CN1513556A - Recombination human Mucl-MBP fusion protein antitumour vaccine and production technology - Google Patents
Recombination human Mucl-MBP fusion protein antitumour vaccine and production technology Download PDFInfo
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Abstract
An anticancer vaccine of recombinant human MOC1-MBP fusion protein is disclosed, in which MBP is used as its adjuvant. The MBP gene and MUC1 gene are fused together. The MBP substituted for other fusion protein to induce CTL reaction. The pMAL-P2 is the carrier for effectively expressing maltose fusion protein. The serial repetitive sequence of MUC1 is inserted to downstream of malE gene.
Description
Technical field:
The present invention relates to the vaccine technologies of active specific immunotherapy in the tumor biotherapy, a kind of recombined human MUC1-MBP fusion rotein anti-tumor vaccine and production technology especially are provided, to reach prevention and treatment tumor purpose, belong to the genetic engineering fusion protein technical field.
Background technology:
MUC1 is the important member of Mucins mucin family, is present in the tumor cell surface in normal glandular tube epithelial cell and source thereof, is made of polypeptide nuclear core (nuclear core peptide) and side shoot sugar chain.Normal structure MUC1 is different with tumor tissues, and the former is distributed in the glandular tube epithelial cell secretion utmost point, and isolate relative with immunocyte, glycosylation is abundant; And the latter extensively distributes and is expressed in the cancerous cell surface unusually galore, and glycosylation is incomplete, therefore exposes epi-position hidden under the normal condition, becomes the target spot that immunocyte is attacked.
At present, be that the vaccine of target spot comprises albumen or polypeptide vaccine, saccharide vaccines and dna vaccination with MUC1, wherein protein vaccine shows good prospects for application.Livingston research group MUC1 tandem repetitive sequence by synthetic different length in 1996, the 30aa repetitive sequence is connected with KLH, immunized mice induces the MUC1 specific antibody that height is tired, although do not induce CTL, but obviously suppressed growth of tumor, and used polypeptide immune can not produce any immunne response separately.U.S. Biomira company is developing the polypeptide vaccine based on MUC1, called after BLP-25, the clinical II phase experimental stage that has entered nonsmall-cell lung cancer.Discover that simple polypeptide can not induce any immunne response, only be prepared into fusion rotein after, induction of immunity reaction that can be in various degree.And the amalgamation protein vaccine of development in the past be artificial synthetic polypeptide mostly, and majority can not inducing cytotoxic T cell shortcomings such as (CTL) a little less than being faced with immunogenicity.
Maltose-binding protein (MBP) is colibacillary composition, and by escherichia coli malE gene code, it has strong immunogenicity.
Summary of the invention:
The present invention utilizes engineered method, a kind of production technology of recombined human MUC1-MBP fusion rotein anti-tumor vaccine is provided, preparation MUC1-MBP fusion rotein-tumor vaccine is the Therapeutic Method of active specific immunotherapy in the tumor biotherapy, to reach prevention and treatment tumor purpose.
The present invention is with MBP gene and MUC1 gene fusion together, develops a kind of genetic engineering fusion protein vaccine.Replace other fusion rotein to induce ctl response with MBP.PMAL-p2 is the prokaryotic expression carrier that efficiently expresses the maltose fusion rotein, we insert the tandem repetitive sequence of MUC1 gene in the downstream of malE gene, on the one hand can be in escherichia coli the albumen of directly efficient abduction delivering and MBP fusion, save the step of chemosynthesis fusion rotein on the other hand.
Technical solution of the present invention is as follows:
1. 450 bases of this MUC1 gene are inserted pMAL-p2 prokaryotic expression carrier malE gene downstream according to proper reading frame, MUC1 is connected together with the MBP gene, identify the correctness of this recombinant vector pMAL-MUC1 by enzyme action.(Fig. 1)
2. express the recombiant plasmid pMAL-MUC1 transformed into escherichia coli DH5 α of MUC1-MBP fusion rotein, under the inducing of IPTG, expressed the fusion rotein MBP-MUC1 of molecular weight 62KD, identify its correctness by SDS-PAGE and Western blot, and obtain stable expression strain pMAL-MUC1/DH5 α.(Fig. 2, Fig. 3)
3, conventional gene sequencing is measured the gene order of MUC1-MBP fusion rotein.
