CN109706176A - Transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method can be targeted - Google Patents
Transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method can be targeted Download PDFInfo
- Publication number
- CN109706176A CN109706176A CN201910183537.9A CN201910183537A CN109706176A CN 109706176 A CN109706176 A CN 109706176A CN 201910183537 A CN201910183537 A CN 201910183537A CN 109706176 A CN109706176 A CN 109706176A
- Authority
- CN
- China
- Prior art keywords
- aav
- egfp
- transfection
- virus
- carrier
- 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
Abstract
The present invention relates to technical field of bioengineering, transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method can especially be targeted, it is characterized in that, obtain appropriate pAAV-GFAP-EGFP expression plasmid, pRep2/Cap6 (pRC6), pRC8 and pHelper carries out the production of AAV virus, it obtains suitable AAV virus and carries out gradient centrifugation purification, transfection obtains AAV carrier, three days after transfection, cell precipitation is resuspended in lysis buffer, and the freezing-thawing and cracking recycled by 3 obtains cell lysate A, carry out purified virus carrier integrity verification, analyze the cell-specific of EGFP expression, the ratio that SGC transduces after assessment vector injection, obtain result.The present invention successfully constructs the AAV carrier containing GFAP promoter, AAV carrier has been approved by the FDA in the United States to use in human body because safety is higher, the carrier specific can transfect SGCs in DRG, it is created condition for subsequent gene treatment, improve safety, the progress for promoting the treatment means of neuropathic pain, alleviates the burden of patient.
Description
Technical field
The present invention relates to gene engineering technology fields, more particularly to can target transfection dorsal root ganglion satellite spongiocyte gland
Viral vectors construction method.
Background technique
Pain is to perplex the big persistent ailment of the mankind, is classified as the fifth-largest vital sign, 73% people by World Health Organization within 2000
It will appear serious pain in life, the most common neurogenic pain aches after having discogenic pain and neurotrosis
Bitterly, clinically only lumbago just has up to 159 kinds of causes of disease, and as the arrival of aging and the vehicles are increasing, backbone is moved back
Neurogenic pain caused by becoming and damaging is increasing, and because of its cause of disease complexity, diagnostic criteria defines difficulty, treatment method
Inaccurate, sing misdiagnosis and mistreatment phenomenon is serious, hardly results in early stage, effective, specification diagnosis and treatment, clinical efficacy has been seriously affected, to trouble
Person family and society bring heavy burden, extreme influence people's health level and social development, and therefore, neurogenic pain is ground
Studying carefully just becomes one of advanced subject；
Chronic neuropathic pain model, which lacks, at present specifically and effectively treats, and dorsal root ganglion (DRG) is used as pain
The incoming primary neuronal of signal and the only way which must be passed are the most suitable positions of pain medication and molecular therapy, while DRG makees
There is very high safety and tolerance for therapy target, U.S. FDA approved in 2012 is carried out transforaminal approach and is directed to
The pain associated treatment of DRG, and clinical test is it has also been found that transforaminal approach DRG injection treatment and electronic stimulation seldom occur
The complication such as neurotrosis, safety with higher；
Gene therapy has confirmed the potential new method of tool that chronic ache can be treated for one kind, sends out in research before
Now have become a kind of effective method using the transfection of adeno-associated virus (AAV) row gene, target gene can be transferred to
Peripheral sensory neuron after silk division, is maintained with the smallest toxicity to the long-term efficacy of neuropathic pain, and in last October,
U.S. FDA is used for clinical treatment in approval adeno-associated virus (AAV), and early period is we have discovered that be directed to dorsal root ganglion neurons
Cell carries out the gene gene transfection for treating of AAV mediation, and influences pain with interference peptide aptamers specific bond neuron surface and pass
The signal path passed, and good result is obtained in animal experiment；
At present to DRG gene therapy mainly for neuronal cell, and have ignored neuromere Satellite spongiocyte
(SGCs) effect in pain signal transmitting, neuromere Satellite spongiocyte (SGCs) is on pain signal transmission path
One pith, SGC proliferation and activation after neurotrosis influence Sensory transmission, including gene table by number of mechanisms
The change reached, increase SGCs between and SGCs and interneuronal coupling；Induce chemotactic factor (CF)/cell of microglial activation
Factor release, to SGC specific molecular (such as connection protein 43 (Cx43), glutamine synthelase (GS), in ATP responsive type
To rectifier potassium channel 10 (Kir4.1), excitability amino acid transporter 1 (EAAT1), purine energy ionic P2X7 receptor
(P2X7R) and GFAP) gene modification, can significantly change normal and neurotrosis rodent to the anti-of feeling
It answers, therefore, the gene therapy in dorsal root ganglion (DRG) for SGC can provide new thinking for the treatment of chronic ache,
And at home and abroad still belong to blank for the specific transfection technology of SGCs in DRG, so transfection back can be targeted by needing to design now
Root neural section satellite spongiocyte adenovirus vector construct method solves the above problems.
