Background technology
In Taiwan, cervical cancer incidence one is to being in the woman cancer first place.When general uterine cervix epithelium variation or early stage cervical cancer took place, almost without any symptom, though more the back is respond well for early treatment, Prevention is better than cure, and therefore relevant scholar is a purpose to look for the method that can effectively prevent cervical cancer all.
Confirmed at present the human papillomavirus (human papillomavirus, HPV) closely bound up with the formation of cervical cancer, be the topmost virulence factor of cervical cancer.The dna sequence dna of some hypotype of HPV (as 16,18 types) is found in the cancer cells of 75%~100% cervical cancer case, but its carcinogenic mechanism is unclear fully as yet.Recently find the early stage virogene product E6 and the E7 albumen of 16, the 18 and 31 high-risk hypotypes of HPV, very easily with the product of Rb and p53 gene its cytostatic function that combines and neutralize, this phenomenon has illustrated that HPV acts on separately, also needs the collaborative of environmental factors simultaneously; And E7 albumen continues performance in cervical cancer cell and cancerous tissue cell, and is playing the part of important role in the process of keeping the transforming tissue malignant phenotype.
Tumor immunity is based on cellular immunization, and humoral immunization is auxilliary.The reacting cells of participating in cellular immunization mainly contain cytotoxic T cell (cytotoxic T lymphocytes, CTL), natural killer cell (NK) and scavenger cell.CTL is situated between by cell after white plain 2 (IL-2) startup, by can be by the T cell recognition, be positioned at compatible complex body (the majorhistocompatibility compolex of tissue on the tumour cell that antigen is present condition, MHC) appearance of the first type molecule, and discharge some lytic enzyme, tumour cell is killed, and suppressed the propagation of tumour cell.The provide protection of CTL is obvious especially when resisting the tumour that causes as HPV, and therefore, the approach that if can come across tumour cell by the complex body that increases HPV antigen and MHC I type molecule brings out and belongs to CTL such as the CD8 that possesses the HPV antigen-specific
+The propagation of T cell then might directly be carried out immunoprophylaxis or treatment at suppressing tumour cell.
The existing cervical cancer that studies confirm that can be come advance preventing by the injection vaccine, and owing in the HPV virus structure closely bound up with forming cervical cancer, E7 albumen often is found in cancer lesion tissue or the preceding injured tissues of cancerization, therefore has the potentiality as vaccine development target; In addition, though present developing dna vaccination has long-acting characteristic, it is expensive, and considering of high risk (virus is brought out sudden change easily) itself is still the main cause that limits its development; Yet, when using E7 albumen as the gene therapy of cervical cancer, the normal because proteic poor antigen characteristic of HPV virus E7, and can't bring out good immune response, and then the effect of desired prevention or treatment can't take place.
Generally speaking, the specific antigens of tumour must combine with MHC-I type molecule in host after treatment, just can be presented to cell surface to start CD8
+The T lymphocyte.Studies show that and contain HPV16 E7 gene in the cervical cancer tissue, but lack the specific complex body to present the specific MHC-I type molecule of E7 genes encoding antigen and presents cell surface, make HPV16 E7 albumen in host, not be presented and escape from host's immunosurveillance in antigen.In addition, generally when the protein of vaccine is in entering living body, how can be thought exogenous antigen by cell, and the preceding i.e. decomposition that is not also playing a role, and lowered the effect of protein vaccine, therefore a kind of transmission system must be developed and can the method that living body produces the validity cell immune response can be brought out simultaneously again effectively with the complete tenuigenin inside of sending to of proteantigen.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can bring out the fusion rotein of cellular immunization effect at particular cancers, especially be difficult for bringing out immunoreactive poor antigen virus, the present invention can be reached the purpose that suppresses cancerization cell proliferation by the process of effective transmission system and activated cell immunity, and reduces the cancerization degree even reach the effect of preventing cancer.
Fusion rotein of the present invention can induce the provide protection of CTL and antibody in the living somatocyte of accepting fusion rotein, and then impels infected cells to be annihilated owing to antigenic presenting; The present invention also provides a kind of medical composition that contains the cervical cancer inhibiting fusion rotein, with the preceding inhibition cancerization cell proliferation aspire to cell generation canceration, reaches and suppresses the purpose that cancer takes place.
The fusion rotein of cervical cancer inhibiting provided by the invention, T cell vaccine or contain the medical composition of this fusion rotein comprises: the E7 protein polypeptide fragment of one section human papillary virus 16 type (type 16 for human papillomavirus, HPV); One section polypeptide fragment with shift function; And the polypeptide fragment of one section tool carboxyl terminal part.
