CN105316361B - Carry HPV 16 saltant type E7m58The recombined glandulae correlation viral vectors and its construction method of antigen gene and application - Google Patents

Abstract

A kind of E7 of HPV 16 of the present invention_m58Antigen gene recombined glandulae correlation viral vectors (rAAV) and its construction method are applied with it, it by the HPV-16 E7 gene mutation of the HPV 16 with oncogenicity is the HPV-16 E7 gene without oncogenicity to be, then by this be mutated after gene insertion rejected in the gland relevant viral vector of structural gene and obtained.The E7 of saltant type HPV 16 that the rAAV of the present invention can be carried_m58Antigen gene is conveyed into monocytic dendritic cell shape cell line, is used for the effector cell of stimulating immune system.It is demonstrated experimentally that the growth of the E7 positive cells of HPV 16 can effectively be suppressed with patient's body by the rAAV of the present invention DC infected the CTL induced in vitro or kill the positive cells of the E7 of HPV 16, including tumour and non-tumor cell.The recombined glandulae correlation viral vectors or its Related product of the present invention, which can be used for preparing treatment HPV 16, to be infected and its related antineoplastic.

Description

Carry HPV 16 saltant type E7m58The restructuring gland related diseases of antigen gene Poisonous carrier and its construction method and application

Technical field

The present invention relates to the carrier in biological technical field and its application, and HPV is carried more particularly to one kind 16 types (Human Papillomavirus Type 16, HPV-16) saltant type E7_m58The recombinant adeno-associated virus of antigen gene carries Body (rAAV) and its construction method and its application in preparing anti-HPV-16 and infecting and its be diseases related.

Background technology

The gene structure of adeno-associated virus (AAV) has been accredited.Nineteen eighty-three, Samulski et al. describes AAV end End repeated fragment (end fragment of upstream 5 ', the end fragment of downstream 3 ') (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, Hermonat et al. describes AAV low infectious particles (Lip) gene and coating (Cap) gene (Hermonat PL, 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 Year, Labow et al. identify between the end fragment of upstream 5 ' and replication protein (Rep) gene p5 promoters (Labow MA, Hermonat PL, Berns KI.Positive and negative autoregulation of the adeno- associated virus type 2 genome.J Virol.160:251-258.)。

A kind of the defects of AAV is non-pathogenic venereal disease poison is, it is necessary to the gene outcome auxiliary of other viruses (such as adenovirus), It can be assembled into infective virion.The base-pair (bp) of AAV full-length genomes about 4700, both ends are repetition end wafer Section (TR), the middle structural gene for virus, including the Rep gene relevant with virus replication and peplos (Cap) gene.By In the unstability that AAV viruses itself be present and its defects of carry allogenic gene (therapeutic gene) limited length etc., It is therefore desirable to carry out genetic recombination to it to form recombinant adeno-associated virus (recombinant adeno-associated Virus, rAAV).Existing numerous studies show, the structural gene in AAV genomes is deleted, can substantially increase allogenic gene Capacity.In addition, will have in medicative allogenic gene insertion rAAV, can be prepared into infective rAAV viruses Particle.

1984, U.S. Paul L.Hermonat took the lead in proving that AAV carriers can be used for the gene therapy of human diseases (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.).At present, mainly American-European countries exists Carry out the clinical test of the gene therapy human diseases based on AAV.Counted according to U.S.'s grain and drug administration, existing 10 Gene Therapy Clinical Trials of the remainder based on AAV are carried out, and mainly suffer from the AAV virus injections for carrying therapeutic gene In person's body, make its expression treatment gene in vivo, so as to reach the purpose for the treatment of disease.There is pa golden mainly for the disease for the treatment of The non-tumours such as gloomy syndrome, rheumatic arthritis, hemophilia, heart failure, the silent syndrome of progressive myatrophy and Olds sea Property disease.On November 2nd, 2012, the Glybera products of European Union approval UniQure companies used in 27 member states of European Union, this It is first granted gene therapy medicament of western countries, it is to utilize adeno-associated virus I types (AAV-1) foreign gene-carrying For treating lipoprotein lipase deficiency hereditary disease (LPLD) genomic medicine.

HPV (HPV) is a kind of papilloma vacuolating virus A category for belonging to papovaviridae.At least divide at present Separate out more than 130 kinds of hypotype.According to different subtype, high-risk-type and low risk can be divided into.Wherein endanger maximum to the mankind is high-risk Type, including the malignant tumour such as HPV-16,18,30,31,33,35,39 and cervical carcinoma, the carcinoma of the rectum, carcinoma of mouth, carcinoma of tonsil are close It is related.Wherein more than 99% cervical carcinoma is as caused by high-risk HPV, and caused by cervical carcinoma more than half is HPV-16.

HPV belongs to the small DNA virus of double-strand closed loop, includes about 8000 base-pairs.Including 8 early stage opening code-reading frames Frame (E1-E8), 2 late period single open reading frames and 1 non-coding Chang Kong area.In early stage open reading frame, there is carcinogenesis E6 and E7 genes cell growth stimulate it is mostly important, E6, E7 coding cancer protein E6, E7 albumen respectively with tumor suppressor gene P53 and Rb is combined, and causes uncontrolled cellular proliferation, and tumor suppressor gene is lost to DNA injury repair function, causes precancerous lesion and cancer The generation of disease.

Shown according to an investigation result of national healths of the 2003-2004 from the U.S. and nutrient research problem, 14-59 Year women the total infection rates of HPV be 26.8%.The popularity of the HPV infection of China there are about 20 every year there has been no formal report More than ten thousand cervical carcinoma newly detected cases, morbidity and mortality have increase trend, and the rejuvenation of cervical cancer pathogenesis age, can be with Speculate that HPV infection rate is pessimistic.But the definite method for curing HPV infection is there is no at present, and HPV vaccines at this stage are possible to pre- Anti- HPV-16 and HPV-18 infection, but the crowd for having infected is invalid.To reach healing purpose, optimal treatment is thorough Remove infected cell in bottom.And there is HPV-16 HPV-16 E7s in infected cell.Therefore, HPV-16 HPV-16 E7s are thin The ideal target of born of the same parents' immunization therapy.But HPV-16 HPV-16 E7s are a kind of carcinogenic proteins, in malignant tumours such as cervical carcinomas Generation in play it is main effect one of.Therefore, in vivo with external use wild type HPV-16 HPV-16 E7 immune response stimulatings Generation, certain risk in terms of security be present.Therefore, it is necessary to remove the oncogenicity of wild HPV-16 HPV-16 E7s, eliminating should Risk.