Summary of the invention
The purpose of the present invention is to provide can target transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct side
Method, to solve the problems mentioned in the above background technology.
To achieve the goals above, present invention employs following technical solutions:
Design can target transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method, comprising the following steps:
Step 1: appropriate pAAV-GFAP-EGFP expression plasmid, pRep2/Cap6 (pRC6), pRC8 and pHelper are obtained
Carry out AAV virus production, prepare enough lysis buffers (50mM Tris-HCl, 150mM NaCl, 2mM MgCl 2,
PH8.0), the benzonase (Sigma-Aldrich) of 100U/ml, the 40%PEG8000 (Sigma- containing 2.5N NaCl
Aldrich storage solutions), phosphate buffered saline (PBS) (PBS), 40ml Quick- containing 1mM CaCl 2 and 2mM KCl
Seal centrifuge tube, No. 18 syringe needles, Centricon Plus-20 (Regenerated Cellulose 100,000MWCO,
Millipore, Bill-erica, MA), contain 5% D-sorbite (Sigma-Aldrich) PBS, amicillin resistance base
Cause, sodium dodecyl sulfate-polypropylene acrylamide gel (SDS-PAGE), Pierce Silver stain kit (Fisher
Scientific, Rockford, IL), the anti-Vp1 of mouse, -2, -3 antibody (1:1000 dilution；Research Diagnostic
Inc.), (400 holes are coated with poly-vinegar acid to 1%Alcian blue (Electron Microscopy Sciences) pretreatment copper mesh
Ethylene methacrylic rouge and thin carbon film；Electron Microscopy Sciences, Hatfield, PA), 1% uranyl acetate
(Electron Microscopy Sciences), 600 transmission electron microscope of JEOL 2100 and Hitachi；
Step 2: suitable AAV virus is obtained, using Optirep (Sigma-Aldrich, St Louis, MO, USA) ladder
Spend centrifugal purification；
Step 3: by with pRC6 or pRC8 and pHelper plasmid by CaPO4 cotransfection pAAV-GFAP-EGFP to 293T
AAV carrier is generated in cell；
Step 4: three days after transfection, cell precipitation is resuspended in lysis buffer (50mM Tris-HCl, 150mM
NaCl, 2mM MgCl 2, pH8.0) in, and the freezing-thawing and cracking recycled by 3 obtains cell lysate A, by cell lysate A
Clarification in 30 minutes is centrifuged by 2500g and obtains cell lysate B, by the benzonase of cell lysate B and 100U/ml
(Sigma-Aldrich) it is incubated 1 hour at 37 DEG C and obtains cell lysate C, by the 40%PEG8000 containing 2.5N NaCl
(Sigma-Aldrich) storage solutions are added in cell lysate to final concentration of 8%, then incubate solution on ice
2 hours, with 2500g centrifugation 30 minutes, and the phosphate buffered saline (PBS) containing 1mM CaCl 2 and 2mM KCl will be deposited in
(PBS) separation in；
Step 5: purified virus carrier integrity verification is carried out；
Step 6: the gland relevant viral vector for taking 2 μ l to construct slowly is injected to SD rat DRG by micro syringe, infuses
Syringe needle is slowly withdrawn from after shooting away complete 5min, DRG embedded section is logical using GFP antibody by animal euthanasia within 5 weeks after virus transfection
Cross the cell-specific of immunofluorescence analysis EGFP expression；
Step 7: the Primary Sensory Neuron cell space week that EGFP is marked in Tubb3 after AAV6-GFAP-EGFP Successful transfection
Enclose expression annular in shape, wherein EGFP signal be located at the Tubb3 positive neuronal cell and NKA α 1 mark neuron plasma membrane it
Outside, height common location is dyed with GFAP；
Step 8: the ratio that SGC transduces after assessment vector injection, to the annular SGC row immuning tissue for surrounding neuron
It learns detection: observing the annular of the EGFP positive around the DRG of injection of AAV 6-GFAP-EGFP, the neuron for having 70% ± 8%
SGC, and only minority pericaryon EGFP is positive (~1%), what the viral vectors for obtaining this project building can be specific
The result expressed in SGCs.
