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (mannitol or sorbitol), sodium chloride and the like in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of an agent which delays absorption, for example, aluminum monostearate or gelatin.

The pharmaceutical composition can be used for treating or inhibiting carcinoma of large intestine, pancreatic cancer, gastric cancer, lung cancer, hepatocarcinoma, cholangiocarcinoma, breast cancer, ovarian cancer, medullary thyroid carcinoma, and urinary system tumor.

Compared with the prior art, the invention has at least one of the following beneficial effects:

(1) the anti-human carcinoembryonic antigen antibody of the invention is a fully humanized anti-human carcinoembryonic antigen antibody which can be used for detecting various tumors and treating and preventing human diseases, has very high affinity, can be specifically combined with human carcinoembryonic antigen, effectively detects various tumors, and has important effect on treating related tumors.

(2) Compared with the existing anti-human carcinoembryonic antigen antibody, the affinity coefficient Kd of the anti-human carcinoembryonic antigen antibody and the human CEA protein is obviously improved (by 44 percent).

(3) The anti-human carcinoembryonic antigen antibody of the invention also has obviously improved inhibiting effect on tumor cells, and under the same condition, after being treated by the anti-CEA-1 full-length antibody IgG-CEA-1 for 40 days, the tumor size is only about 50 percent of that of a control group.

Drawings

FIG. 1 is a schematic structural diagram of a single-chain antibody against human CEA protein in example 1;

FIG. 2 is a specific ELISA assay for the anti-human CEA protein single chain antibody # HCEA-1 of example 2;

FIG. 3 shows the results of ELISA assay of human CEA protein single chain antibody # HCEA-1 in example 3 for sera of various tumor patients.

FIG. 4 is a graph showing the results of the change in tumor size with time after the anti-CEA-1 full length antibody (IgG-CEA-1) or nonspecific human IgG protein or PBS phosphate buffer was injected subcutaneously into SCID mice in example 4 and treated.

Detailed Description

The following is a detailed description with reference to specific examples.

Example 1 screening of anti-human CEA protein Single chain antibody

In this example, a single-chain antibody library was constructed, and a single-chain antibody against human CEA protein was screened against the single-chain antibody library.

The method disclosed by Chinese patent document with publication number CN 1444651A is adopted to construct a human single-chain antibody library, and the anti-human CEA protein single-chain antibody is screened from the human single-chain antibody library, and the specific implementation process is as follows:

1. amplifying to obtain human antibody heavy chain and light chain variable region DNA

Poly A + RNA (purchased from Clontech) from human bone marrow, human fetal liver, human spleen and human peripheral blood leukocytes was used as a template for reverse transcription of poly A + RNA into cDNA using oligo (dT) and random primers (random primers) using a reverse transcriptase kit (purchased from Clontech) according to the protocol guidelines provided by the Clontech kit.

The above cDNA was used as a template, and PCR amplification was performed using a series of primers recognizing the human antibody heavy chain variable region (VH) and light chain variable region (VL) genes to obtain the DNA sequences of all heavy chain variable regions and light chain variable regions in human antibodies. A series of primers recognizing the variable region genes of the heavy and light chains of the human antibody have the following sequences:

the first group is 5' -end primers (SEQ ID Nos. 11-17) for amplifying human antibody heavy chain variable region (VH) genes, and comprises:

VH1b：5’-CCATACGATGTTCCAGATTACCAGGTGCAGCTGCAGGAGTC(C/G)G-3’；

VH2b：5’-CCATACGATGTTCCAGATTACCAGGTACAGCTGCAGCAGTCA-3’；

VH3b：5’-CCATACGATGTTCCAGATTACCAGGTGCAGCTACAGCAGTGG G-3’；

VH4b：5’-CCATACGATGTTCCAGATTACGAGGTGCAGCTG(G/T)TGGAG(A/T)C

(C/T)-3’；

VH5b：5’-CCATACGATGTTCCAGATTACCAGGTCCAGCT(G/T)GT(A/G)CAGTCTG

G-3’；

VH6b：5’-CCATACGATGTTCCAGATTACCAG(A/G)TCACCTTGAAGGAGTCTG-3’；

VH7b：5’-CCATACGATGTTCCAGATTACCAGGTGCAGCTGGTG(C/G)A(A/G)TCTG

G-3’；

the second group is 3' -end primers (SEQ ID Nos. 18-23) for amplifying human antibody heavy chain variable region (VH) genes, comprising:

VH1f：5’-GCCGCCTGATCCACCACCGCCTGAGGAGAC(A/G)GTGACCAGGGTG-3’；

VH2f：5’-GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACCAGGGTT-3’；

VH3f：5’-GCCGCCTGATCCACCACCGCCTGAAGAGACGGTGACCATTGT-3’；

VH4f：5’-GCCGCCTGATCCACCACCGCCTGAGGAGACGGTGACCGTGGTCC-3’；

VH5f：5’-GCCGCCTGATCCACCACCGCCGGTTGGGGCGGATGCACTCC-3’；

VH6f：5’-GCCGCCTGATCCACCACCGCC(C/G)GATGGGCCCTTGGTGGA(A/G)GC-3’；

the third group is 5' -end primers (SEQ ID Nos. 24 to 32) for amplifying a variable region (V.lambda.) of a human antibody lambda-light chain, comprising:

