CN1517437B - Vaccine for specificity treating tumour or endocellular infection and application - Google Patents
Vaccine for specificity treating tumour or endocellular infection and application Download PDFInfo
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Abstract
A recombinant virus carrier is disclosed. An expression box of heat shock-protein-antigenic peptide's fusion protein is inserted in the genome of said virus. Said expression box contains sequentially promoter sequence, the code sequence of heat shock protein-antigenic peptide's fusion protein, and termination codon. Said virus can selectively infect and crack the tumor cells or intracellar microbial infectious cells and excessively express the fusion protein of specific antigen, so effectively stimulating the immunoresponse reaction.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to a kind of recombinant viral vector, this expression vector is cells infected or tissue and do not infect healthy tissues or cell in infected tumor or the born of the same parents optionally, and effectively efficiently expressing specific antigen coalescence protein in killing tumor cells or the cells infected, thereby inducing the specific immune response of human body generation at tumour or cells infected.The construction process and the treatment that the invention still further relates to this expression vector are used.
Background technology
Still main at present dependence of the treatment of malignant tumour performed the operation, radiotherapy and chemotherapy, and the metabolic that these methods all can not solve tumour effectively shifts and recurrence.In addition, the treatment of infecting in the born of the same parents for hepatitis B etc. does not have the comparatively satisfied medicine of some curative effects now yet except that Interferon, rabbit etc.
Yet exist the tumour or the hepatitis B self-healing phenomenon of significant proportion in the actual life.This mainly is that human body produces due to the immunity of tumour or microbial infection.Therefore if can mediate the immune response of human body to tumour or microbial infection effectively, be the unique a kind of effective ways that can thoroughly totally kill cancer cells or remove microbial infection.Effectively the appearance of immune response and memory cell is the effective means of fundamentally preventing and treating recurrence, and the persistence of its effect also is radiotherapy, and chemotherapy or other drug treatment institute are not replaceable.
Attempt utilizes the treatment of viral infections tumour that nearly 50 years history is arranged, and the American National ICR utilized adenovirus treatment cancer in 1956, though effectively, have repeatedly serious.Concentrate at present morely and utilize adenovirus carrier to express with cytokine, suicide gene and P53 are the cancer suppressor gene of representative.This wherein the adenovirus treatment head and neck cancer tumour of utilizing of U.S. ONXY company obtain progress preferably.Therefore be considered at first only in the tumour cell of P53 inactivation, duplicate, killing tumor cells optionally.Yet duplicating, breeding of existing clear and definite statement of facts adenovirus is and P53 gene and unconnected.In addition, this virus belongs to replication defect type, so it does not possess the function of repeated infection tumour cell.
Adenovirus is a kind of security therapeutic viruses that can be applicable to the people, and wherein adenovirus 2 types and 5 type researchs is comparatively clear, is widely used for preventing respiratory tract infection and 4 types and 7 type adenovirus are made into coated tablet in septic yanks.
The infection of virus pair cell is to mediate by the fibril albumen (FIBER) on the coat protein of the acceptor of cell surface and virus.For example hsv is to utilize PROTEIN C to be incorporated into cell surface FGF acceptor.E in the Sindbis virus coat
2An amino acid whose replacement of-glycoprotein just can cause its host range to change.Hepatitis B virus is by annexin V (Annexin V) combination of PRES1 albumen and surface of hepatocytes.In addition, viral life cycle begin depend on also whether host cell has the required specific gene activating transcription factor of virus.
Adenovirus is the infection in conjunction with this virus of initiation by the CAR acceptor of fibril albumen and cell surface.The limitation that adenovirus is used for the treatment of is that the host range of adenovirus is more extensive, can infect normal cell and mutant without distinction.Along with molecular biological progress, people have carried out detailed research to the 26S Proteasome Structure and Function of the gene of virus, can effectively modify the structure gene of virus, and change its host range, make it can infect specific expressed certain antigenic tumour or infected cells specially, this has also solved the problem of many surface of tumor cells CAR expression of receptor disappearances.At present, developed the infection scope (W098/07877, United States Patent (USP) 5,559,099 etc.) that multiple different methods changes adenovirus.
Tumour can form and cause the reason that infects in the pathogenic micro-organism born of the same parents, is successfully to have escaped the immunosurveillance system of human body.The aim of immunotherapy is exactly to destroy such immune evasion, induces the effective immune response of human body to tumour or infected cell, just activates CTL cell and activation T-HELP cell.
The antigen that studies show that heat shock protein, superantigen, LLO equimolecular energy binding specificity in recent years participates in the submission antigen molecule, the anti-infective or anti tumor immune response of mediation human body.Especially in big quantity research, find that HSP plays an important role in antineoplastic immunity to HSP.
HSP is the class protein that various molecular weight are arranged, HSP90 protein family (HSP110 wherein, GP96, HSP90), HSP70 family derives from the HSP65 of mycobacterium tuberculosis var bovis (Mycobacterium Bovis) and the HSP70 of mycobacterium tuberculosis (Mycobacterium Tuberlosis) in addition and all can be used to do antigenic submission.In fact HSP itself immunogenicity not, but when its binding specificity antigen peptide, just can show immunogenicity, and be and pass T-cell.Antigenics company obtains the patent of the non-covalent binding peptide mixture of heat shock protein GP96 and utilizes it to treat the kidney patient, and three phase experiment effects are good, yet its inconvenience is that it is a kind of individualized vaccine.
Up to the present, still do not treat the target medicine that infects in tumour or the born of the same parents effectively.Therefore this area presses for exploitation effectively treatment tumour and the interior target medicine that infects of born of the same parents.
Summary of the invention
Purpose of the present invention just provides medicine and method for making and the purposes that infects in a kind of specific treatment tumour or the born of the same parents.
In a first aspect of the present invention, a kind of recombinant viral vector is provided, insert the expressing fusion protein box of heat shock protein(HSP)-antigenic peptide in this viral genome, described expression cassette comprises successively: the fusion rotein encoding sequence and the terminator codon of promoter sequence, heat shock protein(HSP)-antigenic peptide.
In a preference, described virus vector is selected from: adenovirus, adeno-associated virus (AAV), neofield eqpidemic disease poison, reovirus, Sindbis virus or poxvirus.
In another preference, described virus vector is an adenovirus, and has inserted described expression cassette in adenoviral gene group E3 zone position.
In another preference, the E3 district of described adenovirus is disappearance all, only keeps ADP albumen, thereby makes adenovirus keep the ability of lysing cell.
In another preference, also inserted the encoding sequence of neuregulin in the fibril protein-coding region of described adenovirus, thereby realized specific infection.
In another preference, the E1 district early promoter of described adenovirus is replaced by the ErbB2 promotor, thereby adenovirus is bred in tumour cell specifically.
In another preference, described heat shock protein(HSP) is selected from: Hsp70, Hsp65, Hsp110 or gp96.
In another preference, described antigenic peptide is tumour antigen or pathogen antigen.More preferably, described antigenic peptide is selected from: surface antigen, HBV cAg or its combination of the E6 epi-position of the Th cell epitope structural domain of the t cell epitope of hepatitis B virus core antigen t cell epitope, ErbB2, ErbB3, CEA epi-position, PSA epi-position, WT epi-position, HPV, the E7 epi-position of HPV, HBV.
