CN112521507B - Anti-human c-Met human-mouse chimeric monoclonal antibody and application thereof - Google Patents
Anti-human c-Met human-mouse chimeric monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention provides an anti-human c-Met human mouse chimeric monoclonal antibody and application thereof, belonging to the technical field of enzyme-linked immunity, wherein the nucleotide sequences of a 1D20 heavy chain variable region and a 1D20 light chain variable region of the c-Met targeted human mouse chimeric monoclonal antibody are sequentially shown as SEQ ID No.1 and SEQ ID No. 2. The c-Met-targeted human mouse chimeric monoclonal antibody provided by the invention can be specifically combined with the c-Met with high affinity, is chimeric with a human antibody constant region, can generate a slight immunological rejection reaction compared with a mouse monoclonal antibody when applied in a human body, and improves the application value of a product.
Description
The invention is a divisional application of an invention patent with the application number of CN202010795032.0 and the name of anti-human c-Met human mouse chimeric monoclonal antibody and the application thereof, and the application date is 2020, 08 and 10 days.
Technical Field
The invention belongs to the technical field of enzyme-linked immunity, and particularly relates to an anti-human c-Met human-mouse chimeric monoclonal antibody and application thereof.
Background
Hepatocyte Growth Factor (HGF) is a polypeptide growth factor that has strong mitogenic, epithelial migration inducing, invasion inducing, and angiogenesis inducing effects, and its biological activity is mediated by its receptor c-Met. C-Met is a protein product encoded by C-Met protooncogene, has tyrosine kinase activity, is related to various oncogene products and regulatory proteins, participates in the regulation and control of cell information transduction and cytoskeleton rearrangement, and is an important factor of cell proliferation, differentiation and movement. It is presently believed that C-Met is closely associated with the development and metastasis of a variety of cancers, and many tumor patients have C-Met overexpression and gene amplification during their tumorigenesis and metastasis.
HGF and c-Met play a crucial role in the formation and the evolution of lung cancer, therefore, when the abnormally activated HGF/Met signal pathway is blocked, a series of changes such as morphological change, slow proliferation, reduced tumorigenesis, reduced invasive ability and the like of tumor cells occur, and the HGF/c-Met is an effective molecular target in tumor treatment.
The monoclonal antibody can be directly used for the diagnosis, prevention and treatment of human diseases and the research of immune mechanism, and opens up wide prospects for the immunodiagnosis and immunotherapy of human malignant tumors. The mouse antibody can be recognized by the human immune system to cause human anti-mouse antibody reaction, so that the curative effect of the monoclonal antibody medicament is weakened, and serious adverse reaction is caused. The chimeric antibody replaces the constant region sequence of the corresponding antibody gene of a mouse with the constant region sequence of the humanized antibody generating gene, greatly reduces the immunogenicity reaction generated by the murine antibody, ensures that 70 percent of the components of the antibody are human components, prevents monoclonal antibody molecules from being rapidly eliminated by an immune system as heterologous proteins, and improves the drug effect of monoclonal antibody drugs.
Disclosure of Invention
In view of the above, the present invention aims to provide an anti-human c-Met human mouse chimeric monoclonal antibody and an application thereof, wherein the anti-human c-Met human mouse chimeric monoclonal antibody provided by the present invention can be specifically combined with c-Met with high affinity, and is chimeric with a human antibody constant region, and when the monoclonal antibody is applied in a human body, the monoclonal antibody can generate a more slight immunological rejection reaction compared with a mouse monoclonal antibody, so that the application value of the product is improved, and the monoclonal antibody can be applied to diagnosis and treatment of c-Met high-expression tumor diseases.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides an anti-human C-Met human mouse chimeric monoclonal antibody, wherein the nucleotide sequence of a 1D20 heavy chain variable region of the anti-human C-Met human mouse chimeric monoclonal antibody is shown as SEQ ID No.1, the nucleotide sequence of a 1D20 light chain variable region is shown as SEQ ID No.2, the nucleotide sequence of a 3C36 heavy chain variable region is shown as SEQ ID No.3, the nucleotide sequence of a 3C36 light chain variable region is shown as SEQ ID No.4, the nucleotide sequence of a 3D63 heavy chain variable region is shown as SEQ ID No.5, and the nucleotide sequence of a 3D63 light chain variable region is shown as SEQ ID No. 6.
Preferably, the amino acid sequence of the 1D20 heavy chain variable region is shown in SEQ ID No. 7.
Preferably, the amino acid sequence of the variable region of the 1D20 light chain is shown in SEQ ID No. 8.
Preferably, the amino acid sequence of the 3C36 heavy chain variable region is shown in SEQ ID No. 9.
Preferably, the amino acid sequence of the 3C36 light chain variable region is shown in SEQ ID No. 10.
Preferably, the amino acid sequence of the 3D63 heavy chain variable region is shown in SEQ ID No. 11.
Preferably, the amino acid sequence of the variable region of the 3D63 light chain is shown in SEQ ID No. 12.
