CN105296437B - A kind of slow virus method for concentration - Google Patents
A kind of slow virus method for concentration Download PDFInfo
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- CN105296437B CN105296437B CN201510388253.5A CN201510388253A CN105296437B CN 105296437 B CN105296437 B CN 105296437B CN 201510388253 A CN201510388253 A CN 201510388253A CN 105296437 B CN105296437 B CN 105296437B
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- 241000700605 Viruses Species 0.000 title claims abstract description 58
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- 229920000058 polyacrylate Polymers 0.000 claims abstract description 34
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 230000003612 virological effect Effects 0.000 claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000011550 stock solution Substances 0.000 claims abstract description 11
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229960000935 dehydrated alcohol Drugs 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 5
- 229960004756 ethanol Drugs 0.000 claims abstract description 4
- 235000019441 ethanol Nutrition 0.000 claims abstract description 4
- 238000001179 sorption measurement Methods 0.000 claims abstract description 3
- 239000012141 concentrate Substances 0.000 claims description 2
- 229920001577 copolymer Polymers 0.000 claims description 2
- 239000000178 monomer Substances 0.000 claims description 2
- 229920001495 poly(sodium acrylate) polymer Polymers 0.000 claims description 2
- NNMHYFLPFNGQFZ-UHFFFAOYSA-M sodium polyacrylate Chemical group [Na+].[O-]C(=O)C=C NNMHYFLPFNGQFZ-UHFFFAOYSA-M 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 9
- 238000012545 processing Methods 0.000 abstract description 3
- 238000013459 approach Methods 0.000 abstract description 2
- 230000008719 thickening Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 19
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- 239000012895 dilution Substances 0.000 description 19
- 230000000694 effects Effects 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000002245 particle Substances 0.000 description 5
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 4
- 108091027967 Small hairpin RNA Proteins 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000004055 small Interfering RNA Substances 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 206010041047 Slow virus infection Diseases 0.000 description 1
- 102000001400 Tryptase Human genes 0.000 description 1
- 108060005989 Tryptase Proteins 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000001415 gene therapy Methods 0.000 description 1
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- 238000005213 imbibition Methods 0.000 description 1
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- 238000001727 in vivo Methods 0.000 description 1
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- 210000004165 myocardium Anatomy 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of slow virus method for concentration, comprising the following steps: (1) polyacrylate globule is directly placed into dehydrated alcohol and is impregnated 20-24 hours, suck 50-60 DEG C of ethyl alcohol postposition degree to complete drying；(2) the polyacrylate globule of step (1) preparation is added into virus stock solution used for 1:5-15g/mL according to the mass volume ratio of every gram of polyacrylate globule and virus stock solution used；(3) to the water 1-3h in polyacrylate globule viral adsorption stoste, polyacrylate is removed, viral concentration liquid is obtained.It is that material is concentrated that the present invention, which prepares concentration link using polyacrylate in slow virus, gets rid of the dependence to high equipment and consumptive material, the slow virus after concentration can reach or approach the requirement used substantially；Material price used in it is low, and operating process is simple, and can carry out processing while Multi-example, is applied in combination with the prior art, further increases thickening efficiency.
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of new slow virus method for concentration.
Background technique
Foreign gene or the shRNA of external source can be effectively integrated on host chromosome by slow virus technology, to reach
To the effect of persistence expression aim sequence.It is thin that neuronal cell, liver cell, cardiac muscle can be effectively infected in terms of infection ability
A plurality of types of cells such as born of the same parents, tumour cell, endothelial cell, stem cell, to reach good gene therapy effect.For
The cell of some more difficult transfections, such as primary cell, stem cell, undifferentiated cell can be greatly improved using slow virus carrier
The transduction efficiency of target gene or purpose shRNA, and target gene or purpose shRNA are integrated into the probability of host cell gene group
It greatly increases, more convenient can quickly realize that target gene or stablizing for purpose shRNA are expressed.
Equally, in the research tested in vivo, slow virus has also also showed that good channel genes effect, it is contemplated that
The coming years will be more and more widely used.
The basic process of slow virus preparation includes vector construction and purification containing target gene or interference sequence；With it is auxiliary
Plasmid co-transfection 293T cell is helped, culture collected culture supernatant after 48-72 hours；The purifying and concentration of lentiviral particle；Concentration
The packing and cryo-conservation of liquid.