In another preference, the expressing fusion protein box of described heat shock protein(HSP)-antigenic peptide coding has the fusion rotein of aminoacid sequence shown in the SEQ ID NO:1,2 or 3.
In a second aspect of the present invention, a kind of pharmaceutical composition is provided, it comprises the recombinant viral vector and the pharmaceutically acceptable vehicle of fusion rotein of the expression heat shock protein(HSP)-antigenic peptide of the present invention of significant quantity.
In a third aspect of the present invention, a kind of fusion rotein of heat shock protein(HSP)-antigenic peptide is provided, wherein, described heat shock protein(HSP) is selected from: Hsp70, Hsp65, Hsp110 or gp96, and described antigenic peptide is selected from: the Th cell epitope structural domain of the t cell epitope of hepatitis B virus core antigen t cell epitope, ErbB2, ErbB3 or its combination.
In another preference, described fusion rotein has aminoacid sequence shown in the SEQ ID NO:1,2 or 3.
Description of drawings
Fig. 1 has shown the gene structure figure of a kind of recombinant viral vector of the present invention.
Fig. 2 has shown fusion rotein of the present invention and the recombinant viral vector restraining effect to tumour.
Embodiment
The inventor is extensive studies through going deep into, and finds that antigenic peptide and heat shock protein(HSP) merge formed fusion rotein, and submission antigen induces human body to overcome immune evasion effectively, thereby produces the T-cell immune response.The recombinant viral vector that contains described fusion rotein encoding sequence not only can excite individual immune response effectively, and target ground infects specific cells and propagation therein, thereby treats infection in tumour and the born of the same parents effectively.Finished the present invention on this basis.
The fusion rotein of heat shock protein(HSP)-antigenic peptide
In the present invention, term " fusion rotein of heat shock protein(HSP)-antigenic peptide " refers to merge the fusion rotein that forms by heat shock protein(HSP) and antigenic peptide.The position of heat shock protein(HSP) and antigenic peptide is not particularly limited in the fusion rotein, can be heat shock protein(HSP)-antigenic peptide, or antigenic peptide-heat shock protein(HSP).In addition, in fusion rotein, can contain one or more antigenic peptides.For example, contain 1-5 identical or different antigenic peptide.
Can be used for antigenic peptide of the present invention and comprise tumour antigen and pathogen antigen.Tumour antigen or pathogen antigen are meant the specific antigens that is expressed in tumour cell or infected cell surface, as ErbB2, ErbB3, CEA, PSA, HPV E6, HP VE7, WT, HBV face antigen etc., it be also included within the virus replication or surface receptor molecular folding process in come across specific antigens cell surface, that can mediate the T-cell response, as ErbB2 ICD, hepatitis B virus core antigen etc.
Antigenic peptide can be the antigen of total length, also can be an antigenic part, for example can cause the epi-position of immunne response.In addition, antigenic peptide can also be the fusion rotein of a plurality of epi-positions, as comprises the part of T-cell antigen epitope or the fusion form of a plurality of T-cell antigen epitopes.
Can be used for heat shock protein(HSP) of the present invention and be not particularly limited, can be any heat shock protein(HSP).Representational example comprises (but being not limited to): HSP90 protein family (HSP110, GP96, HSP90), HSP70 family, the HSP65 of mycobacterium tuberculosis var bovis (Mycobacterium Bovis) and mycobacterium tuberculosis (Mycobacterium Tuberlosis) HSP70.Preferred heat shock protein(HSP) is the heat shock protein(HSP) of people or mycobacterium.
Can be used for heat shock protein(HSP) of the present invention can be total length, also can be its function fragment, for example contains the fragment of 161-361 amino acids among people or the mycobacterium tuberculosis Hsp70.
Except Hsp was the albumen of preferred induce immune response, other can submission antigens and/or induce human body to overcome immune evasion, thereby produced or promote the material of T-cell or Th cell immune response, also can be used for the present invention.Representational example comprises PADRE carrier, tetanus element (TT), Measles virus fusion rotein (MVF), malaria peptide of (but being not limited to): LLO, synthetic etc.
The expressing fusion protein box of heat shock protein(HSP)-antigenic peptide
In the present invention, term " the fusion rotein encoding sequence of the heat shock protein(HSP)-antigenic peptide " encoding sequence of fusion rotein of heat shock protein(HSP)-antigenic peptide that refers to encode.Encoding sequence can be DNA or RNA.
In the present invention, term " the expressing fusion protein box of heat shock protein(HSP)-antigenic peptide " refers to contain the fusion rotein encoding sequence of heat shock protein(HSP)-antigenic peptide and the module of expressing required element, expresses required assembly and comprises promotor and terminator codon.In addition, the expressing fusion protein box of heat shock protein(HSP)-antigenic peptide can also contain other regulating and controlling sequences, comprising but be not limited to: enhanser, secretion signal peptide sequence, intron, internal ribosome entry site (IRES), polyadenylation signal sequence etc.
In the present invention, the promotor that goes for the expressing fusion protein box of heat shock protein(HSP)-antigenic peptide can be any common promotor, and it can be constitutive promoter or inducible promoter.Preferably, this promotor is the strong promoter of composing type, for example CMV promotor etc. other be applicable to the promotor of eukaryotic expression.
Recombinant viral vector
In the present invention, the expressing fusion protein box of heat shock protein(HSP)-antigenic peptide is inserted with routine techniques in the genome of various virus vector, just be built into recombinant viral vector of the present invention.
Can be used for virus vector of the present invention and be not particularly limited, representational example comprises (but being not limited to): adenovirus, adeno-associated virus (AAV), neofield eqpidemic disease poison, reovirus, Sindbis virus or poxvirus.
A kind of preferred virus vector is an adenovirus.In the present invention, available recombinant adenovirus can be the adenovirus of any serotype, in addition, and the adenovirus type of should employing having studied comparatively thoroughly, for example Ad2 and Ad5 type adenovirus.
Adenovirus is a kind of nonencapsulated dna virus, and virion is icosahedron, and diameter is 80-90nm.Adenovirus major has three kinds of structural protein: six adjacent bodies, the surface of 20 bodies of formation; Penton is arranged in 12 drift angles of protein enclosure; Fibril albumen, a fiber initiation on each penton.
The adenoviral gene group is the linear double-stranded DNA of duplex, and 36000 base pairs (bp) are arranged approximately.Adenovirus hominis has nearly 50 kinds of serotypes, and wherein the research of Ad2 and Ad5 genome structure and genetic expression is comparatively extensive, now determines the complete nucleotide sequence of this amphitypy adenovirus, and the adenovirus carrier that makes up comes from this two kinds of serotypes mostly at present.
E1 district in the adenoviral gene group promptly is activated in viral genome one enters host cell nuclear, thereby the coding initiation factor regulate the early ambulant of virus, so the E1 district is essential by virus replication.
