CN109288875A - A kind of oncolytic virus preparation and preparation method thereof being injected intravenously - Google Patents
A kind of oncolytic virus preparation and preparation method thereof being injected intravenously Download PDFInfo
- Publication number
- CN109288875A CN109288875A CN201811026737.5A CN201811026737A CN109288875A CN 109288875 A CN109288875 A CN 109288875A CN 201811026737 A CN201811026737 A CN 201811026737A CN 109288875 A CN109288875 A CN 109288875A
- Authority
- CN
- China
- Prior art keywords
- cell
- oncolytic virus
- membrane vesicles
- cell membrane
- preparation
- 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.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
- A61K35/761—Adenovirus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to the oncolytic virus preparations and preparation method thereof that one kind can be injected intravenously, the present invention is by target polypeptide gene transfecting eukaryotic cells, and target polypeptide is shown on cell membrane, after obtaining the genetic engineering engineered cells strain for stablizing expression target polypeptide, the nanoscale cell membrane vesicle of size uniformity is extracted using the cell strain, oncolytic virus (OV) is loaded into engineered Cell membrane vesicles again, synthesis can be injected intravenously while the oncolytic virus complex of target tumor delivering.Oncolytic virus is loaded in inside Cell membrane vesicles by the present invention, it can be effectively reduced neutralization of the neutralizing antibody to oncolytic virus in body, Cell membrane vesicles have targeting after genetic engineering is transformed simultaneously, it can be realized the targeted delivery of oncolytic virus and the enrichment in tumor locus, while reducing the systemic side effects of oncolytic virus.
Description
Technical field
The present invention relates to the preparation methods for the oncolytic virus vaccine that one kind can be injected intravenously.
Background technique
Oncolytic viral therapy is a kind of ideal treatment method for the treatment of cancer, is capable of the thin in tumour of selectivity using virus
Born of the same parents replicate the characteristic of amplification, and oncolytic virus vaccine all achieves good therapeutic effect in multinomial preclinical and clinical test.
Recently, U.S. FDA has approved the oncolytic virus vaccine (T-VEC) of Amgen for skin and lymph node melanoma lesion
Treatment.In addition, also reporting the feelings for using oncolytic virus in localized cancer gene therapy in several human clinical trial's reports
Condition.However, enorganic antiviral immunity and elder generation can be caused when oncolytic virus is directly entered the blood circulation system of body
Its immune response and oncolytic virus are easy that the factors such as non-specific isolation occur in histoorgan, so that traditional oncolytic virus
Vaccine can not enter body by way of intravenous injection, this constitutes great obstacle to the development of cancer virus therapy.Cause
This, there is an urgent need to develop completely new oncolytic virus delivery system to solve the above problems.
Summary of the invention
The main object of the present invention is to provide a kind of preparation method of oncolytic virus complex that can be injected intravenously,
Include the following steps: the polypeptide gene transfecting eukaryotic cells of targets neoplastic cells, and the corresponding targeting of the polypeptide gene is more
Peptide is shown on the cell membrane of eukaryocyte, obtains the genetic engineering engineered cells strain for stablizing expression target polypeptide；Then it utilizes
The cell strain extracts the nanoscale cell membrane vesicle of size uniformity, then oncolytic virus is loaded into the nanoscale cell membrane vesicle
In, obtain the oncolytic virus complex that can be injected intravenously.
Preferably, polypeptide gene includes at least one of PreS1, GPC3, HA, VEGF, PD-1/PD-L1, Trail.
Preferably, the method for polypeptide gene transfecting eukaryotic cells includes electric robin, lipofection, PEI transfection, phosphoric acid
At least one of calcium infection protocol, slow virus or Adenovirus Transfection method.
Preferably, the type of oncolytic virus includes oncolytic adenovirus, Coxsackie virus, herpes simplex virus (HSV), morbilli
At least one of virus, Newcastle virus, poliovirus, parvovirus, reovirus, retrovirus.
It preferably, include liposome extrusion, electric robin, ultrasound by the method that oncolytic virus is loaded into Cell membrane vesicles
At least one of method, freeze-thaw method, saponification method.
Preferably, eukaryocyte, including mdck cell, VERO cell, S2 cell, insect cell, embryonic stem cell, patient
At least one of cell being separately cultured self.