Preferably, pAAV-GFAP-EGFP expression plasmid contains self-complementary (sc) AAV2 opposing end in the step 1
Repetitive sequence (ITR), and transcribed by the EGFP that GFAP promoter drives chimeric interon to enhance downstream, the Rep2/Cap6
(pRC6) and pRC8 (Viromics, Fremont, CA, USA) respectively containing from AAV6 or AAV8 AAV2 duplication (rep) and
Capsid (cap) protein gene；The pHelper (Viromics) of the encoding adenovirus auxiliary gene.
Preferably, in the step 2 Optiprep purify AAV method the following steps are included:
S1: 40ml Quick-Seal centrifuge tube row Optiprep gradient centrifugation (Beckman Instruments, Palo are used
Alto, CA, USA), gradient is respectively 10ml, and 15%；8ml, 25%；6ml, 40%, 2ml, 60%；
S2: 15 milliliters of AAV viruses are subjected to cracking gradient centrifugation, in 70Ti rotor (Beckman at 70 DEG C
Instruments it is centrifuged 70 minutes in) with 18 DEG C, is mentioned with No. 18 syringe needles from 1/2 part of lower layer between 40-60% with 40%
Take virus；
S3: with Centricon Plus-20 (Regenerated Cellulose 100,000 MWCO, Millipore,
Bill-erica, MA) viral suspension obtained is concentrated, and final virus formulation is maintained at containing 5% D-sorbite
(Sigma-Aldrich) it in PBS, and is stored at -80 DEG C；
S4: the carrier relative to standard plasmid is measured with PicoGreen method (Invitrogen, Carlsbad, CA, USA)
Genome, and the dotted trace of row also after the denaturation of AAV carrier, are primer row PCR detection using ampicillin resistance gene,
The AAV plasmid pollution in the AAV vector gene group of purifying is not found, calculates virus titer, and unit is the genome copies of every ml
Number (GC/ml), the titre of AAV6-EGFP and AAV8-EGFP carrier is respectively 2.161013GC/ml and 2.561013GC/ml.
Preferably, in the step 5 carry out purified virus carrier integrity verification method the following steps are included:
B1: assessing the purity of carrier by sodium dodecyl sulfate-polypropylene acrylamide gel (SDS-PAGE) electrophoresis first,
Then Silver stain is carried out using Pierce Silver stain kit (Fisher Scientific, Rockford, IL)；
B2: the anti-Vp1 of mouse, (the 1:1000 dilution of -2, -3 antibody are used；Research Diagnostic Inc.) pass through mark
Quasi- immunoblotting program validation viral capsid proteins；
B3: with NIH ImageJ software (http://rsbweb.nih.gov/ij/) quantitative Vp1:Vp2:Vp3 albumen ratio
Example and relative to non-carrier albumen visible on gel purity, carry out negative staining and electron microscope (EM) scanning it is pure to assess
The granule content of the AAV carrier of change；
B4: with 1%Alcian blue (Electron Microscopy Sciences) pretreatment copper mesh, (400 holes are coated with
Formvar and thin carbon film；Electron Microscopy Sciences, Hatfield, PA) and load 5ml disease
Poisonous carrier washs grid, is dyed with 1% uranyl acetate (Electron Microscopy Sciences), and use JEOL
2100 and Hitachi, 600 transmission electron microscope observation；
B5: particle fraction is determined by directly counting electron micrograph.