In slow virus preparation process, concentration link is one of most important link, one side concentration technique and slow virus
Yield of the particle from first liquid to concentrate is related, and influences the titre index of final finished slow virus (titre is that slow virus is most closed
The technical indicator of key)；Another aspect concentration technique also influences the integrality of lentiviral particle virion during the preparation process,
And then influence the infect efficiency of virus.
Conventional slow virus concentration technique includes ultracentrifugation, ultrafiltration, PEG concentration and anion-exchange column method at present.It is super
Fast centrifugal process needs to buy expensive supercentrifuge (100,000 to hundreds of thousands member), and ultrafiltration and anion-exchange column method require to purchase
Expensive consumptive material is bought, generates very big burden for preparing more number and large volume of slow virus liquid, and PEG method is dense
The time that compression process needs is long, it is also desirable to use centrifuge (but speed is lower), to the no effect of the removal of impurity.
Summary of the invention
The purpose of the present invention is to provide a kind of fast and convenient, reagent consumptive material is cheap, without expensive equipment just
It is able to achieve the method for lentiviral particle being effectively concentrated.
The technical solution of the present invention is as follows:
A kind of slow virus method for concentration, comprising the following steps:
(1) preparation of polyacrylate globule: polyacrylate globule being directly placed into dehydrated alcohol and impregnates 20-24h,
50-60 DEG C of ethyl alcohol postposition degree is sucked to complete drying；Soaking time preferably for 24 hours, preferably 50 DEG C of drying temperature；
(2) virus stock solution used is concentrated: being 1:5- according to the mass volume ratio of every gram of polyacrylate globule and virus stock solution used
The polyacrylate globule of step (1) preparation is added into virus stock solution used by 15g/mL；The preferred 1:10g/mL of mass volume ratio；
(3) polyacrylate is removed, is obtained to the water 1-3h in polyacrylate globule viral adsorption stoste, preferably 2h
Viral concentration liquid.
It can also include step (4) to be further concentrated: step (3) viral concentration liquid super filter tube being taken to be centrifuged 20-
30min2-3 times.
Polyacrylate is the high-molecular compound of crosslinking, is in spacial framework, by between chemical crosslinking and macromolecular chain
Mutually wind physical crosslinking constitute.When polymer encounters water, dissociate into immediately positively charged low molecule ion and
Electronegative macroion.Low molecule ion is contacted with water and is moved, and macroion chain is had left, due to negatively charged
Mutual electric repulsion between macroion makes high score subnet beam gradually stretch (or swelling) by mutually winding state and comes, thus
Cause network structure is inside and outside to generate osmotic pressure, hydrone is spread with penetration mode to network structure, forms colloidal sol.Therefore polypropylene
Hydrochlorate has very powerful water imbibition.It is the new skill that material carries out slow virus concentration that the present invention, which creatively uses polyacrylate,
Art, and achieve the effect of good concentrating virus particles.
Compared with the prior art, beneficial effects of the present invention are as follows:
1. the present invention slow virus preparation important link-concentration link innovatively use polyacrylate be material into
Row concentration, gets rid of the dependence to high equipment and consumptive material, and the slow virus after concentration can reach or approach wanting of using substantially
It asks, can be applied to different types of cellular invasion；
2. material price used in the present invention is low, operating process is simple, and can carry out processing while Multi-example, is not set
The limitation of standby processing capacity；
3. this method can be applied in combination with the prior art, thickening efficiency is further increased, is had a extensive future.
Specific embodiment
Embodiment:
The polyacrylate that the present embodiment uses is Sodium Polyacrylate cross-linked copolymer, has following physicochemical property:
| Appearance | Circular granular solid |
| Diameter | 2-2.5mm |
| Water content | < 10% |
| Water retention | > 97% |
| Bulk density | 0.8-0.85g/mL |
| PH value | 6.5-7.5 |
| Residual monomer | <200ppm |
1. cell inoculation: by collected by trypsinisation 293T cell, with the complete medium of DMEM+10% fetal calf serum with
2-3×106Cell number is inoculated on each 100mm culture dish (Corning company).Cell is placed in containing 5%CO237 DEG C of temperature
It is incubated for 12-16 hours in case, transfection changes 10mL fresh medium into first 2 hours.