E2 district encoded protein is relevant with duplicating of viral DNA, comprises viral DNA end conjugated protein (TP), DNA conjugated protein (DBP) and archaeal dna polymerase, and DBP is relevant with transcriptional control.
E3 district encoded polypeptides is relevant with the immune response of virus escape host anti-virus.E3 district disappearance does not have obvious influence to the infectivity of adenovirus.
E4 district coded product participates in viral dna replication, district's genetic expression in evening, viral protein is synthetic and host protein synthesizes activities such as termination.Main late gene product is a virus structural protein.
In a single day recombinant viral vector of the present invention contains described expression cassette, and just submission antigen effectively induces human body to overcome immune evasion, thereby produces the purpose of T-cell immune response.In addition, can also further improve for recombinant viral vector of the present invention, so as to make that virus can the target sexuality be dyed, proliferated specifically and keep advantage such as cracking specific cells.
In order to realize that the target sexuality dyes, can in the genome of recombinant viral vector of the present invention, comprise the protein-bonded encoding sequence of target, the conjugated protein surface specific antigen that can be incorporated into tumour cell or infected cell specifically of this target.Representational target is conjugated protein to comprise (but being not limited to): the single-chain antibody of specific antigens, single domain antibody (single-domain antibody), hypervariable region polypeptide (hypervariable region polypeptide) is (as anti-HER2, anti-HER3, anti-HBV surface antigen etc.) and the native ligand (as EGF, neuregulin etc.) of acceptor.For example, for adenovirus, just can insert 5 ' end, one side of the proteic coding region of adenovirus fibril by target is conjugated protein (as neuregulin) encoding sequence, or the proteic a part of coding region of displacement fibril.
In order to realize proliferated specifically, can be used in the specific cells effectively initial promotor (as the ErbB2 promotor of in tumour cell, effectively transcribing) and replace the early promoter of virus.In a preference of the present invention, the E1 district early promoter of adenovirus is replaced with the ErbB2 promotor, thereby adenovirus is bred in tumour cell specifically.
In order to keep the cracking specific cells, can keep virus for the required albumen of lysing cell (as the ADP albumen of adenovirus) coding region.In a preference of the present invention, with the whole disappearances in the E3 district of adenovirus, only keep ADP albumen, thereby make adenovirus keep the ability of lysing cell, strengthen result of treatment.
Pharmaceutical composition
After making the fusion rotein recombinant viral vector of heat shock protein(HSP)-antigenic peptide of the present invention, just can with the recombinant viral vector of required purity and optional physiologically acceptable carrier, vehicle or stablizer (Remington ' s Pharmaceutical Sciences, 16 editions, Osol, A., Ed., [1980]), mix with lyophilized form or aqueous solution form, thereby make the pharmaceutical composition that contains recombinant viral vector of the present invention.Pharmaceutical composition of the present invention mainly is a therapeutic composition.
Acceptable carrier, vehicle or stablizer should be nontoxic under used dosage and concentration for the recipient, and can contain damping fluid such as phosphoric acid salt, Citrate trianion or other organic acid damping fluids; Antioxidant such as vitamins C; Various additives such as sequestrant such as EDTA.As modal example, water and physiological saline just can be used as the carrier in the recombinant viral vector preparation of the present invention.
The pharmaceutical composition (preparation) that contains novel recombinant viral vector of the present invention can be applied to the individuality of needs treatment with usual manner, and these modes comprise (but being not limited to): intratumor injection, intramuscularly, subcutaneous injection, intravenous injection etc.For topical, a kind of preferably mode is directly preparation of the present invention to be expelled to around focus or its, for example in the tumor mass or near it.Dosage is 10
6～10
12The PFU/ individuality, preferred 10
8～10
10The PFU/ individuality, accurate dose and mode are done concrete decision by the doctor according to patient's healthy state and overall immune level.
In addition, except adopting virus, also can be with expression cassette of the present invention at BCG, salmonella typhi is expressed in Listeria (Listeria) and the bacillus pumilis, by methods such as mucosal immunities fusion rotein is applied to individuality.Perhaps, with in the virus infection, inject fusion rotein of the present invention or dna molecular in the body.
In treatment, also can be respectively with expressing virus identical fusion rotein of the present invention, different genera or different serotypes, induce and immune strengthening, thus the immune response of avoiding virus vector to bring, or avoid because the immunological memory influence treatment that patient's early infection forms.
The recombinant viral vector of the present invention's preparation has huge using value and advantage, is mainly reflected in following several respects:
(1) excites individual immune response effectively, infect thereby treat effectively in tumour and the born of the same parents;
(2) target ground infected tumor cell or infected cell;
(3) in tumour cell or infected cell, breed specifically;
(4) utilize virus vector to realize intracellular immunity, be beneficial to immunotherapy.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Structure the ErbB2 acceptor gene is crossed expression the tumour cell selective proliferative adenovirus since in tumour the ErbB2 acceptor gene cross expression, so ErbB2 promotor effective initial transcribing in tumour cell.In the present embodiment, replace the early promoter in adenovirus E 1 district with the ErbB2 promotor, thus make up can be in tumour cell the adenovirus (referring to Fig. 1, the left side, below) of selective proliferative.Method is as follows:
Plasmid PXCI and PBHG10 available from Microbix Biosystems (Toronto, Canada), plasmid PXCI gland-containing virus Ad5 bp22-5790 fragment wherein.PBHG10 contains except adenovirus E
1District (bp188-1339) and E
3The full gene group of adenovirus outside the district (bp28133-30818).
Utilize PCR method, gene 551 and T of 552 interdigits increase at plasmid PXCI create an AgeI site, and plasmid is numbered PXCI-T.
Synthetic following primer:
5’ccgtgacaaggtaaacacaacacatcc 3’(SEQ ID NO：4)
5’ttaccggtcccaatggaggggaat 3’(SEQ ID NO：5)
With extractive tire liver gene group DNA is template, carries out pcr amplification, obtains the about 544bp of PCR product (promotor of ErbB2 acceptor gene).This PCR product is behind sspBI and AgeI enzymolysis, and between the sspBI and AgeI site of the PXCI-T plasmid that the insertion same enzyme is cut, the gained plasmid is called PXCI-ErbB
2P.
PXCI-ErbB
2P and PBHG10 cotransfection 293 (or 911) cell, plaque select, PCR identifies and confirms to obtain recombinant adenovirus ad by recombinant virus then
5ErB
2P.
Embodiment 2
Structure has cracking tumour cell function and specificity is induced at ErbB
2Acceptor is crossed the recombinant adenovirus of the t cell immune response of expressing tumor cell
In the present embodiment, the full gene in leaving out the E3 district of adenoviral gene group only keeps the ADP coding region, thereby makes up the adenovirus of cracking tumour cell function.Expression cassette with heat shock protein(HSP)-antigenic peptide inserts adenovirus then, and structure obtains specificity and induces the adenovirus (referring to Fig. 1, in the middle of the below) of crossing the t cell immune response of expressing tumor cell at the ErbB2 acceptor.
2.1 make up the plasmid that the E3 district only keeps the ADP coding region
Plasmid PBHG
11Available from Microbix Biosystems (Toronto, Canada).