In fusion rotein of the present invention, the segmental nucleotides sequence of E7 protein polypeptide of wherein human papillary virus 16 types is classified SEQ.ID.NO.1 as; The displacement polypeptide fragment that is suitable for can be selected from the polypeptide fragment of any tool shift function commonly used, is preferably the part from Rhodopseudomonas extracellular toxin (pseudomonasaeruginosa exotoxin A); And the polypeptide fragment of tool carboxyl terminal part also can be selected from the sequence of any tool carboxyl terminal commonly used, preferable from the ectotoxic part of Rhodopseudomonas, and the polypeptide fragment of this carboxyl terminal part comprises the aminoacid sequence of KDEL, and its nucleotides sequence is classified SEQ.ID.NO.2 as.Wherein this SEQ.ID.NO.2 is positioned at the carboxyl terminal of this fusion rotein, the polypeptide fragment that this section has a shift function is positioned at the amino terminal of this fusion rotein, and the E7 protein polypeptide fragment of the human papillary virus of this section 16 types has at this section between the polypeptide fragment and this SEQ.ID.NO.2 of shift function.
The present invention also provides a kind of and brings out through aforesaid fusion rotein, and to the immune constituent of E7 polypeptide polypeptide tool specificity identification, wherein this E7 peptide sequence is SEQ.ID.NO.1; Utilize antibody composition of the present invention, can detect the appearance of E7 polypeptide fragment in the living body, and with antigen-antibody mode bond.
The present invention utilizes the characteristic of bacillary toxin, make and have proteinic bacteriotoxin and engage with the surface of cell membrane susceptor of target cell (antigen presents cell), make protein enter cell, and the placement property of performance bacteriotoxin nature, again with protein belt to tenuigenin; At this moment, be positioned at cytoplasmic exogenous protein and can be produced into little polypeptide, and then combine, and be presented in the outside that antigen presents cell with organizing compatible complex body II or I type molecule (MhcII or MhcI), with MhcII or the I person of combination again by CD4
+T or CD8
+The identification of T cell, and bring out a series of immune response, make fusion rotein of the present invention reach the effect that immunity promotes.
Medical composition of the present invention can comprise the adjuvant that is fit to according to known technology use in the skill; Dispersion agent or wetting Agent for Printing Inks (as Tween 80) and suspension agent allotment aseptic injection, for example, the aseptic injection aqueous solution or oiliness suspension.Aseptic injection preparation also can be used for thinner or the aseptic parenteral solution in the solvent or the suspension of nontoxicity injection, for example, and the solution in 1,3 butylene glycol.In spendable acceptable carrier and solvent, have N.F,USP MANNITOL, water, Ringer solution, with etc. open sodium chloride solution.In addition, Chang Yong use sterilization, fixed oil are as solvent or suspension medium (for example acid of synthetic list or Diglyceride class).Lipid acid (for example oleic acid and its glyceride derivative), and acceptable oils (for example sweet oil or Viscotrol C, the especially ethylating kenel of its polyoxy) on the natural medicaments can be used in injectable formulation.These oil solutions or suspension also can contain long-chain alcohol thinner or dispersion agent or carboxymethyl cellulose or similar dispersion agent.Other general interfacial agent that uses for example Tweens or Spans or other similar emulsifying agent or bioavailability toughener (generally being used to make pharmaceutically acceptable solid, liquid or other dosage form) also can be used for the purpose of allocating.
The composition that oral administration medicine supplying is used can be any oral acceptable dosage form of, including but not limited to capsule, ingot, emulsion, and waterborne suspension, dispersion agent and solution.In the example of the lozenge of oral use, the general carrier that uses comprises newborn candy and W-Gum.Generally also often add lubricant, for example, Magnesium Stearate.For for the capsule form oral administration medicine supplying, spendable thinner comprises lactose and W-Gum.When oral dispensing aqueous dispersant or emulsion, can make active ingredient and emulsifying agent or suspension agent combined suspension or be scattered in the oil phase.If need, can add specific sweeting agent, flavour agent or tinting material.Nasal cavity spray or inhalation composition can skill according to medicine prescription in known technology preparation, and can be made into normal saline solution, use phenylcarbinol or other sanitas that is fit to, increase known dissolving agent or dispersion agent in absorption enhancer, fluorocarbon and/or other skill of bioavailability.Contain the suppository form dispensing that the composition of benzazolyl compounds can also rectal administration.
