CN105316361A - Recombinant adeno-associated virus vector carrying mutant-type human papillomavirus type 16 E7<m58> antigenic gene and construction method and application of recombinant adeno-associated virus vector - Google Patents
Recombinant adeno-associated virus vector carrying mutant-type human papillomavirus type 16 E7<m58> antigenic gene and construction method and application of recombinant adeno-associated virus vector Download PDFInfo
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Abstract
The invention discloses an rAAV (recombinant adeno-associated virus) vector of an HPV-16 (human papillomavirus type 16) E7<m58> antigenic gene and a construction method and application of the rAAV vector. The rAAV vector is obtained by mutating a tumorigenic HPV-16 E7 antigenic gene into a non-tumorigenic E7 antigenic gene and then inserting the mutated gene into an adeno-associated virus vector with a structural gene removed. The mutant-type HPV-16 E7<m58> antigenic gene carried by the rAAV vector can be delivered into a monocyte-dendritic cell system by the rAAV vector so as to be used for stimulating effector cells of an immune system. Through experiments, a CTL (cytotoxic T lymphocyte) which is induced by a DC (dendritic cell) infected by the rAAV vector is capable of effectively inhibiting growth of HPV-16 E7 positive cells or killing the HPV-16 E7 positive cells including tumor cells and non-tumor cells in vitro and in vivo. The rAAV vector or other related products can be used for preparing antitumor drugs for treating HPV-16 infection and associated diseases.
Description
Technical field
The present invention relates to the carrier in biological technical field and application thereof, particularly relate to one and carry HPV 16 (HumanPapillomavirusType16, HPV-16) saltant type E7
m58the recombined glandulae correlation viral vectors (rAAV) of antigen gene and construction process thereof and its prepare anti-HPV-16 infect and diseases related in application.
Background technology
The gene structure of adeno-associated virus (AAV) is identified.Nineteen eighty-three, the people such as Samulski describe the terminal repetition fragment (upstream 5 ' end fragment of AAV, downstream 3 ' end fragment) (SamulskiRJ, SrivastavaA, BernsKI, MuzyczkaN.Rescueofadeno-associatedvirusfromrecombinantpl asmids:genecorrectionwithintheterminalrepeatsofAAV.Cell. 33:135-143.).1984, the people such as Hermonat describe low infectious particles (Lip) gene and coating (Cap) gene (HermonatPL of AAV, LabowMA, WrightR, BernsKI, MuzyczkaN.Geneticsofadeno-associatedvirus:isolationandpr eliminarycharacterizationofadeno-associatedvirustype2mut ants.JVirol.51:329-339.Hermonat, P.L., andMuzyczka, N.Useofadeno-associatedvirusasamammalianDNAcloningvector: transductionofneomycinresistanceintomammaliantissuecultu recells.Proc.Natl.Acad.Sci.U.S.A.81:6466-6470.).1986, the people such as Labow identify the p5 promotor (LabowMA be positioned between upstream 5 ' end fragment and replication protein (Rep) gene, HermonatPL, BernsKI.Positiveandnegativeautoregulationoftheadeno-asso ciatedvirustype2genome.JVirol.160:251-258.).
AAV is a kind of defective virus of non-virulent, needs the gene product of other virus (as adenovirus) to assist, just can be assembled into and have infective virion.AAV full-length genome about 4700 base pair (bp), two ends are for repeating terminal fragment (TR), and centre is the structure gene of virus, comprises the Rep gene relevant with virus replication and peplos (Cap) gene.Owing to there is the unstable of AAV virus self and carrying the defect of the aspects such as allogenic gene (therapeutic gene) limited length, therefore be necessary that carrying out gene recombination to it forms recombinant adeno-associated virus (recombinantadeno-associatedvirus, rAAV).Existing large quantity research shows, the structure gene in AAV genome is deleted, obviously can increase the capacity of allogenic gene.In addition, medicative for tool allogenic gene is inserted in rAAV, can be prepared into and there is infective rAAV virion.
1984, U.S. PaulL.Hermonat takes the lead in proving that AAV carrier can be used for the gene therapy (Hermonat of human diseases, P.L., andMuzyczka, N.Useofadeno-associatedvirusasamammalianDNAcloningvector: transductionofneomycinresistanceintomammaliantissuecultu recells.Proc.Natl.Acad.Sci.U.S.A.81:6466-6470.).At present, mainly American-European countries in the clinical trial carrying out the gene therapy human diseases based on AAV.According to U.S.'s grain and drug administration's statistics, the Gene Therapy Clinical Trials of existing 10 remainders based on AAV carries out, mainly the AAV virus of carrying therapeutic gene is injected in patient body, make its expression treatment gene in vivo, thus reach the object of disease therapy.Disease mainly for treatment has the non-neoplastic disease such as Parkinsonism, rheumatic arthritis, hemophilia, heart failure, the silent syndromes of progressive myatrophy and Olds sea.The Glybera product of European Union's approval on November 2nd, 2012 UniQure company uses in 27 member statess of European Union, this is the granted gene therapy medicaments of western countries first, and it is the genomic medicine utilizing adeno-associated virus I type (AAV-1) foreign gene-carrying to be used for the treatment of lipoprotein lipase deficiency inherited disease (LPLD).
Human papillomavirus (HPV) is that a kind of papilloma vacuolating virus A belonging to papovaviridae belongs to.At least isolate more than 130 kind of hypotype at present.According to different subtype, high-risk-type and low risk can be divided into.Wherein endanger maximum to the mankind to be high-risk-type, to comprise HPV-16, the malignant tumour such as 18,30,31,33,35,39 and cervical cancer, the rectum cancer, oral carcinoma, carcinoma of tonsil is closely related.Wherein the cervical cancer of more than 99% is caused by high-risk HPV, and cervical cancer more than half is caused by HPV-16.
HPV belongs to the small DNA virus of double-strand closed loop, comprises about 8000 base pairs.Comprising 8 early stage open reading frames (E1-E8), 2 late period single open reading frame and 1 non-coding Chang Kong district.In early days in open reading frame, E6 and the E7 gene pairs Growth of Cells with carcinogenesis stimulates the most important, cancer protein E6, E7 albumen of E6, E7 coding is combined with Suppressor p53 and Rb respectively, cause uncontrolled cellular proliferation, cancer suppressor gene, to the injury repairing afunction of DNA, causes the generation of precancerous lesion and cancer.