Preferably, the AAV virus formulation batch is same batch.
It is proposed by the present invention to target transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method, beneficial to effect
Fruit is: the present invention successfully constructs the AAV carrier containing GFAP promoter, and AAV carrier is higher by U.S. FDA because of safety
For using in human body, which specific can transfect SGCs in DRG for approval, create condition for subsequent gene treatment.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.
Embodiment one: transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method can be targeted, including following
Step:
Step 1: appropriate pAAV-GFAP-EGFP expression plasmid, pRep2/Cap6 (pRC6), pRC8 and pHelper are obtained
The production of AAV virus is carried out, the pAAV-GFAP-EGFP expression plasmid contains the repetition of self-complementary (sc) AAV2 opposing end
Sequence (ITR), and transcribed by the EGFP that GFAP promoter drives chimeric interon to enhance downstream, the Rep2/Cap6
(pRC6) and pRC8 (Viromics, Fremont, CA, USA) respectively containing from AAV6 or AAV8 AAV2 duplication (rep) and
Capsid (cap) protein gene；It is slow to prepare enough cracking by the pHelper (Viromics) of the encoding adenovirus auxiliary gene
Benzonase (the Sigma- of fliud flushing (50mM Tris-HCl, 150mM NaCl, 2mM MgCl 2, pH8.0), 100U/ml
Aldrich), the storage solutions of the 40%PEG8000 (Sigma-Aldrich) containing 2.5N NaCl contain 2 and of 1mM CaCl
Phosphate buffered saline (PBS) (PBS), 40ml Quick-Seal centrifuge tube, No. 18 syringe needles, Centricon Plus-20 of 2mM KCl
(Regenerated Cellulose 100,000 MWCO, Millipore, Bill-erica, MA), contain 5% D-sorbite
(Sigma-Aldrich) PBS, ampicillin resistance gene, sodium dodecyl sulfate-polypropylene acrylamide gel (SDS-
PAGE), Pierce Silver stain kit (Fisher Scientific, Rockford, IL), the anti-Vp1 of mouse, -2, -3 antibody
(1:1000 dilution；Research Diagnostic Inc.), 1%Alcian blue (Electron Microscopy
Sciences) (400 holes are coated with Formvar and thin carbon film to pretreatment copper mesh；Electron Microscopy
Sciences, Hatfield, PA), 1% uranyl acetate (Electron Microscopy Sciences), 2100 and of JEOL
600 transmission electron microscope of Hitachi；
Step 2: suitable AAV virus is obtained, using Optirep (Sigma-Aldrich, St Louis, MO, USA) ladder
It spends centrifugal purification: using 40ml Quick-Seal centrifuge tube row Optiprep gradient centrifugation (Beckman Instruments, Palo
Alto, CA, USA), gradient is respectively 10ml, and 15%；8ml, 25%；6ml, 40%, 2ml, 60%, by 15 milliliters of AAV viruses into
Row cracking gradient centrifugation, is centrifuged 70 minutes, with 18 with 18 DEG C in 70Ti rotor (Beckman Instruments) at 70 DEG C
Number syringe needle between 40-60% and 40% lower layer's 1/2 extracting section virus；With Centricon Plus-20
The virus that (Regenerated Cellulose 100,000MWCO, Millipore, Bill-erica, MA) concentration obtains suspends
Liquid, and final virus formulation is maintained in the PBS containing 5% D-sorbite (Sigma-Aldrich), and at -80 DEG C
Storage；The vector gene group relative to standard plasmid is measured with PicoGreen method (Invitrogen, Carlsbad, CA, USA),
And the dotted trace of row also after the denaturation of AAV carrier, is primer row PCR detection using ampicillin resistance gene, does not find
AAV plasmid pollution in the AAV vector gene group of purifying, calculates virus titer, and unit is the genome copy numbers (GC/ of every ml
