CN102268453B - LMP-1 recombinant adeno-associated virus (rAAV) vector and construction method as well as application thereof - Google Patents
LMP-1 recombinant adeno-associated virus (rAAV) vector and construction method as well as application thereof Download PDFInfo
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Abstract
The invention discloses an LMP-1 recombinant adeno-associated virus (rAAV) vector and a construction method as well as application thereof. The rAAV is obtained by replacing an AAV structure gene in an AAV vector by a tumor antigen gene LMP-1 or a mutant gene thereof. A wild type or mutant type carcinoma embryonic antigen (CEA) gene carried by the rAAV disclosed by the invention can be conveyed into a mononuclear cell-dendritic cell system to be used for stimulating the effector cells of an immune system. The experiment proves that the cytotoxic T lymphocyte (CTL) induced by the dendritic cell (DC) infected by the rAAV disclosed by the invention can effectively inhibit the growth of malignant tumor cells or kill tumor cells in the body of a patient. The rAAV vector or the related products thereof disclosed by the invention can be used for preparing drugs for treating nasopharyngeal carcinoma.
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 the biological field, particularly relate to a kind of LMP-1 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 the 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 rich fertile gene international corporation of U.S. 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, mainly be 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, mainly be that the AAV virus of carrying therapeutic gene is injected in the patient body, make its in vivo expression treatment gene, 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 brings out autoimmune response easily in patient body, even causes serious toxic side effect.
AAV is a kind of defective virus of non-virulent, needs the gene product of other virus (such as adenovirus) auxiliary, just can be assembled into to have infective virion.About 4700 base pairs of AAV genome total length (bp), 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.Owing to have the unstable of 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 among the rAAV, is prepared into to have infective rAAV virion, is injected in the patient body, makes its infectosome inner cell, and then the expression treatment gene, thereby reaches the effect for the treatment of disease.At present, mainly be the treatment that rAAV is applied to 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, unstable such as 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 height, carry allogenic gene LMP-1 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 LMP-1 gene of Epstein-Barr virus or the recombined glandulae correlation viral vectors that its mutated genes obtains.
LMP-1 recombined glandulae correlation viral vectors of the present invention is that the basis transformation at known gland relevant viral vector obtains, described adeno-associated virus structure gene is Rep and Lip/Cap gene, tumour specific antigen gene LMP-1 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 the described recombined glandulae correlation viral vectors be replaced with one or several promotor in scavenger cell virus (cytomegalovirus, CMV) promotor, beta actin promoter and the SV40 viral promotors.
Second purpose of the present invention provides the construction process of above-mentioned LMP-1 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 the gland relevant viral vector is rejected, replace this rejecting gene with aforementioned specific antigen gene LMP-1 or its mutated genes m LMP-1 again, obtain the LMP-1 recombined glandulae correlation viral vectors.
In the construction process of above-mentioned LMP-1 recombined glandulae correlation viral vectors, for improving the transcriptional level of goal gene, also can further the p5 promotor in the described recombined glandulae correlation viral vectors be replaced with one or several promotor in scavenger cell viral promotors, beta actin promoter and the SV40 viral promotors.
The product relevant with LMP-1 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 such as monocyte-dendritic cell system, T lymphocyte series etc. (as described in the LMP-1 recombined glandulae correlation viral vectors genes involved or its mutated genes can in the clones such as monocyte-dendritic cell system or T lymphocyte series, obtain to express under the effect at transcripting promoter) etc.
Medicinal use aspect, another object of the present invention provide a kind of antitumor drug.
The activeconstituents of antitumor drug provided by the present invention is above-mentioned LMP-1 recombined glandulae correlation viral vectors or the product relevant with LMP-1 recombined glandulae correlation viral vectors of the present invention.
As take LMP-1 recombinant adeno-associated virus of the present invention as carrier, tumour antigen-wild-type and/or mutant tumour specific antigen gene are imported monocyte-dendritic cell system, and induce producing dendritic shape cell, to reach outside the patient body and immunostimulating purpose in the body, 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 nasopharyngeal carcinoma etc.
