CN104013973A - Tumor DNA (Deoxyribose Nucleic Acid) vaccine and virus vector vaccine taking mucoprotein 1 and surviving as targets - Google Patents

Abstract

The invention relates to the field of tumor DNA (Deoxyribose Nucleic Acid) vaccines and virus vector vaccines, and in particular relates to recombinant DNA vectors VR-S8 and VR-MS, recombinant adenoviruses AD-S8 and Ad-MS and a recombinant poxvirus MVA-MS vaccine, as well as application of DNA vaccines and optimized combination of immunization ways between virus vector vaccines to preparation of antitumor vaccines.

Description

Take tumor DNA vaccine and the vector-viral vaccine that MUC-1 and survivin be target spot

The application is for dividing an application, and the applying date of its original application is December in 2009 4 days, and application number is 200910252427.X, and name is called " take MUC-1 and survivin are target spot tumor DNA vaccine and vector-viral vaccine ".

Technical field

The present invention relates to tumor DNA vaccine and vector-viral vaccine field.Specifically, the present invention relates to recombinant DNA carrier VR-S8 and VR-MS, recombinant adenovirus Ad-S8 and Ad-MS and recombinant poxvirus MVA-MS vaccine, and the optimum organization of the immunization ways purposes in preparing anti-tumor vaccine between DNA vaccination and vector-viral vaccine and vector-viral vaccine.

Background technology

Survivin (Survivin) is apoptosis inhibit albumen (IAP) family member, has the dual-use function of anti-apoptosis and regulating cell division, and wide expression, in various embryonal tissues and cancerous cell, but is not being expressed in normal terminally differentiated cells.These features make survivin become tumor generation, a novel targets in the fields such as development and treatment.

Current research shows that survivin has potential value in therapy of tumor: its tumor specific expression, and shorten life cycle etc. and to be proportionate with prognosis mala, drug resistance and patient.The immunotherapy of tumors carrying out for survivin and mutant etc. thereof has become the focus that people pay close attention to.At present both at home and abroad strategy have (referring to Li, F.et al., 2006; Li, F., 2003; Altieri, D.C., 2008): the tumor target gene therapy that (1) utilizes the tumor specific expression of survivin promoter to carry out; (2) for the therapeutic strategy of survivin itself: a) antisense of survivin or the cDNA of total length or antisense nucleotide expression vector strategy, be relatively applicable to functional study and the front clinical research of survivin, but may have potential danger with the survivin of total length; B) survivin function negative mutant (dominant negative mutant, DNM) strategy, be mainly SurvT34A and SurvC84A, but this scheme may not be suitable for direct treatment of cancer; C) survivin ribozyme scheme, shears Survivin mRNA to reduce the expression of survivin with Survivin mRNA specific ribozyme, and this scheme is also a kind of effective research tool, but is not suitable for cancer clinical treatment; D) survivin RNAi technology, current research shows: RNAi can effectively make mammalian gene expression reticent, can be used for gene functional research, but expensive its clinical practice of costs constraints; E) with small organic molecule or little peptide, suppress the expression of survivin or disturb its function; F) with the immunization therapy of survivin specificity epitope, this is a very attractive field, but only uses the little peptide of survivin, and immunogenicity may be not high.

Because survivin has the function of anti-apoptosis, if design vaccine by its total length, may there is potential safety hazard, according to the advantage of domestic and international strategy and drawback, intend adopting the anti-apoptosis function disappearance of survivin spliced body to design vaccine, in the situation that guaranteeing its anti-apoptosis function disappearance, preserve length to improve immunogenicity as far as possible.Therefore the present invention adopts the anti-apoptosis function disappearance spliced body of survivin to design vaccine.Survivin works with dimeric forms in vivo, and three aminoacid that its N-end is the 6th, the 7th and the 10th are to form dimeric key amino acid (referring to Shi, Y.et al., 2000); The 5th of survivin is that the specific antigen epi-position of HLA-A2 is (referring to Andersen to the 14th amino acids, M.H.et al., 2001), based on these 2 the present invention, designed first spliced body S8 (cutting 7 aminoacid of survivin N-end), wish to break its dimeric structure of ring, thereby make it lack the function of anti-apoptosis.Therefore the S8 of take designs the safety that vaccine may improve vaccine as target spot.

MUC-1 (MUC1) is mucin family member, is transmembrane glycoprotein, is present in the tumor cell surface in normal glandular tube epithelial cell and source thereof, polypeptide core and side shoot sugar chain, consists of.Its core peptide extracellular fragment contains the Tandem repeat (VNTR) that number does not wait.In normal structure, MUC1 is distributed in the glandular tube epithelial cells utmost point, and isolate relative with immunocyte, glycosylation is abundant; And at tumor tissues, it extensively distributes and is expressed in extremely galore cell surface, glycosylation is incomplete, therefore expose hidden under normal circumstances epi-position, become the target spot that immunocyte is attacked, and express to strengthen, can reach Normocellularly more than 100 times, so MUC1 is comparatively ideal anti-tumor target molecule.

PDTRP sequence in MUC1VNTR is the epi-position of B cell and T cell common identification, can combine with MUC1 specific antibody and cytotoxic T lymphocyte (CTL), therefore claim that this immunogenic position of tool is immundominance domain, therefore, what major part was utilized MUC1 research immunization therapy at present is all to concentrate on VNTR region (referring to Singh, R.et al., 2007; Persson, J.et al., 2006; Xing, P.X.et al., 1992; Tang, C.K.et al., 2008; Tang, C.K.et al., 2008).From finding that MUC1 has carried out a lot of vaccine research work with it till now, and there are some vaccines to enter mankind's clinical research stage, but great majority are all traditional polypeptide class, protide or dendritic cell (DC) class vaccines, all there is the problems such as the poor or preparation difficulty of immunogenicity in these vaccines mostly; Entering clinical gene vaccine is mainly recombinant poxvirus vaccine, although toxicity do not detected, directly with recombinant poxvirus vaccine, just exempt from-strengthen or have larger potential safety hazard, and the clinical effectiveness producing neither be very desirable; DNA vector vaccine is because less immunogenic is at present also substantially in the preclinical study stage.

Inventor's early-stage Study shows to increase immune effect that the number of VNTR can strengthen MUC1 to a certain extent (referring to Zhang S et al., 2008), the immunne response causing as the MUC1VNTR tandem repetitive sequence of 33 copies is obviously better than the immunne response that the MUC1VNTR tandem repetitive sequence of 2 copies causes, therefore the present invention selects the tandem repetitive sequence MUC1VNTR that contains 33 to design vaccine as antigen, and itself and S8 amalgamation and expression are built to amalgamation and expression vaccine to strengthen broad spectrum activity and the effectiveness of vaccine immunity, at present survivin and MUC1VNTR use in conjunction are carried out to oncotherapy and yet there are no report.

Summary of the invention

The invention provides a kind of DNA fragmentation S8, the sequence of described S8 fragment is SEQ ID NO:8.

The invention provides a kind of recombiant plasmid VR-S8, the skeleton carrier of wherein said recombiant plasmid is the VR1012 as Figure 1A, in the S8 fragment described in the multiple clone site of described VR1012.Preferably, the S8 fragment of inserting is between the multiple clone site SalI and BamHI of VR1012.

The invention provides the DNA fragmentation MS shown in a kind of SEQ ID NO:9, the DNA fragmentation 33M that described sequence MS comprises a MUC1VNTR who contains 33 copies and described S8 fragment.

The invention provides a kind of recombiant plasmid VR-MS, the skeleton carrier that it is characterized in that described recombiant plasmid is VR1012, has inserted described MS fragment in the multiple clone site of described VR1012.Preferably, the MS fragment of inserting is between the multiple clone site SalI and BamHI of VR1012.

The invention provides a kind of recombinant adenovirus Ad-S8, it is characterized in that inserting described S8 fragment in adenovirus.Preferably, the skeleton carrier of described recombinant adenovirus is pBHGlox Δ E1,3Cre, and the S8 fragment of inserting is between the multiple clone site EcoRI and BglII of described recombinant adenovirus

The invention provides a kind of recombinant adenovirus Ad-MS, it is characterized in that inserting described MS fragment in adenovirus.Preferably, the skeleton carrier of described recombinant adenovirus is pBHGlox Δ E1,3Cre, and the MS fragment of inserting is positioned at the multiple clone site BglII place of described recombinant adenovirus.

