CN103212066B - A kind of preparation method of cyclobutane pyrimidine dimmer photolyase liposome - Google Patents
A kind of preparation method of cyclobutane pyrimidine dimmer photolyase liposome Download PDFInfo
- Publication number
- CN103212066B CN103212066B CN201310080376.3A CN201310080376A CN103212066B CN 103212066 B CN103212066 B CN 103212066B CN 201310080376 A CN201310080376 A CN 201310080376A CN 103212066 B CN103212066 B CN 103212066B
- Authority
- CN
- China
- Prior art keywords
- photolyase
- cyclobutane pyrimidine
- eluent
- pyrimidine dimmer
- dimmer
- 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.)
- Expired - Fee Related
Links
Abstract
The invention discloses a kind of preparation method of cyclobutane pyrimidine dimmer photolyase liposome, comprise preparation three steps of the expression of cyclobutane pyrimidine dimmer photolyase, the purification of cyclobutane pyrimidine dimmer photolyase and cyclobutane pyrimidine dimmer photolyase liposome; The method adopts Q post to be separated cyclobutane pyrimidine dimmer photolyase, medium low price, and is separated the better effects if removing glutathione-S-transferase, and the industrialization being more suitable for cyclobutane pyrimidine dimmer photolyase is produced; Cyclobutane pyrimidine dimmer photolyase is also prepared as liposome by the present invention, makes the barrier of cyclobutane pyrimidine dimmer photolyase liposome energy permeates cell membranes or skin enter cell, greatly improves the repairing effect of DNA damage.
Description
Technical field
The invention belongs to DNA light repairase preparation field, particularly a kind of preparation method of cyclobutane pyrimidine dimmer photolyase liposome.
Background technology
Cyclobutane pyrimidine dimmer photolyase (cuclobutanepyrimidinedimmerphotolyase, CPDase) is the main tool that organism utilizes light DNA plerosis and damages.CPDase is extensively distributed among various species, is the monomeric protein of a kind of molecular weight between 55-65kD, not yet finds CPDase in human body.At present in the world, along with environmental disruption and ultraviolet arrive the dosage on ground and the increase of intensity, the sickness rate of dermatosis especially skin carcinoma rises year by year, reason is that the ultraviolet in sunlight can damage the macromole in organism, DNA is made to produce the photoproducts such as cyclobutane pyrimidine dimer (cuclobutanepyrimidinedimmer, CPD).CPD damage is difficult to be repaired by DNA damage repair systems such as NER approach in human body, this will certainly make usually to be exposed to the accumulation occurring CPD photoproduct in the crowd under daylight, the accumulation of this damage will affect copying of DNA and transcribe, cause the generation of immunosuppressant and erythema, accumulation a large amount of for a long time can cause the sudden change of DNA, and then cause canceration, and CPDase effectively can remove the plastidogenetic CPD photoproduct of ultraviolet radiation, is preventing having good effect in ultraviolet injury.
At present, the preparation method about CPDase has Patents and document to report both at home and abroad.The preparation method of external existing bibliographical information separate sources CPDase, comprise gene clone, protein expression, purification, these CPDase method for preparing purified, the method of purifying protein that main employing is traditional, namely utilizes the physicochemical characteristics such as the dissolubility of this protein, isoelectric point, IP, electrically charged number, hydrophobicity to carry out substep purification.The U.S.'s " journal of biological chemistry " (J.Biol.Chem.1984,259,6028-6032) report, when preparing colibacillary CPDase, adopt gene clone method, by gene constructed for escherichia coli CPDase to recombiant plasmid, and make this recombiant plasmid can in expression strain overexpression; Thalline after collection, broken expression, the soluble supernatant ammonium sulfate precipitation of collected by centrifugation, carries out substep with Phenyl-Sepharose column chromatography, hydroxyapatite column, diethyl amino ethyl group-sepharose column chromatography, DNA-cellulose chromatography and Phenyl-Sepharose column chromatography successively subsequently and purifies.According to the U.S.'s " mutation research " (MutationResearch, 1996,33,97-104) report, when purification fruit bat CPDase, build a kind of CPDase gene recombination plasmid, after recombinant plasmid transformed escherichia coli, overexpression in expression strain, bacterium liquid is after broken, centrifugal, get soluble supernatant through ammonium sulfate precipitated protein matter, carry out two step affinitive layer purification protein through Blue-Sepharose column chromatography and DNA-cellulose chromatography subsequently; But Blue-Sepharose and DNA-cellulose column substrate price very expensive, in separation process, also can there is the impact of some non-specific adsorption protein.Therefore, although above-mentioned purification process can obtain very pure sample, its purification step is numerous and diverse; purification cycle is long, because repeatedly changing chromatographic column in purge process, makes sample handling processes complexity, protein recovery low; the principle used is expensive, is not suitable for scale batch and prepares protein.Most importantly, the CPDase of preparation the barrier of permeates cell membranes or skin can not enter cell, thus causes DNA damage repairing effect not obvious.