4. cultivate pMAL-MUC1/DH5 α in a large number, extract the whole cell supernatant, obtained purer MUC1-MBP fusion rotein and MBP albumen by gel permeation chromatography → starch sugar affinity chromatograph → ultrafiltration through ultrasonication.Obtained evaluation by SDS-PAGE. (Fig.4)
5.MUC1-MBP the active mensuration of antineoplastic immune.
(1) mensuration of anti-MUC1 specific antibody:, detect polyclonal antibody by ELISA with MUC 1-MBP fusion protein immunization C57 mice.
(2) mensuration of the special CTL cytotoxic activity of anti-MUC1: measure CTL cell toxicant killing activity by mtt assay.
(4) immunized mice becomes the tumor experiment: give the C57 mice of immunity with human breast cancer cell (MCF7) injection of high expressed MUC1,3-5 observes pulmonary and local tumor growing state after week.
MUC1-MBP fusion rotein nucleotide sequence:
Feature: 1710 bases of length
Type nucleic acid
The chain strand
1.atgaaaaaat?tattattcgc?aattccttta?gtggtacctt?tctattctca?ctcggccgat
61?atgaaaatcg?aagaaggtaa?actggtaatc?tggattaacg?gcgataaagg?ctataacggt
121?ctcgctgaag?tcggtaagaa?attcgagaaa?gataccggaa?ttaaagtcac?cgttgagcat
181?ccggataaac?tggaagagaa?attcccacag?gttgcggcaa?ctggcgatgg?ccctgacatt
241?atcttctggg?cacacgaccg?ctttggtggc?tacgctcaat?ctggcctgtt?ggctgaaatc
301?accccggaca?aagcgttcca?ggacaagctg?tatccgttta?cctgggatgc?cgtacgttac
361?aacggcaagc?tgattgctta?cccgatcgct?gttgaagcgt?tatcgctgat?ttataacaaa
421?gatctgctgc?cgaacccgcc?aaaaacctgg?gaa?gagatcc?cggcgctgga?taaagaactg
481?aaagcgaaag?gtaagagcgc?gctgatgttc?aacctgcaag?aaccgtactt?cacctggccg
541?ctgattgctg?ctgacggggg?ttatgcgttc?aagtatgaaa?acggcaagta?cgacattaaa
601?gacgtgggcg?tggataacgc?tggcgcgaaa?gcgggtctga?ccttcctggt?tgacctgatt
661?aaaaacaaac?acatgaatgc?agacaccgat?tactccatcg?cagaagctgc?ctttaataaa
721?ggcgaaacag?cgatgaccat?caacggcccg?tgggcatggt?ccaacatcga?caccagcaaa
781?gtgaattatg?gtgtaacggt?actgccgacc?ttc?aagggtc?aaccatccaa?accgttcgtt
841?ggcgtgctga?gcgcaggtat?taa?cgccgcc?agtccgaaca?aagagctggc?aaaagagttc
901?ctcgaaaact?atctgctgac?tgatgaaggt?ctggaagcgg?ttaataaaga?caaaccgctg
961?ggtgccgtag?cgctgaagtc?ttacgaggaa?gagttggcga?aagatccacg?tattgccgcc
1021?actatggaaa?acgcccagaa?aggtgaaatc?atgccgaaca?tcccgcagat?gtccgctttc
1081?tggtatgccg?tgcgtactgc?ggtgatcaac?gccgccagcg?gtcgtcagac?tgtcgatgaa
1141?gccctgaaag?acgcgcagac?taattcgagc?tcgaacaaca?acaacaataa?caataacaac
1201?aacctcggga?tcgagggaag?gatttcagaa?ttcggatccc?ccgggctgca?ggaattcccc
1261.gcccccccag?cccacggagt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1321.gcccccccag?cccacggtgt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1381.gccccccaag?cccacggtgt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1441.gcccccccag?cccacggtgt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1501.gcccccccag?cccacggtgt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1561.gcccccccac?tccacggtgt?cacctcggcc?ccggagaaca?ggccggcccc?gggctccacc
1621.gcgcccgcag?cccacggtgt?cacctcggcc?ccggagagca?ggccggaatt?cgatatcaag
1681.cttggcactg?gccgtcgttt?tacaacgtcg?tga
The present invention is applicable to following medical application:
1. prevent and treat various adenocarcinoma and metastatic carcinoma thereof.
