CN108285907A - A kind of application of bifunctional vector in inhibiting HBV infection - Google Patents
A kind of application of bifunctional vector in inhibiting HBV infection Download PDFInfo
- Publication number
- CN108285907A CN108285907A CN201810007824.XA CN201810007824A CN108285907A CN 108285907 A CN108285907 A CN 108285907A CN 201810007824 A CN201810007824 A CN 201810007824A CN 108285907 A CN108285907 A CN 108285907A
- Authority
- CN
- China
- Prior art keywords
- psiren
- seq
- hbx
- expression
- shpd
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to gene engineering technology fields, and in particular to application of PD L1 and HBx target gene the silence RNA dual-expression vectors in inhibiting HBV infection.Compared with single function carrier, the pSIREN shPD L1 shHB that the present invention is built are more preferable to the inhibition of HepG2.2.15 cell Proliferations, effect is also advantageously promoted to Apoptosis, intracellular anti-apoptotic proteins expression is detected by Western Blot, it was found that bifunctional vector group can obviously lower the expression of Bcl 2 and Bcl xl, quantitative PCR detection intracellular anti-apoptotic proteins expression conditions find that bifunctional vector group can obviously lower the mRNA expression of Bcl 2 and Bcl xl.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to PD-L1 and HBx target gene silence RNA dual-expression vectors
Application in inhibiting HBV infection.
Background technology
Hepatitis type B virus (Hepatitis B virus, HBV) is accredited in 1966 earliest, is a kind of main process
The addicted to liver property DNA virus that the humeral path ways such as alimentary canal or blood are propagated.Chronic HBV infection is to lead to hepatitis, hepatic sclerosis and liver
One major reason of cancer.
RNA interferes (RNA interference, RNAi), refers to by small non-coding RNA induction mRNA degradations, Translational repression
Or heterochromatin is promoted to be formed, and then cryptiogene expression, generate the phenomenon that corresponding function phenotype lacks.The birth of the technology
It is raw allow people efficiently, specifically silencing virus mRNA to inhibit viral antigen to generate is sick according to the needs of oneself
Poison infection and treating correlative diseases provide a handle sharp weapon.HBV is the double-stranded DNA virus for the infection mankind's minimum being currently known,
But due to the gene order with repetition, therefore its sequence in the gene is close, and utilization rate is high, can encode a variety of of viral life needs
Ingredient, meanwhile, HBV is using itself pgRNA (pregenome RNA) during self-replacation come reverse transcription synthetic DNA.So
RNAi not only can target HBV gene with silence and inhibit its protein expression, can also silence HBVpgRNA suppressing virus replications, therefore it is slow
Property HBV infection be suitable for RNAi technology treatment.
In addition, T cell plays vital antivirus action in HBV infection, but in HBV chronic infectious patients
In vivo, the hypofunction of T cell and tend to apoptosis, a kind of state of exhaustion is presented.The study found that compared with Healthy People, CHB
The apparent up-regulation of co-suppression molecule PD-L1 expression of patient's surface of hepatocytes, also, numerous studies are found, it is anti-using blocking property
Body blocks PD-L1/PD-1 signal paths that can reverse the exhaustion of CD8+T cell functions and enhances antiviral ability.Therefore,
Using RNAi perturbation techniques to the targeted therapy of the immunologic test point molecule may for we Anti-HBV activity treatment in bring it is new
Dawn.
Invention content
The object of the present invention is to which by building PD-L1 and HBx target gene silence RNA dual-expression vectors, transfection is felt by virus
After the cell of dye can silencing virus self component inhibit the immunosuppression molecule on its life cycle and silence host surface come
Enhance the antiviral immunity reaction of body, and then effective treatment is played to the infection of Chronic HBV virus.
To achieve the above object, the invention is realized by the following technical scheme：
The present invention provides a kind of application of bifunctional vector in inhibiting HBV, the bifunctional vector target PD-L1 and
The RNAi bifunctional vectors of HBx.