Human dendritic cell (Dedritic Cells, DC) is that human body is most important, is also most important antigen presenting cell. Largely no matter in vivo or external research is verified, and it is anti-infective and anti-swollen that DC cells can induce or stimulate generation to have The cell immune response of knurl.

The content of the invention

It is an object of the present invention to provide a kind of human papilloma virus 16 type for carrying no pathogenicity (i.e. without oncogenicity) (HPV-16) recombinant adeno-associated virus (rAAV) carrier of saltant type HPV-16 E7 gene.

RAAV carriers provided by the present invention, it is that will reject adeno-associated virus (AAV) structural gene Rep and Cap and carry AAV p5 promoters, cytomegalovirus (cytomegalovirus, CMV) promoter, beta actin promoters and The AAV carriers of any one promoter in SV40 virus early promoters are as the carrier that sets out, by saltant type HPV-16 E7_m58 Antigen gene insertion is set out in carrier, obtains a kind of brand-new rAAV carriers, that is, carries HPV-16 E7_m58The restructuring of antigen gene Gland relevant viral vector (referred to as " E7_m58Recombined glandulae correlation viral vectors " or AAV/HPV-16 E7_m58)。

The AAV carriers are successfully built by the inventor of present patent application, and construction method is referring to Chinese patent ZL201110125683.X。

Here, saltant type HPV-16 E7_m58Antigen gene is by Protocols in Molecular Biology, by wild type HPV-16 E7 Antigen gene is mutated, specifically by HPV-16 (U.S.'s NCI gene pools：KC935953 the half of the 58th, HPV-16 E7 albumen) Cystine (G) changes into glycine (C), by the way that the thymidine (T) of HPV-16 E7 gene opens reading frames (nt736) is replaced It is changed to guanine (G), i.e., the TGC (nt736-738) of encoding aminothiopropionic acid changes into the GGC of coding glycine, acquisition can be with The saltant type HPV-16 HPV-16 E7 genes without oncogenicity are expressed, are named as HPV-16 E7_m58Gene, the HPV-16 E7_m58Base The nucleotide sequence of cause is as shown in sequence 1 in sequence table.

Due to HPV-16 E7_m58Immunogenicity is uninfluenced, can be inserted into gland relevant viral vector described above, should P5 promoter of the AAV carriers with AAV, cytomegalovirus (cytomegalovirus, CMV) promoter, beta fleshes move egg One kind of white promoter and SV40 viruses four kinds of promoters of early promoter, by the brand-new carrying HPV-16 E7 of acquisition_m58Antigen The recombined glandulae correlation viral vectors of gene are referred to as " E7_m58Recombined glandulae correlation viral vectors " or AAV/HPV-16 E7_m58。

Designed more than, wild type HPV-16 HPV-16 E7s gene can be also inserted in above-mentioned gland relevant viral vector, obtained The recombined glandulae correlation viral vectors of wild type HPV-16 HPV-16 E7 genes must be carried, are referred to as " E7 recombinant adeno-associated virus load The nucleotide sequence of body " or AAV/HPV-16 E7, HPV-16 E7 genes is as shown in sequence 2 in sequence table.But due to wild type HPV-16 HPV-16 E7s have oncogenicity, therefore the present invention does not recommend to be used for clinical practice, is only used for studying.

Second object of the present invention is to provide above-mentioned AAV/HPV-16 E7 and AAV/HPV-16 E7_m58It is related to recombinate gland The construction method of viral vector.

Construction method provided by the present invention, comprises the following steps：

1) using conventional Protocols in Molecular Biology method, HPV-16 HPV-16 E7 genes are first obtained, then dashed forward Become, i.e., the 58th cysteine of HPV-16 E7 albumen is changed into glycine, detailed process is by HPV-16 E7 gene opens The thymidine (T) of reading frame (nt736) replaces with guanine (G), i.e., changes the TGC (nt736-738) of encoding aminothiopropionic acid It is changed into encoding the GGC of glycine, that is, obtains the saltant type HPV-16 E7 of no oncogenicity_m58Gene；

2) by HPV-16 E7_m58Antigen gene or the insertion of wild type HPV-16 HPV-16 E7s gene are by adeno-associated virus knot In the gland relevant viral vector that structure gene Rep and Cap are rejected, obtain carrying HPV-16 E7_m58The restructuring gland of antigen gene is related Viral vector (AAV/HPV-16 E7_m58) or carry HPV-16 HPV-16 E7 genes recombined glandulae correlation viral vectors (AAV/HPV- 16 E7)。

E7 in above-mentioned carrier_m58Or the promoter of HPV-16 E7 genetic transcription can select AAV p5 promoters, macrophage Appointing in viral (cytomegalovirus, CMV) promoter, beta actin promoters and SV40 virus early promoters Meaning one.

A further object of the present invention is to provide and recombined glandulae correlation viral vectors AAV/HPV-16 E7_m58With restructuring gland related diseases Product related poisonous carrier AAV/HPV-16 E7, including recombinant adeno-associated virus plasmid, recombinant adeno-associated virus particle and by this Invention recombinant gland relevant viral vector infection or the cell line of transfection, the cell line include monocyte (Monocytes, Mo), BMDC system (Dendritic cells, DC) and lymphocyte.Correlation in the recombined glandulae correlation viral vectors Gene-HPV-16 E7_m58Antigen gene or HPV-16 HPV-16 E7s gene can be in monocytes or BMDC above-mentioned Expressed in the presence of transcripting promoter.

Described and recombined glandulae correlation viral vectors AAV/HPV-16 E7 and AAV/HPV-16 E7_m58The system of related product Preparation Method, it is respectively：

The preparation of recombined glandulae correlation viral vectors plasmid：By recombined glandulae correlation viral vectors DNA-AAV/HPV-16 E7 Or AAV/HPV-16 E7_m58Gene engineering colibacillus (E.coli) DH5 α competent cells are directed respectively into, with containing 100 μ g/mL The LB flat boards of ampicillin carry out resistance screening, picking white single bacterium colony, extract plasmid and purify, obtain AAV/HPV-16 E7 plasmids and AAV/HPV-16 E7_m58Plasmid.

The preparation of recombinant adeno-associated virus：With the recombined glandulae correlation viral vectors plasmid AAV/HPV-16 E7 plasmids or AAV/HPV-16 E7_m58AAV viruses are obtained with pHelper plasmid co-transfection AAV-HEK293 cells, are respectively designated as AAV/ HPV-16 E7 viruses and AAV/HPV-16 E7_m58Virus.

The preparation of recombinant gland relevant viral vector infection or the cell line of transfection：With the recombinant adeno-associated virus rAAV/ HPV-16 E7 viruses or rAAV/HPV-16 E7_m58Virus infects respectively or successively or transfection monocyte (Mo), dendron shape are thin Born of the same parents (DC) or lymphocyte obtain.

In terms of practical use, infected it is a further object to provide a kind of anti-HPV-16 infection and by HPV-16 The medicine and its correlation technique of the cellular immunotherapy of caused tumour.

The active component of the medicine is above-mentioned carrying HPV-16 E7_m58The recombined glandulae correlation viral vectors of antigen gene (AAV/HPV-16 E7_m58) or with the present invention carry HPV-16 E7_m58The recombined glandulae correlation viral vectors correlation of antigen gene Product (because wild type HPV-16 HPV-16 E7s have oncogenicity, therefore does not consider that its is medicinal).

With the E7 of the present invention_m58Recombinant adeno-associated virus is carrier, by HPV-16 saltant types E7_m58Antigen gene imports monokaryon Cell, and producing dendritic shape cell is induced, BMDC can be also introduced directly into, expresses E7_m58Antigen protein, to reach patient The purpose of in vitro and in vivo immunostimulation, to treat HPV-16 infection and associated malignancies, or with the BMDC Cytotoxic T lymphocyte caused by stimulation (Cytotoxic T lymphocytes, CTL) treatment HPV-16 infection and phase Close malignant tumour.

HPV-16 infection associated malignancies be the positive uterine neck papilloma lesion of HPV-16 HPV-16 E7s, cervical carcinoma, Male sex organ Bowen ' s diseases, Buschke-Lo＆4＆wenstein tumor, carcinoma of penis, cancer of anus, the carcinoma of the rectum, carcinoma of mouth, carcinoma of tonsil and mammary gland Cancer etc..

Medicine provided by the present invention can use the formulations such as solvent or pulvis.

The selection of the solvent is diversified, such as cell culture fluid (base), physiological saline or phosphate buffer .

When needs, one or more pharmaceutically acceptable carriers can also be added in said medicine.The load Body includes conventional diluent, sorbefacient and surfactant of pharmaceutical field etc..

Application method can be first to isolate the monocyte (Mo) of patient's body, then after infecting by this medicine or transfect Mo, body Outer induction Mo turns into the BMDC (DC) with antigen presentation function.This medicine can also infect or transfect DC, it is likely that leading Cause intakes or working ability of the DC to antigen poor, so as to cause unsatisfactory curative effect.The DC obtained can feed back patient's body, reach Therapeutic purposes.Or will expression HPV-16 saltant types E7_m58The ripe DC of antigen stimulates caused cytotoxic T lymphocyte (CTL) feed back patient, with obtain more preferably the effect of.

The dosage of said medicine is generally DC：1-5×10⁶/ every time, CTL:1-5×10⁸/ every time, monthly 2 times, the course for the treatment of is led to It is often 3 months.Dosage and the course for the treatment of can all adjust according to actual conditions.

To improve curative effect, medicine of the invention can also be carried out with antibiotic, immunostimulant, targeting and chemotherapeutics etc. Combined therapy.

Present invention also offers a kind of method of the positive tumour cell of killing HPV-16 infection cells and HPV-16 E7.

This method may include following steps：

1) monocyte (Mo) that will be separated from patient's body, or the Mo isolated are induced the BMDC that turns into (DC) HPV-16 E7, are carried_m58Recombined glandulae correlation viral vectors (the AAV/HPV-16 E7 of antigen gene_m58) infect or turn Dye, or by the product treatment related to recombined glandulae correlation viral vectors of the present invention, the cell after each being handled；

2) in the DC input patient's bodies after being handled in step 1), to activate the immune response of patient's body, reach and kill The purpose of the cell of the HPV-16 that goes out infection and the tumour cell of the HPV-16 E7 positives；Or by not processed T lymphocytes with DC mixed culture after the processing, stimulates and produces HPV-16 HPV-16 E7 specificity cell toxicity T lymphocytes (CTL), then The Peptide-specific CTL is inputted into patient's body, kills the cell of HPV-16 infection and the tumour cell that HPV-16 E7 are positive； Or the cell and HPV-16 E7 sun that HPV-16 infection in processed CTL and processed DC input patient's bodies, will be killed The tumour cell of property.

The method for killing tumour can be specifically applied in clinical treatment, including is given a patient and fed back HPV-16 HPV-16 E7 specificity cell toxicity T lymphocyte, the cell origin comes from the spontaneous T lymphocytes of patient should with deriving from Caused by the monocyte of patient-BMDC mixed culture.Before mixed culture, these are in monocyte-dendron shape Cell carries HPV-16 E7 by the present invention_m58The recombinant gland relevant viral vector infection of antigen gene or transfection, or by The product treatment related to recombined glandulae correlation viral vectors of the present invention；

Or give a tumor patient and feed back the monocyte-BMDC for deriving from patient.Before feedback, These have carried HPV-16 E7 in monocyte-BMDC by the present invention_m58The recombinant adeno-associated virus of antigen gene Carrier infects or transfection, or by the product treatment related to recombined glandulae correlation viral vectors of the present invention；

Or, it is above-mentioned from the T lymphocytes of patient and from the patient's to give a tumor patient feedback again Spontaneous monocyte-BMDC.Before feedback, these T lymphocytes carry HPV-16 by the present invention E7_m58At the recombinant gland relevant viral vector infection of antigen gene or the product of the monocyte of transfection-BMDC correlation Reason.These monocyte-BMDCs carry HPV-16 E7 by the present invention_m58The recombinant adeno-associated virus of antigen gene Carrier infects or transfection.

The HPV-16 E7 that recombinant adeno-associated virus (rAAV) carrier of the present invention can be carried_m58Antigen gene is conveyed into In monocyte-BMDC system, HPV-16 E7 are carried_m58The cell of antigen gene is used for stimulating immune system Effector cell (is not limited to T lymphocytes and bone-marrow-derived lymphocyte).It is demonstrated experimentally that the dendron shape infected by the rAAV of the present invention is thin The tumour that born of the same parents and the cytotoxic T lymphocyte induced can effectively kill the HPV-16 HPV-16 E7s positive in patient's body is thin Born of the same parents or the cell of HPV-16 infection.Thus, rAAV carriers of the invention or the product related to rAAV carriers of the present invention can by with In preparing antineoplastic.The present invention has important theoretical and practical significance in the clinical treatment of malignant tumour and application, Have a extensive future.