VL1b：5’-GGCAGCGGTGGTGGAGGCAGTCAGTCTGT(C/G)(C/G/T)TGACGCAGCCGCC-3’；

VL2b：5’-GGCAGCGGTGGTGGAGGCAGTTCCTATG(A/T)GCTGAC(A/T)CAGCCAC-3’；

VL3b：5’-GGCAGCGGTGGTGGAGGCAGTTCCTATGAGCTGA(C/T)(A/G)CAGC(C/T)ACC-3’；

VL4b：5’-GGCAGCGGTGGTGGAGGCAGTCAGCCTGTGCTGACTCA(A/G)(C/T)C-3’；

VL5b：5’-GGCAGCGGTGGTGGAGGCAGTCAG(A/G/T)CTGTGGTGAC(C/T)CAGGAGCC-3’；

VL6b：5’-GGCAGCGGTGGTGGAGGCAGTCAGCC(A/T)G(G/T)GCTGACTCAGCC(A/C)CC-3’；

VL7b：5’-GGCAGCGGTGGTGGAGGCAGTTCCTCTGAGCTGA(C/G)TCAGGA(C/G)CC-3’；

VL8b：5’-GGCAGCGGTGGTGGAGGCAGTCAGTCTG(C/T)(C/T)CTGA(C/T)TCAGCCT-3’；

VL9b：5’-GGCAGCGGTGGTGGAGGCAGTAATTTTATGCTGACTCAGCCCC-3’；

the fourth group is 3' -end primers (SEQ ID Nos. 33 to 34) for amplifying a variable region (V.lambda.) of a human antibody lambda-light chain, comprising:

VL1f：5’-GGGGTTTTTCAGTATCTACGATAGGACGGT(C/G)A(C/G)CTTGGTCC-3’；

VL2f：5’-GGGGTTTTTCAGTATCTACGAGAGGACGGTCAGCTGGGTGC-3’；

the fifth group is primers (SEQ ID Nos. 35 to 38) for amplifying the human antibody k-light chain variable region (Vk) gene at the 5' -end, including:

VK1b：5’-GGCAGCGGTGGTGGAGGCAGTGACATCC(A/G)G(A/G/T)TGACCCAGTCTCC-3’；

VK2b：5’-GGCAGCGGTGGTGGAGGCAGTGAAATTGT(A/G)(A/T)TGAC(A/G)CAGTCTCC-3’；

VK3b：5’-GGCAGCGGTGGTGGAGGCAGTGATATTGTG(A/C)TGAC(C/G/T)CAG(A/T)CTCC-3’；

VK4b：5’-GGCAGCGGTGGTGGAGGCAGTGAAACGACACTCACGCAGTCTC-3’；

the sixth group is 3' -end primers (SEQ ID Nos. 39 to 42) for amplifying a human antibody k-light chain variable region (Vk) gene, comprising:

VK1f：5’-GGGGTTTTTCAGTATCTACGATTTGATTTCCACCTTGGTCC-3’；

VK2f：5’-GGGGTTTTTCAGTATCTACGATTTGATCTCCA(C/G)CTTGGTCC-3’；

VK3f：5’-GGGGTTTTTCAGTATCTACGATTTGATATCCACTTTGGTCC-3’；

VK4f：5’-GGGGTTTTTCAGTATCTACGATTTAATCTCCAGTCGTGTCC-3’。

when the heavy chain variable region (VH) in the human antibody is amplified, a first group of primers and a second group of primers are combined, namely 42 PCR reactions are carried out; when the lambda-light chain variable region (V lambda) in the human antibody is amplified, 18 PCR reactions are carried out by using the combination of the third group of primers and the fourth group of primers; when a k light chain variable region (Vk) in a human antibody is amplified, 16 PCR reactions are performed using a combination of the fifth set of primers and the sixth set of primers.

The first set of primers contained the upstream homologous sequence (underlined) of the multiple cloning site of the yeast two-hybrid vector pACT2(Hua SB, Luo Y, Qiu M, Chan E, Zhou H, Zhu L. (1998) Gene.215: 143-; the fourth group and the sixth group of primers comprise downstream homologous sequences (underlined parts) of the multiple cloning site of the yeast double-hybrid vector pACT 2; the second, third and fifth primers contain linker peptide sequences (underlined) for linking the heavy chain variable region and the light chain variable region of the antibody.

During amplification, the PCR reaction system and the reaction conditions are the same, and the PCR reaction system is as follows:

the components are mixed evenly and then placed in a PCR instrument for reaction. The reaction conditions are as follows: melting at 94 ℃ for 1 min, annealing at 50 ℃ for 1 min, extension at 72 ℃ for 2.5 min, and cycling 30 times.

