CN104587490A - Therapeutic HBV minicircle DNA vaccine and preparation method thereof - Google Patents
Therapeutic HBV minicircle DNA vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention provides a novel therapeutic HBV minicircle DNA vaccine. The vaccine comprises a minicircle DNA plasmid pMCS2.S for coding a HBV outer envelope protein preS2.S gene and a minicircle DNA plasmid pMCIIF for coding a hIL2-IFN gamma fusion protein gene. The plasmids pMCS2.S and pMCIIF do not contain a replication element and an antibiotic element of a prokaryotic plasmid, can be dissociated from host cell genome DNA, can stably and enduringly express an exogenous target gene, successfully realizes expression of an exogenous gene in COS-7 cells and induction of production of Th1 type immune response and specific killing response aiming at high-level IFN gamma of hepatitis b viruses in Balb/c mice.
Description
Technical field
The present invention relates to biotechnology and DNA vaccination preparation field, specifically, relate to a kind of therapeutic HBV minicircle dna vaccine and preparation method thereof.
Background technology
Hepatitis B virus (HBV) is widely current in the world.HBV infection can cause acute and chronic and hepatitis gravis, and closely related with the morbidity of liver cirrhosis, hepatocarcinoma, is the infectious disease of serious harm human health.About there are 3.5 hundred million chronic hepatitis B virus infectons in the current whole world, wherein 75% is distributed in the Asian-Pacific area, and Chronic Asymptomatic the infected of China is more than 1.2 hundred million, and chronic hepatitis B prognosis mala.Current antiviral drugs is nucleoside analog and recombinant interferon class medicine mainly, but effect is not very good.Therefore, be badly in need of a kind of more effective medicine, remove hepatocellular cccDNA virus, patient is fully recovered.
DNA vaccination, is plasmid external source genes of interest being implemented in carrier for expression of eukaryon, can be injected directly into the third generation of vaccine in animal body.Due to object antigen can be expressed in vivo, offered by DC cell by exogenous, endogenous and cross-reacting antigen submission approach, effectively activate CD4
+th1 and CD8
+t cell, is therefore particularly conducive to induction Th1 type immunne response, breaks the immune tolerance state of Chronic Hepatitis B, remove virus, patient is fully recovered.
The structure of selection to DNA vaccination of genophore is most important, on the one hand, the skeleton DNA composition of standard plasmid includes DNA replication dna initiation site, antibiotics opposing gene etc., these become branch to suppress destination gene expression, and vector gene DNA may insert host genome, bring out the danger of insertion mutation and gene silencing; On the other hand, standard plasmid transduction efficiency is low, and genes of interest can only realize Transient Expression, and its molecular weight is comparatively large, easily cause immunoreation and remove by body.Therefore, expressing safely and efficiently is the key that research nucleic acid DNA vaccine gene is treated.
(the Mar A.Kay such as Chen, Cheng-Yi He, Zhi-Ying Chen.A robust systemfor production of minicircle DNA vectors.Nature Biotechnology, (2010) .doi:10.1038/nbt.1708) minicircle dna carrier (MC) technology developed and micro-ring generate strain Escherichia coli ZYCY10P3S2T and contribute to overcoming the above problems.MC is a kind of circular expression cassette, not containing bacterial backbone DNA in standard plasmid, and can the high-caliber genes of interest product of long-term expression in vivo.MC is identical with covalent bond closed loop shape DNA (cccDNA) in persistency and the intracellular stability etc. of structure, gene expression, makes MC become safety, efficiently one of ideal carrier.The preparation method of minicircle dna is after the parental plasmid (PP) containing genes of interest is transformed into Host Strains ZYCY10P3S2T, with the expression of arabinose induction recombinase, its recognition site of recombinase-mediated is recombinated, PP restructuring is made to form the micro-plasmid (miniplasmid containing bacterial backbone, MP) with containing micro-ring (minicircle, MC) DNA of destination gene expression box.By Combined expression I-SceI restriction endonuclease in host bacterial, by MP and PP plasmid enzyme restriction, make it to become linear DNA, then degrade in Host Strains, highly purified minicircle dna can be obtained by conventional plasmid separation method.
Summary of the invention
The object of this invention is to provide a kind of novel therapeutic HBV minicircle dna vaccine.
Another object of the present invention is to provide the preparation method of above-mentioned minicircle dna vaccine.
