CN102277384B - A kind of PSA recombinant adeno-associated virus vector and its construction method and application - Google Patents
A kind of PSA recombinant adeno-associated virus vector and its construction method and application Download PDFInfo
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- CN102277384B CN102277384B CN2011101256030A CN201110125603A CN102277384B CN 102277384 B CN102277384 B CN 102277384B CN 2011101256030 A CN2011101256030 A CN 2011101256030A CN 201110125603 A CN201110125603 A CN 201110125603A CN 102277384 B CN102277384 B CN 102277384B
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Abstract
一种PSA重组腺相关病毒载体(rAAV)及其构建方法与其应用。该rAAV是将腺相关病毒载体中的腺相关病毒结构基因替换为肿瘤抗原基因PSA或其突变型基因得到。本发明的rAAV可将其携带的野生型或突变型的前列腺特异性抗原基因输送入单核细胞-树突状细胞系中，被用于刺激免疫系统的效应细胞。实验证明，被本发明的rAAV感染的DC所诱导的CTL在患者体内可有效地抑制恶性肿瘤细胞的生长或者杀灭肿瘤细胞。本发明的重组腺相关病毒载体或其相关产品可被用于制备治疗前列腺癌的药物。A PSA recombinant adeno-associated virus vector (rAAV) and its construction method and application. The rAAV is obtained by replacing the adeno-associated virus structural gene in the adeno-associated virus vector with the tumor antigen gene PSA or its mutant gene. The rAAV of the present invention can deliver the wild-type or mutant prostate-specific antigen gene carried by it into the monocyte-dendritic cell line, and be used to stimulate the effector cells of the immune system. Experiments have proved that the CTL induced by the DC infected by the rAAV of the present invention can effectively inhibit the growth of malignant tumor cells or kill tumor cells in patients. The recombinant adeno-associated virus vector or related products of the present invention can be used to prepare drugs for treating prostate cancer.
Description
The present invention is for dividing an application.Original application day is on April 23rd, 2008, application number 200880012949.6, and denomination of invention is " one group recombined glandulae correlation viral vectors and construction process and application ".
Technical field
The present invention relates to carrier and application thereof in biological field, particularly relate to a kind of PSA recombined glandulae correlation viral vectors and construction process thereof and its application in the preparation antitumor drug.
Background technology
The gene structure of adeno-associated virus (AAV) is identified.Nineteen eighty-three, the people such as Samulski have described the terminal repetition fragment (upstream 5 ' end fragment of AAV, downstream 3 ' end fragment) (Samulski RJ, Srivastava A, Berns KI, Muzyczka N.Rescue of adeno-associated virus from recombinant plasmids:gene correction within the terminal repeats of AAV.Cell.33:135-143.).1984, the people such as Hermonat have described low infectious particles (lip) gene and coating (cap) gene (the Hermonat PL of AAV, Labow MA, Wright R, Berns KI, Muzyczka N.Genetics of adeno-associated virus:isolation and preliminary characterization of adeno-associated virus type 2 mutants.J Virol.51:329-339.Hermonat, P.L., and Muzyczka, N.Use of adeno-associated virus as a mammalian DNA cloning vector:transduction of neomycin resistance into mammalian tissue culture cells.Proc.Natl.Acad.Sci.U.S.A.81:6466-6470.).1986, the people such as Labow have identified the p5 promotor (LabowMA that is positioned between upstream 5 ' end fragment and rep gene, Hermonat PL, Berns KI.Positive and negative autoregulation of the adeno-associated virus type2 genome.J Virol.160:251-258.).
1984, one of the major technique person in charge of the technology aspect of the U.S. rich fertile gene international corporation Paul professor L.Hermonat takes the lead in proving that the AAV carrier can be used for the gene therapy (Hermonat of human diseases, P.L., and Muzyczka, N.Use of adeno-associated virus as a mammalian DNA cloning vector:transduction of neomycin resistance into mammalian tissue culture cells.Proc.Natl.Acad.Sci.U.S.A.81:6466-6470.).At present, be mainly that American-European countries is carrying out the clinical trial as the gene therapy human diseases on basis take AAV.According to U.S.'s grain and drug administration's statistics, existing ten remainders are carried out take AAV as the Gene Therapy Clinical Trials on basis, be mainly that the AAV virus of carrying therapeutic gene is injected in patient body, make its expression treatment gene in vivo, thereby reach the purpose for the treatment of disease.Disease mainly for treatment has the non-neoplastic diseases such as Parkinsonism, rheumatic arthritis, hemophilia, heart failure, the silent syndromes of progressive myatrophy and Olds sea.But still there are some problems in the AAV virus that is applied to clinical treatment, for example carries the therapeutic gene size and is subject to significant limitation, and virus itself is unstable, causes the therapeutic gene unstable expression, and causes curative effect inconsistent.Although the immunogenicity of AAV virus itself is very weak, expressed therapeutic gene easily brings out autoimmune response in patient body, even cause serious toxic side effect.
AAV is a kind of defective virus of non-virulent, needs the gene product of other virus (as adenovirus) auxiliary, just can be assembled into to have infective virion.AAV genome total length is 4700 base pairs (bp) approximately, and two ends are for repeating terminal fragment (TR), and the centre is the structure gene of virus, comprises the Rep gene relevant with virus replication and viral capsid Cap gene.Due to the unstable that has AAV virus self and carry the aspect defective such as allogenic gene (therapeutic gene) limited length, therefore be necessary that it is carried out gene recombination forms recombinant adeno-associated virus (recombinant adeno-associated virus, rAAV).Existing studies show that in a large number, with the deletion of the structure gene in the AAV genome, can obviously increase the capacity of allogenic gene.In addition, the allogenic gene that will have therapeutic action inserts in rAAV, is prepared into to have infective rAAV virion, is injected in patient body, makes its infectosome inner cell, and then the expression treatment gene, thereby reaches the effect for the treatment of disease.At present, be mainly rAAV to be applied to the treatment of the non-neoplastic diseases such as Parkinsonism, rheumatic arthritis, hemophilia, heart failure, the silent syndromes of progressive myatrophy and Olds sea.But rAAV still comes with some shortcomings, as unstable in recombinant virus, virus titer is low, and the capacity of admitting therapeutic gene is limited (the most about 2000 base pairs of allogenic gene fragment (bp) that generally only can insert, otherwise the stability of rAAV is with destroyed) still.Therefore, need design more rational recombinant adeno-associated virus (rAAV) carrier, to satisfy the needs of practical application.