The present invention is experimentally confirmed, compared with single function vehicle group, the inhibition of bifunctional vector cell proliferation
More preferably, the variation of intracellular signaling pathways albumen is detected by Western Blot, it is found that bifunctional vector group can obviously lower p-
The expression of mTOR albumen；Compared with single function vehicle group, bifunctional vector advantageously promotes effect to Apoptosis, passes through
Western Blot detect intracellular anti-apoptotic proteins expression, it is found that bifunctional vector group can obviously lower Bcl-2 and Bcl-
The expression of xl, quantitative PCR detection intracellular anti-apoptotic proteins expression conditions find that bifunctional vector group can obviously lower Bcl-
The mRNA of 2 and Bcl-xl is expressed.
Description of the drawings
Fig. 1 is the screening preferable PD-L1siRNA sequences of silencing efficiency；Wherein：CTRL groups be not added transfection reagent and
SiRNA groups Lipo2000 is that transfection reagent group is only added；1,2,3 is SEQ ID NO.1, SEQ the ID NO.2 and SEQ of PD-L1
Three siRNA transfection groups shown in ID NO.3.
Fig. 2 is the gene level proof diagram of HBx siRNA silencing efficiencies, wherein：CTRL groups be not added transfection reagent and
SiRNA groups, Lipo2000 are that transfection reagent group is only added, and HBx siRNA are siRNA transfection groups.
Fig. 3 is the protein level proof diagram of HBx siRNA silencing efficiencies, wherein：CTRL groups be not added transfection reagent and
SiRNA groups, Lipo2000 are that transfection reagent group is only added, and HBx siRNA are siRNA transfection groups.
Fig. 4 is gene level of the mono-/bis-function carrier to HBx silencing efficiencies of structure, wherein：Lipo2000 is only to be added
Transfection reagent group, Vector are empty carrier transfection group, and shHBx is pSIREN-shHBx carrier transfection groups, and shPD-L1 is
PSIREN-shPD-L1 carrier transfection groups, Dual are pSIREN-shPD-L1-shHBx bifunctional vector transfection groups.
Fig. 5 is gene level of the mono-/bis-function carrier to HBx silencing efficiencies of structure；Wherein：Lipo2000 is only to be added
Transfection reagent group, Vector are empty carrier transfection group, and shHBx is pSIREN-shHBx carrier transfection groups, and shPD-L1 is
PSIREN-shPD-L1 carrier transfection groups, Dual are pSIREN-shPD-L1-shHBx bifunctional vector transfection groups.
Fig. 6 is that the mono-/bis-function carrier of structure verifies the protein level of HBx silencing efficiencies；Wherein：Lipo2000 is only
Transfection reagent group is added, Vector is empty carrier transfection group, and shHBx is pSIREN-shHBx carrier transfection groups, and shPD-L1 is
PSIREN-shPD-L1 carrier transfection groups, Dual are pSIREN-shPD-L1-shHBx bifunctional vector transfection groups.
Fig. 7 is gene level protein level verification of the mono-/bis-function carrier to HBx silencing efficiencies of structure, wherein：
Lipo2000 is that transfection reagent group is only added, and Vector is empty carrier transfection group, and shHBx transfects for pSIREN-shHBx carriers
Group, shPD-L1 are pSIREN-shPD-L1 carrier transfection groups, and Dual transfects for pSIREN-shPD-L1-shHBx bifunctional vectors
Group.
Fig. 8 is the proliferation that dual-expression vector can inhibit HepG2.2.15 cells；Wherein：Lipo2000 is that transfection is only added
Reagent set, Vector are empty carrier transfection group, and shHBx is pSIREN-shHBx carrier transfection groups, shPD-L1 pSIREN-
ShPD-L1 carrier transfection groups, Dual are pSIREN-shPD-L1-shHBx bifunctional vector transfection groups.
Fig. 9, which is dual-expression vector, can lower the relevant signaling pathway protein of endogenous multiplication；Wherein：Lipo2000 is only to add
Enter transfection reagent group, Vector is empty carrier transfection group, and shHBx is pSIREN-shHBx carrier transfection groups, and shPD-L1 is
PSIREN-shPD-L1 carrier transfection groups, Dual are pSIREN-shPD-L1-shHBx bifunctional vector transfection groups.