The invention also provides a recombinant expression vector, which comprises the heavy chain variable region and/or the light chain variable region of the anti-human c-Met human mouse chimeric monoclonal antibody in the technical scheme;
the nucleotide sequence of the 1D20 heavy chain variable region of the antihuman C-Met human mouse chimeric monoclonal antibody is shown as SEQ ID No.1, the nucleotide sequence of the 1D20 light chain variable region is shown as SEQ ID No.2, the nucleotide sequence of the 3C36 heavy chain variable region is shown as SEQ ID No.3, the nucleotide sequence of the 3C36 light chain variable region is shown as SEQ ID No.4, the nucleotide sequence of the 3D63 heavy chain variable region is shown as SEQ ID No.5, and the nucleotide sequence of the 3D63 light chain variable region is shown as SEQ ID No. 6.
The invention also provides a host cell which is obtained by transforming the recombinant expression vector in the technical scheme.
The invention also provides a method for producing the anti-human c-Met human mouse chimeric monoclonal antibody, which comprises the following steps: culturing the host cell according to the technical scheme, and collecting the anti-human c-Met human mouse chimeric monoclonal antibody from the culture.
The invention provides an anti-human C-Met human mouse chimeric monoclonal antibody, wherein the nucleotide sequence of a 1D20 heavy chain variable region of the anti-human C-Met human mouse chimeric monoclonal antibody is shown as SEQ ID No.1, the nucleotide sequence of a 1D20 light chain variable region is shown as SEQ ID No.2, the nucleotide sequence of a 3C36 heavy chain variable region is shown as SEQ ID No.3, the nucleotide sequence of a 3C36 light chain variable region is shown as SEQ ID No.4, the nucleotide sequence of a 3D63 heavy chain variable region is shown as SEQ ID No.6, and the nucleotide sequence of a 3D63 light chain variable region is shown as SEQ ID No. 6.
Drawings
FIG. 1 shows the results of serum titer measurements after immunization of different mice;
FIG. 2 is a phage display vector construction;
FIG. 3 shows the result of phage ELISA detection;
FIG. 4 is a schematic diagram of the construction of 3D63 human-mouse chimeric monoclonal antibody vector;
FIG. 5 shows Protein A purified 3D63 chimeric antibody;
FIG. 6 shows SDS-PAGE detection of 3D63 purification of antibody A to antibody non-reduced state B to antibody reduced state;
FIG. 7 shows the WB detection results of 3D63 chimeric mouse monoclonal monomers.
Detailed Description
The invention provides an anti-human C-Met human mouse chimeric monoclonal antibody, wherein the nucleotide sequence of a 1D20 heavy chain variable region of the anti-human C-Met human mouse chimeric monoclonal antibody is shown as SEQ ID No.1, the nucleotide sequence of a 1D20 light chain variable region is shown as SEQ ID No.2, the nucleotide sequence of a 3C36 heavy chain variable region is shown as SEQ ID No.3, the nucleotide sequence of a 3C36 light chain variable region is shown as SEQ ID No.4, the nucleotide sequence of a 3D63 heavy chain variable region is shown as SEQ ID No.6, and the nucleotide sequence of a 3D63 light chain variable region is shown as SEQ ID No. 6.
In the invention, the nucleotide sequence of the 1D20 heavy chain variable region is shown as SEQ ID No.1, and specifically comprises the following steps:
GAGGTTCAGCTGCAGCAGTCTGGAGATGATCTGGTAAAGCCTGGGGCCTCAGTGAAGCTGTCCTGCAAGGCTTCTGGTTACTCATTTACTGGCTACTTTATGGACTGGGTGATGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACGTATTAATCCTAACAATGGTGATACTTTTTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCTAGTACAGCCCACATGGAGCTCCGGAGCCTGGCATCTGAGGACTCTGCAGTCTATTATTGTGCAAGAATGAAGCTAATTGGGTCCTATATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。
in the invention, the amino acid sequence of the 1D20 heavy chain variable region is shown as SEQ ID No.7, and is specifically shown as follows:
EVQLQQSGDDLVKPGASVKLSCKASGYSFTGYFMDWVMQSHGKSLEWIGRINPNNGDTFYNQKFKGKATLTVDKSSSTAHMELRSLASEDSAVYYCARMKLIGSYMDYWGQGTSVTVSS, CDRs in bold.
In the invention, the nucleotide sequence of the 1D20 light chain variable region is shown as SEQ ID No.2, and specifically comprises the following steps:
GATATCCAGATGACTCAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGTAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAATC。
in the invention, the amino acid sequence of the 1D20 light chain variable region is shown as SEQ ID No.8, and specifically as follows:
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLFTFGSGTKLEIK, CDRs in bold.
In the invention, the nucleotide sequence of the 3C36 heavy chain variable region is shown as SEQ ID No.3, and specifically comprises the following steps:
GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCCTCTGGCTACACCTTCACCAGCTACTGGATACACTGGGTGAAACAGAGACCTGGACAGGGCCTTGAGTGGATTGGAGAGATTAATCCTAGCAACGGTCATACTTACTACACTGAGAAGTTCAAGATCAAGGCCACAATGACTTTAGACAAATCCTCCAGCACGGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCAGCGGTCTATTTTTGTGGAAGATATCCCAAGGGAGGGTATTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA。
in the invention, the amino acid sequence of the 3C36 heavy chain variable region is shown as SEQ ID No.9, and specifically comprises the following steps:
EVQLQQSGPELVKPGASVKLSCKASGYTFTSYWIHWVKQRPGQGLEWIGEINPSNGHTYYTEKFKIKATMTLDKSSSTAYMQLSSLTSEDSAVYFCGRYPKGGYFDVWGAGTTVTVSS, CDRs in bold.
