CN108424925A - A kind of therapeutic HPV nucleic acid vaccine - Google Patents
A kind of therapeutic HPV nucleic acid vaccine Download PDFInfo
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Abstract
The invention belongs to biomedicine fields, and in particular to a kind of HPV E6/E7 minicircle dna vaccines and preparation method thereof.The nucleotide sequence of E6 and E7 albumen comprising HPV16 and HPV18 hypotypes in the HPV E6/E7 minicircle dnas of the present invention, and by shearing to E6 and E7 sequences and reset and eliminate its carciongenic potency；By increasing the sequence areas Flt3L in E6 and E7 retracing sequences upstream, the immunogenicity of HPV E6/E7 is enhanced.Meanwhile the minicircle dna that the present invention uses has the following advantages：1) unconformity enters host cell gene group, will not induce new mutation, safer compared with viral vectors；2) can in engineered bacterial large amplification, preparation process is simple, and industrialization production is at low cost；3) the more traditional plasmid stabilisation of expression alien gene, efficiently.It is significant for the treatment of HPV infection that the HPV E6/E7 minicircle dnas of the present invention are used to prepare vaccine.
Description
Technical field
The invention belongs to biomedical sectors, and in particular to a kind of HPV E6/E7 minicircle dnas vaccine and preparation method,
And the application in treatment HPV infection.
Background technology
Cervical carcinoma accounts for the second of female gynecological malignant tumour, has become the important illness for threatening women's health, China
The annual number nearly 50,000 for dying of cervical carcinoma, the etiological study of cervical carcinoma is paid attention to by domestic and foreign scholars always, and research finds to draw
The key for playing cervical carcinoma is the infection of HPV.HPV belongs to Papillomaviridae Papillomavirus, is spherical, double-stranded cyclic DNA
Virus.HPV mainly by direct or indirect contact stain article or passes through the Sex transmitted pathogen mankind.HPV's is special infected with host
Anisotropic and tissue specificity can only infect the skin of the mankind and the basal cell that mucous membrane is impaired.
HPV has identified specific hypotype a about more than 200 so far, causes a disease different with prognosis according to it and can be divided into high-risk
Type HPV (HR-HPV) and low risk HPV (LR-HPV), wherein 15 kinds of HR-HPV, including HR-HPV16,18,31,33,35,39,
45, type of 5l, 52,56,53,58 etc., can cause precancerous lesion and cervical carcinoma after infection.Wherein HPV16 and HPV18 is most important
Epidemic strain.16 types of HPV and 18 types of HPV can cause about 70% cases of cervical cancer, 80% cancer of anus, 60% vaginal tumor
With 40% carcinoma of vulva.The infection of only lasting HR-HPV is only the principal element of cervical carcinoma.
The persistent infection of HR-HPV makes HR-HPV DNA be integrated with host's uterine neck basilar memebrane cell DNA, causes HR-HPV
The missing of E2 segments (raq gene participates in transcriptional regulatory, and E2 albumen is the main virus transcription factor), the missing of E2 segments are accelerated
The progress of cervical lesions, increases the possibility of vicious transformation, and main cause is that the missing of E2 segments causes HPV E6/E7mRNA tables
It reaching, E6/E7 albumen Cu Jin ﹑ maintain HR-HPV DNA to be integrated with uterine neck basal cell DNA, make uterine neck basal cell paraplasm,
HPV E6/E7mRNA interfere normal cell-cycle, cause cellular genome unstable, transcribe the E6/E7 albumen of generation, can go out
Tumor suppressor p53, RB and P21 living etc., make normal host cell be converted to pernicious direction, E6/E7 albumen can be such that HPV escapes
The immunosurveillance and interference body immune response of host.E6/E7 albumen is cancer protein, therefore can be opened for E6/E7 albumen
The drug of hair treatment HPV infection, to prevent developing into cervical carcinoma.
DNA vaccination is that external source target gene is implemented in carrier for expression of eukaryon, the third that can be injected directly into animal body
For vaccine.It makes foreign gene in vivo express, the immune system of the antigenic activation body of generation, to inducing specific
Humoral immunity and cellullar immunologic response.
Preventative vaccine Gardasil/Cervarix can effectively prevent HPV infection at present, but for have HPV infection or
Precancerous lesion person has no obvious therapeutic action, therefore it is necessary to develop the therapeutic vaccine for being directed to HPV infection correlation precancerous lesion.
Therefore the present invention develops a kind of HPV E6/E7 minicircle dna vaccines for treating HPV infection, and the carciongenic potency of E6/E7 is
It is eliminated, and enhances its immunogenicity, and minicircle dna (MC) used is safe and efficient ideal carrier.