Preferably, the preparation method of the oncolytic virus vaccine of the intravenous injection, comprising the following steps:
1) method transfected by gene, target polypeptide gene is transferred in eukaryocyte, and passes through target polypeptide gene
In signal peptide will target polypeptide expression on cytoplasma membrane；
2) after transfection for 24 hours, using the G418 of 500~550 μ g/ml, the eukaryocyte of transfection is screened, every 3~5 days
Replace primary screening culture medium；Screening, until resistant clone occurs, culture of being discontinued is gradually increased wait clone；
3) cell is inoculated into Tissue Culture Dish by limiting dilution assay after pancreatin digestion by the cell of picked clones cluster
In, the G418 that 200~250 μ g/ml are added maintains to screen to cell；
4) after monoclonal cell increase, expanded culture, and the limiting dilution assay for repeating step (3) further screens
Stable expression cell strain, the screening through excessive generation obtain the cell strain that can stablize expression target polypeptide；
5) it takes stable cell strain to expand culture, selects the cell of growth logarithmic phase and add after PBS is washed 3~5 times
Enter the hypotonic buffer liquid containing PMSF protease inhibitors, abundant infiltrating cells；
6) cell is collected, cell, which fullys shake, keeps its evenly dispersed, and cell suspension is placed in rotate at 1-5 DEG C and is mixed
It is outstanding, it ruptures cell sufficiently and forms a large amount of Cell membrane vesicles；
7) cell suspension is centrifuged 5~15min at 500~800 × g, supernatant is transferred in new centrifuge tube, 5000
It is centrifuged 5~15min under~8000 × g, supernatant finally moves on to new centrifuge tube, centrifugation 50 under 50000~80000 × g~
70min removes supernatant, and membrane pellet is resuspended with 400~600ul buffer, obtains Cell membrane vesicles, -80 DEG C save backup；
8) oncolytic virus is uniformly mixed with Cell membrane vesicles, mixed liquor is with liposome squeezer in the thin of the aperture 400nm
Film pressure back to squeeze of getting off is no less than 15 bouts, and then changes the aperture 400nm film into the aperture 200nm film, and continuation squeezes not back and forth
Less than 15 bouts, until oncolytic virus particle is loaded into the cavity of Cell membrane vesicles, the cell membrane vesicle of size uniformity is formed
Bubble package oncolytic virus, -80 DEG C save backup；
Preferably, in step 8), oncolytic virus and Cell membrane vesicles mixed proportion are volume ratio 1:0.1~10.
It is a further object of the present invention to provide a kind of oncolytic virus preparations comprising the oncolytic virus that can be injected intravenously
Complex, the oncolytic virus complex are made according to preparation method above-mentioned.
It is a further object of the present invention to provide the oncolytic virus preparations, in the purposes of the therapeutic agent as solid tumor.
Beneficial effects of the present invention:
The invention proposes a kind of novel oncolytic virus vaccine delivery system, the present invention passes through genetic engineering means for target
To peptide modified on cytoplasma membrane, then Cell membrane vesicles are extracted as oncolytic virus using the cell strain for stablizing expression target polypeptide
Delivery vector.And successfully oncolytic virus is loaded into the cavity of Cell membrane vesicles, form the Cell membrane vesicles of size uniformity
Oncolytic virus complex.Traditional oncolytic virus, which is loaded in Cell membrane vesicles, can be effective against body for the anti-of oncolytic virus
The neutralization of virus immunity reaction and neutralizing antibody extends oncolytic virus in the circulation time of blood circulation system, and passes through
Target polypeptide enables oncolytic virus to realize enrichment in tumor locus, improves the therapeutic effect of virus therapy.
1. provide a kind of novel oncolytic viral delivery systems, traditional oncolytic virus granule packaging in Cell membrane vesicles,
The neutralization of enorganic antiviral immunity reaction and neutralizing antibody is effectively reduced, protects oncolytic virus not by immunity of organism
System is removed；
2. Cell membrane vesicles are biological sources, immunogenicity is extremely low, can carry oncolytic virus in body blood cyclic system
The borough chief that unites imitates circulation；
3. the Cell membrane vesicles of genetic engineering transformation have targeting, it is efficiently rich in tumor locus that oncolytic virus can be carried
Collection, reduces the systemic side effects of oncolytic virus；
4. the mode that novel oncolytic viral vaccine can be injected intravenously is administered, overcoming traditional oncolytic virus can only lead to
The deficiency of locally injecting administration mode is crossed, there is good universality；
5. novel oncolytic viral vaccine can (such as immunologic test point PD-1/PD-L1 inhibits with other cancer treatment methods
Agent) it is used in combination, there is huge application prospect.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1: the oncolytic that Cell membrane vesicles (a), oncolytic virus particle (b) and the Cell membrane vesicles of genetic engineering transformation wrap up
The size and pattern (c) of virion, TEM；
Fig. 2: cell in vitro activity experiment detects the oncolytic virus particle of Cell membrane vesicles package to kinds of tumor cells system
Lethal effect；
Fig. 3: external Competitive assays testing inspection cell membrane package oncolytic virus helps oncolytic virus to resist neutralizing antibody
Neutralization；
Fig. 4: tumor bearing nude mice fluorescence imaging detection cell membrane package oncolytic virus particle helps oncolytic virus to be effectively enriched to
Tumor locus；
Fig. 5: with neoplasm growth effect in mouse original position liver cancer model inspection Cell membrane vesicles package oncolytic virus body；
Specific embodiment
Preparation example: the used reagent in following synthesis step is commercial goods.