Summary of the invention
The purpose of this invention is to provide a kind of stability high, carry allogenic gene PSA recombinant adeno-associated virus capacious (rAAV) carrier.
Recombined glandulae correlation viral vectors provided by the present invention is that the adeno-associated virus structure gene in adeno-associated virus (AAV) carrier is replaced with the recombined glandulae correlation viral vectors that prostate-specific-antigen PSA (prostate-specific antigen) gene or its mutated genes obtain;
PSA recombined glandulae correlation viral vectors of the present invention is to transform to obtain on the basis of known gland relevant viral vector, described adeno-associated virus structure gene is Rep and Lip/Cap gene, tumour specific antigen gene PS A is the tumor associated antigen gene, and its mutated genes is the related neoplasms specific antigen gene fragment with identical function.
Known gland relevant viral vector has the p5 promotor, for improving the transcriptional level of goal gene, also can further the p5 promotor in described recombined glandulae correlation viral vectors be replaced with one or several promotor in scavenger cell virus (cytomegalovirus, CMV) promotor, beta actin promoter and SV40 viral promotors.
Second purpose of the present invention is to provide the construction process of above-mentioned PSA recombined glandulae correlation viral vectors.
Construction process provided by the present invention, it is the method for using conventional gene recombination, first the adeno-associated virus structure gene in gland relevant viral vector is rejected, then replaced this rejecting gene with aforementioned specific antigen gene PSA or its mutated genes mPSA, obtain the PSA recombined glandulae correlation viral vectors.
In the construction process of above-mentioned PSA recombined glandulae correlation viral vectors, for improving the transcriptional level of goal gene, also can further the p5 promotor in described recombined glandulae correlation viral vectors be replaced with one or several promotor in scavenger cell viral promotors, beta actin promoter and SV40 viral promotors.
The product relevant to PSA recombined glandulae correlation viral vectors of the present invention; comprise recombinant adeno-associated virus plasmid, recombinant adeno-associated virus particle and by the clone of recombinant gland relevant viral vector infection of the present invention or transfection, all belong to protection scope of the present invention as monocyte-dendritic cell system, T lymphocyte series etc. (as described in the PSA recombined glandulae correlation viral vectors genes involved-prostate specific antigen gene or its mutated genes can obtain to express under the effect at transcripting promoter) etc. in the clones such as monocyte-dendritic cell system or T lymphocyte series.
Medicinal use aspect, another object of the present invention are to provide a kind of antitumor drug.
The activeconstituents of antitumor drug provided by the present invention is above-mentioned PSA recombined glandulae correlation viral vectors or the product relevant to PSA recombined glandulae correlation viral vectors of the present invention.
As take PSA recombinant adeno-associated virus of the present invention as carrier, with tumour antigen-wild-type and/or mutant tumour specific antigen gene importing monocyte-dendritic cell be, and induce producing dendritic shape cell, to reach the immunostimulating purpose of patient's in vitro and in vivo, in order to treating associated malignancies, or stimulate the cytotoxic T lymphocyte that produces (for example but not only be confined to T lymphocyte and bone-marrow-derived lymphocyte) treatment associated malignancies with this dendritic cell.
Described associated malignancies is prostate cancer.
Medicine provided by the present invention can adopt the formulations such as solvent or pulvis.
The selection of described solvent is diversified, all can as cell culture fluid (base), physiological saline or phosphate buffered saline buffer etc.
When needing, can also add one or more pharmaceutically acceptable carriers in said medicine.Described carrier comprises thinner, absorption enhancer and the tensio-active agent etc. of pharmaceutical field routine.
Application method can be the monocyte of first isolating in the tumour patient body, then this medicine is infected or transfection patient's monocyte.Maybe conversion there is the cytotoxic T lymphocyte of generation that dendritic cell stimulates of the maturation of wild-type and/or mutant prostate-specific-antigen PSA to feed back tumour patient.
The consumption of said medicine is generally 100 μ l/5 * 10
6/ each, 2 times per month, be generally 6 months the course for the treatment of.Dosage and the course for the treatment of all can be according to the practical situation adjustment.
For improving curative effect, medicine of the present invention can also carry out combined therapy with microbiotic, immunostimulant and tumor-targeting drug etc.
The present invention also provides a kind of method of killing tumour.
The method of killing tumour provided by the present invention can comprise the following steps:
Recombinant gland relevant viral vector infection or the transfection of 1) spontaneous monocyte-dendritic cell or T lymphocyte in tumour place system (this system can produce by manual simulation's mode or tumour patient body in) being carried the recombined glandulae correlation viral vectors of wild-type tumour specific antigen gene and/or carrying the mutant specific antigen gene by the present invention, or by the product treatment relevant to recombined glandulae correlation viral vectors of the present invention, the cell after being processed separately;
2) with step 1) in monocyte-dendritic cell after processing add in the system of tumour place and kill tumour; Or not processed T lymphocyte and the monocyte after described processing-dendritic cell mixed culture is formed Antigen-specific cytotoxic T lymphocyte, then this Antigen-specific cytotoxic T lymphocyte is added in the system of tumour place kill tumour; Or processed T lymphocyte and not processed monocyte-dendritic cell are added in the system of tumour place kill tumour.