2. Homologous sequence and linker peptide sequence for linking multiple cloning sites of a vector

(1) Taking the human antibody heavy chain variable region DNA obtained by PCR in the step 1 as a template, and taking a primer 7 and a primer 8 as an upstream primer and a downstream primer respectively, wherein the sequences are as follows:

upstream primer (primer 7, SEQ ID No.43, the upstream homologous sequence of the multicloning site of vector pACT2 underlined):

5’-ACCCCACCAAACCCAAAAAAAGAGATCTGTATGGCTTACCCATACGATGTTCCAGATTAC-3’；

the downstream primer (primer 8, SEQ ID No.44, underlined is the linker peptide reverse-strand sequence):

5’-ACTGCCTCCACCACCGCTGCCACCTCCGCCAGATCCTCCGCCGCCTGATCCACCACCGCC-3’；

performing PCR amplification, wherein the reaction conditions and the system are the same as the step 1. After the reaction is completed, the DNA sequence of the heavy chain variable region of the human antibody containing the upstream homologous sequence of the multicloning site of the 5 '-vector pACT 2and the reverse chain sequence of the 3' -connecting peptide is obtained.

(2) Taking the human antibody light chain variable region DNA obtained by PCR in the step 1 as a template, and taking the primer 9 and the primer 10 as a downstream primer and an upstream primer respectively, wherein the sequences are as follows:

upstream primer (primer 10, SEQ ID No.45, underlined sequence of linker peptide):

5’-GGCGGTGGTGGATCAGGCGGCGGAGGATCTGGCGGAGGTGGCAGCGGTGGTGGAGGCAGT-3’；

downstream primer (primer 9, SEQ ID No.46, the underlined part is the downstream homologous sequence of the multicloning site of vector pACT 2):

5’-GAGATGGTGCACGATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCTACGA-3’；

performing PCR amplification, wherein the reaction conditions and the system are the same as the step 1. After the reaction is completed, the DNA sequence of the variable region of the human antibody light chain containing the downstream homologous sequence of the multi-cloning site of the 3 '-vector pACT 2and the sequence of the 5' -connecting peptide cis chain is obtained.

3. Linking Single chain antibody (scFv) DNA

And (3) mixing the human antibody heavy chain variable region DNA and light chain variable region DNA which are obtained by PCR amplification in the step (2) and contain the homologous sequence of the vector pACT2 multiple cloning site and the connecting peptide sequence, and carrying out PCR amplification by taking the mixed DNA as a template and taking a primer 7 and a primer 9 as an upstream primer and a downstream primer respectively, wherein the reaction conditions and the system are the same as those in the step (1).

As shown in FIG. 1, after the reaction was completed, a single chain antibody (scFv) DNA comprising the human antibody heavy chain variable region DNA sequence, the light chain variable region DNA sequence, the vector pACT2 multiple cloning site homologous sequence, and the linker peptide DNA sequence was obtained.

4. Construction of human Single chain antibody Gene library

The single chain antibody (scFv) DNA obtained in step 3 was co-transferred with restriction enzyme treated Yeast two-hybrid vector pACT2 (Yeast Protocol Handbook, PT3024-1) into Yeast strain Y187 (MAT. alpha. URA3-52, HIs3-200, ade2-101, lys2-801, trp1-901, leu2-3,112, GAL4, GAL80, met-, URA3:: GAL1, met-, and EcoRI RI by the method provided by original Clontech_UAS-GAL1_TATA-lac Z, MEL1), and integrating single-chain antibody DNA into pACT2 vector after intracellular homologous recombination, thereby obtaining yeast two-hybrid single-chain antibody library, wherein the single-chain antibody DNA fragment is fused with Gal4 Activation region (AD) on pACT2 vector.

To examine the quality of the single-chain antibody gene library, 21 clones were randomly picked out from the library, and the inserted fragment was subjected to sequencing analysis. The analysis results showed that all clones included single-chain antibody DNA fragments fused to Gal4, and that all single-chain antibody DNA sequences were unique.

The copy number of the antibody DNA in the gene library of the yeast two-hybrid single-chain antibody obtained by homologous recombination is about 1X 10⁸The method can be applied to the yeast two-hybrid technology for screening specific antibodies.

5. Screening for antibodies

(1) Antibody screening

Antibody screening was performed on a yeast two-hybrid single-chain antibody gene library using a DNA encoding the human CEA protein as an antigen gene.

The DNA encoding the mature protein of human CEA (sequence is shown in ID No. M15042 of GenBank and nucleotides 92 to 2095) is recombined into a vector pGBKT7 to construct pGBK-hCEA. pGBK-hCEA encodes Gal4 DNA Binding Domain (BD) and has fused at its C-terminus the human CEA protein.