Invention content
In view of this, the purpose of the present invention is to provide a kind of HPV E6/E7 nucleotide fragments, it is carcinogenic to eliminate E6/E7
Ability, and preserve the immunogenicity of E6/E7.
To achieve the above object, the technical scheme is that：
The present invention has selected the most important epidemic strain HPV16 and HPV18 of HPV, by the core for encoding E6 and E7 albumen to it
The shearing of nucleotide sequence and the carciongenic potency for resetting the albumen for eliminating its coding, while the antigenic determinant of E6 and E7 are remained,
Keep its immunogenicity.
Specially：
The nucleotide sequence of HPV E6/E7 nucleotide fragments such as SEQ ID NO:1、SEQ ID NO:2 or SEQ ID NO:3
It is shown.Wherein SEQ ID NO:1 is the nucleotide fragments of HPV16E6/E7, SEQ ID NO:2 be the nucleotide of HPV18E6/E7
Segment, SEQ ID NO:3 be the nucleotide fragments of HPV16/18E6/E7.
The E6/E7 fusions that three kinds of HPV E6/E7 nucleotide fragments encode the three-dimensional structure deformation of three kinds of amino acid sequences are more
Peptide.
The present invention first analyzes the antigenic determinant of HPV E6, E7, determines its antigenic determinant.Antigen is determined afterwards
Determine the position rearrangement reaction of cluster, and confirms that the fused polypeptide after resetting eliminates carciongenic potency.By screening, above-mentioned SEQ ID NO:1、
SEQ ID NO:2 and SEQ ID NO:The 3 corresponding fused polypeptide of nucleotide sequence, which meets, not only to be retained immunogenicity but also eliminates cause
The requirement of cancer ability.
As a preferred option, the nucleotides sequence of HPV E6/E7 nucleotide fragments is classified as SEQ ID NO:Shown in 3.
The second object of the present invention is to provide a kind of fusion protein of the nucleotide coding of purpose one, by HPV E6/
E7 protein cleavages are reset to obtain, and eliminate the carciongenic potency of E6/E7 albumen, and retain the immunogenicity of E6/E7 albumen.
To achieve the above object, the technical scheme is that：
Fusion protein is encoded to obtain by the HPV E6/E7 nucleotide fragments of purpose one.
The fusion protein of the present invention is the E6/E7 fused polypeptides of three-dimensional structure deformation.
The third object of the present invention is to provide a kind of DNA recombinant fragments including one HPV E6/E7 segments of purpose, and
The fusion protein of DNA fragmentation coding.Above-mentioned DNA fragmentation is exempted from comprising the immunogenicity that can enhance HPV E6/E7 recombinant fragments
Epidemic disease increases propeptide sequence.
To achieve the above object, the technical scheme is that：
One section of DNA recombinant fragment for including one HPV E6/E7 segments of purpose, including signal peptide sequence area, Flt3L sequences
Area, purpose one HPV E6/E7 segments, wherein signal peptide sequence area, the sequence areas Flt3L, purpose one HPV E6/E7 segments according to
Secondary connection；The nucleotide sequence of the sequence areas Flt3L such as SEQ ID NO:Shown in 4.
The E6/E7 fused polypeptides of the present invention are the albumen expressed in nucleus, and immunity is weak.Therefore, by signal peptide
The E6/E7 fusion proteins of the signal inducing peptide three-dimensional structure deformation of sequence expression are secreted to extracellular, to which enhancement antigen is special
Property humoral immune response and cellullar immunologic response.
The signal peptide in the higher eucaryotic cells for including mammal etc. can be used in above-mentioned signal peptide, as (tectotype is fine by tPA
Plasminogen activator), the secretory signal sequence of HSV gDs or usable growth hormone etc..As a preferred option, above-mentioned letter
Number peptide uses tPA.
Further, the nucleotides sequence in tPA signal peptide sequences area of the invention is classified as SEQ ID NO:Shown in 7.
Mucin peptide refers to activation with the relevant cell of immune response (e.g., Dendritic Cells etc.) to enhance immune response
Peptide.Flt3L is the mucin peptide of the present invention, the immunogenicity for enhancing HPV-E6/E7 recombinant fragments.
As a preferred option, the DNA recombinant fragments of purpose three also include bonding pad, and the bonding pad connects Flt3L sequences
Arrange the segment in area and purpose one.
It is further preferred that the nucleotide sequence of above-mentioned bonding pad such as SEQ ID NO:Shown in 5.
As a preferred option, the DNA recombinant fragments of purpose three also include label area, the piece of the label area and purpose one
Section connection and downstream.