The invention provides a kind of recombinant mva virus MVA-MS, it is characterized in that inserting described MS fragment in described recombinant mva virus.Preferably, the MS fragment of inserting is between the multiple clone site SalI and KpnI of described recombinant mva virus.

The invention provides described S8 fragment or described MS fragment in the purposes for the preparation of carrying out in protein vaccine, DNA vaccination, vector-viral vaccine or the dendritic cell vaccine of antineoplastic immune.

In a preferred enforcement side, in above-mentioned vaccine, comprise immunological adjuvant and/or chemotherapeutics, wherein said immunological adjuvant is selected from cytokine interleukin element 2 (IL-2), cytokine grain-giant cell colony stimulating factor (GM-CSF) and non-methylated CpG motif immunostimulation DNA sequence, and wherein said chemotherapeutics is selected from carboplatin, cisplatin, oxaliplatin and paclitaxel.

First the present invention be take S8 and is carried out the research of tumor gene vaccine as target spot, difference constructed dna carrier bacterin (VR-S8) and adenovirus carrier vaccine (Ad-S8), adopt DNA just exempt from-recombinant adenovirus booster immunization (DNA prime-rAd boost) immunization strategy, adopt the plasmid (VR-IL2) of expressing interleukin II (IL2) as immunological adjuvant simultaneously, the immunogenicity and the anti-tumor activity that by immune mouse, detect S8 gene vaccine, result shows: mice can produce specific antibody (seeing Fig. 4) and the CTL (seeing Fig. 5) for S8; This vaccine has certain anti-tumor activity, in preventive experiment, compares with PBS group, and VR-S8/VR-IL2/Ad-S8 vaccine immunity group model mouse tumor growth inhibition ratio is 68.54%, has extended 35.6% (seeing Fig. 6) life cycle.In curative test, compare with PBS group, VR-S8/VR-IL2/Ad-S8 vaccine immunity group model small mouse tumor growth has been subject to obvious inhibition (suppression ratio is 23.42%), but does not have the life cycle (seeing Fig. 7) of obvious lengthening model mice.

It is shot design amalgamation and expression gene vaccine that the present invention adopts survivin and MUC1.Because the polymorphism of MUC1 gene has determined the difference of VNTR number between Different Individual, modal for containing 30-90 repetitive sequence continuously, according to the feature that builds voluntarily MUC1VNTR gene, select with 33 duplicate block sequences (referred to as 33M, the tandem repetitive sequence that this sequence contains 33 VNTR, between each repetitive sequence, there are 6 owing to building the base sequence of the restriction enzyme site of introducing,) build MUC1DNA carrier bacterin (VR-33M), build the DNA vector vaccine (VR-MS) of 33M and S8 amalgamation and expression simultaneously.Whether by immune model mice, detect the immune effect of amalgamation and expression gene vaccine compares and is significantly improved with the vaccine of the two single expression with anti-tumor activity, result shows that the immune effect of survivin and MUC1 amalgamation and expression gene vaccine is obviously better than the immune effect of the vaccine of the two single expression, for example: in to the preventative research of tumor, amalgamation and expression vaccine immunity group is compared tumour inhibiting rate with the independent immune group of survivin vaccine and has been improved 134%, has extended 166% life cycle; With the independent immune group comparison of MUC1, tumour inhibiting rate has improved 25%, has extended 73% (seeing Figure 12) life cycle.In order further to strengthen the immune effect of MS vaccine, built again its vector-viral vaccine (Ad-MS and MVA-MS), adopt the immunization strategy of just exempt from-recombinant adenovirus of DNA booster immunization, adopt the plasmid of expressing IL2 as immunological adjuvant simultaneously, by immune model mice, detect immune effect and the anti-tumor activity of amalgamation and expression gene vaccine, result shows the obviously growth of inhibition mouse tumor of immunization strategy that just exempt from-recombinant adenovirus of DNA is strengthened, and effectively extends the life cycle of mice; IL2 has also demonstrated obvious adjuvant potentiation (seeing Figure 13).

Accompanying drawing explanation

Fig. 1 .DNA carrier and recombinant DNA carrier vaccine collection of illustrative plates.A is VR1012 carrier collection of illustrative plates; B is recombiant plasmid VR-S8 collection of illustrative plates; C is recombiant plasmid VR-33M collection of illustrative plates; D is recombiant plasmid VR-MS collection of illustrative plates.

Fig. 2. recombinant adenoviral vector collection of illustrative plates and preparation flow.A is AdMax ^tMadenovirus vector and recombinant virus preparation flow; B is shuttle vector pDC316 collection of illustrative plates; C is recombinant shuttle vector pDC316-S8 collection of illustrative plates; D is recombinant shuttle vector pDC316-MS collection of illustrative plates.

Fig. 3 .MVA restructuring principle and relevant carriers collection of illustrative plates.A is MVA restructuring schematic diagram; B is shuttle vector pSC11 collection of illustrative plates; C is recombinant shuttle vector pSC11-MS collection of illustrative plates, and MS expression of structural gene framework is recombinated in the TK gene of MVA, and the promoter of gene expression adopts P7.5.

Fig. 4. the detection of the anti-S8 antibody of mouse immune serum.C57/BL mice is through PBS (2), VR-IL2 (3), VR-S8 (4), VR-S8/VR-IL2 (5), after VR-S8/VR-IL2/Ad-S8 (6) immunity, using the serum of mice as primary antibodie, using and express the COS-7 lysis supernatant of S8 as the antigen of detection mice serum, using S8 monoclonal antibody as positive control (1), carry out the result that Western blotting (Western-blot) detects.

Fig. 5. after multi-form S8 DNA gene vaccine, the specificity CTL killing ability of mouse spleen lymphocyte detects.From the lymphocyte by separated immune mouse spleen cell, be effector lymphocyte, and with the murine lung cancer cell strain B16 of specific antigen peptide (H-2d is restricted) labelling as target cell, the CTL of employing on-radiation LDH method for releasing detection immunized mice replys.

Fig. 6. multi-form S8 gene vaccine is to MS _f ⁺the immunoprophylactic effect of B16 (purchased from ATCC) tumor cell tumor growth.C57BL/6 mice through PBS matched group, VR-IL2, VR-S8, VR-S8/VR-IL2 and VR-S8/VR-IL2/Ad-S8 through every other week immunity four times after one week, inoculation MS _f ⁺b16 tumor cell, measured tumor size (A) to 26 days, and existence situation is observed to 50 days (B) after tumor inoculation.

Fig. 7. multi-form S8 gene vaccine is to MS _f ⁺the Immunotherapy of B16 tumor cell tumor growth.Giving C57BL/6 mouse hypodermic inoculation MS _f ⁺after B16 tumor cell, give respectively PBS matched group, carboplatin (Carboplatin), VR-S8/VR-IL2/Ad-S8 and VR-S8/VR-IL2/Ad-S8/ carboplatin vaccine, measure tumor size (A) to 27 days, existence situation is observed to 50 days (B) after tumor inoculation.

Fig. 8. the detection of the anti-S8 of mouse immune serum, 33M and MS antibody.C57/BL mice, after PBS (1), VR-S8 (2), VR-33M (3), VR-MS (4) immunity, is usingd the serum of mice as primary antibodie, using express MS COS-7 cell lysate as antigen, carry out the result of Western blotting detection.

The vaccine-induced mice of Fig. 9 .MS vaccine and single expression survivin and MUC1 produces the comparison of specificity CTL killing ability.From the lymphocyte by separated immune mouse spleen cell, be effector lymphocyte, and with the murine lung cancer cell B16 of survivin (A) and MUC1 (B) specific antigen peptide (H-2d is restricted) labelling as target cell, the CTL that adopts on-radiation LDH method for releasing to detect immunized mice replys.

Figure 10. the detection of the anti-MS antibody of mouse immune serum.C57/BL mice is after PBS (1), VR-IL2 (2), VR-MS (3), VR-MS/VR-IL2 (4), VR-MS/VR-IL2/Ad-MS (5) immunity, using the serum of mice as primary antibodie, using express MS COS-7 cell lysate as antigen, carry out the result of Western blotting detection.