After the DNA sequence encoding the human CEA protein was verified, pGBK-hCEA plasmid DNA was transformed into yeast strain AH109(MATa, trp1-901, leu2-3,112, ura3-52, his3-200, GAL4, GAL80, LYS2:: GAL1_UAS-GAL1_TATA-HIS3,GAL2_UAS-GAL2_TATA-ADE2,URA3::MEL1_UAS-MEL1_TATA-lac Z); AH109 yeast with pGBK-hCEA plasmid can grow on synthetic medium (SD/-W) without tryptophan.

An equal amount of MATa-type yeast cells containing pGBK-hCEA (AH109 strain) and MAT-type yeast cells containing a single-chain antibody gene library (Y187 strain) were co-cultured, and the two types of cells were mated and bound. Since the pACT2 vector carrying the single chain antibody gene library contained the Leu2 gene and pGBK-hCEA contained the Trp1 gene, yeast cells containing both plasmids could be grown in a yeast synthetic medium (SD/-LW) that did not contain leucine and serine.

The presence of the single-chain antibody scFv interacting with the human CEA protein activates the reporter genes ADE 2and HIS3 integrated in the genome of the strain, allowing the yeast cells to grow on medium lacking adenine, histidine, leucine and tryptophan (SD/-AHLW) and form colonies on this plating medium.

(2) Specific antibody screening

1) Galactosidase assay

Since cells in which scFv interacts with the human CEA protein are present also activate the other reporter gene lac Z integrated in the genome of the strain, detection of the expression of galactosidase in the yeast cell can determine the presence of scFv/CEA protein in the yeast cell.

A total of 67 colonies were picked on screening medium (SD/-AHLW) and lac Z expression was detected using the galactosidase assay. The detection method comprises the following steps:

inoculating yeast with scFv/CEA protein interaction to a screening medium (SD/-AHLW) plate for growth;

secondly, transferring the yeast colonies to Whatman No. five filter paper;

thirdly, soaking the filter paper with the yeast cell colony into liquid nitrogen to break the cell;

fourthly, the filter paper is taken out of the liquid nitrogen and is placed in a drying oven at the temperature of 30 ℃; repeating the steps (3) and (4) twice;

fifthly, spreading the filter paper in a proper amount of X-gal solution, and placing the filter paper in a 37 ℃ incubator for about 15 minutes; if blue color is shown at the colony, it is positive for galactosidase, i.e., lac Z gene is activated for expression.

Wherein the formula of the X-gal solution is as follows:

16.1g/L Na₂HPO₄·7H₂O；

5.50g/L NaH₂PO₄·H₂O；

0.75g/L KCl；

0.246g/L MgSO₄·7H₂O；

35mg/L X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)；

5 mM-mercaptoethanol; the pH was 7.0.

And (3) detection results: of the 33 colonies selected above, 17 were positive for galactosidase, indicating that the reporter gene lacZ was activated in these colonies.

2) Specific binding assay

The specificity analysis of the scFv of the 17 galactosidase positive colonies described above was performed to verify whether the single chain antibody scFv specifically binds to the human CEA protein.

Plasmid DNA of pACT2 containing scFv was extracted from the 17 galactosidase-positive yeasts, and then transformed into AH109 yeast cells together with empty vector DNA of pGBKT7, plasmid DNA of pGBK-hCEA, and plasmid DNA of pGBK-Lam (encoding Gal4 DNA binding domain fused at its C-terminus with human lamin C), respectively; the transformed yeast cells were grown by plating on SD/-LW plate medium and then transferred to SD/-AHLW plate medium; the colonies that grew out were analyzed for galactosidase, and the colonies that were confirmed to be positive were regarded as containing nonspecific scFv and were knocked out.

After the specificity analysis, a single-chain antibody with specificity to the human CEA protein is obtained, the number of the single-chain antibody is hCEA-1, an ABI automatic sequencer is used for sequencing analysis of the single-chain antibody, and the result shows that:

the DNA sequence of the heavy chain variable region of hCEA-1 is shown in SEQ ID No. 3;

the heavy chain variable region amino acid sequence (shown as SEQ ID No. 1) of the hCEA-1 is as follows: QVQLQESGPGLVKPSETLSLTCTVSGDSISGDYWSWIRQPPGKGLEWIGEIHHSGSTNYNPSLKSRVSISLDTSKNQFSLKLSSVTAADTAVYYCAA RAAMDRSFDFWGQGTLVTVSSGSASAPT

Wherein, the underlined parts are three hypervariable regions of CDRH1, CDRH 2and CDRH3(SEQ ID Nos. 5-7) in sequence;

the DNA sequence of the light chain variable region of hCEA-1 is shown in SEQ ID No. 4;

the light chain variable region amino acid sequence (SEQ ID No.2) of hCEA-1 is: QSVLTQPPSVSGAPGQRVTISCTG SGSNIGAGYDVHWYQQRPGTAPKLLIYLNNNRPSGVPDRFSGSKSGTSASLAIPGLQAEDEADYYCQSYDKSLRGG LFGGGTKLTVL。

Wherein, the underlined parts are three hypervariable regions of CDRL1, CDRL 2and CDRL3(SEQ ID Nos. 8-10) in sequence.