Medicine provided by the present invention can adopt the formulations such as solvent or pulvis.
The selection of described solvent is diversified, all can such as cell culture fluid (base), physiological saline or phosphate buffered saline buffer etc.
When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, absorption enhancer and the tensio-active agent etc. of pharmaceutical field routine.
Application method can be the monocyte of isolating first in the tumour patient body, this medicine is infected or transfection patient's monocyte again.Maybe conversion there is the cytotoxic T lymphocyte of generation that dendritic cell stimulates of maturation of the LMP-1 of wild-type and/or mutant Epstein-Barr virus 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 may further comprise the steps:
Recombinant gland relevant viral vector infection or the transfection of 1) spontaneous monocyte-dendritic cell or T lymphocyte in the 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 with recombined glandulae correlation viral vectors of the present invention, the cell after obtaining separately processing;
2) with step 1) in monocyte-dendritic cell after processing add in the system of tumour place and kill tumour; Or with not processed T lymphocyte and the monocyte after the described processing-dendritic cell mixed culture formation Antigen-specific cytotoxic T lymphocyte, will kill tumour in this Antigen-specific cytotoxic T lymphocyte adding tumour place system again; Or will kill tumour in processed T lymphocyte and the not processed monocyte-dendritic cell adding tumour place system.
The method of killing tumour of the present invention can specifically be applied in the 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 with 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 with 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 with recombined glandulae correlation viral vectors of the present invention.
The invention provides a kind of stability height, carry allogenic gene LMP-1 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 LMP-1 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 the 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, growth or the killing off tumor cells that in patient body, can effectively be suppressed the associated malignancies cell by the rAAV of the present invention dendritic cell that infects and the cytotoxic T lymphocyte of being induced, thereby recombined glandulae correlation viral vectors of the present invention or the product relevant with 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 F-1 is that the enzyme of rAAV/LMP-1 is cut the result, and Fig. 2 F-2 is the PCR detected result of rAAV/LMP-1.
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/LMP-1.
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/LMP-1 infects peripheral blood lymphocytes.
Fig. 7 is the detected result that the DC of recombinant adeno-associated virus rAAV/LMP-1 infection expresses CD80, CD83 and CD86 level.
Fig. 8 is the IFN-γ expression level detected result of the CTL that induces of DC that rAAV/LMP-1 infects.
Fig. 9 is that the specific detection result is tested and killed and wounded to the CTL killing tumor cell that the DC of rAAV/LMP-1 infection induces.
Figure 10 is the iconography observed result of routine Nasopharyngeal Carcinoma Patients metastatic lesion changing conditions before and after the CTL treatment that the DC that rAAV/LMP-1 infects induces
Figure 11 is the changing conditions of four routine Nasopharyngeal Carcinoma Patients CK19 tumor associated antigen level in the CTL treatment Patients Before And After serum that the DC that rAAV/LMP-1 infects induces.
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, 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.
The primer, the synthetic determined dna sequence that reaches of dna sequence dna are finished by American I nvitrogen company.
The 6th part: recombined glandulae correlation viral vectors rAAV/LMP-1
Structure and the detection of embodiment 6-1, recombined glandulae correlation viral vectors rAAV/LMP-1 and rAAV/mLMP-1
Material and source thereof:
A. carry the pBR322 plasmid (called after pBR-AAV2) of AAV 2 type complete genome DNAs: by one of rich major technique person in charge of the technology aspect who irrigates gene international corporation of U.S. 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. KB cell: separate obtaining or buy from the commercial channel from the cancerous tissue of Nasopharyngeal Carcinoma Patients, immunohistochemical methods confirms that LMP-1 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 Epstein-Barr virus LMP-1 mRNA sequence of publishing in U.S.'s gene pool (U.S. NCI gene pool: AF304432) design.