In addition, the affinity chromatograph method that specificity is high is the important means of protein preparation, describes the affine preparation of CPDase in patent of invention 200310106550.3, comprises amplification CPDase gene, is building up in the vector plasmid with histidine-label; Overexpression CPDase, collected by centrifugation bacterium liquid, the broken thalline of high pressure, supernatant centrifugalize gone out adds in conjunction with nickel ion and with containing in the equilibrated nickel ion affinity chromatograph post of the buffer of 0.005-0.05mol/l imidazoles, with the buffer containing 0.005-0.05mol/l imidazoles wash away can not in conjunction with or the foreign protein of non-specific binding, then with containing buffer or the nickel ion chelating agen eluting of 0.5-1.0mol/l imidazoles, collect elution samples, dialysis, lyophilization, namely obtains CPDase.It is histidine-tagged that CPDase prepared by this method contains external source, and this label may impact the normal configuration of CPDase and function, and brings potential potential safety hazard to follow-up application.Abroad when the affinity chromatograph method purification another kind light repairase adopting specificity high, the external source label introduced is removed.According to the U.S.'s " journal of biological chemistry " (J.Bio.Chem1997, 272, 32591-32598) report, when purification xenopous laevis belongs to (6-4) light repairase, gene recombinaton is building up to one containing in the recombiant plasmid of glutathione-s-transferase gene, overexpression in expression strain, the product of its protein expression is the fused protein of c-terminus with glutathione-S-transferase, very pure fused protein is obtained by Glutathione-Sepharose Affinity Chromatography post single step purification, then glutathione-S-transferase is excised with thrombin, finally utilize DNA affinity column purification.The DNA affinity column that this method utilizes is expensive, and condition is not easy to control, and is unfavorable for that this proteomic scaleization is produced.The CPDase for preparing of these methods the barrier of permeates cell membranes or skin can not enter cell equally, thus causes DNA damage repairing effect not obvious.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of cyclobutane pyrimidine dimmer photolyase liposome; the method overcome existing method purification chromatography base for post matter expensive, be unwell to the defect that scale prepares cyclobutane pyrimidine dimmer photolyase; and improve the ability of cyclobutane pyrimidine dimmer photolyase permeates cell membranes or skin barrier, greatly improve DNA damage repairing effect.
In order to reach foregoing invention object, the technical solution used in the present invention is: the preparation method providing a kind of cyclobutane pyrimidine dimmer photolyase liposome, is characterized in that, comprises the following steps:
The expression of a, cyclobutane pyrimidine dimmer photolyase: be cloned into cyclobutane pyrimidine dimmer photolyase gene from organism, this is gene constructed in prokaryotic expression carrier pGEX4T-1, be converted into E. coli expression strains E.coliBL21 competent cell again, obtain bacterium liquid; In the LB culture medium of 3 ~ 50ml containing ampicillin, add the bacterium liquid of LB culture volume 1 ~ 5%, 37 DEG C are cultured to OD
600be 0.6 ~ 0.8; Adding IPTG again to final concentration is 0.1 ~ 0.8mol/L, 23 ~ 28 DEG C of abduction delivering 3 ~ 4h, centrifugation thalline under 10000 ~ 12000 acceleration of gravity conditions, collect thalline, thalline is carried out ultrasonic disruption, thalline liquid after fragmentation centrifugal segregation bacterial chip under 10000 ~ 12000 acceleration of gravity conditions, collects the supernatant that centrifugalize goes out, obtains cyclobutane pyrimidine dimmer photolyase crude product; Wherein, the condition of ultrasonic disruption is: under the power of 200 ~ 400W, working time 1 ~ 5s, intermittent time 2-5s, ultrasonication 20-40min;