Figure 10 is the apoptosis that dual-expression vector can promote HepG2.2.15 cells；Wherein：Lipo2000 is only to be added to turn
Transfection reagent group, Vector are empty carrier transfection group, and shHBx is pSIREN-shHBx carrier transfection groups, shPD-L1 pSIREN-
ShPD-L1 carrier transfection groups.
Figure 11 is the figure that dual-expression vector can obviously lower intracellular anti-apoptotic proteins protein level.
Figure 12 is the figure that dual-expression vector can obviously lower intracellular anti-apoptotic proteins gene level.
Specific implementation mode
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific implementation mode, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, component and/or combination thereof.
The present invention provides a kind of application of bifunctional vector in inhibiting HBV, and the bifunctional vector is to target PD-L1
With the RNAi bifunctional vectors of HBx.
Further, application of the bifunctional vector in inhibiting cell growth.
Further, application of the bifunctional vector in changing in terms of intracellular signaling pathway.
Further, application of the bifunctional vector in promoting Apoptosis.
Further, specific the silence siRNA nucleotide sequences such as SEQ ID NO.1, SEQ ID of PD-L1 genes
Shown in NO.2 and SEQ ID NO.3.In best-of-breed technology scheme of the present invention, the specificity of the PD-L1 genes in the people source of selection is heavy
Silent siRNA sequence is sequence shown in SEQ ID NO.1
Further, used carrier pSIREN.
Further, HBx siRNA nucleotide sequences are as shown in SEQ ID NO.4.
The construction method of the RNAi bifunctional vectors of the PD-L1 and HBx, includes the following steps：
(1) PD-L1siRNA as shown in SEQ ID NO.1 and nucleotide sequence such as SEQ ID NO.4 by nucleotide sequence
Shown in HBx siRNA sequences be connected into long hairpin structure, and add double enzyme site BamH I and EcoRI sequences at sequence both ends
Row, annealing form lhRNA duplex structures；
(2) pSIREN carriers are subjected to BamH I and EcoRI double digestions, and recycle carrier framework, obtained pSIREN and linearly carry
Body；
(3) concatenated lhRNA made from step step (1) is connect with pSIREN linear carriers made from (2), is built into
PSIREN-shPD-L1-shHBx plasmids to get.
Further, step (1) lhRNA in the construction method of the RNAi bifunctional vectors for targeting PD-L1 and HBx
Nucleotide sequence is as shown in NO.5~6 SEQ ID.
In order to enable those skilled in the art can clearly understand technical scheme of the present invention, below with reference to tool
The embodiment of the body technical solution that the present invention will be described in detail.
The screening of the specific silence siRNA sequence of the PD-L1 genes in 1 people source of embodiment
For the sequence of people source PD-L1 genes, design is for the specific silence siRNA of the PD-L1 genes in people source, sequence
Respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.
The above-mentioned siRNA of design is turned using cationic-liposome Lipofectamine2000 (being purchased from invitrogen)
It contaminates in the horizontal higher human glioma cell line U251 cells of expression PD-L1, PD-L1 genes is detected by qPCR technologies
Expression, shown in the result is shown in Figure 1.As shown in Figure 1, sequence is that siRNA silencing efficiencies shown in SEQ ID NO.1 are best,
Therefore PD-L1siRNA sequences shown in screening SEQ ID NO.1 do further functional study.
A kind of 2 HBx siRNA sequences of embodiment
The HBx siRNA sequences, nucleotide sequence is as shown in SEQ ID NO.4.
The HBx siRNA of synthesis are transfected into the people liver of HBV infection using cationic-liposome Lipofectamine2000
Cell line HepG2.2.15 cells, and by qPCR and Western Blot technologies detect HBx genes and albumen expression,
The results are shown in Figure 2.As shown in Figure 2, the siRNA sequence all has preferable silence to the expression of HBx genes and albumen and imitates
Fruit.