In the invention, the nucleotide sequence of the 3C36 light chain variable region is shown as SEQ ID No.4, and specifically comprises the following steps:
GACATTGTGCTAACTCAGTCTCCAGGCACCCTATCTGTGACTCCAGGAGATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAGCTACCTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAAAAGCTGGCCTTTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAATCTAGTGGTGGCGGTGGTTCG。
in the present invention, the amino acid sequence of the 3C36 light chain variable region is shown in SEQ ID No.10, which is specifically as follows:
DIVLTQSPGTLSVTPGDSVSLSCRASQSISSYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSKSWPFTFGSGTKLEIK, CDRs in bold.
In the invention, the nucleotide sequence of the 3D63 heavy chain variable region is shown as SEQ ID No.5, and specifically comprises the following steps:
GAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGGTCTGGCTACACATTCACTGATTATGCTATGCACTGGGTAAAGCAGAGTCATGCAAAGAGTCTAGAGTGGATTGGAGTTAGTAGTAGTTATTATGGTGAGGCTAACTACAACCAGAAGTTCAAGGCCAAGGCCACAATGACTGTAGACAAATCCTCCAGCACAGCCTATATGGAGCTTGCCGGACTGACATCTGAGGATTCTGCCATCTATTACTGTGTAAGACACGACGTGGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。
in the invention, the amino acid sequence of the 3D63 heavy chain variable region is shown as SEQ ID No.11, and specifically comprises the following steps:
EVQLQQSGAELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGVSSSYYGEANYNQKFKAKATMTVDKSSSTAYMELAGLTSEDSAIYYCVRHDVDAMDYWGQGTSVTVSS, CDRs in bold.
In the invention, the nucleotide sequence of the 3D63 light chain variable region is shown as SEQ ID No.6, and specifically comprises the following steps:
GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAATCTAGT。
in the invention, the amino acid sequence of the 3D63 light chain variable region is shown as SEQ ID No.12, and specifically as follows:
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK, CDRs in bold.
The invention also provides a recombinant expression vector, which comprises the heavy chain variable region and/or the light chain variable region of the anti-human c-Met human mouse chimeric monoclonal antibody in the technical scheme;
the nucleotide sequence of the 1D20 heavy chain variable region of the antihuman C-Met human mouse chimeric monoclonal antibody is shown as SEQ ID No.1, the nucleotide sequence of the 1D20 light chain variable region is shown as SEQ ID No.2, the nucleotide sequence of the 3C36 heavy chain variable region is shown as SEQ ID No.3, the nucleotide sequence of the 3C36 light chain variable region is shown as SEQ ID No.4, the nucleotide sequence of the 3D63 heavy chain variable region is shown as SEQ ID No.5, and the nucleotide sequence of the 3D63 light chain variable region is shown as SEQ ID No. 6.
The invention also provides a host cell which is obtained by transforming the recombinant expression vector in the technical scheme.
The invention also provides a method for producing the anti-human c-Met human mouse chimeric monoclonal antibody, which comprises the following steps: culturing the host cell according to the technical scheme, and collecting the anti-human c-Met human mouse chimeric monoclonal antibody from the culture.
In the present invention, the method for preparing the anti-human c-Met human murine chimeric monoclonal antibody preferably comprises the following steps:
1) carrying out double enzyme digestion on the 1D20 heavy chain variable region, the 3C36 heavy chain variable region and the 3D63 heavy chain variable region respectively through BamHI and XbaI to obtain an enzyme digestion 1D20 heavy chain variable region, an enzyme digestion 3C36 heavy chain variable region and an enzyme digestion 3D63 heavy chain variable region;
carrying out double enzyme digestion on the plasmid vector pcDNA3.1(+) by BamHI and XbaI to obtain a linearized plasmid vector pcDNA3.1 (+);
connecting the enzyme digestion 1D20 heavy chain variable region, the enzyme digestion 3C36 heavy chain variable region and the enzyme digestion 3D63 heavy chain variable region to a linearized plasmid vector pcDNA3.1(+) to obtain a recombinant plasmid pcDNA3.1-H;
2) carrying out double enzyme digestion on the 1D20 light chain variable region, the 3C36 light chain variable region and the 3D63 light chain variable region respectively through BamHI and XbaI to obtain an enzyme digestion 1D20 light chain variable region, an enzyme digestion 3C36 light chain variable region and an enzyme digestion 3D63 light chain variable region;
the plasmid vector pcDNA3.1/zeo (+) is subjected to double enzyme digestion by BamHI and XbaI to obtain a linearized plasmid vector pcDNA3.1/zeo (+);
connecting the enzyme digestion 1D20 light chain variable region, the enzyme digestion 3C36 light chain variable region and the enzyme digestion 3D63 light chain variable region to a linearized plasmid vector pcDNA3.1/zeo (+) to obtain a recombinant plasmid pcDNA3.1/zeo-L;
3) transfecting the recombinant plasmid pcDNA3.1-H obtained in the step 1) and the recombinant plasmid pcDNA3.1/zeo-L obtained in the step 2) to 293 cells, culturing for more than 3d, and purifying by utilizing protein A affinity chromatography to obtain the anti-human c-Met human mouse chimeric monoclonal antibody.