The purification of b, cyclobutane pyrimidine dimmer photolyase: the cyclobutane pyrimidine dimmer photolyase crude product obtained by step a joins in glutathione agarose affinity column with the flow velocity of 100 ~ 150ml/h, drip washing is carried out with the flow velocity of 200 ~ 400ml/h with the eluent A of 5 ~ 10 times of bed volumes, carry out gradient elution with the eluent A of 5 ~ 10 times of bed volumes and eluent B again, obtain merging cyclobutane pyrimidine dimmer photolyase; Again according to the consumption of 1-5U/mg protein, joined by thrombin and merge in cyclobutane pyrimidine dimmer photolyase, 37 DEG C of enzymolysis 1 ~ 3h, excision glutathione-S-transferase, obtains enzyme action hydrolyzed solution; Enzyme action hydrolyzed solution is joined in Q post with the flow velocity of 100 ~ 150ml/h, drip washing is carried out with the flow velocity of 200 ~ 400ml/h with the eluent C of 5 ~ 10 times of bed volumes, carry out gradient elution with the eluent C of 5 ~ 10 times of bed volumes and eluent D again, obtain cyclobutane pyrimidine dimmer photolyase; Again cyclobutane pyrimidine dimmer photolyase is carried out desalting column desalting processing, obtain highly purified cyclobutane pyrimidine dimmer photolyase;
Wherein, eluent A is made up of 50mMTris/HCl and 0.15MNaCl; Eluent B is made up of 50mMTris/HCl and 10mMGSH; Eluent C is made up of 10mMPB and 0.15MNaCl; Eluent D is made up of 10mMPB and 1MNaCl;
The preparation of c, cyclobutane pyrimidine dimmer photolyase liposome: take 50 ~ 100mg lecithin and 20 ~ 50mg cholesterol, be dissolved in 7.5 ~ 15ml ether, add the high-purity cyclobutane pyrimidine dimmer photolyase that 0.1 ~ 0.5mg step b is obtained, carry out ultrasonic emulsification again, form Water-In-Oil, carry out distillation again and remove low boiling organic facies, obtain cyclobutane pyrimidine dimmer photolyase liposome suspension; Continue evaporation again, until white suspension is formed; By white suspension with the centrifugal 30 ~ 40min of 15000-20000r/min, collecting precipitation, freezen protective, obtained cyclobutane pyrimidine dimmer photolyase liposome.
In the preparation method of cyclobutane pyrimidine dimmer photolyase liposome of the present invention, described organism is Dunaliella salina.
In the preparation method of cyclobutane pyrimidine dimmer photolyase liposome of the present invention, in step c, the condition of ultrasonic emulsification is: under the power of 100 ~ 300W, working time 1 ~ 3s, intermittent time 2-5s, and ultrasonic time is 20-30min.
In sum: the preparation method of cyclobutane pyrimidine dimmer photolyase liposome provided by the invention has following beneficial effect:
(1) existing glutathione-sepharose column chromatography method is after excising glutathione-S-transferase with thrombin, general employing DNA column chromatography is separated cyclobutane pyrimidine dimmer photolyase and glutathione-S-transferase, this DNA chromatographic column medium is expensive, condition is wayward, and adopt Q post of the present invention to be separated, not only medium low price, and being separated the better effects if removing glutathione-S-transferase, the industrialization being more suitable for cyclobutane pyrimidine dimmer photolyase is produced.
(2) cyclobutane pyrimidine dimmer photolyase is prepared as liposome by the present invention, this lipid physical ability cell membrane merges, cyclobutane pyrimidine dimmer photolyase is sent in nucleus, thus make the barrier of cyclobutane pyrimidine dimmer photolyase liposome energy permeates cell membranes or skin enter cell, greatly improve the repairing effect of DNA damage.
Accompanying drawing explanation
Fig. 1 is cyclobutane pyrimidine dimmer photolyase liposome optical microscope schematic diagram.
Fig. 2 is the survival rate contrast figure of cell after carrying out light reparation through ultraviolet radiation.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail, but they are not to further restriction of the present invention.