The present invention is described in further details with reference to specific embodiment.

Brief description of the drawings

Fig. 1 carries the E7 or saltant type E7 of HPV 16 (HPV-16)_m58The recombinant adeno-associated virus of gene carries The structural representation of body.

Fig. 2 obtains the agar for the target gene HPV-16 E7 DNA that length is 297bp by polymerase chain reaction,PCR (PCR) Sugared detected through gel electrophoresis result.

Fig. 3 HPV-16 E7_m58Gene order and wild type HPV-16 E7 gene order comparison results.

The AAV/HPV-16 E7 of Fig. 4 structures_m58With the restriction analysis result of AAV/HPV-16 E7 carriers.

The preparation method flow chart of Fig. 5 recombinant adeno-associated virus (rAAV).

Fig. 6 recombinant adeno-associated virus AAV/HPV-16 E7_m58With rAAV/HPV-16 E7 infectors for cervical epithelial cellses Oncogenicity observation result.

Fig. 7 AAV/HPV-16 E7_m58Killing HPV-16 HPV-16 E7s sun based on infected tumor patient BMDC The experiment flow of property cell.

Fig. 8 is respectively four kinds of different promoters (p5, CMVp, SV40p and β-actinp) recombinant adeno-associated virus AAV/ HPV-16 E7_m58Infect the Efficiency testing result of monocyte (Mo).

Fig. 9 recombinant adeno-associated virus AAV/HPV-16 E7_m58With AAV/HPV-16 E7 infection DC expression CD80 and Flow Cytometry testing result horizontal CD86.

Figure 10 recombinant adeno-associated virus rAAV/HPV-16 E7_m58Induced with the rAAV/HPV-16 E7 DC infected respectively CTL IFN-γ expression Flow Cytometry testing result.

Figure 11 recombinant adeno-associated virus AAV/HPV-16 E7_m58The cytotoxic T lymphocyte that the DC of infection is induced (CTL) Cytotoxicity in vitro HPV-16 E7 positive cells⁵¹Cr (chromium -51) killing experiments result.

Figure 12 5 is through recombinant adeno-associated virus AAV/HPV-16 E7_m58The pretherapy and post-treatment palaces of CTL that the DC of infection is induced The situation of change of cervical cancer patients serum Cytokeratin 19 antigen (CK19) and squamous cell carcinoma antigen (SCC) level.

Embodiment

Method therefor is conventional method unless otherwise instructed in following embodiments, and specific steps can be found in： 《Molecular Cloning:A Laboratory Manual》(Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor)。

The percent concentration is mass/mass (W/W, unit g/100g) percent concentration, matter unless otherwise instructed Amount/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/100mL) percent concentration.

The primer is synthesized and determined dna sequence is completed by Life Technology companies of the U.S..

The acquirement approach of various biomaterials described in embodiment be only to provide it is a kind of test obtain approach with up to To specifically disclosed purpose, the limitation to biological material source of the present invention should not be turned into.In fact, used biomaterial Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according to carrying in embodiment Show and be replaced.

Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and specific Operating process, embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.

Embodiment 1, structure recombined glandulae correlation viral vectors AAV/HPV-16 E7 and AAV/HPV-16 E7_m58

First, material：

A. four kinds of adeno-associated virus (AAV) carriers：It is respectively the pAAV/p5 with AAV p5 promoters, with macrophage The pAAV/CMVp, the pAAV/SV40p with SV40 virus early promoters of cell virus (CMV) promoter and with people β-flesh PAAV/ β-the actinp of filamentous actin (β-actin) promoter.Four kinds of AAV carriers only promoter is different, remaining gene structure It is identical, i.e., terminal fragment (TR) sequence is completely repeated with the type both ends of AAV 2, and in both ends TR the 75th nucleotides At sequence inserted with 9 nucleotide fragments (CTGCGCTGG, it is therefore an objective to improve restructuring AAV viral (rAAV) stability and Improve the duplicating efficiency of virus) and without any AAV structural genes (Rep and Cap).Four kinds of AAV carriers are by present patent application Inventor successfully builds (construction method is referring to Chinese patent ZL201110125683.X).

B. oncogene in human cervical carcinoma：From the cervical carcinoma cancerous tissue of surgery excision, SABC confirms that HPV-16 E7 resist It is Antigen positive hybridomas.

C. gene magnification nucleotide primer：According to the HPV-16 E7 gene orders (U.S. openly delivered in U.S.'s gene pool NCI gene pools：KC935953) design, sense primer：5 '-ATGCATGGAGATACA-3 ', anti-sense primer： 5’- TTATTGTTTCTGAGAA-3’。

2nd, structure carries the E7 or saltant type E7 of HPV 16 (HPV-16)_m58The restructuring gland related diseases of gene Poisonous carrier

Build the E7 or saltant type E7 of present invention carrying HPV 16 (HPV-16) respectively with following methods_m58 (" allogenic gene of insertion " is human papilloma virus in figure by the recombined glandulae correlation viral vectors of gene, its structural representation such as Fig. 1 The E7 or saltant type E7 of malicious 16 types (HPV-16)_m58Gene) shown in, detailed process comprises the following steps：

A. HPV-16 E7 DNA are obtained, specific method is：Using DNAzol reagents, (U.S. Life Technology are public Department production) and by specification operated：After the positive cervical cancer tissues of HPV-16 HPV-16 E7s are milled repeatedly first, add 10mL DNAzol, after centrifugation obtains supernatant, washed 2 times with 75% ethanol, add absolute ethyl alcohol, centrifuged, sediment is used Deionized water dissolving, DNA concentration is adjusted to 100ng/ μ L.Using 2 μ L DNA solutions as template, sense primer 5 '- PCR expands HPV-16 E7 under ATGCATGGAGATACA-3 ' and-TTATTGTTTCTGAGAA-3 ' of anti-sense primer 5 ' guiding DNA.PCR amplification conditions are：First 94 DEG C 4 minutes；94 DEG C 30 seconds again, 60 DEG C 35 seconds, 72 DEG C 50 seconds, totally 30 circulation；Last 72 DEG C 8 minutes.After reaction terminates, 1.2% agarose gel electrophoresis detection, testing result such as Fig. 2 institutes are carried out to pcr amplification product Show, it is the 297bp specific bands consistent with expected results a length occur.The purpose band is reclaimed and obtained after purification It is 297bp HPV-16 E7 DNA to length.