Ml), the titre of AAV6-EGFP and AAV8-EGFP carrier is respectively 2.161013GC/ml and 2.561013GC/ml, in this experiment
The AAV virus formulation batch is same batch；
Step 3: by with pRC6 or pRC8 and pHelper plasmid by CaPO4 cotransfection pAAV-GFAP-EGFP to 293T
AAV carrier is generated in cell；
Step 4: three days after transfection, cell precipitation is resuspended in lysis buffer (50mM Tris-HCl, 150mM
NaCl, 2mM MgCl 2, pH8.0) in, and the freezing-thawing and cracking recycled by 3 obtains cell lysate A, by cell lysate A
Clarification in 30 minutes is centrifuged by 2500g and obtains cell lysate B, by the benzonase of cell lysate B and 100U/ml
(Sigma-Aldrich) it is incubated 1 hour at 37 DEG C and obtains cell lysate C, by the 40%PEG8000 containing 2.5N NaCl
(Sigma-Aldrich) storage solutions are added in cell lysate to final concentration of 8%, then incubate solution on ice
2 hours, with 2500g centrifugation 30 minutes, and the phosphate buffered saline (PBS) containing 1mM CaCl 2 and 2mM KCl will be deposited in
(PBS) separation in；
Step 5: purified virus carrier integrity verification is carried out: solidifying by sodium dodecyl sulfate-polypropylene amide first
Glue (SDS-PAGE) electrophoresis assess carrier purity, then using Pierce Silver stain kit (Fisher Scientific,
Rockford, IL) carry out Silver stain；Use the anti-Vp1 of mouse, (the 1:1000 dilution of -2, -3 antibody；Research Diagnostic
Inc. viral capsid proteins) are confirmed by standard immunoassay western blot procedure；With NIH ImageJ software (http:// rsbweb.nih.gov/ij/) quantify the ratio of Vp1:Vp2:Vp3 albumen and relative to non-carrier albumen visible on gel
Purity carries out negative staining and electron microscope (EM) scanning to assess the granule content of the AAV carrier of purifying；Use 1%Alcian
Blue (Electron Microscopy Sciences) pre-processes copper mesh, and (400 holes are coated with Formvar and thin carbon
Film；Electron Microscopy Sciences, Hatfield, PA) and 5ml viral vectors is loaded, grid is washed, with 1%
Uranyl acetate (Electron Microscopy Sciences) dyeing, and electricity is transmitted with JEOL 2100 and Hitachi 600
The micro- sem observation of son；Particle fraction is determined by directly counting electron micrograph；
Step 6: the gland relevant viral vector for taking 2 μ l to construct slowly is injected to SD rat DRG by micro syringe, infuses
Syringe needle is slowly withdrawn from after shooting away complete 5min, DRG embedded section is logical using GFP antibody by animal euthanasia within 5 weeks after virus transfection
Cross the cell-specific of immunofluorescence analysis EGFP expression；
Step 7: the Primary Sensory Neuron cell space week that EGFP is marked in Tubb3 after AAV6-GFAP-EGFP Successful transfection
Enclose expression annular in shape, wherein EGFP signal be located at the Tubb3 positive neuronal cell and NKA α 1 mark neuron plasma membrane it
Outside, height common location is dyed with GFAP；
Step 8: the ratio that SGC transduces after assessment vector injection, to the annular SGC row immuning tissue for surrounding neuron
It learns detection: observing the annular of the EGFP positive around the DRG of injection of AAV 6-GFAP-EGFP, the neuron for having 70% ± 8%
SGC, and only minority pericaryon EGFP is positive (~1%), what the viral vectors for obtaining this project building can be specific
The result expressed in SGCs.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (5)
1. transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method can be targeted, which is characterized in that including following
Step:
Step 1: appropriate pAAV-GFAP-EGFP expression plasmid, pRep2/Cap6 (pRC6), pRC8 and pHelper are obtained and is carried out
The production of AAV virus, prepare enough lysis buffers (50mM Tris-HCl, 150mM NaCl, 2mM MgCl 2,
PH8.0), the benzonase (Sigma-Aldrich) of 100U/ml, the 40%PEG8000 (Sigma- containing 2.5N NaCl
Aldrich storage solutions), phosphate buffered saline (PBS) (PBS), 40ml Quick- containing 1mM CaCl 2 and 2mM KCl
Seal centrifuge tube, No. 18 syringe needles, Centricon Plus-20 (Regenerated Cellulose 100,000MWCO,
Millipore, Bill-erica, MA), contain 5% D-sorbite (Sigma-Aldrich) PBS, amicillin resistance base
Cause, sodium dodecyl sulfate-polypropylene acrylamide gel (SDS-PAGE), Pierce Silver stain kit (Fisher
Scientific, Rockford, IL), the anti-Vp1 of mouse, -2, -3 antibody (1:1000 dilution；Research Diagnostic
Inc.), (400 holes are coated with poly-vinegar acid to 1%Alcian blue (Electron Microscopy Sciences) pretreatment copper mesh
Ethylene methacrylic rouge and thin carbon film；Electron Microscopy Sciences, Hatfield, PA), 1% uranyl acetate
(Electron Microscopy Sciences), 600 transmission electron microscope of JEOL 2100 and Hitachi；
Step 2: obtaining suitable AAV virus, using Optirep (Sigma-Aldrich, St Louis, MO, USA) gradient from
Heart purifying；
Step 3: by with pRC6 or pRC8 and pHelper plasmid by CaPO4 cotransfection pAAV-GFAP-EGFP to 293T cell
In generate AAV carrier；
Step 4: three days after transfection, cell precipitation is resuspended in lysis buffer (50mM Tris-HCl, 150mM NaCl, 2mM
MgCl 2, pH8.0) in, and the freezing-thawing and cracking recycled by 3 obtains cell lysate A, and cell lysate A is passed through 2500g
It is centrifuged clarification in 30 minutes and obtains cell lysate B, by the benzonase (Sigma- of cell lysate B and 100U/ml
Aldrich it) is incubated 1 hour at 37 DEG C and obtains cell lysate C, by the 40%PEG8000 (Sigma- containing 2.5N NaCl
Aldrich storage solutions) are added in cell lysate extremely final concentration of 8%, then incubate solution on ice 2 hours,
With 2500g centrifugation 30 minutes, and it will be deposited in the phosphate buffered saline (PBS) containing 1mM CaCl 2 and 2mM KCl (PBS) and divide
From；
Step 5: purified virus carrier integrity verification is carried out；
Step 6: the gland relevant viral vector for taking 2 μ l to construct slowly is injected to SD rat DRG by micro syringe, injected
Syringe needle is slowly withdrawn from after finishing 5min, DRG embedded section is passed through using GFP antibody and exempted from by animal euthanasia within 5 weeks after virus transfection
The cell-specific of epidemic disease fluorescence analysis EGFP expression；
Step 7: EGFP is in around the Primary Sensory Neuron cell space that Tubb3 is marked after AAV6-GFAP-EGFP Successful transfection
Ring-type expression, wherein EGFP signal is located at except the neuronal cell of the Tubb3 positive and the neuron plasma membrane of the label of NKA α 1, with
GFAP dyes height common location；
Step 8: the ratio that SGC transduces after assessment vector injection examines the annular SGC row immunohistochemistry around neuron
It surveys: observing the annular SGC of the EGFP positive around the DRG of injection of AAV 6-GFAP-EGFP, the neuron for having 70% ± 8%,
And only minority pericaryon EGFP is positive (~1%), the viral vectors for obtaining this project building can be specific
The result expressed in SGCs.
2. according to claim 1 target transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method,
It is characterized in that, pAAV-GFAP-EGFP expression plasmid contains the repetition of self-complementary (sc) AAV2 opposing end in the step 1
Sequence (ITR), and transcribed by the EGFP that GFAP promoter drives chimeric interon to enhance downstream, the Rep2/Cap6
(pRC6) and pRC8 (Viromics, Fremont, CA, USA) respectively containing from AAV6 or AAV8 AAV2 duplication (rep) and
Capsid (cap) protein gene；The pHelper (Viromics) of the encoding adenovirus auxiliary gene.
3. according to claim 1 target transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method,
It is characterized in that, in the step 2 Optiprep purify AAV method the following steps are included:
S1: 40ml Quick-Seal centrifuge tube row Optiprep gradient centrifugation (Beckman Instruments, Palo are used
Alto, CA, USA), gradient is respectively 10ml, and 15%；8ml, 25%；6ml, 40%, 2ml, 60%；
S2: 15 milliliters of AAV viruses are subjected to cracking gradient centrifugation, in 70Ti rotor (Beckman at 70 DEG C
Instruments it is centrifuged 70 minutes in) with 18 DEG C, is mentioned with No. 18 syringe needles from 1/2 part of lower layer between 40-60% with 40%
Take virus；
S3: Centricon Plus-20 (Regenerated Cellulose 100,000MWCO, Millipore, Bill- are used
Erica, MA) viral suspension obtained is concentrated, and final virus formulation is maintained at containing 5% D-sorbite (Sigma-
Aldrich it in PBS), and is stored at -80 DEG C；
S4: the vector gene relative to standard plasmid is measured with PicoGreen method (Invitrogen, Carlsbad, CA, USA)
Group, and the dotted trace of row also after the denaturation of AAV carrier, are primer row PCR detection using ampicillin resistance gene, do not send out
AAV plasmid pollution in the AAV vector gene group now purified, calculates virus titer, and unit is the genome copy numbers of every ml
(GC/ml), the titre of AAV6-EGFP and AAV8-EGFP carrier is respectively 2.161013GC/ml and 2.561013GC/ml.
4. according to claim 1 target transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method,
It is characterized in that, in the step 5 carry out purified virus carrier integrity verification method the following steps are included:
B1: the purity of carrier is assessed by sodium dodecyl sulfate-polypropylene acrylamide gel (SDS-PAGE) electrophoresis first, then
Silver stain is carried out using Pierce Silver stain kit (Fisher Scientific, Rockford, IL)；
B2: the anti-Vp1 of mouse, (the 1:1000 dilution of -2, -3 antibody are used；Research Diagnostic Inc.) exempted from by standard
Epidemic disease western blot procedure confirms viral capsid proteins；
B3: with NIH ImageJ software (http://rsbweb.nih.gov/ij/) quantitative Vp1:Vp2:Vp3 albumen ratio and
Relative to the purity of non-carrier albumen visible on gel, negative staining and electron microscope (EM) scanning are carried out to assess purifying
The granule content of AAV carrier；
B4: with 1%Alcian blue (Electron Microscopy Sciences) pretreatment copper mesh, (400 holes are coated with poly-vinegar
Acid methyl ethylene rouge and thin carbon film；Electron Microscopy Sciences, Hatfield, PA) and load 5ml virus load
Body washs grid, is dyed with 1% uranyl acetate (Electron Microscopy Sciences), and with 2100 He of JEOL
600 transmission electron microscope observation of Hitachi；
B5: particle fraction is determined by directly counting electron micrograph.
