CN1368384A - Genetically engineered vaccine of MUC-1 antigen for human breast cancer - Google Patents

Genetically engineered vaccine of MUC-1 antigen for human breast cancer Download PDF

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CN1368384A
CN1368384A CN01102614A CN01102614A CN1368384A CN 1368384 A CN1368384 A CN 1368384A CN 01102614 A CN01102614 A CN 01102614A CN 01102614 A CN01102614 A CN 01102614A CN 1368384 A CN1368384 A CN 1368384A
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ctl
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gene
lymphocyte
breast cancer
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CN1141142C (en
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于永利
李慧
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China National Pharmaceutical Group Chengdu Xinlibang Biopharmaceutical Co., Ltd.
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DIWEIHUAYU BIO-TECHNOLOGY Co Ltd BEIJING
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Abstract

A genetically engineered vaccine of human breast cancer MUC-1 antigen for effectively preventing and curing the breast cancer is prepared through fusing the coding gene of the tubercle bacillus (BCG) hot shock protein 65 (BCG HSP65) with the toxic T lymphocyte (CTL) epitope gene of MUC (antigen cell expressed by human breast cancer cells, and the protein is fused at HSP-MUCl antigen CTL epitope expressed by colibacillus.

Description

Human breast carcinoma MUC-1 antigen gene engineered vaccine
The present invention relates to a kind of genetic engineering recombinant protein vaccine, particularly relate to the recombinant vaccine of a kind of prevention and treatment human breast carcinoma.
Mucin (mucins) is a kind of protein that is expressed in the breast cancer cell surface, and the mucin relevant with invasion by tumor cells power is called as MUC1.The MUC1 of overexpression can weaken cell and intercellular interaction, promote intercellular dissociate and oncocyte shift.The MUC1 molecule that breast cancer cell is expressed has very strong immunogenicity (Segal-Eiras A, et al.Breast cancer associated mucin:a review.Allergol Immunopathol (Madr) 1997 Jul-Aug; 25), be considered to the target spot that immunocyte is attacked.Therefore, the vaccine that adopts the MUC1 molecule to make may have prevention and therapeutical effect to breast carcinoma.In this respect, existing people has done a few thing:
The external immunity of human dendritic cell of usefulness such as Brossart P loading MUC1 cytotoxic T lymphocyte (CTL) epi-position (PDTRPAPGSTAPPAHGVTSA) polypeptide can induce CTL (Brossart P.et al, Iddentification of HLA-A2-rrestricted T cell epitopes derived from theMUC1 tumor antigen for broadly applicable vaccine therapies.Blood 1999 Jun 15 of mammary tumor cells specific; 93 (12): 4309-17).Usefulness MUC-1 mannan (a kind of polymanna of separating from yeast cell wall) conjugate immune mouses such as Lees CJ have been induced the CTL of MHC I class antigen restriction, this CTL can kill and wound breast cancer cell (Lees CJ, et al.Theeffect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1.CancerImmunol Immunother 2000Feb; 48 (11): 644-52).The patient of 16 Metastasis in Breast Cancer of conjugate immunity of MUC1 polypeptide (GVTSAPDTRPAPGSTA) that Reddish M etc. will be made up of 16 aminoacid and KLH (keyhole maple keyhole limpet hemocyanin), induced expressing CTL (the Reddish M that the MUC1 tumor cell has the MH-C I of lethal effect to limit 7 patients, et al.Anti-MUC-1class I restrieted CTLs in metastatic breast cancer patientsimmunized with a synthetic MUC1 peptide, Int J Cancer 1998 Jun 10; 76 (6): 817-23).Apostolopoulous V etc. with MUC1 polypeptide (20 amino acid residue) but-Mannan fusion protein immunization animal inducing specific CTL, make and produced inoculating protective immunity (the Apostolopoulous V of tumor cell by immune animal, et al.Cyclosphosphamide enhances the CTL precursor frequency in mice immunized with MUC1-mannan fusion protein (M-FP), J Immunotherapy, 1998 Mar; 21 (2): 109-13).Karanikas V etc. adopt MUC1-mannan fusion protein immunization 25 breast carcinoma, colon cancer, gastric cancer and rectal cancer patients, have induced the lymphocytic hypertrophy of T (Karanikas V.et al.Antibody and T cell responses of patients withadenocarcinoma immunized with mannan-MUC1 fusion protein.J Clin Invest 1997 Dec1 2 people; 100 (11): 2783-92).Goydos JS etc. are adjuvant with BCG, with 63 adenocarcinoma of breast patients of the synthetic peptide immunity of the MUC1 of 150 amino acid residues, induce MUC1 specific CTL (Goydos JS, et al.A phase I trial of asynthetic mucin peptide vaccine.Induction of specific immune reactivity in patients withadenocarcinoma).