A kind of construction method for the RNAi bifunctional vectors targeting PD-L1 and HBx of embodiment 3
The construction method of the RNAi bifunctional vectors for targeting PD-L1 and HBx, includes the following steps：
(1) PD-L1siRNA as shown in SEQ ID NO.1 and nucleotide sequence such as SEQ ID NO.4 by nucleotide sequence
Shown in HBx siRNA sequences be connected into long hairpin structure, and add double enzyme site BamH I and EcoRI sequences at sequence both ends
Row, annealing form lhRNA duplex structures, and h-shPD-L1-shHBx-F nucleotide sequences are as shown in SEQ ID NO.5；h-shPD-
L1-shHBx-R nucleotide sequences are as shown in SEQ ID NO.6；
(2) pSIREN carriers are subjected to BamH I and EcoRI double digestions, and recycle carrier framework, obtained pSIREN and linearly carry
Body；
(3) concatenated lhRNA made from step step (1) is connect with pSIREN linear carriers made from (2), is built into
PSIREN-shPD-L1-shHBx plasmids to get.
The structure of 1 single function expression vector of comparative example
The construction method of the single function carrier, includes the following steps：
(1) the PD-L1 siRNA as shown in SEQ ID NO.1 and nucleotide sequence such as SEQ ID by nucleotide sequence
Double enzyme site BamH I and EcoRI sequences are added at HBx siRNA sequences both ends shown in NO.4, and annealing forms shRNA double-strands
Structure, h-shPD-L1-F nucleotide sequences are as shown in SEQ ID NO.7；H-shPD-L1-R nucleotide sequences such as SEQ ID
Shown in NO.8；HBx-F nucleotide sequences are as shown in SEQ ID NO.9；HBx-R nucleotide sequences are as shown in SEQ ID NO.10；
(2) pSIREN carriers are subjected to BamH I and EcoRI double digestions, and recycle carrier framework, obtained pSIREN and linearly carry
Body；
(3) shRNA made from step step (1) is connect with pSIREN linear carriers made from (2) respectively, is built into
PSIREN-shHBx single function expression vectors, pSIREN-shPD-L1 single function expression vectors.
The verification of 1 pair of silent carrier silencing efficiency of test example
(1) quantitative PCR
(1) pSIREN-shHBx single functions expression vector and the difunctional expression vector transfections of pSIREN-shPD-L1-shHBx
HepG2.2.15 cells for 24 hours after, extract RNA, detect HBV x genes, it can be seen that single function expression vector and difunctional expression
Carrier all has good silence effect (as shown in Figure 4) compared to empty carrier；
(2) pSIREN-shPD-L1 single functions expression vector and the difunctional expression vectors of pSIREN-shPD-L1-shHBx turn
After contaminating HepG2.2.15 cells for 24 hours, RNA is extracted, detects PD-L1 genes, it can be seen that single function expression vector and difunctional table
Up to carrier compared to empty carrier, good silencing efficiency (as shown in Figure 5) is all had.
(2) Western Blot
PSIREN-shHBx single functions expression vector and the difunctional expression vector transfections of pSIREN-shPD-L1-shHBx
After HepG2.2.15 cells 48h, extract cell holoprotein, detect HBV x protein expressions, it can be seen that single function expression vector and
Difunctional expression vector all has good silencing efficiency (as shown in Figure 6) compared to empty carrier.
(3) flow cytometry
PSIREN-shPD-L1 single functions expression vector and the difunctional expression vector transfections of pSIREN-shPD-L1-shHBx
After HepG2.2.15 cells 48h, cell is collected, streaming instrument detects cell surface PD-L1 expression, it can be seen that single function expression carries
Body and difunctional expression vector all have good silencing efficiency (as shown in Figure 7) compared to empty carrier.