According to the investigation result display of 2003-2004 from the national health of the U.S. and nutrient research problem, 14-59 year women the total infection rate of HPV be 26.8%.The popularity that the HPV of China infects not yet has formal report, and about have the cervical cancer newly detected cases of more than 200,000 every year, M & M has increase trend, and the rejuvenation of cervical cancer pathogenesis age, can infer that HPV infection rate is pessimistic.But there is no the method that the definite HPV of healing infects at present, and the HPV vaccine of present stage likely prevents HPV-16 and HPV-18 to infect, but invalid for the crowd infected.For reaching healing object, optimal treatment thoroughly removes infected cell.And all there is HPV-16E7 antigen in infected cell.Therefore, HPV-16E7 antigen is the ideal target of cellular immunotherapy.But HPV-16E7 antigen is a kind of carcinogenic protein, in the generation of the malignant tumours such as cervical cancer, play one of main effect.Therefore, in vivo with the immunoreactive generation of external use wild-type HPV-16E7 antigenic stimulation, in security, there is certain risk.Therefore, the tumorigenicity of wild HPV-16E7 antigen must be removed, eliminate this risk.
Human dendritic cell (DedriticCells, DC) be human body most important, be also topmost antigen presenting cell.No matter in vivo or external large quantifier elimination verified, and DC cell all can induce or stimulate generation to have anti-infective and antineoplastic cell immune response.
Summary of the invention
An object of the present invention is to provide recombinant adeno-associated virus (rAAV) carrier that one carries HPV 16 (HPV-16) the saltant type HPV-16 E7 gene of no pathogenicity (namely without tumorigenicity).
RAAV carrier provided by the present invention, will reject adeno-associated virus (AAV) structure gene Rep and Cap and with p5 promotor, the cytomegalovirus (cytomegalovirus of AAV, CMV) the AAV carrier of any one promotor in promotor, beta actin promoter and SV40 virus early promoter as the carrier that sets out, by saltant type HPV-16E7
m58antigen gene inserts and sets out in carrier, obtains a kind of brand-new rAAV carrier, namely carries HPV-16E7
m58the recombined glandulae correlation viral vectors of antigen gene (is called for short " E7
m58recombined glandulae correlation viral vectors " or AAV/HPV-16E7
m58).
Described AAV carrier is successfully built by the contriver of present patent application, and construction process is see Chinese patent ZL201110125683.X.
Here, saltant type HPV-16E7
m58antigen gene passes through Protocols in Molecular Biology, wild-type HPV-16E7 antigen gene is suddenlyd change, specifically the halfcystine (G) of the 58th, the HPV-16 E7 albumen of HPV-16 (U.S. NCI gene pool: KC935953) is changed into glycine (C), by the thymus pyrimidine (T) of HPV-16E7 gene open reading frame (nt736) is replaced with guanine (G), TGC (nt736-738) by encoding aminothiopropionic acid changes into the GGC of encodes glycine, acquisition can express the saltant type HPV-16E7 antigen gene without tumorigenicity, called after HPV-16E7
m58gene, described HPV-16E7
m58the nucleotide sequence of gene is as shown in sequence in sequence table 1.
Due to HPV-16E7
m58immunogenicity is uninfluenced, can be inserted in the above gland relevant viral vector, this AAV carrier is with p5 promotor, the cytomegalovirus (cytomegalovirus of AAV, CMV) one of promotor, beta actin promoter and SV40 virus early promoter four kinds of promotors, completely newly carries HPV-16E7 by what obtain
m58the recombined glandulae correlation viral vectors of antigen gene is referred to as " E7
m58recombined glandulae correlation viral vectors " or AAV/HPV-16E7
m58.
Utilize above design, also wild-type HPV-16E7 antigen gene can be inserted in above-mentioned gland relevant viral vector, obtain the recombined glandulae correlation viral vectors carrying wild-type HPV-16E7 antigen gene, be referred to as the nucleotide sequence of " E7 recombined glandulae correlation viral vectors " or AAV/HPV-16E7, HPV-16E7 gene as shown in sequence in sequence table 2.But because wild-type HPV-16E7 antigen has tumorigenicity, therefore the present invention does not recommend for clinical practice, only for research.
Second object of the present invention is to provide above-mentioned AAV/HPV-16E7 and AAV/HPV-16E7
m58the construction process of recombined glandulae correlation viral vectors.
Construction process provided by the present invention, comprises the following steps:
1) conventional Protocols in Molecular Biology method is used, first obtain HPV-16E7 antigen gene, suddenly change again, the 58th halfcystine by HPV-16 E7 albumen changes into glycine, detailed process is for replace with guanine (G) by the thymus pyrimidine (T) of HPV-16E7 gene open reading frame (nt736), TGC (nt736-738) by encoding aminothiopropionic acid changes into the GGC of encodes glycine, namely obtains the saltant type HPV-16E7 without tumorigenicity
m58gene;
2) by HPV-16E7
m58antigen gene or wild-type HPV-16E7 antigen gene insert in the gland relevant viral vector of being rejected by adeno-associated virus structure gene Rep and Cap, obtain carrying HPV-16E7
m58recombined glandulae correlation viral vectors (the AAV/HPV-16E7 of antigen gene
m58) or carry the recombined glandulae correlation viral vectors (AAV/HPV-16E7) of HPV-16E7 antigen gene.
E7 in above-mentioned carrier
m58or the promotor of HPV-16 E7 genetic transcription can select any one in the viral early promoter of the p5 promotor of AAV, cytomegalovirus (cytomegalovirus, CMV) promotor, beta actin promoter and SV40.
A present invention also object is to provide and recombined glandulae correlation viral vectors AAV/HPV-16E7
m58the product relevant with recombined glandulae correlation viral vectors AAV/HPV-16E7, comprise recombinant adeno-associated virus plasmid, recombinant adeno-associated virus particle and the clone by recombinant gland relevant viral vector infection of the present invention or transfection, described clone comprises monocyte (Monocytes, Mo), dendritic cell system (Dendriticcells, DC) and lymphocyte.Genes involved-HPV-16E7 in described recombined glandulae correlation viral vectors
m58antigen gene or HPV-16E7 antigen gene can obtain in monocyte or dendritic cell under the effect of above-mentioned transcripting promoter expresses.
Described with recombined glandulae correlation viral vectors AAV/HPV-16E7 and AAV/HPV-16E7
m58the preparation method of relevant product, is respectively:
The preparation of recombined glandulae correlation viral vectors plasmid: by recombined glandulae correlation viral vectors DNA-AAV/HPV-16E7 or AAV/HPV-16E7
m58quiding gene engineering colon bacillus (E.coli) DH5 α competent cell respectively, resistance screening is carried out with the LB flat board containing 100 μ g/mL penbritins, the single bacterium colony of picking white, extracts plasmid and purifying, obtains AAV/HPV-16E7 plasmid and AAV/HPV-16E7
m58plasmid.