In the present invention, the double-restriction enzyme system of the heavy chain variable region and the light chain variable region preferably comprises, per 80 μ L: target Gene fragment 15. mu. L, BamHI 2. mu. L, XbaI 2. mu.L, 10 XNEBuffer 48. mu.L, 10 XBSA 8. mu.L and ddH at a concentration of 150 ng/. mu.L2O45. mu.L. In this busy situation, the conditions for the double enzyme digestion preferably include: the reaction was carried out at 37 ℃ overnight.
In the present invention, the double enzyme digestion system of the plasmid vector pcDNA3.1(+) and the plasmid vector pcDNA3.1/zeo (+) preferably comprises, per 80. mu.L: linearized plasmid vector 15. mu. L, BamHI 2. mu. L, XbaI 2. mu.L, 10 XNEBuffer 48. mu.L, 10 XBSA 8. mu.L and ddH at a concentration of 150 ng/. mu.L2O45 μ L. In the present invention, the conditions of the double enzyme digestion preferably include: the reaction was carried out at 37 ℃ overnight.
In the present invention, the linked system preferably comprises, per 25. mu.L: target baseThe plasmid vector was linearized by 3. mu.L, 1. mu.L of linearized plasmid vector, 10 XT 4 DNA ligation buffer 2.5. mu. L, T4 DNA ligation 1. mu.L and ddH2O17.5. mu.L. In the present invention, the conditions for the connection preferably include: the metal bath was connected overnight at 16 ℃.
In the present invention, the ratio of the mass of the recombinant plasmid pcDNA3.1-H, the mass of the recombinant plasmid pcDNA3.1/zeo-L and the number of 293 cells is preferably 2. mu.g: 1X 105And (4) respectively. In the present invention, the transfection also preferably comprises mixing the recombinant plasmid with liposome and then transfecting, wherein the volume ratio of the liposome of the recombinant plasmid pcDNA3.1-H is preferably 2 μ g to 10 μ L. The invention does not limit the type of culture medium needed in the transfection process, and the culture medium is selected conventionally in the field.
In the present invention, the protein a affinity chromatography purification preferably comprises: preparing ProteinA packing and packing: 1.5g of protein A-Sepharose CL-4B dry powder was dissolved in 6-7mL of triple distilled water, soaked in equilibration buffer for 15min, and loaded onto a chromatography column. The column was flushed with 10 bed volumes of equilibration buffer at a flow rate of 1mL/min to bring the effluent to a pH of 7.4.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Mouse immunization and potency detection
One week after the last immunization, 50-60 μ L of blood is taken from orbital venous plexus of mice, and after standing overnight at 4 ℃, the upper serum layer is centrifugally separated for later inspection. Taking a proper amount of c-Met antigen protein, diluting the c-Met antigen protein to 5 mu g/mL by using a coating buffer solution, then adding 100 mu L into each hole of a 96-hole plate by using a single-channel pipettor, slightly beating the plate to uniformly mix the sample, sealing the sample by using a preservative film, and coating the sample at 4 ℃ overnight; washing the plate 1 time by using a washing solution according to 200 mu L/hole, and buckling and drying the ELISA plate; then sealing the enzyme label plate by sealing liquid according to 300 mu L/hole, and sealing for 1 hour at room temperature; washing the plate with washing solution at 200 μ L/well for 2 times, adding sample (adding sample diluted by gradient and sample diluent at 100 μ L/well), adding detection antibody at the same time, adding into 96-well plate at 100 μ L/well, and acting at room temperature for 2 hr; washing the plate with washing solution at a rate of 200 μ L/hole for 5 times, adding color developing solution at a rate of 100 μ L/hole, and standing at room temperature for 12 min; adding stop solution into 50 mu L/hole to stop reaction; and (3) detecting by using a microplate reader: the measurement wavelength was 450nm, and the measurement results are shown in FIG. 1. After the determination, 5 mice all form high-titer c-Met-targeted antibodies, wherein the titer of the antibodies detected by the mouse A is the highest, so the mouse A is selected for the subsequent phage library construction.
Example 2
Biopanning variable region sequences
Extracting total RNA from spleen A of a mouse immunized with c-Met extracellular antigen by a trizol method, amplifying variable region genes of a mouse antibody by a mouse antibody scFv gene amplification kit (cargo number: P001Z), connecting heavy chains and light chains of the antibody by a peptide Linker (SEQ ID No. 13: SSGGGGSGGGGGGSSRSS) consisting of a plurality of glycin (Gly) and serine (Ser) to form a single-chain antibody gene fragment scFv, cloning the single-chain antibody gene fragment into a phagemid vector pCANTAB5E, constructing a phage display library, and displaying and expressing the single-chain antibody scFv in the phage display library, wherein the construction diagram of the phage display vector is shown in figure 2. Then, through plasmid extraction, phage plasmids are electrically transformed to build a library, and the library capacity is more than or equal to 108. Then, screening the phage library by using an ELISA plate coated with a specific antigen, and obtaining positive clones by adopting a cyclic mode of 'panning-amplification-enrichment', wherein the number of rounds is generally more than 2. The selected positive clones are further detected through an ELISA experiment, PCR of the positive clones detected through ELISA is cut by enzyme and typed, then sequencing is carried out, a proper variable region sequence is selected according to a sequencing result, and finally clone strains with the best effect of the three strains of 1D20, 3C36 and 3D63 are determined, and the ELISA result is shown in figure 3. The detection results show that the three clone strains of 1D20, 3C36 and 3D63 have extremely strong binding capacity with the C-Met antigen, but have no obvious binding with the control antigen and cell lysate, which indicates that the three clone strains have very good specific targeting function for the C-Met antigen.
Example 3
Construction, expression and purification of human-mouse chimeric monoclonal antibody
Construction of human-mouse chimeric monoclonal antibody vector
1.1 vector construction
The plasmid construction process is shown in FIG. 4, and the specific steps are as follows:
1.1.1PCR amplification of light and heavy chain variable Gene fragments
Designing and synthesizing an upstream primer SH.R containing a restriction enzyme cutting site BamHI and a signal peptide sequence HHS according to the related information of a human heavy chain signal peptide sequence and a pcDNA3.1(+) expression vector; according to the human light chain signal peptide sequence and the relevant information of pcDNA3.1/zeo (+) expression vector, a human light chain signal peptide upstream primer SL.F containing enzyme cutting site BamHI and a signal peptide sequence HLS are designed and synthesized, and primers are obtained by the design of primer design software Oligo 7.
Human IgG heavy chain signal peptide (HHS)
SEQ ID No.14：
5'-atggactggacctggaggatcctcttcttggtggcggccgccacaggcgcgcactcc-3’；
SEQ ID No.15：
SH.F5’-CGGGATCCatggactggacc-3'(BamHI)；
SEQ ID No.16：
Human IgG light chain signal peptide (HLS) 5'-atgttgccatcacaactcattgggtttctgctgctctgggttccagctagccgcggt-3';
SEQ ID No.17：
SL.F5'-CGGGATCCatgttgccatc-3'(BamHI)。
the constructed recombinant plasmid pCANTAB5E-3D63scFv is used as a template, primers are designed, heavy chain variable region primers VH.F and VH.R are used for amplifying heavy chain variable region gene segment VH, and light chain variable region primers VL.F and VL.R are used for amplifying light chain variable region gene segment VL.
Heavy chain variable region primer:
SEQ ID No.18：
VH.F5'-caggcgcgcactccTCTGGGGCTGAACTG-3'；
SEQ ID No.19：
VH.R5'-gcccttggtgctagctgaggagacggtgact-3'；
light chain variable region primer:
SEQ ID No.20：
VL.F5'-cagctagccgcggtGATGTTGTGATGACC-3'；
SEQ ID No.21：
VL.R5'-cgccgccaccgtacgtttgatttccagcttg-3'。
the PCR reaction was added as shown in Table 1 and centrifuged instantaneously to mix the reaction:
TABLE 1PCR reaction System for VH and VL Gene fragments
The PCR reaction conditions are as follows: 5min at 94 ℃ (1 cycle); 1min at 94 ℃ and 1min at 65 ℃; 1min at 72 ℃ (30 cycles); 10min at 72 ℃ (1 cycle). VH and VL were amplified separately.
The PCR product was recovered by agarose gel electrophoresis.
1.1.2PCR amplification of human IgG heavy and light chain constant region Gene fragments
Based on the information on the human IgG heavy and light chain constant region genes and pcDNA3.1(+) and pcDNA3.1/zeo (+) expression vectors, primers containing the restriction enzyme site XbaI were designed to amplify the constant region gene fragments, and primers were obtained by designing primer design software Oligo 7.
Taking human peripheral blood lymphocytes and extracting mRNA, and obtaining cDNA through RT-PCR.
Amplifying a heavy chain constant region CH by using the obtained cDNA as a template and CH.F and CH.R primers; the light chain constant region CL was amplified with cl.f and cl.r primers.
Heavy chain constant region primer
SEQ ID No.22：
CH.F5'-gctagcaccaagggcccatcggtc-3'；
SEQ ID No.23：
CH.R5'-tgctctagatcatttacccggag-3'(XbaI)；
Light chain constant region primer
SEQ ID No.24：
CL.F5'-cgtacggtggcggcgccatctg-3'；
SEQ ID No.25：
CL.R5'-tgctctagatcatttacccggag-3'(XbaI)。
The PCR reaction was added as shown in Table 2 and centrifuged instantaneously to mix the reaction:
TABLE 2PCR reaction System for CH and CL Gene fragments
The PCR reaction conditions are as follows: 5min at 94 ℃ (1 cycle); 1min at 94 ℃ and 1min at 65 ℃; 1min at 72 ℃ (30 cycles); 10min at 72 ℃ (1 cycle). CH and CL are amplified separately.
The PCR product was recovered by agarose gel electrophoresis.
1.1.3 splicing of murine antibody variable regions to human antibody constant regions by nested PCR
Using synthesized heavy chain signal peptide HHS and amplified heavy chain variable region VH and CH as templates, using SH.F and CH.R as primers, and amplifying full heavy chain gene fragments by overlap extension PCR (SOE-PCR); and similarly, using the synthesized light chain signal peptide HLS and the amplified light chain variable region VL and CL as templates, and using SL.F and CL.R as primers to amplify the whole light chain gene segment.
The PCR reaction was added as shown in tables 3 and 4 and centrifuged instantaneously to mix the reaction:
TABLE 3 PCR reaction System for heavy chain Gene fragments
TABLE 4 PCR reaction System for light chain Gene fragments
The PCR reaction conditions are as follows: 5min at 94 ℃ (1 cycle); 1min at 94 ℃ and 1min at 65 ℃; 1min at 72 ℃ (30 cycles); 10min at 72 ℃ (1 cycle). And respectively finishing the amplification splicing of the chimeric antibodies H and L.
The PCR product was recovered by agarose gel electrophoresis.
1.1.4 chimeric antibody heavy and light chains and plasmid pcDNA3.1(+) and pcDNA3.1(+)/zeo
And carrying out double enzyme digestion reaction on the target gene according to restriction enzyme sites BamHI and XbaI on the upper and lower streams of the target gene fragment. Plasmid pcDNA3.1(+) and pcDNA3.1(+)/zeo were double digested with restriction enzyme sites BamHI and XbaI to form a linear plasmid vector.
The enzyme digestion reaction system is shown in table 5 and table 6, and the mixture is added into the reaction system, mixed evenly and placed at 37 ℃ for reaction overnight.
Table 5 double enzyme digestion reaction system of target gene fragment
TABLE 6 double digestion reaction System for plasmid vectors
The product of the digestion reaction was recovered by agarose gel electrophoresis.
1.1.5 ligation of plasmid pcDNA3.1(+) with the heavy chain
The recovered heavy chain gene fragment and linearized plasmid vector pcDNA3.1(+) were ligated with T4 DNA ligase, and the target gene fragment and the linearized plasmid were mixed at a molar ratio of 10:1 to prepare a 25. mu.L reaction system, as shown in Table 7.
TABLE 7 ligation System of pcDNA3.1(+) and heavy chain Gene fragments
1.1.6 ligation of plasmid pcDNA3.1(+)/zeo with the light chain
The recovered light chain gene fragment and linearized plasmid vector pcDNA3.1/zeo (+) were ligated with T4 DNA ligase, and the target gene fragment and the linearized plasmid were mixed at a molar ratio of 10:1 to prepare a 25. mu.L reaction system, as shown in Table 8. The metal bath was used for overnight ligation at 16 ℃.
TABLE 8 ligation system of pcDNA3.1/zeo (+) and light chain gene fragments
2 expression of the recombinant plasmid
One day before transfection, 1X 10 cells were added5And (3) inoculating 293 cells into a 6-well plate, culturing the cells by using an antibiotic-free cell growth solution, performing transfection when the growth abundance of the cells reaches about 90%, replacing a culture medium 1h before transfection, and adding an Opti-MEM serum-free culture medium. 2. mu.g each of the recombinant plasmids pcDNA3.1-H and pcDNA3.1/zeo-L was mixed with 250. mu.l of Opti-MEM, 10. mu.l of liposome was added thereto, mixed with 250. mu.l of Opti-MEM, allowed to stand for 5min, the two mixtures were thoroughly mixed, and allowed to stand for 20 min. Adding the liposome complex into cells, mixing, and culturing in 37 deg.C incubator for 5 hr. The medium was replaced with a cell growth medium and the culture was continued for 3 days.
3 antibody purification
Affinity chromatography purification of 3D63 monoclonal antibody protein a was used for affinity chromatography of the antibody of interest and the purification process was monitored by AKTA explorer 100. Preparing Protein A packing and packing: 1.5g of protein A-Sepharose CL-4B dry powder is dissolved in 6-7mL of triple distilled water, soaked in the equilibrium buffer for 15min and loaded into a chromatographic column. The column was flushed with 10 bed volumes of equilibration buffer at a flow rate of 1mL/min to bring the effluent to a pH of 7.4. The supernatant of 293 cells was collected by centrifugation at 12000g at 4 ℃ for 15 min. 5mL of the supernatant was diluted with the equilibration buffer to 50mL, and the supernatant was filtered through a 0.45 μm filter to prepare a sample. The flow rate of the sample was 1 mL/min. After loading, flow wash was performed with 10 bed volumes of equilibration buffer at a flow rate of 1mL/min until UV reached baseline. The target antibody is then eluted with an elution buffer, and when a baseline rise is observed, i.e., an elution peak appears, the eluate is collected in tubes to neutralize the buffer to neutrality. Collecting eluate until the elution peak returns to baseline, continuously balancing the column bed with 5-10 times of the volume of the column bed of the equilibrium buffer solution, and adjusting the flow rate to 1 mL/min. The bed was equilibrated with 10 bed volumes of triple distilled water, the procedure is shown in FIG. 5. The ProteinA is a surface protein of staphylococcus aureus, can be specifically combined with a high-affinity site of an antibody Fc segment, and is suitable for purifying a monoclonal antibody of a cell culture supernatant. Thus, the antibody was purified using a Protein A affinity column, and the progress of the purification was monitored by AKTK explorer 100. The results of the purification were then examined by SDS discontinuous acrylamide gel electrophoresis and are shown in FIG. 6. Lane M is protein Marker; lanes N-R are the intact antibody bands in the non-reduced state, at a position above 212 kD; lane R is the antibody band in reduced state, and the antibody is reduced to light chain 26KD and heavy chain 50KD fragments. The results showed that the bands were very clear and single, demonstrating that the antibodies were of very high purity.
Example 4
Western-Blot identification of human-mouse chimeric monoclonal antibody
In order to verify whether the prepared human-mouse chimeric antibody has the capacity of targeted detection of the c-Met antigen, Western Blot test verification is carried out by preparing the purified 3D63 monoclonal antibody in example 3, and the test steps are as follows:
the separation gel is prepared according to the specification of the table, and the separation gel solution is poured into the mold. And lightly covering a layer of absolute ethyl alcohol on the separation gel to keep the gel flat, and standing for 10-15 min at room temperature. After the clear boundary between the separation gel and the ethanol is visible to the naked eye, the ethanol is poured off, and the residual ethanol is carefully sucked dry by using absorbent paper. Adding the concentrated gel on the separation gel until reaching the top end of the glass plate, inserting a comb matched with the thickness of the glass plate into the gel, and standing at room temperature for about 15min until the gel is formed. Placing the glass plate in a protein vertical electrophoresis tank, compacting, adding 1 XMOPS-SDS electrophoresis solution, exceeding the scale mark of two pieces of glue outside the electrophoresis tank, and checking whether the protein vertical electrophoresis tank has liquid leakage phenomenon. And (3) taking 20 mu LA549 cell protein extract samples, respectively adding the samples into SDS-PAGE concentrated gel sample holes, adding 5 mu L of protein pre-staining Marker, and recording the sample adding sequence. And setting the electrophoresis power supply to be constant voltage of 120V for electrophoresis for about 1h until the bromophenol blue indicator runs to the bottom of the separation gel, the green edge of the shelf and the protein Marker strip of the required section is completely separated. According to the size of a rubber block in a sample area, a PVDF membrane with the size consistent with that of the rubber block and 6 layers of filter paper with the length and the width smaller than that of the rubber block by 1mm are cut, placed in methanol for 6s, and quickly transferred to an electrotransformation liquid for soaking for standby. According to the principle that the current flows from the anode to the cathode and the film is positive and negative, the semi-dry carbon plate transfer printing tank is placed in the order of filter paper-film-glue-film. And (5) calculating the current I according to a formula, and rotating the membrane for 50 min. After the membrane conversion is finished, taking a dark box of the Western Blot, sealing the PVDF membrane by using a 5% skimmed milk powder solution, and placing the PVDF membrane on a horizontal shaking table for 4 hours. And discarding the skimmed milk powder solution. And washing the membrane by using TBST until no skimmed milk powder solution exists. Purified human-mouse chimeric monoclonal antibody was added as a primary antibody and shaken on a horizontal shaker for 10min to ensure that the PVDF membrane was immersed in the primary antibody solution and allowed to cool overnight at 4 ℃. Discard the primary antibody solution, wash the membrane 3 times with TBST solution, and shake horizontally for 10min3 times each time. The TBST solution was discarded. And pouring the secondary antibody solution into a Western Blot light-proof black box under the light-proof condition, ensuring that the PVDF membrane is immersed in the secondary antibody solution, tightly covering the black box, and shaking the table for 50min in the light-proof manner. Discard the secondary antibody solution. The membranes were washed 2 times with TBST solution for 10min each time. The TBST solution was discarded. The membrane was washed 1 time with TBS solution and horizontally for 5min on a shaker. The results of the membrane-scanning analysis are shown in FIG. 7. H1299 is a c-Met positive cell strain, c-Met protein in the c-Met positive cell strain is knocked out by using a CRISPR technology to be used as a control cell, a WB result shows that a band can be detected in an H1299 cell which is not knocked out, other bands do not exist, the knocked-out cell cannot be obviously increased, the size of the protein detected in the knock-out group is consistent with that of c-Met, and the prepared 3D63 human-mouse chimeric antibody can be specifically combined with the c-Met protein and can be used for experiments such as WB detection and the like.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Beijing ancient cooking peptide source Biotechnology Ltd
<120> anti-human c-Met human mouse chimeric monoclonal antibody and application thereof
<160> 25
<170> SIPOSequenceListing 1.0
<210> 1
<211> 357
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gaggttcagc tgcagcagtc tggagatgat ctggtaaagc ctggggcctc agtgaagctg 60
tcctgcaagg cttctggtta ctcatttact ggctacttta tggactgggt gatgcagagc 120
catggaaaga gccttgagtg gattggacgt attaatccta acaatggtga tactttttac 180
aaccagaagt tcaagggcaa ggccacattg actgtagaca aatcctctag tacagcccac 240
atggagctcc ggagcctggc atctgaggac tctgcagtct attattgtgc aagaatgaag 300
ctaattgggt cctatatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357
<210> 2
<211> 344
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gatatccaga tgactcagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagtaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc tattcacgtt cggctcgggg 300
acaaagttgg aaataaaatc 320
<210> 3
<211> 354
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gaggtccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaagctg 60
tcctgcaagg cctctggcta caccttcacc agctactgga tacactgggt gaaacagaga 120
cctggacagg gccttgagtg gattggagag attaatccta gcaacggtca tacttactac 180
actgagaagt tcaagatcaa ggccacaatg actttagaca aatcctccag cacggcctac 240
atgcaactca gcagcctgac atctgaggac tcagcggtct atttttgtgg aagatatccc 300
aagggagggt atttcgatgt ctggggcgca gggaccacgg tcaccgtctc ctca 354
<210> 4
<211> 342
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gacattgtgc taactcagtc tccaggcacc ctatctgtga ctccaggaga tagcgtcagt 60