Example 2 specific detection

In this example, the single-chain antibody obtained by screening in the above examples was expressed in a bacterial system, purified, and its specificity was measured.

1. Single chain antibody expression and purification

Cloning the coding gene of the single-chain antibody hCEA-1 into an expression vector pET27b (+) to construct pET27b-hCEA 1;

pET27b-hCEA1 was transformed into the expression bacterium E.coli BL21(DE3) and expression was induced with IPTG (0.5mM) according to the method provided by Novagen; in the expressed target protein, the N end of the scFv is a pelB sequence, and the pelB sequence can secrete the expressed scFv into the periplasmic cavity (periplastmic space) of BL21(DE 3); the C end of the scFv contains an HSV tag and a 6 XHis tag, which facilitates the purification of the target protein; the single-chain antibody of anti-human CEA protein is conveniently obtained by separation and purification with Ni-NTA column by the method provided by Qiagen company.

2. ELISA test for specificity of Single chain antibodies to CEA proteins

Human CEA protein was purchased from Shanghai Kexing Biotech, Inc. The ELISA test method was as follows:

(1) coating 96-well plate with CEA protein, and standing at 2-8 deg.C overnight;

(2) sealing the coated 96-pore plate by using SuperBlock;

(3) serially diluting a single-chain antibody hCEA-1 in 0.02% BSA, adding the diluted single-chain antibody hCEA-1 into a 96-hole coated with CEA protein, and combining with the CEA protein;

(4) cleaning a 96-well plate, adding a 5000-fold diluted antibody of a mouse anti-HSV marker to detect the combined single-chain antibody;

(5) cleaning a 96-well plate, and adding a 10000-fold diluted goat anti-mouse IgG antibody-horseradish peroxidase conjugate;

(6) after the 96-pore plate is finally cleaned, horse radish peroxidase substrate TMB reagent is applied for color development treatment;

(7) the reaction was stopped with 0.5M sulfuric acid and the absorption spectrum at 450nm was measured.

The detection result is shown in FIG. 2, which shows that the single-chain antibody # hCEA-1 can effectively bind to CEA protein.

The affinity coefficient Kd value of hCEA-1 single-chain antibody and human CEA protein is 0.786 nM. The affinity of hCEA-1 single-chain antibody is superior to that of hCEA-A (1.132nM) (Chinese patent application No.: 201510039112.2).

Example 3 detection of serum CEA by anti-human CEA protein Single chain antibody

In this embodiment, the anti-human CEA protein single-chain antibody is used to detect CEA tumor marker in serum of tumor patients.

Rabbit polyclonal antibodies against human CEA (Cat. No. sc-20131, H300) were purchased from Santa Cruz Biotechnology, Inc., USA. Human CEA protein was purchased from Shanghai Kexing Biotech, Inc. The tumor patient serum sample and the normal healthy serum sample come from Hangzhou armed police hospital.

The ELISA test method was as follows:

(1) coating a 96-pore plate with a rabbit polyclonal antibody against human CEA, and standing overnight at 2-8 ℃;

(2) sealing the coated 96-pore plate by using SuperBlock;

(3) diluting a serum sample (100-fold dilution) or human CEA protein in 0.02% BSA-physiological saline, adding the diluted serum sample or human CEA protein into the 96-well coated with the rabbit anti-human CEA polyclonal antibody, and binding the polyclonal antibody with the CEA protein in the sample;

(4) after washing the 96-well plate, a single-chain antibody hCEA-1 (0.1. mu.g/ml) diluted in 0.02% BSA-physiological saline was added to the above 96-well;

(5) adding a 5000-fold diluted antibody of the mouse anti-HSV marker to detect the combined single-chain antibody;

(6) cleaning a 96-well plate, and adding a 10000-fold diluted goat anti-mouse IgG antibody-horseradish peroxidase conjugate;

(7) after the 96-pore plate is finally cleaned, horse radish peroxidase substrate TMB reagent is applied for color development treatment;

(8) the reaction was stopped with 0.5M sulfuric acid and the absorbance spectrum at 450nm (OD450nm) was measured.

The detection result is shown in figure 3, which shows that the single-chain antibody hCEA-1 can effectively bind to CEA protein and is used for detecting CEA tumor markers of serum samples of tumor patients.

Example 4 full Length anti-human CEA antibodies inhibit tumor growth in tumor model animals

1. Preparation of full-length recombinant humanized anti-human CEA protein antibody IgG-CEA-1

The full-length recombinant humanized anti-human CEA protein antibody IgG-CEA-1 (the buffer solution is a conventional PBS phosphate buffer solution, and the concentration is 1.0mg/ml) is prepared by Beijing Yiqian Shenzhou biotechnology limited. The full-length IgG-CEA-1 is IgG1 type, and the variable region sequence thereof is identical to that of the single-chain antibody hCEA-1.