Also use the partially digested plasmid PBHG of BamHI with the XbaI complete degestion
11, separate the 14kb fragment, mend with the Klenow enzyme then and put down, connect with ligase enzyme, form plasmid PBHG
64The PBHG that obtains
64Contain from 21562 to 35935 in Nucleotide, remove E
3Ad outside the district (28133-30818bp)
5The full gene of adenovirus.
Synthetic following primer:
5’aaccatggcccgggcagggtataac 3’(SEQ ID NO：6)
5’
atcgatcagtcgtagccatccgc 3’(SEQ ID NO：7)
With plasmid PBHG
11DNA is a template, and pcr amplification obtains the dna fragmentation (adenovirus E3 district early promoter) of about 290bp.The PCR product inserts the NcoI/EcoRv site of plasmid pGEM-5Z (Promega company) after the NcoI enzyme is cut, obtain carrier pGEM5P.
Synthetic following primer:
5’aa
atcgatgaccaacacaaccaacg 3’(SEQ ID NO：8)
5’aa
ctcgaggaatcatgtc 3’(SEQ ID NO：9)
With adenovirus ad5 (wild-type) genomic dna is template, and pcr amplification obtains about 310bp amplified production (adenovirus ADP fragment).The PCR product inserts the ClaI/EcoRv site of carrier pGEM5P through the ClaI enzymolysis, obtains plasmid pGEM5adp.
Synthetic following primer:
5’ctcgagttaattaatttaaatgtccagtttattcagcagc 3’(SEQ ID NO：10)
5’tccgtgtcatatggatacac 3’(SEQ ID NO：11)
With adenovirus Ad5 (wild-type) genomic dna is template, and pcr amplification obtains about 220bp amplified production (being the required downstream sequence of adenovirus E3 district's genetic expression).After the PCR product is cut with XhoI and NdeI enzyme, insert the pGEM5adp that same enzyme is cut, obtain plasmid pGEM5adpSN.
With SrfI and NdeI, purpose fragment under the double digestion is inserted into PBHG then from plasmid pGEM5adpSN
64SrfI and the NdeI site between, obtain plasmid PBHGadpSN.
2.2 make up the plasmid that contains expression cassette
Utilize the commodity plasmid pCI-vector of Promega company, connect manual splice gatcttaattaa (SEQ ID NO:12) and gatcatttaaat (SEQ ID NO:13) respectively in BgIII site, BamHI site, obtain plasmid pCIPS.
Synthetic following primer:
AacttaagAtgaagatctttgggagcctggcatttctggctcgtgcggtcgggatcgac (SEQID NO:14) (fusion has the Hsp70 upstream primer of ErbB2369T cell antigen, contains the Bst98 restriction enzyme site)
Aa
GcggccgcTcatcagccgagccggggtg (SEQ ID NO:15) (containing the NotI restriction enzyme site)
Use ordinary method, extracting mycobacterium tuberculosis H
37RV (ATCC36801) genomic dna is as template.Carry out PCR with above-mentioned primer, amplification obtains PCR product (i.e. the encoding sequence of coding " the t cell epitope 369+ mycobacterium tuberculosis Hsp70 of ErbB2 ").
Amplified production inserts Bst98I and the NotI site of plasmid pCIPS through Bst98 and NotI enzymolysis, confirms through order-checking, obtains plasmid pCIPSEr
2H369.
With plasmid pCIPSEr
2H369 inserts between the PacI and SwaI site of PBHGadPSN through the fragment of PacI/SwaI enzymolysis gained, obtains plasmid PBIadeh369.
2.3 structure recombinant adenovirus
Separate the recombinant virus ad among the embodiment 1
5ErB
2P through the SpeI enzymolysis, isolates left arm.With this left arm and plasmid PBIadeh369 cotransfection 293 cells.The plaque select recombinant adenovirus, and PCR identifies the adenovirus called after ad of acquisition
5E
2Peh369.
Embodiment 3
Structure has the cracking tumour and can induce the recombinant adenovirus of crossing expressing tumor cell Th-cell immune response at the ErbB3 acceptor
In the present embodiment, with the method that is similar to embodiment 2, make up the recombinant adenovirus that contains another fusion rotein (being the fusion rotein of mycobacterium tuberculosis var bovis Hsp65 and people ErbB3 incomplete antigen) expression cassette.This adenovirus energy cracking tumour cell, and can induce at ErbB
3Acceptor is crossed expressing tumor cell Th-cell immune response.Method is as follows:
3.1 make up the synthetic following primer of the plasmid of the fusion gene that contains mycobacterium tuberculosis var bovis Hsp65 and people ErbB3 incomplete antigen:
5’cccttaagatggactgcgtggcagagggcaaagt(SEQ ID NO：16)
5’ggggagcctcgagaatttgcccatatggccaagacaattgcgt (SEQ ID NO：17)
5’aagcggccgctcagaaatcatccatgccaccca(SEQ ID NO：18)
With the mycobacterium tuberculosis var bovis genomic dna of ordinary method extraction and people's tire liver cDNA of ordinary method preparation is template, carries out overlapping PCR with above-mentioned 3 primers, and amplification obtains coding ErbB
3The fusion gene of the heat shock protein 65 of part antigen and prapes mycomycete.
Amplified production after order-checking is confirmed, is cut through Bst98 and NotI enzyme, inserted Bst98 and the NotI site of plasmid pCIPS (embodiment 2), obtain plasmid pCIPSer
3D
4H.
Use PacI and SwaI enzymolysis pCIPSer then
3D
4H separates PacI and SwaI site that recombinant fragment inserts PBHGadpSN, gained plasmid PBTade
3D
4H.
3.2 structure recombinant adenovirus
Separate recombinant adenovirus ad among the embodiment 1
5ErB
2P through the speI enzymolysis, separates left arm.With this left arm and PBTade
3D
4H cotransfection 293 cells.The plaque select recombinant adenovirus, and through the PCR evaluation, the recombinant adenovirus of acquisition is ad
5E
2Pe
3D
4H.
Embodiment 4
Make up the recombinant adenovirus of specific infection ErbB2 high expression tumour cell
In the present embodiment, the encoding sequence of neuregulin is inserted the fibril protein-coding region of adenovirus, thereby make up the recombinant adenovirus (referring to Fig. 1, the right side, below) that obtains specific infection ErbB2 high expression tumour cell.Method is as follows:
4.1 make up the plasmid that contains the proteic fusion gene of neuregulin-fibril
Cut PBHG with XbaI and PacI enzyme
10(Microbix Biosystems) separates small segment, mends flat end with the Klenow enzyme, and the T4DNA ligase enzyme connects, and obtains plasmid pXT1.The full gene of plasmid pXT1 gland-containing virus terminal (30819-35935).