It is further preferred that the nucleotide sequence of above-mentioned label area such as SEQ ID NO:Shown in 6.
As a preferred option, the DNA recombinant fragments tail end of above-mentioned purpose three also includes terminator codon.
In conclusion the optimal connection scheme of the DNA recombinant fragments of purpose three is：TPA signal peptide sequences area-Flt3L sequences
Area-bonding pad the label areas-HPV16/18E6/E7 segments-his-terminator codon is arranged, nucleotides sequence is classified as SEQ ID NO:8 institutes
Show.The fusion protein of DNA fragmentation coding cannot inactivate Tumor suppressor p53, RB and P21 etc., and have immunogenicity, can cause
Immune response.
The fourth object of the present invention is to provide a kind of recombination minicircle dna including three DNA recombinant fragments of purpose, use
Minicircle dna is safe and efficient ideal carrier.
To achieve the above object, the technical scheme is that：
The carrier that the DNA recombinant fragments of purpose three use is minicircle dna, and the minicircle dna used is parental plasmid
The product of (parentalplasmid, PP) locus specificity recombination in vivo.It is inserted in the parental plasmid both sides for carrying eukaryotic expression cassette
Entering and recombinates enzyme recognition site, related recombination expression of enzymes, the recombinase cut off the DNA sequence dna among recognition site in inductor,
Parental plasmid is divided into 2 supercoil molecules, the i.e. miniplasmids (miniplasmid, MP) with copy function and carrying eukaryon
Micro-loop (minicircle, MC) DNA of expression cassette.
Minicircle dna is that the novel small ring of one kind that traditional plasmid is recombinated in Escherichia coli by locus specificity is super
Spiral expression cassette lacks the bacterial sequences such as resistant maker gene, replication origin, enhances the safety in clinical application,
It can the high-caliber transgene product of long-term expression in vivo.MC is in structure, the persistence of transgene expression, intracellular stability
On, it is identical as covalent bond closed loop shape DNA (cccDNA), so that MC is become safe and efficient ideal carrier.
Compared with traditional plasmid and viral vectors, minicircle dna expression alien gene in mammalian cell has a system
Row unique advantage：1) unconformity enters host cell gene group, will not induce new mutation, safer compared with viral vectors；2)
Can in engineered bacterial large amplification, preparation process is simple, and industrialization production is at low cost；3) inhibition in traditional plasmid is eliminated
Signal, the more traditional plasmid stabilisation of expression alien gene, efficiently.
Minicircle dna includes WPRE elements (regulating element after the translation of woodchuck hepatitis virus), and member is adjusted after belonging to transcription
Part can significantly improve the expression of target gene.The optimization of posttranscriptional regulatory element is a kind of plan of DNA vaccination optimization design
Slightly.The expression of the antigen gene of lacking introns can significantly be enhanced, while inducing ideal immune response, had with suitable
The characteristics of regulation and control of the formula mode of action and position flexibility and changeability.Specific mechanism includes that transcript can be promoted to go out core, can also be led to
The poly-adenosine for crossing up-regulation primary transcript finally improves the expression effect of metastatic gene to increase intracellular messenger RNA total amounts
Rate.
Therefore, recombination minicircle dna of the invention has the advantages that safe efficient, preparation is simple, is easy to industrialization.
The fifth object of the present invention is to provide a kind of preparation method of the recombination minicircle dna of purpose four, prepare it is simple,
It is easy to industrialized production.
To achieve the above object, the technical scheme is that：
The preparation method of the recombination minicircle dna of purpose four includes the following steps：
1) design both ends add the HPV E6/E7 recombinant fragments of the purpose three of BglII and SalI restriction enzyme sites, and synthesize, and obtain
Target gene；
2) BglII and SalI while the target gene and ZY781 carriers of double digestion step 1), recycling is used to connect to get parent
This plasmid；
3) Escherichia coli ZYCY10P3S2T is converted with above-mentioned parental plasmid, be incubated overnight, plasmid enzyme restriction and sequence verification are inserted
Enter correct segment；
4) above-mentioned bacterium solution is accessed in the TB culture solutions containing kana, 37 DEG C are incubated overnight, take the bacterium solution that is incubated overnight with
Minicle Induction Mix react 5-8h by isometric mixing, in 32 DEG C, after reaction, centrifuge upgrading grain, that is, obtain
Recombination minicircle dna containing DNA fragmentation described in claim 3；
Wherein, the preparation method of Minicle Induction Mix is in step 4)：It is added into 100ml LB culture mediums
200 μ l of 1M NaOH 4ml and 20% arabinose, through 0.22 μm of membrane filtration after mixing.