(cytotoxic T lymphocyte CTL) is the immunocyte the most efficiently of human body killing tumor cell to cytotoxic T lymphocyte.Whether the vaccine of any excitating organism antineoplastic immune is effective, is decided by whether this vaccine can excite tumour-specific CTL.Ectogenic protide tumor antigen is behind immune human body, usually by antigen presenting cell picked-up, processing, enter MHC II class and offer approach, excite humoral immune reaction (Heikema A, Agsteribbe E, Wilschut J, Huckriede A.Generation of heat shock protein-based vaccines by intracellular loading of gp96with antigenic peptides.Immunol Lett 1997 Jun 1; 57 (1-3): 69-74), but can not effectively activate the generation of the CTL of tumour-specific, thereby can not produce effective tumor prevention and therapeutical effect.Therefore, giving character that the MUC1 specificity activates CTL, just to become research be a key problem in technology of antigenic, prevention and treatment human breast carcinoma vaccine with MUC-1.
(heat shock protein is to be present in the intravital molecular chaperone protein matter family of multiple biology HSP) to heat shock protein.In the process of immunne response, heat shock protein can show following three kinds of basic functions: 1, assist antigenicity substance to enter the antigen presenting cell that comprises dendritic cell; 2, in antigen presenting cell and the antigenic substance of processed interact and to make it to enter MHC I class angtigen presentation approach, and then activation antigen specific CTL; 3, stimulate dendritic cell to express collaborative stimulation molecule (as B7 etc.) and secrete cytokines.
Studies show that in recent years, HSP can make the non-altogether bonded tumor antigen of key process back and the combination of MHC I quasi-molecule and offer to activate the tumor cell that CTL removes efficiently to kill and wound the expression specificity tumor antigen effectively on its surface in antigen presenting cell.Heat shock protein-antigenic peptide complexes that injection is extracted from autologous tumor can suppress the growth of tumor-bearing mice primary tumo(u)r, the rate of transform of reduction tumor, prolong life span (the Tamura Y of tumor-bearing mice, Peng P, Liu K, Daou M, SrivastavaPK.Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations.Science 1997 Oct 3; 278 (5335): 117-20).
The objective of the invention is tubercule bacillus (BCG) heat shock protein 65 (HSP65) genes and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene are merged, develop a kind of genetic engineering recombiant vaccine that human breast carcinoma is had prevention and therapeutical effect.
The present invention is connected the encoding gene of tubercule bacillus (BCG) heat shock protein 65 (BCG HSP65) with people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene, connected mode is that tubercule bacillus HSP65 is positioned at 5 ' end, and MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene is connected in the 3 ' end of BCG HSP65.3 ' end at BCGHSP65-MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene connects 6 polyhistidyl coded sequences, obtains the recombination fusion protein vaccine.The gene order of encoding said fusion protein is the nucleotide sequence shown in the SEQ ID NO:1; The structure of described fusion rotein is the aminoacid sequence shown in the SEQ ID NO:2.Described people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene sequence is the nucleotide sequence shown in the SEQ ID NO:3.
The preparation method of vaccine of the present invention is: the encoding gene that 1, obtains tubercule bacillus (BCG) 65KD heat shock protein (HSP65)
1) cultivates tubercule bacillus (BCG)
2) extract tubercle bacillus gene group DNA
3) with PCR method from tubercule bacillus heat of dissociation shock protein 65 (HSP65) structural gene
4) clone of PCR product
5) fusion gene of the sequence analysis 2 of PCR product, the fusion gene 3 that makes up HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene, clone HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene
1) with NcoI and HindIII digestion PCR product.
2) digestion product separates through the lipolysaccharide gel electrophoresis.
3) the DNA electrophoresis band on the cutting-out agarose gel.
4) the PCR product that reclaims is cloned into procaryotic cell expression carrier (pET-28a (+) plasmid) with NcoI and HindIII point of contact.
5) will contain reorganization pET-28a (+) plasmid transformation escherichia coli of HSP-65 gene.4, measure the sequence of HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene.5, express the fusion gene of HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene.6, purification HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epi-position fusion rotein.7, separate HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene fusion rotein.8, identify the purity of HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene fusion rotein.
This fusion rotein is tubercule bacillus heat shock protein 65-people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene antigen-4 fusion protein gene engineered vaccine.
Embodiment 1 obtains the encoding gene of bacillus calmette-guerin vaccine tubercule bacillus (BCG) 65KD heat shock protein (HSP65)
1, the bacillus calmette-guerin vaccine tubercule bacillus derives from Changchun Biological Products Institute.
2, adopt the logical potato culture of Soviet Union to cultivate the bacillus calmette-guerin vaccine tubercule bacillus, the temperature of cultivation is 37-39 ℃, and the bacillus calmette-guerin vaccine tubercule bacillus that grows presents the lurid Mycoderma of shriveling.Collect Mycoderma, therefrom extract bacillus calmette-guerin vaccine tubercle bacillus gene group DNA.(method of extracting tubercle bacillus gene group DNA is with reference to Molecular Cloning one book (J.Sambrook, Isolation of high-molecular-weight DNA from mammalian cells, 9.16-9.22, ColdSpring Harbor Laboratory Press, Molecular cloning, 1989).)
3, adopt PCR method from tubercule bacillus heat of dissociation shock protein 65 (HSP65) structural gene.5 ' the end primer sequence that adopts is 5 ' CCATG GCC AAG ACA ATT GCG3 ', and 3 ' end primer sequence is 5 ' ACC CAA TTC GCTAGC CAT ATG GAA ATC CAT GCC ACC CAT 3 '.
Described PCR operation sequence is: add following reagent: template cDNA 5 μ l (mmol/L) 10 * PCR buffer (containing magnesium chloride) 5 μ ldNTPs (10mmol/L) 1 μ l5 ' end and 3 ' each 0.5 μ lTaq archaeal dna polymerase (5u/ μ l), 0.25 μ l of end primer (0.01mmol/L) and add deionized water and mix 3 reaction condition: 94C of back adding mineral oil, 30 to final volume 50 μ l in one 500 μ l microcentrifugal tubes "; 55 ℃, 1 '; 72 ℃, behind 2 ', 30 cycle periods, 72 ℃ were extended 10 minutes.
4, the clone of PCR product adopts TA cloning process clone PCR products, and method is seen document (Yu Yongli, numb red brightness, Yang Guizhen: TA clone and double-stranded DNA check order, and introduce the method for a kind of quick clone and analysis PCR product, Chinese Journal of Immunology, 1994,10 (1): 5).
5, the sequence analysis of PCR product
(J.Sambrook according to a conventional method, Polyacrylamide gel electrophoresis 1.21-1.32, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989) extract plasmid, adopt the terminal cessation method of two deoxidations, carry out sequencing inserting fragment.
Embodiment 2 makes up the fusion gene of HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene
Adopt the fusion gene of synthetic HSP-65 of PCR method and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene, template is the encoding gene (concentration is 0.01pmol/L) of tubercule bacillus 65KD heat shock protein (HSP), the PCR primer be, 5 ' end primer sequence is: the sequence of 5 ' ttc gcc atg gcc aag aca att gcg, 3 ', 3 ' end primer is: 5 ' ggc cgc aag ctt cag agc cgg acg gtt gtc cgg agc aga ggt aac acc gtg agc cgg cgg agcggt aga acc cgg agc cgg acg ggt gtc cgg agc aga ggt aac acc gtg agc cgg cgg agc ggt aga acc gaattc gct agc cat atg caa atc 3 '
Reaction condition is: 94 ℃, and 30 "; 55 ℃, 1 '; 72 ℃, behind 4 ' 30 cycle periods, 72 ℃ are extended 10
Minute.
The clone of embodiment 3 HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene fusion gene
1, digest the PCR products with NcoI and HindIII at 37 ℃, the time is 2 hours.
2, the digestion product fine jade separates through the lipolysaccharide gel electrophoresis.Electrophoretic condition is: 1% agarose gel, 1 * TAE buffer, 150-200mA, electrophoresis 0.5-1 hour.20 * TAE buffer: 0.8mol/L Tris base0.4mol/L NaOAc0.04mol/L Na2 EDTA transfers pH8.3 with glacial acetic acid.
3, the DNA electrophoresis band on observation and the cutting-out agarose gel under uviol lamp.The agarose gel that will contain DNA district band downcuts, put-70 ℃ freezing 15 minutes, glue is melted; Add the full phenol that closes of the long-pending TE-of isoploid, 30 seconds of thermal agitation, ℃ freezing 15 minutes then-70; After room temperature is melted, 12, centrifugal 5 minutes of 000rpm moves to upper phase in another pipe, with 2-2.5 times of ethanol precipitation, washing and dry DNA.
4, the PCR product cloning that reclaims is gone into upstream through 6 polyhistidyls (histidine) codon of procaryotic cell expression carrier pET-28a (+) plasmid (U.S. Novagen company) of restricted enzyme NcoI and HindIII digestion.
The digestion reaction of plasmid DNA: 1 μ g plasmid DNA, 1 μ l, 10 * buffer (seeing Promega Corporation product description).1 μ l restricted enzyme NcoI (10 units/μ l), 1 μ l restricted enzyme HindIII (10 units/μ l) mixed back 37 ℃ of incubation 30-120 minutes with distilled water polishing to 10 μ l.
Coupled reaction: plasmid DNA (0.5 μ g/ μ l) 2 μ lDNA insert fragment (300ng/ μ l) 5 μ l10 * connection buffer and (see Protocols and Applications Guide, p57, Promega Corporation, Second Edition, 1991) 1 μ lT4 dna ligase, 1 μ l with distilled water polishing to 10 μ l mix rearmounted 14-16 ℃ water-bath 6-12 hour.
Reorganization pET-28a (+) plasmid transformation escherichia coli that 5, will contain the HSP-65 gene.The preparation of competent cell:
A, escherichia coli are rule on the LB agar culture medium, cultivated 12-16 hour for 37 ℃;
B, next day are got a single bacterium colony in 2ml LB culture medium from agar plate, and 37 ℃ with 225rpm speed concussion cultivation 12-16 hour;
C, get the above-mentioned culture of 1ml and be inoculated in the 100ml LB culture medium, it is about 0.5 (about 3 hours) that 37 ℃ of speed concussions with 225rpm are cultivated until the OD value;
D, with bacterium liquid ice bath 2 hours, then 2,500Xg collected thalline in centrifugal 20 minutes for 4 ℃;
E, add the ice-cold Trituration buffer of 100ml (100mmol/L CaCl2,70mmol/L MgCl2, the 40mmol/L sodium acetate, pH5.5), mixing was put 45 minutes on ice;
F, 1,800Xg, 4 ℃ centrifugal 10 minutes, abandon supernatant, add the ice-cold Trituration buffer suspension cell of 10ml;
G. by every part of 200ul packing, can preserve 1-2 week for 4 ℃.If need long preservation, but glycerol adding to final concentration is 15%, put-70 ℃ standby.