Carrier in the medical composition is necessary for " can accept ", mean with fill a prescription in active ingredient compatible (and be preferably can make prescription stable) and to dock subject sufferer harmless.The example of other carrier comprises colloidal silica, Magnesium Stearate, Mierocrystalline cellulose, sodium lauryl sulphate, reaches D﹠amp; Yellow No. 10 of C.
Embodiment
The row of embodiment one, E7 Nucleotide and KDEL preface are synthetic
In the database of the U.S. state-run biotechnology data center (NCBI), find HPV16 E7 protein sequence (NC 001526, SEQ.ID.NO.3), totally 98 amino acid.
Utilize Taiwan patent application case number 92126644 disclosed methods, HPV16E7 albumen can be showed in a large number by the intestinal bacteria system; The emphasis of upgrading is mainly at the nucleotide fragments with the wild virus strain, not influence the amino acid that it shows originally, and under the situation that can effectively in the escherichia coli host system, show, carrying out the upgrading of single Nucleotide, nucleotides sequence is classified SEQ.ID.NO.1 as behind the upgrading.
Present embodiment synthetic nucleotide fragments, utilize totally 8 pairs of introductions respectively, with polymerase chain reaction (polymerase chain reaction, PCR) carry out the synthetic of nucleotide fragments, the right sequence of all introductions is asked for an interview table 1, has the sequence of bottom line to represent to take place with specific fragment complementary in the table.
At first utilize the polymerization methods of no dna profiling, only utilize F1 and R1 introduction to carry out nucleotide fragments, wherein in 3 ' end parts of this two introduction 15 bases being arranged respectively is to be designed to combination complimentary to one another, again via the read-write of polymerizing enzyme and supply and become a bifilar dna profiling polymerisate; After finishing the PCR first time, get the template DNA of the polymerisate of 1 μ l, add each 4 μ l of F1 and R2 introduction simultaneously,, begin to carry out the PCR second time together with required dNTPs, reaction reagent and Pfu polysaccharase etc. as the PCR second time; Then carry out 8 times PCR altogether, synthesize the nucleotide sequence (SEQ.ID.NO.1) behind the upgrading according to same way as.
Polymerize one section KDEL sequence in the same way, K3F and K3R in its introduction sequence such as the table 1 as the message polypeptide; The KDEL nucleotides sequence that synthesizes is classified SEQ.ID.NO.2 as.
Table 1, introduction are to sequence table
Embodiment two, carrier are constructed
To finish the E7 product that is obtained after the polyreaction among the embodiment one, utilize 5% polypropylene vinegar ammonia (polyacrylamide) agar-agar colloid to separate, and be to extract the target product according to purifying with molecular weight of product; Get pET or pPE (Δ III) carrier (J.R.Chen, C.W.Liao, S, J.T.Mao, and C.N.Weng, Vet.Microbiol.80 (2001) 347-357), utilize restriction enzyme to handle E7 fragment behind two carriers and the purifying simultaneously, carry out separation and purification with 5% polypropylene vinegar ammonia agar-agar colloid equally again, get 0.3kb and contain the fragment of E7 sequence, utilize T4 ligase that E7 fragment and carrier are configured as a segment length and be 7.84kb, contain PE (Δ III) gene that the Pseudomonas aeruginosa exotoxin A does not contain ferment toxicity position, and plastid pPE (Δ the III)-E7 that contains E7 gene Fusion albumen PE (Δ III)-E7, and a segment length is 3.83kb, contains the pE7 plastid of E7 and pET23a.
By the prepared PCR DNA of K3-F K3-R introduction with utilize restriction enzyme digestion and cut and purifying after after, it is 3.78kb that the Sal1-Xho1 position that is engaged to pET23a can obtain a segment length, contains the pKDEL3 plastid that n '-KDELRDELKDEL gives birth to the polypeptide gene more.
With the same manner, with restriction enzyme Sal1 and Pst1 in the pKDEL3 plastid is that 1.47kb contains on PE (Δ the III)-E7 plastid DNA that is engaged to after the KDEL sequence cuts out with the 6.5kb of restriction enzyme Xho11 and Pst1 cutting with a segment length, finishes plastid pPE (DIII)-E7-K3 that a 8.0kb size contains fusion rotein PE (Δ III)-E7-KDEL3 gene.Above-mentioned plastid is constructed schema and is seen also Fig. 1.
The above-mentioned plastid constructed finished is changeed shape (transform) and goes in the intestinal bacteria JM108 bacterial strain to preserve.