Influence of the 2 pairs of silent carriers of test example to HepG2.2.15 cell growths
(1) by pSIREN empty carriers, pSIREN-shHBx single function expression vectors, the expression of pSIREN-shPD-L1 single functions
After carrier and pSIREN-shPD-L1-shHBx expression vectors transfection HepG2.2.15 cells 12h, by cell with 6000, every hole
It is taped against in 96 orifice plates, cell viability is detected after three days.It can be seen that compared with single function vehicle group, bifunctional vector pair
The inhibition of cell Proliferation is more preferable (as shown in Figure 8).
(2) by pSIREN empty carriers, pSIREN-shHBx single function expression vectors, the expression of pSIREN-shPD-L1 single functions
After carrier and pSIREN-shPD-L1-shHBx expression vectors transfect HepG2.2.15 cells 48h, cell is collected, extraction intracellular is complete
Albumen, the variation of Western Blot detection intracellular signaling pathways albumen, it is found that bifunctional vector group can obviously lower mTOR eggs
White activation (as shown in Figure 9).
Influence of the 3 pairs of silent carriers of test example to HepG2.2.15 Apoptosis
(1) by pSIREN empty carriers, pSIREN-shHBx single function expression vectors, the expression of pSIREN-shPD-L1 single functions
After carrier and pSIREN-shPD-L1-shHBx expression vectors transfect HepG2.2.15 cells for 24 hours, the apoptosis situation of cell is detected,
It can be seen that compared with single function vehicle group, bifunctional vector advantageously promotes effect (as shown in Figure 10) to Apoptosis.
(2) by pSIREN empty carriers, pSIREN-shHBx single function expression vectors, the expression of pSIREN-shPD-L1 single functions
After carrier and pSIREN-shPD-L1-shHBx expression vectors transfect HepG2.2.15 cells 48h, cell is collected, extraction intracellular is complete
Albumen, Western Blot detect intracellular anti-apoptotic proteins expression, find bifunctional vector group can obviously lower Bcl-2 and
The expression (as shown in figure 11) of Bcl-xl.
(3) by pSIREN empty carriers, pSIREN-shHBx single function expression vectors, the expression of pSIREN-shPD-L1 single functions
After carrier and pSIREN-shPD-L1-shHBx expression vectors transfect HepG2.2.15 cells for 24 hours, cell is collected, extracts RNA, it is fixed
It measures PCR and detects intracellular anti-apoptotic proteins expression conditions, it is found that bifunctional vector group can obviously lower Bcl-2's and Bcl-xl
MRNA expresses (as shown in figure 12).
SEQUENCE LISTING
<110>Shandong University
<120>A kind of application of bifunctional vector in inhibiting HBV infection
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> si-h-PD-L1-001
<400> 1
catagtagct acagacaga 19
<210> 2
<211> 19
<212> DNA
<213> si-h-PD-L1-002
<400> 2
caagcgaatt actgtgaaa 19
<210> 3
<211> 19
<212> DNA
<213> si-h-PD-L1-003
<400> 3
gaaaggactc acttggtaa 19
<210> 4
<211> 22
<212> DNA
<213> si-HBx
<400> 4
cggcataaat tggtctgttc ac 22
<210> 5
<211> 109
<212> DNA
<213> h-shPD-L1-shHBx-F
<400> 5
ggatcccata gtagctacag acagatctcg gcataaattg gtctgttcac ttcaagagat 60
ctgtctgtag ctactatgag agtgaacaga ccaatttatg ccgtttttt 109
<210> 6
<211> 109
<212> DNA
<213> h-shPD-L1-shHBx-R
<400> 6
gaattcaaaa aacggcataa attggtctgt tcactctcat agtagctaca gacagatctc 60
ttgaagtgaa cagaccaatt tatgccgaga tctgtctgta gctactatg 109
<210> 7
<211> 55
<212> DNA
<213> h-shPD-L1-F
<400> 7
gatccatagt agctacagac agatcaagag tctgtctgta gctactatgt ttttt 55
<210> 8
<211> 55
<212> DNA
<213> h-shPD-L1-R
<400> 8
aattaaaaaa catagtagct acagacagac tcttgatctg tctgtagcta ctatg 55
<210> 9
<211> 55
<212> DNA
<213> HBx-F
<400> 9
gatccggcat aaattggtct gttcactcaa gaggtgaaca gaccaattta tgccg 55
<210> 10
<211> 55
<212> DNA
<213> HBx-R
<400> 10
aattcggcat aaattggtct gttcacctct tgagtgaaca gaccaattta tgccg 55
Claims (9)
1. a kind of application of bifunctional vector in inhibiting HBV infection, which is characterized in that the RNAi for targeting PD-L1 and HBx is bis-
Function carrier.