2. Preparation of human Primary peripheral blood mononuclear cells PBMC

The majority (about 60-70%) of human primary peripheral blood mononuclear PBMC cells are immune T cells. PBMC were derived from peripheral blood of healthy donors and prepared by Ficoll Lymphocyte Separation Medium (Sigma-Aldrich, USA, Histopaque-1077) density gradient centrifugation method according to the supplier's method. PBMC cells prepared as described above were first pre-cultured with anti-CD 3 monoclonal antibody (40ng/ml) (eBioscience, Calif., USA, Cat. No. 16-0037) for 3 days.

3. Preparation of liver cancer tumor cells with human CEA antigen on surface

Human hepatoma tumor cell line Hep3B was derived from american ATCC (Rockville, Maryland, USA). DNA encoding full-length human CEA protein (see GenBank ID No. M15042, nucleotide sequence 1 to 2092), to the C-terminal of which was added Transmembrane (TM) peptide of TGF-beta receptor II (ISLLPPLGVAISVIIIFYCYRVNRQ (SEQ ID No.47)) (see GenBank ID No. NM-001024847.2, nucleotide sequence 965 to 1039) and stop codon TAA. The DNA sequence of the CEA-TM fusion protein was synthesized by Nanjing Kinshire and cloned into pcDNA3.1/Hygromycin plasmid.

The plasmid DNA with CEA-TM fusion protein described above was transfected into Hep3B cells using conventional cell transfection techniques and screened in medium containing 200. mu.g/ml Hygromycin (Sigma Co.) for 14 days; the selected Hep3B/CEA-TM cell line was cultured by dilution to obtain a cell line (Fuller SA et al, 2001.Current Protocols of Molecular biology. Chapter 11, section 11.8). The CEA-TM-expressing cell line was analyzed by anti-CEA antibody (in combination with flow cytometry FACS, and human hepatoma tumor cell line Hep3B/CEA-TM with high CEA-TM expression level was selected.

4. Preparation of human liver cancer tumor Hep3B/CEA-TM mouse model

Tumors were induced in SCID mice according to the methods described in the literature (Pollack VA et al 1999, "Inhibition of Epidermal growth factor receptor phosphorylation in human carbonic mas with CP-358: Dynamics of receptor Inhibition in situ and receptors effects in therapy," J.Pharmacol. exp.Ther.291:739-748 ").

Human liver cancer tumor cell line Hep3B/CEA-TM (about 3x 10)⁶Individual cells) and 50% Matrigel (Beckton-Dickinson, usa) preparations were injected subcutaneously into SCID mice (experimental animal technology ltd, viton, beijing) of 6-7 week malesInducing a tumor; after 15 days of tumor cell inoculation, tumors formed to about 100-200mm³In size, 5X 10 is injected into the tumor⁵Primary human peripheral blood mononuclear cells PBMC (PBMC cells from three healthy donors after mixing).

5、

Three days after PBMC inoculation, these SCID mice were divided into three groups, and (1) anti-CEA-1 full-length antibody (IgG-CEA-1), (2) non-specific human IgG protein (Sigma, USA), (3) phosphate buffered saline PBS was injected subcutaneously into the SCID mice described above, respectively. The antibody dose was 10mg/kg, and the same dose was re-injected every 10 days for a total of three injections. The size of the tumor was determined by measuring two diameters of the tumor with a vernier caliper and calculating the volume of the tumor (length x [ width ] according to the method described in the literature ("Protocols for screening chemical agents and natural products against biological systems." Cancer chemiher. Rep.3: 1-104.)) according to the method described in the literature]²)/2。

FIG. 4 shows the tumor size versus time after three days of PBMC inoculation, after the anti-CEA-1 full-length antibody (IgG-CEA-1) or non-specific human IgG protein or PBS, respectively, was injected subcutaneously into the SCID mice described above.

The detection result shows that the anti-CEA-1 full-length antibody IgG-CEA-1 has stronger tumor inhibition effect on tumor cells compared with a control group. After 40 days of anti-CEA-1 full length antibody IgG-CEA-1 treatment, the tumor size was only about 50% of the control group.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Hangzhou Cixing medical instruments Co., Ltd

<120> anti-human carcinoembryonic antigen antibody, and coding gene and application thereof

<130>

<160> 47

<170> PatentIn version 3.3

<210> 1

<211> 125

<212> PRT

<213> Artificial sequence

<400> 1

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Gly Asp

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile His His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Ser Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Arg Ala Ala Met Asp Arg Ser Phe Asp Phe Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr

115 120 125

<210> 2

<211> 111

<212> PRT

<213> Artificial sequence

<400> 2

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Gly Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Arg Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Leu Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Pro Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Lys Ser