Embodiment 1
The expression of a, cyclobutane pyrimidine dimmer photolyase
Cyclobutane pyrimidine dimmer photolyase gene (international publication number is cloned into from Dunaliella salina, GenBank:EF090912.1), this is gene constructed in prokaryotic expression carrier pGEX4T-1, be converted into E. coli expression strains E.coliBL21 competent cell again, obtain bacterium liquid; The above-mentioned bacterium liquid of 0.03ml is joined 3ml containing in the LB culture medium of ampicillin, 37 DEG C are cultured to OD
600be 0.6; Adding IPTG again to final concentration is 0.1mol/L, 23 DEG C of abduction delivering 3h, centrifugation thalline under 10000 acceleration of gravity conditions, collect thalline, with ultrasonic cell disruption instrument, thalline is carried out ultrasonic disruption, the condition of ultrasonic disruption is: under the power of 200 ~ 400W, working time 1 ~ 5s, intermittent time 2-5s, broken time 20-40min; Then by the centrifugal segregation bacterial chip under 10000 acceleration of gravity conditions of the thalline liquid after fragmentation, collect the supernatant that centrifugalize goes out, obtain cyclobutane pyrimidine dimmer photolyase crude product;
The purification of b, cyclobutane pyrimidine dimmer photolyase
Join in glutathione agarose affinity column by above-mentioned obtained cyclobutane pyrimidine dimmer photolyase crude product with the flow velocity of 100ml/h, the addition of cyclobutane pyrimidine dimmer photolyase crude product is 4 ~ 5 times of bed volume; Carry out drip washing with the eluent A of 5 times of bed volumes with the flow velocity of 200ml/h again, then carry out gradient elution with the eluent A of 5 times of bed volumes and eluent B, obtain merging cyclobutane pyrimidine dimmer photolyase; Again according to the consumption of 1U/mg protein, joined by thrombin and merge in cyclobutane pyrimidine dimmer photolyase, 37 DEG C of enzymolysis 1h, excision glutathione-S-transferase, obtains enzyme action hydrolyzed solution; Again enzyme action hydrolyzed solution is joined in Q post with the flow velocity of 100ml/h, drip washing is carried out with the flow velocity of 200ml/h with the eluent C of 5 times of bed volumes, carry out gradient elution with the eluent C of 5 times of bed volumes and eluent D again, obtain cyclobutane pyrimidine dimmer photolyase; Again cyclobutane pyrimidine dimmer photolyase is carried out desalting column desalting processing, obtain highly purified cyclobutane pyrimidine dimmer photolyase;
Wherein, the preparation method of glutathione agarose affinity column is: void column is loaded glutathione agarose affinity media, with the eluent B drip washing of 5 times of bed volumes, then uses the eluent A drip washing of 5 times of bed volumes, until baseline is constant; Eluent A consists of: 50mMTris/HCl and 0.15MNaCl (pH8.0); Eluent B consists of: 50mMTris/HCl and 10mMGSH (pH8.0);
The preparation method of Q post is: void column is loaded Q, with the eluent D drip washing of 10 times of bed volumes, then uses the eluent C drip washing of 5 times of bed volumes, until baseline is constant; Eluent C consists of: 10mMPB and 0.15MNaCl (pH7.4); Eluent D consists of: 10mMPB and 1MNaCl (pH7.4).
C, the preparation of cyclobutane pyrimidine dimmer photolyase liposome: take 50mg lecithin and 20mg cholesterol, be dissolved in 7.5ml ether, add the above-mentioned obtained high-purity cyclobutane pyrimidine dimmer photolyase of 0.1mg, (power is 100 ~ 300W to carry out ultrasonic emulsification with ultrasonic cell disruption instrument again, working time 1 ~ 3s, intermittent time 2 ~ 5s, broken time 20 ~ 30min), form oil bag and w/o type emulsion, in vacuum rotary evaporator 25 DEG C again, under 53KPa, low boiling organic facies is removed in distillation, obtain cyclobutane pyrimidine dimmer photolyase liposome suspension, under 97KPa condition, continue evaporation again, eliminate residual organic solvents, until adularescent suspension is formed, by white suspension with the centrifugal 30 ~ 40min of 15000r/min, supernatant is poured in centrifuge tube, the cyclobutane pyrimidine dimmer photolyase do not wrapped into is separated with liposome, collecting precipitation, at-20 DEG C of freezen protective, obtained cyclobutane pyrimidine dimmer photolyase liposome.
Embodiment 2
The expression of a, cyclobutane pyrimidine dimmer photolyase
Cyclobutane pyrimidine dimmer photolyase gene (international publication number is cloned into from Dunaliella salina, GenBank:EF090912.1), this is gene constructed in prokaryotic expression carrier pGEX4T-1, be converted into E. coli expression strains E.coliBL21 competent cell again, obtain bacterium liquid; The above-mentioned bacterium liquid of 0.5ml is joined 50ml containing in the LB culture medium of ampicillin, 37 DEG C are cultured to OD
600be 0.8; Adding IPTG again to final concentration is 0.8mol/L, 28 DEG C of abduction delivering 4h, centrifugation thalline under 12000 acceleration of gravity conditions, collect thalline, with ultrasonic cell disruption instrument, thalline is carried out ultrasonic disruption, the condition of ultrasonic disruption is: under the power of 200 ~ 400W, working time 1 ~ 5s, intermittent time 2-5s, broken time 20-40min; Then by the centrifugal segregation bacterial chip under 12000 acceleration of gravity conditions of the thalline liquid after fragmentation, collect the supernatant that centrifugalize goes out, obtain cyclobutane pyrimidine dimmer photolyase crude product;
The purification of b, cyclobutane pyrimidine dimmer photolyase
Join in glutathione agarose affinity column by above-mentioned obtained cyclobutane pyrimidine dimmer photolyase crude product with the flow velocity of 150ml/h, the addition of cyclobutane pyrimidine dimmer photolyase crude product is 4 ~ 5 times of bed volume; Carry out drip washing with the eluent A of 10 times of bed volumes with the flow velocity of 400ml/h again, then carry out gradient elution with the eluent A of 10 times of bed volumes and eluent B, obtain merging cyclobutane pyrimidine dimmer photolyase; Again according to the consumption of 5U/mg protein, joined by thrombin and merge in cyclobutane pyrimidine dimmer photolyase, 37 DEG C of enzymolysis 3h, excision glutathione-S-transferase, obtains enzyme action hydrolyzed solution; Again enzyme action hydrolyzed solution is joined in Q post with the flow velocity of 150ml/h, drip washing is carried out with the flow velocity of 400ml/h with the eluent C of 10 times of bed volumes, carry out gradient elution with the eluent C of 10 times of bed volumes and eluent D again, obtain cyclobutane pyrimidine dimmer photolyase; Again cyclobutane pyrimidine dimmer photolyase is carried out desalting column desalting processing, obtain highly purified cyclobutane pyrimidine dimmer photolyase;
Wherein, the preparation method of glutathione agarose affinity column is: void column is loaded glutathione agarose affinity media, with the eluent B drip washing of 10 times of bed volumes, then uses the eluent A drip washing of 10 times of bed volumes, until baseline is constant; Eluent A consists of: 50mMTris/HCl and 0.15MNaCl (pH8.0); Eluent B consists of: 50mMTris/HCl and 10mMGSH (pH8.0);
The preparation method of Q post is: void column is loaded Q, with the eluent D drip washing of 20 times of bed volumes, then uses the eluent C drip washing of 10 times of bed volumes, until baseline is constant; Eluent C consists of: 10mMPB and 0.15MNaCl (pH7.4); Eluent D consists of: 10mMPB and 1MNaCl (pH7.4).
C, the preparation of cyclobutane pyrimidine dimmer photolyase liposome: take 100mg lecithin and 50mg cholesterol, be dissolved in 15ml ether, add the above-mentioned obtained high-purity cyclobutane pyrimidine dimmer photolyase of 0.5mg, (power is 100 ~ 300W to carry out ultrasonic emulsification with ultrasonic cell disruption instrument again, working time 1 ~ 3s, intermittent time 2 ~ 5s, broken time 20 ~ 30min), form oil bag and w/o type emulsion, in vacuum rotary evaporator 25 DEG C again, under 53KPa, low boiling organic facies is removed in distillation, obtain cyclobutane pyrimidine dimmer photolyase liposome suspension, under 97KPa condition, continue evaporation again, eliminate residual organic solvents, until adularescent suspension is formed, by white suspension with the centrifugal 30 ~ 40min of 20000r/min, supernatant is poured in centrifuge tube, the cyclobutane pyrimidine dimmer photolyase do not wrapped into is separated with liposome, collecting precipitation, at-20 DEG C of freezen protective, obtained cyclobutane pyrimidine dimmer photolyase liposome.
Embodiment 3
The expression of a, cyclobutane pyrimidine dimmer photolyase
Cyclobutane pyrimidine dimmer photolyase gene (international publication number is cloned into from Dunaliella salina, GenBank:EF090912.1), this is gene constructed in prokaryotic expression carrier pGEX4T-1, be converted into E. coli expression strains E.coliBL21 competent cell again, obtain bacterium liquid; The above-mentioned bacterium liquid of 0.3ml is joined 10ml containing in the LB culture medium of ampicillin, 37 DEG C are cultured to OD
600be 0.7; Adding IPTG again to final concentration is 0.9mol/L, 25 DEG C of abduction delivering 3.5h, centrifugation thalline under 12000 acceleration of gravity conditions, collect thalline, with ultrasonic cell disruption instrument, thalline is carried out ultrasonic disruption, the condition of ultrasonic disruption is: under the power of 200 ~ 400W, working time 1 ~ 5s, intermittent time 2-5s, broken time 20-40min; Then by the centrifugal segregation bacterial chip under 10000 acceleration of gravity conditions of the thalline liquid after fragmentation, collect the supernatant that centrifugalize goes out, obtain cyclobutane pyrimidine dimmer photolyase crude product;
The purification of b, cyclobutane pyrimidine dimmer photolyase
Join in glutathione agarose affinity column by above-mentioned obtained cyclobutane pyrimidine dimmer photolyase crude product with the flow velocity of 130ml/h, the addition of cyclobutane pyrimidine dimmer photolyase crude product is 4 ~ 5 times of bed volume; Carry out drip washing with the eluent A of 7 times of bed volumes with the flow velocity of 300ml/h again, then carry out gradient elution with the eluent A of 8 times of bed volumes and eluent B, obtain merging cyclobutane pyrimidine dimmer photolyase; Again according to the consumption of 3U/mg protein, joined by thrombin and merge in cyclobutane pyrimidine dimmer photolyase, 37 DEG C of enzymolysis 2h, excision glutathione-S-transferase, obtains enzyme action hydrolyzed solution; Again enzyme action hydrolyzed solution is joined in Q post with the flow velocity of 130ml/h, drip washing is carried out with the flow velocity of 300ml/h with the eluent C of 8 times of bed volumes, carry out gradient elution with the eluent C of 7 times of bed volumes and eluent D again, obtain cyclobutane pyrimidine dimmer photolyase; Again cyclobutane pyrimidine dimmer photolyase is carried out desalting column desalting processing, obtain highly purified cyclobutane pyrimidine dimmer photolyase;
Wherein, the preparation method of glutathione agarose affinity column is: void column is loaded glutathione agarose affinity media, with the eluent B drip washing of 7 times of bed volumes, then uses the eluent A drip washing of 7 times of bed volumes, until baseline is constant; Eluent A consists of: 50mMTris/HCl and 0.15MNaCl (pH8.0); Eluent B consists of: 50mMTris/HCl and 10mMGSH (pH8.0);
The preparation method of Q post is: void column is loaded Q, with the eluent D drip washing of 15 times of bed volumes, then uses the eluent C drip washing of 8 times of bed volumes, until baseline is constant; Eluent C consists of: 10mMPB and 0.15MNaCl (pH7.4); Eluent D consists of: 10mMPB and 1MNaCl (pH7.4).
C, the preparation of cyclobutane pyrimidine dimmer photolyase liposome: take 80mg lecithin and 40mg cholesterol, be dissolved in 10ml ether, add the above-mentioned obtained high-purity cyclobutane pyrimidine dimmer photolyase of 0.3mg, (power is 100 ~ 300W to carry out ultrasonic emulsification with ultrasonic cell disruption instrument again, working time 1 ~ 3s, intermittent time 2 ~ 5s, broken time 20 ~ 30min), form oil bag and w/o type emulsion, in vacuum rotary evaporator 25 DEG C again, under 53KPa, low boiling organic facies is removed in distillation, obtain cyclobutane pyrimidine dimmer photolyase liposome suspension, under 97KPa condition, continue evaporation again, eliminate residual organic solvents, until adularescent suspension is formed, by white suspension with the centrifugal 30 ~ 40min of 17000r/min, supernatant is poured in centrifuge tube, the cyclobutane pyrimidine dimmer photolyase do not wrapped into is separated with liposome, collecting precipitation, at-20 DEG C of freezen protective, obtained cyclobutane pyrimidine dimmer photolyase liposome.
Test example
The cyclobutane pyrimidine dimmer photolyase liposome (Fig. 1 is cyclobutane pyrimidine dimmer photolyase liposome optical microscope schematic diagram) prepared for embodiment 1; measure the protected effect of cyclobutane pyrimidine dimmer photolyase liposome to ultraviolet injury, under this test adopts CCK8 method to detect ultraviolet, cyclobutane pyrimidine dimmer photolyase liposome is to the protective effect of cell:
Cultured cell is spread 96 orifice plates, and density domination, in 5000/ hole, enters exponential phase after adopting fresh culture cultivation 24h; Add cyclobutane pyrimidine dimer photolyase and cyclobutane pyrimidine dimmer photolyase liposome, and negative control is set.Put into after incubator cultivates 2h and take out, wash three times with fresh culture, remove remaining albumen, then do not add to cultivate and carry out ultraviolet radiation based under super-clean bench, irradiation time is 5s.Fresh culture washs again, avoids ultraviolet to cause the impact of superoxide radical, and the culture medium more renewed, then put into incubator and cultivate 24h.Add cck-8 again, after 1h, detect the absorption value at 450nm place by microplate reader.
Be 100% (control (-)) with the cell number not through ultra-vioket radiation; not adding cyclobutane pyrimidine dimmer photolyase, the cell that adds cyclobutane pyrimidine dimmer photolyase and the add cyclobutane pyrimidine dimmer photolyase liposome cell number after ultra-vioket radiation with not through the ratio of the cell number of ultraviolet radiation for cell survival rate, compare the protected effect of cyclobutane pyrimidine dimmer photolyase to ultraviolet injury.
As shown in Figure 2, the cell survival rate not adding cyclobutane pyrimidine dimmer photolyase is 43%, the cell survival rate adding cyclobutane pyrimidine dimmer photolyase is 62%, and the cell survival rate adding cyclobutane pyrimidine dimmer photolyase liposome is 87%; From above-mentioned data, compared with cyclobutane pyrimidine dimmer photolyase, cyclobutane pyrimidine dimmer photolyase liposome makes the survival rate of DNA damage cell improve 44%, and therefore, cyclobutane pyrimidine dimmer photolyase liposome has obvious repairing effect to DNA cell injury.
Although describe in detail the specific embodiment of the present invention in conjunction with specific embodiments, it is not the restriction to this patent protection domain.In claims limited range, the various amendment that those skilled in the art can make without creative work or adjustment are still by the protection of this patent.
Claims (1)
1. a preparation method for cyclobutane pyrimidine dimmer photolyase liposome, is characterized in that, comprises the following steps:
The expression of a, cyclobutane pyrimidine dimmer photolyase
Cyclobutane pyrimidine dimmer photolyase gene is cloned into from Dunaliella salina, this is gene constructed in prokaryotic expression carrier pGEX4T-1, then be converted into E. coli expression strains E.coliBL21 competent cell, obtain bacterium liquid; The above-mentioned bacterium liquid of 0.03ml is joined 3ml containing in the LB culture medium of ampicillin, 37 DEG C are cultured to OD
600be 0.6; Adding IPTG again to final concentration is 0.1mol/L, 23 DEG C of abduction delivering 3h, centrifugation thalline under 10000 acceleration of gravity conditions, collect thalline, with ultrasonic cell disruption instrument, thalline is carried out ultrasonic disruption, the condition of ultrasonic disruption is: under the power of 200 ~ 400W, working time 1 ~ 5s, intermittent time 2-5s, broken time 20-40min; Then by the centrifugal segregation bacterial chip under 10000 acceleration of gravity conditions of the thalline liquid after fragmentation, collect the supernatant that centrifugalize goes out, obtain cyclobutane pyrimidine dimmer photolyase crude product;
The purification of b, cyclobutane pyrimidine dimmer photolyase
Join in glutathione agarose affinity column by above-mentioned obtained cyclobutane pyrimidine dimmer photolyase crude product with the flow velocity of 100ml/h, the addition of cyclobutane pyrimidine dimmer photolyase crude product is 4 ~ 5 times of bed volume; Carry out drip washing with the eluent A of 5 times of bed volumes with the flow velocity of 200ml/h again, then carry out gradient elution with the eluent A of 5 times of bed volumes and eluent B, obtain merging cyclobutane pyrimidine dimmer photolyase; Again according to the consumption of 1U/mg protein, joined by thrombin and merge in cyclobutane pyrimidine dimmer photolyase, 37 DEG C of enzymolysis 1h, excision glutathione-S-transferase, obtains enzyme action hydrolyzed solution; Again enzyme action hydrolyzed solution is joined in Q post with the flow velocity of 100ml/h, drip washing is carried out with the flow velocity of 200ml/h with the eluent C of 5 times of bed volumes, carry out gradient elution with the eluent C of 5 times of bed volumes and eluent D again, obtain cyclobutane pyrimidine dimmer photolyase; Again cyclobutane pyrimidine dimmer photolyase is carried out desalting column desalting processing, obtain highly purified cyclobutane pyrimidine dimmer photolyase;
Wherein, the preparation method of glutathione agarose affinity column is: void column is loaded glutathione agarose affinity media, with the eluent B drip washing of 5 times of bed volumes, then uses the eluent A drip washing of 5 times of bed volumes, until baseline is constant; Eluent A consists of: 50mMTris/HCl and 0.15MNaCl, and the pH value of this eluent A is 8.0; Eluent B consists of: 50mMTris/HCl and 10mMGSH, and the pH value of this eluent B is 8.0;
The preparation method of Q post is: void column is loaded Q, with the eluent D drip washing of 10 times of bed volumes, then uses the eluent C drip washing of 5 times of bed volumes, until baseline is constant; Eluent C consists of: 10mMPB and 0.15MNaCl, and the pH value of this eluent C is 7.4; Eluent D consists of: 10mMPB and 1MNaCl, and the pH value of this eluent D is 7.4;
C, the preparation of cyclobutane pyrimidine dimmer photolyase liposome: take 50mg lecithin and 20mg cholesterol, be dissolved in 7.5ml ether, add the above-mentioned obtained high-purity cyclobutane pyrimidine dimmer photolyase of 0.1mg, ultrasonic emulsification is carried out again with ultrasonic cell disruption instrument, the condition of ultrasonic emulsification is: power is 100 ~ 300W, working time 1 ~ 3s, intermittent time 2 ~ 5s, broken time 20 ~ 30min, form oil bag and w/o type emulsion, in vacuum rotary evaporator 25 DEG C again, under 53KPa, low boiling organic facies is removed in distillation, obtain cyclobutane pyrimidine dimmer photolyase liposome suspension, under 97KPa condition, continue evaporation again, eliminate residual organic solvents, until adularescent suspension is formed, by white suspension with the centrifugal 30 ~ 40min of 15000r/min, supernatant is poured in centrifuge tube, the cyclobutane pyrimidine dimmer photolyase do not wrapped into is separated with liposome, collecting precipitation, at-20 DEG C of freezen protective, obtained cyclobutane pyrimidine dimmer photolyase liposome.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310080376.3A CN103212066B (en) | 2013-03-14 | 2013-03-14 | A kind of preparation method of cyclobutane pyrimidine dimmer photolyase liposome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310080376.3A CN103212066B (en) | 2013-03-14 | 2013-03-14 | A kind of preparation method of cyclobutane pyrimidine dimmer photolyase liposome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103212066A CN103212066A (en) | 2013-07-24 |
| CN103212066B true CN103212066B (en) | 2016-01-06 |
Family
ID=48810449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310080376.3A Expired - Fee Related CN103212066B (en) | 2013-03-14 | 2013-03-14 | A kind of preparation method of cyclobutane pyrimidine dimmer photolyase liposome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103212066B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08266275A (en) * | 1995-03-29 | 1996-10-15 | Shiseido Co Ltd | Dna repair enzyme |
| CN101144088A (en) * | 2006-09-15 | 2008-03-19 | 四川大学 | Dunaliella salina CPD photolyase, photolyase liposome prepared from the same and preparation method for the photolyase liposome |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2005073373A1 (en) * | 2004-01-29 | 2007-09-13 | 社団法人芝蘭会 | Method for collecting damaged DNA fragments |
| WO2007005596A2 (en) * | 2005-07-01 | 2007-01-11 | Applied Genetics Incorporated Dermatics | Production of cpd glycosylases |
-
2013
- 2013-03-14 CN CN201310080376.3A patent/CN103212066B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08266275A (en) * | 1995-03-29 | 1996-10-15 | Shiseido Co Ltd | Dna repair enzyme |
| CN101144088A (en) * | 2006-09-15 | 2008-03-19 | 四川大学 | Dunaliella salina CPD photolyase, photolyase liposome prepared from the same and preparation method for the photolyase liposome |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103212066A (en) | 2013-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8481298B2 (en) | Separation matrix for viral purification | |
| US9663766B2 (en) | Methods for purifying adenovirus vectors | |
| CN101638427B (en) | Method for purifying virus antigens | |
| Wu et al. | A mixed-mode resin with tryptamine ligand for human serum albumin separation | |
| CN102234332B (en) | Process for separating and purifying recombinant human serum albumin and fusion protein thereof | |
| CN105713080A (en) | Stabilization of bio-sensors for in vivo applications | |
| CN105018435A (en) | Purifying method for virus-like particle | |
| CN113308456B (en) | D-psicose 3-epimerase mutant with enhanced thermal stability | |
| CN103212066B (en) | A kind of preparation method of cyclobutane pyrimidine dimmer photolyase liposome | |
| Huang et al. | A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha | |
| CN103675164B (en) | Method for separating estrogen endocrine disruptors in detection environment | |
| CN107090442B (en) | N-glycosyltransferase BtNGT and application thereof | |
| Chu et al. | High-throughput process development for recombinant human serum albumin separation from Pichia pastoris broth with mixed-mode chromatography | |
| CN107603959A (en) | The method for improving buffer solution salt ionic concentration purified virus | |
| CN109486780A (en) | A kind of ω-transaminase mutant that catalytic efficiency improves | |
| CN105017407A (en) | Purification method for recombinant human epidermal growth factor | |
| CN109134621A (en) | A kind of purification process of HBcAg-VLP or HBcAg-VLP derivative | |
| CN104862331B (en) | A kind of method of solubility expression Rhodococcus equi Disease-causing gene VapA albumen | |
| Gomes et al. | Applications of gellan natural polymer microspheres in recombinant catechol-O-methyltransferase direct capture from a Komagataella pastoris lysate | |
| CN102327608B (en) | Purifying method of encephalitis B vaccine | |
| CN102181468A (en) | Method for efficiently expressing and purifying mature peptide in S.cescerevisiae metallothionein | |
| CN102327607B (en) | Production method of encephalitis B vaccine preparation | |
| Liu et al. | An affinity-based aqueous two-phase mixed micellar system and its purification of Yeast 3′, 5′-bisphosphate nucleotidase | |
| CN105734097B (en) | A kind of MIM-I-BAR protein extraction purification process | |
| CN108676829A (en) | A method of removal acellular pertussis components endotoxin in vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160106 Termination date: 20170314 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |