CN103897067B - A kind of recombinant polypeptide for the treatment of tumour - Google Patents

Abstract

The present invention relates to a kind of recombinant polypeptide for the treatment of tumour.Especially, recombinant polypeptide of the present invention comprises three immunogenic antigens peptides and human epidermal growth factor hEGF mature peptide, is connected between each antigen peptide and hEGF mature peptide by the connection peptides of 2 to 10 amino acid lengths.

Description

A kind of recombinant polypeptide for the treatment of tumour

Technical field

The present invention relates to for a kind of recombinant polypeptide, and it is used for the treatment of the purposes of tumour.Described tumour is selected from the rectum cancer, lung cancer, mammary cancer.

Background technology

At present, clinically pharmacotherapy is still to one of topmost therapeutic modality of cancer patients.But traditional antitumor drug is all reach certain Plasma Concentration by distribution whole body and play curative effect, and the mechanism of action of these medicines be generally vigorous for metabolism, breed cells play toxic action faster, selectivity is poor, thus normal tissue cell also has larger lethal effect, produce obvious toxic-side effects, bring great pain to patient's body and mind, the antineoplaston of these medicines is worth and greatly reduces.In recent years, oncomolecularbiology develop the new model having innovated oncotherapy rapidly, tumor biotherapy, cancer target biotherapy become the new trend of oncotherapy gradually, and obtain some breakthroughs.Tumour cell general specifically expressing quasi-molecule or high expression level to tumor proliferation, attack relevant acceptor, and the usual also unconventionality expression cytokine receptoroid in tumor-associated vessels surface, these molecules and tumor growth and proliferation and Invasion and Metastasis closely related, can be used as the target spot of antineoplaston.Cancer target biotherapy is the specific binding effect utilized between Ag-Ab, ligand-receptor, the active substance of medicine or other killing tumor cells is optionally transported to tumor locus, or antibody itself is as medicine, make therapeutic action or drug effect be confined in specific target cell, tissue or organ as far as possible, and do not affect the function of normal cell, tissue or organ, thus improve a kind of method of curative effect, minimizing toxic side effect.Current laboratory and clinical study results show, the many target spots as tumor biotherapy of cytokine receptoroid, have special, wide spectrum, efficiently feature.Common cancer target, as: EGF-R ELISA (EGFR), vascular endothelial growth factor receptor (VEGFR), CD20 and mTOR kinases etc.

EGFR family targeted therapy is the focus of current therapeutic field of tumor research, and EGFR family is made up of 4 different receptor tyrosine kinases (Receptortyrosinekinase, RTK), is EGFR, HER2, HER3 and HER4 respectively.Their participate in activating the cellular signal transduction pathways of series of complex, wide expression in epithelium, interstitial and nervous tissue, the important physiological process such as growth, division, differentiation of regulating cell in the normal tissue.Wherein the process LAN of EGFR or abnormal activation often cause Normocellular canceration; in the kinds of tumor cells such as nonsmall-cell lung cancer, head and neck cancer, mammary cancer, ovarian cancer, kidney, find that its expression amount is much higher than normal cell, simultaneously the high invasiveness of EGFR and the tumour of high expression level, Concurrent Chemoradiotherapy Sensitivity reduce and prognosis mala relevant.The native ligand of EGFR is EGF, human epidermal growth factor (humanepidermalgrowthfactor, hEGF) small-molecular peptides that mature peptide is made up of 53 amino acid, can stimulate cellular proliferation, break up and move, in wound healing and organ occur, play key player.So far, be that the antineoplaston of target spot enters search procedure or Application Areas mainly contains four classes: 1. macromole monoclonal antibodies, as Cetuximab and Victibix etc. with EGFR; 2. small molecule tyrosine kinase inhibitors class, as Gefitinib, Tarceva and lapatinibditosylate etc.; 3. antisense oligonu-cleotides, as siRNA, miRNA and shRNA etc.; 4. EGFR downstream signaling pathway molecule inhibitor etc.Front two classes enter the more of clinical application, wherein monoclonal antibodies demonstrates stronger anti-tumor activity in experimentation on animals, but clinical study results is undesirable, major cause has several lower aspect: 1) the target monoclonal antibody of mouse is when being applied to human body therapy, stimulate human anti-murine antibodies, not only can in and targeting antibodies, make it be removed fast, and human allergy can be caused to react; 2) Human monoclonal antibody or genetic engineering antibody have problems in avidity and specificity, and target-oriented drug is poor; 3) monoclonal antibody class or monoclonal antibody conjugate class, molecular weight is comparatively large, and capillary vessel penetrativity is low, and inside tumor organizational stress is high, makes the dose of arrival tumour not enough; 4) along with the prolongation for the treatment of time, tumour antigen generation modulation or disappearance, make monoclonal antibody not be incorporated into target cell.In addition, small molecule tyrosine kinase inhibitors class medicine, clinical studies show only 10% ~ 30% patient is responsive to it, and most patient exists the situation of resistance, and along with treatment, most original responsive patient obtains secondary resistance.Major cause is EGFR structural mutation or other many molecular mutations being positioned at EGFR downstream signaling pathway or alternative activation pathway, causes this signal paths continuous activation when not relying on EGF.For the problems referred to above, the target therapeutic agent that positive active development is new both at home and abroad at present, can solve above-mentioned deficiency.Some small molecules recombinant polypeptide class medicines, biogenic, because its molecular weight is little, is easy to through tumour capillary vessel, arrives target site, has good application prospect, but need to improve its targeting.In addition, the small protein target with immunoloregulation function, in conjunction with after tumour cell, can activate body immune system, Promote immunity cell migration, propagation and activation, utilizes body autoimmune mechanism to play long-time antitumor action.