B. HPV-16 E7 are obtained_m58DNA, specific method are：Using point mutation kit (U.S. Strategeng Company), operated according to its kit specification：By HPV-16 E7 genes (U.S.'s NCI gene pools：KC935953 it is) open The thymidine (T) of reading frame (nt736) replaces with guanine (G), i.e., by the TGC (nt736-nt738) of encoding aminothiopropionic acid The GGC of coding glycine is changed into, that is, obtains the saltant type HPV-16 E7 of no oncogenicity_m58Gene.After the completion of, carry out DNA sequences Row measure, HPV-16 E7_m58Gene order is as shown in sequence 1 in sequence table, sequence in HPV-16 E7 gene orders such as sequence table Shown in row 2, HPV-16 E7_m58The comparison result of gene order and HPV-16 E7 gene orders is as shown in figure 3, in nt736 positions Thymidine (T) is replaced by guanine (G), and the TGC (nt736-nt738) of encoding aminothiopropionic acid changes into coding glycine GGC, that is, obtain the saltant type HPV-16 E7 of no oncogenicity_m58Gene, it was demonstrated that gene mutation success, obtain HPV-16 E7_m58It is anti- Protogene.

C. recombined glandulae correlation viral vectors AAV/HPV-16 E7 and AAV/HPV-16 E7 are obtained_m58：Using DNA connection skills Art, respectively by the HPV-16 E7 and E7 of above-mentioned acquisition_m58DNA fragmentation inserts pAAV/p5, pAAV/CMVp, pAAV/ respectively In these four rAAV carriers of SV40p and pAAV/ β-actinp.To insert the genetic fragment, endonuclease reaction, Ran Houjin are carried out first Row coupled reaction.Wherein, endonuclease reaction system is：100ng plasmids and 50ng HPV-16 E7 or E7_m58DNA fragmentation；10U is limited Property restriction endonuclease BamH I and Sal I (being purchased from Promega companies of the U.S.) processed, 2.5 μ 10 × buffer solutions of l C and 19.5 μ l go from Sub- water；Reaction condition is：Water-bath 4 hours at 37 DEG C.Coupled reaction system is：Plasmid after 50ng digestions, 50ng HPV-16 E7 or HPV-16 E7_m58DNA fragmentation, 10IU T4 DNA ligases (are purchased from Promega companies of the U.S.), 1.5 10 × T of μ l₄DNA Connect buffer solution and 11.5 μ l deionized waters；Reaction condition is：4 DEG C 8 hours.Finally respectively obtain carry p5 promoters, CMV promoter, SV40 early promoters or β protein promoters and HPV-16 E7_m58The restructuring of gene or HPV-16 E7 genes Gland relevant viral vector, correspond to four kinds of promoters respectively four kinds carry HPV-16 E7_m58The recombinant adeno-associated virus of gene carries The decorum is referred to as AAV/HPV-16 E7_m58, four kinds carrying HPV-16 E7 genes recombined glandulae correlation viral vectors be referred to as AAV/ HPV-16 E7。

D. recombined glandulae correlation viral vectors plasmid AAV/HPV-16 E7 and AAV/HPV-16 E7 are obtained_m58：Respectively will connection AAV/HPV-16 E7 afterwards_m58With AAV/HPV-16 E7 transformation gene engineerings Escherichia coli (E.coli) DH5 α competent cells (Invitrogen companies of the U.S.), resistance screening, picking white single bacterium are carried out with the LB flat boards containing 100 μ g/mL ampicillins Fall, extract plasmid and purify, respectively obtain AAV/HPV-16 E7_m58Plasmid and AAV/HPV-16 E7 plasmids.

F. plasmids detection：By the AAV/HPV-16 E7 of acquisition_m58Plasmid and AAV/HPV-16 E7 plasmids are with restriction enzyme Whether enzyme BamH I and Sal I (being purchased from Promega companies of the U.S.) carry out restriction analysis, successfully constructed with identification.Endonuclease reaction Condition and method are as described above (two .C).The AAV/HPV-16 E7 of structure_m58With the restriction analysis knot of rAAV/HPV-16 E7 carriers (swimming lane 1-6 is respectively AAV/p5/HPV-16 E7 to fruit as shown in Figure 4_m58、AAV/CMVp/ HPV-16 E7_m58、AAV/SV40p/ HPV-16 E7_m58、AAV/β-actinp/HPV-16 E7_m58, AAV/p5/HPV-16 E7 and AAV/CMVp/HPV-16 E7, Analysis result shows the success of structure restructuring AAV carriers), HPV-16 E7_m58AAV carriers are respectively inserted with HPV-16 E7 genes In, the E7 or saltant type E7 of carrying HPV 16 (HPV-16)_m58The recombined glandulae correlation viral vectors of gene are built into Work(.

Embodiment 2, the preparation of recombinant adeno-associated virus (rAAV) and virus titer measure

Material and its source：

A. the carrying HPV-16 E7 that embodiment 1 is built_m58With the recombined glandulae correlation viral vectors of HPV-16 HPV-16 E7 genes (AAV/HPV-16 E7_m58With AAV/HPV-16 E7).

B. the helper plasmid pHelper of the Rep genes containing AAV and Cap genes：It is built into by the inventor of present patent application Work((Liu, Y., Santin AD., Mane M., Chiriva-Internati, M., Parham GP., Ravaggi A., and Hermonat,P.L.Transduction and Utility of the Granulocyte-Macrophage Colony- Stimulating Factor Gene into Monocytes and Dendritic Cells by Adeno- Associated Virus.Journal of Interferon and Cytokine Research.20:21–30.2000)。

C. containing the adenoviral gene (E1, E2A, E4, VAI and VAII gene) for being integrated in cell chromosome and expressing AAV-HEK293 cells：(Liu, Y., Santin AD., Mane M., Chiriva- are established by the inventor of present patent application Internati,M.,Parham GP.,Ravaggi A.,and Hermonat,P.L.Transduction and Utility of the Granulocyte-Macrophage Colony-Stimulating Factor Gene into Monocytes and Dendritic Cells by Adeno-Associated Virus.Journal of Interferon and Cytokine Research.20:21–30.2000)。

D. lipofectamine Lipofectin：Purchased from Life Technology companies of the U.S..

E.DMEM culture mediums and hyclone (or calf serum)：Purchased from Cellgro companies of the U.S..