Synthetic with following primer:
5’gtgtatccatatgacacggaa 3’(SEQ ID NO：19)
5’ctgtaacactaaccattacactaagccatcttgtaaaatgt 3’(SEQ ID NO：20)
5’caaaactacgtaatggccagcactccaagtgcatactctatg 3’(SEQ ID NO：21)
5’tattcctgcaggacggagcgg3’(SEQ ID NO：22)
With above-mentioned 4 primers, be template with people's tire liver cDNA and adenovirus genomic dna simultaneously, by dual repeatedly PCR, amplification obtains the proteic fusion gene of neuregulin-fibril.The fusion rotein of this fusion gene coding fibril albumen and neuregulin (neuregulin) binding domains.
Amplified production is after order-checking is confirmed, the pXT1 plasmid that the insertion same enzyme is cut behind the NdeI/SfbI enzymolysis obtains plasmid pXT1NG to replace former NdeI/SfbI fragment.
4.2 structure recombinant adenovirus
Adenovirus ad with preparation among the embodiment 2
5E
2Peh369 separates big fragment as left arm through partly hydrolysis of NdeI.With 293 cells that this left arm and pXT1NG cotransfection had transformed with ErbB2, plaque select recombinant adenoviral vector.After PCR identifies, obtain adenovirus WYad
5EB
2369.
Use same program, with the adenovirus ad of preparation among the embodiment 3
5E
2Pe
3D
4H separates big fragment as left arm through partly hydrolysis of NdeI.With this left arm and pXT1NG cotransfection ErbB
2293 cells of gene transformation, the plaque select recombinant adenoviral vector.After PCR identifies, obtain adenovirus WYad
5E
3D
4
Embodiment 5
Structure has liver cell specifically expressing beta-interferon and can induce at the active recombinant adenovirus of hepatitis B virus T-cellular immunization
In the present embodiment, the expression cassette with fusion rotein (mycobacterium tuberculosis var bovis Hsp65+ hepatitis B antigen) inserts the recombinant adenovirus genomic dna.For the enhancing immunity result of treatment, also the beta-interferon expression cassette is inserted adenovirus DNA, thereby obtain to have liver cell specifically expressing beta-interferon and can induce at the active recombinant adenovirus of hepatitis B virus T-cellular immunization.
5.1 make up the plasmid that inserts beta-interferon expression cassette and expressing fusion protein box
Synthetic following joint: tatcga (SEQ ID NO:23) and ttaatttaaa (SEQ ID NO:24) insert carrier PIRES respectively
2AseI and the AflII site of-EGFP (Clontech company) obtain carrier PIRES
2-EGFPCS.
Synthetic following primer
Ttgctagcatga ccaacaagtgtct (containing NheI but point) (SEQ ID NO:25)
Aaggatcctcagtttcggaggtaacctgta (containing the BamHI site) (SEQ ID NO:26)
CDNA is a template with people's tire liver, pcr amplification human beta interferon gene.
Synthetic following primer
Ttccatggcaacacttccggaaactactgttgttagacgagccaagacaattgcgt acgac (fusion has the Hsp65 upstream primer of hepatitis B core T-epitope, contains the NcoI restriction enzyme site) (SEQ ID NO:27)
Aagcggccgctcagaaatccatgccacccat (containing the NotI restriction enzyme site) (SEQ ID NO:28)
Using above-mentioned primer, is template with the genomic dna of mycobacterium tuberculosis var bovis, obtains fusion gene by pcr amplification.The fusion rotein of this fusion gene coding hepatitis B antigen and Hsp65.
Above-mentioned two amplified productions after order-checking is confirmed, are inserted PIRES respectively
2The NheI/BamHI of-EGFPCS and NcoI/NotI site get plasmid PIRESHBCH.
With ClaI and SwaI digested plasmid PIRESHBCH, the former ClaI-SwaI fragment with isolating purpose fragment displacement plasmid pBHGadpSN obtains plasmid PBTadIBHc (this plasmid is used to produce the recombinant adenovirus that tool does not have cytolytic function).
Simultaneously, plasmid PIRESHBCH cuts through PacI and SwaI enzyme, and the former PacI-SwaI fragment with isolating purpose fragment displacement plasmid pBHGadpSN obtains plasmid PBTadIBHcPS (this plasmid is used to produce the recombinant adenovirus with cytolytic function).
5.2 make up with hepatitis B virus promoter replacement E1 district early promoter plasmid
Synthetic following primer:
5’gcgccggcgcacctctctttacgc 3’(SEQ ID NO：29)
5 ' ca
GgtaccCcccgctcagatgggtttcttcgctc
CatGccccaaagccaccc 3 ' (containing the KpnI restriction enzyme site) (SEQ ID NO:30)
With hepatitis B gene group DNA is template, and pcr amplification obtains the fragment of the HBV core promoter about 400bp in district.The PCR product inserts plasmid PXCI behind SgrAI and KpnI enzymolysis, to replace the fragment of former ad5 DNA 187-2049 position, obtain plasmid and be called PXC
△E1ACpE
1B.
5.3 structure recombinant adenovirus
With plasmid PXC
△E1ACp
E1B and PBTadIBHc cotransfection 293 cells, plaque select, PCR identifies and confirms to obtain recombinant adenovirus WYad by recombinant virus then
5HBc.This adenovirus is proliferated specifically in infecting the hepatitis B virus cell.
With plasmid PXC
△E1ACpE
1B and PBTadIBHcPS cotransfection 293 cells, plaque select, PCR identifies and confirms to obtain recombinant adenovirus WYad by recombinant virus then
5HBcPS.This adenovirus is proliferated specifically in infecting the hepatitis B virus cell, and the liver cell of hepatitis B virus is infected in cracking simultaneously.
Embodiment 6
The separation and purification fusion rotein
In the present embodiment, the transient expression purpose fusion rotein in Chinese hamster ovary celI, separation and purification then.Method is as follows:
Distinguish separation and purification plasmid PCIPSEr according to a conventional method
2(embodiment 2, encoding fusion protein ErbB for h369
2369-Hsp70), PCIPSer
3D
4(embodiment 3, encoding fusion protein " ErbB for h
3D4-Hsp65 "), PIRESHBCH (embodiment 5, encoding fusion protein HBC141Hsp65).
With the plasmid of purifying, (Gibco company) transforms the COS7 cell respectively by liposome method.Cultivate after 48 hours, separating and cracking COS7 cell is got lysate 20 μ l and transfer to pvdf membrane behind the SDS-page electrophoresis, through 5% skim-milk, and 37 ℃ of sealings.Add anti--HSP65 antibody (CalBiochem company) or anti--HSP70 antibody (Antigenics company), 4 ℃ are spent the night, and add two anti-and luminous agents next day, and compressing tablet is confirmed fusion rotein.Through scanning and determination of protein concentration, the about 300-400ng/ml of Expression of Fusion Protein.Simultaneously, utilize FPLC method purifying, obtain following 3 kinds of fusion roteins.
Table 1 Expression of Fusion Protein amount
| Fusion rotein | SEQID NO： | Expression amount |
| ErbB 2 369-Hsp70 | 1 | 332ng/ml |
| HBC141Hsp65 | 2 | 300ng/ml |
| ErbB 3D4-Hsp65 | 3 | 380ng/ml |
Embodiment 7
Test immunotherapy effect in the animal model
Get the first-generation mouse of the male mouse mating of female mouse of FVB/N and 57BL/6, in-situ inoculating 10
6Individual mouse mastopathy cell.