The oncolytic adenovirus epidemic disease of the Cell membrane vesicles package of target polypeptide hepatitis B virus envelope pre-s1 protein (preS1) modification
Seedling preparation method, comprising the following steps:
1. the preparation of the Cell membrane vesicles of target polypeptide PreS1 modification:
1) before transfection for 24 hours, with the HepG2 cell of trypsin digestion logarithmic growth phase, to contain the culture medium of 10% serum
Adjustment cell density is every milliliter of 5x 105A cell is reinoculated on 10cm Tissue Culture Dish, 37 DEG C, 5%CO2In incubator
Culture.It can be used to transfect when cell density is up to 70%~80%, cell culture medium be changed to free serum culture before transfection
Base；
2) the eukaryon expression plasmid containing PreS1 gene and commercialization transfection reagent Lipofectamine diluted
2000 (match is silent to fly) are mixed with the ratio of mass/volume ratio 2:1, are stored at room temperature 20 minutes, said mixture is added dropwise
Enter into HepG2 cell culture medium, mix gently, cell is placed in 37 DEG C, 5%CO2Cell incubator in cultivate；
3) after transfection for 24 hours, using the G418 (Geneticin, Geneticin) of 500 μ g/ml, to the eukaryocyte of transfection into
Row screening, every 3~5 days replacement primary screening culture mediums.It can get resistant clone after screening 2 weeks to occur, culture of being discontinued, to
Clone is gradually increased；
4) after pancreatin digests, it is thin by limiting dilution assay to be inoculated into 96 holes by the cell at careful place of picked clones group for cell
In born of the same parents' culture dish, the G418 that 250 μ g/ml are added maintains to screen to cell；
5) after the monoclonal cell of 96 orifice plates increases, is expanded culture, and repeatedly the limiting dilution assay of step (3) into
One step screens stable expression cell strain, and the cell strain that can stablize expression target polypeptide PreS1 is obtained by the screening in 5 generations
HepG2-PreS1；
6) stable cell strain HepG2-PreS1 is taken to expand culture, select growth logarithmic phase cell, PBS washing 3~
After 5 times, suitable 10mM Tris and Mg2+ hypotonic buffer containing PMSF (phenylmethylsulfonyl fluoride) protease inhibitors is added
Liquid, abundant infiltrating cells；
7) cell is collected, cell, which fullys shake, keeps its evenly dispersed, and cell suspension is placed at 4 DEG C and rotates suspension
For 24 hours, it ruptures cell sufficiently and forms a large amount of Cell membrane vesicles；
8) cell suspension is centrifuged 10min at 700 × g, supernatant is transferred in new centrifuge tube, under 7000 × g from
Supernatant is finally moved on to new centrifuge tube by heart 10min, is centrifuged 60min under 70000 × g, removes supernatant, and membrane pellet is with 400
~600ul phosphate buffer (PBS, pH=7.4) is resuspended, and obtains Cell membrane vesicles, -80 DEG C save backup；
2. the preparation of oncolytic adenovirus:
1) pDC315-hTERTp-3E-E1A adenoviral plasmid constructs: a, replacing telomerase promoter (hTERTp)
Promoter CMV in AdMaxTM recombined adhenovirus system in shuttle plasmid pDC315, constructs pDC315-hTERTp-3E matter
Grain；B, using adenovirus E 1 A cDNA segment as template, EcoRI, BamHI restriction enzyme site, PCR amplification E1A are introduced in primer；c,
Plasmid pDC315-hTERTp-3E EcoRI+BamHI recycling large fragment is attached with e1a gene segment, construct shuttle
Plasmid pDC315-hTERTp-3E-E1A；
2) by shuttle plasmid pDC315-hTERTp-3E-E1A and skeleton plasmid pBGHfrt Δ E1,3FLp with mass ratio 1:2
Ratio cotransfection HEK293 cell；
3) it after transfecting, changes a subculture within every 2 days, cell state is observed continuously；
4) after transfecting 1 week, it can be seen that cell plaque occurs, and when cytopathy to when almost falling off fastly from culture dish, collects
Cell and culture medium supernatant；
5) operating procedure provided according to Adenovirus Purification kit Vivapure AdenoPACK 20RT is to oncolytic adenopathy
Poison is purified, and oncolytic adenovirus after purification is saved backup in -80 DEG C；
3. by oncolytic adenovirus (about 1 × 1011PfU it) is uniformly mixed in equal volume with Cell membrane vesicles, mixed liquor liposome
Squeezer pressure no less than 15 bouts back to squeeze under the film in the aperture 400nm, and then change the aperture 400nm film into 200nm hole
Diameter film continues to squeeze no less than 15 bouts back and forth, until oncolytic adenovirus particle is loaded into the cavity of Cell membrane vesicles,
The Cell membrane vesicles for forming size uniformity wrap up oncolytic adenovirus, and -80 DEG C save backup.