Method for transformation:
A, the 200ul competent cell put on ice melt, add 3ul DMSO or beta-mercaptoethanol then, after the mixing, add 3-5 μ l coupled reaction liquid (containing recombiant plasmid), gentle mixing was put 30 minutes on ice;
B, 42 ℃ 45 seconds put back to rapidly in the ice 1-2 minute then;
C, adding 2ml LB culture fluid, 37 ℃ of speed with 225rpm are swayed and were cultivated 1 hour;
D, 4 abandons supernatant at 000Xg centrifugal 10 seconds, with the resuspended thalline of 200ul LB culture fluid;
E, bacterium liquid is laid on contains on the suitable antibiotic LB agar culture plate, smoothen, room temperature was placed 20-30 minute, was inverted in 37 ℃ of incubators to cultivate 12-16 hour.Method with digestion with restriction enzyme is identified recombinant clone.
The preservation of f, plasmid and bacterial strain: the plasmid stored frozen is in-20 ℃.Bacterial strain is stored in-20 ℃ or-70 ℃ in containing 20-50% glycerol culture fluid.
The order-checking of the fusion gene of HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene (is adopted the terminal cessation method of two deoxidations, concrete grammar is seen document: Yu Yongli, the red brightness of fiber crops, loyal .TA clone of Yang Gui and double-stranded DNA order-checking: the method for introducing a kind of quick clone and analysis PCR product. Chinese Journal of Immunology, 1994,10 (1): 5) result shows, the fusion gene of resulting HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene and design in full accord.
The expression of the fusion gene of embodiment 4 HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epitope gene
1, with the microbionation of single bacterium colony in 50ml LB culture medium, in the 250ml conical flask, it is 0.6 that 37 ℃ of water-bath concussions are cultured to OD600.
2, going into IPTG, to make its final concentration be 0.4mM, and 37 ℃ of water-baths concussions were cultivated 2-3 hour.
3, conical flask is in 5 minutes on ice, 4 ℃ centrifugal 5 minutes (5000xg).
4, supernatant is abandoned in suction, collects antibacterial, immediately use or frozen.
The purification of embodiment 5 HSP-65 and people MUC-1 cell antigen toxic T lymphocyte (CTL) epi-position fusion rotein
1, Bacillus coli cells (3g weight in wet base) is thawed under room temperature.Tissue is resuspended in the buffer A that 20ml contains 0.2% NaTDC (DOC) after the of short duration homogenate of historrhexis's device.
The preparation of buffer A (1000ml): final concentration 1mol/L Tris.HCL (pH7.9) 50ml 50mmol/L0.5mol/LEDTA 1ml 0.5mmol/L5mol/L NaCL 10ml 50mmol/L100% glycerol 50ml 5%
2, with desk-top ultrasonic cell disintegration instrument (SANYO, MSE SONIPREP 150), establish the interval of 8 circulations and 50%, ultrasonic 90 seconds broken bacterium in ice bath stir 10-20min.
3, add DOC, making final concentration is 0.2% (that is 20% NaTDC (DOC) that, adds about 240ul.Mixing leaves standstill 10min.20% NaTDC (DOC) stock solution should shift to an earlier date preparation in 1 day, fills a prescription to the anhydrous DOC of 10g, and is soluble in water, transfers volume to 50ml.Get rough lysate (A)
4,13000rpm, 4 ℃ of centrifugal 10min.Incline gently and supernatant.
5, add the 18ml buffer A and 2ml 20%DOC stock solution precipitates in inclusion body.With the abundant resuspended precipitation of historrhexis's device, left standstill at least 10 minutes under the room temperature.With suspension in 4 ℃ of centrifugal 10min, 13000rpm.
6, repeating step 5.
7, add 39.4ml buffer A and 0.6ml 20% sarcosyl (SKL) liquid storage (the SKL net concentration is 0.3%).Vigorous agitation is slowly dissolved precipitation.Left standstill at least 30 minutes.
The anhydrous sarcosyl of sarcosyl (SKL) liquid storage: 10g (SKL) is dissolved among the H2O, transfers volume to 50ml.
8, with suspension in 4 ℃ of centrifugal 10min, 13000rpm.
9, diluting dissolved protein with buffer A+0.3%SKL, to make its concentration be 1mg/ml.With 10 times of dissolved protein dilutions, making its concentration is 0.1mg/ml with buffer A, and the final concentration of SKL is 0.03%.