The method of killing tumour of the present invention can specifically be applied in oncotherapy, comprise that giving a tumour patient feeds back Antigen-specific cytotoxic T lymphocyte, this cell origin comes from patient's spontaneous T lymphocyte and derives from this patient's monocyte-dendritic cell mixed culture and produces.Before mixed culture, recombinant gland relevant viral vector infection or transfection that these have been carried the recombinant adeno-associated virus of wild-type specific antigen gene and/or carried the mutant specific antigen gene by the present invention in monocyte-dendritic cell, or by the product treatment relevant to recombined glandulae correlation viral vectors of the present invention;
Perhaps, give a tumour patient and feed back the monocyte-dendritic cell that derives from the patient.Before feeding back, recombinant gland relevant viral vector infection or transfection that these have been carried the recombinant adeno-associated virus of wild-type specific antigen gene and/or carried the mutant specific antigen gene by the present invention in monocyte-dendritic cell, or by the product treatment relevant to recombined glandulae correlation viral vectors of the present invention;
Again or, give spontaneous monocyte-dendritic cell that tumour patient feeds back the above-mentioned patient's of deriving from T lymphocyte and derives from this patient.Before feeding back, recombinant gland relevant viral vector infection or transfection that these T lymphocytes have been carried the recombinant adeno-associated virus of wild-type specific antigen gene and/or carried the mutant specific antigen gene by the present invention, or by the product treatment relevant to recombined glandulae correlation viral vectors of the present invention.
The invention provides a kind of stability high, carry allogenic gene (PSA) PSA recombinant adeno-associated virus capacious (rAAV) carrier.In rAAV carrier of the present invention, the structure gene Rep of AAV and Lip/Cap gene are replaced by the tumour specific antigen gene PS A of wild-type or mutant.The wild-type that recombined glandulae correlation viral vectors of the present invention can carry it or the specific antigens gene of mutant are conveyed in monocyte-dendritic cell system, carry with the cell of these specific antigens genes be used to effector cell's (being not limited to T lymphocyte and bone-marrow-derived lymphocyte) of stimulating immune system.Experimental results show that, can effectively be suppressed growth or the killing off tumor cells of associated malignancies cell by the rAAV of the present invention dendritic cell that infects and the cytotoxic T lymphocyte of being induced in patient body, thereby recombined glandulae correlation viral vectors of the present invention or the product relevant to recombined glandulae correlation viral vectors of the present invention can be used to prepare antitumor drug.The present invention has important theoretical and practical significance at the clinical treatment of malignant tumour with in using, and has a extensive future.
Below in conjunction with specific embodiment, the present invention is described in further details.
Description of drawings
Fig. 1 is the structural representation of recombined glandulae correlation viral vectors.
Fig. 2 A to Fig. 2 H is that the enzyme of eight kinds of recombined glandulae correlation viral vectors rAAV is cut and the PCR detected result; Wherein Fig. 2 A is that the enzyme of rAAV/PSA is cut the detected result with PCR.
Fig. 3 is the preparation flow figure of recombinant adeno-associated virus rAAV.
Fig. 4 is the virus titer detected result of recombinant adeno-associated virus rAAv/PSA.Fig. 5 kills the tumor experiment flow process for one or more rAAV virus infection tumour patient monocytes that carry tumor antigen gene for the basis.
Fig. 6 is the efficient detected result that recombinant adeno-associated virus rAAV/PSA infects peripheral blood lymphocytes.
Fig. 7 expresses the detected result of CD80, CD83 and CD86 level for the DC that is infected by the rAAV/PSA recombinant adeno-associated virus respectively.
Fig. 8 is the IFN-γ expression level detected result of the CTL that DC induced that infected by the rAAV/PSA recombinant adeno-associated virus.
Fig. 9 is the IFN-γ expression level detected result of the CTL that DC induced that infected by the rAAV/PSA recombinant adeno-associated virus.
Figure 10 is the iconography observed result of example metastatic lesion changing conditions before and after the CTL treatment that the DC that rAAV/PSA infects induces.
Figure 11 is the changing conditions of four examples PSA tumour antigen level in the CTL treatment Patients Before And After serum that the DC that rAAV/PSA infects induces.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions, concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
Described percentage concentration is mass/volume (W/V) percentage concentration or volume/volume (V/V) percentage concentration if no special instructions.
Synthetic and the determined dna sequence of the primer, DNA sequence dna is completed by American I nvitrogen company.
First part: recombined glandulae correlation viral vectors rAAV/PSA
The structure of embodiment 1-1, recombined glandulae correlation viral vectors rAAV/PSA and rAAV/mPSA (mutant) and test material and source thereof:
A. carry the pBR322 plasmid (called after pBR-AAV2) of AAV 2 type complete genome DNAs: by one of the major technique person in charge of the technology aspect of the U.S. rich fertile gene international corporation Paul professor L.Hermonat preparation (Hermonat, P.L., and Muzyczka, N.Use of adeno-associated virus as a mammalian DNA cloning vector:transduction of neomycin resistance into mammalian tissue culture cells.Proc.Natl.Acad.Sci.U.S.A.81:6466-6470.).
B. the primary prostate cancer cell of people: separate obtaining or obtaining from the commercial channel from the cancerous tissue of patients with prostate cancer, immunohistochemical methods confirms that prostate specific antigen is positive.
C. the pCI-neo plasmid that carries the CMV promotor is purchased from U.S. Promega company, and the plasmid pSG424 that carries the SV40 early promoter is purchased from U.S. Clonitic company.
D. gene amplification nucleotide primer: according to the PSA of publishing in U.S.'s gene pool (PSA) gene order design (U.S. NCI gene pool: M26663).