2. application according to claim 1, which is characterized in that the bifunctional vector answering in inhibiting cell growth
With.
3. application according to claim 1, which is characterized in that the bifunctional vector is changing intracellular signaling pathway side
Application in face.
4. application according to claim 1, which is characterized in that application of the bifunctional vector in promoting Apoptosis.
5. application according to claim 1, which is characterized in that the specific silence siRNA nucleotide sequences of PD-L1 genes
As shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.
6. application according to claim 1 or 2, which is characterized in that expression carrier used thereof pSIREN.
7. application according to claim 1, which is characterized in that HBx siRNA nucleotide sequences such as SEQ ID NO.4 institutes
Show.
8. application according to claim 1, which is characterized in that the structure side of the RNAi bifunctional vectors of PD-L1 and HBx
Method includes the following steps：
(1) by nucleotide sequence PD-L1siRNA and nucleotide sequence as shown in SEQ ID NO.1 as shown in SEQ ID NO.4
HBx siRNA sequences be connected into long hairpin structure, and add double enzyme site BamH I and EcoRI sequences at sequence both ends,
Annealing forms lhRNA duplex structures；
(2) pSIREN carriers are subjected to BamH I and EcoRI double digestions, and recycle carrier framework, obtain pSIREN linear carriers；
(3) concatenated lhRNA made from step step (1) is connect with pSIREN linear carriers made from (2), is built into
PSIREN-shPD-L1-shHBx plasmids to get.
9. application according to claim 8, which is characterized in that step (1) lhRNA nucleotide sequences such as SEQ ID NO.5
Shown in~6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810007824.XA CN108285907A (en) | 2018-01-04 | 2018-01-04 | A kind of application of bifunctional vector in inhibiting HBV infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810007824.XA CN108285907A (en) | 2018-01-04 | 2018-01-04 | A kind of application of bifunctional vector in inhibiting HBV infection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108285907A true CN108285907A (en) | 2018-07-17 |
Family
ID=62834923
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810007824.XA Pending CN108285907A (en) | 2018-01-04 | 2018-01-04 | A kind of application of bifunctional vector in inhibiting HBV infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108285907A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101054577A (en) * | 2006-12-07 | 2007-10-17 | 山东大学 | A siRNA and expression carrier capable of inhibiting Bax gene expression and application of the same used as virus hepatitis B treatment medicament |
| CN106715431A (en) * | 2014-09-16 | 2017-05-24 | 吉利德科学公司 | Solid forms of a toll-like receptor modulator |
| CN107298713A (en) * | 2017-08-15 | 2017-10-27 | 联合益康（北京）生物科技有限公司 | A kind of anti-PD L1 antibody and application, preparation method, kit and medicine |
-
2018
- 2018-01-04 CN CN201810007824.XA patent/CN108285907A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101054577A (en) * | 2006-12-07 | 2007-10-17 | 山东大学 | A siRNA and expression carrier capable of inhibiting Bax gene expression and application of the same used as virus hepatitis B treatment medicament |
| CN106715431A (en) * | 2014-09-16 | 2017-05-24 | 吉利德科学公司 | Solid forms of a toll-like receptor modulator |
| CN107298713A (en) * | 2017-08-15 | 2017-10-27 | 联合益康（北京）生物科技有限公司 | A kind of anti-PD L1 antibody and application, preparation method, kit and medicine |
Non-Patent Citations (8)