2. preparing calcium phospate-DNA precipitate: by taking 100mm culture dish 1mL reacts total volume as an example.It is added in sterile water
Resulting slow virus plasmid and helper plasmid (25 μ g of total amount is preferred), add 62 μ l 2M CaCl2, reach three's total volume
500 μ l are mixed.Isometric 2 × HBS salting liquid is added dropwise, while flicking tube wall, makes to mix in time after being often added dropwise to.It is quiet
After setting 12 minutes, the calcium phosphate-DNA suspension of this 1mL is added dropwise in the cell culture medium of above-mentioned cell monolayer immediately, gently
Jog moves plate and mixes.
3. changing liquid within 5-6 hours after transfection.Change the complete medium of DMEM+10% fetal calf serum into.37 DEG C of CO again2Incubator
Middle incubation harvested slow virus supernatant after 48 hours.
4. the concentration of slow virus supernatant:
(1) preparation of polyacrylate globule: polyacrylate globule being directly placed into dehydrated alcohol and is impregnated 24 hours,
It sucks in 50 degree of baking ovens of ethyl alcohol postposition 24 hours, until drying completely.
(2) amount for needing to be added is calculated according to the amount that reduce volume, can be adsorbed according to every gram of polyacrylate globule
10mL moisture calculates dosage, and polyacrylate globule is added into virus stock solution used.
(3) after adsorbing 2 hours, polyacrylate globule is removed, liquid is taken out, carry out titer determination (see following steps
5)
It (4), can be on the basis of step 4 (3), in conjunction with answering for super filter tube for the viral sample for needing further to be concentrated
With, virus liquid is further concentrated to 10 times or so, then can obtain the concentrated effect of 100 times or so of total, specific as follows:
The super filter tube (100KD) of Millipore is taken, the virus liquid 3mL, 4000g being concentrated with 4 (3) steps is added and is centrifuged 20-
30 minutes, taking-up saw volume-diminished to how many, and general centrifugation 2-3 times can be by volume concentration to 300ul or so.
5. slow virus liquid titer determination: by HEK293 cell culture to logarithmic growth phase, viral dilution culture solution is containing 8
The cell culture medium of μ g/mL polybrene and 2% fetal calf serum.First day, after cell tryptase enzymic digestion counts, according to every hole
8000 cell inoculation, 96 orifice plate, 37 DEG C of overnight incubations, the cell long fusion density to 30-50% when infection；Second day, the same day
When transfection, by the virus liquid ice-water bath being stored in -80 DEG C of refrigerators melt, with contain 8 μ g/mL polybrene and 2% tire ox
The cell culture fluid of serum carries out gradient dilution, and each dilution is provided that
1.+225 μ l viral dilution culture medium of 25 μ l virus liquid of number dilution,
2.+225 μ l viral dilution culture medium of No. 1 dilution of 25 μ l of number dilution,
3.+225 μ l viral dilution culture medium of No. 2 dilutions of 25 μ l of number dilution,
4.+225 μ l viral dilution culture medium of No. 3 dilutions of 25 μ l of number dilution,
5.+225 μ l viral dilution culture medium of No. 4 dilutions of 25 μ l of number dilution,
……
The culture medium in 96 orifice plates is carefully sucked, each pipe slow virus dilution is mixed gently, respectively takes 100 μ l that every hole is added thin
In born of the same parents, two repetitions of each dilution are put into 37 DEG C of cell incubator and are incubated overnight；Third day is removed containing slow virus
The complete medium of 100 μ l is added in culture medium；Five, the six days, the fluorecyte quantity in fluorescence microscopy under the microscope each hole,
Virus titer is to express the cell number of fluorescence multiplied by corresponding extension rate.
6. slow-virus infection aim cell: selecting a kind of cell (such as Hela cell) kind 6 orifice plates on the day before virus infection, plant plate
Amount: 2-3.0 × 105Cells/well；The same day is infected, is previously added polybrene (final concentration 8 in used medium when virus infection
μg/mL)；Fresh configuration culture medium: the hole 1mL/ is changed in 6 orifice plates, with MOI=5, slow virus liquid is added in 10,20 gradients, after mixing
It is placed in cell incubator；4 hours or so the fresh culture mediums without polybrene of change after virus is added, 37 DEG C, 5%CO2's
Continue to cultivate in incubator.48-96 hours observation cell growth status, as carried out target gene or interference effect detection, 48
Hour collects cell sample, carries out qPCR detection.