In recent years, some microbe-derived protein moleculars cause the attention of people gradually.After certain microbial pathogen of human infection, often body produces stronger immunne response, mainly because microorganism and human body species variation large, the protein molecular immunogenicity expressed or secrete is high, fully can activate inherent immunity and the adaptive immune system of body, transfer immune defence mechanism, thus the killing and removing of substance of having wholesome effects.At human body, why tumour cell is difficult to is utterly destroyed by body, and one of the main reasons is the normal cell that tumour cell derives from abnormal sudden change, and antigenicity is strong, and immunocyte can not fully identify, activate and play killing ability.Therefore, utilize microbial origin albumen to prepare anti cancer target biotherapeutics and there is good application prospect.Listeria bacteria cytolysin (ListeriolysinO, LLO) is the important albumen of one that listeria bacteria produces, and due to the uniqueness of its primary structure and three-dimensional structure, LLO is taken seriously gradually in the exploitation of antitumor drug.Listeria bacteria (Listeriamonocytogenes, Lm) is a kind of gram-positive bacteria, born of the same parents' endoparasitism, by passive engulf or initiatively adhere to can invade broad variety cell, mainly other phagocytic cells such as scavenger cell.Why listeria bacteria can parasitize cell interior, mainly because LLO can assist Lm to escape from phagolysosome, enters cytoplasm, and the anti-infectious immunity caused replys mainly T cell immunity.LLO itself has very strong immunogenicity, has much dominant CD4+ and CD8+T cell epitope [1-4], body can be induced to produce specific T cell, plays a significant role in anti-listeria infects.Through foreign literature report, confirm to there are 1 dominant CD8+T cell epitope and 2 CD4+T cell epitopes [3,5,6] in LLO molecule.These antigen small peptides, by specific T cell identification, promote T cell amplification and activation.We expect utilizing these dominant antigen epi-positions, build a restructuring small molecular protein, make it both have the effect of target in conjunction with tumour cell, can also Immunestimulatory effect be played simultaneously, promote T cell propagation and be divided into effector cell, playing the effect of targeting anti-tumor.

The target spot considering current neoplasm targeted therapy is selected, and we expect utilizing the native ligand EGF of EGFR as " the target guided missile " that build small recombinant proteins.On the one hand, relative to monoclonal antibody, the small-molecular peptides that EGF is made up of 53 amino acid, is not easily removed by body endothelial system, is easy to through capillary vessel, and can be incorporated into the EGFR of tumour cell high expression level specifically, avidity is high; On the other hand, because tumour patient exists EGFR structural mutation more, EGF can not activate the proliferation signal of tumour cell in conjunction with EGFR, the EGF that the recombinant protein built also can be secreted with tumour cell self competes, the recombinant protein being incorporated into target cell can play Immunestimulatory effect, improve the antigenicity of tumour cell, thus have the specific killing utilizing immunocyte to oncocyte.

Summary of the invention

The invention provides a kind of structure and preparation method of new targeting anti-tumor recombinant protein, this recombinant protein (pLLO-hEGF) not only efficiently in conjunction with tumour cell, and can obviously can play the effect of Tumor suppression growth in transplanted tumor in nude mice experiment.