Tumor-bearing mice is divided into six groups, 8 every group.Press shown in the table 2, inject the mixture of BSA (negative control), Ag and Hsp respectively, Zorubicin (positive control).Test group injection low dosage Ag-Hsp fusion rotein, high dosage Ag-Hsp fusion rotein, and Ag-Hsp fusion rotein and adDNA mixture.
During inoculation, per two days once, injects continuously 14 times, dissects mouse after 30 days and claim knurl heavy.Table 2-3 is listed in gained result and analysis.
Table 2 medicine is to the restraining effect of tumour
Annotate: Ag is artificial synthetic ErbB
2The mol ratio of-369 peptides (sequence is KIFGSLAFL SEQ ID NO:33) and ErbB3D4 peptide (sequence is DCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAH, SEQ ID NO:32) is 1: 1 mixture.
Ag-Hsp is ErbB
2369-Hsp70 (SEQ ID NO:1) and ErbB
3The mixture of D4-Hsp65 (SEQ ID NO:3).
AdDNA is the adenovirus ad of embodiment 2 preparations
5E
2The DNA of Peh369.
Table 3 medicine is to the restraining effect of tumour
| Sample | Dosage regimen | Number of animals | The weight of animals beginning/end | The tumour body weight | Relative tumor proliferation rate |
| Negative control BSA 0.5mg/kg | Subcutaneous | 8 | 21.3/31.6 | 4.45±0.72 | 100％ |
| Ag+hsp thing contrast 0.6mg/kg | Subcutaneous | 8 | 21.5/30.7 | 4.35±0.69 | 98％ |
| Ag-Hsp 0.6mg/kg | Subcutaneous | 8 | 21.6/28.3 | 1.95±0.41 | 43.85％ |
| Ag-Hsp 1.2mg/kg | Subcutaneous | 8 | 21.7/29.1 | 1.71±0.34 | 38.43％ |
| Zorubicin 2mg/kg | The abdominal cavity | 8 | 21.6/28.3 | 1.33±0.31 | 29.89％ |
| Ag-Hsp 1.2mg+adDNA | In the subcutaneous tumors | 8 | 21.4/27.9 | 0.71±0.23 | 15.96％ |
Embodiment 8
Recombinant adenovirus is to hepatitis B infected therapeutic action
Recombinant adenovirus WYad with preparation among the embodiment 5 of purifying
5The DNA of HBc is injected directly in the 6-8 BALB/c mouse quadriceps muscle of thigh in age in week.Every mouse is with 100ug DNA (concentration is 100ug/100ul) immunity, every three all immunity once, and totally three times.Separating spleen cell is then cultivated and is stimulated with hepatitis B virus core antigen.
As a result, tangible T cell proliferation occurred, and the target cell P815 of HBc gene transfection has been had tangible lethal effect (67.4% ± 15.1%).
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉open, wise
＜120〉vaccine and the application of infecting in specific treatment tumour or the born of the same parents
<130>027469
<160>32
<170>PatentIn version 3.1
<210>1
<211>560
<212>PRT
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<400>1
Met Lys Ile Phe Gly Ser Leu Ala Phe Leu Ala Arg Ala Val Gly Ile
1 5 10 15
Asp Leu Gly Thr Thr Asn Ser Val Val Ser Val Leu Glu Gly Gly Asp
20 25 30
Pro Val Val Val Ala Asn Ser Glu Gly Ser Arg Thr Thr Pro Ser Ile
35 40 45
Val Ala Phe Ala Arg Asn Gly Glu Val Leu Val Gly Gln Pro Ala Lys
50 55 60
Asn Gln Ala Val Thr Asn Val Asp Arg Thr Val Arg Ser Val Lys Arg
65 70 75 80
His Met Gly Ser Asp Trp Ser Ile Glu Ile Asp Gly Lys Lys Tyr Thr
85 90 95
Ala Pro Glu Ile Ser Ala Arg Ile Leu Met Lys Leu Lys Arg Asp Ala
100 105 110
Glu Ala Tyr Leu Gly Glu Asp Ile Thr Asp Ala Val Ile Thr Thr Pro
115 120 125
Ala Tyr Phe Asn Asp Ala Gln Arg Gln Ala Thr Lys Asp Ala Gly Gln
130 135 140
Ile Ala Gly Leu Asn Val Leu Arg Ile Val Asn Glu Pro Thr Ala Ala
145 150 155 160
Ala Pro Gly Tyr Gly Leu Asp Lys Gly Glu Lys Glu Gln Arg Ile Leu
165 170 175
Val Phe Asp Leu Gly Gly Gly Thr Phe Asp Val Ser Leu Leu Glu Ile
180 185 190
Gly Glu Gly Val Val Glu Val Arg Ala Thr Ser Gly Asp Asn His Leu
l95 200 205
Gly Gly Asp Asp Trp Asp Gln Arg Val Val Asp Trp Leu Val Asp Lys
210 215 220
Phe Lys Gly Thr Ser Gly Ile Asp Leu Thr Lys Asp Lys Met Ala Met
225 230 235 240
Gln Arg Leu Arg Glu Ala Ala Glu Lys Ala Lys Ile Glu Leu Ser Ser
245 250 255
Ser Gln Ser Thr Ser Ile Asn Leu Pro Tyr Ile Thr Val Asp Ala Asp
260 265 270
Lys Asn Pro Leu Phe Leu Asp Glu Gln Leu Thr Arg Ala Glu Phe Gln
275 280 285
Arg Ile Thr Gln Asp Leu Leu Asp Arg Thr Arg Lys Pro Phe Gln Ser
290 295 300
Val Ile Ala Asp Thr Gly Ile Ser Val Ser Glu Ile Asp His Val Val
305 310 315 320
Leu Val Gly Gly Ser Thr Arg Met Pro Ala Val Thr Asp Leu Val Lys
325 330 335
Glu Leu Thr Gly Gly Lys Glu Pro Asn Lys Gly Val Asn Pro Asp Glu
340 345 350
Val Val Ala Val Gly Ala Ala Leu Gln Ala Gly Val Leu Lys Gly Glu
355 360 365
Val Lys Asp Val Leu Leu Leu Asp Val Thr Pro Leu Ser Leu Gly Ile
370 375 380
Glu Thr Lys Gly Gly Val Met Thr Arg Leu Ile Glu Arg Asn Thr Thr
385 390 395 400
Ile Pro Thr Lys Arg Ser Glu Thr Phe Thr Thr Ala Asp Asp Asn Gln
405 410 415
Pro Ser Val Gln Ile Gln Val Tyr Gln Gly Glu Arg Glu Ile Ala Ala
420 425 430
His Asn Lys Leu Leu Gly Ser Phe Glu Leu Thr Gly Ile Pro Pro Ala
435 440 445
Pro Arg Gly Ile Pro Gln Ile Glu Val Thr Phe Asp Ile Asp Ala Asn
450 455 460
Gly Ile Val His Val Thr Ala Lys Asp Lys Gly Thr Gly Lys Glu Asn
465 470 475 480
Thr Ile Arg Ile Gln Glu Gly Ser Gly Leu Ser Lys Glu Asp Ile Asp
485 490 495
Arg Met Ile Lys Asp Ala Glu Ala His Ala Glu Glu Asp Arg Lys Arg
500 505 510
Arg Glu Glu Ala Asp Val Arg Asn Gln Ala Glu Thr Leu Val Tyr Gln
515 520 525
Thr Glu Lys Phe Val Lys Glu Gln Arg Glu Ala Glu Gly Gly Ser Lys
530 535 540
Val Pro Glu Asp Thr Leu Asn Lys Val Asp Ala Ala Val Ala Glu Ala
545 550 555 560
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Met Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg Ala Lys Thr Ile
1 5 10 15
Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu Arg Gly Leu Asn Ala
20 25 30
Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro Lys Gly Arg Asn Val
35 40 45
Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile Thr Asn Asp Gly Val
50 55 60
Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro Tyr Glu Lys Ile Gly
65 70 75 80
Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr Asp Asp Val Ala Gly
85 90 95
Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln Ala Leu Val Arg Glu
100 105 110
Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro Leu Gly Leu Lys Arg