Two, embodiment:
1. the oncolytic adenovirus Morphology of Virions characterization of Cell membrane vesicles package:
Cell membrane vesicles, oncolytic virus particle and the Cell membrane vesicles that genetic engineering is transformed using transmission electron microscope (TEM)
The size and pattern of the oncolytic virus particle of package are characterized respectively.As shown in Figure 1, empty Cell membrane vesicles partial size is 100
~200nm range (a), exposed oncolytic adenovirus grain diameter is at 70~90nm or so (b), and cell membrane wraps up oncolytic adenopathy
Whole particle size range is expanded after poison, at 100~500nm or so (c).
2. the outer evaluation to tumor cell killing potential of the oncolytic virus granule of Cell membrane vesicles package:
To the killing ability of tumour cell outside oncolytic virus granule in order to verify cell membrane package, by oncolytic adenovirus
Normal liver cell LO2 is added to the concentration of MOI=2, in liver cancer cells (HepG2, Huh7 and Hepa 1-6), is being infected respectively
For 24 hours, when 48h and 72h, with the activity of mtt assay detection cell.As shown in Figure 2, the results showed that the novel oncolytic virion of preparation
Normal liver cell is had little effect, and liver cancer cells (HepG2, Huh7 and Hepa 1-6) cell activity after infection for 24 hours
It begins to decline, illustrates that oncolytic virus starts duplication in liver cancer cells and expands and cause cell cracking dead.The cancer after infecting 72h
The activity of cell drops to 50% or so.The result shows that novel oncolytic viral vaccine prepared by the present invention to normal cell almost
There is no fragmentation effect, and good killing ability is shown to cancer cell.
3. cell membrane wraps up the evaluation that oncolytic adenovirus helps oncolytic virus to resist neutralizing antibody neutralization ability:
In order to evaluate oncolytic virus is resisted neutralizing antibody neutralization ability after cell membrane package, first by oncolytic gland
Virus mouse be immunized three times (it is be immunized weekly primary, every time 1 × 108PfU, continuous immunity three weeks), mouse blood is acquired,
Separation obtains the neutralizing antibody serum of oncolytic adenovirus.With cell culture medium (DMEM) antagonistic Serum (nAb) continuous two-fold dilution,
The antiserum of different dilutions is separately added into cell, then with 1 × 104The Cell membrane vesicles package of pfU has green
The sero-fast cell of adenovirus infection difference dilution of fluorescin GFP gene, exposed adenovirus (having GFP gene) are made
To compare, after 8h, the infection conditions of fluorescence microscopy microscopic observation cell.As shown in figure 3, the adenovirus of Cell membrane vesicles package exists
In the case where antiserum very high (dilution 1:2), it still is able to keep certain infection ability, and control group is diluted in antiserum
Just almost without infection ability in the case where spending for 1:32.As a result after illustrating Cell membrane vesicles package adenovirus well,
Adenovirus can be effectively helped to resist the neutralization of neutralizing antibody.