The preparation of recombinant adeno-associated virus: with described recombined glandulae correlation viral vectors plasmid AAV/HPV-16E7 plasmid or AAV/HPV-16E7
m58aAV virus is obtained, respectively called after AAV/HPV-16E7 virus and AAV/HPV-16E7 with pHelper plasmid co-transfection AAV-HEK293 cell
m58virus.
The preparation of the clone of recombinant gland relevant viral vector infection or transfection: with described recombinant adeno-associated virus rAAV/HPV-16E7 virus or rAAV/HPV-16E7
m58virus infects respectively or successively or transfection monocyte (Mo), dendritic cell (DC) or lymphocyte obtain.
Practical use aspect, another object of the present invention is to provide medicine and the correlation technique thereof that a kind of anti-HPV-16 infects and infected by HPV-16 the cellular immunotherapy of the tumour caused.
The activeconstituents of described medicine is above-mentionedly carry HPV-16E7
m58recombined glandulae correlation viral vectors (the AAV/HPV-16E7 of antigen gene
m58) or carry HPV-16E7 with the present invention
m58the product (because wild-type HPV-16E7 antigen exists tumorigenicity, therefore not considering that it is medicinal) that the recombined glandulae correlation viral vectors of antigen gene is relevant.
With E7 of the present invention
m58recombinant adeno-associated virus is carrier, by HPV-16 saltant type E7
m58antigen gene imports monocyte, and induces producing dendritic shape cell, also directly can import dendritic cell, expresses E7
m58antigen protein, to reach the immunostimulating object of patient's in vitro and in vivo, infect and associated malignancies in order to treat HPV-16, or stimulate the cytotoxic T lymphocyte (CytotoxicTlymphocytes, CTL) produced to treat HPV-16 infection and associated malignancies with this dendritic cell.
Described HPV-16 infects uterine neck papilloma pathology, cervical cancer, male genital organ Bowen ' s disease, Buschke-Lo&4&wenstein tumor, penile cancer, anus cancer, the rectum cancer, oral carcinoma, carcinoma of tonsil and the mammary cancer etc. that associated malignancies is HPV-16E7 antigen positive.
Medicine provided by the present invention can adopt the formulation such as solvent or pulvis.
The selection of described solvent is diversified, as cell culture fluid (base), physiological saline or phosphate buffered saline buffer etc.
When needing, one or more pharmaceutically acceptable carriers can also be added in said medicine.Described carrier comprises thinner, the absorption enhancer and tensio-active agent etc. of pharmaceutical field routine.
Application method can be the monocyte (Mo) first isolated in patient body, then by after the infection of this medicine or transfection Mo, external evoked Mo becomes the dendritic cell (DC) with antigen presentation function.This medicine also can infect or transfection DC, but likely cause DC to the picked-up of antigen or working ability poor, thus cause unsatisfactory curative effect.The DC obtained can feed back in patient body, reaches therapeutic purpose.Maybe will express HPV-16 saltant type E7
m58the DC of the maturation of antigen stimulate the cytotoxic T lymphocyte of generation (CTL) to feed back patient, to obtain better curative effect.
The consumption of said medicine is generally DC:1-5 × 10
6/ each, CTL:1-5 × 10
8/ each, monthly 2 times, the course for the treatment of is generally 3 months.Dosage and the course for the treatment of all can adjust according to practical situation.
For improving curative effect, medicine of the present invention can also carry out combined therapy with microbiotic, immunostimulant, target and chemotherapeutics etc.
Present invention also offers a kind of method killing the tumour cell of HPV-16 cells infected and the HPV-16E7 positive.
The method can comprise the following steps:
1) monocyte (Mo) will be separated in patient body, or isolated Mo is induced the dendritic cell (DC) that becomes, is carried HPV-16E7
m58recombined glandulae correlation viral vectors (the AAV/HPV-16E7 of antigen gene
m58) infect or transfection, or by the product treatment relevant to recombined glandulae correlation viral vectors of the present invention, obtain the cell after processing separately;
2) by step 1) in DC after process input in patient body, to activate the immune response in patient body, reach the object of the tumour cell killing cell that HPV-16 infects and the HPV-16E7 positive; Or by the DC mixed culture after not processed T lymphocyte and described process, stimulate and produce HPV-16E7 Antigen-specific cytotoxic T lymphocyte (CTL), again by this Peptide-specific CTL input patient body, kill the cell of HPV-16 infection and the tumour cell of the HPV-16E7 positive; Maybe processed CTL and processed DC is inputted in patient body, kill the cell of HPV-16 infection and the tumour cell of the HPV-16E7 positive.
The described method killing tumour can be specifically applied in clinical treatment, comprise and give a patient and feed back HPV-16E7 Antigen-specific cytotoxic T lymphocyte, this cell origin comes from the spontaneous T lymphocyte of patient and produces with the monocyte-dendritic cell mixed culture deriving from this patient.Before mixed culture, these are carried HPV-16E7 by the present invention in monocyte-dendritic cell
m58the recombinant gland relevant viral vector infection of antigen gene or transfection, or by the product treatment relevant to recombined glandulae correlation viral vectors of the present invention;
Or, give a tumour patient and feed back the monocyte-dendritic cell deriving from patient.Before feedback, these are carried HPV-16E7 by the present invention in monocyte-dendritic cell
m58the recombinant gland relevant viral vector infection of antigen gene or transfection, or by the product treatment relevant to recombined glandulae correlation viral vectors of the present invention;
Again or, give tumour patient and feed back the T lymphocyte of the above-mentioned patient of deriving from and derive from the spontaneous monocyte-dendritic cell of this patient.Before feedback, these T lymphocytes are carried HPV-16E7 by the present invention
m58the product treatment that the recombinant gland relevant viral vector infection of antigen gene or the monocyte-dendritic cell of transfection are correlated with.These monocyte-dendritic cell are carried HPV-16E7 by the present invention
m58the recombinant gland relevant viral vector infection of antigen gene or transfection.
The HPV-16E7 that recombinant adeno-associated virus of the present invention (rAAV) carrier can be carried
m58antigen gene is conveyed in monocyte-dendritic cell system, carries with HPV-16E7
m58the cell of antigen gene is used to effector cell's (being not limited to T lymphocyte and bone-marrow-derived lymphocyte) of stimulating immune system.Experiment proves, the dendritic cell infected by rAAV of the present invention and the cytotoxic T lymphocyte of inducing effectively can kill the cell that the tumour cell of HPV-16E7 antigen positive or HPV-16 infect in patient body.Thus, rAAV carrier of the present invention or the product relevant to rAAV carrier of the present invention can be used to prepare antitumor drug.The present invention has important theoretical and practical significance in the clinical treatment and application of malignant tumour, has a extensive future.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 carries E7 or the saltant type E7 of HPV 16 (HPV-16)
m58the structural representation of the recombined glandulae correlation viral vectors of gene.
Fig. 2 obtains by polymerase chain reaction,PCR (PCR) the agarose gel electrophoresis detected result that length is the goal gene HPV-16E7DNA of 297bp.
Fig. 3 HPV-16E7
m58gene order and wild-type HPV-16E7 gene order comparison result.
The AAV/HPV-16E7 that Fig. 4 builds
m58with the restriction analysis result of AAV/HPV-16E7 carrier.
Preparation method's schema of Fig. 5 recombinant adeno-associated virus (rAAV).
Fig. 6 recombinant adeno-associated virus AAV/HPV-16E7
m58with the tumorigenicity observations of rAAV/HPV-16E7 infector for cervical epithelial cells.
Fig. 7 AAV/HPV-16E7
m58the experiment flow killing HPV-16E7 antigen-positive cell based on infected tumor patient dendritic cell.
Fig. 8 is respectively the recombinant adeno-associated virus AAV/HPV-16E7 of four kinds of different promoters (p5, CMVp, SV40p and β-actinp)
m58infect the Efficiency testing result of monocyte (Mo).
Fig. 9 recombinant adeno-associated virus AAV/HPV-16E7
m58with the Flow Cytometry detected result of DC expression CD80 and the CD86 level that AAV/HPV-16E7 infects.
Figure 10 recombinant adeno-associated virus rAAV/HPV-16E7
m58the Flow Cytometry detected result of the IFN-γ expression level of the CTL that the DC infected respectively with rAAV/HPV-16E7 induces.
Figure 11 recombinant adeno-associated virus AAV/HPV-16E7
m58cytotoxic T lymphocyte (CTL) the Cytotoxicity in vitro HPV-16E7 positive cell that the DC infected induces
51cr (chromium-51) killing experiments result.
Figure 125 example is through recombinant adeno-associated virus AAV/HPV-16E7
m58the changing conditions of cervical cancer patient serum Cytokeratin 19 antigen (CK19) and squamous cell carcinoma antigen (SCC) level before and after the CTL treatment that the DC infected induces.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions, concrete steps can be see: " MolecularCloning:ALaboratoryManual " (Sambrook, J., Russell, DavidW., MolecularCloning:ALaboratoryManual, 3rdedition, 2001, NY, ColdSpringHarbor).
Described percentage concentration is mass/mass (W/W if no special instructions, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, Unit/mL/100mL) percentage concentration.
The primer synthesis and determined dna sequence complete by LifeTechnology company of the U.S..
The approach that obtains of the various biomaterials be described in embodiment is only to provide a kind of approach of testing acquisition to reach concrete disclosed object, should not become the restriction to biological material source of the present invention.In fact, the source of used biomaterial is widely, and any biomaterial that can obtain with moral ethics that keeps on the right side of the law can replace use according to the prompting in embodiment.
Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and concrete operating process, and embodiment will contribute to understanding the present invention, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1, structure recombined glandulae correlation viral vectors AAV/HPV-16E7 and AAV/HPV-16E7
m58
One, material:
A. four kinds of adeno-associated virus (AAV) carriers: be namely respectively the pAAV/p5 with AAVp5 promotor, the pAAV/CMVp with cytomegalovirus (CMV) promotor, there is the pAAV/SV40p of SV40 virus early promoter and there is the pAAV/ β-actinp of people's beta-actin (β-actin) promotor.These four kinds of AAV carriers only promotor are different, remaining gene structure is identical, namely there is repetition terminal fragment (TR) sequence that AAV2 type two ends are complete, and be all inserted with 9 nucleotide fragments (CTGCGCTGG, object improves the stability of restructuring AAV virus (rAAV) and improves the duplicating efficiency of virus) at the 75th the nucleotide sequence place of two ends TR and without any AAV structure gene (Rep and Cap).These four kinds of AAV carriers are successfully built (construction process is see Chinese patent ZL201110125683.X) by the contriver of present patent application.
B. oncogene in human cervical carcinoma: the cervical cancer cancerous tissue deriving from excision, immunohistochemical methods confirms HPV-16E7 antigen positive.
C. gene amplification nucleotide primer: design according to the HPV-16E7 gene order published in U.S.'s gene pool (U.S. NCI gene pool: KC935953), upstream primer: 5 '-ATGCATGGAGATACA-3 ', downstream primer: 5 '-TTATTGTTTCTGAGAA-3 '.
Two, the E7 or the saltant type E7 that carry HPV 16 (HPV-16) is built
m58the recombined glandulae correlation viral vectors of gene
E7 or the saltant type E7 that the present invention carries HPV 16 (HPV-16) is built respectively by following method
m58the recombined glandulae correlation viral vectors of gene, its structural representation is as Fig. 1 (E7 that in figure, " allogenic gene of insertion " is HPV 16 (HPV-16) or saltant type E7
m58gene) shown in, detailed process comprises the following steps:
A. HPV-16E7DNA is obtained, concrete grammar is: adopt DNAzol reagent (production of LifeTechnology company of the U.S.) and by specification operate: after first the cervical cancer tissues of HPV-16E7 antigen positive being milled repeatedly, add 10mLDNAzol, after centrifugal acquisition supernatant liquor, by 75% washing with alcohol 2 times, then add dehydrated alcohol, centrifugal, by throw out deionized water dissolving, DNA concentration is adjusted to 100ng/ μ L.With 2 μ LDNA solution for template, pcr amplification HPV-16E7DNA under the guiding of upstream primer 5 '-ATGCATGGAGATACA-3 ' and downstream primer 5 '-TTATTGTTTCTGAGAA-3 '.Pcr amplification condition is: first 94 DEG C 4 minutes; Again 94 DEG C 30 seconds, 60 DEG C 35 seconds, 72 DEG C 50 seconds, totally 30 circulations; Last 72 DEG C 8 minutes.After reaction terminates, carry out 1.2% agarose gel electrophoresis detection to pcr amplification product, detected result as shown in Figure 2, occurs that a length is the 297bp specific band consistent with expected results.Being reclaimed and obtain length after purifying by this object band is 297bpHPV-16E7DNA.
B. HPV-16E7 is obtained
m58dNA, concrete grammar is: adopt point mutation test kit (Strategeng company of the U.S.), operate according to its test kit specification sheets: the thymus pyrimidine (T) of HPV-16E7 gene (U.S. NCI gene pool: KC935953) opening code-reading frame (nt736) is replaced with guanine (G), TGC (nt736-nt738) by encoding aminothiopropionic acid changes into the GGC of encodes glycine, namely obtains the saltant type HPV-16E7 without tumorigenicity
m58gene.After completing, carry out determined dna sequence, HPV-16E7
m58gene order as shown in sequence in sequence table 1, HPV-16E7 gene order as shown in sequence in sequence table 2, HPV-16E7
m58the comparison result of gene order and HPV-16E7 gene order as shown in Figure 3, guanine (G) is replaced by the thymus pyrimidine (T) of nt736 position, the TGC (nt736-nt738) of encoding aminothiopropionic acid changes into the GGC of encodes glycine, namely obtains the saltant type HPV-16E7 without tumorigenicity
m58gene, proves transgenation success, obtains HPV-16E7
m58antigen gene.