115 120 125
Gly Ile Glu Lys Ala Val Glu Lys Val Thr Glu Thr Leu Leu Lys Gly
130 135 140
Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ala Ala Thr Ala Ala Ile
145 150 155 160
Ser Ala Gly Asp Gln Ser Ile Gly Asp Leu Ile Ala Glu Ala Met Asp
165 170 175
Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu Glu Ser Asn Thr Phe
180 185 190
Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg Phe Asp Lys Gly Tyr
195 200 205
Ile Ser Gly Tyr Phe Val Thr Asp Pro Glu Arg Gln Glu Ala Val Leu
210 215 220
Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys
225 230 235 240
Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gly Ala Gly Lys Pro Leu
245 250 255
Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val
260 265 270
Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val Ala Val Lys Ala Pro
275 280 285
Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln Asp Met Ala Ile Leu
290 295 300
Thr Gly Gly Gln Val Ile Ser Glu Glu Val Gly Leu Thr Leu Glu Asn
305 310 315 320
Ala Asp Leu Ser Leu Leu Gly Lys Ala Arg Lys Val Val Val Thr Lys
325 330 335
Asp Glu Thr Thr Ile Val Glu Gly Ala Gly Asp Thr Asp Ala Ile Ala
340 345 350
Gly Arg Val Ala Gln Ile Arg Gln Glu Ile Glu Asn Ser Asp Ser Asp
355 360 365
Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala Lys Leu Ala Gly Gly
370 375 380
Val Ala Val Ile Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu
385 390 395 400
Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val
405 410 415
Glu Glu Gly Ile Val Ala Gly Gly Gly Val Thr Leu Leu Gln Ala Ala
420 425 430
Pro Thr Leu Asp Glu Leu Lys Leu Glu Gly Asp Glu Ala Thr Gly Ala
435 440 445
Asn Ile Val Lys Val Ala Leu Glu Ala Pro Leu Lys Gln Ile Ala Phe
450 455 460
Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu Lys Val Arg Asn Leu
465 470 475 480
Pro Ala Gly His Gly Leu Asn Ala Gln Thr Gly Val Tyr Glu Asp Leu
485 490 495
Leu Ala Ala Gly Val Ala Asp Pro Val Lys Val Thr Arg Ser Ala Leu
500 505 510
Gln Asn Ala Ala Ser Ile Ala Gly Leu Phe Leu Thr Thr Glu Ala Val
515 520 525
Val Ala Asp Lys Pro Glu Lys Glu Lys Ala Ser Val Pro Gly Gly Gly
530 535 540
Asp Met Gly Gly Met Asp Phe
545 550
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Met Asp Cys Val Ala Glu Gly Lys Val Cys Asp Pro Leu Cys Ser Ser
1 5 10 15
Gly Gly Cys Trp Gly Pro Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn
20 25 30
Tyr Ser Arg Gly Gly Val Cys Val Thr His Cys Asn Phe Leu Asn Gly
35 40 45
Glu Pro Arg Glu Phe Ala His Met Ala Lys Thr Ile Ala Tyr Asp Glu
50 55 60
Glu Ala Arg Arg Gly Leu Glu Arg Gly Leu Asn Ala Leu Ala Asp Ala
65 70 75 80
Val Lys Val Thr Leu Gly Pro Lys Gly Arg Asn Val Val Leu Glu Lys
85 90 95
Lys Trp Gly Ala Pro Thr Ile Thr Asn Asp Gly Val Ser Ile Ala Lys
100 105 110
Glu Ile Glu Leu Glu Asp Pro Tyr Glu Lys Ile Gly Ala Glu Leu Val
115 120 125
Lys Glu Val Ala Lys Lys Thr Asp Asp Val Ala Gly Asp Gly Thr Thr
130 135 140
Thr Ala Thr Val Leu Ala Gln Ala Leu Val Arg Glu Gly Leu Arg Asn
145 150 155 160
Val Ala Ala Gly Ala Asn Pro Leu Gly Leu Lys Arg Gly Ile Glu Lys
165 170 175
Ala Val Glu Lys Val Thr Glu Thr Leu Leu Lys Gly Ala Lys Glu Val
180 185 190
Glu Thr Lys Glu Gln Ile Ala Ala Thr Ala Ala Ile Ser Ala Gly Asp
195 200 205
Gln Ser Ile Gly Asp Leu Ile Ala Glu Ala Met Asp Lys Val Gly Asn
210 215 220
Glu Gly Val Ile Thr Val Glu Glu Ser Asn Thr Phe Gly Leu Gln Leu
225 230 235 240
Glu Leu Thr Glu Gly Met Arg Phe Asp Lys Gly Tyr Ile Ser Gly Tyr
245 250 255
Phe Val Thr Asp Pro Glu Arg Gln Glu Ala Val Leu Glu Asp Pro Tyr
260 265 270
Ile Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys Asp Leu Leu Pro
275 280 285
Leu Leu Glu Lys Val Ile Gly Ala Gly Lys Pro Leu Leu Ile Ile Ala
290 295 300
Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val Val Asn Lys Ile
305 310 315 320
Arg Gly Thr Phe Lys Ser Val Ala Val Lys Ala Pro Gly Phe Gly Asp
325 330 335
Arg Arg Lys Ala Met Leu Gln Asp Met Ala Ile Leu Thr Gly Gly Gln
340 345 350
Val Ile Ser Glu Glu Val Gly Leu Thr Leu Glu Asn Ala Asp Leu Ser
355 360 365
Leu Leu Gly Lys Ala Arg Lys Val Val Val Thr Lys Asp Glu Thr Thr
370 375 380
Ile Val Glu Gly Ala Gly Asp Thr Asp Ala Ile Ala Gly Arg Val Ala
385 390 395 400
Gln Ile Arg Gln Glu Ile Glu Asn Ser Asp Ser Asp Tyr Asp Arg Glu
405 410 415
Lys Leu Gln Glu Arg Leu Ala Lys Leu Ala Gly Gly Val Ala Val Ile
420 425 430
Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu Arg Lys His Arg
435 440 445
Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val Glu Glu Gly Ile
450 455 460
Val Ala Gly Gly Gly Val Thr Leu Leu Gln Ala Ala Pro Thr Leu Asp
465 470 475 480
Glu Leu Lys Leu Glu Gly Asp Glu Ala Thr Gly Ala Asn lle Val Lys
485 490 495
Val Ala Leu Glu Ala Pro Leu Lys Gln Ile Ala Phe Asn Ser Gly Leu
500 505 510
Glu Pro Gly Val Val Ala Glu Lys Val Arg Asn Leu Pro Ala Gly His
515 520 525
Gly Leu Asn Ala Gln Thr Gly Val Tyr Glu Asp Leu Leu Ala Ala Gly
530 535 540
Val Ala Asp Pro Val Lys Val Thr Arg Ser Ala Leu Gln Asn Ala Ala
545 550 555 560
Ser Ile Ala Gly Leu Phe Leu Thr Thr Glu Ala Val Val Ala Asp Lys
565 570 575
Pro Glu Lys Glu Lys Ala Ser Val Pro Gly Gly Gly Asp Met Gly Gly
580 585 590
Met Asp Phe
595
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ccgtgacaag gtaaacacaa cacatcc 27
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ttaccggtcc caatggaggg gaat 24
<210>6
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aaccatggcc cgggcagggt ataac 25
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atcgatcagt cgtagccatc cgc 23
<210>8
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aaatcgatga ccaacacaac caacg 25
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aactcgagga atcatgtc 18
<210>10
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ctcgagttaa ttaatttaaa tgtccagttt attcagcagc 40
<210>11
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gatcttaatt aa 12
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gatcatttaa at 12
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aacttaagat gaagatcttt gggagcctgg catttctggc tcgtgcggtc gggatcgac 59
<210>15