Embodiment three, protein purification
The above-mentioned plastid constructed finished is changeed shape (transform) and goes in e. coli bl21 (DE3) the pLys bacterial strain, then intestinal bacteria are incubated in the 200ml LB nutrient solution that contains 200 μ g/ml microbiotic ampicillin, OD550 reaches 0.3 up to bacterial concentration; (isopropylthio-β-D-galactoside, Promege USA), continue to cultivate 2 hours, and the cell that is grown is carried out centrifugal collection then to add 1mM IPTG; Mode with the freeze-thaw repeatable operation makes the membrane structure of the cell that has target protein slightly loose, add lysate 10ml and (contain the 0.3mg/ml N,O-Diacetylmuramidase this moment, the PMSF of 1mM and the DNaseI of 0.06mg/ml), placed room temperature following 20 minutes, then add 1ml 10%TritonX-100 again, placed room temperature following 10 minutes, afterwards collect protein with centrifugal 10 minutes of 12000xg; Clean with urea respectively again with 1M and 2M; At last, collected protein inclusion body (Inclusion body) is dissolved among the 8M urea of 8ml.
Then (Novagen, USA), carry out following experiment according to the description of test book: the protein inclusion body that will be dissolved among the 8M urea is poured into earlier with 4mlNTA-Ni2 with commercially available pET His-Tag purification system again
+In the equipped agar-agar resin tubing string of finishing; Again the protein of gluing in tubing string is swept away collection with the damping fluid (containing 6M urea, 0.3m NaCl, 20mM phosphate buffered saline buffer and 20mM Tris-HCl) of different pH (8.0,7.0,6.5,6.0,5.4 and 3.5); At last with SDS-PAGE analysing protein purity and quantitative.
Embodiment four, tumor cell line (TC-1) preparation
With HPV16-E6, E7 and the ras oncogene main lung epithelial cell in order to the mouse of cancerization C57BL/6 strain, this cancerization cell strain is TC-1, utilizes known manner to keep and produce the TC-1 cell strain.This cell strain is incubated among the RPMI 1640, add 10% (v/v) foetal calf serum simultaneously, the penicillin of 50 units/ml or streptomycin, L-bran vinegar propylhomoserin (L-Glutamine) 2mM, Sodium.alpha.-ketopropionate (sodium pyruvate) 1mM, nonessential amino acid of 2mM and 0.4mg/ml G418, culture condition is 37 ℃, keeps 5% CO in the incubator
2
Desire is used the same day of tumor cell line, with trypsin treatment (trypsin) and collect cultured cells, and with 1X HBSS buffer solution for cleaning cell 2 times, the last concentration of using to desire with 1X HBSS diluting cells again.
The vivo tumor test of embodiment five, mouse cancerization pattern
With the protein example desiring to test: E7, PE (Δ III), PE (Δ III)-E7, PE (Δ III)-E7-KDEL3 respectively with 1: 10 dilution proportion in phosphate buffered saline buffer, making concentration is 0.1mg/ml, be incubated at earlier 37 ℃ 2 hours, before the use again with 10%ISA206 adjuvant (Sepec, France) mix in the concussion mode, form four kinds of different vaccines; Get the above-mentioned vaccine that contains antigen amount 0.1mg respectively, carry out the immunity of mouse simultaneously, and respectively at appending once immunity of reinforcement after 2 weeks; The last time immunity one week of back, get 5 * 10
4TC-1 tumor cell injection to the right leg subcutaneous position of immune mouse is grown with induced tumor, injects one group simultaneously without mice immunized, to observe the self-sow situation of tumour.After carry out at the induced tumor growth experiment 60 days, per 1 to 2 week is carried out the range estimation and the palpation of tumor growth.
The result accepts the mouse after PE (Δ III)-E7-KDEL3 fusion rotein (■) immunity as shown in Figure 2, and during the induced tumor growth test, the mouse that participates in this test group does not all have tumour and takes place 100%, and continues to have observed and be all same phenomenon in 60 days; Opposite, accept E7, PE (Δ III), PE (Δ III)-fusion roteins such as E7, and the mouse group of not accepting the fusion rotein injection, behind the induced tumor growth test, the longest have only 20 days and keep no tumour note down.By the result as can be seen, contain the fusion rotein of PE (Δ III) and KDEL3 sequence and E7 antigen fragment, bring out at the TC-1 cell strain and can effectively bring into play the immune effect that suppresses tumor growth in the mouse cancerization test model.