2. the treatment of residual focus behind the tumor operation.
3.BCG strengthen the inductive immunity of MUC1-MBP as adjuvant.
4. use with IL-2 or GM-CSF compatibility.
5. use with the chemotherapeutics compatibility.
Oncotherapy takes composite treatment to comprise operation, radiation and chemotherapy at present.But be faced with subject matter is residual focus, can not thoroughly effect a radical cure.Along with etiology, cytobiology, development of molecular biology to tumor, people constantly deepen the understanding of tumor and host immune relation, tumor biotherapy, especially the tumor vaccine that with the tumor antigen is foundation development is subjected to common concern, effectively not only antitumor action is strong for tumor vaccine, and do not have the toxic and side effects of radiotherapy chemotherapy, make the residual focus that solves in the oncotherapy become possibility.
Following zoopery has confirmed that pharmaceutical preparation of the present invention has prevention and treats effects such as various adenocarcinoma and metastatic carcinoma thereof:
1. the inductive antibody response of MUC 1-MBP
Adopt competitive ELISA antagonism MUC1 specific antibody to measure.The result shows that the protein induced mice of recombinant human MUC1 has produced the specific antibody of anti-human MUC1.PBS matched group and pure product MBP group immunized mice there is no anti-MUC1 specific antibody, but occur high anti-MBP antibody of tiring in latter's serum, show that MBP induces Mus to produce the favourable carrier of MUC1 antibody.Tiring of the anti-MUC1 antibody of MUC1-MBP immune group is 1:5760 ± 3221.(Fig.5 Fig.6) with the MUC1-MBP fusion protein immunization C57 mice of reorganization, has produced high special anti-human MUC1 antibody of tiring.Adjuvant BCG has obviously strengthened antibody response.
2. the special ctl response of the inductive anti-MUC of fusion rotein MUC1-MBP
Having measured mice spleen CTL activity by mtt assay, used MCF-7 and Mus Lewis lung cancer cell as target cell respectively, is 100: 1 o'clock imitating the target ratio, and the BCG group is compared killing activity with the PBS matched group is not had significant difference; MUC1-MBP immune group mouse boosting cell is respectively 47.7 ± 4.3% and 67.5 ± 6.5% to MCF-7 cell and Lewis lung cancer cell killing activity, compares P＜0.01 with PBS matched group and BCG group, and significant difference is arranged; When using K562, compare P.＞0.05, there was no significant difference between MUC1-MBP immune group, BCG group and PBS control group mice splenocyte killing activity group as target cell.This result shows, still be that every Mus of Mus Lewis lung cancer cell all induces the special CTL activity of MUC1 for human MCF-7 no matter, and kill rate increases (Fig.7) along with imitating increasing of target ratio).This result shows that not only there is the B cell epitope in MUC 1-MBP fusion rotein, also has the CTL epi-position simultaneously.
Produced the special CTL of intensive anti-MUC1 with fusion rotein MUC1-MBP immunity C57 Mus, it all shows tangible killing activity at human MCF7 and Mus Lewis lung cancer cell.Because the material that we use in experiment is a splenocyte, therefore the killing activity of being measured may comprise non-specific killing and wounding (NK), for getting rid of this possibility, we with K562 in contrast, the MUC1 immune group is compared with the PBS matched group, though the killing activity to K562 slightly increases, therefore not statistically significant can get rid of the possibility of inducing the NK cytoactive.In addition, be to get rid of inductive non-specific the killing and wounding of BCG, we are with the independent immune mouse of BCG, and the killing activity of measuring splenocyte is not seen yet and increased.The above results confirms that all the MUC1-MBP immunized mice induced the special CTL activity of MUC1.The CTL specific killing is subjected to the restriction of self MHC usually, and can kill and wound the mankind mastopathy cell through the inductive CTL of MUC1-MBP fusion protein immunization Mus, shows that its inductive killing and wounding is not limited by MHC.