10, at 4 ℃ of dissolved protein prepared products (volume is about 400ml) buffer A (volume is about 4000ml) was dialysed 8 hours, fully stir simultaneously.Renew bright buffer and repetition then.
11, shift out protein solution through dialysis from bag filter (bore 1cm), 4 ℃, the centrifugal 20min of 8000rpm remove all aggregations.Leave and take supernatant, do the fusion gene fusion rotein of the t cell epitope of tweezer-Saphrose-4-B chromatography HSP-65 and prostate specific antigen (PSA).
12, adopt conventional SDS-PAGE method (J.Sambrook, Polyacrylamide gelelectrophoresis 6.36-6.49, Cold Spring Harbor Laboratory Press, Molecularcloning, 1989).Identify the proteic purity of gene fusion of the t cell epitope of HSP-65 and prostate specific antigen (PSA), purity is: 98%.
Suppress MCF-7 cell growth experiment in embodiment 6 bodies
One, material
1, MCF-7 cell: MCF7 is that (American Type Culture Collection ATCC), expresses the MUC-1 molecule to the MCF-7 cell, can form the original position entity tumor mice.
2, laboratory animal: the female BALB/c in 6 weeks, each 20 of vaccine group and matched groups.
3, experimental technique
1) adopts DMEM culture medium culturing MCF7 cell.In the DMEM culture medium, contain 10% inactivated fetal bovine serum, 2mML-glutathion, 100 units/ml penicillin, 100 μ g/ml streptomycins.Before injection, cell is given a baby a bath on the third day after its birth inferior with serum-free DMEM.Every injected in mice 1 * 10 7Individual MCF7 cell, injection site are subcutaneous between mice two scapulas.After 6 weeks, measure the area of mouse tumor piece basilar part.
2) at inoculated tumour before 30 days and before 15 days, vaccine group is lumbar injection 100 μ g human breast carcinoma recombinant vaccines respectively; Matched group respectively lumbar injection with vaccine group with the volume normal saline.
4, experimental result and conclusion
The meansigma methods of animal tumor piece basilar part area: vaccine group is 61mm 2, matched group is 300mm 2
Conclusion: this human breast carcinoma recombinant vaccine can obviously suppress human breast cancer cell and grow in the mice body.
SEQ ID NO：1 ( i ) ( A ) ：1800 ( B ) ： ( C ) ： ( D ) ： ( ii ) ：DNA ( iii ) ：SEQ ID NO：1 1 ATGGCCAAGA CAATTGCGTA CGACGAAGAG GCCCGTCGCG GCCTCGAGCG GGGCTTGAAC 60 61 GCCCTCGCCG ATGCGGTAAA GGTGACATTG GGCCCCAAGG GCCGCAACGT CGTCCTGGAA 120121 AAGAAGTGGG GTGCCCCCAC GATCACCAAC GATGGTGTGT CCATCGCCAA GGAGATCGAG 180181 CTGGAGGATC CGTACGAGAA GATCGGCGCC GAGCTGGTCA AAGAGGTAGC CAAGAAGACC 240241 GATGACGTCG CCGGTGACGG CACCACGACG GCCACCGTGC TGGCCCAGGC GTTGGTTCGC 300301 GAGGGCCTGC GCAACGTCGC GGCCGGCGCC AACCCGCTCG GTCTCAAACG CGGCATCGAA 360361 AAGGCCGTGG AGAAGGTCAC CGAGACCCTG CTCAAGGGCG CCAAGGAGGT CGAGACCAAG 420421 GAGCAGATTG CGGCCACCGC AGCGATTTCG GCGGGTGACC AGTCCATCGG TGACCTGATC 480481 GCCGAGGCGA TGGACAAGGT GGGCAACGAG GGCGTCATCA CCGTCGAGGA GTCCAACACC 540541 TTTGGGCTGC AGCTCGAGCT CACCGAGGGT ATGGGGTTCG ACAAGGGCTA CATCTCGGGG 600601 TACTTCGTGA CCGACCCGGA GCGTCAGGAG GCGGTCCTGG AGGACCCCTA CATCCTGCTG 660661 GTCAGCTCCA AGGTGTCCAC TGTCAAGGAT CTGCTGCCGC TGCTCGAGAA GGTCATCGGA 720721 GCCGGTAAGC CGCTGCTGAT CATCGCCGAG GACGTCGAGG GCGAGGCGCT GTCCACCCTG 780781 GTCGTCAACA AGATCCGCGG CACCTTCAAG TCGGTGGCGG TCAAGGCTCC CGGCTTCGGC 840841 GACCGCCGCA AGGCGATGCT GCAGGATATG GCCATTCTCA CCGGTGGTCA GGTGATCAGC 900901 GAAGAGGTCG GCCTGACGCT GGAGAACGCC GACCTGTCGC TGCTAGGCAA GGCCCGCAAG 960961 GTCGTGGTCA CCAAGGACGA GACCACCATC GTCGAGGGCG CCGGTGACAC CGACGCCATC 10201021 GCCGGACGAG TGGCCCAGAT CCGCCAGGAG ATCGAGAACA GCGACTCCGA CTACGACCGT 10801081 GAGAAGCTGC AGGAGCGGCT GGCCAAGCTG GCCGGTGGTG TCGCGGTCAT CAAGGCCGGT 11401141 GCCGCCACCG ACGTCGAACT CAAGGAGCGC AAGCACCGCA TCGAGGATGC GGTTCGCAAT 12001201 GCCAAGGCCG CCGTCGAGGA GGGCATCGTC GCCGGTGGGG GTGTGACGCT GTTGCAAGCG 12601261 GCCCCGACCC TGGACGAGCT GAAGCTCGAA GGCGACGAGG CGACCGGCGC CAACATCGTG 1320
Figure A0110261400121
SEQ ID NO：2 ( i ) ( A ) ：599 ( B ) ： ( C ) ： ( D ) ： ( ii ) ： ( iii ) ：SEQ ID NO：2 1 Met Ala Lys Thr Ihr Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu 15 16 Glu Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu 30 31 Gly Pro Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala 45 46 Pro Thr Ihr Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu 60 6l Leu Glu Asp Pro Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu 75 76 Val Ala Lys Lys Thr Asp Asp Val Ala Gly Asp Gly Thr Thr Thr 90 91 Ala Thr Val Leu Ala Gln Ala Leu Val Arg Glu Gly Leu Arg Asn 105106 Val Ala Ala Gly Ala Asn Pro Leu Gly Leu Lys Arg Gly Ile Glu 120121 Lys Ala Val Glu Lys Val Thr Glu Thr Leu Leu Lys Gly Ala Lys 135136 Glu Val Glu Thr Lys Glu Gln Ile Ala Ala Thr Ala Ala Ile Ser 150151 Ala Gly Asp Gln Ser Ile Gly Asp Leu Ile Ala Glu Ala Met Asp 165166 Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu Glu Ser Asn Thr 180181 Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg Phe Asp Lys 195196 Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Pro Glu Arg Gln Glu 210211 Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val 225226 Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gly 240241 Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu 255256 Ala Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys 270271 Ser Val Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala 285286 Met Leu Gln Asp Met Ala Ile Leu Thr Gly Gly Gln Val Ile Ser 300301 Glu Glu Val Gly Leu Thr Leu Glu Asn Ala Asp Leu Ser Leu Leu 315316 Gly Lys Ala Arg Lys Val Val Val Thr Lys Asp Glu Thr Thr Ile 330331 Val Glu Gly Ala Gly Asp Thr Asp Ala Ile Ala Gly Arg Val Ala 345346 Gln Ile Arg Gln Glu Ile Glu Asn Ser Asp Ser Asp Tyr Asp Arg 360361 Glu Lys Leu Gln Glu Arg Leu Ala Lys Leu Ala Gly Gly Val Ala 375376 Val Ile Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu Arg 390391 Lys His Arg Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val 405406 Glu Glu Gly Ile Val Ala Gly Gly Gly Val Thr Leu Leu Gln Ala 420421 Ala Pro Thr Leu Asp Glu Leu Lys Leu Glu Gly Asp Glu Ala Thr 435436 Gly Ala Asn Ile Val Lys Val Ala Leu Glu Ala Pro Leu Lys Gln 450451 Ile Ala Phe Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu Lys 465466 Val Arg Asn Leu Pro Ala Gly His Gly Leu Asn Ala Gln Thr Gly 480481 Val Tyr Glu Asp Leu Leu Ala Ala Gly Val Ala Asp Pro Val Lys 495496 Val Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Gly Leu 510511.Phe Leu Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Glu 525526 Lys Ala Ser Val Pro Gly Gly Gly Asp Met Gly Gly Met Asp Phe 540541.His Met Ala Ser Glu Phe Gly Ser Thr Ala Pro Pro Ala His Gly 555556 Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 570571 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro Ala 585586 Leu Lys leu Ala Ala Ala Leu Glu His His His His His His 600SEQ ID NO：3 ( i ) ( A ) ：120 ( B ) ： ( C ) ： ( D ) ： ( ii ) ：DNA ( iii ) ：SEQ ID NO：3