F.PCR DIG labelling kits and DIG hybridization check kits：Purchased from Roche companies of Switzerland.

G.DNA copy number standards：Respectively 10¹²Copy number (copies)/μ L to 10⁶(copies)/μ L, purchased from the U.S. Promega companies.

First, the preparation of recombinant adeno-associated virus (rAAV)

Method Prepare restructuring adeno-associated virus (rAAV) as shown in Figure 5.To prepare the disease of a disk 10.0cm Tissue Culture Dish Exemplified by poison, when AAV-HEK293 cells are grown in carbon dioxide cell incubator accounts for culture dish 70 % of area, carry out Following operation：

A. operated according to Lipofectin operation instruction：By 1.0 μ g rAAV, 1.0 μ g pHelper plasmids, 4.0 DMEM culture mediums of the μ L Lipofectin and 50.0 μ L containing 5% hyclone (or calf serum) mixes, and is stored at room temperature 20 points Clock.

B. mixed liquor is added in Tissue Culture Dish, continues to be placed in carbon dioxide cell incubator and cultivated at 37 DEG C.

C.72 after hour, all cells and nutrient solution in culture dish are harvested.

D. after acutely vibrating 1 minute, centrifugation, supernatant, i.e. rAAV virus liquids are retained.

E. by the rAAV virus liquid filtration sterilizations of collection.

2nd, the virus titer measure of recombinant adeno-associated virus (rAAV)

Using the spot hybridization of routine, the rAAV viruses obtained to step 1 carry out virus titer measure, specific method Comprise the following steps ()：.

A. using conventional DNA phenol/chloroform extraction methods, extraction rAAV virions DNA.

B. nylon membrane is placed in Dot blot instrument, adds the rAAV virion DNA through alkaline denaturation, and add DNA and copy Shellfish number standard, is vacuumized.

C. after taking out nylon membrane drying, ultraviolet is fixed.

D. the specific probe of DIG marks is prepared with PCR DIG labelling kits and with reference to kit specification, it is used DNA probe is the specific probe for HPV-16 E7 genes, and probe is the " HPV16-E7 obtained in the step C of embodiment 1 DNA.After PCR amplifications terminate, 1.2% agarose gel electrophoresis is carried out to pcr amplification product, detects PCR amplifications under ultraviolet light Product, as a result there is positive band, show that probe marks successfully.

E. with DIG hybridization checks kit and with reference to kit specification, to various rAAV virions in hybrid heater DNA carries out DNA hybridization.

All rAAV virus titers of experimental result are 10¹²-10¹⁴Copy/mL, show that the rAAV virus titers of acquisition are higher, It is fully available for scientific research and clinical practice.

Embodiment 3, recombinant adeno-associated virus AAV/HPV-16 E7_m58With AAV/HPV-16 E7 infectors for epithelium of cervix uteri The oncogenicity observation of cell

Material and its source：

A.rAAV viruses：The recombinant adeno-associated virus AAV/HPV-16 E7 that embodiment 2 obtains_m58With AAV/HPV-16 E7.

B.Keratinocyte-SCF cell culture mediums：Purchased from Life Technology companies of the U.S..

C. primary cervical epithelial cellses：Separated and obtained from normal cervical epithelial tissue with conventional method.

Oncogenicity observation experiment

By primary cervical epithelial cellses in 10.0cm Tissue Culture Dish is put into, 10mL is added immediately Keratinocyte-SCF cell culture mediums, put in CO2gas incubator and cultivated at 37 DEG C.After cell is completely adherent, take Go out culture dish, after removing 7mL nutrient solutions, recombinant adeno-associated virus AAV/HPV-16 E7 are separately added into according to 100MOI dosage_m58 Or AAV/HPV-16 E7, it is refitted in CO2gas incubator.After 8 hours, culture dish is taken out, removes nutrient solution, it is new to add 10mL Fresh Keratinocyte-SCF cell culture mediums, are refitted in CO2gas incubator and are cultivated at 37 DEG C.Change training within every 2 days Support base.The cellular morphology of timing observation daily changes 2 times.Until there is knurl change in cell.

As a result as shown in fig. 6, two kinds of rAAV are expressed in cell, still, by AAV/HPV-16 E7_m58The cell of infection Knurl change does not occur, shows that (three culture dishes are separately added into band CMV promoter, SV40 early promoters to no oncogenicity from top to bottom Or the AAV/HPV-16 E7 of β protein promoters_m58)；And the cell infected by AAV/HPV-16 E7 occurs obvious knurl and become, have (three culture dishes are separately added into CMV promoter, SV40 early promoters or β protein promoters oncogenicity from top to bottom rAAV/HPV-16 E7)。

Embodiment 4, tumour antigen import the killing tumor experiment of monocyte-BMDC system

Material and its source：

A.rAAV viruses：The AAV/HPV-16 E7 that embodiment 2 obtains_m58With AAV/HPV-16 E7.

B.AIM-V cell culture mediums：Purchased from Life Technology companies of the U.S..

C. cell factor：Granulocyte colony stimulating factor (GM-CSF) and interleukin 2,4,7 are purchased from R＆D companies of the U.S..

Cell positive D.HPV-16 E7：Separation obtains or preserves center from American. tissue cell from tumor tissues (ATCC) obtain, it is thin that malignant tumour includes cervical cancer cell, breast cancer cell, penis cancer cell, anus cancer cell and carcinoma of mouth Born of the same parents.

First, tumor experiment is killed

As shown in fig. 7, the present invention is carried into HPV-16 E7 or HPV-16 E7_m58RAAV viruses (the AAV/ of antigen gene HPV-16 E7_m58Or AAV/HPV-16 E7) killing HPV-16 HPV-16 E7s based on infected tumor's patient's monocyte (Mo) Positive cell experiment comprises the following steps：

A. tumor patient 50-150mL peripheral bloods are taken, with haemocyte separator (or lymphocyte separation medium) according to a conventional method PMNC (PBMC) is obtained, after being mixed with AIM-V culture mediums, Tissue Culture Flask is added, is placed in constant temperature titanium dioxide Cultivated 2 hours at 37 DEG C in carbon incubator.

B. suspension cell is removed, retains attached cell (monocyte).Suspension cell is PBLC, by its with After AIM-V culture mediums mix, continue to cultivate standby.

C. rAAV viruses are added, addition is about 100MOI, while adds GM-CSF (600IU/mL), continues culture 4 Hour.

D. old culture medium is removed, AIM-V culture mediums of the supplement containing GM-CSF and IL-4 (600IU/mL), continues to cultivate.

E. after cultivating 5 days, the BMDC (DC) of maturation is harvested, and is mixed with the PBLC cultivated, IL-2 (10IU/mL) and IL-7 (200IU/mL) is added in AIM-V culture mediums, continues to cultivate.

F. cultivate to after 7-9 days, the cytotoxic T lymphocyte (CTL) for harvesting activation is detected.

2nd, the detection of BMDC (DC) and cytotoxic T lymphocyte (CTL)

The Efficiency testing of A.rAAV infection PMBCs (Mo)

Decoration method is marked using the fluorescence antibody of routine, (U.S. is purchased from for HPV-16 E7 specific fluorescent antibodies BD companies) markers step one obtain the monocyte (Mo) infected by rAAV of the present invention or immature BMDC (DC), then the quantity of flow cytomery positive cell is carried out.Wherein, rAAV infects the Efficiency testing knot of monocyte (Mo) Fruit is as shown in figure 8, carry the AAV/HPV-16 E7 of four kinds of different promoters (p5, CMVp, SV40p and β-actinp)_m58With AAV/HPV-16 E7 infection Mo efficiency is each about 90%, i.e., 90 about percent Mo can be infected by rAAV viruses, it was demonstrated that The rAAV of the present invention has higher efficiency of infection.

B. the detection of the CD molecular levels of BMDC (DC)

DC expression CD80 and CD86 level and DC function is proportionate.With with step A identical detection methods, that is, divide Not Cai Yong fluorescence labeling the antibody (being purchased from U.S. company BD) for this two kinds of CD molecules step 1 is obtained DC expression CD80 and CD86 level is detected.AAV/HPV-16 E7_m58Or AAV/HPV-16 E7 infection DC expression CD80 and As shown in Figure 9 (using the rAAV with CMV promoter as representative), the CD expressed by infected DC divides testing result horizontal CD86 It is sub horizontal higher, it was demonstrated that the present invention carries HPV-16 E7 or HPV-16 E7_m58The monocyte institute of the rAAV infection of antigen gene The DC of induction activated cell immune response it is powerful.

C. the horizontal detection of the interferon (IFN-γ) of cytotoxic T lymphocyte (CTL) expression

CTL function and its expression of the ability of killing tumor cell and IFN-γ are proportionate.With with step A The level for the CTL expression IFN-γs that similar method detection is induced by the rAAV of the present invention DC infected is (with HPV-16 E7 albumen The CTL that the DC of stimulation and non-stimulated DC are induced is control), after DC terminates with PBLC mixed culture, receive Cell is obtained, carries out cell fluorescence dye marker using traditional Intracellular cytokine staining methods, antibody used is the fluorescence mark for IFN-γ Remember antibody (being purchased from U.S. company BD), finally utilize flow cytomery result.Wherein, by AAV/HPV-16 E7_m58Or The IFN-γ expression for the CTL that the DC of AAV/HPV-16 E7 infection is induced is as shown in Figure 10 (with band β-actin promoters RAAV be representative), by rAAV infect DC stimulate caused by CTL expression IFN-γ level it is higher, it was demonstrated that it is of the invention AAV/HPV-16 E7_m58Or the CTL that the DC of AAV/HPV-16 E7 infection is induced kills the activity of target cell compared with (function is strong by force Greatly).

D. cytotoxic T lymphocyte (CTL) killing tumor cell is tested

Mixed culture terminate after, by step 1 by AAV/HPV-16 E7_m58Or the DC institutes of AAV/HPV-16 E7 infection The cytotoxic T lymphocyte (CTL) of induction presses 20:1 (lymphocyte:Tumour cell) respectively with cervical cancer cell, breast cancer After cell, penis cancer cell, anus cancer cell and cancer cell of oral cavity mixing, using traditional⁵¹Cr (chromium -51) fragmentation test, inspection Survey the activity of CTL killing tumor cells.

By the rAAV DC infected the CTL Cytotoxicity in vitro HPV-16 E7 positive cells induced⁵¹Cr (chromium -51) killings are real Result such as Figure 11 is tested (with the AAV/HPV-16 E7 with CMV promoter_m58Exemplified by, ordinate represents killing rate) shown in, sent out by this The CTL that the DC of bright rAAV infection is induced can more effectively crack the positive target cell of (killing) HPV-16 HPV-16 E7s, killing Rate is 40%-60% or so, and to lung (lung) negative HPV-16 E7, mammary gland (breast), liver (liver), kidney (k- Cells cell) is compares, no lethal effect, it was demonstrated that has antigen special by the rAAV of the present invention DC infected the CTL induced The opposite sex, i.e., to the cell of antigen negative without lethal effect.

Above-mentioned testing result shows, saltant type HPV-16 E7 are carried by the present invention_m58The restructuring gland related diseases of antigen gene Poison (AAV/HPV-16 E7_m58) cell that the CTL that are induced of DC of infection are positive to HPV-16 HPV-16 E7s has powerful killing (cracking) effect, has a higher specificity (i.e. targeting lethal effect), and to the negative cell of HPV-16 HPV-16 E7s without killing Wound acts on, the killing ability indistinction with the CTL of wild type HPV-16 HPV-16 E7s induction, and without oncogenicity, available for preparing Antineoplastic.

The clinical trial of the positive oncotherapy of embodiment 5, HPV-16 HPV-16 E7s

Using recombinant adeno-associated virus-BMDC technology, i.e., by AAV/HPV-16 E7 of the present invention in embodiment 4_m58 Cytotoxic T lymphocyte (CTL) that the DC of infection is induced feeds back 5 cervical cancer patients, all patients it is verified that its Cervical cancer tissues are HPV-16 E7 positive.Infusion amount is 2 × 10⁸-5×10⁸.Treatment course：Usually 3 months are a treatment Journey, monthly 2 times, the state of an illness can be kept to monthly 1-2 times after improving, and can further be kept to treat once every 1-3 months.Treatment results are summarized (B as shown in table 1:Blood serum tumor markers are reduced or disappeared.Q:Quality of life of patients improves.Such as pain relief or disappearance, appetite Increase etc..C:CT or PET-CT show that cancer focus or metastatic lesion are obviously reduced or disappeared.), adverse reaction：1 treatment Slight influenza sample reaction occurs in the short time afterwards, but patient can bear, and symptom disappears in a short time, is not observed serious Adverse reaction and toxic reaction.5 through recombinant adeno-associated virus AAV/HPV-16 E7_m58The CTL treatments that the DC of infection is induced The situation of change of front and rear cervical cancer patient serum Cytokeratin 19 antigen (CK19) and squamous cell carcinoma antigen (SCC) level is as schemed Shown in 12, after treatment, the serum Keratin 19 antigen (CK19, cyfra21-1) and squamous cell carcinoma antigen of 3 patients (SSC) (CK19 levels, normal value are decreased obviously<3.3ng/ml；SCC is horizontal, normal value<5.0ng/ml), or even recover normal. When present patent application is submitted, 5 patients are survived.Clinical trial results further prove, are infected by rAAV of the present invention The CTL that DC is induced can play the effect of certain in patient's body, can effectively suppress the pernicious swollen of the HPV-16 E7 positives Tumour cell is killed in the growth of oncocyte, and security is higher, available for preparing antineoplastic.