In order to realize the object of the invention, a kind of therapeutic HBV minicircle dna vaccine of the present invention, described vaccine comprises double-mass model, the minicircle dna plasmid namely containing coding HBV peplos albumen preS2.S gene and the minicircle dna plasmid containing coding hIL2-IFN γ antigen-4 fusion protein gene.Reproduction element all not containing prokaryotic plasrnid in described double-mass model and antibiotics element.
The preparation method of above-mentioned vaccine comprises the following steps:
1) respectively with plasmid preS2.S/pcDNA3.1 and hIL2-IFN γ/pcDNA3.1 for template, design primer, carry out pcr amplification, obtain object fragment;
2) with BglII and SalI double digestion object fragment and ZY781 carrier simultaneously, reclaim, connect, object fragment is cloned into respectively in the multiple clone site of carrier ZY781, Ji get parental plasmid preS2.S/ZY781 and hIL2-IFN γ/ZY781;
3) respectively with above-mentioned parental plasmid transformation of E. coli ZYCY10P3S2T, incubated overnight, plasmid enzyme restriction and sequence verification insert correct fragment;
4) respectively above-mentioned bacterium liquid access is contained in the TB culture fluid of kana by the inoculum concentration of 0.25 ‰, 37 DEG C, 250rpm incubated overnight, the bacterium liquid getting incubated overnight mixes by equal-volume with Minicle Induction Mix, and in 32 DEG C, 250rpm reacts 5-8h, after reaction terminates, centrifugal upgrading grain, obtains the minicircle dna plasmid pMCS2.S containing coding HBV peplos albumen preS2.S gene, and the minicircle dna plasmid pMCIIF containing coding hIL2-IFN γ antigen-4 fusion protein gene;
5) respectively strain fermentation cultivation is carried out to the positive colony containing above-mentioned plasmid pMCS2.S and plasmid pMCIIF, after fermentation ends, centrifugal upgrading grain, purification.
Wherein, step 1) described in primer comprise:
Forward primer 5 '-GAAGATCTGGCGGGTTGACATTGATTATTGACTAG-3 ' and
Downstream primer 5 '-ACGCGTCGACCCATAGAGCCCACCGCAT-3 ';
Step 4) described in the preparation method of Minicle Induction Mix be: in 100ml LB, add 1M NaOH 4ml and 20% arabinose 200 μ l, through 0.22 μm of membrane filtration after mixing.
The present invention also provides the medicine for preventing and/or treating hepatitis B containing described vaccine.
The present invention also provides described vaccine for the preparation of the application prevented and/or treated in the medicine of hepatitis B.
Therapeutic HBV minicircle dna vaccine provided by the invention, comprises the minicircle dna plasmid pMCS2.S containing coding HBV peplos albumen preS2.S gene and the minicircle dna plasmid pMCIIF containing coding hIL2-IFN γ antigen-4 fusion protein gene.All not containing reproduction element and the antibiotics element of prokaryotic plasrnid in plasmid pMCS2.S and pMCIIF, can be free on outside host cell gene group DNA and express external source genes of interest stably and lastingly, successfully achieve at COS-7 cells exogenous gene, induction Balb/c mice produces Th1 type immunne response for the high-level IFN γ of hepatitis B virus and specific killing response.
Accompanying drawing explanation
Fig. 1 is that minicircle dna plasmid of the present invention generates schematic diagram.
Fig. 2 is that minicircle dna plasmid pMCS2.S and pMCIIF of the present invention builds flow chart.
Fig. 3 is the electrophoresis result after plasmid of the present invention and plasmid enzyme restriction; Wherein, 1.DL2000marker; 2.ZY781 empty carrier; 3.pMCS2.S plasmid; 4.preS2.S/ZY781 plasmid; After 5.pMCS2.S plasmid double digestion; After 6.preS2.S/ZY781 plasmid double digestion; 7.pMCIIF plasmid; 8.hIL2-IFN γ/ZY781 plasmid; After 9.pMCIIF plasmid double digestion; After 10.hIL2-IFN γ //ZY781 plasmid double digestion.
Fig. 4 is the expression of IL2 after pMCIIF and hIL2-IFN γ/pcDNA3.1 plasmid transfection COS-7 cell of the present invention.
Fig. 5 is the expression of IFN γ after pMCIIF and hIL2-IFN γ/pcDNA3.1 plasmid transfection COS-7 cell of the present invention.