85 90 95

Leu Arg Gly Gly Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 3

<211> 375

<212> DNA

<213> Artificial sequence

<400> 3

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtga ctccatcagt ggtgactact ggagttggat ccggcagccc 120

ccagggaagg gactggagtg gattggggaa atccatcata gtgggagcac caactacaat 180

ccttccctca agagtcgagt cagcatatca ctagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcggc tcgtgcagct 300

atggaccgct cttttgactt ctggggccag ggaaccctgg tcaccgtctc ctcagggagt 360

gcatccgccc caact 375

<210> 4

<211> 333

<212> DNA

<213> Artificial sequence

<400> 4

cagtctgtgt tgacgcagcc gccctcagtg tctggggccc cagggcagag ggtcaccatc 60

tcctgcactg ggagcggctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120

cgtccaggaa cagcccccaa actcctcatc tatcttaaca acaatcggcc ctcaggggtc 180

cctgatcgat tctctgggtc caagtctggc acctcagcct ccctggccat ccctggactc 240

caggctgagg atgaggctga ttattactgc cagtcctatg acaagagcct gaggggagga 300

ctattcggcg gagggaccaa gctgaccgtc cta 333

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<400> 5

Gly Asp Ser Ile Ser Gly Asp

1 5

<210> 6

<211> 5

<212> PRT

<213> Artificial sequence

<400> 6

His His Ser Gly Ser

1 5

<210> 7

<211> 11

<212> PRT

<213> Artificial sequence

<400> 7

Ala Ala Arg Ala Ala Met Asp Arg Ser Phe Asp

1 5 10

<210> 8

<211> 14

<212> PRT

<213> Artificial sequence

<400> 8

Thr Gly Ser Gly Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial sequence

<400> 9

Leu Asn Asn Asn Arg Pro Ser

1 5

<210> 10

<211> 11

<212> PRT

<213> Artificial sequence

<400> 10

Gln Ser Tyr Asp Lys Ser Leu Arg Gly Gly Leu

1 5 10

<210> 11

<211> 43

<212> DNA

<213> Artificial sequence

<400> 11

ccatacgatg ttccagatta ccaggtgcag ctgcaggagt csg 43

<210> 12

<211> 42

<212> DNA

<213> Artificial sequence

<400> 12

ccatacgatg ttccagatta ccaggtacag ctgcagcagt ca 42

<210> 13

<211> 43

<212> DNA

<213> Artificial sequence

<400> 13

ccatacgatg ttccagatta ccaggtgcag ctacagcagt ggg 43

<210> 14

<211> 42

<212> DNA

<213> Artificial sequence

<400> 14

ccatacgatg ttccagatta cgaggtgcag ctgktggagw cy 42

<210> 15

<211> 44

<212> DNA

<213> Artificial sequence

<400> 15

ccatacgatg ttccagatta ccaggtccag ctkgtrcagt ctgg 44

<210> 16

<211> 43

<212> DNA

<213> Artificial sequence

<400> 16

ccatacgatg ttccagatta ccagrtcacc ttgaaggagt ctg 43

<210> 17

<211> 44

<212> DNA

<213> Artificial sequence

<400> 17

ccatacgatg ttccagatta ccaggtgcag ctggtgsart ctgg 44

<210> 18

<211> 43

<212> DNA

<213> Artificial sequence

<400> 18

gccgcctgat ccaccaccgc ctgaggagac rgtgaccagg gtg 43

<210> 19

<211> 43

<212> DNA

<213> Artificial sequence

<400> 19

gccgcctgat ccaccaccgc ctgaggagac ggtgaccagg gtt 43

<210> 20

<211> 42

<212> DNA

<213> Artificial sequence

<400> 20

gccgcctgat ccaccaccgc ctgaagagac ggtgaccatt gt 42

<210> 21

<211> 44

<212> DNA

<213> Artificial sequence

<400> 21

gccgcctgat ccaccaccgc ctgaggagac ggtgaccgtg gtcc 44

<210> 22

<211> 41

<212> DNA

<213> Artificial sequence

<400> 22

gccgcctgat ccaccaccgc cggttggggc ggatgcactc c 41

<210> 23

<211> 42

<212> DNA

<213> Artificial sequence

<400> 23

gccgcctgat ccaccaccgc csgatgggcc cttggtggar gc 42

<210> 24

<211> 44

<212> DNA

<213> Artificial sequence

<400> 24

ggcagcggtg gtggaggcag tcagtctgts btgacgcagc cgcc 44

<210> 25

<211> 43

<212> DNA

<213> Artificial sequence

<400> 25

ggcagcggtg gtggaggcag ttcctatgwg ctgacwcagc cac 43

<210> 26

<211> 45

<212> DNA

<213> Artificial sequence

<400> 26

ggcagcggtg gtggaggcag ttcctatgag ctgayagcag cyacc 45

<210> 27

<211> 41

<212> DNA

<213> Artificial sequence

<400> 27

ggcagcggtg gtggaggcag tcagcctgtg ctgactcary c 41

<210> 28

<211> 44

<212> DNA

<213> Artificial sequence

<400> 28

ggcagcggtg gtggaggcag tcagdctgtg gtgacycagg agcc 44

<210> 29

<211> 44

<212> DNA

<213> Artificial sequence

<400> 29

ggcagcggtg gtggaggcag tcagccwgkg ctgactcagc cmcc 44

<210> 30

<211> 44

<212> DNA

<213> Artificial sequence

<400> 30

ggcagcggtg gtggaggcag ttcctctgag ctgastcagg ascc 44

<210> 31

<211> 42