Make up the recombined glandulae correlation viral vectors (as shown in Figure 1) that the present invention carries LMP-1 gene or its mutated genes with following method, detailed process may further comprise the steps:
One, the structure of recombined glandulae correlation viral vectors
The reconstruction of A.pBR-AAV2 plasmid, concrete grammar is: use first 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 the 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 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 above-mentioned preparation of 1 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, 9 nucleotide fragments are inserted in the plasmid, reaction system is: 500ng plasmid, 9 nucleotide sequences of 300ng, 10IU T again
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 the sequence table) and primer 2: TGTCAGAAGCACTGACTGC (SEQ ID NO:2 in the 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 behind the 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 the 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 behind the purifying.
C.PCR amplification beta actin promoter, total length LMP-1 cDNA and part LMP-1 cDNA fragment (difference called after A (from 5 ' end 1-268 bit base), B (from 5 ' end 347-433 bit base), C (from 5 ' end 511-1370 bit base), present embodiment is as an example of above-mentioned fragment example but be not limited to above-mentioned fragment, the LMP-1cDNA fragment that other and LMP-1cDNA have identical function all can be used for making up recombined glandulae correlation viral vectors of the present invention), concrete grammar is: adopt the separate nucleic acid technology, from KB cell, separate total DNA and mRNA (but also synthetic or commercial channel acquisition), then take total DNA as template, pcr amplification beta actin promoter under the guiding of primer 5:CCCGGGCCCAGCACCCCAAG (SEQ ID NO:5 in the sequence table) and primer 6:CATCCATGGTGAGCTGCG (SEQ ID NO:6 in the 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 behind the purifying.Again with synthetic its cDNA of mRNA reverse transcription and as template, pcr amplification total length LMP-1 cDNA under the guiding of primer 7:ATGGAACGCGACCTTGAGAG (SEQ ID NO:7 in the sequence table) and primer 8:TTAGTCATAGTAGCTTAG (SEQ ID NO:8 in the sequence table), the pcr amplification condition is: first 94 ℃ 4 minutes; Again 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute 30 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, the specific band that occurs an expection at the 1370bp place, with obtaining total length LMP-1 cDNA behind this purpose band recovery and the purifying, obtain LMP-1 cDNA Segment A (primer 7 and primer 9:TCATCAGTAGGAGTAGACC (SEQ ID NO:9 in the sequence table)) with above-mentioned same procedure, LMP-1 cDNA fragment B (primer 10:TCACCCTCCTACTTCATCG (SEQ ID NO:10 in the sequence table) and primer 11:CAAGTAAGCAGCCAAAGATG (SEQ ID NO:11 in the sequence table)), LMP-1 cDNA fragment C (primer 12:TCTTAGGTCTCTGGATCTAC (SEQ ID NO:12 in the sequence table) and primer 8).
D. adopt the DNA interconnection technique, with CMV promotor, SV40 early promoter, beta actin promoter, total length LMP-1 cDNA or the part LMP-1 cDNA fragment of above-mentioned amplification successively inserting step A 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 Xba I and BamH I enzyme respectively carrying the plasmid of promotor and the LMP-1cDNA 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/LMP-1) of CMV promotor, SV40 early promoter, beta actin promoter and total length LMP-1 cDNA, and the recombined glandulae correlation viral vectors (difference called after rAAV/AmLMP-1, rAAV/BmLMP-1 and rAAV/CmLMP-1, unified called after rAAV/mLMP-1) that carries CMV promotor, SV40 early promoter, beta actin promoter and A or B or C Different L MP-1cDNA fragment (mutant).
E. the DNA-rAAV/LMP-1 after will connecting and rAAV/mLMP-1 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/LMP-1 plasmid and rAAV/mLMP-1 plasmid.
Two, the detection of recombined glandulae correlation viral vectors
The purified rAAV/LMP-1 plasmid and the rAAV/mLMP-1 plasmid that first step 1 are obtained (are followed successively by Xba I﹠amp with restriction enzyme; BamHI, Xba I﹠amp; Not I, EcoR V﹠amp; Sal I, EcoR V﹠amp; BamHI, Pst I) carry out enzyme and cut, used restriction enzyme is all available from U.S. Promega company.(CMV promotor, SV40 early promoter, the beta actin promoter successively inserting step A pBR-AAV2 carrier through reconstruction is obtained with the negative contrast of rAAV carrier without the LMP-1 gene simultaneously, this carrier is cut with restriction enzyme Pst I enzyme), after reaction finishes, enzyme is cut product carry out the detection of 1.2% agarose gel electrophoresis, the wherein detected result of rAAV/LMP-1 plasmid (1.rAAV/LMP-1 (Xba I and BamH I restriction endonuclease) shown in Fig. 2 F-1.(2.rAAV/LMP-1 Xba I and NotI restriction endonuclease).(3.rAAV/LMP-1 EcoR V and Sal I restriction endonuclease).(4.rAAV/LMP-1 EcoR V and BamHI restriction endonuclease).(5.rAAV/LMP-1 Pst I restriction endonuclease).6.DNA molecular weight standard.), cut the specific band that has obtained 1370bp through XbaI and BamHI enzyme, cut the specific band that has obtained 1370kb through Xba I and NotI enzyme, cut the specific band that has obtained 2213bp through EcoR V and Sal I enzyme, cut the specific band that has obtained 876bp through EcoR V and BamHI enzyme, cut the specific band that has obtained 684bp and 1003bp through Pst I enzyme, conform to expected results.The enzyme of rAAV/mLMP-1 plasmid is cut detected result and is also conformed to expected results.Again rAAV/LMP-1 plasmid and rAAV/mLMP-1 plasmid are done further detection with the method for gene amplification (PCR), wherein the detected result of rAAV/LMP-1 plasmid (1.DNA molecular weight standard shown in Fig. 2 F-2.2.LMP-1cDNA pcr amplification product.3. positive control.), obtained the specific band of the expection of 1370bp through amplification.The PCR detected result of rAAV/mLMP-1 plasmid also conform to expected results (size of amplified production is followed successively by rAAV/AmLMP-1:268bp, rAAV/BmLMP-1:87bp, rAAV/CmLMP-1:860bp).Above-mentioned detected result shows and has obtained on position and the sequence all correct recombined glandulae correlation viral vectors rAAV/LMP-1 that carries the LMP-1 gene and and the recombined glandulae correlation viral vectors rAAV/mLMP-1 that carries the LMP-1 mutated genes.
The preparation of embodiment 6-2, recombinant adeno-associated virus (rAAV) and virus titer are measured
Material and source thereof:
A. the recombined glandulae correlation viral vectors rAAV/LMP-1 that carries the LMP-1 gene that makes up of embodiment 6-1 and and the recombined glandulae correlation viral vectors rAAV/mLMP-1 (rAAV/AmLMP-1, rAAV/BmLMP-1, rAAV/CmLMP-1 and rAAV/DmLMP-1) that carries the LMP-1 mutated genes.
B. contain the Rep gene of AAV and the helper plasmid pHelper of Lip/Cap gene: make up (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)/and μ l, 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 carriers (rAAV/LMP-1 or rAAV/mLMP-1), 1.0 μ g pHelper plasmid, 4.0 μ l Lipofectin and 50.0 μ l contain the DMEM substratum mixing of 10% foetal calf serum (or calf serum), room temperature left standstill 20 minutes.
B. mixed solution is added in the Tissue Culture Dish, continue to place carbon dioxide cell incubator to 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/LMP-1, rAAV/AmLMP-1, rAAV/BmLMP-1, the rAAV/CmLMP-1 that carry tumor associated antigen gene LMP-1 full length gene LMP-1 cDNA or part LMP-1 cDNA fragment (A, B, C 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/LMP-1, rAAV/AmLMP-1, rAAV/BmLMP-1, rAAV/CmLMP-1) that step 1 obtains are carried out virus titer mensuration, and concrete grammar may further comprise the steps: only used dna probe is the specific probe for the tumor associated antigen gene.
A. adopt conventional DNA phenol/chloroform extraction method, extract rAAV virion DNA.
B. nylon membrane is placed the Dot blot instrument, add 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 " LMP-1cDNA that obtains among the embodiment 6-1 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 the 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/LMP-1 as shown in Figure 4, the virus titer of rAAV/LMP-1 is 10
11-10
10Copy/mL, the virus titer of three kinds of rAAV/AmLMP-1 is 10
11-10
10Copy/mL.
Embodiment 6-3, tumor associated antigen import the tumor experiment of killing of monocyte-dendritic cell system
Material and source thereof:
A.rAAV virus: rAAV/LMP-1 and rAAV/mLMP-1 (rAAV/AmLMP-1, rAAV/BmLMP-1, rAAV/CmLMP-1).
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, kills tumor experiment
As shown in Figure 5, one or more rAAV virus infection tumour patient monocytes that carry tumor associated antigen gene (LMP-1 gene and mutated genes thereof) may further comprise the steps for the basic whole process of killing tumor experiment with the present invention:
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), behind AIM-V substratum mixing, add Tissue Culture Flask, place the constant temperature CO2gas incubator to cultivate 2 hours.
B. remove suspension cell, keep attached cell (monocyte, monocyte, Mo).Suspension cell is peripheral blood lymphocyte, with behind itself and the AIM-V substratum mixing, continues to cultivate for subsequent use.
C. add the rAAV virus that a kind of (or multiple, effect is better) embodiment of the invention 6-2 obtains, 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, gather in the crops ripe dendritic cell (DC), and mix with the peripheral blood lymphocyte of cultivating, in the AIM-V substratum, add IL-2 (20IU/mL) and IL-7 (500IU/mL), 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 LMP-1, carry out again the quantity that flow cytometer detects positive cell.Wherein, the efficient detected result of recombinant adeno-associated virus rAAV/LMP-1 infection peripheral blood lymphocytes as shown in Figure 6, the efficient that rAAV/LMP-1 infects peripheral blood lymphocytes is 87%, the efficient that the various rAAV (VrAAV/AmLMP-1, VrAAV/BmLMP-1, VrAAV/CmLMP-1) that carry tumor associated antigen constructed and preparation infect peripheral blood lymphocytes all is about 90%, namely about peripheral blood lymphocytes of 90 percent 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 LMP-1 albumen and non-stimulated DC are as contrast.Wherein, DC expression CD80, the CD83 that recombinant adeno-associated virus rAAV/LMP-1 infects and the detected result of CD86 level are as shown in Figure 7, carried the expressed CD molecular level of DC that the rAAV (rAAV/AmLMP-1, rAAV/BmLMP-1, rAAV/CmLMP-1) of tumor associated antigen infects apparently higher than contrast by VrAAV/LMP-1 and other, after the rAAV that carries tumor associated antigen LMP-1 and mutated genes 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.Express the level of IFN-γ (take the non-stimulated CTL that DC induced as contrast with detecting the CTL that DC induced that is infected by rAAV of the present invention with the similar method of steps A.), 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, used antibody 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/LMP-1 as shown in Figure 8, carried level that the CTL that DC induced that the rAAV (rAAV/AmLMP-1, rAAV/BmLMP-1, rAAV/CmLMP-1) of tumor associated antigen infects expresses IFN-γ apparently higher than contrast by rAAV/LMP-1 and other, prove that the CTL that DC induced of the rAAV infection of carrying tumor associated antigen-LMP-1 and mutated genes thereof that is made up by the present invention and prepare 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 (rAAV/LMP-1, rAAV/AmLMP-1, rAAV/BmLMP-1, rAAV/CmLMP-1) in the step 1 by 20: 1 (lymphocyte: tumour cell) with after nasopharyngeal carcinoma cell mixes, adopt traditional mtt assay with
51Cr (chromium-51) fragmentation test, the activity of detection CTL killing tumor cell.Wherein the tumor cell destruction statistics of the CTL that induces of the DC that infects of rAAV/LMP-1 as shown in Figure 9, with compare with the non-stimulated CTL that DC was induced, more effectively cracking of the CTL that DC induced (killing and wounding) tumour cell that the rAAV that carries tumor associated antigen-LMP-1 and mutated genes thereof that is made up by the present invention and prepare infects, kill rate can reach more than 50%.
Take mammary cancer, colorectal carcinoma, adenocarcinoma of lung and the prostate cancer cell of antigen negative as contrast, use again in the above-mentioned identical method detecting step one specificity of the cytotoxic T lymphocyte killing tumor cell that DC induced that is infected by rAAV (rAAV/LMP-1, rAAV/AmLMP-1, rAAV/BmLMP-1, rAAV/CmLMP-1).Wherein, the tumor cytotoxicity specific detection result of the CTL that the DC that rAAV/LMP-1 infects induces as shown in Figure 9, to the cancer cells of LMP-1 antigen negative without lethal effect, the CTL that the DC that proof the present invention makes up and the rAAV that carries tumor associated antigen-LMP-1 and mutated genes thereof of preparation infects induces has antigen-specific, namely to the cell of antigen negative without lethal effect.
Above-mentioned detected result shows, carried CTL that DC (being referred to as rAAV-DC) that the rAAV of tumor associated antigen-LMP-1 and mutated genes thereof infects induced by the present invention the malignant tumours such as nasopharyngeal carcinoma of LMP-1 antigen positive are had preferably curative effect, can be used for preparing antitumor drug.
The clinical experiment of embodiment 6-4, oncotherapy
One, curative effect and survival time are detected
Use recombinant adeno-associated virus-dendritic cell technology, the CTL that is about to be induced by the DC (rAAV-DC) of one or both infection among the rAAV of the present invention (rAAV/LMP-1, rAAV/AmLMP-1, rAAV/BmLMP-1 and rAAV/CmLMP-1) among the embodiment 6-3 feeds back 8 routine Nasopharyngeal Carcinoma Patients, 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 shown in table 6-1.Q: quality of life of patients improves.Such as pain relief or disappearance, appetite increases etc.C:CT or PET-CT demonstration cancer focus or metastatic lesion obviously reduce or disappear.), untoward reaction: can occur slight influenza sample reaction after the 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 the treatment: patient begins to accept the survival time (when death is calculated to death) after the rAAV-DC treatment shown in table 6-2 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 6-1 recombinant adeno-associated virus-dendritic cell technology (rAAV-DC)
Treat the statistics of the curative effect of 8 routine Nasopharyngeal Carcinoma Patients
The course for the treatment of and the survival time statistics of table 6-2 8 routine Nasopharyngeal Carcinoma 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 the 8 routine radiotherapy in patients with nasopharyngeal carcinomas in the step 1 is carried out iconography observation, wherein the iconography observed result of routine IV phase transitivity Nasopharyngeal Carcinoma Patients metastatic lesion changing conditions before and after the CTL treatment that the DC that rAAV/LMP-1 infects induces as shown in figure 10, the result is through rAAV (rAAV/LMP-1 of the present invention, rAAV/AmLMP-1, rAAV/BmLMP-1 and rAAV/CmLMP-1) in the DC (rAAV-DC) of one or both infection CTL treatment of inducing after, 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 related antigen level
The level of serum tumor related antigen CK19 (data from the detected result of experiment hospital) before and after the 8 routine radiotherapy in patients with nasopharyngeal carcinomas in the detecting step one.Wherein, the changing conditions of four examples CK19 tumor associated antigen level in the CTL treatment front and back patients with nasopharyngeal carcinoma that the DC that rAAV/LMP-1 infects induces as shown in figure 11.After the CTL treatment that the DC (rAAV-DC) of one or both infection of result in rAAV of the present invention (rAAV/LMP-1, rAAV/AmLMP-1, rAAV/BmLMP-1 and rAAV/CmLMP-1) induces, its serum tumor related antigen CK19 level all obviously descends, show that the knurl lifting capacity obviously reduces (tumour cell obviously reduces) in the patient body, 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, growth or the killing off tumor cells that in patient body, can effectively be suppressed the associated malignancies cell by LMP-1 recombinant adeno-associated virus the rAAV of the present invention dendritic cell that infects and the cytotoxic T lymphocyte of being induced, thereby, LMP-1 recombined glandulae correlation viral vectors of the present invention or the product relevant with LMP-1 recombined glandulae correlation viral vectors of the present invention can be used to prepare antitumor drug, in malignant tumour such as prostate cancer, the epithelial cell malignant tumour, mammary cancer, colorectal carcinoma, cancer of the stomach, adenocarcinoma of lung, lung cancer, ovarian cancer, nasopharyngeal carcinoma, cervical cancer, lung squamous cancer, have great importance in the clinical treatment of liver cancer etc. and the application.