Figure 11. after the multi-form immunity of MS gene vaccine, the specificity CTL killing ability of mouse spleen lymphocyte detects.From the lymphocyte by separated immune mouse spleen cell, be effector lymphocyte, and with the murine lung cancer cell B16 of survivin (A) and MUC1 (B) specific antigen peptide (H-2d is restricted) labelling as target cell, the CTL that adopts on-radiation LDH method for releasing to detect immunized mice replys.

The vaccine of Figure 12 .MS vaccine and single expression survivin and MUC1 is to the comparison of model mice immunoprophylaxis effect.C57BL/6 mice through PBS matched group, VR-S8, VR-33M and VR-MS through every other week immunity three times after one week, inoculation MS _f ⁺b16 tumor cell, measured tumor size (A) to 26 days, and existence situation is observed to 50 days (B) after tumor inoculation.

Figure 13. multi-form MS gene vaccine is to MS _f ⁺the Immunotherapy of B16 tumor cell tumor growth.Giving C57BL/6 mouse hypodermic inoculation MS _f ⁺after B16 tumor cell, give respectively PBS matched group, carboplatin, VR-MS/VR-IL2/Ad-MS, VR-MS/VR-IL2/MVA-MS and VR-MS/VR-IL2/Ad-MS/ carboplatin group, measure tumor size (A) to 27 days, existence situation is observed to 50 days (B) after tumor inoculation.

The specific embodiment

One. the preparation method of above-mentioned fragment 33M and S8 and condition

1.33M preparation

MUC1VNTR is a repeated fragment that contains 60 bases, is for No. GenBank NM_002456 (SEQ ID NO:1).According to the base sequence of a VNTR, two pairs of primers have been designed, adopt synthetic 1 VNTR (1m) of overlap extension pcr (SOE PCR) duplicate block fragment, then utilize restricted enzyme SalI and XhoI enzyme action to produce the characteristic of identical sticky end, connect successively, build 33M genetic fragment.Concrete grammar is as follows:

1) overlap extension pcr

PCR reaction condition is: 95 ℃ of 20s, 55 ℃ of 20s, 72 ℃ of 30s, carry out 30 circulations, utilize primer P1 (SEQ ID NO:2) and P2 (SEQ ID NO:3) to amplify the 1 copy MUC1VNTR (being abbreviated as m) without ATG, take P2 and P3 (SEQ ID NO:4) to copy MUC1VNTR (being abbreviated as Am) fragment for primer amplification goes out containing 1 of ATG.

2) structure of pGEM-T-33M, enzyme action are identified and sequence analysis

Above-mentioned PCR product m is connected with pGEM-T-easy (Promega company) carrier respectively with Am, obtain respectively pGEM-T-m and two plasmids of pGEM-T-Am that contain described m and Am fragment, then utilize restricted enzyme SalI and XhoI enzyme action to produce the characteristic of identical sticky end, connect successively and obtain the pGEM-T-33M plasmid that contains described 33M fragment, after SalI and the evaluation of XhoI enzyme action, carry out sequence analysis, sequencing result is correct.Detailed process first obtains object segment m by pGEM-T-m with SalI and XhoI double digestion, and then is inserted into the XhoI site of pGEM-T-m, obtains pGEM-T-2m.And then pGEM-T-2m is obtained to object segment 2m with SalI and XhoI double digestion, and then be inserted into the XhoI site of pGEM-T-2m, obtain pGEM-T-4m, in like manner obtain pGEM-T-32m, and then pGEM-T-Am is obtained to object segment Am with SalI and XhoI double digestion, and then be inserted into the SalI site of pGEM-T-32m, obtained plasmid pGEM-T-33m.

2.S8 preparation

Survivin, is for No. GenBank NM_001168, according to the sequential design of survivin two pairs of primers, adopt RT-PCR technology from total RNA of 293 cells, to amplify the genetic fragment of S8, sequence is the base sequence shown in SEQ NO:8.Concrete grammar is as follows:

1) RT-PCR technology

Adopt TRIzol RNA to extract total RNA that test kit (Gibco company) extracts 293 cells, adopt Super-Script reverse transcription test kit (Gibco company) therefrom to obtain the cDNA of 293 cells, then take it as template, take P4 (SEQ ID NO:5) and P5 (SEQ ID NO:6) carries out PCR (reaction condition is 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min, carries out 30 circulations) amplification survivin cDNA as primer.

2) structure of pGEM-T-S8, enzyme action are identified and sequence analysis

The PCR product of survivin cDNA is connected with pGEM-T-easy carrier and obtains the pGEM-T-Surv plasmid that contains survivin cDNA.After sequence analysis confirms correctly, take pGEM-T-Surv as template, take P6 (SEQ ID NO:7) and P5 passes through pcr amplification S8 fragment (SEQ ID NO:8) as primer, forward primer P6 introduces the restriction enzyme site of EcoRI, XbaI and SalI, and reverse primer P5 has introduced the restriction enzyme site of BamHI.S8PCR product is connected into pGEM-T-easy carrier and obtains the pGEM-T-S8 plasmid that contains described S8 fragment, after SalI and the evaluation of BamHI enzyme action, carry out sequence analysis, sequencing result is correct.

Two. the preparation method of recombiant vaccine and corresponding condition

1.DNA carrier bacterin

1) structure of VR-33M

VR1012 carrier (VR1012 be Vical company exploitation through U.S. FDA official approval can be for the carrier of human body gene vaccine clinical trial, the clinical trial having completed shows that its application at human body is safe, the building process of this carrier is referring to document (Human Gene Therapy1996,7:1205-1217)) contain CMV promoter, kalamycin resistance gene, the conventional components such as intron A and BGH PolyA translation termination signal form, be total to 4913bp, Figure 1A is shown in by collection of illustrative plates.

The above-mentioned pGEM-T-33M plasmid making is reclaimed to test kit (Beijing Tian Gen biochemical technology company limited) with glue after SalI/XhoI double digestion and reclaim 33M genetic fragment, carrier for expression of eukaryon VR1012 carrier is reclaimed to test kit (Beijing Tian Gen biochemical technology company limited) with glue after SalI single endonuclease digestion and reclaim carrier segments, utilization has this character of identical sticky end by reclaimed genes of interest fragment and carrier segments after SalI and XhoI enzyme action, with T4DNA ligase, at 16 ℃, connect 3h.With gained, connect product transformed competence colibacillus escherichia coli Top10 (invitrogen company), the LB of coating kalamycin resistance is dull and stereotyped, and at 37 ℃ overnight incubation, select positive bacteria and fall within 5mlLB culture medium and at 37 ℃, cultivate 16h, 1, the centrifugal results thalline of 2000rpm, with plasmid extraction kit (Beijing Tian Gen biochemical technology company limited), extract plasmid, with SalI/BamHI, carry out double digestion and order-checking evaluation, result is correct, obtained recombiant plasmid VR-33M, Fig. 1 C is shown in by collection of illustrative plates.

2) structure of VR-S8

PGEM-T-S8 and carrier for expression of eukaryon VR1012 carrier, respectively through SalI/BamHI double digestion, are reclaimed to test kit (Beijing Tian Gen biochemical technology company limited) with glue and reclaim genes of interest fragment S8 and VR1012 carrier segments, use T ₄dNA ligase, 16 ℃ connect 3h.With gained, connect product transformed competence colibacillus escherichia coli Top10 (invitrogen company), the LB of coating kalamycin resistance is dull and stereotyped, and at 37 ℃ overnight incubation, select positive bacteria and fall within 5mlLB culture medium and at 37 ℃, cultivate 16h, 1, the centrifugal results thalline of 2000rpm, with plasmid extraction kit (Beijing Tian Gen biochemical technology company limited), extract plasmid, with SalI/BamHI, carry out double digestion and order-checking evaluation, result is correct, obtained recombiant plasmid VR-33M, Figure 1B is shown in by collection of illustrative plates.

3) structure of VR-MS

PGEM-T-33M is reclaimed after SalI/XhoI double digestion to genes of interest fragment 33M, VR-S8 carrier is reclaimed to carrier segments after SalI single endonuclease digestion, and utilization has this character of identical sticky end and will reclaim genes of interest fragment and carrier segments T after SalI and XhoI enzyme action ₄dNA ligase, 16 ℃ connect 3h.Obtain VR-33M-S8, be called for short VR-MS plasmid, with SalI/BamHI, carry out double digestion and order-checking evaluation, result is correct, has obtained recombiant plasmid VR-MS, and Fig. 1 D is shown in by collection of illustrative plates.

MS sequence is the base sequence as shown in SEQ ID NO:9.

2. the acquisition of recombinant adenovirus vaccine (Ad-S8, Ad-MS)

Adenovirus is a kind of uncanned double-stranded DNA virus, and genome is about 36kb, and capsid is 20 body structures of rule, diameter 80-110nm.Respectively there is the inverted terminal repeat (ITR) of one section of 100bp at the two ends of adenoviral gene group, are the initiation sites copying.In 3 ' side of left end ITR, there is packaging signal (ψ) the mediation adenoviral gene group of the about 300bp of a segment length to be packaged into viral capsid.For adenovirus, the sequence that only comprises the ITR at two ends and the approximately 0.5kb of packaging signal (ψ) is cis acting element, must be self-contained by adenovirus vector, and other more than 30 plant albumen can be by trans the supplying of helper virus (or cell).

The adenovirus system adopting in this experiment is AdMax ^tMadenovirus carrier system (Microbix company), this system is site-specific recombination system, locus specificity restructuring is to occur in two restructuring on DNA chain specific site, and it is that specificity site and site-specific protein factor are that recombinase participates in catalysis that the generation of restructuring needs one section of homologous sequence.The restructuring of recombinase between can only catalysis specificity site, can not other any two homologies of catalysis or non-homogeneous sequence between restructuring, thereby restructuring has specificity and high conservative.Accordingly, locus specificity restructuring claims again conservative restructuring, and this regrouping process does not need RecA enzyme to participate in.At present application is more Cre/loxP, FLP/FRT and BP/attBP system, wherein Cre, FLP and BP are specificity recombinase, belong to recombinase lambda integrase family, the response type of their catalysis, target site and recombination mechanism are quite similar, loxP, FRT and attBP are specificity site, also have similar structure.What AdMaxTM Adenovirus carrier was applied is Cre/loxP system, Cre gene expression product is 343 amino acid whose single glycoprotein polyprotein precursors, molecular weight is 38kDa, it is that required specificity recombinase occurs in locus specificity restructuring, its recognition site is called as loxP (locus of crossing-over P1), be the DNA sequence of one section of 34bp, by the inverted repeat of two 13bp and the asymmetric intervening sequence of a 8bp (being called again core sequence), form 5 '-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-3 '.LoxP site base is reset the rear criss-cross structure that forms, and core sequence forms a single-stranded loop, and it is the substrate of Cre recombinase cutting, is also DNA identification and the site of recombinating, and symmetrical 13bp inverted repeat is the sequence of Cre recombinase combination.

AdMaxTM Adenovirus carrier system is to consist of shuttle vector and skeleton carrier (pBHGlox Δ E1,3Cre), respectively contains a LoxP site of arranging in the same way, and wherein skeleton carrier also contains the Cre gene by CMV promoter regulation.By shuttle vector and skeleton carrier cotransfection packaging cell line 293, the entrained Cre gene of skeleton carrier starts to express, mediate the restructuring between two LoxP sites, cut off reproduction element, resistant gene and Cre gene in the antibacterial on skeleton carrier, complete the insertion of genes of interest simultaneously.Although but lack and be formed with the necessary packaging signal of infection ability adenovirus particles (ψ) because skeleton carrier carries most adenovirus genomic dna, shuttle vector contains ψ, therefore only between two carriers, there is restructuring, just can complete adenovirus biocycle, be formed with the recombinant adenovirus granule of infection ability; In addition, skeleton carrier lacks E1 gene, therefore needs 293 cells of transfection constitutive expression E1 albumen.By skeleton carrier and shuttle vector, (shuttle vector selected of this research is pDC316, Fig. 2 B is shown in by collection of illustrative plates) cotransfection 293 cells, utilize the E1 albumen in 293 cells, 10 sky, left and right just can produce the recombinant adenovirus plaque (seeing Fig. 2 A) that contains exogenous gene, picking plaque, carry out PCR (reaction condition is: 95 ℃ of 30s, 53 ℃ of 30s, 72 ℃ of 1min, carry out 30 circulations) and identify, choose that correct clone increases in a large number, purification and titer determination.

1) the shuttle back and forth acquisition of recombiant plasmid pDC316-S8 and pDC316-MS

PGEM-T-S8 is obtained to S8 object fragment with recovery after EcoRI/BamHI double digestion, after using EcoRI/Bgl П double digestion, reclaims as carrier pDC316, utilize BamHI and Bgl П to there is the character of identical sticky end, will reclaim genes of interest fragment and carrier segments T ₄dNA ligase, 16 ℃ connect 3h.With gained, connect product transformed competence colibacillus escherichia coli Top10 (Invitrogen company), the LB of coating kalamycin resistance is dull and stereotyped, and at 37 ℃ overnight incubation, selecting positive bacteria falls within 5mlLB culture medium and cultivates 16h at 37 ℃, 1, the centrifugal results thalline of 2000rpm, with plasmid extraction kit (Beijing Tian Gen biochemical technology company limited), extract plasmid, with EcoRI/Bgl П, carry out double digestion and order-checking evaluation, result is correct, has obtained recombiant plasmid pDC316-S8 (Fig. 2 C is shown in by collection of illustrative plates) plasmid.VR-MS is obtained to MS object fragment with recovery after Bgl П single endonuclease digestion, after using Bgl П single endonuclease digestion, reclaims as carrier pDC316, recovery genes of interest fragment is connected with carrier segments, correct through double digestion and order-checking qualification result, obtain pDC316-MS (Fig. 2 D is shown in by collection of illustrative plates) plasmid.

2) acquisition of Ad-S8 and Ad-MS recombinant virus

By pBHGlox Δ E1, after 3Cre and pDC316-S8 or pDC316-MS cotransfection 293 cells, after screening, amplification, purification, obtain the recombinant adenovirus Ad-S8 or the Ad-MS that contain respectively S8 and MS fragment.

3. the acquisition of recombinant MVA vaccine (MVA-MS)

Improvement Ankara vaccine (MVA, Modified Vaccinia Ankara) has the advantage such as safe, exogenous gene capacity is large is widely used in immunotherapy of tumors it.MVA contains thymidine kinase gene (TK) (referring to Fig. 3 A); PSC11 (referring to Fig. 3 B) is its shuttle plasmid, have that exogenous gene can be inserted in polyclone enzyme action site, left arm and the right arm (TKL, TKR) with thymidine kinase can carry out homologous recombination with MVA, contain lacZ gene simultaneously, can be for the locus coeruleus screening of recombinant MVA (rMVA).Described MVA system (containing shuttle plasmid pSC11) is purchased from ATCC.

1) the shuttle back and forth acquisition of recombiant plasmid pSC11-MS

VR-MS and pSC11 are reclaimed after SalI/KpnI double digestion, will reclaim genes of interest fragment MS and pSC11 carrier segments T ₄dNA ligase, 16 ℃ connect 3h.With gained, connect product transformed competence colibacillus escherichia coli Top10 (invitrogen company), the LB of coating kalamycin resistance is dull and stereotyped, and at 37 ℃ overnight incubation, selecting positive bacteria falls within 5mlLB culture medium and cultivates 16h at 37 ℃, the centrifugal results thalline of 12000rpm, with plasmid extraction kit (Beijing Tian Gen biochemical technology company limited), extract plasmid, with SalI/KpnI, carry out double digestion and order-checking evaluation, result is correct, has obtained recombiant plasmid pSC11-MS (seeing Fig. 3 C).

2) acquisition of MVA-MS recombinant virus

First the MVA infection tk-ts13 cell with titre 0.05pfu/cell (purchased from ATCC, is for No. ATCC CRL-1632 ^tM, be the mutant of the BHK-21 that do not contain TK gene).Infect after 2 hours, utilize the method for Lipofection2000 (INVITROGEN) that pSC11-MS is transfected in the tk-ts13 cell that has infected MVA.MVA is in the process of tk-ts13 time multiplexed cell system, because pSC11-MS has TKL and the TKR with the TK DNA homolog of MVA, so have a certain proportion of MVA virus to recombinate with pSC11-MS, result MS and LacZ reading frame are recombined in MVA viral genome.Form MVA-MS recombinant virus as shown in Figure 3 C.

Infected cell culture is after three days above, collecting cell, and cell lysis, ultrasonic Treatment, the centrifugal 10min of 2000rpm removes cell residue and retains supernatant.Get a certain amount of supernatant, as kind of a poison, in 6 orifice plates, virus is diluted to 10 successively ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, infect tk-ts13 cell 2h.By isopyknic at 45 ℃ 2% low melting-point agarose and 2 * DMEM-10/BrdU (BrdU of pre-temperature, 5-bromine uracil, can be by TK phosphorylation, the BrdU of phosphorylation can be incorporated in wild type MVA, producing lethal mutation, thereby can make wild type MVA reduce gradually) culture medium mixes, and every hole adds 3ml culture medium, at room temperature solidify, at 37 ℃ in containing 5%CO ₂incubator in cultivate 48h.Spread layer-selective culture medium, composition than lower floor's agar many 4%X-gal of 1/120 volume.Overnight incubation, selects blue single speckle, and multigelation three breakup releasing virus, carries out the screening of next round, and step is identical, so carries out 6 and takes turns above screening, last until obtain only containing the clone strain of recombinant virus MVA-MS.

Three. immune effect of vaccine

1.S8 immune effect of vaccine

1.1S8 the immunogenicity of vaccine

This part experiment is inquired into survivin DNA vaccinations and recombinant adenovirus vaccine from humoral immunization and two aspects of cellular immunization, with and with the IL-2 plasmid immunne response of immune induction mice altogether.Experiment is divided into groups C57/BL/6 mice by table 1,1 group at the 0th, 2,4,6 weeks injection PBS; 2-4 group was injected VR-S8 vaccine and VR-IL2 adjuvant at the 0th, 2,4,6 weeks; At the 0th, 2,4 weeks, inject VR-S8DNA vaccine and VR-IL2 adjuvant for 5 groups, at the 6th week injection recombinant adenovirus vaccine.Investigate the immune effect of the only immunity of survivin DNA single, the adjuvant effect of IL-2 plasmid, and the immunological enhancement of recombinant adenovirus.

Table 1S8 vaccine immunogenicity

1.1.1 the Western blotting of humoral immunization-antibody detects

After last immunity 1 week, mice is carried out to tail venous blood sampling, by institute's separation of serum for detection of specific antibody.The cracking supernatant of collecting the COS-7 cell of expressing S8, after SDS-PAGE separation, is transferred on nitrocellulose filter, as the antigen that detects mice serum.From each experimental group, get the immune serum of dilution in 1: 20 (diluent is 1% milk of PBS preparation) as first antibody, using survivin monoclonal antibody as positive control, using and use the anti-Mus IgG of horse (immuno research company) of alkali phosphatase enzyme mark as second antibody, through western blotting, detect the specificity of each immune serum survivin antibody.

Result as shown in Figure 4, PBS (swimming lane 2) group and IL2 (swimming lane 3) group do not dye band, and other experimental group testing results are all positive, show that C57/BL/6 mice, after survivin gene vaccine immunity, has produced the specific antibody of anti-survivin antigen in its serum.The S8 antibody titer that separately immunity produces a little less than, add the antibody titer producing after IL-2 to strengthen to some extent, after recombinant adenovirus Ad-S8 booster immunization, antibody titer obviously strengthens.

1.1.2 cellular immunization-cytotoxic T lymphocyte (CTL) activity

Within after last immunity two weeks, put to death mice, get spleen separated lymphocyte.Take from the lymphocyte by separated immune mouse spleen cell is effector lymphocyte, and to use survivin specific antigen peptide (S _88-96) the B16 cell (purchased from ADCC) of labelling, mix by a certain percentage above-mentioned effector lymphocyte and target cell, adopt inactive lactic acid dehydrogenase (LDH) method for releasing detection kit Cyto Tox96 (Promega) to detect the kill and wound situation of lymphocyte to target cell, be used for characterizing the specific CTL response strength that described vaccine is induced in Mice Body.

The present invention is according to the H-2D of SYFPEITHI algorithm (www.syf-peithi.de) prediction survivin ^bmHCI restricted epitope peptide, selects and H-2D ^bhigher antigenic peptides (the S of binding constant value of MHCI _88-96) as antigenic peptides, carry out above-mentioned experiment.

Experimental result as shown in Figure 5, as seen from Figure 5, is compared with PBS matched group, and VR-IL2 group does not detect survivin specific CTL (p > 0.05); VR-S8 group is only CTL can be detected at 100: 1 o'clock to reply at effect target ratio, but there is no statistical significance; VR-S8/VR-IL2 combined immunization group is specific CTL all to have been detected at 100: 1 and 33: 1 o'clock to reply (p < 0.05) at effect target ratio, than the CTL of the independent immune group of VR-S8, replys and increases but there is no statistical significance (p > 0.05); VR-S8/VR-IL2/Ad-S8 group has been injected again a pin recombinant adenovirus vaccine Ad-S8 booster immunization after having injected three pin DNA vaccinations, result is specific CTL all to have been detected at 100: 1,33: 1 and 10: 1 o'clock to reply (p < 0.01) at effect target ratio, compares CTL reply also obviously raising (p < 0.05) with VR-S8/VR-IL2 group.

By above-mentioned humoral immunization and cellular immunization result, can find out that independent application VR-S8 gene vaccine can produce specific antibody and the CTL immunne response for survivin by inducing mouse.After immunological adjuvant VR-IL2 plasmid and VR-S8DNA vaccine are injected altogether, the special humoral immunization that inducing mouse produces and cell immune response all strengthen to some extent, show that VR-IL2 can strengthen the immunne response of VR-S8 gene vaccine, has played the effect of immunological adjuvant; Recombinant adenovirus vaccine carries out antibody titer and CTL that booster immunization can obviously improve VR-S8/VR-IL2 combined immunization group and replys level after three pin DNA vaccination immunity, has played the effect of booster immunization.

The tumor protection of 1.2S8 vaccine

1.2.1 preventative

According to the known survivin gene vaccine of result above, can produce specific humoral immunity and cellular immunization by inducing mouse; the DNA vaccination of this section employing survivin and recombinant adenovirus vaccine and IL2 carry out the prevention experiment of model mice tumor; to investigate survivin gene vaccine, whether can in Mice Body, induce generation protectiveness anti-tumor immune response, and whether VR-IL2 plasmid adjuvant and prime-boost immunization ways can promote this protective effect.

Table 2 survivin vaccine prevention effect

C57BL/6 mice is divided into five groups (15/group) at random by table 3.1st, 2,3,4 groups respectively at 0,2,4,6 week immune DNA vaccination; At 0,2,4 week immune DNA vaccination, at the 6th week, inject recombinant adenovirus vaccine booster immunization for the 5th group.All mices all carry on the back bottom subcutaneous vaccination MS in right side of mice in last immunity after one week _f ⁺b16 tumor cell, investigates into the variation of tumor time and tumor size, other five groups of variations of simultaneously observing mouse survival rate.Experiment grouping and immunity time are as shown in table 4.

1.2.1.1 the inhibitory action of tumor growth

Experimental result shows, compares with PBS immune group, and the model mice tumor growth of VR-S8, VR-S8/VR-IL2 and VR-S8/VR-IL2/Ad-S8 immune group obviously slows down, and along with slow down adding with the reinforcement of recombinant adenovirus Ad-S8 of IL2 adjuvant successively.Fig. 6 A tests and respectively organizes gross tumor volume situation of change for prevention, as seen from the figure: VR-IL2 immune group and the comparison of PBS matched group, tumor growth is without significant change; VR-S8 immune group and PBS group relatively, the poor growth of mouse tumor, there were significant differences for the two (after inoculated tumour 23 to 26 days, p<0.05); With the independent immune group comparison of VR-S8, VR-S8/VR-IL2 altogether immune group tumor growth obviously slowly (after inoculated tumour 20 to 26 days, p<0.05); With VR-S8/VR-IL2 altogether immune group compare, VR-S8/VR-IL2/Ad-S8 group tumor growth obviously slow down (after inoculated tumour 20 to 23 days, p<0.05; 24 to 26 days, p<0.01).

1.2.1.2 model mice life extends situation

PBS group is 33.92 ± 1.36 days mean survival time (MST), to after inoculated tumour cell 40 days time mice all dead, VR-IL2 group the average survival time 35.40 ± 1.70 days, all dead to 48 days mices after inoculated tumour cell, with PBS group without obviously difference; VR-S8 group the average survival time 37.47 ± 1.60 days, to after inoculated tumour cell 50 days, has 6.7% mouse survival, and increase in life span is 10.5%, but relatively there is no significant difference with PBS group; And VR-S8/VR-IL2 organizes the average survival time 39.67 ± 1.70 days, to the observation period, have 6.7% mouse survival, increase in life span is 17%, compares with PBS group, life obviously extends (p<0.05), but relatively there is no notable difference with VR-S8 group; And VR-S8/VR-IL2/Ad-S8 organizes the average survival time 46.01 ± 1.60 days, to the observation period, still have 53.3% mouse survival, increase in life span is 35.6%, compares with other groups, life obviously extends (p<0.01), sees Fig. 6 B.

From above-mentioned preventive experiment result, can find out, VR-S8 vaccine is the growth of inhibition mouse tumor obviously, but effective lengthening model survival time of mice; But add after IL2 adjuvant (VR-S8/VR-IL2 immune group), the life cycle that not only significantly the growth of inhibition mouse tumor can also effectively improve model mice, so VR-IL2 has played the effect of immunological adjuvant; After VR-S8/VR-IL2 immunity three times, the growth of inhibition mouse tumor the life cycle of very significant lengthening model mice with energy highly significant after recombinant adenovirus booster immunization (VR-S8/VR-IL2/Ad-S8 immune group), illustrate that prime-boost strategy can obviously strengthen the immune effect of vaccine.

1.2.2 therapeutic

According to the result of model mouse prevention experiment; S8DNA vaccine is under IL-2 adjuvant is auxiliary; with recombinant adenovirus vaccine Ad-S8 booster immunization, can very effectively bring into play antitumor protective effect; therefore in the treatment experiment of model mice tumor; select VR-S8 and VR-IL2 after immune three pins, to use recombinant adenovirus Ad-S8 booster immunization altogether, investigate VR-S8/VR-IL2/Ad-S8 gene vaccine to the inhibitory action of model mice tumor growth in vivo and the prolongation effect to mice with tumor life.Meanwhile, with chemotherapeutics carboplatin, do positive control, whether by the mode of VR-S8/VR-IL2/Ad-S8 vaccine and carboplatin administration use in conjunction, inquire into the two has synergism in the treatment of model mice tumor.

Table 3 survivin vaccine therapy effect

(note: the administration time of the carboplatin in table 3 is the 1st, 6,11,16 days)

C57BL/6 mice is divided into VR-S8/VR-IL2/Ad-S8, VR-S8/VR-IL2/Ad-S8 and carboplatin synergy group (VR-S8/VR-IL2/Ad-S8/Carb), carboplatin positive controls (carboplatin) and matched group (PBS), 15 every group at random by table 3.At the 0th day, in right side of mice, carry on the back bottom near the about 0.7cm of root of the tail portion place's subcutaneous vaccination MS _f ⁺b16 tumor cell, gives respectively gene vaccine and PBS for the 1st, 8,15 days, and administering mode is intramuscular injection; The administration time of carboplatin is the 1st, 6,11,16 days, and administering mode is lumbar injection.Since about the 10th day, observe the growth of tumor and big or small variation, other four groups of variations of simultaneously observing mouse survival rate.Tumour inhibiting rate group experiment mice starts death the 28th day left and right control group mice, puts to death whole mices, peels off tumor, measures tumor weight, calculates tumour inhibiting rate; Organize experiment mice life cycle and observe the existence situation of mice, until mice is all dead, the time is about 50 days.

1.2.2.1 the inhibitory action of tumor growth

Fig. 7 A tests and respectively organizes gross tumor volume situation of change for treatment.With the comparison of PBS matched group, VR-S8/VR-IL2/Ad-S8, carboplatin and VR-S8/VR-IL2/Ad-S8/ carboplatin group tumor growth obviously slow down, and after inoculated tumour 21 to 27 days, are p<0.05); VR-S8/VR-IL2/Ad-S8 and carboplatin use in conjunction group and the two independent set of applications comparison, tumor growth rate obviously slows down (after inoculated tumour 21 to 27 days, be p<0.05), result shows that carboplatin and vaccine use in conjunction can obviously improve the inhibitory action of vaccine to mice with tumor tumor growth.

1.2.2.2 model mice life extends situation

PBS group is 31.21 days mean survival time (MST), to after inoculated tumour cell 38 days time mice all dead; VR-S8/VR-IL2/Ad-S8 group the average survival time 33.64 days, to after inoculated tumour cell 44 days time mice all dead, relatively there is no significant difference with PBS group; Carboplatin group the average survival time natural law is respectively 35.29, has 7.1% mouse survival during to the observation period, and increase in life span is 13.07%, with relatively all obviously prolongations (being p<0.05) of mice life of matched group; VR-S8/VR-IL2/Ad-S8 and carboplatin use in conjunction group the average survival time natural law are 39.29 days, during to the observation period, there is 21.4% mouse survival, increase in life span is 25.89%, with relatively all obviously prolongations (being p<0.01) of mice life of matched group; The independent set of applications comparison of carboplatin and vaccine use in conjunction group and vaccine, mice life obviously extends, and shows that carboplatin can obviously improve vaccine to the mice with tumor prolongation effect of life cycle, as Fig. 7 B.

2.MS immune effect

The immunogenicity of 2.1MS vaccine

2.1.1MS the comparison of the immune effect of vaccine and survivin vaccine and MUC1 vaccine

This part experiment is inquired into the two the vaccine of immunogenicity and single expression of survivins and MUC1 fusion gene vaccine and is compared whether there is obvious enhancing from two aspects of humoral immunization and cellular immunization.Experiment is divided into groups C57/BL/6 mice by table 4,5 every group, at 0,2,4 weeks, give respectively and PBS for first group, the 2nd, 3,4 groups at 0,2,4 weeks the corresponding DNA vaccination of immunity respectively, all mices drew neck execution at the 6th week, get spleen is CTL.Investigate survivin and MUC1 fusion gene immune effect of vaccine and whether be better than the two immune effect of independent application.

The comparison of the immune effect of table 4MS vaccine and survivin vaccine and MUC1 vaccine

2.1.1.1 the Western blotting of humoral immunization-antibody detects

After last immunity 1 week, mice is carried out to tail venous blood sampling, separation of serum is for detection of specific antibody.The cracking supernatant of collecting the COS-7 cell of expressing MS, after SDS-PAGE separation, is transferred on nitrocellulose filter, as the antigen that detects mice serum.From each experimental group, get the immune serum of dilution in 1: 20 (diluent is 1% milk of PBS preparation) as first antibody.With the anti-Mus IgG of horse (immuno research company) of alkali phosphatase enzyme mark, as second antibody, through western blotting, detect the titre of survivin and MUC1 specific antibody in each immune group mice serum.

As shown in Figure 8, situation for each group immune mouse generation antibody, VR-S8 (swimming lane 2), VR-33M (swimming lane 3) and VR-MS (swimming lane 4) group mice have all produced the specific antibody of anti-survivin and MUC1 as seen from the figure, the antibody titer that wherein VR-33M group produces is the strongest, and the antibody titer that VR-MS group produces is weaker than the antibody titer that VR-33M group produces.This is unexpected result, infer its reason may be MUC1 this as transmembrane protein and major part outside born of the same parents, therefore producible antibody titer is higher; And survivin is intracellular protein, survivin and MUC1 may hinder MUC1 to cell membrane transporter after merging, extend the holdup time of described fusion rotein in born of the same parents, may improve the probability of protease to its hydrolysis, thereby reduce described fusion rotein (MS) stability in vivo, it is degraded rapidly, and then antibody titer is declined.But this process may provide a large amount of little peptides that can be combined with MHC molecule for cellular immunization approach, and then promote cellullar immunologic response.

2.1.1.2 cellular immunization-cytotoxic T lymphocyte (CTL) activity

Take from the lymphocyte by separated immune mouse spleen cell is effector lymphocyte, and with survivin or the peptide-labeled B16 cell of MUC1 specific antigen, mix by a certain percentage above-mentioned effector lymphocyte and target cell, adopt inactive lactic acid dehydrogenase (LDH) method for releasing detection kit Cyto Tox96 (Promega) to measure the cleaved ratio of target cell B16, be used for characterizing the specific CTL response strength that this vaccine is induced in Mice Body.Result as shown in Figure 9.

Fig. 9 is the situation that each immune group (VR-S8, VR-33M and VR-MS) mice of table 4 correspondence produces survivin and MUC1 specific CTL.A figure is usingd the B16 cell of hatching with the special CTL restricted epitope peptide of survivin as target cell, detects the situation that produces survivin specific CTL with different vaccine-induced mices.As seen from the figure, compare with PBS matched group, VR-S8 group does not detect survivin specific CTL (p > 0.05); VR-MS group detects survivin specific CTL (p < 0.05), but compares the difference on not statistically significant with VR-S8 group; And VR-33M group does not detect CTL and replys, illustrate that the CTL of generation is plain specific for survival.

B figure is usingd the B16 cell of hatching with the special CTL restricted epitope peptide of MUC1 as target cell, detects the situation that produces MUC1 specific CTL with different vaccine-induced mices.As seen from the figure, compare with PBS matched group, VR-33M and VR-MS group have all produced the specific CTL of MUC1 (p < 0.05), and the CTL that VR-MS group produces replys level, the level of replying producing a little more than VR-33M group, but difference on not statistically significant; And VR-S8 group does not detect CTL and replys, illustrate that the CTL of generation is that MUC1 is specific.

Visible, VR-33M, VR-MS gene vaccine all can produce special CTL immunne response by inducing mouse.

2.1.2 first exempt from-booster immunization (Prime-boost) immunization strategy and the immunological enhancement of IL2 adjuvant to MS vaccine

This part experiment adopts the immunization strategy of just exempt from-recombinant adenovirus of DNA booster immunization, and IL2 immunological adjuvant is inquired into the situation of survivin and the vaccine-induced mice generation of MUC1 fusion gene immunne response, the adjuvant effect of investigation prime-boost immunization strategy to the potentiation of immune effect of vaccine and IL2 plasmid from humoral immunization and two aspects of cellular immunization.

Table 5 is exempt from-booster immunization immunization strategy and the immunological enhancement of IL2 adjuvant to MS vaccine just

C57/BL/6 mice is divided into groups by table five, every group 5,1st, at the 0th, 2,4,6 weeks, inject respectively DNA vaccination for 2,3 groups, 4th, at the 0th, 2,4 weeks, inject respectively DNA vaccination for 5 groups, the 6th week injection recombinant adenovirus vaccine, within the 8th week, put to death all mices, get spleen, detect survivin and MUC1 specific CTL.

2.1.2.1 the Western blotting of humoral immunization-antibody detects

After last immunity 1 week, mice is carried out to tail venous blood sampling, separation of serum is for detection of specific antibody.The cracking supernatant of collecting the COS-7 cell of expressing MS, after SDS-PAGE separation, is transferred on nitrocellulose filter, as the antigen that detects mice serum.From each experimental group, get the immune serum of dilution in 1: 20 (diluent is 1% milk of PBS preparation) as first antibody.The anti-Mus IgG of horse (immuno research company) of usining with alkali phosphatase enzyme mark is as second antibody, through western blotting, detects the titre of survivin and MUC1 specific antibody in each immune group mice serum.

Figure 10 is the production of survivin and MUC1 specific antibody in each immune group mice serum of table 5 correspondence.As seen from Figure 10, mice through PBS (swimming lane 1) and IL2 (swimming lane 2) immunity does not produce survivin and MUC1 specific antibody, and the mice of VR-MS (swimming lane 3), VR-MS/Ad-MS (swimming lane 4) and VR-MS/IL2/Ad-MS (swimming lane 5) immunity has all produced survivin and MUC1 specific antibody, and antibody titer strengthens successively, show that recombinant adenovirus Ad-MS and IL2 have all played the effect that strengthens immune effect.

2.1.2.2 cellular immunization---cytotoxic T lymphocyte (CTL) activity

It is effector lymphocyte that the present invention be take from the lymphocyte by separated immune mouse spleen cell, and with survivin or peptide-labeled B16 cell or the stable expression cell line MS of MUC1 specific antigen _f ⁺b16 (purchased from ATCC), as target cell, detects generation specific CTL in Mice Body and replys situation.Result as shown in figure 11.

Figure 11 is the situation that each immune group (PBS, VR-IL2, VR-MS, VR-MS/Ad-MS and VR-MS/VR-IL2/Ad-MS) mice of table 5 correspondence produces survivin and MUC1 specific CTL.A figure is usingd the B16 cell of hatching with the special CTL restricted epitope peptide of survivin as target cell, detects the situation that different vaccine-induced mices produces survivin specific CTL.As seen from the figure, compare with PBS matched group, VR-MS, VR-MS/Ad-MS and VR-MS/VR-IL2/Ad-MS group have all detected survivin specific CTL (p < 0.05), the CTL level of replying producing raises successively, and all has statistical significance (being followed successively by p < 0.05); And VR-IL2 group does not detect CTL and replys, illustrate that the CTL of generation is plain specific for survival.

B figure is usingd the B16 cell of hatching with the special CTL restricted epitope peptide of MUC1 as target cell, detects the situation that different vaccine-induced mices produces MUC1 specific CTL.Compare with PBS matched group as seen from the figure, VR-MS, VR-MS/Ad-MS, VR-MS/VR-IL2/Ad-MS group have all produced the specific CTL of MUC1 (p < 0.05), the CTL level of replying producing raises successively, and all has statistical significance (being followed successively by p < 0.05); And VR-IL2 group does not detect CTL and replys, illustrate that the CTL of generation is that MUC1 is specific.

In sum, VR-MS, VR-MS/Ad-MS, VR-MS/VR-IL2/Ad-MS gene vaccine all can produce for survivin or the special CTL immunne response of MUC1 by inducing mouse, and the CTL level producing obviously raises successively, illustrates that the immunization strategy of just exempt from-recombinant adenovirus of DNA booster immunization can obviously strengthen the immune effect of vaccine; IL2 has also played the potentiation of immunological adjuvant.

The protective effect of 2.2MS vaccine to model mice

2.2.1 prophylactic action

Adopt DNA vaccination VR-S8, VR-33M and VR-MS to carry out the prevention experiment of model tumor, whether the protection of investigating survivin and MUC1 amalgamation and expression vaccine on mouse tumor is significantly improved compared with the two the protection of vaccine of single expression.

C57BL/6 mice is divided into four groups (10/group).PBS, VR-S8, VR-33M and VR-MS group be respectively at 0,2,4,6 week immune DNA vaccination and PBS, all mices all last immunity after one week in the right leg outer side inoculation of mice MS _f ⁺b16 tumor cell, investigates into the variation of tumor time and tumor size, other four groups of variations of observing mouse survival rate.Experiment grouping and immunity time are as follows:

Table 6MS vaccine is preventative to model mice tumor

2.2.1.1 the inhibitory action of tumor growth

Figure 12 A is for respectively organizing gross tumor volume situation of change in prevention experiment.VR-S8, VR-33M immune group are compared with PBS matched group, the poor growth of mouse tumor, and (22 days from inoculated tumour start, p<0.05) significant difference; And VR-MS immune group is compared with PBS matched group, the growth of mouse tumor is obviously slowed down, and (22 days start, p<0.01) for beginning in 20 days from inoculated tumour, p<0.05 significant differences; VR-MS immune group is compared with the independent immune group of VR-S8, gross tumor volume obviously reduce (after inoculated tumour, 22 start, p<0.05,24 days start, p<0.01), but with VR-33M group relatively without significant difference.

2.2.1.2 the existence situation of mice

PBS group is 31.6 days mean survival time (MST), to after inoculated tumour cell 38 days time mice all dead; VR-S8 group the average survival time 35.7 days, all dead to 45 days mices after inoculated tumour cell, with PBS group without obviously difference; VR-33M group the average survival time 37.89 days, to after inoculated tumour cell 50 days, has 11.1% mouse survival, relatively has significant difference (p<0.05) with PBS group; And VR-MS organizes the average survival time 42.5 days, finish to still have 37.5% mouse survival to the observation period, increase in life span is 34.49% (p<0.01), compares with VR-S8 group and VR-33M group, and life obviously extends (p<0.05).See Figure 12 B.

From above-mentioned preventive experiment result, can find out, VR-MS vaccine is obviously better than VR-S8 vaccine to the growth inhibited effect of model mice tumor with to the model mice life situation that extends; Compare with VR-33M vaccine, although be not significantly improved aspect tumor growth inhibition, can obvious lengthening model mice life.At present the evaluation of tumor vaccine has been started to tend to the raising to the prolongation of tumor patient life and quality of life, by tumor, dwindling even to disappear becomes people's mindset already as treatment of solid tumors evaluation criterion.And when being indiscriminately imitated tumor vaccine and evaluate, people but find that it can not reflect the true curative effect of vaccine.Although because some tumor vaccine has effectively extended patient's life, improved quality of life, gross tumor volume may not obviously dwindle.Therefore, we think that VR-MS vaccine is significantly improved to the protection of tumor.

2.2.2 therapeutic effect

According to the known IL2 adjuvant of experimental result above, can obviously improve immune effect and the protection to mouse tumor of vaccine; The immunization strategy of just exempt from-recombinant adenovirus of DNA booster immunization can significantly strengthen immune effect and the protection to mouse tumor of DNA vaccination; The immune effect of survivin and MUC1 amalgamation and expression vaccine and tumor protection power obviously improve while using the two more separately.Therefore this section is according to above-mentioned conclusion, in the treatment experiment of model mice tumor, select after immune three pins, to use recombinant adenovirus Ad-MS booster immunization altogether with VR-MS and VR-IL2, investigate VR-MS/VR-IL2/Ad-MS gene vaccine to the inhibitory action of model mice tumor growth in vivo and the prolongation effect to mice with tumor life.Meanwhile, with chemotherapeutics carboplatin, do positive control, whether by the mode of VR-MS/VR-IL2/Ad-MS immunity and carboplatin administration use in conjunction, inquire into the two has synergism in the treatment of model mice tumor.In order to investigate the immune strengthening effect of MVA-MS, in experiment, design again VR-MS/VR-IL2/MVA-MS immune group simultaneously, and then compared the power of Ad-MS and MVA-MS immune strengthening effect.

C57BL/6 mice is divided into VR-MS/VR-IL2/Ad-MS, VR-MS/VR-IL2/MVA-MS, VR-MS/VR-IL2/Ad-MS and carboplatin synergy group (MS/IL2/Ad-MS/ carboplatin), carboplatin positive controls and matched group (PBS), 15 every group at random by table 7.At the 0th day, in right side of mice, carry on the back bottom subcutaneous vaccination MS _f ⁺b16 tumor cell, gave respectively gene vaccine and PBS at the 1st, 8,15 days, and administering mode is intramuscular injection; The administration time of carboplatin is the 1st, 6,11,16 days, and administering mode is lumbar injection.Since about the 10th day, observe the growth of tumor and big or small variation, other four groups of variations of simultaneously observing mouse survival rate.Tumour inhibiting rate group experiment mice starts death the 28th day left and right control group mice, puts to death whole mices, peels off tumor, measures tumor weight, calculates tumour inhibiting rate; Organize experiment mice life cycle and observe the existence situation of mice, until mice is all dead, the time is about 50 days.

Table 7MS vaccine is to the therapeutic of model mice tumor (tumour inhibiting rate experiment)

(note: the administration time of the carboplatin in table 7 is the 1st, 6,11,16 days)

2.2.2.1 the inhibitory action of tumor growth

Figure 13 A tests and respectively organizes gross tumor volume situation of change for treatment.With the comparison of PBS matched group, VR-MS/VR-IL2/Ad-MS, VR-MS/VR-IL2/MVA-MS, carboplatin and VR-MS/VR-IL2/Ad-MS/ carboplatin group all can obviously suppress the growth (after inoculated tumour 21 to 27 days, be p<0.05) of tumor; VR-MS/VR-IL2/Ad-MS and carboplatin use in conjunction group and independent group comparison of applying the two, tumor growth rate slightly slows down, but there is no statistical significance, and the two use in conjunction fails to play collaborative or cumulative effect; VR-MS/VR-IL2/Ad-MS group does not relatively have significant difference with VR-MS/VR-IL2/MVA-MS group.

2.2.2.2 the existence situation of mice

PBS group is 31.21 days mean survival time (MST), to after inoculated tumour cell 38 days time mice all dead, VR-MS/VR-IL2/MVA-MS and VR-MS/VR-IL2/Ad-MS group the average survival time are respectively 34.21 and 35.28 days, relatively there is no significant difference with PBS group; Carboplatin group the average survival time natural law is 35.29, has 7.1% mouse survival during to the observation period, and increase in life span is respectively 13.07%, and with matched group comparison, mice life all obviously extends (p<0.05); VR-MS/VR-IL2/Ad-MS and carboplatin use in conjunction group the average survival time natural law are 38.36, during to the observation period, there is 28.6% mouse survival, increase in life span is 22.91%, with relatively all obviously prolongations (being p<0.01) of mice life of matched group, obviously extend (p<0.05) with the set of applications comparison mice life of the two single expression vaccine; VR-MS/VR-IL2/Ad-MS group is compared and be there is no notable difference with VR-MS/VR-IL2/MVA-MS group, as shown in Figure 13 B.

From above-mentioned curative test result, can find out, VR-MS/VR-IL2/Ad-MS, VR-MS/VR-IL2/MVA-MS be the growth of inhibition mouse tumor but effective life cycle of lengthening model mice obviously; VR-MS/VR-IL2/Ad-MS vaccine and carboplatin use in conjunction be the remarkable growth of inhibition mouse tumor effective life cycle of lengthening model mice not only; Vaccine is applied and is compared separately with the two with carboplatin combined immunization, although suppressing not embody obvious advantage aspect tumor growth, the life cycle of energy significant prolongation model mice.Traditional tumor vaccine evaluation criterion is tended to dwindling of gross tumor volume, but the clinical practice of oncotherapy proves some tumor vaccine, can effectively extend patient's life, improves the quality of living, but may not necessarily obviously reduce gross tumor volume.Therefore, we think that the protection that the independent application of carboplatin and vaccine use in conjunction and the two compares model mouse tumor is significantly improved.

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Claims (9)

1. the DNA fragmentation S8 fragment that a sequence is SEQ ID NO:8 is in the purposes for the preparation of carrying out in protein vaccine, DNA vaccination, vector-viral vaccine or the dendritic cell vaccine of antineoplastic immune.

2. a recombiant plasmid VR-S8 is in the purposes for the preparation of carrying out in the DNA vaccination of antineoplastic immune, the skeleton carrier of wherein said recombiant plasmid is the VR1012 as Figure 1A, and in the multiple clone site of described VR1012, inserting a kind of sequence is the DNA fragmentation S8 fragment of SEQ ID NO:8.

3. purposes claimed in claim 2, the S8 fragment wherein inserted is between the multiple clone site SalI and BamHI of described VR1012.

4. the purposes described in claim 2 or 3, in wherein said vaccine, also comprise recombinant adenovirus Ad-S8, and wherein with VR-S8, carry out just exempting from, with Ad-S8, carry out booster immunization, wherein said recombinant adenovirus Ad-S8 inserts the DNA fragmentation S8 fragment that a kind of sequence is SEQ ID NO:8 in adenovirus.

5. the purposes described in claim 4, the skeleton carrier of wherein said recombinant adenovirus is pBHGlox Δ E1,3Cre, the S8 fragment of inserting is between the multiple clone site EcoRI and BglII of described recombinant adenovirus.

6. the purposes described in any one in claim 2-5, also comprises immunological adjuvant in wherein said vaccine, and wherein said immunological adjuvant and described VR-S8 together give.

7. the purposes described in claim 6, wherein said immunological adjuvant is selected from cytokine interleukin element 2 (IL-2), cytokine grain-giant cell colony stimulating factor (GM-CSF) and non-methylated CpG motif immunostimulation DNA sequence.

8. the purposes described in any one in claim 2-7, also comprises chemotherapeutics in wherein said vaccine.

9. the purposes described in claim 8, wherein said chemotherapeutics is selected from carboplatin, cisplatin, oxaliplatin and paclitaxel.