ctttcctgca gggccagcca aagtattagc agctacctac actggtatca acaaaaatca 120
catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc 180
aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 240
gaagattttg gaatgtattt ctgtcaacag agtaaaagct ggcctttcac gttcggctcg 300
gggacaaagt tggaaataaa atctagtggt ggcggtggtt cg 342
<210> 5
<211> 351
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaggtccagc tgcagcagtc tggggctgaa ctggtgaggc ctggggtctc agtgaagatt 60
tcctgcaagg ggtctggcta cacattcact gattatgcta tgcactgggt aaagcagagt 120
catgcaaaga gtctagagtg gattggagtt agtagtagtt attatggtga ggctaactac 180
aaccagaagt tcaaggccaa ggccacaatg actgtagaca aatcctccag cacagcctat 240
atggagcttg ccggactgac atctgaggat tctgccatct attactgtgt aagacacgac 300
gtggatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a 351
<210> 6
<211> 342
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca accagcctcc 60
atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120
ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180
tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240
agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttcct 300
cggacgttcg gtggaggcac caagctggaa atcaaatcta gt 342
<210> 7
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Val Gln Leu Gln Gln Ser Gly Asp Asp Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Phe Met Asp Trp Val Met Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Asn Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala His
65 70 75 80
Met Glu Leu Arg Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Met Lys Leu Ile Gly Ser Tyr Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val Ser Ser
115
<210> 8
<211> 106
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 9
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly His Thr Tyr Tyr Thr Glu Lys Phe
50 55 60
Lys Ile Lys Ala Thr Met Thr Leu Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Gly Arg Tyr Pro Lys Gly Gly Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 10
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Asp Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Val Thr Pro Gly
1 5 10 15
Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr
65 70 75 80
Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Lys Ser Trp Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 11
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Val
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile
35 40 45
Gly Val Ser Ser Ser Tyr Tyr Gly Glu Ala Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Ala Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ala Gly Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Val Arg His Asp Val Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110
Val Thr Val Ser Ser
115
<210> 12
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr 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
85 90 95
Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 13
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg
1 5 10 15
Ser Ser
<210> 14
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atggactgga cctggaggat cctcttcttg gtggcggccg ccacaggcgc gcactcc 57
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cgggatccat ggactggacc 20
<210> 16
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
atgttgccat cacaactcat tgggtttctg ctgctctggg ttccagctag ccgcggt 57
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cgggatccat gttgccatc 19
<210> 18
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
caggcgcgca ctcctctggg gctgaactg 29
<210> 19
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gcccttggtg ctagctgagg agacggtgac t 31
<210> 20
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cagctagccg cggtgatgtt gtgatgacc 29
<210> 21
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cgccgccacc gtacgtttga tttccagctt g 31
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gctagcacca agggcccatc ggtc 24
<210> 23
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tgctctagat catttacccg gag 23
<210> 24
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cgtacggtgg cggcgccatc tg 22
<210> 25
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tgctctagat catttacccg gag 23
Claims (6)
1. The anti-human c-Met human mouse chimeric monoclonal antibody is characterized in that the nucleotide sequence of a 1D20 heavy chain variable region of the anti-human c-Met human mouse chimeric monoclonal antibody is shown as SEQ ID No.1, and the nucleotide sequence of a 1D20 light chain variable region is shown as SEQ ID No. 2.
2. The chimeric monoclonal antibody against human c-Met mouse of claim 1, wherein the amino acid sequence of the heavy chain variable region of 1D20 is represented by SEQ ID No. 7.
3. The chimeric monoclonal antibody against human c-Met mouse of claim 1, wherein the amino acid sequence of the variable region of the light chain of 1D20 is represented by SEQ ID No. 8.
4. A recombinant expression vector comprising the nucleotide sequences of the heavy chain variable region and the light chain variable region of the anti-human c-Met human murine chimeric monoclonal antibody of claim 1;
the nucleotide sequence of the 1D20 heavy chain variable region of the anti-human c-Met mouse chimeric monoclonal antibody is shown as SEQ ID No.1, and the nucleotide sequence of the 1D20 light chain variable region is shown as SEQ ID No. 2.
5. A host cell transformed with the recombinant expression vector of claim 4.
6. A method of producing a chimeric human murine monoclonal antibody against human c-Met comprising: culturing the host cell according to claim 5, and collecting the human mouse chimeric monoclonal antibody against human c-Met from the culture.
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| MX2019014316A (en) * | 2017-05-30 | 2020-01-27 | Chong Kun Dang Pharmaceutical Corp | A novel anti-c-met antibody and use thereof. |
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Also Published As
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| CN111892656B (en) | 2021-02-19 |
| CN112521507A (en) | 2021-03-19 |
| CN112625131B (en) | 2021-08-17 |
| CN111892656A (en) | 2020-11-06 |
| CN112625131A (en) | 2021-04-09 |
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