7. determination of recovery rates: slow virus liquid is concentrated using method of the invention, virus liquid can be concentrated 10 at 2 hours or so
Times or so, the rate of recovery calculates=(volume after titre * recycling after recycling)/(stoste titre * stoste volume) * 100%, actually measured
The rate of recovery is close with classical high speed centrifugation method in 70-80%.
The concentration of slow virus liquid is carried out using polyacrylate globule, method is simple, it is only necessary to add to virus liquid according to volume
Enter appropriate polyacrylate globule, virus liquid can be made to be concentrated 10 times or so, reach 1*108TU/mL, this titre can meet greatly
Needed for multiple infection cell.The rate of recovery is close with classical supercentrifugal process in 70-80% or so, without use high speed from
Scheming.If necessary to the higher slow virus liquid of titre, then polyacrylate globule absorption method and ultrafiltration can be combined, energy
Virus liquid titre is set to reach 5*108Or 1*109TU/mL or so, wherein centrifuge only needs low speed, and the short time can reach effect,
It is centrifuged without high speed, long-time.
Claims (5)
1. a kind of slow virus method for concentration, which comprises the following steps:
(1) preparation of polyacrylate globule: polyacrylate globule is put into dehydrated alcohol and impregnates 18-24h, sucks ethyl alcohol
50-60 DEG C of postposition dries to complete；The polyacrylate globule is Sodium Polyacrylate cross-linked copolymer, is had following physical and chemical special
Property: appearance: circular granular solid；Diameter: 2-2.5mm；Water content < 10%；Water retention > 97%；Bulk density: 0.8-0.85g/
mL；PH value 6.5-7.5；Residual monomer < 200ppm；
(2) virus stock solution used is concentrated: being 1:5-15g/mL according to the mass volume ratio of every gram of polyacrylate globule and virus stock solution used
The polyacrylate globule of step (1) preparation is added into virus stock solution used；
(3) to the water 1-3h in polyacrylate globule viral adsorption stoste, polyacrylate globule is removed, viral concentration is obtained
Liquid.
2. slow virus method for concentration according to claim 1, which is characterized in that polyacrylate globule exists in step (1)
The time impregnated in dehydrated alcohol is 20-24h, and drying temperature is 50-55 DEG C.
3. slow virus method for concentration according to claim 1, which is characterized in that in step (2) polyacrylate globule with
The mass volume ratio of virus stock solution used is 1:8-10g/mL.
4. slow virus method for concentration according to claim 1, which is characterized in that polyacrylate globule is inhaled in step (3)
The time of water in attached virus stock solution used is 1.5-2.5h.
5. slow virus method for concentration according to claim 1, which is characterized in that further include step (4): taking step (3) sick
Malicious concentrate with super filter tube be centrifuged 20-30min, 2-3 times.
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| CN112048523A (en) * | 2019-06-05 | 2020-12-08 | 南京艾德免疫治疗研究院有限公司 | Method for preparing high-titer lentiviral vector by conventional centrifugation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892327A (en) * | 2010-07-20 | 2010-11-24 | 复旦大学 | A Method for Concentrating Viruses in Large Volume Water Samples |
| CN103242468A (en) * | 2013-04-15 | 2013-08-14 | 北京恒聚化工集团有限责任公司 | Beaded sodium polyacrylate and preparation method thereof |
-
2015
- 2015-07-03 CN CN201510388253.5A patent/CN105296437B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892327A (en) * | 2010-07-20 | 2010-11-24 | 复旦大学 | A Method for Concentrating Viruses in Large Volume Water Samples |
| CN103242468A (en) * | 2013-04-15 | 2013-08-14 | 北京恒聚化工集团有限责任公司 | Beaded sodium polyacrylate and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 聚丙烯酸水凝胶在蛋白质浓缩分离中的应用;吕志芳等;《应用化学》;20091031;第212-215页，参见摘要、第212页第1段及第213页第5段 |
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