The present invention passes through biotechnology expression and purification, Single polypeptide chain, 147 amino acid.N holds 94 amino acid to comprise three immunodominant antigen peptides from listeria bacteria cytolysin, is connected between antigen peptide by flexible peptide linker, and antigenicity is strong, effectively can activate body immune system, promotes lymphopoiesis and activation.C holds 53 amino acid to be complete sequence of human epidermal growth factor EGF mature peptide, and EGF is the native ligand of EGFR, specificly can be incorporated into EGFR.Most tumour cell high expression level EGFR is the important target spot of antitumor drug.This recombinant protein utilizes the characteristic of EGF specific combination EGFR, and target is attached to tumor cell surface, increases the antigenicity of tumour cell, antigen peptide chemotactic, activating immune cell that its N holds, killing and wounding of remarkable reinforcing effect cells against tumor cells.

The technical solution adopted for the present invention to solve the technical problems is: at the design aspect of recombinant protein, flexible peptide linker is adopted to connect between three dominant antigen peptides of LLO, be conducive to being degraded submission by antigen presenting cell, connection peptides is adopted to be connected between antigen peptide with hEGF, be conducive to the correct folding of hEGF three-D space structure, thus play recombinant protein target and act in conjunction with " the target guided missile " of oncocyte.The DNA sequence dna of recombinant protein adopts chemical synthesis synthesis, express and adopt prokaryotic expression system E.ColiBL (21), pET-30a (+) carrier, IPTG is inductor, Induction Process adopts low temperature, lower concentration inductor and induces for a long time, solve protokaryon system protein expression inclusion body problem, recombinant protein is in a large number with solvable activity form expression.Recombinant protein purification adopts Co2+ affinity column to cross column purification, and Thrombin proteolytic enzyme enzyme is cut, PBS wash-out target protein, solves the problem that there is more bacillary foreign protein in albumen pronucleus expression purge process.Intracellular toxin purification column is removed in employing, removes the pyrogen lipopolysaccharides (LPS) in purifying protein, makes purifying protein reach the application level of cell and experiment in vivo.Adopt the method for cell climbing sheet immunochemistry dyeing, a large amount of target of fluorescence microscope recombinant protein is in conjunction with tumor cell surface; Mtt assay is adopted to prove the ability of recombinant protein without potential stimulus cancer cell multiplication; The immunogenicity of the methods analyst recombinant protein adopting stimulation human peripheral blood mononuclearcell (PBMC) to breed, recombinant protein can stimulate lymphopoiesis, and Proliferative Activated lymphocyte has obvious killing activity to multiple EGFR high expression tumour cell; The Human colorectal cancer cells system HCT116 of high expression level EGFR is inoculated in nude mice and sets up Transplanted tumor model, in the lymphocyte tumor-side injection nude mouse of recombinant protein activation, can obviously Tumor suppression growth.

Advantage of the present invention is: compared with current antineoplastic target medicine, this restructuring cancer suppressor protein, and manufacture is simple, cost is low, and output is high, as a kind of biological response modifier, get final product target in conjunction with tumour, lymphopoiesis can be stimulated again to activate, strengthen killing and wounding tumour cell.

The invention provides a kind of recombinant polypeptide, described recombinant polypeptide comprises three immunogenic antigens peptides and human epidermal growth factor hEGF mature peptide, is connected between each antigen peptide and hEGF mature peptide by the connection peptides of 2 to 10 amino acid lengths.

Further, according to recombinant polypeptide of the present invention, wherein said three immunogenic antigens peptides identify the epi-position of a CD8+T cell and the epi-position of two CD4+T cells respectively.

Further, according to recombinant polypeptide of the present invention, wherein three immunogenic antigens peptides are selected from the polypeptide shown in SEQIDNO:3-5 respectively.

Further, according to recombinant polypeptide of the present invention, wherein said connection peptides has 6 amino acid lengths.

Further, according to recombinant polypeptide of the present invention, the aminoacid sequence of wherein said connection peptides is as shown in SEQIDNO:8.

Further, according to recombinant polypeptide of the present invention, the aminoacid sequence of wherein said recombinant polypeptide is as shown in the 15th amino acids of SEQIDNO:2 to the 161st amino acids.

On the other hand, the invention provides the nucleic acid molecule of code book invention recombinant polypeptide.

Further, according to nucleic acid molecule of the present invention, the sequence of wherein said nucleic acid is as shown in SEQIDNO:1.

On the other hand, the invention provides according to recombinant polypeptide of the present invention, the purposes in the medicine for the preparation for the treatment of tumour.

Further, according to purposes of the present invention, wherein said tumour is selected from the rectum cancer, lung cancer, mammary cancer.