4. the evaluation that cell membrane wraps up targeted delivery effect in oncolytic virus granule:
In order to evaluate the internal targeted delivery effect of cell membrane package oncolytic virus, by 1 × 105Express targeting proteins
The HepG2 cell inoculation of the receptor of PreS1 grows to a certain size to tumour on nude mice, the Cell membrane vesicles that PreS1 is modified
In the adenovirus Ad5-GFP tail vein injection to above-mentioned nude mouse of package, per injection 1 × 108PfU injects a needle every other day, altogether
3 needles are injected, exposed Ad5-GFP is control group.Second day after third needle, fluorescence imaging is carried out to all nude mices, such as
Shown in Fig. 4, the adenovirus Ad5-GFP of the Cell membrane vesicles package of PreS1 modification can be effectively enriched in tumor locus, and exposed
Adenovirus Ad5-GFP fallen quickly by organism metabolism.
5. the evaluation that Cell membrane vesicles wrap up neoplasm growth effect in oncolytic virus body:
By 1 × 105Liver of the liver cancer cells Hepa1-6 in-situ inoculating with luciferase reporter group to C57 mouse
Dirty position, be inoculated with liver cancer cells after third day, respectively to mouse tail vein injection cell membrane package oncolytic adenovirus (1 ×
108PfU), exposed oncolytic adenovirus (1 × 108PfU), empty Cell membrane vesicles (1 × 108) and physiological saline (100 μ), every other day
Injection is primary, co-injection 4 times, carries out living body fluorescent imaging to corresponding mouse at the 4th, 6,8,10 and 14 day respectively.Such as Fig. 5 institute
Show, the oncolytic adenovirus of Cell membrane vesicles package prepared by the present invention can effectively inhibit the growth of tumour cell, at the 14th day
When tumour cell disappear substantially；The mouse of exposed oncolytic adenovirus group, ghost membrane vesicle group and PBS group, it is intracorporal swollen
Tumor is increasing, inhibits tumor growth effect poor.
Claims (9)
1. the preparation method of the oncolytic virus vaccine of intravenous injection, comprising the following steps:
1) method transfected by gene, target polypeptide gene is transferred in eukaryocyte, and by target polypeptide gene
Signal peptide expresses target polypeptide onto cytoplasma membrane；
2) after transfection for 24 hours, using the G418 of 500~550 μ g/ml, the eukaryocyte of transfection is screened, is replaced within every 3~5 days
Primary screening culture medium；Screening, until resistant clone occurs, culture of being discontinued is gradually increased wait clone；
3) cell is inoculated into Tissue Culture Dish by limiting dilution assay, is added after pancreatin digestion by the cell of picked clones cluster
The G418 for entering 200~250 μ g/ml maintains to screen to cell；
4) after monoclonal cell increase, expanded culture, and the limiting dilution assay for repeating step (3) further screens stabilization
Expression cell strain, the screening through excessive generation obtain the cell strain that can stablize expression target polypeptide；
5) it takes stable cell strain to expand culture, selects the cell of growth logarithmic phase, after PBS is washed 3~5 times, addition contains
There are the hypotonic buffer liquid of PMSF protease inhibitors, abundant infiltrating cells；
6) cell collecting, cell, which fullys shake, keeps its evenly dispersed, and cell suspension is placed at 1-5 DEG C and rotates suspension,
It ruptures cell sufficiently and forms a large amount of Cell membrane vesicles；
7) cell suspension is centrifuged 5~15min at 500~800 × g, supernatant is transferred in new centrifuge tube, 5000~
It is centrifuged 5~15min under 8000 × g, supernatant finally moves on to new centrifuge tube, centrifugation 50 under 50000~80000 × g~
70min removes supernatant, and membrane pellet is resuspended with 400~600ul buffer, obtains Cell membrane vesicles, -80 DEG C save backup；
8) oncolytic virus is uniformly mixed with Cell membrane vesicles, mixed liquor is with liposome squeezer in the thin of the aperture 350-450nm
Film pressure back to squeeze of getting off is no less than 15 bouts, and then changes film into the aperture 150-250nm film, continues to squeeze no less than 15 back and forth
Bout forms the Cell membrane vesicles package of size uniformity until oncolytic virus particle is loaded into the cavity of Cell membrane vesicles
Oncolytic virus, -80 DEG C save backup.
2. the preparation method for the oncolytic virus complex that one kind according to claim 1 can be injected intravenously, it is characterised in that:
Target polypeptide gene described in step 1) includes at least one of PreS1, GPC3, HA, VEGF, PD-1/PD-L1, Trail.
3. the preparation method for the oncolytic virus complex that one kind according to claim 1 can be injected intravenously, it is characterised in that:
The method of target polypeptide gene transfecting eukaryotic cells described in step 1) includes electric robin, lipofection, PEI transfection, phosphorus
At least one of sour calcium infection protocol, slow virus or Adenovirus Transfection method.
4. the preparation method for the oncolytic virus complex that one kind according to claim 1 can be injected intravenously, which is characterized in that
Eukaryocyte described in step 1), including mdck cell, VERO cell, S2 cell, insect cell, embryonic stem cell, patient are certainly
At least one of the cell that body is separately cultured.
5. the preparation method for the oncolytic virus complex that one kind according to claim 1 can be injected intravenously, it is characterised in that:
The type of step 8) oncolytic virus include oncolytic adenovirus, Coxsackie virus, herpes simplex virus, measles virus, Newcastle virus,
At least one of poliovirus, parvovirus, reovirus, retrovirus.
6. the preparation method of the oncolytic virus vaccine of intravenous injection according to claim 1, comprising the following steps:
1) it before transfection for 24 hours, with the HepG2 cell of trypsin digestion logarithmic growth phase, is adjusted with the culture medium containing 10% serum
Cell density is every milliliter of 5x 105A cell is reinoculated on 10cm Tissue Culture Dish, 37 DEG C, 5%CO2Culture in incubator.
It can be used to transfect when cell density is up to 70%~80%, cell culture medium be changed to serum free medium before transfection；
The eukaryon expression plasmid containing PreS1 gene that has diluted and transfection reagent Lipofectamine 2000 with quality/
The ratio of volume ratio 2:1 is mixed, and is stored at room temperature 20 minutes, and said mixture is added dropwise to HepG2 cell culture medium
In, it mixes gently, cell is placed in 37 DEG C, 5%CO2Cell incubator in cultivate；
2) after transfection for 24 hours, using the G418 of 500~550 μ g/ml, the eukaryocyte of transfection is screened, is replaced within every 3~5 days
Primary screening culture medium；Screening, until resistant clone occurs, culture of being discontinued is gradually increased wait clone；
3) cell is inoculated into Tissue Culture Dish by limiting dilution assay, is added after pancreatin digestion by the cell of picked clones cluster
The G418 for entering 200~250 μ g/ml maintains to screen to cell；
4) after monoclonal cell increase, expanded culture, and the limiting dilution assay for repeating step (3) further screens stabilization
Expression cell strain, the screening through excessive generation obtain the cell strain that can stablize expression target polypeptide；
5) it takes stable cell strain to expand culture, selects the cell of growth logarithmic phase, after PBS is washed 3~5 times, addition contains
There are the hypotonic buffer liquid of PMSF protease inhibitors, abundant infiltrating cells；
6) cell collecting, cell, which fullys shake, keeps its evenly dispersed, and cell suspension is placed at 1-5 DEG C and rotates suspension,
It ruptures cell sufficiently and forms a large amount of Cell membrane vesicles；
7) cell suspension is centrifuged 5~15min at 500~800 × g, supernatant is transferred in new centrifuge tube, 5000~
It is centrifuged 5~15min under 8000 × g, supernatant finally moves on to new centrifuge tube, centrifugation 50 under 50000~80000 × g~
70min removes supernatant, and membrane pellet is resuspended with 400~600ul buffer, obtains Cell membrane vesicles, -80 DEG C save backup；
8) oncolytic virus is uniformly mixed with Cell membrane vesicles, mixed liquor is with liposome squeezer under the film in the aperture 400nm
It squeezes back and forth and is no less than 15 bouts, and then change the aperture 400nm film into the aperture 200nm film, continue extruding back and forth and be no less than
15 bouts form the Cell membrane vesicles packet of size uniformity until oncolytic virus particle is loaded into the cavity of Cell membrane vesicles
Oncolytic virus is wrapped up in, -80 DEG C save backup.
7. the preparation method of the oncolytic virus vaccine of intravenous injection according to claim 1 or 6, which is characterized in that step
8) in, oncolytic virus and Cell membrane vesicles mixed proportion are volume ratio 1:0.1~10.
8. a kind of oncolytic virus preparation, including the oncolytic virus complex that can be injected intravenously, the oncolytic virus complex root
It is made according to preparation method as claimed in any one of claims 1 to 6.
9. oncolytic virus preparation according to claim 8, in the purposes of the therapeutic agent as solid tumor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811026737.5A CN109288875B (en) | 2018-09-04 | 2018-09-04 | Oncolytic virus preparation capable of being injected intravenously and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811026737.5A CN109288875B (en) | 2018-09-04 | 2018-09-04 | Oncolytic virus preparation capable of being injected intravenously and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109288875A true CN109288875A (en) | 2019-02-01 |
| CN109288875B CN109288875B (en) | 2020-09-01 |
Family
ID=65166427
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811026737.5A Active CN109288875B (en) | 2018-09-04 | 2018-09-04 | Oncolytic virus preparation capable of being injected intravenously and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109288875B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109943538A (en) * | 2019-03-14 | 2019-06-28 | 北京领柯生物科技有限公司 | The recombinant virus and its modification scheme and application that a kind of tumour-specific is promoted |
| CN110215514A (en) * | 2019-04-22 | 2019-09-10 | 上海瑞可新生物科技有限公司 | A kind of genetically engineered cell film nano vesicle and its preparation and application |
| CN110433287A (en) * | 2019-08-15 | 2019-11-12 | 南方医科大学 | Virus-carrier complexes and its preparation method and application for anti-curing oncoma |
| CN112941039A (en) * | 2021-02-01 | 2021-06-11 | 南京大学 | Novel vesicular oncolytic virus and application thereof in preparation of antitumor drugs |
| CN113832111A (en) * | 2020-06-23 | 2021-12-24 | 南京大学 | Method for preparing novel oncolytic virus by exosome-like technology |
| CN113832114A (en) * | 2020-06-23 | 2021-12-24 | 南京大学 | Novel oncolytic adenovirus EM/VSV-G Ad5-P and application thereof in preparation of antitumor drugs |
| CN114269936A (en) * | 2019-05-31 | 2022-04-01 | 阿维塔斯有限公司 | Method for delivering viruses to cell populations |
| CN114404571A (en) * | 2022-01-20 | 2022-04-29 | 中山大学·深圳 | Chemotherapy drug loaded and TIGIT over-expressed engineered drug loaded cell membrane vesicle, and preparation method and application thereof |
| CN115252582A (en) * | 2022-08-19 | 2022-11-01 | 复旦大学 | Preparation and application of erythrocyte membrane heterozygosis pH liposome coated oncolytic virus preparation |
| CN115252576A (en) * | 2022-08-01 | 2022-11-01 | 沈阳药科大学 | Oncolytic virus intravenous delivery system based on engineered bacterial outer membrane vesicles and construction method and application thereof |
| EP3999116A4 (en) * | 2019-07-16 | 2023-08-16 | Coastar Therapeutics Inc. | Process of making membrane lipid coated nanoparticles |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104958324A (en) * | 2015-05-29 | 2015-10-07 | 黄波 | Oncolytic virus preparation and preparing method thereof |
| CN105148264A (en) * | 2015-09-25 | 2015-12-16 | 厦门大学 | Universal influenza vaccine and preparation method thereof |
-
2018
- 2018-09-04 CN CN201811026737.5A patent/CN109288875B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104958324A (en) * | 2015-05-29 | 2015-10-07 | 黄波 | Oncolytic virus preparation and preparing method thereof |
| US20180078508A1 (en) * | 2015-05-29 | 2018-03-22 | Hubei Soundny Biotechnology Co., Ltd. | Oncolytic virus formulation and preparation method thereof |
| CN105148264A (en) * | 2015-09-25 | 2015-12-16 | 厦门大学 | Universal influenza vaccine and preparation method thereof |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109943538B (en) * | 2019-03-14 | 2021-05-25 | 北京领柯生物科技有限公司 | Recombinant virus with improved tumor specificity and modification scheme and application thereof |
| CN109943538A (en) * | 2019-03-14 | 2019-06-28 | 北京领柯生物科技有限公司 | The recombinant virus and its modification scheme and application that a kind of tumour-specific is promoted |
| CN110215514A (en) * | 2019-04-22 | 2019-09-10 | 上海瑞可新生物科技有限公司 | A kind of genetically engineered cell film nano vesicle and its preparation and application |
| CN110215514B (en) * | 2019-04-22 | 2023-09-05 | 上海瑞可新生物科技有限公司 | Genetically engineered cell membrane nano vesicle and preparation and application thereof |
| CN114269936A (en) * | 2019-05-31 | 2022-04-01 | 阿维塔斯有限公司 | Method for delivering viruses to cell populations |
| EP3999116A4 (en) * | 2019-07-16 | 2023-08-16 | Coastar Therapeutics Inc. | Process of making membrane lipid coated nanoparticles |
| CN110433287A (en) * | 2019-08-15 | 2019-11-12 | 南方医科大学 | Virus-carrier complexes and its preparation method and application for anti-curing oncoma |