<212> DNA

<213> Artificial sequence

<400> 31

ggcagcggtg gtggaggcag tcagtctgyy ctgaytcagc ct 42

<210> 32

<211> 43

<212> DNA

<213> Artificial sequence

<400> 32

ggcagcggtg gtggaggcag taattttatg ctgactcagc ccc 43

<210> 33

<211> 41

<212> DNA

<213> Artificial sequence

<400> 33

ggggtttttc agtatctacg ataggacggt sascttggtc c 41

<210> 34

<211> 41

<212> DNA

<213> Artificial sequence

<400> 34

ggggtttttc agtatctacg agaggacggt cagctgggtg c 41

<210> 35

<211> 44

<212> DNA

<213> Artificial sequence

<400> 35

ggcagcggtg gtggaggcag tgacatccrg dtgacccagt ctcc 44

<210> 36

<211> 44

<212> DNA

<213> Artificial sequence

<400> 36

ggcagcggtg gtggaggcag tgaaattgtr wtgacrcagt ctcc 44

<210> 37

<211> 44

<212> DNA

<213> Artificial sequence

<400> 37

ggcagcggtg gtggaggcag tgatattgtg mtgacbcagw ctcc 44

<210> 38

<211> 43

<212> DNA

<213> Artificial sequence

<400> 38

ggcagcggtg gtggaggcag tgaaacgaca ctcacgcagt ctc 43

<210> 39

<211> 41

<212> DNA

<213> Artificial sequence

<400> 39

ggggtttttc agtatctacg atttgatttc caccttggtc c 41

<210> 40

<211> 41

<212> DNA

<213> Artificial sequence

<400> 40

ggggtttttc agtatctacg atttgatctc cascttggtc c 41

<210> 41

<211> 41

<212> DNA

<213> Artificial sequence

<400> 41

ggggtttttc agtatctacg atttgatatc cactttggtc c 41

<210> 42

<211> 41

<212> DNA

<213> Artificial sequence

<400> 42

ggggtttttc agtatctacg atttaatctc cagtcgtgtc c 41

<210> 43

<211> 60

<212> DNA

<213> Artificial sequence

<400> 43

accccaccaa acccaaaaaa agagatctgt atggcttacc catacgatgt tccagattac 60

<210> 44

<211> 60

<212> DNA

<213> Artificial sequence

<400> 44

actgcctcca ccaccgctgc cacctccgcc agatcctccg ccgcctgatc caccaccgcc 60

<210> 45

<211> 60

<212> DNA

<213> Artificial sequence

<400> 45

ggcggtggtg gatcaggcgg cggaggatct ggcggaggtg gcagcggtgg tggaggcagt 60

<210> 46

<211> 57

<212> DNA

<213> Artificial sequence

<400> 46

gagatggtgc acgatgcaca gttgaagtga acttgcgggg tttttcagta tctacga 57

<210> 47

<211> 25

<212> PRT

<213> Artificial sequence

<400> 47

Ile Ser Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val Ile Ile Ile

1 5 10 15

Phe Tyr Cys Tyr Arg Val Asn Arg Gln

20 25

Claims (9)

1. An anti-human carcinoembryonic antigen antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of three hypervariable regions of a heavy chain variable region, namely CDRH1, CDRH 2and CDRH3, are respectively as follows: GDSISGD, HHSGS, AARAAMDRSFDF; the amino acid sequences of three hypervariable regions CDRL1, CDRL 2and CDRL3 of the light chain variable region are TGSGSNIGAGYDVH, LNNNRPS, QSYDKSLRGGL respectively.

2. The anti-human carcinoembryonic antigen antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID No.1, and the amino acid sequence of the light chain variable region is represented by SEQ ID No. 2.

3. The anti-human carcinoembryonic antigen antibody of claim 2, wherein the anti-human carcinoembryonic antigen antibody is a whole antibody or an antigen-binding portion of a whole antibody.

4. The anti-human carcinoembryonic antigen antibody of claim 3, wherein the whole antibody is of the IgG1 type; the antigen binding portion is Fab fragment, Fab 'fragment, F (ab')₂Fragments or single chain antibodies.

5. The gene encoding the anti-human carcinoembryonic antigen antibody of claim 2, wherein the nucleotide sequence of the gene encoding the heavy chain variable region is represented by SEQ ID No.3, and the nucleotide sequence of the gene encoding the light chain variable region is represented by SEQ ID No. 4.

6. A recombinant vector comprising the coding gene of claim 5.

7. An expression system comprising the encoding gene of claim 5.

8. The use of the anti-human carcinoembryonic antigen antibody of any one of claims 1 to 4 in the preparation of an anti-tumor drug, a tumor in vitro diagnostic reagent, or a tumor imaging diagnostic reagent.

9. A pharmaceutical composition comprising a pharmaceutically effective amount of the anti-human carcinoembryonic antigen antibody of any one of claims 1 to 4.

Images