The sixth object of the present invention is to provide the recombination minicircle dna of purpose four a kind of and treats HPV infection drug preparing
Purposes.
To achieve the above object, the technical scheme is that：
The recombination minicircle dna of the present invention has eliminated the carcinogenicity of HPV E6/E7, and enhances HPV E6/E7 recombinant fragments
Immunogenicity, the minicircle dna as carrier can stablize expression in vivo, and safer, therefore, can be used for preparing treatment
The drug of HPV infection.
As a preferred option, the drug of above-mentioned treatment HPV infection is recombination minicircle dna nucleic acid vaccine.
The nucleotide fragments that the seventh object of the present invention is to provide a kind of purpose one are being used to prepare treatment HPV infection
The purposes of drug.
As a preferred option, the drug of above-mentioned treatment HPV infection is nucleic acid vaccine.
The DNA recombinant fragments that the eighth object of the present invention is to provide a kind of purpose three are being used to prepare treatment HPV infection
Drug purposes.
As a preferred option, the drug of above-mentioned treatment HPV infection is nucleic acid vaccine.
The nineth object of the present invention is to provide a kind of fusion protein of purpose two in the medicine for being used to prepare treatment HPV infection
The purposes of object.
The tenth object of the present invention is to provide a kind of fusion protein of purpose three in the medicine for being used to prepare treatment HPV infection
The purposes of object.
The tenth object of the present invention one is to provide a kind of drug, four recombination minicircle dna for the purpose of active ingredient, and
Including assigning the carrier of its pharmacy.
As a preferred option, said medicine is nucleic acid vaccine.
The beneficial effects of the present invention are：HPV E6/E7 segments provided by the invention, pass through the shearing to E6 and E7 sequences
Its carciongenic potency is eliminated with resetting；The recombinant fragment of the HPV E6/E7 of the present invention is increased by HPV E6/E7 segments upstream
The sequence areas Flt3L enhance the immunogenicity of HPV E6/E7.The minicircle dna that the present invention uses has the following advantages：1) not whole
It is incorporated into host cell gene group, new mutation will not be induced, it is safer compared with viral vectors；2) can largely expand in engineered bacterial
Increase, preparation process is simple, and industrialization production is at low cost；3) the more traditional plasmid stabilisation of expression alien gene, efficiently.The present invention's
HPV E6/E7 minicircle dnas can be used for preparing the nucleic acid vaccine for the treatment of HPV infection, significant for the prevention of cervical carcinoma.
Specific implementation mode
It detailed description of a preferred embodiment of the present invention will be given below.The reality of actual conditions is not specified in preferred embodiment
Proved recipe method, usually according to normal condition, illustrated embodiment are but not to be to preferably be illustrated to present disclosure
Present disclosure is only limitted to illustrated embodiment.So those skilled in the art according to foregoing invention content to embodiment party
Case carries out nonessential modifications and adaptations, still falls within protection scope of the present invention.
The preparation of 1 minicircle dna of embodiment
1. reagent prepares
1) TB culture solutions：Peptone 12g, yeast extract 24g and glycerine 4ml are dissolved in 900ml water.Wait for each component
High pressure sterilization after dissolving.60 DEG C are cooled to, adds 100ml through high pressure sterilization or with the 170mmol/L of 0.22 μm of membrane filtration
The mixed liquor of KH2PO4 and 0.72mol/L K 2HPO4 to get.
2)Minicle Induction Mix：1M NaOH 4ml and 20% arabinose, 200 μ are added into 100ml LB
L, through 0.22 μm of membrane filtration after mixing.
2. recombinating the preparation of minicircle dna
1) design both ends add the SEQ ID NO of BglII and SalI restriction enzyme sites:HPV E6/E7 recombinant fragments shown in 8,
And synthesize, obtain target gene；
2) by above-mentioned purpose genetic fragment and ZY781 carriers BglII and SalI while double digestion, after glue recycling routinely
Target fragment is cloned into the multiple cloning sites of carrier ZY781 by method, obtains recombinant plasmid, as parental plasmid (PP)；
3) Escherichia coli ZYCY10P3S2T is converted with above-mentioned parental plasmid, be incubated overnight, extract plasmid enzyme restriction electricity after plasmid
Correct segment is inserted into swimming and plasmid order-checking verification；
4) in the TB culture solutions that the correct Escherichia coli access of above-mentioned sequencing is contained to 50 μ g/ml kana, 37 DEG C, 250rpm
It is incubated overnight；
5) take the bacterium solution being incubated overnight with Minicle InductionMix by mixing in equal volume, in 32 DEG C, 250rpm is anti-
Answer 5h-8h.After having reacted, plasmid is extracted in centrifugation, as recombinates minicircle dna plasmid.
The recombination minicircle dna of acquisition has eliminated the carcinogenicity of HPV E6/E7, and enhances exempting from for HPV E6/E7 recombinant fragments
Epidemic focus, the minicircle dna as carrier can stablize expression in vivo, and safer, therefore, can be used for preparing treatment
The nucleic acid vaccine of HPV infection.
3. recombinating a large amount of preparations of minicircle dna
1) bacterial strain mass propgation：Step 3) in the preparation of above-mentioned recombination minicircle dna has been transferred to parental plasmid and has been sequenced just
True Escherichia coli ZYCY10P3S2T bulk fermentation cultures；
2) minicircle dna generates：In the TB culture solutions that the access of above-mentioned Escherichia coli is contained to 50 μ g/ml kana, 37 DEG C,
250rpm is incubated overnight afterwards with Minicle Induction Mix by mixing in equal volume, and in 32 DEG C, 250rpm reacts 5h-8h.Instead
After having answered, centrifugation extracts plasmid, as recombinates minicircle dna.
The recombination minicircle dna being prepared can be used as the accounting vaccine of therapeutic HPV infection.
The HPV E6/E7 minicircle dna vaccines of the present invention have safe and stable excellent in vivo for treating HPV infection
Point, and therapeutic effect is apparent.It recombinates minicircle dna to prepare simply, cost is relatively low, is suitable for industrialization production.
Finally illustrate, the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to compared with
Good embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the right of invention.
<110>The Jiangsu bio tech ltd Run Jie
<120>A kind of therapeutic HPV nucleic acid vaccine
<160> 8
<170> PatentIn Version 2.1
<210> 1
<211>729
<212> DNA
<213> HPV16 E6/E7
<400> 1
aggaagctgc cccagctgtg caccgagggc ggcggcggca gcggcggcgg cggcagcggc 60
ggcggcggca gctacaacaa gcccctgtgc gacctgctga tcaggtgcat caactgccag 120
aagcccctgt gccccggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 180
aacccctacg ccgtgtgcga caagtgcctg aagttctaca gcaagggcgg cggcggcagc 240
ggcggcggcg gcagcggcgg cggcggcagc cagaccacca tccacgacat catcctggag 300
tgcgtgtact gcaagcagca gctgctgagg agggaggtgt acgacttcgc cttcagggac 360
ctgtgcatcg tgtacggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 420
agcgagtaca ggcactactg ctacagcctg tacggcaggt gcatgagctg ctgcaggacc 480
ctgcacgagt acatgctgga cctgggcggc ggcggcagcg gcggcggcgg cagcggcggc 540
ggcggcagcc actacaacat cgtgaccttc tgctgcaagt gcgacagcac cctgaggctg 600
tgcgtgcaga gcacccacgt ggacatcggc ggcggcggca gcggcggcgg cggcagcggc 660
ggcggcggca gcctgatggg caccctgggc atcgtgtgcc ccatcaccac cgacctgtac 720
tgctacgag 729
<210> 2
<211>777
<212> DNA
<213> HPV18 E6/E7
<400> 2
aggccctaca agctgcccga cctgtgcacc gagctgtaca acctgctgat caggtgcctg 60
aggtgccaga agcccggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 120
gccttcaagg acctgttcgt ggtgtacagg gacagcatcc cccacgccgc ctgccacaag 180
tgcatcgact tctacagcag gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 240
ggcagcctgc aggacatcga gatcacctgc gtgtactgca agaccgtgct ggagctgacc 300
gaggtgttcg agggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcgag 360
ctgaggcact acagcgacag cgtgtacggc tacaggggcc agtgccacag ctgctgcaac 420
cccaaggcca ccctgcagga catcgtgctg cacctggacg gcgtgaacca ccagcacctg 480
cccgccggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag cgacctgagg 540
gccttccagc agctgttcct gaacaccctg agcttcgtgt gcccctgggg cggcggcggc 600
agcggcggcg gcggcagcgg cggcggcggc agcaggcaca ccatgctgtg catgtgctgc 660
aagtgcgagg ccaggatcga gctggtggtg gagagcggcg gcggcggcag cggcggcggc 720
ggcagcggcg gcggcggcag caacgagatc cccgtggacc tgctgtgcca cgagcag 777
<210> 3
<211>1506
<212> DNA
<213> HPV16/18 E6/E7
<400> 3
aggaagctgc cccagctgtg caccgagggc ggcggcggca gcggcggcgg cggcagcggc 60
ggcggcggca gctacaacaa gcccctgtgc gacctgctga tcaggtgcat caactgccag 120
aagcccctgt gccccggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 180
aacccctacg ccgtgtgcga caagtgcctg aagttctaca gcaagggcgg cggcggcagc 240
ggcggcggcg gcagcggcgg cggcggcagc cagaccacca tccacgacat catcctggag 300
tgcgtgtact gcaagcagca gctgctgagg agggaggtgt acgacttcgc cttcagggac 360
ctgtgcatcg tgtacggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 420
agcgagtaca ggcactactg ctacagcctg tacggcaggt gcatgagctg ctgcaggacc 480
ctgcacgagt acatgctgga cctgggcggc ggcggcagcg gcggcggcgg cagcggcggc 540
ggcggcagcc actacaacat cgtgaccttc tgctgcaagt gcgacagcac cctgaggctg 600
tgcgtgcaga gcacccacgt ggacatcggc ggcggcggca gcggcggcgg cggcagcggc 660
ggcggcggca gcctgatggg caccctgggc atcgtgtgcc ccatcaccac cgacctgtac 720
tgctacgaga ggccctacaa gctgcccgac ctgtgcaccg agctgtacaa cctgctgatc 780
aggtgcctga ggtgccagaa gcccggcggc ggcggcagcg gcggcggcgg cagcggcggc 840
ggcggcagcg ccttcaagga cctgttcgtg gtgtacaggg acagcatccc ccacgccgcc 900
tgccacaagt gcatcgactt ctacagcagg ggcggcggcg gcagcggcgg cggcggcagc 960
ggcggcggcg gcagcctgca ggacatcgag atcacctgcg tgtactgcaa gaccgtgctg 1020
gagctgaccg aggtgttcga gggcggcggc ggcagcggcg gcggcggcag cggcggcggc 1080
ggcagcgagc tgaggcacta cagcgacagc gtgtacggct acaggggcca gtgccacagc 1140
tgctgcaacc ccaaggccac cctgcaggac atcgtgctgc acctggacgg cgtgaaccac 1200
cagcacctgc ccgccggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 1260
gacctgaggg ccttccagca gctgttcctg aacaccctga gcttcgtgtg cccctggggc 1320
ggcggcggca gcggcggcgg cggcagcggc ggcggcggca gcaggcacac catgctgtgc 1380
atgtgctgca agtgcgaggc caggatcgag ctggtggtgg agagcggcgg cggcggcagc 1440
ggcggcggcg gcagcggcgg cggcggcagc aacgagatcc ccgtggacct gctgtgccac 1500
gagcag 1506
<210> 4
<211> 471
<212> DNA
<213>Flt3L sequences
<400> 4
atcacccagg actgctcctt ccaacacagc cccatctcct ccgacttcgc tgtcaaaatc 60
cgtgagctgt ctgactacct gcttcaagat tacccagtca ccgtggcctc caacctgcag 120
gacgaggagc tctgcggggg cctctggcgg ctggtcctgg cacagcgctg gatggagcgg 180
ctcaagactg tcgctgggtc caagatgcaa ggcttgctgg agcgcgtgaa cacggagata 240
cactttgtca ccaaatgtgc ctttcagccc ccccccagct gtcttcgctt cgtccagacc 300
aacatctccc gcctcctgca ggagacctcc gagcagctgg tggcgctgaa gccctggatc 360
actcgccaga acttctcccg gtgcctggag ctgcagtgtc agcccgactc ctcaaccctg 420
ccacccccat ggagtccccg gcccctggag gccacagccc cgacagcccc g 471
<210> 5
<211> 18
<212> DNA
<213>Bonding pad
<400> 5
ggcggcggca gcggcgat 18
<210> 6
<211> 18
<212> DNA
<213>HIS labels
<400> 6
catcatcacc atcatcat 18
<210> 7
<211> 69
<212> DNA
<213>Signal peptide tPa
<400> 7
atggatgcta tgaaacgggg cctgtgctgc gtgctgctcc tgtgcggcgc tgtgtttgtg 60
agccctagc 69
<210> 8
<211>2085
<212> DNA
<213>HPV16/18 E6/E7 recombinant fragments
<400> 8
atggatgcta tgaaacgggg cctgtgctgc gtgctgctcc tgtgcggcgc tgtgtttgtg 60
agccctagca tcacccagga ctgctccttc caacacagcc ccatctcctc cgacttcgct 120
gtcaaaatcc gtgagctgtc tgactacctg cttcaagatt acccagtcac cgtggcctcc 180
aacctgcagg acgaggagct ctgcgggggc ctctggcggc tggtcctggc acagcgctgg 240
atggagcggc tcaagactgt cgctgggtcc aagatgcaag gcttgctgga gcgcgtgaac 300
acggagatac actttgtcac caaatgtgcc tttcagcccc cccccagctg tcttcgcttc 360
gtccagacca acatctcccg cctcctgcag gagacctccg agcagctggt ggcgctgaag 420
ccctggatca ctcgccagaa cttctcccgg tgcctggagc tgcagtgtca gcccgactcc 480
tcaaccctgc cacccccatg gagtccccgg cccctggagg ccacagcccc gacagccccg 540
ggcggcggca gcggcgatag gaagctgccc cagctgtgca ccgagggcgg cggcggcagc 600
ggcggcggcg gcagcggcgg cggcggcagc tacaacaagc ccctgtgcga cctgctgatc 660
aggtgcatca actgccagaa gcccctgtgc cccggcggcg gcggcagcgg cggcggcggc 720
agcggcggcg gcggcagcaa cccctacgcc gtgtgcgaca agtgcctgaa gttctacagc 780
aagggcggcg gcggcagcgg cggcggcggc agcggcggcg gcggcagcca gaccaccatc 840
cacgacatca tcctggagtg cgtgtactgc aagcagcagc tgctgaggag ggaggtgtac 900
gacttcgcct tcagggacct gtgcatcgtg tacggcggcg gcggcagcgg cggcggcggc 960
agcggcggcg gcggcagcag cgagtacagg cactactgct acagcctgta cggcaggtgc 1020
atgagctgct gcaggaccct gcacgagtac atgctggacc tgggcggcgg cggcagcggc 1080
ggcggcggca gcggcggcgg cggcagccac tacaacatcg tgaccttctg ctgcaagtgc 1140
gacagcaccc tgaggctgtg cgtgcagagc acccacgtgg acatcggcgg cggcggcagc 1200
ggcggcggcg gcagcggcgg cggcggcagc ctgatgggca ccctgggcat cgtgtgcccc 1260
atcaccaccg acctgtactg ctacgagagg ccctacaagc tgcccgacct gtgcaccgag 1320
ctgtacaacc tgctgatcag gtgcctgagg tgccagaagc ccggcggcgg cggcagcggc 1380
ggcggcggca gcggcggcgg cggcagcgcc ttcaaggacc tgttcgtggt gtacagggac 1440
agcatccccc acgccgcctg ccacaagtgc atcgacttct acagcagggg cggcggcggc 1500
agcggcggcg gcggcagcgg cggcggcggc agcctgcagg acatcgagat cacctgcgtg 1560
tactgcaaga ccgtgctgga gctgaccgag gtgttcgagg gcggcggcgg cagcggcggc 1620
ggcggcagcg gcggcggcgg cagcgagctg aggcactaca gcgacagcgt gtacggctac 1680
aggggccagt gccacagctg ctgcaacccc aaggccaccc tgcaggacat cgtgctgcac 1740
ctggacggcg tgaaccacca gcacctgccc gccggcggcg gcggcagcgg cggcggcggc 1800
agcggcggcg gcggcagcga cctgagggcc ttccagcagc tgttcctgaa caccctgagc 1860
ttcgtgtgcc cctggggcgg cggcggcagc ggcggcggcg gcagcggcgg cggcggcagc 1920
aggcacacca tgctgtgcat gtgctgcaag tgcgaggcca ggatcgagct ggtggtggag 1980
agcggcggcg gcggcagcgg cggcggcggc agcggcggcg gcggcagcaa cgagatcccc 2040
gtggacctgc tgtgccacga gcagcatcat caccatcatc attaa 2085
Claims (19)
1.HPV E6/E7 nucleotide fragments, which is characterized in that its nucleotide sequence such as SEQ ID NO:1、SEQ ID NO:2 or
SEQ ID NO:Shown in 3.
2. fusion protein, which is characterized in that the fusion protein is compiled by HPV E6/E7 nucleotide fragments described in claim 1
Code obtains.
3. one section of DNA recombinant fragment for including HPV E6/E7 segments described in claim 1, which is characterized in that include signal peptide sequence
Arrange area, the sequence areas Flt3L, HPV E6/E7 segments described in claim 1, wherein signal peptide sequence area, the sequence areas Flt3L, power
Profit requires the HPV E6/E7 segments described in 1 to be sequentially connected；
The nucleotide sequence of the sequence areas Flt3L such as SEQ ID NO:Shown in 4.
4. DNA recombinant fragments according to claim 3, which is characterized in that the DNA recombinant fragments also include bonding pad,
The bonding pad connection sequence areas Flt3L and HPV E6/E7 segments described in claim 1.
5. DNA recombinant fragments according to claim 4, which is characterized in that the nucleotide sequence of the bonding pad such as SEQ
ID NO:Shown in 5.
6. DNA recombinant fragments according to claim 3, which is characterized in that also include label area, the label area with it is described
HPV E6/E7 segments connection and downstream.
7. DNA recombinant fragments according to claim 6, which is characterized in that the nucleotide sequence of the label area such as SEQ
ID NO:Shown in 6.
8. DNA recombinant fragments according to claim 3, which is characterized in that the DNA fragmentation tail end also includes termination codon
Son.
9. DNA recombinant fragments according to claim 3, which is characterized in that the signal peptide sequence area is selected from tectotype
Activator of plasminogen tPa, HSV gDs and growth hormone the corresponding nucleotide sequence of secreting signal peptide in one kind or more
Kind.
10. fusion protein, which is characterized in that the fusion protein is encoded to obtain by the DNA recombinant fragments described in claim 3.
11. recombinating minicircle dna, which is characterized in that the minicircle dna includes the DNA recombinant fragments described in claim 3.
12. the preparation method of the recombination minicircle dna described in claim 11, which is characterized in that include the following steps：
1) design both ends add the HPV E6/E7 recombinant fragments of the claim 3 of BglII and SalI restriction enzyme sites, and synthesize, and obtain mesh
Gene；
2) BglII and SalI while the target gene and ZY781 carriers of double digestion step 1), recycling is used to connect to get parent's matter
Grain；
3) Escherichia coli ZYCY10P3S2T is converted with above-mentioned parental plasmid, be incubated overnight, plasmid enzyme restriction and sequence verification are inserted into just
True segment；
4) above-mentioned bacterium solution is accessed in the TB culture solutions containing kana, pressed with Minicle Induction Mix after 37 DEG C of cultures etc.
Volume mixture reacts 5-8h in 32 DEG C, after reaction, centrifuges upgrading grain, that is, obtains containing DNA fragmentation described in claim 3
Recombination minicircle dna；
Wherein, the preparation method of Minicle Induction Mix is in step 4)：1M is added into 100ml LB culture mediums
200 μ l of NaOH 4ml and 20% arabinose, through 0.22 μm of membrane filtration after mixing.
13. the recombination minicircle dna described in claim 11 is in the purposes for being used to prepare the drug for treating HPV infection.
14. nucleotide fragments described in claim 1 are in the purposes for the drug for being used to prepare treatment HPV infection.
15. the DNA recombinant fragments described in claim 3 are in the purposes for being used to prepare the drug for treating HPV infection.
16. the fusion protein described in claim 2 is in the purposes for being used to prepare the drug for treating HPV infection.
17. fusion protein according to any one of claims 10 is in the purposes for the drug for being used to prepare treatment HPV infection.
18. a kind of drug for treating HPV infection, which is characterized in that the drug is the recombination minicircle dna described in claim 11
And assign the carrier of its pharmacy.
19. drug according to claim 18, which is characterized in that the drug is nucleic acid vaccine.
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| CN201811560858.8A CN109706144A (en) | 2018-03-07 | 2018-03-07 | A kind of therapeutic HPV nucleic acid vaccine |
| CN201810186910.1A CN108424925A (en) | 2018-03-07 | 2018-03-07 | A kind of therapeutic HPV nucleic acid vaccine |
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| CN201810186910.1A CN108424925A (en) | 2018-03-07 | 2018-03-07 | A kind of therapeutic HPV nucleic acid vaccine |
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| CN201810186910.1A Pending CN108424925A (en) | 2018-03-07 | 2018-03-07 | A kind of therapeutic HPV nucleic acid vaccine |
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| CN110564751A (en) * | 2019-09-03 | 2019-12-13 | 深圳新诺微环生物科技有限公司 | Design and application of micro-ring DNA vaccine |
| CN114134165A (en) * | 2022-01-27 | 2022-03-04 | 北京循生生物医学研究有限公司 | Novel HPV therapeutic nucleic acid vaccine |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564751A (en) * | 2019-09-03 | 2019-12-13 | 深圳新诺微环生物科技有限公司 | Design and application of micro-ring DNA vaccine |
| WO2021042947A1 (en) * | 2019-09-03 | 2021-03-11 | 深圳新诺微环生物科技有限公司 | Minicircle dna vaccine design and use |
| CN114134165A (en) * | 2022-01-27 | 2022-03-04 | 北京循生生物医学研究有限公司 | Novel HPV therapeutic nucleic acid vaccine |
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| US11911455B2 (en) | 2022-01-27 | 2024-02-27 | Beijing Aeonvital Biomedicine Research Co., Ltd. | HPV therapeutic nucleic acid vaccine |
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