CN106963945A - A kind of reinforced HPV HPV 16/18 divalence DC vaccines - Google Patents

A kind of reinforced HPV HPV 16/18 divalence DC vaccines Download PDF

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CN106963945A
CN106963945A CN201710187380.8A CN201710187380A CN106963945A CN 106963945 A CN106963945 A CN 106963945A CN 201710187380 A CN201710187380 A CN 201710187380A CN 106963945 A CN106963945 A CN 106963945A
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hpv
vaccines
divalence
reinforced
imdc
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刘明录
李言志
万磊
强邦明
冯建海
金海峰
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Jinan Xingyi Medical Technology Co Ltd
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Jinan Xingyi Medical Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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  • Life Sciences & Earth Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention discloses a kind of reinforced HPV HPV 16/18 divalence DC vaccines, including the BMDC through genetic modification, recombination sequence in the BMDC through genetic modification is by granular leukocyte macrophage stimulus factor, HPV HPV 16 connects with HPV 18 L1 transcriptional domains, the DC vaccines are made up of the DC surface antigens through genetic modification, the granular leukocyte macrophage stimulus factor expressed including enhancement antigen, HPV 16 and HPV 18 L1 antigen proteins, the invention, which belongs to, prevents and treats mating type vaccine, HPV 16 can effectively be prevented, relevant disease caused by HPV 18 infection and HPV infection.

Description

A kind of reinforced HPV HPV-16/18 divalence DC vaccines
Technical field
It is a kind of reinforced HPV in particular the present invention relates to biological and new medical technology field HPV-16/18 divalence DC vaccines.
Background technology
HPV (Human papillomavirus, HPV) is a kind of papilloma for belonging to papovaviridae Vacuolating virus A belongs to, it is easy to infects human epidermal and mucous membrane scaly epithelium, is to cause cervical carcinoma and genital condylomas and part anus Door cancer, the principal element of carcinoma of mouth.Persistent infection high-risk HPV -16, HPV-18 may cause cervical carcinoma, the carcinoma of the rectum, oral cavity The malignant tumours such as cancer, carcinoma of tonsil.Cervical carcinoma is the clear and definite malignant tumour of the currently the only cause of disease, 99.7% cases of cervical cancer because HPV infection is caused, and the World Health Organization and international cancer center also confirm that high-risk HPV persistent viral infection is the master of cervical carcinoma The cause of disease is wanted, therefore, HPV vaccines turn into vaccine of cervical cancer by people's habituation again.According to incompletely statistics, the annual whole world has 50 Ten thousand women suffer from cervical carcinoma because infecting HPV, wherein 200,000 die from the tumour, HPV viruse is with AIDS, cancer by world health group Knit and be classified as the big incurable disease of the mankind three.
HPV-16/18 can cause 70% cases of cervical cancer, it is also possible to cause the genital condylomas of masculinity and femininity, property life Work is the main channel that it is propagated, and conventional safeguard measure, such as sheath can not block the propagation of HPV viruse completely, Thus vaccinate just into mode best prevention HPV.
HPV viruse genome is the double chain DNA molecule of closed hoop, is about 7.2-8kb, encodes 8 ORFs (Open Reading Frame), be divided into early transcription area (Early Region, E areas), late transcription area (Late Region, L areas) and control zone (Long Control Region) 3 functional areas, the early transcription head of district about 4.5Kb, encode six non-structural Property early protein：E1, E2, E4-E7, participate in the functions such as duplication, transcription, translational control and the cell transformation of virus；Late transcription The head of district about 2.5Kb, coding core capsid protein L 1 and secondary capsid protein L2, constitute the capsid of virus；Control zone is about 800- 900bp, between E8 and L1, the area contain HPV genomic DNAs replication orgin and HPV gene expressions needed for regulation and control member Part, is responsible for the duplication and expression of regulating DNA.Virion is made up of L1, L2 capsid protein, diameter about 45-55nm, in without coating The 20 symmetrical nucleocapsid structures of face body, there are 72 shell particulates on surface.