5. according to claim 1 target transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method,
It is characterized in that, the batch of AAV virus formulation described in this experiment is same batch.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910183537.9A CN109706176A (en) | 2019-03-12 | 2019-03-12 | Transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method can be targeted |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910183537.9A CN109706176A (en) | 2019-03-12 | 2019-03-12 | Transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method can be targeted |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109706176A true CN109706176A (en) | 2019-05-03 |
Family
ID=66266786
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910183537.9A Pending CN109706176A (en) | 2019-03-12 | 2019-03-12 | Transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method can be targeted |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109706176A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134181A (en) * | 2021-03-02 | 2022-03-04 | 河南大学 | Expression unit, recombinant lentivirus expression vector, recombinant lentivirus, preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101883859A (en) * | 2007-10-05 | 2010-11-10 | 吉尼松公司 | Use peripheral injection of AAV vectors to carry out extensive gene delivery to motor neuron |
| CN108330147A (en) * | 2017-01-20 | 2018-07-27 | 上海吉凯基因化学技术有限公司 | A kind of foundation of recombined glandulae correlation viral vectors production technology |
-
2019
- 2019-03-12 CN CN201910183537.9A patent/CN109706176A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101883859A (en) * | 2007-10-05 | 2010-11-10 | 吉尼松公司 | Use peripheral injection of AAV vectors to carry out extensive gene delivery to motor neuron |
| CN108330147A (en) * | 2017-01-20 | 2018-07-27 | 上海吉凯基因化学技术有限公司 | A kind of foundation of recombined glandulae correlation viral vectors production technology |
Non-Patent Citations (2)
| Title |
|---|
| HONGFEI XIANG等: "Glial fibrillary acidic protein promoter determines transgene expression in satellite glial cells following intraganglionic adeno-associated virus delivery in adult rats", 《JOURNAL OF NEUROSCIENCE RESEARCH》 * |
| HONGWEI YU等: "Intraganglionic AAV6 Results in Efficient and Long-Term Gene Transfer to Peripheral Sensory Nervous System in Adult Rats", 《PUBLIC LIBRARY OF SCIENCE ONE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134181A (en) * | 2021-03-02 | 2022-03-04 | 河南大学 | Expression unit, recombinant lentivirus expression vector, recombinant lentivirus, preparation method and application thereof |
| CN114134181B (en) * | 2021-03-02 | 2024-01-12 | 河南大学 | Expression unit, recombinant lentivirus expression vector, recombinant lentivirus, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20240124894A1 (en) | Adeno-associated virus vector | |
| Khabou et al. | Noninvasive gene delivery to foveal cones for vision restoration | |
| AU2018200657B2 (en) | Methods of predicting ancestral virus sequences and uses thereof | |
| US10081659B2 (en) | Adeno-associated vectors for enhanced transduction and reduced immunogenicity | |
| KR102522661B1 (en) | Variant adeno-associated viruses and methods of use | |
| Khabou et al. | Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant‐7m8 | |
| CN105163764B (en) | The method and composition of dual glycan combination AAV carrier | |
| CN110520536A (en) | For passing through the method and composition of the gene transfer of vasculature | |
| TW202104592A (en) | Methods of redosing gene therapy vectors | |
| CN108137655A (en) | Escape the method and composition of the viral vectors of antibody | |
| CN107828820A (en) | For to the adeno-associated virus particle of nervous system cell quiding gene | |
| WO2009108274A2 (en) | Methods and compositions for adeno-associated virus (aav) with hi loop mutations | |
| EP2550021A2 (en) | Methods of increasing efficiency of vector penetration of target tissue | |
| JP2023055843A (en) | Method and composition for introducing targeted gene | |
| CN111601884A (en) | AAV vectors | |
| KR20210006357A (en) | Antibody-avoiding virus vector | |
| CN109706176A (en) | Transfection dorsal root ganglion satellite spongiocyte adenovirus vector construct method can be targeted | |
| CN113648432B (en) | rAAV2/Retro as delivery system for retina photoreceptor cells and application thereof in preparation of medicament for treating retina diseases | |
| WO2012109214A1 (en) | Targeted transduction of aav vectors | |
| JP2023535232A (en) | Compositions and methods for the treatment of eye diseases | |
| CN113383082A (en) | Recombinant nucleic acid comprising an alpha herpes virus promoter sequence | |
| WO2022226301A1 (en) | Chimeric heart tropic aav capsids | |
| WO2023187822A1 (en) | A system for an ocular gene therapy and a process for preparing thereof | |
| WO2023164447A1 (en) | Chimeric lung tropic aav capsids | |
| Zhang et al. | 95. Incorporation of AAV Serotype 2 Viral Protein 2 into Other AAV Serotype Capsids |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190503 |