The rAAV/HPV-16 E7 of table 1_m58- BMDC technology treats the statistics knot of the effect of 5 cervical carcinoma cancer patients Fruit

Numbering	Clinical stages	RAAV-DC treatment courses	Time-to-live after treatment	Therapeutic effect
							(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

It is demonstrated experimentally that by HPV-16 E7 of the present invention_m58The BMDC of recombinant adeno-associated virus infection and induced Cytotoxic T lymphocyte can effectively suppress the cell of HPV16 infection and relative malignant tumour in patient's body Tumour cell is killed in the growth of cell, thus, HPV-16 E7 of the invention_m58Recombined glandulae correlation viral vectors or with this hair Bright/HPV-16 E7_m58The related product of recombined glandulae correlation viral vectors can be used for the cell and its phase for preparing anti-HPV16 infection The antineoplastic of pass, have great importance in clinical treatment and application.

Claims (5)

1. one kind carries HPV 16 saltant type E7_m58The recombined glandulae correlation viral vectors of antigen gene, it is by purpose Genic mutation type HPV-16 E7_m58In antigen gene insertion gland relevant viral vector, obtain and carry HPV-16 E7_m58Antigen gene Recombined glandulae correlation viral vectors, referred to as " E7_m58Recombined glandulae correlation viral vectors " or AAV/HPV-16 E7_m58；

The saltant type HPV-16 E7_m58Antigen gene is by the half of the 58th, the HPV-16 E7 albumen of HPV-16 HPV-16 E7 genes Cystine changes into gene corresponding to glycine, i.e. replaces HPV-16 E7 gene open reading frames nt736 thymidine t For guanine g, or the tgc of encoding aminothiopropionic acid is changed into the ggc of coding glycine, the prominent of no oncogenicity can be expressed by obtaining Modification HPV-16 E7_m58Gene；The HPV-16 E7_m58The nucleotide sequence of gene is as shown in sequence 1 in sequence table；

The gland relevant viral vector is the gland relevant viral vector for rejecting adeno-associated virus AAV structural genes Rep and Cap, its The promoter of carrying is that p5 promoters, cytomegalovirus CMV promoter, people beta actin promoters or SV40 viruses are early Any one in phase promoter；

The method of the recombined glandulae correlation viral vectors, comprises the following steps：

1) it is glycine by the 58th cysteine mutation of HPV-16 HPV-16 E7 albumen, i.e., reads HPV-16 E7 gene opens Code frame nt736 thymidine t replaces with guanine g, i.e., the tgc of encoding aminothiopropionic acid is changed into the ggc of coding glycine, Obtain the saltant type HPV-16 E7 without oncogenicity_m58Gene；

2) by HPV-16 E7_m58Antigen gene inserts the E7_m58In recombined glandulae correlation viral vectors, obtain carrying HPV-16 E7_m58The recombined glandulae correlation viral vectors AAV/HPV-16 E7 of antigen gene_m58。

2. with recombined glandulae correlation viral vectors AAV/HPV-16 E7 described in claim 1_m58Related product, including restructuring gland phase Close virus particle, recombinant adeno-associated virus particle and by the recombinant gland relevant viral vector infection or the cell line of transfection, carefully Born of the same parents system includes monocyte Mo and BMDC DC.

3. prepare claim 2 described in recombined glandulae correlation viral vectors AAV/HPV-16 E7_m58The method of Related product, respectively For：Carry HPV-16 E7_m58The preparation of the recombined glandulae correlation viral vectors plasmid of antigen gene：HPV-16 E7 will be carried_m58It is anti- The recombined glandulae correlation viral vectors DNA of protogene is AAV/HPV-16 E7_m58Quiding gene engineering colon bacillus (E.coli) DH5 α competent cells, resistance screening is carried out with the LB flat boards containing 100 μ g/mL ampicillins, picking white single bacterium colony, extracts matter Grain simultaneously purifies, and obtains AAV/HPV-16 E7_m58Plasmid；

Carry HPV-16 E7_m58The preparation of the recombinant adeno-associated virus of antigen gene：With the carrying HPV-16 E7_m58Antigenic site The recombined glandulae correlation viral vectors plasmid and pHelper plasmids of cause transfect AAV-HEK293 cells and obtain rAAV viruses, life jointly Entitled AAV/HPV-16 E7_m58Virus；

Carry HPV-16 E7_m58The preparation of the recombinant gland relevant viral vector infection of antigen gene or the cell line of transfection：With institute State recombinant adeno-associated virus AAV/HPV-16 E7_m58Virus infects or transfected monocyte Mo or BMDC respectively or successively DC is obtained.

4. a kind of medicine of the cellular immunotherapy of anti-HPV-16 infection and the tumour as caused by infecting HPV-16, its activity into It is divided into and HPV-16 E7 is carried described in claim 1_m58The recombined glandulae correlation viral vectors AAV/HPV-16 E7 of antigen gene_m58Or With carrying HPV-16 E7 described in claim 3_m58The related product of the recombined glandulae correlation viral vectors of antigen gene, including AAV/ HPV-16 E7_m58Plasmid, AAV/HPV-16 E7_m58Virus, by HPV-16 E7_m58Recombinant adeno-associated virus infects respectively or successively Or the monocyte Mo or BMDC DC that transfection obtains.

5. medicine according to claim 4, it is characterised in that：The HPV-16 infection associated malignancies are HPV-16 The positive uterine neck papilloma lesion of HPV-16 E7, cervical carcinoma, male sex organ Bowen ' s diseases, Buschke-Lo＆4＆wenstein tumor, carcinoma of penis, anus Door cancer, the carcinoma of the rectum, carcinoma of mouth, carcinoma of tonsil or breast cancer.