Figure A0110261400141

Claims (4)

1, the genetic engineering recombinant protein vaccine of a kind of prevention and treatment human breast carcinoma is characterized in that this vaccine contains that MUC1 cell antigen toxic T lymphocyte (CTL) epitope gene that the encoding gene and the human breast cancer cell of tubercule bacillus (bacillus calmette-guerin vaccine) heat shock protein 65 (BCG HSP65) are expressed is connected and the genetic engineering recombination fusion protein that obtains; The gene order of encoding said fusion protein is the nucleotide sequence shown in the SEQ ID NO:1.
2. the described genetic engineering recombinant protein vaccine of claim 1, the structure of wherein said fusion rotein is the aminoacid sequence shown in the SEQ ID NO:2.
3. the described genetic engineering recombinant protein vaccine of claim 1, wherein said encoding gene and the mode that is connected of human breast cancer cell MUC1 cell antigen toxic T lymphocyte (CTL) epitope gene of expressing with tubercule bacillus heat shock protein 65 is: tubercule bacillus HSP 65 is positioned at 5 ' end, MUC1 cell antigen toxic T lymphocyte (CTL) epitope gene that human breast cancer cell is expressed is connected in the 3 ' end of BCG HSP65, at 3 ' end connection, the 6 polyhistidyl coded sequences of BCG HSP65-MUC-1 cytotoxic T lymphocyte (CTL) epitope gene.
4. the described genetic engineering recombinant protein vaccine of claim 1 is characterized in that MUC1 cell antigen toxic T lymphocyte (CTL) the epitope gene sequence that described human breast cancer cell is expressed is the nucleotide sequence shown in the SEQ ID NO:3.
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WO2006010308A1 (en) * 2004-07-30 2006-02-02 Second Military Medical University Preparation and application of therapeutic vaccines of tumour
WO2006042465A1 (en) * 2004-10-19 2006-04-27 Beijing Hydvax Biotechnology Co., Ltd. A vaccine composition containing a recombinant fusion protein and a adjuvant as well as its application
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WO2006010308A1 (en) * 2004-07-30 2006-02-02 Second Military Medical University Preparation and application of therapeutic vaccines of tumour
WO2006042465A1 (en) * 2004-10-19 2006-04-27 Beijing Hydvax Biotechnology Co., Ltd. A vaccine composition containing a recombinant fusion protein and a adjuvant as well as its application
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CN103146734B (en) * 2013-03-12 2014-10-01 中国人民解放军军事医学科学院军事兽医研究所 Anti-burn and scald infection multiple organ failure Pseudomonas aeruginosa toxin vaccine
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CN106279435B (en) * 2016-08-16 2019-06-07 新乡医学院 Target anti-tumor vaccine, encoding gene, expression vector, expression engineering bacteria and the application of VEGF and mucin1
CN112996816A (en) * 2018-06-29 2021-06-18 Go医疗股份有限公司 anti-sugar-MUC 1 antibodies and uses thereof

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