| CN113832114A (en) * | 2020-06-23 | 2021-12-24 | 南京大学 | Novel oncolytic adenovirus EM/VSV-G Ad5-P and application thereof in preparation of antitumor drugs |
| CN113832111A (en) * | 2020-06-23 | 2021-12-24 | 南京大学 | Method for preparing novel oncolytic virus by exosome-like technology |
| CN112941039A (en) * | 2021-02-01 | 2021-06-11 | 南京大学 | Novel vesicular oncolytic virus and application thereof in preparation of antitumor drugs |
| CN114404571A (en) * | 2022-01-20 | 2022-04-29 | 中山大学·深圳 | Chemotherapy drug loaded and TIGIT over-expressed engineered drug loaded cell membrane vesicle, and preparation method and application thereof |
| CN115252576A (en) * | 2022-08-01 | 2022-11-01 | 沈阳药科大学 | Oncolytic virus intravenous delivery system based on engineered bacterial outer membrane vesicles and construction method and application thereof |
| CN115252576B (en) * | 2022-08-01 | 2024-04-02 | 沈阳药科大学 | Oncolytic virus intravenous delivery system based on engineering bacterial adventitia vesicles, construction method and application thereof |
| CN115252582A (en) * | 2022-08-19 | 2022-11-01 | 复旦大学 | Preparation and application of erythrocyte membrane heterozygosis pH liposome coated oncolytic virus preparation |
| CN115252582B (en) * | 2022-08-19 | 2024-01-02 | 复旦大学 | Preparation and application of erythrocyte membrane heterozygous pH liposome coated oncolytic virus preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109288875B (en) | 2020-09-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109288875A (en) | A kind of oncolytic virus preparation and preparation method thereof being injected intravenously | |
| US6183746B1 (en) | Immunogenic peptides from the HPV E7 protein | |
| JP2019509032A (en) | A novel recombinant adeno-associated virus capsid that is resistant to existing human neutralizing antibodies | |
| CN110461368A (en) | Adeno-associated virus virion and its application method with variant capsids | |
| CN104087591A (en) | Modified factor viii and factor ix genes and vectors for gene therapy | |
| EP0922105B1 (en) | Infectious papillomavirus pseudoviral particles | |
| Haeseleer et al. | Gene transfer mediated by recombinant baculovirus into mouse eye | |
| US20210332341A1 (en) | Codon optimized gla genes and uses thereof | |
| KR20220004114A (en) | Variant AAV Capsids for Intravitreal Delivery | |
| Aboody-Guterman et al. | Green fluorescent protein as a reporter for retrovirus and helper virus-free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo | |
| Ellsworth et al. | Clade F AAVHSCs cross the blood brain barrier and transduce the central nervous system in addition to peripheral tissues following intravenous administration in nonhuman primates | |
| CN110423281A (en) | For treating the fusion protein, viral vectors and drug of senile macular degeneration | |
| JP2022505816A (en) | Miniaturized dystrophins and their use | |
| CN104136613B (en) | Carrier, method with virulent gene and application thereof | |
| WO2018161418A1 (en) | Humanized mouse model for hepatitis b constructed by using stem cells and application thereof | |
| US20220064673A1 (en) | Codon optimized rpgrorf15 genes and uses thereof | |
| CN108410893A (en) | A kind of tumour cell specific response expression vector started by NF- κ B and its expression product and application | |
| Sweet et al. | The local origin of the febrile response induced in ferrets during respiratory infection with a virulent influenza virus | |
| CN110982802B (en) | Recombinant human SGK3 protein kinase hydrogel and preparation method and application thereof in promoting myocardial regeneration | |
| CN106520834B (en) | A kind of recombinant retroviral vector and application thereof | |
| JP2022512667A (en) | KIR7.1 Gene Therapy Vector and How to Use It | |
| Hrusková et al. | Bcr-Abl fusion sequences do not induce immune responses in mice when administered in mouse polyomavirus based virus-like particles | |
| US20050256067A1 (en) | Method of preparing a treatment product, treatment product and a plasmid construct | |
| AU2021337580B2 (en) | Codon optimized REP1 genes and uses thereof | |
| CN112587530B (en) | Novel pharmaceutical application of Baricitinib |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200812 Address after: Unit 04, 8th floor, building B13, Xiamen biomedical industrial park, 2074 wengjiao West Road, Haicang District, Xiamen City, Fujian Province Applicant after: Xiamen Hong Pu Fu Biotechnology Co., Ltd Address before: Xiamen City, Fujian Province, 361000 South Siming Road No. 422 Applicant before: XIAMEN University |
|
| TA01 | Transfer of patent application right |