Sequence table
＜110〉Health Power Biomed (Tianjin) Inc.
＜120〉a kind of LMP-1 recombined glandulae correlation viral vectors and construction process and application
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Claims (6)
1. LMP-1 recombined glandulae correlation viral vectors, be LMP-1 gene or its mutated genes that the adeno-associated virus structure gene in the gland relevant viral vector is replaced with Epstein-Barr virus, the p5 promotor replaced with CMV promotor, beta actin promoter and SV40 viral promotors obtain; Described LMP-1 mutated genes is: by primer 7:ATTCCGCCGGAGAGCTGTG and the lower LMP-1cDNA Segment A that increases and obtain of primer 9:TCATCAGTAGGAGTAGACC guiding, guide the lower LMP-1cDNA fragment B that increases and obtain by primer 10:TCACCCTCCTACTTCATCG and primer 11:CAAGTAAGCAGCCAAAGATG, or by primer 13:TCTTAGGTCTCTGGATCTAC and the lower LMP-1 cDNA fragment C that increases and obtain of primer 8:CCCAGGACACAGAGAGAGGAC guiding;
Its construction process may further comprise the steps:
The reconstruction of A.pBR-AAV2 plasmid: with restriction enzyme Bst98 I and Hpa I the genomic structure gene Rep of adeno-associated virus AAV and Lip/Cap gene in the pBR-AAV2 plasmid are excised fully first, the nucleotide sequence CGAATTCATGCGATATCGTT that then will contain restriction enzyme EcoR I and EcoR V restriction enzyme site inserts in the plasmid, keeps the complete TR sequence in two ends or the 75th the nucleotide sequence place of the genomic two ends TR of AAV all inserted the fragment CTGCGCTGG that is comprised of 9 Nucleotide;
B. adopt gene amplification amplification CMV promotor and SV40 early promoter;
C.PCR amplification beta actin promoter, total length LMP-1cDNA and described LMP-1cDNA fragment;
D. adopt the DNA interconnection technique, CMV promotor with above-mentioned amplification, the SV40 early promoter, the beta actin promoter, total length LMP-1cDNA or described LMP-1cDNA fragment successively inserting step A through the reconstruction the pBR-AAV2 carrier in, for inserting promotor, at first carry out endonuclease reaction, then carry out ligation, obtain carrying the CMV promotor, the SV40 early promoter, the LMP-1 recombined glandulae correlation viral vectors of beta actin promoter and total length LMP-1cDNA, called after rAAV/LMP-1, or carry the CMV promotor, the SV40 early promoter, the mutant LMP-1 recombined glandulae correlation viral vectors of beta actin promoter and LMP-1cDNA fragment, called after rAAV/mLMP-1.
2. method that makes up the LMP-1 recombined glandulae correlation viral vectors may further comprise the steps:
The reconstruction of A.pBR-AAV2 plasmid: with restriction enzyme Bst98 I and Hpa I the genomic structure gene Rep of adeno-associated virus AAV and Lip/Cap gene in the pBR-AAV2 plasmid are excised fully first, the nucleotide sequence CGAATTCATGCGATATCGTT that then will contain restriction enzyme EcoR I and EcoR V restriction enzyme site inserts in the plasmid, keeps the complete TR sequence in two ends or the 75th the nucleotide sequence place of the genomic two ends TR of AAV all inserted the fragment CTGCGCTGG that is comprised of 9 Nucleotide;
B. adopt gene amplification amplification CMV promotor and SV40 early promoter;
The LMP-1cDNA fragment of mentioning in C.PCR amplification beta actin promoter, total length LMP-1cDNA and the claim 1;
D. adopt the DNA interconnection technique, CMV promotor with above-mentioned amplification, the SV40 early promoter, the beta actin promoter, total length LMP-1cDNA or described LMP-1cDNA fragment successively inserting step A through the reconstruction the pBR-AAV2 carrier in, for inserting promotor, at first carry out endonuclease reaction, then carry out ligation, obtain carrying the CMV promotor, the SV40 early promoter, the LMP-1 recombined glandulae correlation viral vectors of beta actin promoter and total length LMP-1cDNA, called after rAAV/LMP-1, or carry the CMV promotor, the SV40 early promoter, the mutant LMP-1 recombined glandulae correlation viral vectors of beta actin promoter and LMP-1cDNA fragment, called after rAAV/mLMP-1.