Embodiment six, cellular immunization experiment
As the mouse test of above-mentioned vaccine immunity, get its spleen scavenger cell behind the sacrifice test mice behind the Yu Yizhou, the spleen scavenger cell that belongs to different vaccines processing contains 3.5 * 10 approximately
5Enchylema with contain E7 polypeptide (46-57 amino acid position) the 1 μ g/ml that organizes compatible complex body (MHC class I) antigen decision bit, cultivated together 16 hours, utilize CD8 again
+The plain IFN-γ of cell surface marker (surface marker) and cell within a cell combines staining reaction and flow cytometer (FACScan) analytical results, observes to belong to the narrow spectrum CD8 of E7 between each immune group
+The generation difference situation of T cell precursor.CD8
+Cell surface marker (surface marker) sees also Cheng, et al., Hum Gene Ther, 13:553-568,2002. with the dyeing process of the plain IFN-γ of cell within a cell and the method for inspection of use flow cytometer (FACScan)
In present embodiment, confirm that for the first time PE (Δ III)-E7-KDEL3 fusion rotein is influential for bringing out of E7 specificity immunity.As shown in Figure 3, in this group of injection PE (Δ III)-E7-KDEL3 fusion rotein, can find that IFN-γ manufacturing provides the narrow spectrum CD8 of E7
+T cell precursor is than the next height of any one group of experimental group (blank group 10.0 ± 1.4, E7 group 14.0 ± 2.1, PE (Δ III) group 12.0 ± 2.1, PE (Δ III)-E7 group 36.0 ± 2.8, PE (III)-E7-KDEL3 group 564.0 ± 28.0, p<0.01, AVONA).By the result as can be known PE (III)-E7-KDEL3 group can bring out than the E7 group and be higher than 40 times the narrow spectrum CD8 of tool E7
+T cell precursor.
Embodiment seven, E7 specificity detection of antibodies
As the immune animal method of embodiment five, carry out the immunity of mouse with E7, PE (Δ III), PE (Δ III)-E7, PE (Δ the III)-E7-KDEL3 fusion rotein of 0.1mg, and append vaccine of reinforcement respectively at after 1 week and 2 weeks; Immunity carries out collecting in back 7 days mice serum the last time.
In each hole of 96 porose discs, be covered with HPV16-E7 (the 0.5 μ g/ml) fusion rotein of 100 μ l, cultivate overnight down in 4 ℃; Be overlying in the culture hole with the PBS resistance that contains 20% foetal calf serum again; Mice serum is carried out serial dilution with PBS, then add in the culture hole, in 37 ℃, cultivated 2 hours; After cleaning culture hole with the PBS that contains 0.05%Tween 20 again, the bond that added 1: 2000 has the anti-ageing mouse IgG of rabbit antibody (the peroxidase-conjugated rabbit anti-mouse IgG antibody of peroxidase, Zymed, San Francisco, CA) in culture hole, under room temperature, cultivated 1 hour again; The post-flush culture plate, and (Pierce, Rockford IL) launch color, at last with the H of 1M to add 1-Step Turbo TMB-ELISA
2SO
4Termination reaction; Utilize the ELISA interpretoscope under the absorption spectrum of 450nm, to carry out result's interpretation.
In present embodiment, further try to achieve PE (Δ III)-E7-KDEL3 and can promote the power valency of anti-E7 antibody with experiment.As shown in Figure 4, the antibody of the anti-E7 that mouse after the immunity is produced, the numerical value that in serum, records, with 1: 100 extension rate, blank group is 0.629 ± 0.093, E7 group 0.882 ± 0.086, PE (Δ III) group 0.69 ± 0.06, PE (Δ III)-E7 group 0.93 ± 0.06, PE (Δ III)-E7-KDEL3 is then up to 3.593 ± 0.54 (p<0.01, AVONA), can obviously understand, can make mouse produce the anti-E7 antibody that is higher than other group with PE (Δ III)-KDEL/E7 fusion protein immunization mouse.
Experimental result of the present invention shows that fully PE (III)-E7-KDEL3 fusion rotein has the narrow spectrum immune response of the E7 of bringing out, and comprising increases E7 specificity CD8
+T lymphocyte and E7 specificity antibody.
The gained data are all with mean value and standard error value (Mean ± SEM) expression.Comparative data utilization " statistical package for social sciences " between the experimental group (Statistical Package for Social Sciences, SPSS 9.0, SPSS Inc, Chicago, IL) carry out the ANOVA analysis of variance, be lower than 0.05 as the error probability, represent that then these data have apparent otherness.
The foregoing description is only given an example for convenience of description, and the interest field that the present invention advocated should be as the criterion so that claim is described certainly, but not only limits to the foregoing description.
<110〉Healthbanks Biotech Co., Ltd.
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