Accompanying drawing explanation

Figure 1A to Figure 1B is the primary structure of restructuring cancer suppressor protein pLLO-hEGF.Figure 1A: complete before proteolytic cleavage primary amino acid sequences composition, Thrombin restriction enzyme site in different small peptide implication and purge process; Figure 1B: the restructuring final aminoacid sequence of cancer suppressor protein and the solid show of functions small peptide: 1.N terminal sequence, 2.C terminal sequence, 3.CD8+T cell epitope and both sides basic amine group acid sequence, 4.CD4+T cell epitope 1,5.CD4+T cell epitope 2,6. connection peptides.

Fig. 2 A represents construction of recombinant plasmid schematic flow sheet, the order-checking qualification result of Fig. 2 B recombinant plasmid.

Fig. 3 A-Fig. 3 B recombinant protein in a small amount abduction delivering and great expression enzyme cuts purification result.Fig. 3 A:5ml recombinant bacterium 37 DEG C, 1mMIPTG, induction 4h, the expression of recombinant protein, arrow place is the target protein of induction; Fig. 3 B:2L recombinant bacterium 23 DEG C, 0.5mMIPTG, induction 8 ~ 10h, through Co2+ column purification after ultrasonic degradation, Thrombin enzyme is cut, the purifying of recombinant protein, and arrow place is the target protein of purifying.

Fig. 4 A to Fig. 4 F represents it is that recombinant protein target is in conjunction with tumour cell effect observation.Fig. 4 A to 4B: recombinant protein is incorporated into colorectal cancer cell HCT116 and HT29; Fig. 4 C to 4D: recombinant protein is incorporated into lung cell A549 and NCI-H157; Fig. 4 E to 4F: recombinant protein is incorporated into breast cancer cell SK-BR-3 and MDA-MB-231.

Fig. 5 A to Fig. 5 L represents MTT proliferation experiment, proves that (EGF is protein control, and PBS is blank, if 4 concentration gradients: 10 [-10] M/L for the ability of the non-stimulated cancer cell multiplication of recombinant protein; 10 [-9] M/L; 10 [-8] M/L; 10 [-7] M/L).Fig. 5 A to 5D: under recombinant protein effect, the growth curve of colorectal cancer cell HCT116 and HT29; Fig. 5 E to 5H: under recombinant protein effect, the growth curve of lung cell A549 and NCI-H157; Fig. 5 I to 5L: under recombinant protein effect, the growth curve of breast cancer cell SK-BR-3 and MDA-MB-231.

Fig. 6 A to Fig. 6 G is that recombinant protein (pLLO-hEGF) stimulates lymphopoiesis activation and effector cell to the fragmentation effect of different tumour cell.Fig. 6 A to 6B: recombinant protein stimulates CD3+T cell proliferation; Fig. 6 C to 6D: recombinant protein stimulates CD3+CD4+T cell proliferation; Fig. 6 E to 6F:ConA stimulates CD3+CD8+T cell proliferation; Fig. 6 G: cytotoxicity experiment shows that lymphocyte that recombinant protein activates is to the kill rate of different tumour cell.

Fig. 7 A to 7F shows that recombinant protein suppresses the effect of nude mice tumor growth in vivo.Fig. 7 A to 7B: each group average mice body weight variation tendency; Fig. 7 C to 7D: each group mouse interior tumor growth (volume) situation and each group knurl weight; Fig. 7 E: each group mouse HCT116 solid tumor schematic diagram; Fig. 7 F: each group mouse tumor lump paraffin section HE coloration result; Note: 1.: PBS control group; 2.: recombinant protein activated lymphocyte injection group (d0); 3.: Control group lymphocyte injection group (d0); 4.: recombinant protein activated lymphocyte injection group (d7); 5.: Control group lymphocyte injection group (d7).

Embodiment

Below in conjunction with accompanying drawing, the invention will be further described.

Embodiment 1:

Design 336bpDNA sequence, comprising three dominant antigen peptide nucleotide sequences from LLO and hEGF nucleotide sequence, is connection peptides nucleotide sequence between different peptide section encoding sequence.Implementation sequence adopts chemical synthesis synthetic, is implemented in template plasmid: A-336_pMA-T(Amp ⁺).In fig. 2, adopt alkaline lysis extracting expression plasmid pET-30a (+), getting 1 μ g respectively joins in 50 μ l competent cell DH5a and 50 μ l competent cells BL21 (DE3), after rotating mixing gently, ice bath 30min, 42 DEG C of heat-shocked 60s, put 2min on ice, add LB substratum 800 μ l respectively, after putting 37 DEG C of shaking table 200rpm, jolting 90min, put the centrifugal 5min of whizzer 3000rpm, remove supernatant, residual 100 μ l substratum, after careful resuspended thalline, coat with glass stick on the flat board of Kana resistance.Flat board is placed in 37 DEG C of incubators, is inverted and cultivates 16h.Picking mono-clonal bacterium is in LB liquid nutrient medium enlarged culturing.Wherein transform DH5a bacterium and can preserve plasmid pET-30a (+), transform BL21 (DE3) bacterium does not insert goal gene unloaded bacterium as plasmid pET-30a (+).Choose restriction enzyme site BamHI and XhoI, purpose of design gene PCR primer: Forward5 '-GCGGATCCAGAAAAGGTTAC-3 ', Reverse5 '-GCTCGAGTTAGCGCAGTTC-3 '.Take A-336_pMA-T as template PCR amplifications goal gene, amplified production and empty plasmid pET-30a (+) carry out double digestion (BamHI, XhoI) simultaneously.PCR primer 37 DEG C of enzymes cut 5h, plasmid enzyme restriction 2h.Digestion products capable 1%TAE agarose gel electrophoresis 30min, cuts glue and reclaims.With PCR primer: the molar concentration rate of plasmid=5:1, connect 2h by T4DNA ligase enzyme room temperature.20 μ l linked systems transform 100 μ l competent cells BL21 (DE3), after 42 DEG C of heat shock 60s, 37 DEG C of shaking table joltings, are laid on Kana resistant panel, after 16h is cultivated in 37 inversions, and picking mono-clonal.Mono-clonal is with after LB substratum enlarged culturing, and extracting plasmid as template, with above-mentioned PCR primer amplifying target genes.Plasmid BamHI and XhoI that can amplify goal gene carries out double digestion checking, and the mono-clonal that can cut out goal gene send biotech firm to carry out DNA sequencing.DNA sequencing result input GenBank database, carry out Blast comparison with the goal gene sequence of design, the order-checking plasmid of 100%match is the recombinant plasmid (Fig. 2 B) successfully constructed.

Embodiment 2:

As shown in Figure 3A, the abduction delivering of recombinant protein (pLLO-hEGF).First the correct recombinant monoclonal bacterium liquid of order-checking is inoculated in the LB substratum that 5ml/ props up in the ratio in 1:100, adds Kana microbiotic in the ratio of 1:1000,37 DEG C, 250rpm shaking table shaking culture.Treat the OD of bacterium liquid ₆₀₀0.6 ~ 0.8 time, get 1 5ml bacterium, as a small amount of abduction delivering of recombinant protein.Add inductor IPTG in bacterium liquid, make its final concentration be 1mM, continue 37 DEG C, 250rpm shaking culture 4h.Recombinant protein, when 37 DEG C of abduction deliverings, mainly exists with inclusion bodies.Bacterium liquid before respectively getting 1ml induction and after induction, the centrifugal 1min of 10000rpm, add 100 μ l1XSDS sample-loading buffers, 100 DEG C are boiled 10min, the centrifugal 5min of 12000rpm after cooling, get supernatant carries out 12% SDS-PAGE as loading sample, gel, through 0.05% coomassie brilliant blue staining 2h, is observed after destainer decolouring.Induce expression of recombinant proteins in a small amount, occur at 16kDa place significantly inducing band (Fig. 3 B).A large amount of induction expression of recombinant proteins and purifying, induce different from a small amount of, through groping experiment condition, determining low temperature, lower concentration inductor and inducing for a long time, target protein can be made to express with soluble form.First get 4 OD ₆₀₀the 5ml bacterium of 0.6 ~ 0.8, add 4 respectively respectively containing in the Erlenmeyer flask of 500mlLB substratum, add Kana microbiotic in 1:1000 ratio simultaneously, 37 DEG C, 250rpm shaking culture 3 hours.Add IPTG, final concentration is 0.5mM, adopts 23 DEG C, 220rpm, low temperature induction 8 ~ 10h.Receive bacterium liquid, 4 DEG C, the centrifugal 30min of 5500rpm, abandon supernatant, precipitates and cross post damping fluid resuspended (TALON1XEquilibration/Wash) with 20ml, put-80 DEG C frozen, then flowing water melts, 3 cracking thalline repeatedly.Cracking thalline, through supersound process, reduces viscosity, and power is that 400w(surpasses 10s, stops 5s, is total to 30min).4 DEG C, the centrifugal 40min of 12000rpm, abandon precipitation and stay supernatant, containing soluble recombinant protein in supernatant.Supernatant, after 0.45 μm of membrane filtration, prepared TALONCo ²⁺affinity chromatography column purification.Co ²⁺affinity column is through activating process in advance: the MES solution of 20 times of column volumes crosses post, and pillar is lived again; The 1XEquilibration/Wash of 10 ~ 20 times of column volumes crosses post, balance filler resin.After application of sample crosses post, wash away the foreign protein be combined with pillar with 50 ~ 80mlTALON1XWashBuffer.Post is crossed again, balance resin with the 1XEquilibration/Wash of 10 times of column volumes.Then Thrombin(20U/ml is diluted with PBS), add in chromatography column.Chromatography column is placed in 20 DEG C, after enzyme cuts 12 ~ 16h, crosses post collect purifying protein (1ml/ props up) with 10mlPBS.38 μ l purifying proteins add 2 μ l1MDTT and 8 μ l6XSDS sample-loading buffers, and 100 DEG C are boiled 10min, the SDS-PAGE of sample capable 12%, observe, have obvious purifying protein band (Fig. 3-3) at 15kDa place after dyed and desolventing technology.Purifying protein is through 0.22 μm of membrane filtration, ToxinEraser ^tMafter EndotoxinRemovalKit goes intracellular toxin, BCA method to survey concentration, can be used for injection in LS and animal body.