C. recombined glandulae correlation viral vectors AAV/HPV-16E7 and AAV/HPV-16E7 is obtained
m58: adopt DNA interconnection technique, respectively by HPV-16E7 and E7 of above-mentioned acquisition
m58dNA fragmentation inserts in these four kinds of rAAV carriers of pAAV/p5, pAAV/CMVp, pAAV/SV40p and pAAV/ β-actinp respectively.For inserting this gene fragment, first carrying out endonuclease reaction, then carrying out ligation.Wherein, endonuclease reaction system is: 100ng plasmid and 50ngHPV-16E7 or E7
m58dNA fragmentation; 10U restriction enzyme BamHI and SalI (purchased from American Promega company), 2.5 μ l10 × damping fluid C and 19.5 μ l deionized waters; Reaction conditions is: water-bath 4 hours at 37 DEG C.Ligation system is: 50ng enzyme cut after plasmid, 50ngHPV-16E7 or HPV-16E7
m58dNA fragmentation, 10IUT4DNA ligase enzyme (purchased from American Promega company), 1.5 μ l10 × T
4dNA connects damping fluid and 11.5 μ l deionized waters; Reaction conditions is: 4 DEG C 8 hours.Finally obtain respectively carrying p5 promotor, CMV promoter, SV40 early promoter or β protein promoter and HPV-16E7
m58the recombined glandulae correlation viral vectors of gene or HPV-16E7 gene, four kinds of corresponding four kinds of promotors are carried HPV-16E7 respectively
m58the recombined glandulae correlation viral vectors of gene is referred to as AAV/HPV-16E7
m58, four kinds of recombined glandulae correlation viral vectors carrying HPV-16E7 gene are referred to as AAV/HPV-16E7.
D. recombined glandulae correlation viral vectors plasmid AAV/HPV-16E7 and AAV/HPV-16E7 is obtained
m58: respectively by the AAV/HPV-16E7 after connection
m58with AAV/HPV-16E7 transformation gene engineering intestinal bacteria (E.coli) DH5 α competent cell (American I nvitrogen company), resistance screening is carried out with the LB flat board containing 100 μ g/mL penbritins, the single bacterium colony of picking white, extract plasmid and purifying, obtain AAV/HPV-16E7 respectively
m58plasmid and AAV/HPV-16E7 plasmid.
F. plasmids detection: by the AAV/HPV-16E7 obtained
m58whether plasmid and AAV/HPV-16E7 plasmid carry out restriction analysis with restriction enzyme BamHI and SalI (purchased from American Promega company), successfully construct to identify.Endonuclease reaction condition and method are as above-mentioned (two .C).The AAV/HPV-16E7 built
m58(swimming lane 1-6 is respectively AAV/p5/HPV-16E7 as shown in Figure 4 with the restriction analysis result of rAAV/HPV-16E7 carrier
m58, AAV/CMVp/HPV-16E7
m58, AAV/SV40p/HPV-16E7
m58, AAV/ β-actinp/HPV-16E7
m58, AAV/p5/HPV-16E7 and AAV/CMVp/HPV-16E7, analytical results shows to build the success of restructuring AAV carrier), HPV-16E7
m58insert respectively in AAV carrier with HPV-16E7 gene, carry E7 or the saltant type E7 of HPV 16 (HPV-16)
m58the recombined glandulae correlation viral vectors of gene successfully constructs.
The preparation of embodiment 2, recombinant adeno-associated virus (rAAV) and virus titer measure
Material and source thereof:
What A. embodiment 1 built carries HPV-16E7
m58with the recombined glandulae correlation viral vectors (AAV/HPV-16E7 of HPV-16E7 antigen gene
m58and AAV/HPV-16E7).
B. containing the Rep gene of AAV and the helper plasmid pHelper of Cap gene: successfully construct (Liu by the contriver of present patent application, Y., SantinAD., ManeM., Chiriva-Internati, M., ParhamGP., RavaggiA., andHermonat, P.L.TransductionandUtilityoftheGranulocyte-MacrophageCol ony-StimulatingFactorGeneintoMonocytesandDendriticCellsb yAdeno-AssociatedVirus.JournalofInterferonandCytokineRes earch.20:21 – 30.2000).
C. containing being integrated in cell chromosome and the adenoviral gene (E1 expressed, E2A, E4, VAI and VAII gene) AAV-HEK293 cell: set up (Liu by the contriver of present patent application, Y., SantinAD., ManeM., Chiriva-Internati, M., ParhamGP., RavaggiA., andHermonat, P.L.TransductionandUtilityoftheGranulocyte-MacrophageCol ony-StimulatingFactorGeneintoMonocytesandDendriticCellsb yAdeno-AssociatedVirus.JournalofInterferonandCytokineRes earch.20:21 – 30.2000).
D. lipofectamine Lipofectin: purchased from American LifeTechnology company.
E.DMEM substratum and foetal calf serum (or calf serum): purchased from American Cellgro company.
F.PCRDIG labelling kit and DIG hybridization check test kit: purchased from Roche company of Switzerland.
G.DNA copy number standard: be respectively 10
12copy number (copies)/μ L to 10
6(copies)/μ L, purchased from American Promega company.
One, the preparation of recombinant adeno-associated virus (rAAV)
Method prepares recombinant adeno-associated virus (rAAV) as shown in Figure 5.To prepare the virus of a dish 10.0cm Tissue Culture Dish, when AAV-HEK293 cell grow in carbon dioxide cell incubator account for culture dish area 70% time, proceed as follows:
A. operate according to the operation instruction of Lipofectin: 1.0 μ grAAV, 1.0 μ gpHelper plasmids, 4.0 μ LLipofectin and 50.0 μ L are mixed containing the DMEM substratum of 5% foetal calf serum (or calf serum), room temperature leaves standstill 20 minutes.
B. mixed solution is added in Tissue Culture Dish, continue to be placed in carbon dioxide cell incubator and cultivate at 37 DEG C.
Hour C.72 after, all cells in results culture dish and nutrient solution.
D. thermal agitation is after 1 minute, centrifugal, retains supernatant, i.e. rAAV virus liquid.
The rAAV virus liquid filtration sterilization of E. will collect.
Two, the virus titer of recombinant adeno-associated virus (rAAV) measures
Adopt conventional spot hybridization, carry out virus titer mensuration to the rAAV virus that step one obtains, concrete grammar comprises the following steps ():.
A. adopt conventional DNA phenol/chloroform extraction method, extract rAAV virion DNA.
B. nylon membrane is placed in Dot blot instrument, adds the rAAV virion DNA through alkaline denaturation, and add DNA copy number standard, vacuumize.
C., after taking out nylon membrane drying, ultraviolet is fixed.
D. with PCRDIG labelling kit and reference reagent box specification sheets prepare DIG mark specific probe, DNA probe used is the specific probe for HPV-16E7 gene, and probe is the " HPV16-E7DNA obtained in embodiment 1 step C.After pcr amplification terminates, carrying out 1.2% agarose gel electrophoresis to pcr amplification product, detect pcr amplification product under ultraviolet light, there is positive band in result, shows probe mark success.
E. use DIG hybridization check test kit and reference reagent box specification sheets, in hybrid heater, DNA hybridization is carried out to various rAAV virion DNA.
The all rAAV virus titers of experimental result are 10
12-10
14copy/mL, shows that the rAAV virus titer obtained is higher, can be used for scientific research and clinical practice completely.
Embodiment 3, recombinant adeno-associated virus AAV/HPV-16E7
m58observe with the tumorigenicity of AAV/HPV-16E7 infector for cervical epithelial cells
Material and source thereof:
A.rAAV virus: the recombinant adeno-associated virus AAV/HPV-16E7 that embodiment 2 obtains
m58and AAV/HPV-16E7.
B.Keratinocyte-SCF cell culture medium: purchased from American LifeTechnology company.
C. primary cervical epithelial cells: be separated from normal cervical epithelial tissue by ordinary method and obtain.
Tumorigenicity observation experiment
Primary cervical epithelial cells is being put into 10.0cm Tissue Culture Dish, is adding 10mLKeratinocyte-SCF cell culture medium immediately, putting in CO2gas incubator and cultivate at 37 DEG C.After cell is completely adherent, take out culture dish, after removing 7mL nutrient solution, add recombinant adeno-associated virus AAV/HPV-16E7 respectively according to 100MOI dosage
m58or AAV/HPV-16E7, be refitted in CO2gas incubator.After 8 hours, take out culture dish, remove nutrient solution, add the Keratinocyte-SCF cell culture medium that 10mL is fresh, be refitted in CO2gas incubator and cultivate at 37 DEG C.Every 2 days replaced medium.Timing every day observation of cell metamorphosis 2 times.Until cell occurs that knurl becomes.
Result as shown in Figure 6, two kinds of rAAV all at cells, but, by AAV/HPV-16E7
m58there is not knurl and become in the cell infected, shows that (three culture dish add the AAV/HPV-16E7 of band CMV promoter, SV40 early promoter or β protein promoter from top to bottom respectively without tumorigenicity
m58); And by the cell that AAV/HPV-16E7 infects, obvious knurl is occurred and become, there is tumorigenicity (three culture dish add the rAAV/HPV-16E7 of band CMV promoter, SV40 early promoter or β protein promoter from top to bottom respectively).
Embodiment 4, tumour antigen importing monocyte-dendritic cell system kills tumor experiment
Material and source thereof:
A.rAAV virus: the AAV/HPV-16E7 that embodiment 2 obtains
m58and AAV/HPV-16E7.
B.AIM-V cell culture medium: purchased from American LifeTechnology company.
C. cytokine: granulocyte colony stimulating factor (GM-CSF) and interleukin II, 4,7 purchased from American R & D companies.
The cell of the D.HPV-16E7 positive: be separated from tumor tissues and obtain or preserve center (ATCC) acquisition from American. tissue cell, malignant tumour comprises cervical cancer cell, breast cancer cell, penile cancer cell, anus cancer cell and cancer cell of oral cavity.
One, tumor experiment is killed
As shown in Figure 7, the present invention is carried HPV-16E7 or HPV-16E7
m58rAAV virus (the AAV/HPV-16E7 of antigen gene
m58or AAV/HPV-16E7) cell experiment killing HPV-16E7 antigen positive based on infected tumor patient monocyte (Mo) comprises the following steps:
A. tumour patient 50-150mL peripheral blood is got, peripheral blood mononuclear cell (PBMC) is obtained according to a conventional method with hemocyte separometer (or lymphocyte separation medium), after mixing with AIM-V substratum, add Tissue Culture Flask, be placed in constant temperature CO2gas incubator and cultivate 2 hours at 37 DEG C.
B. remove suspension cell, retain attached cell (monocyte).Suspension cell and peripheral blood lymphocyte, after itself and AIM-V substratum being mixed, continue cultivation for subsequent use.
C. add rAAV virus, add-on is about 100MOI, adds GM-CSF (600IU/mL) more simultaneously, continues cultivation 4 hours.
D. remove old substratum, supplement the AIM-V substratum containing GM-CSF and IL-4 (600IU/mL), continue to cultivate.
E. cultivate after 5 days, the dendritic cell (DC) that results are ripe, and mix with cultivated peripheral blood lymphocyte, in AIM-V substratum, add IL-2 (10IU/mL) and IL-7 (200IU/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)
A.rAAV infects the Efficiency testing of peripheral blood lymphocytes (Mo)
Adopt conventional fluorescence antibody mark staining, by the monocyte (Mo) infected by rAAV of the present invention obtained for HPV-16E7 specific fluorescent antibody (purchased from American BD company) markers step one or immature dendritic cell (DC), then carry out the quantity of flow cytomery positive cell.Wherein, rAAV infects the Efficiency testing result of monocyte (Mo) as shown in Figure 8, carries the AAV/HPV-16E7 of four kinds of different promoters (p5, CMVp, SV40p and β-actinp)
m58the efficiency infecting Mo with AAV/HPV-16E7 is all about 90%, and namely the Mo of 90 about percent by rAAV virus infection, can 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 the level of CD80 and CD86 and the function of DC is proportionate.By the detection method identical with steps A, the fluorescently-labeled antibody for these two kinds of CD molecules (purchased from American BD company) is namely adopted to detect the level that the DC that step one obtains expresses CD80 and CD86 respectively.AAV/HPV-16E7
m58or the DC that AAV/HPV-16E7 infects expresses the detected result of CD80 and CD86 level as shown in Figure 9 (with the rAAV with CMV promoter for representative), CD molecular level expressed by infected DC is higher, proves that the present invention carries HPV-16E7 or HPV-16E7
m58the activated cell of the DC that the monocyte that the rAAV of antigen gene infects is induced is immunoreactive powerful.
C. the detection of IFN-γ (IFN-γ) level expressed of cytotoxic T lymphocyte (CTL)
The expression level of the function of CTL and the ability of killing tumor cell and IFN-γ is proportionate.Detect by the method similar with steps A the DC infected by rAAV of the present invention the CTL that induces express the level (DC stimulated with HPV-16E7 albumen and non-stimulated DC the CTL that induces for contrasting) of IFN-γ, after DC and peripheral blood lymphocyte mixed culture terminate, harvested cell, traditional Intracellular cytokine staining methods is adopted to carry out cell fluorescence dye marker, antibody used is the fluorescent-labeled antibody (purchased from American BD company) for IFN-γ, finally utilizes flow cytomery result.Wherein, by AAV/HPV-16E7
m58or the IFN-γ expression level of CTL that the DC that infects of AAV/HPV-16E7 induces as shown in Figure 10 (with the rAAV with β-actin promotor for representative), to stimulate by the DC that rAAV infects the CTL of generation to express the level of IFN-γ higher, prove by AAV/HPV-16E7 of the present invention
m58or the CTL that the DC of AAV/HPV-16E7 infection induces kills and wounds the activity of target cell comparatively by force (powerful).
D. cytotoxic T lymphocyte (CTL) killing tumor cell test
After mixed culture terminates, by step one by AAV/HPV-16E7
m58or after the DC cytotoxic T lymphocyte (CTL) of inducing that infects of AAV/HPV-16E7 mixes with cervical cancer cell, breast cancer cell, penile cancer cell, anus cancer cell and cancer cell of oral cavity respectively by 20:1 (lymphocyte: tumour cell), adopt traditional
51cr (chromium-51) fragmentation test, detects the activity of CTL killing tumor cell.
By the DC that rAAV infects the CTL Cytotoxicity in vitro HPV-16E7 positive cell of inducing
51cr (chromium-51) killing experiments result is if Figure 11 is (with the AAV/HPV-16E7 with CMV promoter
m58for example, ordinate zou represents kill rate) shown in, the CTL that induces by the DC that rAAV of the present invention infects can the target cell of more effectively cracking (killing and wounding) HPV-16E7 antigen positive, kill rate is about 40%-60%, and be contrast to the cell of the lung (lung) of HPV-16E7 feminine gender, mammary gland (breast), liver (liver), kidney (k-cells), without lethal effect, prove the DC infected by rAAV of the present invention the CTL that induces there is antigen-specific, namely to the cell of antigen negative without lethal effect.
Above-mentioned detected result shows, is carried saltant type HPV-16E7 by the present invention
m58recombinant adeno-associated virus (the AAV/HPV-16E7 of antigen gene
m58) DC that the infects cell of CTL to HPV-16E7 antigen positive of inducing have and powerful kill and wound (cracking) effect, there is higher specificity (i.e. targeting lethal effect), and to the cell of HPV-16E7 antigen negative without lethal effect, with the killing ability indistinction of the CTL of wild-type HPV-16E7 antigen induction, and without tumorigenicity, can be used for preparing antitumor drug.
The clinical experiment of the oncotherapy of embodiment 5, HPV-16E7 antigen positive
Application recombinant adeno-associated virus-dendritic cell technology, namely in embodiment 4 by AAV/HPV-16E7 of the present invention
m58the cytotoxic T lymphocyte (CTL) that the DC infected induces feeds back 5 routine cervical cancer patients, and it is that HPV-16E7 is positive that all patients have been proved its cervical cancer tissues.Infusion amount is 2 × 10
8-5 × 10
8.Treat the course for the treatment of: being generally 3 months is a course for the treatment of, monthly 2 times, the state of an illness can be kept to monthly 1-2 time after improving, and can be kept to further and treat once every 1-3 month.Treatment result sums up (B: blood serum tumor markers reduces or disappears as shown in table 1.Q: quality of life of patients improves.As pain relief or disappearance, appetite increase etc.C:CTorPET-CT shows cancer focus or metastatic lesion obviously reduces or disappears.), untoward reaction: there will be the reaction of slight influenza sample in the short period of time after 1 example treatment, but patient all can bear, and symptom disappears in a short time, does not observe serious adverse reaction and toxic reaction.5 examples are through recombinant adeno-associated virus AAV/HPV-16E7
m58before and after the CTL treatment that the DC infected induces, the changing conditions of cervical cancer patient serum Cytokeratin 19 antigen (CK19) and squamous cell carcinoma antigen (SCC) level as shown in figure 12, after treatment, the serum Keratin 19 antigen (CK19 of 3 routine patients, cyfra21-1) and squamous cell carcinoma antigen (SSC) obviously to decline (CK19 level, normal value <3.3ng/ml; SCC level, normal value <5.0ng/ml), even recover normal.When submitting present patent application, 5 routine patients are all survived.Clinical trial results proves further, the CTL that induces by the DC that rAAV of the present invention infects can play certain curative effect in patient body, effectively can suppress growth or the killing off tumor cells of the malignant cell of the HPV-16E7 positive, and security is higher, can be used for preparing antitumor drug.
Table 1 rAAV/HPV-16E7
m58-dendritic cell technology treats the statistics of the curative effect of 5 routine cervical cancer cancer patients
| Numbering | Clinical stages | RAAV-DC treats the course for the treatment of | Survival time after treatment | Result for the treatment of |
| (moon) | (moon) | |||
| 1 | III | 9 | 17 | B,Q,C |
| 2 | III | 11 | 26 | B,Q,C |
| 3 | III | 8 | 13 | Q |
| 4 | IV | 8 | 12 | B,Q |
| 5 | IV | 6 | 14 | Q, |
| Amount to | III—IV | 42 | 82 |
industrial applicability
Experiment proves, by HPV-16E7 of the present invention
m58the growth of the dendritic cell that recombinant adeno-associated virus infects and the cell that the cytotoxic T lymphocyte of inducing can effectively suppress HPV16 to infect in patient body and relative malignant cell or killing off tumor cells, thus, HPV-16E7 of the present invention
m58recombined glandulae correlation viral vectors or with the present invention/HPV-16E7
m58the product that recombined glandulae correlation viral vectors is correlated with can be used to prepare the cell of anti-HPV16 infection and relevant antitumor drug thereof, has great importance in clinical treatment and application.
Claims (10)
1. one kind is carried HPV 16 saltant type E7
m58the recombined glandulae correlation viral vectors of antigen gene is by goal gene saltant type HPV-16E7
m58antigen gene or wild-type HPV-16E7 antigen gene insert in gland relevant viral vector, obtain and carry HPV-16E7
m58the recombined glandulae correlation viral vectors of antigen gene (is called for short " E7
m58recombined glandulae correlation viral vectors " or AAV/HPV-16E7
m58) or the recombined glandulae correlation viral vectors that carries HPV-16E7 antigen gene (be called for short " E7 recombined glandulae correlation viral vectors " or AAV/HPV-16E7).
2. recombined glandulae correlation viral vectors according to claim 1, is characterized in that: described saltant type HPV-16E7
m58antigen gene the halfcystine (G) of the 58th, the HPV-16 E7 albumen of HPV-16E7 antigen gene is changed into gene corresponding to glycine (C), namely, the thymus pyrimidine (T) of HPV-16E7 gene open reading frame (nt736) is replaced with guanine (G), the TGC (nt736-738) of encoding aminothiopropionic acid is changed into the GGC of encodes glycine, obtain the saltant type HPV-16E7 that can express without tumorigenicity
m58gene.
3. recombined glandulae correlation viral vectors according to claim 1 and 2, is characterized in that: described HPV-16E7
m58the nucleotide sequence of gene is as shown in sequence in sequence table 1.
4. according to arbitrary described recombinant adeno-associated virus (rAAV) carrier of claim 1-3, it is characterized in that: described gland relevant viral vector is the gland relevant viral vector of rejecting adeno-associated virus (AAV) structure gene Rep and Cap, its promotor of carrying is p5 promotor, cytomegalovirus (cytomegalovirus, CMV) promotor, people beta actin promoter or SV40 virus early promoter in any one, described in carry HPV-16E7
m58the recombined glandulae correlation viral vectors of antigen gene is referred to as " E7
m58recombined glandulae correlation viral vectors " or AAV/HPV-16E7
m58.
5. build a method for the arbitrary described recombined glandulae correlation viral vectors of claim 1-4, comprise the following steps:
1) be glycine by the 58th of HPV-16E7 antigen protein the cysteine mutation, thymus pyrimidine (T) by HPV-16E7 gene open reading frame (nt736) replaces with guanine (G), TGC by encoding aminothiopropionic acid changes into the GGC of encodes glycine, obtains the saltant type HPV-16E7 without tumorigenicity
m58gene;
2) by HPV-16E7
m58antigen gene or wild-type HPV-16E7 antigen gene insert described E7
m58in recombined glandulae correlation viral vectors, obtain carrying HPV-16E7
m58the recombined glandulae correlation viral vectors of antigen gene (is referred to as AAV/HPV-16E7
m58) or carry the recombined glandulae correlation viral vectors (be referred to as AAV/HPV-16E7) of HPV-16E7 antigen gene.
6. with the arbitrary described recombined glandulae correlation viral vectors AAV/HPV-16E7 of claim 1-4
m58the product relevant with recombined glandulae correlation viral vectors AAV/HPV-16E7, comprise recombinant adeno-associated virus plasmid, recombinant adeno-associated virus particle and the clone by described recombinant gland relevant viral vector infection or transfection, clone comprises monocyte (Monocyte, and dendritic cell (DendriticCells, DC) Mo).
7. prepare described in claim 6 with recombined glandulae correlation viral vectors AAV/HPV-16E7 and AAV/HPV-16E7
m58the method of related products, is respectively:
Carry HPV-16E7 or HPV-16E7
m58the preparation of the recombined glandulae correlation viral vectors plasmid of antigen gene: will HPV-16E7 or HPV-16E7 be carried
m58recombined glandulae correlation viral vectors DNA-AAV/HPV-16E7 or AAV/HPV-16E7 of antigen gene
m58quiding gene engineering colon bacillus (E.coli) DH5 α competent cell; resistance screening is carried out with the LB flat board containing 100 μ g/mL penbritins; the single bacterium colony of picking white, extracts plasmid and purifying, obtains AAV/HPV-16E7 plasmid or AAV/HPV-16E7
m58plasmid;
Carry HPV-16E7 or HPV-16E7
m58the preparation of the recombinant adeno-associated virus of antigen gene: carry HPV-16E7 or HPV-16E7 with described
m58recombined glandulae correlation viral vectors plasmid and the pHelper plasmid common transfection AAV-HEK293 cell of antigen gene obtain rAAV virus, respectively called after AAV/HPV-16E7 virus or AAV/HPV-16E7
m58virus;
Carry HPV-16E7 or HPV-16E7
m58the preparation of the recombinant gland relevant viral vector infection of antigen gene or the clone of transfection: with described recombinant adeno-associated virus AAV/HPV-16E7 virus or AAV/HPV-16E7
m58virus infects respectively or successively or transfection monocyte (Mo) or dendritic cell (DC) obtain.
8. anti-HPV-16 infects and is infected by HPV-16 a medicine for the cellular immunotherapy of the tumour caused, and its activeconstituents carries HPV-16E7 for described in one of claim 1-4
m58recombined glandulae correlation viral vectors (the AAV/HPV-16E7 of antigen gene
m58) or claim 6 described in carry HPV-16E7
m58the product that the recombined glandulae correlation viral vectors of antigen gene is relevant, comprises AAV/HPV-16E7
m58plasmid, AAV/HPV-16E7
m58virus, by HPV-16E7
m58the monocyte (Mo) that recombinant adeno-associated virus infects respectively or successively or transfection obtains or dendritic cell (DC).
9. medicine according to claim 8, is characterized in that: described HPV-16 infects uterine neck papilloma pathology, cervical cancer, male genital organ Bowen ' s disease, Buschke-Lo&4&wenstein tumor, penile cancer, anus cancer, the rectum cancer, oral carcinoma, carcinoma of tonsil and the mammary cancer etc. that associated malignancies is HPV-16E7 antigen positive.
10. kill a method for the cell of HPV-16 infection and the tumour cell of the HPV-16E7 positive, comprise the following steps:
1) described in arbitrary by claim 1-4 for monocyte spontaneous in patient body, HPV-16E7 is carried
m58recombined glandulae correlation viral vectors (the AAV/HPV-16E7 of antigen gene
m58) infect or transfection, or by product treatment relevant to recombined glandulae correlation viral vectors of the present invention described in claim 6, obtain the cell after processing separately;
2) by step 1) in monocyte (Mo) dendritic cell (DC) of inducing after process input in patient body, activating cytotoxic T-lymphocyte (CTL), this CTL kills the cell of HPV-16 infection and the tumour cell of the HPV-16E7 positive; Or the Mo-DC mixed culture after not processed T lymphocyte and described process is formed the CTL of HPV-16E7 antigen-specific, then kill the cell of HPV-16 infection and the tumour cell of the HPV-16E7 positive by this CTL input patient body; Maybe processed T lymphocyte and processed Mo-DC are inputted in patient body, kill the cell of HPV-16 infection and the tumour cell of the HPV-16E7 positive.
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