Build with following method the recombined glandulae correlation viral vectors (as shown in Figure 1) that the present invention carries prostate specific antigen (prostate-specific antigen, PSA) gene or its mutated genes, detailed process comprises the following steps:
One, the structure of recombined glandulae correlation viral vectors
The reconstruction of A.pBR-AAV2 plasmid, concrete grammar is: first use restriction enzyme Bst98 I and Hpa I (available from U.S. Promega company) that the genomic structure gene Rep of adeno-associated virus AAV and Lip/Cap gene in the pBR-AAV2 plasmid are excised fully, reaction system is: 1 μ g pBR-AAV2,10U Bst98I, 10U Hpa I, 2.5 μ l 10 * damping fluid D and 19.5 μ l deionized waters; Reaction conditions was: 37 ℃ of lower water-baths 4 hours.Then, the nucleotide sequence (CGAATTCATGCGATATCGTT) that will contain restriction enzyme EcoR I and EcoR V restriction enzyme site inserts in plasmid, and reaction system is: 500ng plasmid, the nucleotide sequence of 300ng EcoR I and EcoR V, 10IU T
4DNA ligase (available from U.S. Promega company), 1.5 μ l 10 * T
4DNA connects damping fluid and 11.5 μ l deionized waters; Reaction conditions was: 4 ℃ of lower water-baths 8 hours.Then, keep the complete TR sequence in two ends or the 75th the nucleotide sequence place of the genomic two ends TR of AAV is all inserted the fragment that is comprised of 9 Nucleotide: CTGCGCTGG, purpose is to improve the stability of rAAV virus and the duplicating efficiency that improves virus, method is: the TR that at first uses restriction enzyme Ban I (available from U.S. Promega company) cutting two ends, reaction system is: the plasmid of the 1 above-mentioned preparation of μ g, 10U Ban I, 1.5 μ l 10 * damping fluid G and 11.5 μ l deionized waters; Reaction conditions is: 37 ℃ of lower water-baths 4 hours, then 9 nucleotide fragments are inserted in plasmid, reaction system is: 500ng plasmid, 9 nucleotide sequences of 300ng, 10IU T
4DNA ligase (available from U.S. Promega company), 1.5 μ l 10 * T
4DNA connects damping fluid and 11.5 μ l deionized waters; Reaction conditions was: 4 ℃ of lower water-baths 8 hours.
B. adopt gene amplification (polymerase chain reaction,PCR, PCR) amplification CMV promotor, SV40 early promoter.Concrete grammar is: first take pCI-neo plasmid (available from U.S. Promega company) as template, pcr amplification CMV promotor under the guiding of primer 1:AGATCTTCAATATTGGCCAT (SEQ ID NO:1 in sequence table) and primer 2: TGTCAGAAGCACTGACTGC (SEQ ID NO:2 in sequence table), the pcr amplification condition is: first 94 ℃ 4 minutes; Again 94 ℃ 30 seconds, 60 ℃ 35 seconds, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 8 minutes, after reaction finishes, the PCR product is carried out 1.2% agarose gel electrophoresis detects, the specific band of an expection appears at 740bp place, this purpose band is reclaimed also obtains the CMV promotor after purifying.Again take pSG424 plasmid (available from U.S. Clonitic company) as template, pcr amplification SV40 early promoter under the guiding of primer 3:GAACCAGCTGTGGAATGTGTC (SEQ ID NO:3) and primer 4:TCAGGAAGCTTAGATCTAGC (SEQ ID NO:4 in sequence table), the pcr amplification condition is: first 94 ℃ 4 minutes; Again 94 ℃ 30 seconds, 60 ℃ 35 seconds, 72 ℃ 40, second, totally 30 circulations; Last 72 ℃ 8 minutes, after reaction finishes, the PCR product is carried out 1.2% agarose gel electrophoresis detects, the specific band of an expection appears at 359bp place, this purpose band is reclaimed also obtains the SV40 early promoter after purifying.
C.PCR amplification beta actin promoter, total length PSA cDNA and part PSA cDNA fragment are (according to U.S. NCBI gene pool (M26663), difference called after A (from 5 ' end 25-264 bit base), B (from 5 ' end 265-435 bit base), C (from 5 ' end 436-663 bit base), D (from 5 ' end 664-810 bit base), the present embodiment is as an example of above-mentioned fragment example but be not limited to above-mentioned fragment, the PSA cDNA fragment that other and PSA cDNA have identical function all can be used for building recombined glandulae correlation viral vectors of the present invention), concrete grammar is: adopt the separate nucleic acid technology, separate total DNA and mRNA (also can obtain from the commercial channel or synthetic this total DNA and mRNA) from the primary prostate cancer cell of people, then take total DNA as template, pcr amplification beta actin promoter under the guiding of primer 5:CCCGGGCCCAGCACCCCAAG (SEQ ID NO:5 in sequence table) and primer 6:CATCCATGGTGAGCTGCG (SEQ ID NO:6 in sequence table), the pcr amplification condition is: first 94 ℃ 4 minutes, again 94 ℃ 30 seconds, 58 ℃ 35 seconds, 72 ℃ 1 minute 20 seconds, totally 30 circulations, last 72 ℃ 8 minutes, after reaction finishes, the PCR product is carried out 1.2% agarose gel electrophoresis detects, the specific band of an expection appears at 1176bp place, this purpose band is reclaimed also obtains the beta actin promoter after purifying.again with synthetic its cDNA of mRNA reverse transcription and as template, pcr amplification total length PSA cDNA under the guiding of primer 7:ATTCCGCCGGAGAGCTGTG (SEQ ID NO:7 in sequence table) and primer 8:CCCAGGACACAGAGAGAGGAC (SEQ ID NO:8 in sequence table), the pcr amplification condition is: first 94 ℃ 4 minutes, again 94 ℃ 30 seconds, 59 ℃ 35 seconds, 72 ℃ 1 minute 15 seconds, totally 30 circulations, last 72 ℃ 8 minutes, after reaction finishes, the PCR product is carried out 1.2% agarose gel electrophoresis to be detected, specific band in an expection of 1006bp place's appearance, this purpose band is reclaimed also obtain total length PSA cDNA after purifying, obtain PSA cDNA Segment A (primer 9:ATGTGGGTCCCGGTTGTCTTC (SEQ ID NO:9 in sequence table) and primer 10:TGTGGCCGACCCAGCAAG (SEQ ID NO:10 in sequence table)) with above-mentioned same procedure, PSA cDNA fragment B (primer 11:GCCTGTTTCATCCTGAAG (SEQ ID NO:11 in sequence table) and primer 12:GAGCTCGGCAGGCTCTGAC (SEQ ID NO:12 in sequence table)), C (primer 13:ACGGATGCTGTGAAGGTC (SEQ IDNO:13 in sequence table) and primer 14:AGCACCTGCTCGGGTGATTC (SEQ ID NO:14 in sequence table)), D (primer 15:GGTGCTCAGGGGTTGGCCAC (SEQ ID NO:15 in sequence table)).
D. adopt the DNA interconnection technique, with CMV promotor, SV40 early promoter, beta actin promoter, total length PSA cDNA or the part PSA cDNA fragment of above-mentioned amplification inserting step A successively in the pBR-AAV2 carrier of reconstruction, for inserting promotor, at first carry out endonuclease reaction, then carry out ligation, wherein, the endonuclease reaction system is: 1 μ g plasmid; 10U restriction enzyme BamH I and Sal I (available from U.S. Promega company), 2.5 μ l 10 * damping fluid C and 19.5 μ l deionized waters; Reaction conditions is: 37 ℃ of lower water-baths 4 hours, the ligation system was: the plasmid after the 500ng enzyme is cut, 300ng promoter DNA, 10IU T
4DNA ligase (available from U.S. Promega company), 1.5 μ l 10 * T
4DNA connects damping fluid and 11.5 μ l deionized waters; Reaction conditions was: 4 ℃ of lower water-baths 8 hours.Then, cut with restriction enzyme EcoR I and EcoR V enzyme respectively carrying the plasmid of promotor and the PSA cDNA of total length.Endonuclease reaction and to carry out system and the condition of ligation same as described above.Obtain respectively at last carrying the recombined glandulae correlation viral vectors (called after rAAV/PSA) of CMV promotor, SV40 early promoter, beta actin promoter and total length PSA cDNA, and the recombined glandulae correlation viral vectors (difference called after rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA, unified called after rAAV/mPSA) that carries CMV promotor, SV40 early promoter, beta actin promoter and A or B or C or the different PSA cDNA of D fragment (mutant).
E. the DNA-rAAV/PSA after connecting and rAAV/mPSA be quiding gene engineering colon bacillus (E.coli) DH5 α competent cell (American I nvitrogen company) respectively, carry out resistance screening with the LB flat board that contains 100 μ g/mL penbritins, the single bacterium colony of picking white, extract plasmid and purifying, obtain rAAV/PSA plasmid and rAAV/mPSA plasmid.
Two, the detection of recombined glandulae correlation viral vectors
The purified rAAV/PSA plasmid that first step 1 is obtained and rAAV/mPSA plasmid carry out enzyme with restriction enzyme (being followed successively by Nsi I, EcoR V and EcoR I, Not I and Bam H I, Pst I) to be cut, and restriction enzyme used is all available from U.S. Promega company.(the pBR-AAV2 carrier through reconstruction of CMV promotor, SV40 early promoter, beta actin promoter inserting step A is successively obtained with the negative contrast of rAAV carrier without the PSA gene simultaneously, this carrier is cut with restriction enzyme Hind III enzyme), after reaction finishes, enzyme is cut product carry out 1.2% agarose gel electrophoresis detection, the wherein detected result of rAAV/PSA plasmid (1.DNA molecular weight standard as shown in Fig. 2 A.2. without the rAAV carrier (Hind III restriction endonuclease) of PSA gene.(4.rAAV/PSA Nsi I restriction endonuclease).(5.rAAV/PSA EcoR V and EcoR I restriction endonuclease).(6.rAAV/PSA Not I and BamH I restriction endonuclease).(7.rAAV/PSA Pst I restriction endonuclease).), cut the specific band that obtains 6.2kb through Nsi I enzyme, cut through EcoR V and EcoR I enzyme the specific band that has obtained 1.1kb and 2.0kb, cut through Not I and BamH I enzyme the specific band that obtains 2.0kb, cut through Pst I enzyme the specific band that has obtained 1.3kb and 2.5kb, conform to expected results.The enzyme of rAAV/mPSA plasmid is cut detected result and is also conformed to expected results.Again rAAV/PSA plasmid and rAAV/mPSA plasmid are done further detection with the method for gene amplification (PCR), wherein the detected result of rAAV/PSA plasmid (3.PCR gene amplification product as shown in Fig. 2 A.), obtained the specific band of the expection of 1006bp through amplification.The PCR detected result of rAAV/mPSA plasmid also conform to expected results (size of amplified production is followed successively by 240bp, 171bp, 228bp, 147bp).Above-mentioned detected result shows and has obtained on position and the sequence all correct recombined glandulae correlation viral vectors rAAV/PSA that carries prostate specific antigen (PSA) gene and and the recombined glandulae correlation viral vectors rAAV/mPSA that carries prostate specific antigen (PSA) mutated genes.
The preparation of embodiment 1-2, recombinant adeno-associated virus (rAAV) and virus titer are measured
Material and source thereof:
A. the recombined glandulae correlation viral vectors rAAV/PSA that carries prostate specific antigen (PSA) gene that builds of embodiment 1-1 and and the recombined glandulae correlation viral vectors rAAV/mPSA (rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA) that carries prostate specific antigen (PSA) mutated genes.
B. contain the Rep gene of AAV and the helper plasmid pHelper of Lip/Cap gene: build (Liu by hospital attached to a medical college gene therapy center professor Liu Yong of U.S. University of Arkansas, Y., Chiriva-Internati, M., Grizzi, F.Salati, E., Roman, J.J., Lim S., and Hermonat, P.L.Rapid induction of cytotoxic T cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector.Cancer Gene Therapy 8:948-957.).
C. contain the adenoviral gene (E1 that is integrated in cell chromosome and expresses, E2A, E4, VAI and VAII gene) the AAV-HEK293 cell: set up (Liu by U.S. University of Arkansas hospital attached to a medical college gene therapy center, Y., Chiriva-Internati, M., Grizzi, F.Salati, E., Roman, J.J., Lim S., and Hermonat, P.L.Rapid induction of cytotoxic T cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector.Cancer Gene Therapy 8:948-957.).
D. lipofectamine Lipofectin: available from American I nvotrogen company.
E.DMEM substratum and foetal calf serum (or calf serum): available from U.S. Cellgro company.
F.PCR DIG labelling kit and DIG hybridization check test kit: available from Switzerland Roche company.
G.DNA copy number standard: be respectively 10
12Copy number (copies)/μ l to 10
6(copies)/μ l is available from U.S. Promega company.
One, the preparation of recombinant adeno-associated virus (rAAV)
With reference to Fig. 3, prepare recombinant adeno-associated virus (rAAV) with following method, take the virus for preparing a dish 10.0cm Tissue Culture Dish as example, when the AAV-HEK293 cell grows in carbon dioxide cell incubator when accounting for culture dish area 70%, proceed as follows:
A. the operation instruction according to Lipofectin operates: with 1.0 μ g rAAV/PSA or rAAV/mPSA, 1.0 μ g pHelper plasmid, 4.0 μ l Lipofectin and 50.0 μ l contain the DMEM substratum mixing of 5% foetal calf serum (or calf serum), standing 20 minutes of room temperature.
B. mixed solution is added in Tissue Culture Dish, continue to be placed in carbon dioxide cell incubator and cultivate.
C.72 hour after, all cells and nutrient solution in the results culture dish.
D. thermal agitation is after 1 minute, and is centrifugal, keeps supernatant, i.e. rAAV virus liquid.
E. with the rAAV virus liquid filtration sterilization of collecting.With the rAAV virus difference called after rAAV/PSA, rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA, the rAAV/DmPSA that carry tumor antigen gene-prostate specific antigen full length gene PSA cDNA or part PSA cDNA fragment (A, B, C, D, mutated genes) that obtains.
Two, the virus titer of recombinant adeno-associated virus (rAAV) is measured
Adopt conventional spot hybridization, the various rAAV viruses (rAAV/PSA, rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA, rAAV/DmPSA) that step 1 obtains are carried out virus titer mensuration, and concrete grammar comprises the following steps: only DNA probe used is the specific probe for the PSA gene.
A. adopt conventional DNA phenol/chloroform extraction method, extract rAAV virion DNA.
B. nylon membrane is placed in the Dot blot instrument, adds through the rAAV of alkaline denaturation virion DNA, and add DNA copy number standard, vacuumize.
C. after taking out the nylon membrane drying, ultraviolet ray is fixing.
D. prepare the specific probe of DIG mark with PCR DIG labelling kit and reference reagent box specification sheets, probe is " the PSA cDNA that obtains in embodiment 1-1 step C.Pcr amplification carries out 1.2% agarose gel electrophoresis to pcr amplification product after finishing, and detects pcr amplification product under ultraviolet ray, and positive band appears in result, shows the probe mark success.
E. use DIG hybridization check test kit and reference reagent box specification sheets, in hybrid heater, various rAAV virion DNA are carried out DNA hybridization.
Wherein, the detected result of rAAV/PSA as shown in Figure 4, the virus titer of rAAV/PSA is 10
12-10
11Copy/Ml.The virus titer of four kinds of rAAV/mPSA also can reach 10
12-10
11Copy/mL.
Embodiment 1-3, tumour antigen import the tumor experiment of killing of monocyte-dendritic cell system
Material and source thereof:
A.rAAV virus: rAAV/PSA and rAAV/mPSA (rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA).
B.AIM-V cell culture medium: available from American I nvitrogen company.
C. cytokine: granulocyte colony stimulating factor (GM-CSF), interleukin II, 4,7 (IL-2,4,7) and tumour necrosis factor (TNF-α) are available from U.S. R﹠amp; D company.
One, kill tumor experiment
As shown in Figure 5, the rAAV virus infection tumour patient monocyte that carries prostate specific antigen gene and mutated genes thereof of the present invention is comprised the following steps for the basic whole process of killing tumor experiment:
A. get tumour patient 50-150 milliliter peripheral blood, obtain according to a conventional method peripheral blood mononuclear cell (PBMC) with hemocyte separometer (or lymphocyte separation medium), after AIM-V substratum mixing, add Tissue Culture Flask, be placed in the constant temperature CO2gas incubator and cultivated 2 hours.
B. remove suspension cell, keep attached cell (monocyte, monocyte, Mo).Suspension cell is peripheral blood lymphocyte, with after itself and AIM-V substratum mixing, continues to cultivate standby.
C. the rAAV virus that adds a kind of (or multiple, effect is better) embodiment of the present invention 1-2 to obtain, add-on is about 100-1000MOI, adds GM-CSF (800IU/mL) simultaneously again, continues to cultivate 4 hours.
D. remove old substratum, replenish and to contain GM-CSF, the AIM-V substratum of IL-4 (800IU/mL) and TNF-α (20IU/mL) continues to cultivate.
E. after cultivating 5 days, the dendritic cell (DC) that results are ripe, and mix with the peripheral blood lymphocyte of cultivating, add IL-2 (20IU/mL) and IL-7 (500IU/mL) in the AIM-V substratum, continue to cultivate.
F. after being cultured to 7-9 days, the cytotoxic T lymphocyte (CTL) that results activate detects.
Two, the detection of dendritic cell (DC) and cytotoxic T lymphocyte (CTL)
The efficient that A.rAAV infects peripheral blood lymphocytes detects
Adopt conventional fluorescence antibody mark staining, use the monocyte that is infected by rAAV of the present invention or immature DC for specificity fluorescent antibody (available from U.S. company BD) markers step one acquisition of tumor associated antigen-prostate specific antigen (PSA), then carry out the quantity that flow cytometer detects positive cell.Wherein, the efficient detected result of recombinant adeno-associated virus rAAV/PSA infection peripheral blood lymphocytes as shown in Figure 6, the efficient that rAAV/PSA infects peripheral blood lymphocytes is 88.4%, the efficient that the various rAAV (rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA) that carry tumour antigen constructed and preparation infect peripheral blood lymphocytes all is about 90%, namely approximately 90 percent peripheral blood lymphocytes can by the rAAV virus infection, prove that rAAV of the present invention has higher efficiency of infection.
B. the detection of the CD molecular level of dendritic cell (DC)
DC expresses CD80, CD83 and the level of CD86 and the function of DC and is proportionate.With the detection method identical with steps A, the level that the DC that namely adopts respectively fluorescently-labeled antibody for these three kinds of CD molecules (available from U.S. company BD) that step 1 is obtained expresses CD80, CD83 and CD86 detects, and the DC that stimulates take PSA albumen and non-stimulated DC are as contrast.Wherein, the detected result that the DC that recombinant adeno-associated virus rAAV/PSA infects expresses CD80, CD83 and CD86 level as shown in Figure 7, the expressed CD molecular level of the DC that is infected by rAAV/PSA is apparently higher than contrast.The DC that rAAV/mPSA (rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA) infects is also apparently higher than contrast.After the rAAV that carries tumor associated antigen-prostate specific antigen and mutant thereof of proof structure and preparation infected peripheral blood lymphocytes, the DC's that induces was powerful.
C. the detection of IFN-γ (IFN-γ) level of cytotoxic T lymphocyte (CTL) expression
The expression level of the function of CTL and the ability of killing tumor cell thereof and IFN-γ is proportionate.(DC that stimulates take PSA albumen and the non-stimulated CTL that DC induced are as contrast with detecting with the similar method of steps A the level that the CTL that DC induced that is infected by rAAV of the present invention expresses IFN-γ.), after DC and peripheral blood lymphocyte mixed culture finished, harvested cell adopted traditional Intracellular cytokine staining methods to carry out the cell fluorescence dye marker, antibody used is for the fluorescent-labeled antibody of IFN-γ (available from U.S. company BD), utilizes at last the flow cytometer detected result.Wherein, the IFN-γ expression level of the CTL that DC induced that is infected by rAAV/PSA as shown in Figure 8, the CTL that DC induced that is infected by rAAV/PSA expresses the level of IFN-γ apparently higher than contrast.The DC that rAAV/mPSA (rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA) infects is also apparently higher than contrast.The CTL that DC induced that proof is built by the present invention and the rAAV that carries tumour antigen-prostate specific antigen and mutant thereof for preparing infects is powerful.
D. cytotoxic T lymphocyte (CTL) killing tumor cell test
After mixed culture finishes, with the cytotoxic T lymphocyte that DC induced that infected by rAAV/PSA in step 1 by 20: 1 (lymphocyte: tumour cell) with after prostate cancer cell mixes, adopt traditional mtt assay with
51Cr (chromium-51) fragmentation test, the activity of detection CTL killing tumor cell.The tumor cell destruction statistics of the CTL that DC induced that is wherein infected by rAAV/PSA as shown in Figure 9, the more effectively cracking of the CTL that DC induced (killing and wounding) that the rAAV that carries tumour antigen-prostate specific antigen and mutant thereof that is built by the present invention and prepare infects is from four routine patients with prostate cancer (A, B, C, D) isolate prostate cancer cell in, kill rate can reach 43-52%.The CTL that DC induced that is infected by rAAV/mPSA (rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA) also obtains similar kill rate.
With lung (lung), mammary gland (breast), liver (liver), the cell of kidney (k-cells) is contrast, then uses in above-mentioned identical method detecting step one by the specificity of the cytotoxic T lymphocyte killing tumor cell that DC induced of rAAV (rAAV/PSA, rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA) infection.wherein, the tumor cytotoxicity specific detection result of the CTL that DC induced that is infected by rAAV/PSA as shown in Figure 9, the CTL that DC induced that the rAAV that carries tumor associated antigen-prostate specific antigen and mutant thereof that is built by the present invention and prepare infects is to lung (lung), mammary gland (breast), the cell of liver (liver) and kidney (k-cells) is without lethal effect, the CTL that DC induced that proof is built by the present invention and the rAAV that carries tumor associated antigen-prostate specific antigen and mutant thereof for preparing infects has antigen-specific, namely to the cell of antigen negative without lethal effect.
Above-mentioned detected result shows, carried by the present invention the CTL that DC (being referred to as rAAV-DC) that the rAAV of tumour antigen-prostate specific antigen and mutant thereof infects induced prostate cancer is had curative effect preferably, can be used for preparing antitumor drug.