Embodiment 3:

In Fig. 4 A-4F, recombinant protein (pLLO-hEGF) can target in conjunction with kinds of tumor cells.Can the methods analyst recombinant protein that immunocytochemistry be adopted to dye in conjunction with the tumour cell of high expression level EGFR.Prepare the cell climbing sheet of different tumour cell, comprise colorectal cancer cell HCT116 and HT29(Fig. 4 A and 4B), lung cell A549 and NCI-H157(Fig. 4 C and 4D), breast cancer cell SK-BR-3 and MDA-MB-231(Fig. 4 E and 4F).PBS washes cell climbing sheet 3 times, each 2min.4% paraformaldehyde room temperature fixes 30min, and pincet is clamped slide immersion PBS and washed 3 times, each 5min.3%BSA room temperature closes 30min, and PBS washes 2 times, each 5min.If 3 groups: anti-EGFRRabbitmAb group, i.e. positive controls; EGF control group; Recombinant protein group.EGF group and recombinant protein group need add EGF and recombinant protein respectively, hatch 8h at 4 DEG C.The anti-employing goat anti-rabbit igg of anti-EGFRRabbitmAb group two-FITC, press 1:100 dilution with the PBS containing 1%BSA, incubated at room 3h, PBS wash 3 times, each 5min.EGF group and recombinant protein group are adding after albumen hatches respectively, and PBS washes 2 times, each 5min.Primary antibodie all uses mouse anti human EGF antibody, presses 1:200 dilution, 4 DEG C of overnight incubation with the PBS containing 1%BSA.Two anti-employing sheep anti-mouse igg-FITC, press 1:100 dilution with the PBS containing 1%BSA, incubated at room 3h, PBS wash 3 times, each 5min.Often organize and all contaminate core mountant mounting with DAPI, observation of cell fluorescence (20X10 doubly) under fluorescent microscope.Compare with EGF control group with anti-EGFRmAb control group, recombinant protein can effectively in conjunction with target cell, and binding ability is suitable.To the different tumour cells of high expression level EGFR, recombinant protein all can effectively combine.

Embodiment 4:

As shown in Fig. 5 A to 5L, different concns recombinant protein (pLLO-hEGF) on the growing multiplication of multiple EGFR high expression tumour cell without impact, namely without the effect of potential promotion cancer cell multiplication.By the tumour cell in T25 culturing bottle with 0.25% trysinization get off, with corresponding substratum (HCT116:5%FBSIMDM; HT29:5%FBSIMDM/F12; A549:10%FBSMcCoy ' 5A; NCI-H157:10%FBSRPMI1640; SK-BR-3:10%FBSRPMI1640; MDA-MB-231:10%FBSL15) make individual cells suspension, ScepterTM type cell counter counts, and different tumour cell inoculates 96 orifice plates (HCT116:1000/ hole with different concns; HT29:2000/ hole; A549:1000/ hole; NCI-H157:2000/ hole; SK-BR-3:2500/ hole; MDA-MB-231:3000/ hole), every pore volume 100 μ l, respectively with 4 96 orifice plates, gets 4 time points and detects cell proliferative conditions.After cell inoculation 24h, take EGF as contrast, add different concns recombinant protein, concentration gradient is: 10 ^[-10]m/L; 10 ^[-9]m/L; 10 ^[-8]m/L; 10 ^[-7]m/L, wherein≤10 ^[-10]m/L is normal body fluid physiological concentration range, and each concentration gradient establishes 6 multiple holes.Add MTT respectively at 24h, 48h, 72h and 96h of cultivating after adding recombinant protein and carry out color reaction, every hole adds 100 μ lMTT solution (5mg/ml, 0.5mg/ml is mixed with) with aseptic PBS, continue to hatch 4h, stop cultivating, carefully inhale with negative pressure of vacuum pump and abandon culture supernatant in hole, every hole adds 100 μ lDMSO, shaking table vibration 10min, crystallisate is fully dissolved, and microplate reader detects the absorbance in each hole, OD570 place.See Fig. 5, identical with EGF, under different concns, the effect (PBS is contrast) of all non-stimulated tumor cell proliferation of recombinant protein, colorectal cancer cell HCT116 and HT29(Fig. 5 A-5D to high expression level EGFR), lung cell A549 and NCI-H157(Fig. 5 E-5H), breast cancer cell SK-BR-3 and MDA-MB-231(Fig. 5 I-5L), different concns recombinant protein on its growth curve all without impact.

Embodiment 5:

In Fig. 6 A to 6G, recombinant protein (pLLO-hEGF) can stimulate human PBMC breed and activate.The Healthy People PBMC recovering frozen from liquid nitrogen or isolate PBMC from healthy volunteer's peripheral blood, then with containing rhIL-2(100U/ml), mycillin (100U/ml), glutamine, 10% import foetal calf serum (FBS) RPMI1640 perfect medium resuspended.Scepter ^tMtype cell analysis calculating instrument counts, and adjustment cell concn is 2X10 ⁶/ ml, with every hole 1ml, is laid on 24 or 48 orifice plates, is placed in 37 DEG C, 5%CO ₂incubator is cultivated.If three groups: blank group (Control), recombinant protein stimulating group (Antigen), positive controls (ConA).Recombinant protein concentration is set to 10 μ g/ml, and ConA concentration is set to 5 μ g/ml.Respectively at stimulating the 3rd day, the 7th day and the 14th day that cultivates, count CD3 in each group of cell absolute number and flow cytometer detection cell colony ⁺, CD3 ⁺cD4 ⁺, CD3 ⁺cD8 ⁺the per-cent of T cell, and calculate its absolute number.In Fig. 6, compared with control group, recombinant protein group (pLLO-hEGF) CD3 ⁺, CD3 ⁺cD4 ⁺t cell is the 14th day time, and its per-cent and absolute number all obviously increase (Fig. 6 A-6D).ConA group compared with Control group, its CD3 ⁺, CD3 ⁺cD8 ⁺t cell all obviously increases (Fig. 6-1,6-3) at the 7th day and the 14th day per-cent and absolute number, but CD3 ⁺cD4 ⁺t cell per-cent obviously reduces (Fig. 6 C-6D) the 14th time.CD3 ⁺cD4 ⁺the amplification of T cell and activation are the important steps of specific immune response process, and recombinant protein (pLLO-hEGF) can stimulate CD3 ⁺cD4 ⁺the amplification of T cell, starts immune response.ConA is nonspecific short T cell mitogenic agent, is also called T cell mitogen, can not starts specific immune response.Stimulate the lymphocyte of 14 days for effector cell with recombinant protein, observe the lethal effect to different tumour cell.By different tumor cell inoculation to 96 orifice plate, add recombinant protein (10 μ g/ml) simultaneously, after 3h cell attachment, add the stimulation lymphocyte of 14 days, effect target ratio (Effector:Target, E:T) is set to 50:1, adds MTT, put 37 DEG C, 5%CO after 12h ₂incubator, detects every hole OD570 value after 4h.Recombinant protein (pLLO-hEGF) can effective killing tumor cell, is respectively: colorectal cancer cell: HCT11669.89 ± 1.05, HT2960.40 ± 2.33 to the kill rate (%) of different tumour cell; Lung carcinoma cell: A54949.45 ± 1.72, NCI-H15785.26 ± 4.82; Breast cancer cell: SK-BR-347.90 ± 1.39(Fig. 6 G).

Embodiment 6:

As shown in Fig. 7 A-7F, the lymphocyte of recombinant protein activation can suppress the growth of transplanted tumor in nude mice.For HCT116 cell, 30 SPF level BALB/c-nude mices (4 ~ 6w), inoculate HCT116 cell, 2X10 under the armpit of left side ⁶cell/only.If 5 groups, often organize 6, be respectively: 1. PBS control group; 2. recombinant protein-lymphocyte injection group 1; 3. Control lymphocyte injection group 1; 4. recombinant protein-lymphocyte injection group 2; 5. Control lymphocyte injection group 2.4. and 5. 2. and 3. wherein group is while inoculated tumour cell, and tumor-side injection lymphocyte, group is at inoculated tumour cell tumor-side injection lymphocyte after 7 days.Later every 7 days, 1. group injection PBS, 100 μ l/ only, 2., 3., 4., 5. organize equal tumor-side injection 1 lymphocyte, and different group lymphocyte is cultivation and injects after 14 days, often organizes and all injects 3 lymphocytes.Lymphocyte serum-free RPMI1640 substratum is resuspended, and only adds rhIL-2(200U/), adjustment cell concn, 2X10 ⁶cell/100 μ l, every nude mice injects 100 μ l.2. and 4. group is injecting lymphocytic while, and abdominal injection recombinant protein 40 μ g/ only, maintains lymphocyte active state in vivo.Within after inoculated tumour every 3 days or 4 days, weigh in, measure that knurl is long and knurl is wide, within 25 days, afterwards after disconnected neck execution mouse, aseptic nipper separate entities knurl, weighs, statistical analysis 2., 3., 4., 5. group have indifference compared with control group, i.e. tumor-inhibiting action.Knurl block 10% neutral formalin stationary liquid is fixed, and changes 70% ethanol and fix after 48h.Then through dehydration, waxdip, embedding, paraffin embedded tissues is made.Paraffin slicing machine is cut into the tissue slice that thickness is 3 μm, through HE dyeing after slide glass paster 24h, observes knurl stove endolymph cellular infiltration and tumor cell necrosis degree.As illustrated in figures 7 a-b, and 4. 2. organize nude mouse and focus on body weight apparently higher than initial 0 day when putting to death for the 25th day, and all the other each group does not all have significant difference (One-wayANOVAAnalysis), illustrate in recombinant protein group lymphocyte injection tumor-bearing mice body, its life quality can be improved, there is no obvious toxic-side effects.As shown in figs. 7 c-7d, calculate tumor growth in vivo curve, 2. and 4. organize tumor-bearing mice tumor growth rate and be significantly less than 1. group; Put to death mouse and weigh knurl weight, 2. and 4. organize knurl representation work and is less than 1. group (P<0.05), and 3. with 5. group compared with 1. group, no significant difference (P>0.05).Illustrate that recombinant protein stimulates the lymphocyte of activation to have the effect (Fig. 7 E) obviously suppressing tumor growth in vivo.Each group of mouse tumor tissue paraffin section de HE dyeing Microscopic observation result display: 1. organize tumor tissue necrosis region few (4X), growth of tumour cell is vigorous, cell is downright bad because growing very fast generation ischemic hypoxia, cell becomes spindle shape (20X) by circle, tumor cell growth is fine and close, in invasive growth (40X); 2. tumor tissue appearance necrosis in various degree, 3., 4., is 5. organized, wherein 2. and 4. organize necrosis region more, oncocyte is nodal-like growth, occur large stretch of necrotic area (4X), necrotic area oncolysis is downright bad, and form is imperfect, HE dyeing is in orange (20X), a large amount of lymphocytic infiltration, has more present lymphocyte inoculation position, in the form of sheets or streak distribution (40X) (Fig. 7 F).HE coloration result shows that lymphocyte that recombinant protein activates can killing tumor cell in a large number, and its effect is better than control group does not stimulate lymphocyte, and recombinant protein, as a kind of biological response modifier, can play the effect of targeting anti-tumor.

Reference

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Claims (8)

1. a recombinant polypeptide, described recombinant polypeptide comprises three immunogenic antigens peptides and human epidermal growth factor hEGF mature peptide, is connected between each antigen peptide and hEGF mature peptide by the connection peptides of 2 to 10 amino acid lengths;

Wherein said three immunogenic antigens peptides identify the epi-position of a CD8+T cell and the epi-position of two CD4+T cells respectively; And

Wherein three immunogenic antigens peptides are selected from the polypeptide shown in SEQIDNO:3-5 respectively.

2. recombinant polypeptide according to claim 1, wherein said connection peptides has 6 amino acid lengths.

3. recombinant polypeptide according to claim 2, the aminoacid sequence of wherein said connection peptides is as shown in SEQIDNO:8.

4. recombinant polypeptide according to claim 3, the aminoacid sequence of wherein said recombinant polypeptide is as shown in the 15th amino acids of SEQIDNO:2 to the 161st amino acids.

5. the nucleic acid molecule of the recombinant polypeptide of any one of claim 1-4 of encoding.

6. nucleic acid molecule according to claim 5, the sequence of wherein said nucleic acid molecule is as shown in SEQIDNO:1.

7. the purposes of the recombinant polypeptide according to any one of claim 1-4 in the medicine for the preparation for the treatment of tumour.

8. purposes according to claim 7, wherein said tumour is selected from the rectum cancer, lung cancer, mammary cancer.