| Title |
|---|
| COELHO MA 等: "Homo sapiens CD274 molecule (CD274), transcript variant 1, mRNA", 《GENBANK DATABASE》 * |
| WENZHENG JIANG 等: "Blockade of B7-H1 enhances dendritic cell-mediated T cell response and antiviral immunity in HBV transgenic mice", 《VACCINE》 * |
| YIN,J. 等: "Hepatitis B virus isolate b270-s precore/core protein (C) gene, partial cds", 《GENBANK DATABASE》 * |
| 兰培祥 等: "治疗性双功能表达载体通过纠正肝细胞内源性免疫耐受逆转机体系统性免疫耐受", 《第八届全国免疫学学术大会》 * |
| 关妘 等: "HBV DNA聚合酶（HBp）通过下调Fas表达抑制肝癌细胞的凋亡", 《现代免疫学》 * |
| 房良华 主编: "《现代肿瘤免疫靶向治疗》", 30 November 2010, 东南大学出版社 * |
| 曾其毅 主编: "《肝细胞癌防控与分子医学》", 31 January 2017, 辽宁科学技术出版社 * |
| 蒋涛 等: "ＲNAi沉默肺腺癌A549细胞系PD-L1基因表达对其增殖和凋亡的影响", 《现代肿瘤医学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wei et al. | MyD88 as a target of microRNA-203 in regulation of lipopolysaccharide or Bacille Calmette-Guerin induced inflammatory response of macrophage RAW264. 7 cells | |
| Kumar | MicroRNA in HCV infection and liver cancer | |
| Mollaie et al. | RNAi and miRNA in viral infections and cancers | |
| Kim et al. | Viral envelope protein 53R gene highly specific silencing and iridovirus resistance in fish cells by AmiRNA | |
| Panda et al. | MiRNA-SARS-CoV-2 dialogue and prospective anti-COVID-19 therapies | |
| CN107365785B (en) | Gene expression vector for regulating and controlling intracellular NF-kB activity and regulating and controlling method and application thereof | |
| Zhai et al. | Borna disease virus encoded phosphoprotein inhibits host innate immunity by regulating miR-155 | |
| Oikawa et al. | Role of endothelial microRNA-23 clusters in angiogenesis in vivo | |
| CN106176795B (en) | Application of the prawn miR-35 in prawn is antiviral and inhibition human tumour shifts | |
| McCoy | The role of miRNAs in cytokine signaling | |
| CN107523569B (en) | Application of PDCD1 gene and related medicaments thereof | |
| CN102206645B (en) | Mediating method of RNAi (ribonucleic acid interference) utilizing lentiviral vector | |
| Rashid et al. | The diverse roles of miRNAs in HIV pathogenesis: Current understanding and future perspectives | |
| CN108285907A (en) | A kind of application of bifunctional vector in inhibiting HBV infection | |
| CN101948859B (en) | shRNA expression vector expressed by specificity inhibitor SET protein, construction method and application thereof | |
| CN101948544B (en) | FAT10 gene siRNA recombination analogue virus as well as preparation method and application thereof | |
| Chung et al. | Silencing E1A mRNA by RNA interference inhibits adenovirus replication | |
| Pandita et al. | Role of miRNAs in regulating virus replication | |
| US8957199B2 (en) | Oligoribonucleotide or peptide nucleic acid capable of inhibiting activity of hepatitis C virus | |
| CN102813926B (en) | Application of miR-7 expression inhibitor in preparing medicines used for treating systemic lupus erythematosus | |
| CN103173446A (en) | Construction method of recombinant adeno-associated virus vector of targeting interference SOCS1 gene and application of construction method | |
| Chaudhary et al. | Interplay of host and viral factors in inflammatory pathway mediated cytokine storm during RNA virus infection | |
| Kumar et al. | A Review of miRNA Regulation in Japanese Encephalitis (JEV) Virus Infection | |
| Zhou et al. | Establishment of a screening system for selection of siRNA target sites directed against hepatitis B virus surface gene | |
| CN107184984A (en) | Application of the RPL10 inhibitor in the medicine for preparing treatment oophoroma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |