CN110055227A - For the wild type p53 adenovirus of hyperplastic scar treatment and the preparation method of purifying - Google Patents
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- CN110055227A CN110055227A CN201910191222.9A CN201910191222A CN110055227A CN 110055227 A CN110055227 A CN 110055227A CN 201910191222 A CN201910191222 A CN 201910191222A CN 110055227 A CN110055227 A CN 110055227A
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Abstract
The invention discloses the preparation methods of the wild type p53 adenovirus and purifying treated for hyperplastic scar, and preparation process is simple and convenient, and curative effect is splendid, the therapeutic effect of hyperplastic scar patient is improved, and cure rate is high, it is without any side effects, preparation method is simple, easy practice and extension.
Description
Technical field
The present invention relates to virus preparation and the technical fields of purifying medical skill, the more particularly, to open country of hyperplastic scar treatment
Raw type p53 adenovirus and the preparation method of purifying.
Background technique
Wound surface of burn patients healing often forms hyperplastic scar (Hypertrophic Scar, HS).HS is as a kind of tight
The fibrotic skin diseases of weight not only influence beauty, endanger the physical and mental health of patient, but also may cause serious limbs device
Official's dysfunction is one of the main problem for influencing life quality of patients with burn, therefore, is brought to family and society heavy
Burden.Although clinical at present, there are many treat HS scheme, the current special efficacy still improved without prevention cicatrization and scar
Drug, therefore, HS are the problems of clinical treatment, and pathogenesis is also the key points and difficulties of domestic and foreign scholars' research.
Recombinant P 53 adenovirus can significantly reduce fibroblasts from hyperplastic scar type i collagen, III Collagen Type VI and α-
The expression of SMA；Dramatically increase the apoptosis of fibroblasts from hypertrophic scars；Inhibit the generation of fibroblast fibrosis；It can be bright
The aobvious appearance for improving scar, improves dermal collagen fiber structure at the formation for reducing scar；Have to skin hyperplasia cicatrical fibrosisization
There is significant curative effect.In view of disadvantages described above, it is really necessary to the systems of P53 adenovirus and purifying designed for hyperplastic scar treatment
Preparation Method.
Summary of the invention
Technical problem to be solved by the present invention lies in: the wild type p53 adenovirus for hyperplastic scar treatment is provided
And the preparation method of purifying, to solve the problems, such as that background technique proposes.
In order to solve the above technical problems, the technical scheme is that the wild type p53 gland treated for hyperplastic scar
Virus and the preparation method of purifying sequentially include the following steps:
(1) material prepares: the incasing cells of cell strain 293, adenovirus, is anchorage dependence type into epithelioid cell, growth
Culture medium is DMEM (containing 10%FBS), bacterial strain, coli strain DH5 α, for expanding viral vectors and auxiliary package carrier
Plasmid will construct successful adenovirus recombinant plasmid and packaging plasmid and be extracted using the plasmid extraction kit of Qiagen company.
Resulting Plasmid DNA is dissolved in sterile TE, is measured its concentration and purity with UV Absorption method, is guaranteed proposed Plasmid DNA
A260/A280 is between 1.8~2.0；
(2) cell divides disk: the day before transfection, and the cell grown is passed in proper proportions in 6cm culture dish,
Prepare transfection when cell grows to 70%~80%；
(3) liquid is changed before transfecting: the preceding 1~2h of transfection will need the cell transfected to renew fresh culture medium；
(4) it transfects: taking sterile 1.5ml EP pipe or 15ml centrifuge tube, rotaring redyeing system according to the form below:
| DMEM | 0.5ml |
| Expression plasmid | 2μg |
| pBHG(delta)E1,3 cre | 4μg |
| HG transgene reagent | 18μl |
After mixing, after being placed at room temperature for 15min-20min, drops evenly and changed in the culture dish of liquid in advance, be placed on CO2
It is cultivated in incubator；
(5) add Enhancing buffer: after 10~12h of transfection, dropping evenly 100 × Enhancing buffer promotion
Transfection, 120 μ l/ wares；
(6) it changes liquid: after 18~20h of transfection, carefully sopping up cell culture fluid and abandon in the waste liquid cup for filling thimerosal, then
Fresh cell culture medium is added to continue to cultivate；
(7) collection virus: will observe whether virus plaque forms before collection virus, 7 days or so after infection can be micro-
Small plaque is seen under mirror, is generally formed in 10 to 21 days, collects cell and supernatant, multigelation collect virus three times, with
This virus is P1 generation virus；
(8) virus amplification: with P1 for 293 cell of adenovirus infection, being carried out continuously three generations's infection, until P4 generation carries out adenovirus
Massive amplification, virus is collected after plaque test and purification and concentration are carried out to virus；
(9) viral purification: viral purification is combined method purified virus, CsCl gradient using CsCl density gradient centrifugation-dialysis
The preparation method is as follows: be added 2.0ml density be 1.40g/ml CsCl solution, being then slowly added into 3.0ml density is
The CsCl solution of 1.30g/ml, the virus for adding 5ml suspend, and 20000rpm, room temperature is centrifuged 2 hours.Density is collected to exist
Into bag filter, (bag filter is boiled using preceding with the EDTA-Na2 of 10mM virus band between 1.30g/ml and 1.40g/ml
10min).In elution buffer (50g sucrose, 10ml 1M Tris-HCl, PH8.0,2ml 1M MgCl2 are settled to 1L), 4
DEG C stirring dialysed overnight, a dialyzate is changed in centre.Virus is collected, virus titer is measured；
(10) virus resuspension and preservation: viral pellet is resuspended in appropriate PBS, uses in one week and is then placed in 4 degree of refrigerators preservations, such as
Need storage for a long time that need to be placed in -80 degree even Liquid nitrogen storage；
(11) titer determination: surveying previous 96 orifice plate of natural gift of titre, 96 orifice plates taken out from incubator, determines under the microscope every
The equal well-grown of the cell in hole prepares 10 sterile Ep pipes, the complete culture solution of 990 μ l is added in first Ep pipe,
The complete culture solution of 900 μ l is respectively added in 9 remaining pipes, the dilution of virus liquid to be measured: 10 μ l adenoviral stocks being taken to be added 990
1:100 dilution (10-2) is done in the Ep pipe of μ l；Then with this as the starting point, then 100 μ l dilutions is taken to be added in the Ep pipe of 900 μ l
1:10 dilution (10-3) is done, until being diluted to 10-14, inhales and abandons old culture solution, then successively by the diluted virus of 10-7 to 10-14
Liquid is added in 96 orifice plates, and each dilution occupies a line, and 90 μ l viral dilutions are added in the every hole in the hole 1-10 of every a line, and every
Complete medium of the 90 μ l without virus is added as control in the hole 11-12 of a line, and 96 orifice plates are placed in 37 DEG C, and 5%
Continue to cultivate in CO2 cell incubator, cytopathic phenomena is observed after 10d, and count to the hole CPE, calculate every a line
Positive rate calculates virus titer (Spearman-Karber Method): virus titer=10^ (x+0.8) (PFU/ml), x=
10-1 to 10-14 successively CPE positive rate summation under dilution, formula use condition: negative control does not have CPE and growth inhibition existing
As, the Kong Jun that minimum diluted concentration virus crude extract is added has CPE, after measured, virus titer 1x1011pfu/ml。
Compared with prior art, this is used for the wild type p53 adenovirus of hyperplastic scar treatment and the preparation method of purifying,
Preparation process is simple and convenient, and curative effect is splendid, improves the therapeutic effect of hyperplastic scar patient, and cure rate is high, does not recur,
Without any side effects, preparation method is simple, easy practice and extension.
Detailed description of the invention
Fig. 1 is preparation flow figure of the present invention
Specific embodiment
Hereinafter, a variety of specific details are elaborated, in order to provide to the saturating of the concept for constituting described embodiment basis
Thorough understanding.However, it will be apparent to those skilled in the art that described embodiment can be in these no specific details
In some or all situations get off practice.In other cases, well-known processing step is not specifically described.
Embodiment
(12) preparation method for the P53 adenovirus treated for hyperplastic scar and purifying that the present embodiment is enumerated, is pressed
The method of stating is made, and material prepares: the incasing cells of cell strain 293, adenovirus, is anchorage dependence type into epithelioid cell, growth
Culture medium is DMEM (containing 10%FBS), bacterial strain, coli strain DH5 α, for expanding viral vectors and auxiliary package carrier
Plasmid will construct successful adenovirus recombinant plasmid and packaging plasmid and be extracted using the plasmid extraction kit of Qiagen company.
Resulting Plasmid DNA is dissolved in sterile TE, is measured its concentration and purity with UV Absorption method, is guaranteed proposed Plasmid DNA
A260/A280 is between 1.8~2.0, the day before transfection, and the cell grown is passed in proper proportions to 6cm and is cultivated
In ware, prepare transfection when cell grows to 70%~80%, transfecting preceding 1~2h will need the cell transfected to renew fresh culture
Base takes sterile 1.5ml EP pipe or 15ml centrifuge tube, rotaring redyeing system according to the form below:
| DMEM | 0.5ml |
| Expression plasmid | 2μg |
| pBHG(delta)E1,3 cre | 4μg |
| HG transgene reagent | 18μl |
After mixing, after being placed at room temperature for 15min-20min, drops evenly and changed in the culture dish of liquid in advance, be placed on CO2
It is cultivated in incubator, after transfecting 10~12h, drops evenly 100 × Enhancing buffer and promote transfection, 120 μ l/ wares turn
Then plus fresh cell culture medium it after contaminating 18~20h, carefully sops up cell culture fluid and abandons in the waste liquid cup for filling thimerosal,
Continue to cultivate, to observe whether virus plaque forms before collection virus, can see under the microscope within 7 days or so after infection small
Plaque is generally formed in 10 to 21 days, collects cell and supernatant, multigelation collect virus three times, with this virus for P1 generation
Virus is carried out continuously three generations's infection with P1 for 293 cell of adenovirus infection, until P4 generation carries out the massive amplification of adenovirus, to sky
Spot collects virus and carries out purification and concentration to virus after being formed, viral purification uses CsCl density gradient centrifugation-dialysis
Combination method purified virus, CsCl gradient the preparation method is as follows: be added 2.0ml density be 1.40g/ml CsCl solution, then
It is slowly added to the CsCl solution that 3.0ml density is 1.30g/ml, the virus for adding 5ml suspends, 20000rpm, room temperature centrifugation 2
Hour.Collecting virus band of the density between 1.30g/ml and 1.40g/ml, (bag filter is using preceding using 10mM into bag filter
EDTA-Na2 boil 10min).In elution buffer (50g sucrose, 10ml 1M Tris-HCl, PH8.0,2ml 1M MgCl2
It is settled to 1L) in, 4 DEG C of stirring dialysed overnights, a dialyzate is changed in centre.Virus is collected, virus titer, appropriate PBS weight are measured
Outstanding viral pellet need to be placed in -80 degree even Liquid nitrogen storage if you need to store for a long time using 4 degree of refrigerators preservations are then placed in one week,
Previous 96 orifice plate of natural gift of titre is surveyed, 96 orifice plates are taken out from incubator, determine the equal well-grown of the cell in every hole under the microscope, it is quasi-
Standby 10 sterile Ep pipes, the complete culture solution of 990 μ l is added in first Ep pipe, is respectively added 900 in remaining 9 pipe
The dilution of virus liquid to be measured: the complete culture solution of μ l takes 10 μ l adenoviral stocks to be added in the Ep pipe of 990 μ l and does 1:100 dilution
(10-2)；Then with this as the starting point, then take 100 μ l dilutions to be added in the Ep pipe of 900 μ l to do 1:10 dilution (10-3), until
It is diluted to 10-14, inhales and abandons old culture solution, then successively the diluted virus liquid of 10-7 to 10-14 is added in 96 orifice plates, it is each dilute
Degree of releasing occupies a line, and 90 μ l viral dilutions are added in the every hole in the hole 1-10 of every a line, and the hole 11-12 of every a line is added
96 orifice plates are placed in 37 DEG C, continue to train in 5%CO2 cell incubator by complete medium of the 90 μ l without virus as control
It supports, cytopathic phenomena is observed after 10d, and count to the hole CPE, calculate the positive rate of every a line, calculate virus titer
(Spearman-Karber Method): virus titer=10^ (x+0.8) (PFU/ml), x=10-1 to 10-14 successively dilutes
Lower CPE positive rate summation is spent, formula use condition: negative control does not have CPE and growth inhibition phenomenon, and minimum diluted concentration is added
The Kong Jun of viral crude extract has CPE, after measured, virus titer 1x1011pfu/ml.
The preparation method of the wild type p53 adenovirus and purifying treated for hyperplastic scar, preparation process are simply square
Just, and curative effect is splendid, improves the therapeutic effect of hyperplastic scar patient, and cure rate is high, without any side effects, preparation method
Simply, easy practice and extension.
The present invention is not limited to above-mentioned specific embodiment, those skilled in the art from the above idea,
Without creative labor, the various transformation made are within the scope of the present invention.
Claims (1)
1. for the wild type p53 adenovirus of hyperplastic scar treatment and the preparation method of purifying, it is characterised in that: press following step
It is rapid to carry out:
(1) material prepares: the incasing cells of cell strain 293, adenovirus, is anchorage dependence type into epithelioid cell, grown cultures
Base is DMEM (contain 10%FBS), bacterial strain, coli strain DH5 α, for expanding viral vectors and auxiliary package carrier plasmid,
Successful adenovirus recombinant plasmid and packaging plasmid will be constructed to extract using the plasmid extraction kit of Qiagen company.It is resulting
Plasmid DNA is dissolved in sterile TE, is measured its concentration and purity with UV Absorption method, is guaranteed the A260/ of mentioned Plasmid DNA
A280 is between 1.8~2.0；
(2) cell divides disk: the day before transfection, the cell grown is passed in proper proportions in 6cm culture dish, when thin
Born of the same parents prepare transfection when growing to 70%~80%；
(3) liquid is changed before transfecting: the preceding 1~2h of transfection will need the cell transfected to renew fresh culture medium；
(4) it transfects: taking sterile 1.5ml EP pipe or 15ml centrifuge tube, rotaring redyeing system according to the form below:
It after mixing, after being placed at room temperature for 15min-20min, drops evenly and was changed in the culture dish of liquid in advance, be placed on CO2 culture
It is cultivated in case；
(5) add Enhancing buffer: after 10~12h of transfection, dropping evenly 100 × Enhancing buffer and promote to turn
Dye, 120 μ l/ wares；
(6) it changes liquid: after 18~20h of transfection, carefully sopping up cell culture fluid and abandon in the waste liquid cup for filling thimerosal, then plus newly
Fresh cell culture medium continues to cultivate；
(7) collection virus: will observe whether virus plaque forms before collection virus, 7 days or so after infection can be under the microscope
See small plaque, generally formed in 10 to 21 days, collect cell and supernatant, multigelation collect virus three times, with this disease
Poison is P1 generation virus；
(8) virus amplification: with P1 for 293 cell of adenovirus infection, being carried out continuously three generations's infection, until P4 generation carries out the big of adenovirus
Amount amplification collects virus after plaque test and carries out purification and concentration to virus；
(9) viral purification: viral purification is combined method purified virus, the system of CsCl gradient using CsCl density gradient centrifugation-dialysis
Preparation Method is as follows: the CsCl solution that 2.0ml density is 1.40g/ml is added, being then slowly added into 3.0ml density is 1.30g/ml
CsCl solution, add 5ml virus suspend, 20000rpm, room temperature be centrifuged 2 hours.Collect density in 1.30g/ml and
Virus band between 1.40g/ml is into bag filter (bag filter boils 10min with the EDTA-Na2 of 10mM using preceding).Saturating
It analyses in buffer (50g sucrose, 10ml 1M Tris-HCl, PH8.0,2ml 1M MgCl2 are settled to 1L), 4 DEG C of stirrings were dialysed
A dialyzate is changed in night, centre.Virus is collected, virus titer is measured；
(10) virus resuspension and preservation: viral pellet is resuspended in appropriate PBS, uses in one week and is then placed in 4 degree of refrigerators preservations, if you need to length
Time storage need to be placed in -80 degree even Liquid nitrogen storage；
(11) titer determination: previous 96 orifice plate of natural gift of titre is surveyed, 96 orifice plates are taken out from incubator, determine every hole under the microscope
The equal well-grown of cell prepares 10 sterile Ep pipes, the complete culture solution of 990 μ l is added in first Ep pipe, remaining 9
The complete culture solution of 900 μ l is respectively added in a pipe, the dilution of virus liquid to be measured: 10 μ l adenoviral stocks being taken to be added 990 μ l's
1:100 dilution (10-2) is done in Ep pipe；Then with this as the starting point, then take 100 μ l dilutions to be added in the Ep pipe of 900 μ l to do 1:
10 dilutions (10-3) inhale until being diluted to 10-14 and abandon old culture solution, then successively add the diluted virus liquid of 10-7 to 10-14
Enter in 96 orifice plates, each dilution occupies a line, and the every hole in the hole 1-10 of every a line is added 90 μ l viral dilutions, and every a line
The hole 11-12 be added 90 μ l without virus complete medium as control, 96 orifice plates are placed in 37 DEG C, 5%CO2Carefully
Continue to cultivate in born of the same parents' incubator, cytopathic phenomena is observed after 10d, and count to the hole CPE, calculate the positive of every a line
Rate calculates virus titer (Spearman-Karber Method): virus titer=10^ (x+0.8) (PFU/ml), x=10-1
CPE positive rate summation under to 10-14 successively dilution, formula use condition: negative control does not have CPE and growth inhibition phenomenon,
The Kong Jun that minimum diluted concentration virus crude extract is added has CPE, after measured, virus titer 1x1011pfu/ml。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1136826A (en) * | 1993-10-29 | 1996-11-27 | 德克萨斯州立大学董事会 | Recombinant P53 adenovirus method and compositions |
| CN1147768A (en) * | 1994-04-25 | 1997-04-16 | 德克萨斯州立大学董事会 | Compositions comprising DNA damaging agents and P53 |
| CN1471977A (en) * | 2003-05-10 | 2004-02-04 | Gene recombined medicine of adenovirus carrier and p53 gene for treating proliferative diseases | |
| CN104846013A (en) * | 2015-04-15 | 2015-08-19 | 广州福宸生物技术有限公司 | Replication-defective human adenovirus type 55 vector and preparation method and application thereof |
-
2019
- 2019-03-13 CN CN201910191222.9A patent/CN110055227A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6143290A (en) * | 1992-10-13 | 2000-11-07 | The Board Of Regents, University Of Texas System | Tumor regression by adenovirus expression of wild-type p53 |
| CN1136826A (en) * | 1993-10-29 | 1996-11-27 | 德克萨斯州立大学董事会 | Recombinant P53 adenovirus method and compositions |
| CN1147768A (en) * | 1994-04-25 | 1997-04-16 | 德克萨斯州立大学董事会 | Compositions comprising DNA damaging agents and P53 |
| CN1471977A (en) * | 2003-05-10 | 2004-02-04 | Gene recombined medicine of adenovirus carrier and p53 gene for treating proliferative diseases | |
| CN104846013A (en) * | 2015-04-15 | 2015-08-19 | 广州福宸生物技术有限公司 | Replication-defective human adenovirus type 55 vector and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| B G HUYGHE等: "Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography", 《HUMAN GENE THERAPY》 * |
| 齐桓: ""以AdMax载体系统构建和制备肾癌相关抗原G250基因重组腺病毒表达载体"", 《南方医科大学学报》 * |
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