CN103880956A - Anti-MUC1 monoclonal antibody as well as light chain and heavy chain variable regions thereof - Google Patents
Anti-MUC1 monoclonal antibody as well as light chain and heavy chain variable regions thereof Download PDFInfo
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Abstract
The invention discloses an anti-MUC1 monoclonal antibody as well as light chain and heavy chain variable regions thereof. Light chain and heavy chain variable regions of the anti-MUC1 monoclonal antibody FMU-MUC1-No.1 having a bioactivity are provided, an amino acid sequence of the light chain variable region is shown in SEQ ID NO. 1 and an amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2. A group of mouse anti-MUC1 mAbs are prepared through a genetic engineering technology and a hybridoma technology; a hybridoma cell line capable of stably excreting high-specificity anti-MUC1 mAb is screened; ascites is prepared to obtain high-specificity and high-affinity anti-MUC1 mAb FMU-MUC1-No. 1. Uniquenesses and CDR sequences of a sequence of a gene sequence and a corresponding protein sequence are confirmed; the monoclonal antibody provides support for research and development of anti-MUC1 chimeric or humanized genetic engineering antibodies or molecular diagnosis and treatment reagents.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to a kind of monoclonal antibody, be particularly related to the anti-MUC1 monoclonal antibody of high specific (FMU-MUC1-No.1) and light chain and variable region of heavy chain, comprise its aminoacid sequence and nucleotide sequence and preparation method for antibody and its diagnosis and treatment purposes to mammary cancer etc.Background technology
Mammary cancer is the common cancer of serious harm women's health, and worldwide, mammary cancer accounts for 10% of all cancer morbidities, account for women's cancer morbidity 32%, women's cancer mortality 15%.The whole world approximately has 1,200,000 women to suffer from mammary cancer every year.In the developed country such as North America, West Europe, breast cancer incidence accounts for women's malignant tumour first place.China is one of the fastest country of breast cancer incidence rate of growth, breast cancer incidence is just with annual 3% speed increase in recent years, become the first killer of Urban Women, although have operation and the complex treatment measure including radiotherapy, chemotherapy, endocrine therapy etc., but breast cancer relapse, transporting patient are dead because not curing eventually, therefore, need to seek new diagnosis and methods for the treatment of.The appearance of monoclonal antibody technique is the important breakthrough of field of immunology.Utilizing monoclonal antibody target pathological tissues or cell-surface antigens, become the Critical policies of mammary cancer molecular diagnosis and immunotherapy, is its key and select desirable target molecule.The distinctive structure and function feature of MUC1, becomes the desirable target antigen of one of mammary cancer molecular diagnosis and immunotherapy.
1, MUC1 molecule and biological function
MUC1 has another name called multiform mucins (polymorphic epithelial mucin, PEM), it is one of cell surface molecule contacting with body immune system at first, it is a kind of I type transmembrane protein of high glycosylation, mainly be expressed under normal circumstances the epithelial cell lumen of gland face in Various Tissues, organ, be top and express, polar contribution; At cancer cells surface MUC1 unconventionality expression, be nonpolar distribution.Research shows: MUC1 is unconventionality expression in the kinds of tumors tissues such as mammary cancer, and closely related with generation, development and the prognosis of tumour.
1.1MUC1 gene
The cDNA expression library clone that MUC1 gene builds from clones such as mammary cancer the earliest obtains, and is positioned the long-armed 21 band places of lq21(1 karyomit(e)), this gene is also the mucoprotein gene of unique coding cross-film.People's MUC1 gene cDNA total length is 1821bp, contains 7 exons, and different exon effects are different.A key character of MUC1 gene is its polymorphism (polymorphism), be in its 2nd exon, to contain tandem repetitive sequence (the variable number of tandem repeats that quantity does not wait, VNTRs), from 20～125 not etc., epitope is positioned at VNTRs district.Each VNTR structure contains 60 bases, is rich in GC, 20 amino acid of encoding, and they are GVTSAPDTRPAPGSTAPPAH.Wherein APDTRPA position is the epi-position of T cell and B cell corecognition, and the immunogenicity of MUC1 is played a decisive role.
1.2MUC1 molecular structure
MUC1 is a kind of glycoprotein of high molecular, is made up of core peptide and sugar chain, and sugar chain accounts for 50～90%, is connected with the Ser/Thr of polypeptide backbone VNTR mainly with O type glycosidic link.Tandem repeat is the typical structure of core peptide, and each tumor-necrosis factor glycoproteins VNTR contains 2 Serine Ser, 3 Threonine Thr, and these are all glycosylated potential sites.At MUC1 C-terminal, comprise the intracellular region being formed by 72 amino acid, 28 cross-film district and 58 extracellular region three parts that amino acid forms that amino acid forms.Wherein born of the same parents' inner segment and the structure of cross-film section between different genera have high conservative property, point out its important biological function.Extracellular fragment contains the tandem repetitive sequence being made up of 20 amino acid, has determined space structure specificity and the immunogenicity of MUC1.Under normal circumstances, MUC1 extracellular fragment is covered with intensive, hyperbranched sugar chain, and in the time of cell generation canceration, the sugar chain of MUCI becomes sparse, thereby epitope peptide backbone comes out, and has immunogenicity.
1.3MUC1 is at expression of tumor tissue such as mammary cancer
Under normal circumstances, the mainly nearly tube chamber of epithelial cell or the expression of lumen of gland face in Various Tissues, the organs such as mammary gland, pancreas, digestive tube, respiratory tract, reproductive tract of MUC1, is top and expresses, polar contribution, concentrate and be positioned at epithelioglandular chamber face, kytoplasm has no expression, is difficult for by immune system recognition.But MUC1 unconventionality expression in tumour cell: (1) MUC1 significantly raises in tumor cell surface expression amount, is overexpression, is the more than 100 times of normal expression amount, and is proportionate with the grade malignancy of tumour; (2) MUC1 expresses and is nonpolar on tumour cell, is distributed in the surface of cell, and also visible in kytoplasm; (3) in tumour cell because glycosyl transferase activity increases, cause glycosylation incomplete, sugar chain shortens, and normal hidden core peptide epi-position is exposed, make it have immunogenicity, and occurred new sugar chain epi-position (as sugared epi-positions such as Tn, STn, TF).MUC1 is weak positive expression in normal breast epithelial cell and benign tumor tissue, is polar contribution, and core peptide epi-position is covered by periphery sugar chain, can not be identified; On cancerous tumor cell surface, because glycosylation hapten peptide epitopes is exposed.Research discovery, MUC1 positive expression rate in breast cancer tissue exceedes 90%.In addition MUC1 wide expression in the tumor tissue cells such as carcinoma of the pancreas, colorectal cancer, lung cancer, ovarian cancer, thyroid carcinoma.
The biological function of 1.4MUC1
Early stage studies have shown that the mucoprotein main biological function of MUC1 is the epithelial cell of lumen of gland to be played to lubricated, provide protection and stickiness maintain etc.Along with deepening continuously that MUC1 is studied, find that MUC1 molecule has several functions, as stick-anti-adhesion, immune activation-immunosuppressive action etc.MUC1 also has the signal transduction between mediated cell and participates in the functions such as Organism immunoregulation.Up-to-date research shows: due to MUC1 gene and messenger RNA(mRNA), to translate rear regulation and control relevant, and therefore its unconventionality expression may and shift relevant with the growth of cancer cells.
2, MUC1 antibody present Research
Monoclonal antibody (monoclonal antibody, mAb) research is one of the most popular research direction of 2l century field of biology, and Cynthia etc. have described a kind of preparation method and purposes of MUC1 monoclonal antibody.The invention antigen such as Daniel Cheek in conjunction with and/or the anti-mucin antibody of evident characteristics and for improvement of the antigen of anti-mucin antibody in conjunction with and/or know method for distinguishing, for the immunotherapy of cancer.The inventions such as D.B Rubinstein provide the while in conjunction with the α subunit of complete MUC1 albumen and the antibody of β subunit, and prepare and use the method for these antibody.One youth of gentry of western village waits discovery: use 2 of MUC1,3ST glycopeptide carries out immunity as antigen to animal, and gained antibody can be identified the specific MUC1 sugar chain of cancer specifically, and then can identify, kill and wound the cancer cells of expressing MUC1 sugar chain.The specific recognition of the specific recognition of the cancer dependency structure of the antibody that this invention provides to MUC1 and the healthy tissues dependency structure to MUC1 the two there were significant differences.
Domestic scholars Zhang Lixin etc. obtains human milk's membrana granulosa (HMFGM) from normal people Ruzhong through high speed centrifugation, through further fragmentation, degreasing and purifying, obtain containing the mucinous component of MUC1, and after SDS-PAGE, Western-blot and ELISA qualification, how anti-immunizing rabbit preparation is.Result shows, what prepare how anti-ly measures and tire as 1:64000～1:128000 through ELISA.Horse faithful and upright person waits the mouse with breast cancer cell line ZR75 immunity Bulb/c, obtains the hybridoma cell line BM109 of the anti-ZR75 monoclonal antibody of constant secretion, has certain specificity.The 8R-MUC1-6Histag that the employing escherichia coli prokaryotic expression systems such as Li Yuan are expressed is that immunogen is prepared MUC1 monoclonal antibody, but the monoclonal antibody None-identified tumor cell surface MUC1 obtaining with this expresses, can not meet the requirement of preparation highly sensitive and high specific ELISA detection kit.For obtaining the MUC1 monoclonal antibody of high specific, use Bac-to-Bac baculovirus expression system instead and express MUC1 albumen, to becoming the desirable antigen of preparation MUC1 monoclonal antibody.Tang Yan etc. are taking 8R-MUC1 core peptide recombinant protein as antigen immune rabbit, determine that anti-MUC1 polyclonal antibody tires, with Western blot and ELISA blocking test qualification antibodies specific with ELISA method.Result MUC1 core peptide polyclonal antibody is tired as 1:256000, can with MUC1 core peptide specific binding, but compared with monoclonal antibody, polyclonal antibody specificity is poor.
3, antibody improvement and application
Antibody monomer molecule is heavy chain (H chain) with two the identical light chain (L chain) identical by two, the tetrapeptide chain structure being formed by connecting by interchain disulfide bond.H chain and L chain comprise amino (N) end and carboxyl (C) end, are made up of hypervariable region/complementary determining region (HVR/CDR) and skeleton district (FR) near the variable region (V district) of N end; Be constant region (C district) near C end.The protein folding that variable region of heavy chain (VH) and variable region of light chain (VL) form is antigen-binding site, and CDR/HVR is wherein the position of antibody and the complementary combination of epitope, and the reaction after antigen-antibody identification is caused in C district.Antibody can divide for people source, mouse source etc. according to FR/C district difference, mouse antibody has immunogenicity while use in human body, easily cause the immune response of human body, these immune responses can cause the allergy of removing to mouse antibody and immunocomplex mediation.In order to overcome the defect of mouse antibody, need to build specific chimeric antibody, single-chain antibody or the humanized antibody of high-affinity.
In transformation process, most importantly first must obtain the mouse parental antibody with good specificity, avidity, clone its light chain and heavy chain variable region gene, then these two genes are transformed, build recombinant antibodies gene.Therefore, filter out high specific, the anti-MUC1mAb of mouse of high-affinity, therefrom clone light, heavy chain variable region gene, further development is there is to the MUC1 genetic engineering antibody preparation of complete independent intellectual property right, as the important means of mammary cancer molecular diagnosis and immunotherapy to fill up the blank of China in this field, not only there is earth shaking significance for promoting China's breast cancer prevention adaptibility to response, and in the carcinoma of the pancreas of MUC1 positive expression, lung cancer, the molecular diagnosis of gastroenteric tumor and thyroid carcinoma and immunotherapy aspect also have very important actual application value.
Summary of the invention
The problem that the present invention solves is to provide anti-MUC1 monoclonal antibody and light chain and variable region of heavy chain, this antibody be combined with mammary tumor cells specific, not with the antibody of normal breast Cell binding, provide support for building the chimeric or humanized genetic engineering antibody of the anti-MUC1 of high specific.
The present invention is achieved through the following technical solutions:
A kind of anti-MUC1 monoclonal antibody, comprises light chain and heavy chain, and 3 complementary determining regions (CDR) sequence of described variable region of light chain is respectively:
CDR1：Gly-Phe-Thr-Phe-Ser-Asn-Tyr-Trp；
CDR2：Ile-Arg-Leu-Ile-Ser-Asn-Asn-Tyr-Val-Pro；
CDR3：Ser-Phe-Gly-Asn-Ser-Phe-Ala-Tyr；
3 complementary determining regions (CDR) sequence of the variable region of described heavy chain is respectively:
CDR1：Ser-Ser-Val-Ser-Tyr；
CDR2：Leu-Thr-Ser；
CDR3：Gln-Gln-Trp-Ser-Ser-Asn-Pro-Leu-Thr。
The aminoacid sequence of the variable region of light chain of described anti-MUC1 monoclonal antibody is as SEQ.ID.NO.1, and the aminoacid sequence of variable region of heavy chain is as shown in SEQ.ID.NO.2.
The gene order of described coding MUC1 monoclonal antibody variable region of light chain is as shown in SEQ.ID.NO.3, and the gene order of encoding heavy chain variable region is as shown in SEQ.ID.NO.4.
The light chain of described anti-MUC1 monoclonal antibody and variable region of heavy chain are applied to the preparation building for genetic engineering antibody or molecular diagnosis and the treatment reagent of MUC1.
Compared with prior art, the present invention has following useful technique effect:
1, anti-MUC1 monoclonal antibody provided by the invention is the anti-MUC1 monoclonal antibody of a kind of high specific: be combined with MUC1 positive expression tumor cell specifics such as mammary cancer, show high-affinity, high specific; Be combined with mammary tumor cells specific, not with the antibody of normal breast Cell binding.Can be further used for the research of the aspect such as molecular diagnosis and immunotherapy of mammary cancer, for early diagnosis and the immunotherapy of mammary cancer provide novel method, New Policy.
2, the present invention clones light chain, heavy chain variable region gene and the aminoacid sequence of anti-MUC1 monoclonal antibody, and sequential analysis has confirmed the uniqueness of this antibody sequence.
3, analyze and obtain light chain, CDR district, variable region of heavy chain, provide support for building the chimeric or humanized genetic engineering antibody of the anti-MUC1 of high specific, high-affinity on this basis.
Brief description of the drawings
Fig. 1 is the immunofluorescence detected result of FMU-MUC1-No.1mAb;
Fig. 2 is the flow cytometry qualification result of FMU-MUC1-No.1mAb;
Fig. 3 is the Western blot qualification result of FMU-MUC1-No.1mAb;
Fig. 4 is FMU-MUC1-No.1mAb ImmunohistochemistryResults Results;
Fig. 5 is FMU-MUC1-No.1mAb gene PCR result;
Fig. 6 is FMU-MUC1-No.1mAb chain variable region gene homology sequence detected result;
Fig. 7 is FMU-MUC1-No.1mAb heavy chain variable region gene homology sequence detected result;
Fig. 8 is FMU-MUC1-No.1mAb variable region of light chain amino acid identity sequential detection result;
Fig. 9 is FMU-MUC1-No.1mAb variable region of heavy chain amino acid identity sequential detection result;
Figure 10 is FMU-MUC1-No.1mAb light chain and weight chain variable region amino acid sequence structure.
Embodiment
The present invention, with synthetic MUC1 tumor-necrosis factor glycoproteins immunity Balb/c mouse, filters out the anti-MUC1 monoclonal antibody of energy stably excreting high specific FMU-MUC1-No.1mAb hybridoma cell strain, and preparation ascites obtains the anti-MUC1mAb of high specific; And confirm uniqueness and the CDR sequence thereof of this gene order and corresponding protein sequence; For anti-MUC1 is chimeric or humanized genetic engineering antibody provides support.Below in conjunction with preparation method, antibodies specific and active detection of concrete monoclonal antibody, and determining of the detection of sequence and uniqueness elaborate to the present invention, and the explanation of the invention is not limited.The present invention specifically implements according to the following steps:
1, preparation and the specificity identification thereof of anti-MUC1 monoclonal antibody FMU-MUC1-No.1mAb
Prepare with antigen 1.1MUC1 small peptide is synthetic
Synthetic two tumor-necrosis factor glycoproteinss of MUC1 are C GVTSA PDTRP APGST APPAH GVTSA PDTRP APGST APPAH.Composition sequence N → C, purity >95%.After polypeptide is synthetic, for strengthening its immunity, polypeptide and keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) are cross-linked.The small peptide of MUC1 tumor-necrosis factor glycoproteins synthesizes and is completed by Zi Yu bio tech ltd, Shanghai with KLH, BSA albumen coupling.The immunogen of conduct preparation monoclonal antibody after synthetic two tumor-necrosis factor glycoproteins small peptides of MUC1 and KLH, BSA albumen coupling.
The preparation of 1.2 monoclonal antibodies, purifying
Press method for preparing monoclonal antibody (cell and molecular immunology experimental technique first version, P9-P17), with MUC1 antigen immune Balb/c mouse (every mouse 20 μ g/ time, purchased from The Fourth Military Medical University's Experimental Animal Center).Initial immunity, uses Freund's complete adjuvant, and follow-up immunization uses Freund's incomplete adjuvant, and every minor tick 3 weeks, is subcutaneous multi-point injection, altogether immunity 4 times.Last immunity 7～10 days afterwards blood sampling is surveyed it and is tired, and detects immune effect.Behind 2～3 weeks, interval, through abdominal injection antigen booster immunization again, after 3 days, put to death animal and get spleen and carry out cytogamy.
The murine myeloma cell SP2/0 growing that takes the logarithm counts, and prepares immune spleen cell suspension simultaneously.Myeloma cell is mixed and carries out cytogamy in 1:10 ratio with splenocyte.After merging, cell suspension adds 96 orifice plates that contain feeder cell (normal Balb/c Turnover of Mouse Peritoneal Macrophages), 37 DEG C, 5%CO
2incubator is cultivated.After clone occurs, indirect ELISA detects, and selects positive colony.Adopt limiting dilution assay to carry out cloning to the cell that contains positive colony hole, until obtain can stably excreting antibody hybridoma cell line (in-vitro cultivation exceedes 6 months).
Induction produces ascites and antibody purification: obtain can the hybridoma cell strain of stably excreting antibody after, prepare routinely ascites (cell and molecular immunology experimental technique first version, P9-P17).Ascites, after 45% saturated ammonium sulphate, adopts QFF anion exchange chromatography purifying, and the FMU-MUC1-No.1mAb purity of purifying reaches 95%.The operation of Sigma detection kit specification sheets is pressed in the qualification of the IgG hypotype of antibody.The antibody of its secretion is carried out to Ig subclass mensuration (result is IgG1 subclass, κ type light chain).
The titration of 1.3 anti-MUC1 monoclonal antibody FMU-MUC1-No.1
With the relative affinity of mAb after ascites and purifying before indirect ELISA method mensuration purifying.Wherein envelope antigen is synthetic MUC1 immunogen, mAb after the ascites that testing sample is serial dilution and purifying, and detecting antibody is sheep anti mouse-HRP enzymic-labelled antibody, substrate uses ABTS.The high-affinity FMU-MUC1-No.1mAb filtering out, titer of ascites is 1 × 10
-6, after purifying, tire and reach 1 × 10
-8, reach 1 × 10 and generally adopt indirect ELISA to detect titer of ascites
-5above antibody can use.
The immunofluorescence of 1.4 anti-MUC1 monoclonal antibody FMU-MUC1-No.1 detects
Of the anti-MUC1mAb of preparation, breast cancer cell (MCF-7) and liver cancer cell (HepG2) are made the expression of immunofluorescence analysis MUC1.Step is as follows: take out the long cover glass that has cell, abandon nutrient solution, wash 2 times with PBS, add 40g/L paraformaldehyde, room temperature fix 10min, after PBS washing, adds the BSA/PBS of 1mL100g/L, and room temperature is sealed 1h.Use the BSA/PBS of 100g/L to dilute anti-FMU-MUC1-No.1, incubated at room 3h.Use the goat anti-mouse IgG room temperature lucifuge of TRITC (TRITC) mark of the BSA/PBS dilution of 100g/L to hatch 1h.PBS washes 6 times.Add the DAPI of 1 μ g/mL PBS dilution, hatch 2min.Wash 4 times lucifuge, natural air drying with PBS.After mounting, observe.
Shown in detected result Fig. 1, can see that breast cancer cell has sent fluorescence (right side), and liver cancer cell does not send fluorescence (left side).
Immunofluorescence dyeing detects and confirms: anti-MUC1 monoclonal antibody can be combined with the mammary tumor cells specific of MUC1 positive expression, expresses negative liver cancer cell be not combined with MUC1.
The flow cytometry qualification of 1.5 anti-MUC1 monoclonal antibody FMU-MUC1-No.1
Breast cancer cell MCF-7 is made to single cell suspension with 0.25% tryptic digestion, counting 1 × 10
6individual cell, with PBS washing 2 times, the centrifugal 5min of 1000r/min, abandoning supernatant, then adds monoclonal antibody FMU-MUC1-No.1 (200 μ g/mL) to hatch 45min, uses homotype monoclonal antibody IgG-PE to do negative control.With PBS washing 2 times, then add sheep anti mouse PE fluorescence two anti-lucifuges to hatch 30min.PBS washing 2 times, adds paraformaldehyde stationary liquid 400 μ L, upper machine testing and analysis, and use instrument is flow cytometer.
Fluidic cell detected result as shown in Figure 2, can be seen that combining FMU-MUC1-No.1 cell has afterwards fallen target area (right figure), and not see that contrast IgG drops into target area (left figure); Result shows: compared with control group, and anti-MUC1 monoclonal antibody and MUC1 positive expression breast cancer cell MCF-7 high specific association reaction.
The Western blot qualification of 1.6 anti-MUC1 monoclonal antibody FMU-MUC1-No.1
Collect breast cancer cell (MCF-7), liver cancer cell (HepG2), lysing cell, analyzes the expression of MUC1 in different tumour cells with the FMU-MUC1-No.1mAb of anti-MUC1 preparing by Western blot step after SDS-PAGE.
Western blot step: collecting cell, by sample preparation after lysis, transferring film after SDS-PAGE electrophoresis.37 DEG C of sealing 2h of skim-milk of 50g/L, TBST washes film 3 times; The mAb (1:2000) that adds the anti-MUC1 of preparation, 4 DEG C are spent the night, and TBST washes film.Add goat anti-mouse IgG to hatch 1h; Add ECL substrate, develop.
Result shows as shown in Figure 3, can see that FMU-MUC1-No.1 can identify specific albumen (swimming lane 1) after breast cancer cell cracking, and fail to identify the albumen in liver cancer cell lysate, result shows: anti-MUC1 monoclonal antibody can specific recognition breast cancer tissue cell lysate in natural MUC1 albumen, relative molecular weight is 220kDa, is not combined with the negative liver cancer cell lysate of expressing of MUC1.
The immunohistochemical methods qualification of 1.7 anti-MUC1 monoclonal antibody FMU-MUC1-No.1
Breast cancer tissue's section dewaxing, aquation.PBS washes 5min.To cut into slices and immerse in 0.01mol/L citrate buffer (pH6.0), pressure kettle 2min or heating in water bath, naturally cooling, PBS washes; 30mL/L hydrogen peroxide is hatched 10min; PBS washes section; Drip anti-MUC1 primary antibodie, in 37 DEG C of water baths, hatch 1h.PBS washes 5 times; Drip biotin labeled goat anti-mouse IgG, hatch 30min for 37 DEG C.PBS washes 5 times; DAB colour developing 1～3 minute; Hematorylin lining dyes, dehydration, transparent, mounting, om observation.In experiment using blank test and alternate test as negative control, to guarantee the special and reliability of experiment.
Mammary cancer paraffin organization section immunohistochemical staining analytical results as shown in Figure 4, result confirms: anti-MUC1mAb and breast cancer cell high specific are strong in (Fig. 4 A, B, C, positive staining mainly appears on breast cancer cell membrane, part endochylema dyeing), and be not combined (Fig. 4 D) with Ai Pang normal galactophore tissue.
2, the clone of anti-MUC1 monoclonal antibody FMU-MUC1-No.1mAb light chain and heavy chain variable region gene
The cultivation of 2.1FMU-MUC1-No.1mAb hybridoma
The hybridoma of recovery (cell cultures, first version, P88) secretion FMU-MUC1-No.1mAb, uses containing the RPMI1640 of 10% calf serum and cultivates based on 37 DEG C, 5%CO according to a conventional method
2in incubator, be cultured to logarithmic phase.
Synthesizing of the extraction of 2.2 total RNA and cDNA the first chain
Adopt TRIZOL Reagent(purchased from GIBCO company of the U.S.) extract total RNA, concrete operation step by specification carries out; CDNA the first chain synthetic agent box is purchased from GIBCO company of the U.S., carries out synthetic cDNA the first chain of reverse transcription obtaining by specification after total RNA.
VL and the VH gene of 2.3RT-PCR method amplification FMU-MUC1-No.1mAb
Single stage method RT-PCR amplification kit is purchased from TakaRa company, by VL and the VH gene of test kit specification sheets amplification FMU-MUC1-No.1mAb;
Primer is as follows:
The primer (being degeneracy base in bracket) of amplification antibody Fd fragment and light chain full-length gene
Murine heavy chain V district 5 ' end primer:
VH1:5’-GGG?
GAT?ATC?CAC?CAT?GG(AG)ATG(CG)AG?CTG(TG)GT(CA)AT(CG)CT?CTT-3’
VH2:5’-GGG?
GAT?ATC?CAC?CAT?G(AG)A?CTT?CGG?G(TC)T?GAG?CT(TG)GGT?TTT-3’
VH3:5’-GGG?
GAT?ATC?CAC?CAT?GGC?TGT?CTT?GGG?GCT?GCT?CTT?CT-3’
VH4:5’-GGG?
GAT?ATC?CAC?CAT?GAT(AG)GT?GTT(AG)AG?TCT?T(CT)T?GT(AG)CCT?G3’
Fd3':5'-AGG?CTT?
ACT?AGT?ACA?ATC?CCT?GGG?CAC?AAT-3'；
Mouse light chain V district 5 ' end primer
VL1:5’-GGG?
GAT?ATC?CAC?CAT?GGA?GAC?AGA?CAC?ACT?CCT?GCT?AT-3’
VL2:5’-GGG?
GAT?ATC?CAC?CAT?GGA?TTT?TCA?AGT?GCA?GAT?TTT?CAG-3’
VL3:5’-GGG?
GAT?ATC?CAC?CAT?GGA?G(AT)C?ACA(GT)(AT)C?TCG?GGTCTT?T(GA)T?A-3’
VL4:5’-GGG?
GAT?ATC?CAC?CAT?G(GT)C?CCC(AT)(AG)C?TCA?G(CT)T?C(CT)C?T(TG)G?T-3’
VL5:5’-GGG?
GAT?ATC?CAC?CAT?GAA?GTT?GCC?TGT?TAG?GCT?GTT?G-3’
Light chain 3' holds primer:
MLC-3’:5'-GCG?CCG?
TCT?AGA?ATT?AAC?ACT?CAT?TCC?TGT?TGA?A-3'；
The clone of 2.4PCR amplified production and screening
PCR product is through 1.5% agarose gel electrophoresis; reclaim test kit (can take charge of purchased from Shanghai China Shun biotechnology is limited) with a small amount of glue and reclaim pcr amplified fragment; with DNA ligation kit (purchased from TakaRa company) by this fragment by specification; utilization adds A tail and inserts in pMD-T18 carrier (purchased from TakaRa company); connector Transformed E .coli(is purchased from Chinese common micro-organisms DSMZ; CGMCC, Beijing), be seeded in 37 DEG C of overnight incubation in Amp resistance LB agar culture dish.Anti-MUC1 monoclonal antibody FMU-SEB-No.1mAb gene PCR result as shown in Figure 5.
In picking LB agar culture dish, clone, in Amp resistance LB substratum, 37 DEG C are shaken bacterium and spend the night, taking l μ l bacterium liquid as template, by the above-mentioned primer for light chain, variable region of heavy chain design, with the positive E.coli clone of PCR method screening restructuring.
Positive obtain restructuring E.coli clone is shaken to bacterium, bacterium liquid is delivered Shanghai biotechnology Services Co., Ltd and is completed gene sequencing, the gene order of variable region of light chain is as shown in SEQ ID NO.3, and the gene order of variable region of heavy chain is as shown in SEQ ID NO.4.
3, the nucleotide sequence of FMU-MUC1-No.1mAb light chain and variable region of heavy chain and homology analysis
After 3.1 definite order-checkings are errorless, in GenBank+EMBL+DDBJ+PDB database, carry out nucleotide sequence homology analysis (Blastn).
In GenBank+EMBL+DDBJ+PDB database, carry out nucleotide sequence homology analysis (Blastn).
As shown in Figure 6, the highest contrast sequence of FMU-MUC1-No.1mAb chain variable region gene and homology can reach 289/299 (98%);
As shown in Figure 7, the highest contrast sequence of FMU-MUC1-No.1mAb heavy chain variable region gene and homology can reach 283/286 (99%).
Homology analysis shows, the nucleotide sequence of light, the variable region of heavy chain of coding FMU-MUC1-No.1mAb, although have certain homology with other gene order, do not find and the identical gene order of the present invention, show that the present invention has uniqueness in gene order.
Variable region gene is translated into aminoacid sequence by 3.2, carries out amino acid sequence analysis
The aminoacid sequence of monoclonal antibody variable region of light chain is as SEQ ID NO.1, and the aminoacid sequence of variable region of heavy chain is as shown in SEQ ID NO.2.
In non-redundant Genbank CDS translations+PDB+SwissProt+PIR+PRF Protein Data Bank, carry out amino acid sequence homology analysis (Blastp).
Analytical results shows:
As shown in Figure 8, the highest contrast sequence of FMU-MUC1-No.1mAb light-chain amino acid sequence and homology can reach 91/99 (92%);
As shown in Figure 9, the highest contrast sequence of FMU-MUC1-No.1mAb heavy chain amino acid sequence and homology can reach 81/98 (83%).
Homology analysis shows, FMU-MUC1-No.1mAb is light, the aminoacid sequence of variable region of heavy chain, although have certain homology with other Argine Monohydrochloride sequence, do not find and the identical aminoacid sequence of the present invention, show that the present invention also has uniqueness on aminoacid sequence.
3.3 utilize IMGT/V-QUEST to analyze variable region structure, determine CDR district.
To check order gained FMU-MUC1-No.1mAb light chain and weight chain variabl area sequence, in IMGT/V-QUEST website, (http://imgt.cines.fr/IMGT_vquest/vquest) analyzes, and draws QiCDR district.
3 complementary determining regions (CDR) sequence of variable region of light chain, as shown in SEQ ID NO.1 line part, is specially:
CDR1：Gly-Phe-Thr-Phe-Asn-Tyr-Trp；
CDR2：Ile-Arg-Leu-Ile-Ser-Asn-Asn-Tyr-Val-Pro；
CDR3：Ser-Phe-Gly-Asn-Ser-Phe-Ala-Tyr；
3 complementary determining regions (CDR) sequence of variable region of heavy chain, as shown in SEQ ID NO.2 line part, is specially:
CDR1：Ser-Ser-Val-Ser-Tyr；
CDR2：Leu-Thr-Ser；
CDR3：Gln-Gln-Trp-Ser-Ser-Asn-Pro-Leu-Thr。
4. genetic engineering antibody design
Based on expression, purifying and the sequential analysis of anti-MUC1 monoclonal antibody FMU-MUC1-No.1mAb, the following biological products of design construction
1) structure of single-chain antibody: can FMU-MUC1-No.1mAb of the present invention is light, heavy chain variable region gene connects by linker, insert protokaryon or carrier for expression of eukaryon, transform Host Strains or transfecting eukaryotic cells, for the preparation of the single-chain antibody that can have to MUC1 targeting.
2) structure of people-mouse-anti MUC1 chimeric antibody: can FMU-MUC1-No.1mAb of the present invention is light, heavy chain variable region gene inserts in universal chimeric antibody expression vector, obtain the carrier transfecting eukaryotic cells containing mosaic gene, for the preparation of the chimeric antibody that can have to MUC1 targeting.
3) structure of humanized antibody: can FMU-MUC1-No.1mAb of the present invention is light, heavy chain variable region gene CDR district is transplanted in the skeleton district (FR) of human antibody variable region, form complementary determining region (CDR) grafted antibody (CDR-grafted antibody), also claim reshaped antibody (reshaping antibody) or humanized antibody (humanized antibody).
Utilize CDR implantation technique engineered antibody, can obtain and both keep mouse parent mAb specificity, more approach again the novel antibody of people's antibody, for the preparation of the humanized antibody that can have to MUC1 targeting.
4) can be according to the aminoacid sequence of gene order of the present invention and coding thereof, preparation is for other biological products of MUC1 functional epitope.
5) the ELISA diagnostic kit that can apply high specific of the present invention, high-affinity FMU-MUC1-No.1mAb formation determination MUC1 and express the tumours such as positive mammary cancer and carcinoma of the pancreas, lung cancer, gastrointestinal cancer, thyroid carcinoma, is expected to have a wide range of applications in the early diagnosis inspection of the MUC1 positive expression tumours such as mammary cancer.
Claims (4)
1. an anti-MUC1 monoclonal antibody, comprises light chain and heavy chain, it is characterized in that, 3 complementary determining regions (CDR) sequence of the variable region of described light chain is respectively:
CDR1：Gly-Phe-Thr-Phe-Ser-Asn-Tyr-Trp；
CDR2：Ile-Arg-Leu-Ile-Ser-Asn-Asn-Tyr-Val-Pro；
CDR3：Ser-Phe-Gly-Asn-Ser-Phe-Ala-Tyr；
3 complementary determining regions (CDR) sequence of the variable region of described heavy chain is respectively:
CDR1：Ser-Ser-Val-Ser-Tyr；
CDR2：Leu-Thr-Ser；
CDR3：Gln-Gln-Trp-Ser-Ser-Asn-Pro-Leu-Thr。
2. anti-MUC1 monoclonal antibody as claimed in claim 1, is characterized in that, the aminoacid sequence of described variable region of light chain is as SEQ.ID.NO.1, and the aminoacid sequence of variable region of heavy chain is as shown in SEQ.ID.NO.2.
3. anti-MUC1 monoclonal antibody as claimed in claim 1, is characterized in that, the gene order of encoded light chain variable region is as shown in SEQ.ID.NO.3, and the gene order of encoding heavy chain variable region is as shown in SEQ.ID.NO.4.
Anti-MUC1 monoclonal antibody claimed in claim 1 build for the application in genetic engineering antibody or molecular diagnosis and the treatment reagent of MUC1.
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| US11673966B2 (en) | 2017-01-18 | 2023-06-13 | Nanocruise Pharmaceutical Ltd. | Monoclonal and humanized antibodies to a cancer glycopeptide |
| CN112190688A (en) * | 2020-09-09 | 2021-01-08 | 广州医科大学附属第一医院（广州呼吸中心） | Application of mucin MUC1 in preparation of medicine with effect of delaying lung aging |
| CN116555187A (en) * | 2023-06-27 | 2023-08-08 | 山东省成体细胞产业技术研究院有限公司 | Mucin1 chimeric antigen receptor modified T cell and application thereof |
| CN116555187B (en) * | 2023-06-27 | 2023-09-08 | 山东省成体细胞产业技术研究院有限公司 | Mucin1 chimeric antigen receptor modified T cell and application thereof |
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