The clinical experiment of embodiment 1-4, oncotherapy
One, curative effect and survival time are detected
Use recombinant adeno-associated virus-dendritic cell technology, the CTL that the DC (rAAV-DC) that is about to be infected respectively by rAAV of the present invention (rAAV/PSA, rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA) in embodiment 1-3 is induced feeds back 20 routine patients with prostate cancer, and infusion amount is 1 * 10
9-5 * 10
9Treat the course for the treatment of: be generally 6 months, 2-3 time per month, the state of an illness can be kept to 1-2 time per month after improving, and further can be kept to and treat once every 1-3 month.(B: blood serum tumor markers reduces or disappears result for the treatment of (reaction after the rAAV-DC treatment) statistics as shown in table 1-1.Q: quality of life of patients improves.As pain relief or disappearance, appetite increases etc.C:CT or PET-CT shows that cancer focus or metastatic lesion obviously reduce or disappear.), untoward reaction: can occur slight influenza sample reaction after majority of cases treatment in the short period of time, but patient can bear all, and symptom disappears in a short time, do not observe serious adverse reaction and toxic reaction.(survival time after treatment: patient begins to accept the survival time (when death is calculated to death) after the rAAV-DC treatment as shown in table 1-2 for the course for the treatment of and survival time statistics.), the equal unprovoked rAAV-DC of death treatment causes, and this group patient great majority are in cancer whole latter stage, and part patient causes immunologic function, liver and kidney failure because of excessive chemicotherapy.Above-mentioned statistics further proves, the CTL that the DC (being referred to as rAAV-DC) that is infected by rAAV of the present invention is induced can bring into play certain curative effect in patient body, growth or the killing off tumor cells that can effectively suppress malignant cell, and security is higher, can be used for preparing antitumor drug.
Table 1-1 recombinant adeno-associated virus-dendritic cell technology (rAAV-DC)
Treat the statistics of the curative effect of 20 routine patients with prostate cancer
The course for the treatment of and the survival time statistics of table 1-2 20 routine cancer patients
Two, the changing conditions of Tumor Patient Before and After Treatment iconography aspect and blood serum tumor markers aspect
The changing conditions of A, Tumor Patient Before and After Treatment iconography aspect
metastatic lesion changing conditions before and after 20 routine patients with prostate cancer treatments in step 1 is carried out iconography observation, wherein a routine IV phase through the iconography observed result of metastatic lesion changing conditions before and after the CTL treatment that the DC that rAAV/PSA infects induces as shown in Figure 10 A (before treatment and after treating 6 months), after the CTL treatment that the DC (rAAV-DC) that result infects through rAAV of the present invention induces, patient's metastatic lesion obviously disappears, further prove, the CTL that DC induced that is infected by rAAV of the present invention can suppress growth or the killing off tumor cells of malignant cell effectively in patient body, can be used for preparing antitumor drug.
The changing conditions of B, treatment Patients Before And After serum tumor antigen levels
in the CTL treatment Patients Before And After serum that the DC that four examples infect through rAAV/PSA induces, the changing conditions of PSA tumour antigen level (data from the detected result of experiment hospital) is as shown in Figure 11 A, after the CTL treatment that the DC (rAAV-DC) that result infects through rAAV of the present invention induces, its serum tumor antigen PSA level all obviously descends, show that in patient body, the knurl lifting capacity obviously reduces (tumour cell obviously reduces), further prove, the CTL that DC induced that is infected by rAAV of the present invention can suppress growth or the killing off tumor cells of malignant cell effectively in patient body, can be used for preparing antitumor drug.
Industrial applicability
Experimental results show that, can effectively be suppressed growth or the killing off tumor cells of associated malignancies cell by PSA recombinant adeno-associated virus the rAAV of the present invention dendritic cell that infects and the cytotoxic T lymphocyte of being induced in patient body, thereby, PSA recombined glandulae correlation viral vectors of the present invention or the product relevant to PSA recombined glandulae correlation viral vectors of the present invention can be used to prepare antitumor drug, have great importance at the clinical treatment of malignant tumour such as prostate cancer etc. with in using.
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| CNA2008100932231A Withdrawn CN101307329A (en) | 2007-04-23 | 2008-04-23 | A Her-2/neu recombinant adeno-associated virus vector and its construction method and application |
| CNA2008100932227A Withdrawn CN101307328A (en) | 2007-04-23 | 2008-04-23 | A kind of CK19 recombinant adeno-associated virus vector and its construction method and application |
| CN2011101256651A Active CN102268453B (en) | 2007-04-23 | 2008-04-23 | LMP-1 recombinant adeno-associated virus (rAAV) vector and construction method as well as application thereof |
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| CN2011101256435A Active CN102268455B (en) | 2007-04-23 | 2008-04-23 | CEA (chorioembryonic antigen) recombinant adeno-associated viral vector, constructing method thereof and application thereof |
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| CNA2008100932227A Withdrawn CN101307328A (en) | 2007-04-23 | 2008-04-23 | A kind of CK19 recombinant adeno-associated virus vector and its construction method and application |
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| CA3192967A1 (en) | 2020-10-15 | 2022-04-21 | Aiquan CHANG | Recombinant adeno-associated virus vectors with cd14 promoter and use thereof |
| CN116355058A (en) * | 2020-11-12 | 2023-06-30 | 天津大学 | Epstein-Barr virus epitope and its application |
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Also Published As
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|---|---|
| CN102268454A (en) | 2011-12-07 |
| CN101255442A (en) | 2008-09-03 |
| CN102277384A (en) | 2011-12-14 |
| CN102268457A (en) | 2011-12-07 |
| CN101307329A (en) | 2008-11-19 |
| CN101307326A (en) | 2008-11-19 |
| CN102268455A (en) | 2011-12-07 |
| CN102268456A (en) | 2011-12-07 |
| CN102268458A (en) | 2011-12-07 |
| CN102268456B (en) | 2013-02-06 |
| CN102268453A (en) | 2011-12-07 |
| CN101307327A (en) | 2008-11-19 |
| CN102268457B (en) | 2013-06-26 |
| CN102268455B (en) | 2013-09-18 |
| CN101255441A (en) | 2008-09-03 |
| WO2008128440A1 (en) | 2008-10-30 |
| CN101680002A (en) | 2010-03-24 |
| CN101307328A (en) | 2008-11-19 |
| CN102268458B (en) | 2013-04-03 |
| CN101680002B (en) | 2011-11-23 |
| CN102268453B (en) | 2013-04-03 |
| CN101307330A (en) | 2008-11-19 |
| CN102268454B (en) | 2013-07-24 |
| CN101307325A (en) | 2008-11-19 |
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