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aagcggccgc tcatcagccg agccggggtg 30
<210>16
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cccttaagat ggactgcgtg gcagagggca aagt 34
<210>17
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ggggagcctc gagaatttgc ccatatggcc aagacaattg cgt 43
<210>18
<211>33
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aagcggccgc tcagaaatca tccatgccac cca 33
<210>19
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gtgtatccat atgacacgga a 21
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ctgtaacact aaccattaca ctaagccatc ttgtaaaatg t 41
<210>21
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caaaactacg taatggccag cactccaagt gcatactcta tg 42
<210>22
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tattcctgca ggacggagcg g 21
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tatcga 6
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ttgctagcat gaccaacaag tgtct 25
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aaggatcctc agtttcggag gtaacctgta 30
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ttccatggca acacttccgg aaactactgt tgttagacga gccaagacaa ttgcgtacga 60
c 61
<210>28
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aagcggccgc tcagaaatcc atgccaccca t 31
<210>29
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gcgccggcgc acctctcttt acgc 24
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caggtacccc ccgctcagat gggtttcttc gctccatgcc ccaaagccac cc 52
<210>31
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<222>(1)..(9)
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Lys Ile Phe Gly Ser Leu Ala Phe Leu
1 5
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<222>(1)..(54)
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<400>32
Asp Cys Val Ala Glu Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly
1 5 10 15
Gly Cys Trp Gly Pro Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr
20 25 30
Ser Arg Gly Gly Val Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu
35 40 45
Pro Arg Glu Phe Ala His
50
Claims (9)
1. recombinant viral vector, it is characterized in that, insert the expressing fusion protein box of heat shock protein(HSP)-antigenic peptide in this viral genome, described expression cassette comprises successively: the fusion rotein encoding sequence and the terminator codon of promoter sequence, heat shock protein(HSP)-antigenic peptide, described virus vector is selected from: adenovirus and adeno-associated virus (AAV).
2. recombinant viral vector as claimed in claim 1 is characterized in that described virus vector is an adenovirus.
3. recombinant viral vector as claimed in claim 1 is characterized in that described virus vector is an adenovirus, and has inserted described expression cassette in adenoviral gene group E3 zone position.
4. recombinant viral vector as claimed in claim 1 is characterized in that, described heat shock protein(HSP) is selected from: Hsp70, Hsp65, Hsp110 or gp96.
5. recombinant viral vector as claimed in claim 1 is characterized in that, described antigenic peptide is tumour antigen or pathogen antigen.
6. recombinant viral vector as claimed in claim 5, it is characterized in that described antigenic peptide is selected from: surface antigen, HBV cAg or its combination of the E6 epi-position of the Th cell epitope structural domain of the t cell epitope of hepatitis B virus core antigen t cell epitope, ErbB2, ErbB3, CEA epi-position, PSA epi-position, WT epi-position, HPV, the E7 epi-position of HPV, HBV.
7. recombinant viral vector as claimed in claim 1 is characterized in that, the expressing fusion protein box coding of described heat shock protein(HSP)-antigenic peptide has the fusion rotein of aminoacid sequence shown in the SEQ ID NO:1,2 or 3.
8. a pharmaceutical composition is characterized in that, it comprises the recombinant viral vector and the pharmaceutically acceptable vehicle of fusion rotein of the described expression heat shock protein(HSP)-antigenic peptide of claim 1 of significant quantity.
9. pharmaceutical composition as claimed in claim 8 is characterized in that described virus vector is an adenovirus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03114903 CN1517437B (en) | 2003-01-15 | 2003-01-15 | Vaccine for specificity treating tumour or endocellular infection and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03114903 CN1517437B (en) | 2003-01-15 | 2003-01-15 | Vaccine for specificity treating tumour or endocellular infection and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1517437A CN1517437A (en) | 2004-08-04 |
| CN1517437B true CN1517437B (en) | 2010-06-09 |
Family
ID=34284022
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03114903 Expired - Fee Related CN1517437B (en) | 2003-01-15 | 2003-01-15 | Vaccine for specificity treating tumour or endocellular infection and application |
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| CN1727362B (en) * | 2004-07-30 | 2010-12-01 | 中国人民解放军第二军医大学 | Preparation and application of vaccine for curing tumor in positive carcino-embryonic antigen |
| CN103372209B (en) * | 2012-04-24 | 2014-09-17 | 中国科学院微生物研究所 | Application of antibody of gp96 protein in preparation of cancer cell inhibitor |
| WO2019061297A1 (en) * | 2017-09-29 | 2019-04-04 | 苏州工业园区唯可达生物科技有限公司 | Cd4 helper t-cell epitope fusion peptide and vaccine thereof |
| CN116688107A (en) * | 2019-09-06 | 2023-09-05 | 北京微九九科技有限公司 | Preparation method of tumor vaccine and tumor vaccine prepared by using same |
| CN114099639B (en) * | 2021-11-25 | 2024-03-01 | 徐州医科大学 | H1-pHSP65 nanometer vaccine, preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6338952B1 (en) * | 1988-06-15 | 2002-01-15 | Whitehead Institute For Biomedical Research | Stress proteins and uses therefor |
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| US6338952B1 (en) * | 1988-06-15 | 2002-01-15 | Whitehead Institute For Biomedical Research | Stress proteins and uses therefor |
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