Fig. 6 is each experimental group ELISPOT speckle schematic diagram directly perceived in the embodiment of the present invention 3; Wherein, a. saline control group; B.PHA positive controls; C.pcDNA3.1/preS2.S plasmid experimental group; D.pcDNA3.1/preS2.S+pcDNA3.1/hIL2-IFN γ double-mass model experimental group; E.pMCS2.S micro-ring plasmid experimental group; The two micro-ring plasmid experimental group of f.pMCS2.S+pMCIIF.
Fig. 7 is each experimental group ELISPOT speckle statistical result in the embodiment of the present invention 3.
Detailed description of the invention
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001) condition of, or according to manufacturer's description advising.
The preparation of embodiment 1 therapeutic HBV minicircle dna vaccine
Comprise the following steps:
1. design of primers and pcr amplification object fragment
Based on recombiant plasmid preS2.S/pcDNA3.1 and hIL2-IFN γ/pcDNA3.1, (construction method is shown in: He Xiaoqiang, Chen Guangming, Huang Ying, Deng. the structure of therapeutic double-mass model HBV DNA vaccination and qualification. PLA's medical journal, 2003,28 (6): 493-498) sequence, at the enhancer upstream of genes of interest and polyA downstream design primer, for the DNA fragmentation of increase enhancer, promoter and polyA regulating and controlling sequence containing genes of interest and upstream and downstream thereof.The primer sequence of design is as follows:
F:5’-GAAGATCTGGCGGGTTGACATTGATTATTGACTAG-3’
R:5’-ACGCGTCGACCCATAGAGCCCACCGCAT-3’
Respectively with plasmid preS2.S/pcDNA3.1 and hIL2-IFN γ/pcDNA3.1 for template, carry out following PCR reaction respectively: in 25 μ l systems containing 10 × buffer 2.5 μ l, dNTP (2.5mmol/L) 2 μ l, forward primer (10pM) 0.5 μ l, downstream primer (10pM) 0.5 μ l, template 5 μ l, archaeal dna polymerase (2.5U/ μ l) 0.4 μ l, distilled water 14.2 μ l.
Pcr amplification program: 95 DEG C of 5min; 95 DEG C of 50s, 55 DEG C of 50s, 72 DEG C of 60s, totally 30 circulations; 72 DEG C of 5min.
2. the structure of parental plasmid
Agarose gel electrophoresis detects pcr amplification result, cut object fragment band, glue is used to reclaim test kit (Takara MinBEST Agarose Gel Extraction kit Ver3.0, D823A) after reclaiming object product, the while of use BglII and SalI (NEB company of the U.S.), double digestion object fragment and ZY781 carrier are (see Chen ZY, He CY, Ehrhardt A, Kay MA.minicircle DNA vectors devoid of bacterial DNA result in persistent andhigh-level transgene expression in vivo.Mol Ther.2003, 8 (3): 495-500, presented by Shenzhen Institutes of Advanced Technology, Chinese Academy of Science professor Chen Zhiying).According to a conventional method object fragment is cloned into respectively in the multiple clone site of carrier ZY781, Ji get parental plasmid preS2.S/ZY781 and hIL2-IFN γ/ZY781.
Transformation of E. coli ZYCY10P3S2T is (see Mar A.Kay, Cheng-Yi He, Zhi-Ying Chen.A robust system for production of minicircle DNAvectors.Nature Biotechnology, (2010) .doi:10.1038/nbt.1708, presented by Shenzhen Institutes of Advanced Technology, Chinese Academy of Science professor Chen Zhiying), and carry out liquid culture amplification.After enzyme action qualification and order-checking determine that Insert Fragment sequence is correct, carry out the operation that following parental plasmid reassembles into minicircle dna.
3. parental plasmid reassembles into minicircle dna
(1) respectively above-mentioned bacterium liquid access is contained in the TB culture fluid of 50 μ g/ml kana by the inoculum concentration of 0.25 ‰, 37 DEG C, 250rpm incubated overnight.
The preparation method of TB culture fluid is as follows: peptone 12g, yeast extract 24g and glycerol 4ml are dissolved in 900ml water.Autoclaving after each components dissolved.Be cooled to 60 DEG C, then add 100ml through autoclaving or the 170mmol/L KH with 0.22 μm of membrane filtration
2pO
4with 0.72mol/L K
2hPO
4mixed liquor, to obtain final product.
(2) the bacterium liquid Minicle Induction Mix getting incubated overnight mixes by equal-volume, and in 32 DEG C, 250rpm reacts 5h, can extend reaction 1-3h if desired.Reaction finishes after bundle, centrifugal, extracts plasmid, be minicircle dna plasmid with plasmid extraction kit (Takara miniBEST Plasmid Purification kit ver 4.0, Code No.9760).
The preparation method of Minicle Induction Mix is as follows: in 100ml LB, add 1MNaOH 4ml and 20% arabinose 200 μ l, through 0.22 μm of membrane filtration after mixing.
The preparation of 4.HBV minicircle dna vaccine: respectively strain fermentation cultivation is carried out to the positive colony obtained in step 2, carry out minicircle dna generation by step 3 method, finally extract plasmid, purification, to obtain final product.
As seen from Figure 1, reassemble into micro-ring plasmid pMCS2.S by parental plasmid preS2.S/ZY781, size becomes about 2500bp from about 6000bp, all occurs two specific bands after enzyme action, illustrate that parental plasmid's enzyme action qualification is correct, and successfully reassemble into micro-ring plasmid.
Known through checking order, genes of interest and enhancers upstream, promoter and downstream polyA regulating and controlling sequence have been cloned in parental plasmid, and Sequence Identification 100% is correct.Sequencing result is shown in SEQID No.1 and SEQ ID No.2.
Minicircle dna plasmid pMCS2.S and pMCIIF builds flow chart and sees Fig. 2, and the electrophoresis result after each plasmid and plasmid enzyme restriction is shown in Fig. 3.
Embodiment 2 therapeutic HBV minicircle dna vaccine Ex vivo cell transfection and detection of expression
Cultivate COS-7 cell strain according to a conventional method, use trypsinization attached cell, be resuspended in the RPMI1640 culture fluid containing serum, by 3 × 10
5individual/porocyte is inoculated on 6 orifice plates, in 37 DEG C, and 5%CO
2incubator cultivates 24h.Treat that cell fusion degree reaches about 90-95%, get 4 μ g pMCS2.S, pMCIIF, preS2.S/pcDNA3.1, hIL2-IFN γ/pcDNA3.1 plasmids respectively, cell transfecting is carried out with reference to Invitrogen company lipofectamine Lipofectamine TM2000 operating instruction, each plasmid do two parallel, set simultaneously plasmid-free transfectional cell group as contrast.Continue to cultivate, and after 24h, 48h, 72h, collect supernatant, by the expression (detecting when 24h, 48h, 72h) of hepatitis B virus surface antigen diagnostic kit (detecting during 48h) and people IL2 and IFN γ detection kit detection by quantitative genes of interest.Table 1 and the display of table 2 result, plasmid-free transfectional cell matched group does not all detect object product, plasmid pMCS2.S, pMCIIF of the present invention all can carry out destination gene expression in COS-7 cell, and higher than the expression of preS2.S/pcDNA3.1 and hIL2-IFN γ/pcDNA3.1 plasmid (Fig. 4-5).
Qualitative detection preS2.S albumen result after table 1 pMCS2.S and preS2.S/pcDNA3.1 plasmid transfection COS-7 cell
| Sample | OD value | COV | OD/COV | Result |
| Blank well | 0 | --- | 0 | Negative |
| Negative control (test kit is supporting) | 0.001 | 0.101 | 0.01 | Negative |
| Positive control (test kit is supporting) | 3.259 | -- | 32.267 | Positive |
| Plasmid-free transfection group | 0.091 | 0.901 | Negative | |
| pcDNA3.1/preS2.S | 3.090±0.018 | -- | 30.594±0.178 | Positive |
| Micro-ring plasmid pMCS2.S | 3.871±0.012 | -- | 38.327±0.119 | Positive |
Criterion: COV:cut-off value reference value; NCx: negative control mean OD value; NCn: negative control OD value n=3; PC: positive control OD value; Calculate NCx=(NC1+NC2+NC3) ÷ 3, if NCx < 0, then calculate by 0; Calculate COV=NCx+0.100; When OD/COV >=1.0 are HBsAg positive findings; When OD/COV < 1 is HBsAg negative findings.
The expression contents of detection by quantitative IL2 and IFN γ after table 2 pMCIIF and hIL2+IFN γ/pcDNA3.1 plasmid transfection COS-7 cell
Embodiment 3 therapeutic HBV minicircle dna vaccine interior animal experiment and immune effect detect
Laboratory animal, immunizing dose, grouping and observing time: healthy Balb/c mice 40,18-20g, 4-6 week age, SPF level, is divided into 5 groups at random, often organize 8.First group is saline control group (100 μ l/ only); Second group is pcDNA3.1/preS2.S plasmid (20 μ g/ only); 3rd group is pcDNA3.1/preS2.S+pcDNA3.1/hIL2+IFN γ (10 μ g+10 μ g/ only) double-mass model; 4th group is the present invention's micro-ring vaccine pMCS2.S (20 μ g/ only); 5th group is the present invention's micro-ring vaccine pMCS2.S+pMCIIF (10 μ g+10 μ g/ only).Adopt and inject at body electric pulse mice bilateral tibialis anterior.Take a blood sample at the eyeball rear vein beard puncture method that adopts for the 0th, 2,4 week of inoculation respectively, with the centrifugal 10min separation of serum of 3000r/min and in-20 DEG C of preservations, treat that last property ELISA detects Anti-HBs antibody level, the positive mice number that calculating antibody occurs.After inoculating 4 weeks, execution mice is got spleen separation lymphocyte and carries out ELISPOT experiment.
Detection method: (1) humoral immunization detects: get the strict by specification of mice serum (Shanghai Shiye Kehua Biotechnology Co., Ltd) and carry out ELISA detection; (2) cellular immunization detects: 1. cervical dislocation puts to death mice, puts in the beaker filling 75% ethanol, makes mice chaeta complete wetting more than about 3 minutes, then cut mouse peritoneal, takes out spleen; 2. the spleen of separation is put on 200 eye mesh screens of underlying sterile beaker and (has cultivation drop), spleen tissue is twisted gently into pieces with grinding rod (useable glass plunger generation), grinding squeezes out splenocyte, and the RPMI-1640 (IC) getting 5ml serum-free slowly sweeps away cell in beaker.3. slowly being added by 5ml splenocyte suspension is equipped with in the 14ml centrifuge tube of 2.5ml lymphocyte separation medium, the centrifugal 20min of 2000-2500r/min, collects after being separated lymphocyte, centrifuge washing cell 2 times, the centrifugal 6min of each 1500r/min; 4. with the appropriate splenocyte containing the resuspended separation of 5%FCS-1640 complete culture solution, platform expects that blue dyeing counting every mice is separated lymphocyte total viable cell, adjustment cell density to 3 × 10
5/ ml, 37 DEG C, 5%CO
2cultivate in incubator.
ELISPOT detects: strictly press the test kit operation instructions method operation of BD company, set phytohemagglutinin (PHA) as positive control simultaneously.Meet following condition and then regard as ELISPOT detection HBsAg specificity T h1 cell positive: 1. under certain cell density condition, in ELISPOT check-out console, same sample HBsAg and non-HBsAg stimulates the ratio in each 3 multiple holes speckle mean (SFC) to be not less than 2; 2. immunization experiment group sample lymphocytes density is 3 × 10
5when/hole is detected, HBsAg and non-HBsAg stimulates the ratio of each 3 multiple hole SFC to be not less than 5, and as the positive evaluation criterion of mice specific cellular immunity, the cellullar immunologic response positive rate of HBV minicircle dna vaccine immunity group should be not less than 60%.
Statistical result process, between group, mean and percentage difference significance statistics adopt t inspection respectively and look into statistics abridged table method.
Serum-Anti-HBsAg antibody >=10mIU is considered to collective protection valid density.The results are shown in Table 3, Fig. 6-7.Normal saline group mice serum Anti-HBs antibody concentration < 10mIU, Anti-HBsAg antibody mice number is 0; PreS2.S/pcDNA3.1 group, double-mass model preS2.S/pcDNA3.1+hIL2-IFN γ/pcDNA3.1 group and pMCS2.S, pMCS2.S+pMCIIF mice all can at 2 weeks induction Anti-HBs antibody, stronger immunoreation is produced after 4 weeks, pMCS2.S combines adjuvant pMCIIF more remarkable effect, and positive rate reaches 100%; The amount that minicircle dna vaccine pMCS2.S+pMCIIF inducing mouse produces Anti-HBs antibody is higher than double-mass model DNA vaccination pcDNA3.1/preS2.S+pcDNA3.1/hIL2-IFN γ.
Table 3 humoral immunization and cellular immunization testing result
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Sequence table
<110> Hospital No.458 of P.L.A.
<120> therapeutic HBV minicircle dna vaccine and preparation method thereof
<160> 4
<170> PatentIn version 3.3
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ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240
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<211> 35
<212> DNA
<213> artificial sequence
<400> 3
gaagatctgg cgggttgaca ttgattattg actag 35
<210> 4
<211> 28
<212> DNA
<213> artificial sequence
<400> 4
acgcgtcgac ccatagagcc caccgcat 28
Claims (5)
1. therapeutic HBV minicircle dna vaccine, is characterized in that, described vaccine comprises double-mass model, the minicircle dna plasmid namely containing coding HBV peplos albumen preS2.S gene and the minicircle dna plasmid containing coding hIL2-IFN γ antigen-4 fusion protein gene.
2. vaccine according to claim 1, is characterized in that, the reproduction element all not containing prokaryotic plasrnid in described double-mass model and antibiotics element.
3. the preparation method of vaccine described in claim 1 or 2, is characterized in that, comprises the following steps:
1) respectively with plasmid preS2.S/pcDNA3.1 and hIL2-IFN γ/pcDNA3.1 for template, design primer, carry out pcr amplification, obtain object fragment;
2) with BglII and SalI double digestion object fragment and ZY781 carrier simultaneously, reclaim, connect, object fragment is cloned into respectively in the multiple clone site of carrier ZY781, Ji get parental plasmid preS2.S/ZY781 and hIL2-IFN γ/ZY781;
3) respectively with above-mentioned parental plasmid transformation of E. coli ZYCY10P3S2T, incubated overnight, plasmid enzyme restriction and sequence verification insert correct fragment;
4) respectively above-mentioned bacterium liquid access is contained in the TB culture fluid of kana by the inoculum concentration of 0.25 ‰, 37 DEG C, 250rpm incubated overnight, the bacterium liquid getting incubated overnight mixes by equal-volume with Minicle Induction Mix, and in 32 DEG C, 250rpm reacts 5-8h, after reaction terminates, centrifugal upgrading grain, obtains the minicircle dna plasmid pMCS2.S containing coding HBV peplos albumen preS2.S gene, and the minicircle dna plasmid pMCIIF containing coding hIL2-IFN γ antigen-4 fusion protein gene;
5) respectively strain fermentation cultivation is carried out to the positive colony containing above-mentioned plasmid pMCS2.S and plasmid pMCIIF, after fermentation ends, centrifugal upgrading grain, purification;
Wherein, step 1) described in primer comprise:
Forward primer 5 '-GAAGATCTGGCGGGTTGACATTGATTATTGACTAG-3 ' and
Downstream primer 5 '-ACGCGTCGACCCATAGAGCCCACCGCAT-3 ';
Step 4) described in the preparation method of Minicle Induction Mix be: in 100mlLB, add 1M NaOH 4ml and 20% arabinose 200 μ l, through 0.22 μm of membrane filtration after mixing.
4. the medicine for preventing and/or treating hepatitis B containing vaccine described in claim 1.
5. vaccine described in claim 1 is for the preparation of the application prevented and/or treated in the medicine of hepatitis B.
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| CN108949791A (en) * | 2017-10-26 | 2018-12-07 | 深圳新诺微环生物科技有限公司 | Minicircle dna expresses the therapeutic engineered antibody of anti-HPV and its application |
| CN108949791B (en) * | 2017-10-26 | 2021-09-07 | 深圳新诺微环生物科技有限公司 | Micro-ring DNA expression anti-HPV therapeutic engineering antibody and application thereof |
| CN108424926A (en) * | 2018-03-07 | 2018-08-21 | 江苏润洁生物科技有限公司 | A kind of nucleic acid vaccine for preventing HPV infection |
| CN108424925A (en) * | 2018-03-07 | 2018-08-21 | 江苏润洁生物科技有限公司 | A kind of therapeutic HPV nucleic acid vaccine |
| CN109536521A (en) * | 2018-11-26 | 2019-03-29 | 吉林农业大学 | A kind of minicircle dna and the preparation method and application thereof |
| 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 |
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