3. the product relevant with the described LMP-1 recombined glandulae correlation viral vectors of claim 1 is for the LMP-1 recombined glandulae correlation viral vectors plasmid that prepared by the described LMP-1 recombined glandulae correlation viral vectors of claim 1, LMP-1 recombinant adeno-associated virus or by the clone of LMP-1 recombinant gland relevant viral vector infection or transfection.
4. the preparation method of the product that the described LMP-1 recombined glandulae correlation viral vectors of claim 3 is relevant is respectively:
The preparation of recombined glandulae correlation viral vectors plasmid: increase step e after the described method A-D step of claim 2: the DNA-rAAV/LMP-1 after will connecting or rAAV/mLMP-1 be quiding gene engineering colon bacillus (E.coli) DH5 α competent cell 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/LMP-1 plasmid and rAAV/mLMP-1 plasmid;
The preparation of LMP-1 recombinant adeno-associated virus: obtain LMP-1 restructuring rAAV virus or mutant LMP-1 restructuring rAAV virus, respectively called after rAAV/LMP-1 virus and rAAV/mLMP-1 virus with described LMP-1 recombined glandulae correlation viral vectors plasmid and pHelper plasmid co-transfection AAV-HEK293 cell;
The preparation of the clone of LMP-1 recombinant gland relevant viral vector infection or transfection: infect respectively or successively or transfection monocyte, dendritic cell DC and T lymphocyte CTL obtain with described LMP-1 recombinant adeno-associated virus, described clone comprises monocyte-dendritic cell system and T lymphocyte series.
5. the application of LMP-1 recombined glandulae correlation viral vectors claimed in claim 1 in the anti-medicine for nasopharyngeal of preparation.
The rAAV/LMP-1 for preparing of product claimed in claim 3 or claim 4 method or rAAV/mLMP-1 plasmid, rAAV/LMP-1 or rAAV/mLMP-1 virus, by the LMP-1 recombinant adeno-associated virus infects respectively or successively or transfection obtains monocyte or DC or the application of CTL in the anti-medicine for nasopharyngeal of preparation.
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| CN106591370A (en) * | 2015-10-19 | 2017-04-26 | 南京华贞生物医药科技有限公司 | Virus vector for treating autoimmune related diseases and diabetes, construction method and applications thereof |
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| Publication number | Publication date |
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| CN101307325A (en) | 2008-11-19 |
| CN102268456B (en) | 2013-02-06 |
| CN101307327A (en) | 2008-11-19 |
| CN101255442A (en) | 2008-09-03 |
| CN102268457A (en) | 2011-12-07 |
| CN101307328A (en) | 2008-11-19 |
| CN102268453A (en) | 2011-12-07 |
| CN102268454B (en) | 2013-07-24 |
| CN102268454A (en) | 2011-12-07 |
| CN101307330A (en) | 2008-11-19 |
| CN102268455A (en) | 2011-12-07 |
| CN102277384B (en) | 2013-06-26 |
| WO2008128440A1 (en) | 2008-10-30 |
| CN102268456A (en) | 2011-12-07 |
| CN101680002A (en) | 2010-03-24 |
| CN101680002B (en) | 2011-11-23 |
| CN101307326A (en) | 2008-11-19 |
| CN102277384A (en) | 2011-12-14 |
| CN102268458A (en) | 2011-12-07 |
| CN102268457B (en) | 2013-06-26 |
| CN101255441A (en) | 2008-09-03 |
| CN101307329A (en) | 2008-11-19 |
| CN102268455B (en) | 2013-09-18 |
| CN102268458B (en) | 2013-04-03 |
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