3.MUC1-MBP inductive active immunity is for MCF-7 (MCF7) growth inhibited
In the mice with tumor experiment, using tumor cell is that MCF7 does animal model.Giving C57 injected in mice MCF7 cell with 3-10 days behind the vaccine immunity, after three weeks, matched group all grows the tumor that differs in size at the back injection site, and wherein that the volume maximum is 2250mm
3, the tumor center's ulceration that has; The Mus state is become thin, the gloomy tarnish of hair color, this Mus death after 28 days.Matched group tumor average volume 537mm
3 And, only have 6 to grow 18-31mm at the back through fusion rotein MUC1-MBP immunized mice
3Size tumor.Vaccine immunity group tumor average volume 14mm
3Statistical procedures result shows that experimental group compared significant difference (p＜0.001) with matched group.The inductive immunity of human recombinant MUC1-MBP fusion rotein has obviously suppressed the growth of human adenocarcinoma cell.
The growth of table 1.MUC1 immunized mice injection MCF7R human breast carcinoma
Gross tumor volume (mm
3)
NO
Matched group vaccine immunity group
1 1500 18
2 456 26
3 502 0
4 722 31
5 268 0
6 151 19
7 353 24
8 198 0
9 821 27
10 397 0
Good effect of the present invention is: the present invention is by inducing body specific immune response, especially cellullar immunologic response, reaching the antineoplastic purpose.Having overcome in the past can not inducing cytotoxic T cell shortcomings such as (CTL) a little less than the amalgamation protein vaccine immunogenicity.This vaccine is to utilize engineered method to prepare in escherichia coli, and is with low cost, is suitable for commercially producing.
Description of drawings:
The recombinate analysis of pMAL-MUC1 endonuclease bamhi of Fig. 1.
With BamHI and HindIII digestion pMAL-MUC1, pMAL and pBS-MUC1.
The 1st row: pMAL-MUC1, the 2nd row: pMAL, the 3rd row: pBS-MUC1,
The 4th row: DNA standard substance.
The expression of MUC1-MBP fusion rotein in bacillus coli DH 5 alpha of Fig. 2 reorganization.
First row: protein standard substance, secondary series: the DH5 α of unconverted, the 3rd row: without the inductive pMAL/DH5 α of IPTG, the 4th row: be listed as the 8th row from the 4th and be respectively the pMAL/DH5 α that induced through IPTG 1 to 5 hour.The 9th row:, be listed as the 14th row from the 10th and be respectively the pMAL-MUC1/DH5 α that induced through IPTG 1 to 5 hour without the inductive pMAL-MUC1/DH5 α of IPTG.
The expression of Fig. 3 MUC1-MBP is by the analysis of Westemblot.
First row: the total protein of pMAL-MUC1/DH5 α, secondary series: the matter week chamber albumen of pMAL-MUC1/DH5 α, the 3rd row: without the total protein of the inductive pMAL-MUC1/DH5 α of IPTG, the 4th row: the total protein of pMAL/DH5 α.
The MUC-MBP of Fig. 4 purification is by the analysis of SDS-PAGE.
First row: protein standard substance, secondary series: the MUC1-MBP of purification, the 3rd row: the MBP of purification.
The reaction of anti-MUC1 antibody of Fig. 5 and MUC1-MBP.
At first, with antiserum and excessive MBP reaction, seal anti-MBP antibody and fusion rotein MUC1-MBP reaction site in the antiserum, and then measure anti-MUC1 antibody by ELISA.
Tiring of the anti-MUC1 antibody of Fig. 6
Respectively by MUC1-MBP (◆), MBP (▲), PBS (■) and BCG (●).Tiring of anti-MUC1 antibody measured by competitive ELISA in the C57 mice serum of injection.Make adjuvant with BCG in the immunologic process.
The special ctl response of the anti-MUC1 of Fig. 7 MUC1-MBP immunized mice spleen.
(●) is the MUC1-MBP immune group,, (◆) is the PBS matched group, (■) is the BCG matched group.
A be Lewis lung cancer as target cell, B be MCF7 as target cell, C is that K562 is as target cell.
The specific embodiment:
Embodiment
The structure of one .MUC1-MBP fusion protein expression vector
1. conventional plasmid extraction and purification. (referring to molecular cloning experiment guide, j.Sa nurse Brooker, chief editor Science Press publishes 1998)
2. the separation of endonuclease bamhi and recovery.
(1) with EcoRI and HindIII double digestion pMAL-P2 and PSK-MUC1 plasmid, 25-37 ℃ 1-3 hour.
(2) electrophoresis DNA isolation fragment on the 0.7-1.5% agarose gel reclaims carrier segments pMAL-p2 and 450bp purpose fragment MUC1 with freeze-thaw method or test kit.
(3) electrophoresis is identified carrier segments and the purpose fragment that reclaims on the 0.7-1.5% agarose gel.
3.pMAL-p2 carrier is connected with MUC1 is segmental
PMAL-p2 carrier segments 1-10 μ l
MUC1 fragment 5-20 μ l
EcoRI 2-4μl
HindIII 2-4μl
T4 ligase 1-2 μ l
By above-mentioned amount, in 50-100 μ l reaction system, react an evening under 16 ℃ the condition. by the T4 ligase MUC1 fragment is connected on the carrier segments of BamHI and HindIII double digestion by correct reading frame, takes out the conventional transformed into escherichia coli DH5 of 1-10 μ l α then.
4. conventional preparation competence is (referring to molecular cloning experiment guide, j.Sa nurse Brooker, chief editor Science Press publishes 1998)
5. escherichia coli transform
(1) plasmid of adding 1-5 μ l in the competence of every pipe 250 μ l rotates mixing, ice bath 30 minutes gently.
Suffered a shock 2 minutes for (2) 42 ℃.
(3) 2 minutes .. of ice bath
Centrifugal 10 minutes of (4) 4 ℃ of 5000rpm.
(5) abandon supernatant and remain the resuspended thalline of 100 μ l. be laid on the LB agar plate surface that contains penbritin.
(6) 37 ℃ of constant temperature are inverted incubated overnight.
The screening of 6 recombinant clones and evaluation
Transform bacterium colony through the ampicillin screening, picking different clones cultivate in a small amount, extracts plasmid as previously mentioned, with EcoRI and HindIII double digestion plasmid, pass through 1% agarose gel electrophoresis DNA isolation then, determine the MUC1 fragment by inserting segmental molecular weight size.
The expression of two .MUC1-MBP fusion rotein recombinant vectors (pMAL-MUC1) and the abduction delivering of identifying 1 MUC 1 gene
The pMAL-MUC1 carrier transformed into escherichia coli DH5 α that (1) will correctly connect, and it is laid in the LB culture medium flat plate that contains ampicillin 25-37 ℃ of incubated overnight.
(2) the different colony inoculation of picking is in the LB that contains ampicillin cultivates, in 25-37 ℃ of incubated overnight.
(3) every kind of overnight culture of 50 μ l is inoculated in 5ml respectively and contains on the LB culture medium of ampicillin, 25-37 ℃ aerobic culture 3-4 hour, with 0.1mM-1.0mM IPTG inducing culture 3-6 hour, the centrifugation antibacterial abandoned supernatant, precipitation is stored in-20 ℃.
(4) resuspended precipitation adds the load sample liquid mixing of equal volume then, and 80-100 ℃ is boiled 5 minutes, centrifugal, gets supernatant 10-30 μ l and joins SDS-PAGE and go up electrophoresis and identify proteic expression.
2.pMAL-MUC1 the great expression in escherichia coli
(1) frozen pMAL-MUC1/DH5 α is inoculated in the 5-150ml LB culture fluid, 25-37 ℃ of jolting spent the night.
(2) next day, culture is inoculated in the 500-2000ml LB culture fluid
(3) centrifugation. abandon supernatant, precipitation is stored in-20 ℃.
(4) adding 0.1-1.0mM IPTG continues to cultivate 3-8 hour.
(5) centrifugation. abandon supernatant. precipitation is stored in-20 ℃.
(6) take by weighing the weight .-20 ℃ of preservation of wet bacterium.
3. polyacrylate hydrogel electrophoresis (SDS-PAGE)
(1) electrophoresis glass plate device is installed,
(2) preparation 5% concentrates glue and 10% separation gel,
(3) bacterial precipitation is resuspended in 1 * load sample liquid, 100 ℃ are boiled 5 minutes broken antibacterials,
(4) electrophoresis tank is installed
(5) get supernatant 10-30 μ l and add 10%SDS-PAGE
(6) establish constant voltage 100-200V, electrophoresis 1-2h
(7) unload gel, Coomassie brilliant blue R-250 jolting dyeing 1h.
(8) at 50% methanol, 7% acetic acid, 1h. decolours in the aqueous solution
5.Western?blot
(1) when the SDS-PAGE electrophoresis closes to an end,, dries with the graphite cake of distilled water flushing half dry type transfer groove.
(2) cut glue, 6 filter paper and a nitrocellulose filter, size is identical.Soaked 5 minutes with shifting liquid.
(3) transfer groove is installed, is kept flat the graphite cake base, on graphite cake, place 3 and then nitrocellulose filter is placed on the filter paper, guarantee alignment through shifting the filter paper that liquid soaks.
(4) gel is lain against on the cellulose membrane, 3 filter paper of another group are placed on the gel.
(5) the transfer groove lid is anchored on the graphite cake, connects power supply, establish electric current 60-100mA and shifted 1-2 hour.
(6) after transfer finishes,, sealed 1-2 hour putting into the 1-3%BSA confining liquid after the transfer membrane flushing.
(7) get 2 times express developed, wash film 3 times.
(8) transfer membrane is put into hybridization bag, add one-level antibody mouse-anti people MUC1 antibody, jolting 1-2 hour with 2-8 μ l/ml concentration.
(9) wash film as (7).
(10) add secondary antibody mountain sheep anti mouse IgG-HRP, 60 times of dilutions.Reacted 1-2 hour.
(11) wash film.
(12) added substrate reactions 5 minutes, substrate solution: diaminobenzidine (DAB) 7mg, 30%H
2O
210 μ l are dissolved in TBS solution.
Three. conventional gene sequencing
MUC1-MBP fusion rotein nucleotide sequence: see accompanying drawing 8
Feature: 1710 bases of length
Type nucleic acid
The chain strand
The purification of four .MUC1-MBP fusion rotein and MBP
1. escherichia coli ultrasonication
Every gram weight in wet base bacterium adds 10-30ml TE buffer, ultrasonication, audio frequency 15-35KHz, 10-20 time, each 12 seconds, 10 seconds of interval.4 ℃, the centrifugal 10-30min of 4000-10000rpm leaves and takes supernatant, and the SDS-PAGE electrophoresis is identified.
2.Amyrose affinity chromatograph
(1) gets amylose resin 4ml and wash 3 times 10ml/ time with distillation.
Pack into the glass column of 1.5cm diameter of the amylose resin that (2) will wash, bed volume 4ml.
(3) the antibacterial supernatant 8ml of ultrasonication dress post.
(4) (10mM 2ME 1mMEDTA) washes 3 times for 20mM Tris PH 7.4,0.2M NaCL with washing liquid.
(5) measure protein content OD 280/260.
(6) the SDS-PAGE electrophoresis is identified the purity of fusion rotein.Its purity 95%.
5.Sephadex G-25 desalination or super rate desalination
(1) Sephadex G-25 adds water-soluble rising an evening.
(2) pack on the elongated pillar.
(3) pack into the protein solution of a bed volume accessed the post effluent.
(4) PBS washes 3 times, accesses eluent.
(5) SDS-PAGE electrophoresis, Western blot, ELISA identify purified albumen.
6. quantification of protein. utilize Coomassie brilliant blue G-250 staining to measure protein concentration.
(1) standard protein solution, BSA 300 μ g/ml.
(2) dyeing liquor: 0.01% Coomassie brilliant blue G-250,4.7% ethanol, 8.5%H3PO4,4 ℃ of preservations.
(3) getting 10 μ l MUC1 protein solutions dilution 100, extraordinarily to go into 900 μ l H2O stand-by.
(4) the BSA solution of preparation variable concentrations, 0,7,15,30,60,90 μ g/ml.
(5) get above-mentioned solution and solution 1ml to be measured respectively, add the 3ml dyeing liquor, room temperature 15 minutes.
(6) OD595 colorimetric determination.
The mensuration of five .MUC1-MBP fusion rotein anti-tumor activities
1. the female C57 mice of animal 18-20g is available from the high-new Company of Animals Ltd. in Changchun
2. immune programme for children
Every Mus injected dose 100-500 μ l wherein contains MUC1-MBP and BCG, and BCG is as adjuvant.Subcutaneous multiple spot of nape portion and both sides groin subcutaneous injection, every other week once, immunity twice was altogether got the supraorbital vein blood examination in 2-10 days in second all immunity back and is surveyed antibody titer.
3.C57 mouse immune
(1) 40 of C57 mices are divided into 4 groups, 3 groups is matched group, is respectively PBS, PBS+BCG, PBS+BCG+MBP; One group is experimental group: MUGl-MBP+BCG
(2) route of administration: subcutaneous multiple spot of nape portion and both sides groin subcutaneous injection.
(3) dosage: MUC1 10-100 μ g/, BCG 1-5mg/ only.
(4) immune programme for children: once twice totally every other week.
(5) antibody titer is measured in supraorbital vein blood samplings in 2-10 days of last immunity.
4. antibody titer is measured
(1) after competitive ELISA takes place to seal the binding site of anti-MBP antibody with excessive MBP and antiserum, reacts with MUC1-MBP and MBP respectively, measure the existence of anti-MUC1 antibody by the difference of change color.
1) at first uses the MUC1-MBP and the MBP wrapper sheet of identical molal quantity, an evening.
2) dilution antiserum.
3) be the antiserum of the MBP of 5,10,20 μ g/ml and the dilution 1-3h that reacts with final concentration.
4) wash ELISA Plate.
5) confining liquid sealing 1.5h.
6) add ELISA Plate reaction 1-2 hour with the antiserum of crossing among the MBP.
7) add substrate reactions 5 minutes, add stop buffer.
8) microplate reader is measured OD490.
(2) aminoacid sequence of synthetic MUC1 is directly measured anti-MUC1 antibody with ELISA.
5. mice CTL determination of cytotoxic activity
(1) mice after the immunity is killed the aseptic spleen of getting in back.
(2) preparation splenocyte suspension.
(3) modulation target cell MCF-7 and Lewis lung cancer cell concentration.
(4) the effect target ratio according to 200: 1,100: 1,50: 1 and 25: 1 adds in 96 orifice plates 200 μ l/well, light shaking mixing, 37 ℃, 5%CO
2Incubator was cultivated 4-10 hour.
(5) add MTT 5-20 μ l/well, continue to cultivate 4 hours.
(6) add 100-200 μ l/well DMSO Rong Xie Jia Za.
(7) measure OD490 with microplate reader.
6. immunized mice becomes the tumor experiment
By 2-10 days after twice immunity of above-mentioned immune programme for children, with 3-7 Grey roentgenization mice, give MCF7 breast cancer cell back subcutaneous injection then, the injection cell number is 1-5 * 10
7/ only.Under clean environment, raise, measure the size of tumor after three weeks.
Claims (7)
1, recombined human MUC1-MBP fusion rotein anti-tumor vaccine, its nucleotide sequence: length is 1710 bases, and type is a nucleic acid, and chain is a strand,
1.atgaaaaaat?tattattcgc?aattccttta?gtggtacctt?tctattctca?ctcggccgat
61?atgaaaatcg?aagaaggtaa?actggtaatc?tggattaacg?gcgataaagg?ctataacggt
121?ctcgctgaag?tcggtaagaa?attcgagaaa?gataccggaa?ttaaagtcac?cgttgagcat
181?ccggataaac?tggaagagaa?attcccacag?gttgcggcaa?ctggcgatgg?ccctgacatt
241?atcttctggg?cacacgaccg?ctttggtggc?tacgctcaat?ctggcctgtt?ggctgaaatc
301?accccggaca?aagcgttcca?ggacaagctg?tatccgttta?cctgggatgc?cgtacgttac
361?aacggcaagc?tgattgctta?cccgatcgct?gttgaagcgt?tatcgctgat?ttataacaaa
421?gatctgctgc?cgaacccgcc?aaaaacctgg?gaa?gagatcc?cggcgctgga?taaagaactg
481?aaagcgaaag?gtaagagcgc?gctgatgttc?aacctgcaag?aaccgtactt?cacctggccg
541?ctgattgctg?ctgacggggg?ttatgcgttc?aagtatgaaa?acggcaagta?cgacattaaa
601?gacgtgggcg?tggataacgc?tggcgcgaaa?gcgggtctga?ccttcctggt?tgacctgatt
661?aaaaacaaac?acatgaatgc?agacaccgat?tactccatcg?cagaagctgc?ctttaataaa
721?ggcgaaacag?cgatgaccat?caacggcccg?tgggcatggt?ccaacatcga?caccagcaaa
781?gtgaattatg?gtgtaacggt?actgccgacc?ttc?aagggtc?aaccatccaa?accgttcgtt
841?ggcgtgctga?gcgcaggtat?taa?cgccgcc?agtccgaaca?aagagctggc?aaaagagttc
901?ctcgaaaact?atctgctgac?tgatgaaggt?ctggaagcgg?ttaataaaga?caaaccgctg
961?ggtgccgtag?cgctgaagtc?ttacgaggaa?gagttggcga?aagatccacg?tattgccgcc
1021?actatggaaa?acgcccagaa?aggtgaaatc?atgccgaaca?tcccgcagat?gtccgctttc
1081?tggtatgccg?tgcgtactgc?ggtgatcaac?gccgccagcg?gtcgtcagac?tgtcgatgaa
1141?gccctgaaag?acgcgcagac?taattcgagc?tcgaacaaca?acaacaataa?caataacaac
1201?aacctcggga?tcgagggaag?gatttcagaa?ttcggatccc?ccgggctgca?ggaattcccc
1261.gcccccccag?cccacggagt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1321.gcccccccag?cccacggtgt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1381.gccccccaag?cccacggtgt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1441.gcccccccag?cccacggtgt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1501.gcccccccag?cccacggtgt?cacctcggcc?ccggacacca?ggccggcccc?gggctccacc
1561.gcccccccac?tccacggtgt?cacctcggcc?ccggagaaca?ggccggcccc?gggctccacc
1621.gcgcccgcag?cccacggtgt?cacctcggcc?ccggagagca?ggccggaatt?cgatatcaag
1681.cttggcactg?gccgtcgttt?tacaacgtcg?tga
2, the production technology of recombined human MUC1-MBP fusion rotein anti-tumor vaccine may further comprise the steps:
1) 450 bases of this MUC1 gene is inserted pMAL-p2 prokaryotic expression carrier malE gene downstream according to proper reading frame, MUC1 is connected together with the MBP gene, identify the correctness of this recombinant vector pMAL-MUC1 by enzyme action;
2) the recombiant plasmid pMAL-MUC1 transformed into escherichia coli DH5a of expression MUC1-MBP fusion rotein, under the inducing of IPTG, expressed the fusion rotein MBP-MUC1 of molecular weight 62KD, identify its correctness by SDS-PAGE and Western blot, and obtain stable expression strain pMAL-MUC1/DH5a;
3) conventional gene sequencing is measured the gene order of MUC1-MBP fusion rotein;
4) cultivate pMAL-MUC1/DH5a in a large number, extract the whole cell supernatant, obtained purer MUC1-MBP fusion rotein and MBP albumen by gel permeation chromatography → starch sugar affinity chromatograph → ultrafiltration through ultrasonication.
3, recombined human MUC1-MBP fusion rotein anti-tumor vaccine is used for prevention and treats various adenocarcinoma and metastatic carcinoma thereof.
4, recombined human MUC1-MBP fusion rotein anti-tumor vaccine is used for the treatment of residual focus behind the tumor operation.
5. recombined human MUC1-MBP fusion rotein anti-tumor vaccine is used for BCG as the inductive immunity of adjuvant enhancing MUC1-MBP.
6. recombined human MUC1-MBP fusion rotein anti-tumor vaccine is used for using with IL-2 or GM-CSF compatibility.
7. recombined human MUC1-MBP fusion rotein anti-tumor vaccine is used for using with the chemotherapeutics compatibility.
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