DC is commonly called as BMDC (dendritic cells, DCs), and many dendron samples or pseudopodium sample are stretched out during because of maturation Projection and gain the name, be that in vivo functionality is most strong, can uniquely activate the professional antigen of Resting T cells be in delivery cell (Antigen Presenting Cells, APC), antigen can be absorbed, processed and presented, is startup, regulation and control and maintains the immune of T cell mediation The key link of reaction.
Current DC vaccines have been applied to the monitoring and treatment of Partial tumors, and the vaccine preparation method based on DC mainly has It is several below：(1) DC of antigen polypeptide sensitization, it is this such as using the tumour specific antigen polypeptide sensitization DC of purifying or restructuring Method targeting is good.But the antigen polypeptide having determined at present is limited, single antigen polypeptide is only defined in specific HLA classes Type, and tumour cell is easy to escape body by antigenic mutation and is directed to the immunization that is designated as of single antigen.(2) whole antigens The DC of sensitization, DC is stimulated using the pyrolysis product of virus infected cell (or tumour cell), protein extract, apoptosis product, this Class vaccine includes all viral antigens, is also easy to prepare, can trigger polyclonal CTL immune responses.But the program Need infected tissue's amount larger, and optimal stimulus amount is difficult to determine.In addition, virus infected cell is also anti-containing organism itself Original, there is the danger for inducing autoimmune response.(3) gene transfection DC, that is, utilize the RNA/DNA sensitization DC of encoding viral antigen. The method is by all or part of DNA sequence dnas of PCR amplicon virus antigens, then by nucleic acid with viral or non-viral mediation Method is imported in DC, and purpose antigen can form lasting expression in the cell.This method can after customer service antigenic stimulus DC, because antigen- Adverse effect produced by the immune response that MHC complex dissociations or MHC molecule degraded are induced it.
DC vaccines can not only induce a large amount of effector T cells of generation to migrate to viral infection site, moreover it is possible to keep effector T cell Long-term existence at easy infection position, plays a part of immunosurveillance.
The content of the invention
The invention provides a kind of reinforced HPV HPV-16/18 divalence DC vaccines to solve above-mentioned background The weak point of technical problem.
The solution of the present invention is：
A kind of reinforced HPV HPV-16/18 divalence DC vaccines, including the dendron shape through genetic modification are thin Recombination sequence in born of the same parents, the BMDC through genetic modification is by granulocytes-macrophages stimulating factor, people's nipple Tumor virus HPV-16 connects with HPV-18 L1 transcriptional domains.
As preferred technical scheme, described granulocytes-macrophages stimulating factor is sequence table SEQ ID NO.1 institutes The nucleotide sequence shown.
As preferred technical scheme, the L1 antigen sequences of the HPV16 are the nucleosides shown in sequence table SEQ ID NO.2 Acid sequence.
As preferred technical scheme, the L1 antigen sequences of the HPV18 are the nucleosides shown in sequence table SEQ ID NO.3 Acid sequence.
As preferred technical scheme, in addition to by the method for DC cell loading humanization recombinations：By the restructuring Gene order is inserted into Lentiviral pLent-C-GFP, obtain pLent- (GM-CSF)-(HPV16-L1)- (HPV18-L1)-GFP carriers；Meanwhile, by human umbilical cord blood mononuclear cell in GMP laboratories, the human umbilical cord blood mononuclear cell warp ImDC is induced into, the HPV16/18-L1 sequences for being packaged in slow virus are infected into imDC, and by imDC through maturation factor induced maturation Obtain DC vaccines.
ImDC preparation method is induced into as preferred technical scheme, in addition to the human umbilical cord blood mononuclear cell：Receive The healthy human umbilical cord blood 50ml of collection, is isolated in mononuclearcell, culture medium, 37 DEG C using lymphocyte separation medium, 5% CO2Under the conditions of after culture 2-3 hour, outwell suspension dead cell, leave and take attached cell, and add cell factor rhIL-4, end is dense Degree：50ng/ml, rhGM-GSF final concentration 100ng/ml, induction mononuclearcell are divided into DC, and training was more renewed every 48 hours Base is supported, culture obtains imDC on the 5th day.
As preferred technical scheme, the imDC induces the DC vaccines for obtaining maturation, the imDC the 6th through maturation factor It adds maturation factor TNF-α, and induction obtains the DC cells of maturation.
A kind of reinforced HPV HPV-16/18 divalence DC vaccines are used to prepare prophylactic divalence DC epidemic diseases The purposes of seedling.
As preferred technical scheme, the prevention disease is cervical carcinoma, but is not limited to cervical carcinoma, good to include prevention Other malignant tumours caused by the high-risk-type HPV infection such as the carcinoma of the rectum, carcinoma of mouth, carcinoma of tonsil.
By adopting the above-described technical solution, a kind of reinforced HPV HPV-16/18 divalence DC vaccines, bag Include recombination sequence in the BMDC through genetic modification, the BMDC through genetic modification by granulocyte- Macrophage stimulation factor, HPV HPV-16 connect with HPV-18 L1 transcriptional domains；The present invention HPV-16, HPV- 18 L1 areas antigen load is used as guiding son enhancing L1 sequences in ripe DC cells, and using granulocytes-macrophages stimulating factor The expression of row；The DC cells can be combined antigen with MHC molecule, form L1 polypeptides-MHC molecule compound, and submission is thin to T Born of the same parents, so as to start the reaction of MHC-I classes specific CTL and MHC-II class specific Cs D4+Thl reactions, are carried out to HPV16 and HPV18 Accuracy, specificity, targeting, initiatively hit and kill.Meanwhile, DC also passes through the costimulatory molecules (CD80/B7- of its height expression 1st, CD86/B7-2, CD40 etc.) secondary signal necessary to T cell activation is provided, so that the person's that thoroughly breaks HPV infection is immune Tolerance status, make the immune system of patient as normal person's infection cell virus, produce the specific antibody for HPV, come Confrontation, attack simultaneously finally remove HPV；Due to having cut intake of the DC cells to antigen, process, the DC vaccines improve T Lymphocyte can provide HPV invasion again the long term immune memory work(of protectiveness to the phagocytosis efficiency of HPV infection cell Energy.
Brief description of the drawings：
Fig. 1 is HPV16/18 recombinations module diagram of the present invention；
Fig. 2 be Lentiviral pLent- (GM-CSF)-(HPV16-L1)-(HPV18-L1) of the present invention- GFP schematic diagram, wherein, sequence clockwise is positive genetic fragment, and sequence counterclockwise is reverse genetic fragment；
Fig. 3 is the imDC Stereo microscope downward view figures of Cord blood induction of the present invention；
Fig. 4 is maturation DC cell Stereo microscope downward view figures of the invention；
Fig. 5 is the amount streaming figure of the imDC surface moleculars mark CD83+ expression of Cord blood induction of the present invention；
Fig. 6 is the amount streaming figure of present invention maturation DC cell surface molecules mark CD83+ expression；
Fig. 7 is that the imDC surface moleculars of Cord blood induction of the present invention mark the streaming figure of CD86+ expression quantity；
Fig. 8 is the streaming figure that present invention maturation DC cell surface molecules mark CD86+ expression quantity；
Fig. 9 is pLent- (HPV16-L1)-(the HPV18-L1)-GFP and pLent- (GM- that the present invention is measured by qPCR CSF)-(HPV16-L1)-(HPV18-L1)-GFP respectively as Lentiviral cause DC cell surfaces HPV16-L1 and HPV18-L1 relative expression quantity, wherein, * is represented, and there are data to have significant difference, and * *, which represent data, has pole significant difference.
Embodiment
In order that the technical means, the inventive features, the objects and the advantages of the present invention are easy to understand, tie below Specific embodiment is closed, the present invention is expanded on further.
Embodiment one：ImDC induction
(1) healthy human umbilical cord blood 50ml is collected, mononuclearcell, culture medium are isolated using lymphocyte separation medium In (being purchased from TaKaRa companies, GT-T551), 37 DEG C, 5% CO2Under the conditions of culture 2-3 hours after, outwell suspension dead cell, stay Attached cell is taken, and adds cell factor rhIL-4 (final concentrations：50ng/ml), rhGM-GSF (final concentration 100ng/ml), induction Mononuclearcell is divided into DC, more renews culture medium every 48 hours, and culture obtains imDC on the 5th day, seen by inverted microscope The ripe preceding cellular morphologies of DC are examined, Flow cytometry cell surface molecule marks CD83+ and CD86+ expression, comprehensive identification The imDC of induction.
Embodiment two：HPV-16, HPV-18 L1 sequence modifications maturation DC cells
(1) prepared by the packaging of slow virus：Using gene clone technology, PCR amplification HPV-16, HPV-18 L1 full length genes CDNA sequence, is connected in Lentiviral pLent-C-GFP, is built the Lentiviral of L1 antigens, is passed through PCR With nucleic acid sequencing identification.Carry out virus titer measure.
(2) the L1 slow virus carriers of structure are infected into imDC, the imDC of infection is cultivated maturation factor TNF- is added after 6h α, induction obtains the DC cells of maturation, and cellular morphology, Flow cytometry cell after DC maturations are observed by inverted microscope Surface molecular marks CD83+ and CD86+ expression, the mDC of comprehensive identification induction.The mDC can direct injection be used as prevention HPV16 With HPV18 vaccine.
DC cell surfaces HPV16-L1 and HPV18-L1 are expressed for checking whether there is GM-CSF, qPCR determines pLent- (HPV16-L1)-(HPV18-L1)-GFP and pLent- (GM-CSF)-(HPV16-L1)-(HPV18-L1)-GFP is respectively as slow The influence of virus expression carrier HPV16/18 intracellular to DC mRNA relative expressions.HPV16-L1 qPCR primers are： HPV16-Fw：5 '-GAATTCATTTGCCAGATCCA-3 ', HPV16-Rv：5’-ATACCAACACCCAATGGTTG-3’； HPV18-L1 qPCR primers are：HPV18-Fw：5’-GGGTGCAGTTACCTGACCCA3’,HPV18-Rv:5’- AGGCCAACACCTAAAGGCTG-3’。
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent circle.
Sequence table
GM-CSF signal peptide nucleic acid artificial sequences
SEQ ID NO.1
<110>Shandong Xing Rui bio tech ltd
<120>A kind of reinforced HPV HPV-16/18 divalence DC vaccines
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 2
<211> 75
<212> DNA
<213>Artificial sequence
<400> 2
atgctgctgc tggtgaccag cctgctgtgc gagctggagc cccaccccgc ctttctgctg 60
atccccgaca tccag 75
HPV-16-L1 nucleotide sequence
SEQ ID NO.2
<110>Shandong Xing Rui bio tech ltd
<120>A kind of reinforced HPV HPV-16/18 divalence DC vaccines
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1416
<212> DNA
<213>Artificial sequence
<400> 1
atgtcattat ggcttccttc agaggctacc gtatacttac ctcctgttcc agttagtaaa 60
gtcgtttcta cagatgaata cgttgctaga actaatattt attatcatgc tggtacttct 120
agattgttgg ctgttggtca tccatatttt ccaattaaaa aaccaaataa taataaaatt 180
ttggttccaa aagtttctgg tttgcaatat agagttttta gaattcattt gccagatcca 240
aataaatttg gttttccaga tacttctttt tataatccag atactcaaag attggtttgg 300
gcttgtgttg gtgttgaagt tggtagaggt caaccattgg gtgttggtat ttctggtcat 360
ccattgttga ataaattgga tgatactgaa aatgcttctg cttatgctgc taatgctggt 420
gttgataata gagaatgtat ttctatggat tataaacaaa ctcaattgtg tttgattggt 480
tgtaaaccac caattggtga acattggggt aaaggttctc catgtactaa tgttgctgtt 540
aatccaggtg attgtccacc attggaattg attaatactg ttattcaaga tggtgatatg 600
gttgatactg gttttggtgc tatggatttt actactttgc aagctaataa atctgaagtt 660
ccattggata tttgtacttc tatttgtaaa tatccagatt atattaaaat ggtttctgaa 720
ccatatggtg attctttgtt tttttatttg agaagagaac aaatgtttgt tagacatttg 780
tttaatagag ctggtgctgt tggtgaaaat gttccagatg atttgtatat taaaggttct 840
ggttctactg ctaatttggc ttcttctaat tattttccaa ctccatctgg ttctatggtt 900
acttctgatg ctcaaatttt taataaacca tattggttgc aaagagctca aggtcataat 960
aatggtattt gttggggtaa tcaattgttt gttactgttg ttgatactac tagatctact 1020
aatatgtctt tgtgtgctgc tatttctact tctgaaacta cttataaaaa tactaatttt 1080
aaagaatatt tgagacatgg tgaagaatat gatttgcaat ttatttttca attgtgtaaa 1140
attactttga ctgctgatgt tatgacttat attcattcta tgaattctac tattttggaa 1200
gattggaatt ttggtttgca accaccacca ggtggtactt tggaagatac ttatagattt 1260
gttacttctc aagctattgc ttgtcaaaaa catactccac cagctccaaa agaagatcca 1320
ttgaaaaaat atactttttg ggaagttaat ttgaaagaaa aattttctgc tgatttggat 1380
caatttccat tgggtagaaa atttttgttg caagcttag 1419
HPV-18-L1 nucleotide sequence
SEQ ID NO.3
<110>Shandong Xing Rui bio tech ltd
<120>A kind of reinforced HPV HPV-16/18 divalence DC vaccines
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 2
<211> 1416
<212> DNA
<213>Artificial sequence
<400> 2
atggctttgt ggcggcctag tgacaatacc gtatatcttc cacctccttc tgtggcaaga 60
gttgtaaata ccgatgatta tgtgactcgc acaagcatat tttatcatgc tggcagctct 120
agattattaa ctgttggtaa tccatatttt agggttcctg caggtggtgg caataagcag 180
gatattccta aggtttctgc ataccaatat agagtattta gggtgcagtt acctgaccca 240
aataaatttg gtttacctga taatagtatt tataatcctg aaacacaacg tttagtgtgg 300
gcctgtgttg gagtggaaat tggccgtggt cagcctttag gtgttggcct tagtgggcat 360
ccattttata ataaattaga tgacactgaa agttcccatg ccgccacgtc taatgtttct 420
gaggacgtta gggacaatgt gtctgtagat tataagcaga cacagttatg tattttgggc 480
tgtgcccctg ctattgggga acactgggct aaaggcactg cttgtaaatc gcgtccttta 540
tcacagggcg attgcccccc tttagaactt aaaaacacag ttttggaaga tggtgatatg 600
gtagatactg gatatggtgc catggacttt agtacattgc aagatactaa atgtgaggta 660
ccattggata tttgtcagtc tatttgtaaa tatcctgatt atttacaaat gtctgcagat 720
ccttatgggg attccatgtt tttttgctta cggcgtgagc agctttttgc taggcatttt 780
tggaataggg caggtactat gggtgacact gtgcctccat ccttatatat taaaggcaca 840
ggtatgcgtg cttcacctgg cagctgtgtg tattctccct ctccaagtgg ctctattgtt 900
acctctgact cccagttatt taataaacca tattggttac ataaggcaca gggtcataac 960
aatggtgttt gctggcataa tcaattattt gttactgtgg tagataccac tcgcagtacc 1020
aatttaacaa tatgtgcttc tacacagtct cctgtacctg ggcaatatga tgctaccaaa 1080
tttaagcagt atagcagaca tgttgaggaa tatgatttgc agtttatttt tcagttgtgt 1140
actattactt taactgcaga tgttatgtcc tatattcata gtatgaatag cagtatttta 1200
gaggattgga actttggtgt tccccccccg ccaactacta gtttggtgga tacatatcgt 1260
tttgtacaat ctgttgctat tacctgtcaa aaggatgctg caccggctga aaataaggat 1320
ccctatgata agttaaagtt ttggaatgtg gatttaaagg aaaagttttc tttagactta 1380
gatcaatatc cccttggacg taaatttttg gttcagtag 1419

Claims (9)

1. a kind of reinforced HPV HPV-16/18 divalence DC vaccines, it is characterised in that：Including through genetic modification Recombination sequence in BMDC, the BMDC through genetic modification by granulocytes-macrophages stimulate because Son, HPV HPV-16 connect with HPV-18 L1 transcriptional domains.
2. a kind of reinforced HPV HPV-16/18 divalence DC vaccines as claimed in claim 1, its feature exists In：Described granulocytes-macrophages stimulating factor is the nucleotide sequence shown in sequence table SEQ ID NO.1.
3. a kind of reinforced HPV HPV-16/18 divalence DC vaccines as claimed in claim 1, its feature exists In：The L1 antigen sequences of the HPV16 are the nucleotide sequence shown in sequence table SEQ ID NO.2.
4. a kind of reinforced HPV HPV-16/18 divalence DC vaccines as claimed in claim 1, its feature exists In：The L1 antigen sequences of the HPV18 are the nucleotide sequence shown in sequence table SEQ ID NO.3.
5. a kind of reinforced HPV HPV-16/18 divalence DC vaccines as claimed in claim 1, its feature exists In, in addition to by the method for DC cell loading humanization recombinations：The recombination sequence is inserted into slow virus expression In carrier pLent-C-GFP, pLent- (GM-CSF)-(HPV16-L1)-(HPV18-L1)-GFP carriers are obtained；Meanwhile, by navel Band blood mononuclear cell is in GMP laboratories, and the human umbilical cord blood mononuclear cell will be packaged in slow virus through inducing into imDC HPV16/18-L1 sequences infect imDC, and imDC is obtained into DC vaccines through maturation factor induced maturation.
6. a kind of reinforced HPV HPV-16/18 divalence DC vaccines as claimed in claim 5, its feature exists In, in addition to the human umbilical cord blood mononuclear cell induces into imDC preparation method：Healthy human umbilical cord blood 50ml is collected, is utilized Lymphocyte separation medium is isolated in mononuclearcell, culture medium, 37 DEG C, 5% CO2Under the conditions of culture 2-3 hours after, outwell Suspension dead cell, leaves and takes attached cell, and add cell factor rhIL-4, final concentration：50ng/ml, rhGM-GSF final concentration 100ng/ml, induction mononuclearcell is divided into DC, more renews culture medium every 48 hours, and culture obtains imDC on the 5th day.
7. a kind of reinforced HPV HPV-16/18 divalence DC vaccines as claimed in claim 5, its feature exists In：The imDC induces the DC vaccines for obtaining maturation through maturation factor, and the imDC adds maturation factor TNF-α, induction on the 6th day Obtain the DC cells of maturation.
Prevent 8. the divalence DC vaccines of reinforced HPV HPV-16/18 described in claim 1 a kind of are used to prepare The purposes of the divalence DC vaccines of disease.
9. the purposes of prophylactic divalence DC vaccines is prepared as claimed in claim 8, it is characterised in that：The prevention disease For cervical carcinoma, but it is not limited to the high-risk-type HPV infections such as cervical carcinoma, in addition to the prevention carcinoma of the rectum, carcinoma of mouth, carcinoma of tonsil and draws The other malignant tumours risen.
CN201710187380.8A 2017-03-27 2017-03-27 A kind of reinforced HPV HPV 16/18 divalence DC vaccines Pending CN106963945A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109957582A (en) * 2017-12-26 2019-07-02 上海尚泰生物技术有限公司 A kind of preparation method of the cytotoxic T lymphocyte of a variety of KRAS mutation epitopes of target tumor
CN110734477A (en) * 2019-11-18 2020-01-31 维塔恩(广州)医药有限公司 Papillomavirus related antigen short peptide and application thereof
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