CN103834677A - Recombinant expression method for human cryptochrome protein I (hCRY1) and application of human cryptochrome protein I (hCRY1) in preparation of radiotherapy protective agent - Google Patents

Abstract

The invention relates to a recombinant expression method for a human cryptochrome protein I (hCRY1) and an application of the hCRY1 in the preparation of a radiotherapy protective agent. The method comprises the steps of recombinant expression of the protein hCRY1 in a prokaryotic organism, purification, activation, functional detection and the like. According to the method, about 20 to 25 milligrams of hCRY1 can be averagely obtained from every liter of bacterial liquid, the yield is far higher than that of any other conventional method, and the cost is calculated to be far lower than that of a eukaryotic expression and purification method and a polypeptide chemical synthesis method. Moreover, a protein product obtained by the method is proved to have ray absorption and radiation injury protection functions, is an active protein potential to be developed into a radiotherapy protection medicament, and can be used as the radiotherapy protective agent.

Description

The recombinant expression method of people's cryptochrome protein I (hCRY1) and in the application of preparing in radiation therapy protective reagents

Technical field

The present invention relates to the recombinant expression method of hCRY1 albumen and in the application of preparing in radiation therapy protective reagents.

Background technology

Cancer is the maximum enemy of human health, and the number that cancer is died from the whole world every year approaches 1,000 ten thousand people.In the clinical treatment of cancer, operation, radiotherapy and chemotherapy are three kinds of topmost methods for the treatment of, wherein radiotherapy because of its indication more wide in range, selectivity is larger, more than 70% malignant tumor patient all needs to accept radiotherapy in certain stage of its treatment.

Radiocurable object is to greatest extent radiation dose to be focused in diseased region (target area); killing off tumor cells; and normal surrounding tissue or organ are subject to less or avoid unnecessary irradiation, some vitals, as brain stem, crystal, spinal cord, kidney, sexual gland etc., need special protection.No matter be conventional radiotheraphy technology or modern accurate radiotherapy technology, all can not accomplish to protect healthy tissues completely in treatment tumour, for example, need the tumour of irradiating to have more vitals or healthy tissues around; Tumour and healthy tissues or vitals are interlaced; Tumor tissues holds vitals etc.Therefore, find some methods of protecting as much as possible normal skin, tissue, organ and just become the key point that alleviates patient suffering, raising result for the treatment of in Patients During Radiotherapy.For this reason present patent application express and purifying restructuring hCRY1---a kind of have the protein of repairing uv irradiating damage function, and preliminary identification the effect of this albumen in gamma absorption, ultraviolet injury protection.

Modern time biology thinks, at occurring in nature from protistan to higher organism, and all vital movements of the mankind all exist according to certain rule operation, periodic vital movement phenomenon.Prove through a large amount of experimental studies, this vital movement phenomenon is obvious rhythmic activity, is called biorhythm.The cryptochrome gene (Cry) of finding for 1993 is exactly one of Circadian Gene of finding of current people.Cryptochrome is the Photoreceptors that one can be experienced blue light and near-ultraviolet light (330-390nm) region, in the plant of almost all kinds, all finds to some extent, has the homologous gene of coding cryptochrome simultaneously in insect, Mammals.Different types of cryptochrome is all made up of with the apoprotein of replying optical signal the chromophore that accepts optical signal, and apoprotein part has two structural domains.The PHR structural domain that is wherein positioned at N end is high conservative, and this structural domain and photodestruciton enzyme are quite similar, be the calmodulin binding domain CaM of chromophore, but it does not have photodestruciton enzymic activity; The DAS structural domain of C end is a larger non-conservative region of length variations in different plant species, is generally responsible for the output of signal.

Up to now, the cryptochrome of finding in animal body mainly contains two kinds of CRY1 and CRY2, they and photodestruciton enzyme family regulate the adaptation reaction of organism for ultraviolet ray and blue light light exposure jointly, participate in the regulation and control that DNA light is repaired, and know with growth, growth, the magnetic strength of animal and physiological rhythm all has close contacting.Compared with CRY2, it is mostly that CRY1 participates in is that light dependent form is transcribed inhibition, the rhythm and pace of moving things associated protein direct effects such as it and Baml1/CLOCK albumen dimer, Per1, and in rhythmicity physiological period, directly play negative regulation effect.

The radiation exposure such as ultraviolet, X-ray can cause producing in cell DNA double splitting of chain, DNA double splitting of chain inducing cell produces H2AX FOCI, the latter plays an important role in the reparation of DNA double splitting of chain, therefore in the observation of this experiment by immunofluorescence and quantization cell, the fluorescence intensity of H2AX FOCI detects the degree of injury of cell, and has further confirmed the function of hCRY1 in Protective agent on radiation injury.

Because the CRY1 content of native state in animal is low, separate a large amount of natural products of acquisition very difficult.And the synthetic high cost of polypeptide lacks the application prospect as drug manufacture.Up to now, also cross the Prokaryotic expression, purification method of animal CRY1 albumen without bibliographical information.Therefore the technological line of a set of Prokaryotic expression, purification hCRY1 has been sought in this research; by building its prokaryotic expression carrier pET28a (+)-hCRY1; in E.coliBL21 (DE3), express; and separation and purification goes out the recombinant protein with gamma absorption and ultraviolet injury protection activity of higher degree; for the further research hCRY1 condition of providing convenience, also for being become to new radiation therapy protective reagents, hCRY1 exploitation provides theoretical basis.

Summary of the invention

The present invention relates to a kind of expression method of the hCRY1 of restructuring albumen, particularly a kind of prokaryotic expression method of the hCRY1 albumen of recombinating.

The prokaryotic expression method of restructuring hCRY1 albumen of the present invention comprises the following steps: construction expression plasmid, induction expression of recombinant proteins, purification of recombinant proteins, recombinant protein activity and Function detection.

Wherein preferred, the step of above-mentioned construction expression plasmid comprises: hCRY1 is connected into prokaryotic expression carrier, as pET28a (+).Be specially: obtain hCRY1 gene by pcr amplification, then cut and be connected with T4DNA ligase enzyme afterwards with restriction enzyme XhoI and BamlI enzyme respectively with carrier pET28a (+), finally obtain recombinant expression plasmid pET28a (+)-hCRY1.Wherein, preferred, hCRY1 albumen is as shown in SEQ ID NO:1.

Wherein preferred, the step of above-mentioned induction expression of recombinant proteins comprises: the expression plasmid building is proceeded to abduction delivering, the wherein preferred E.coli BL21 of host cell (DE3) after host cell; Wherein preferably proceeding to after host cell, screening by SDS-PAGE the clone cell that expression amount is high.Be specially: pET28a (+)-hCRY1 is proceeded in competent cell E.coli BL21 (DE3) and coated plate cultivation, choose several (5) different single colony clone next day and access respectively in the LB substratum containing kantlex, shake after bacterium spends the night and be transferred to and fresh carry out amplification culture containing in the LB substratum of kantlex with 1:100; Centrifugal receipts bacterium after shaking bacterium and induce 6h in Erlenmeyer flask; By PBS washing resuspended for received thalline, carry out ultrasonication after adding proteinase inhibitor PMSF; From every part of broken sample, get 20 μ l samples and carry out SDS-PAGE analysis, pick out that target protein band is brighter, the clone of weak (being that expression amount is high) of foreign protein band, protect bacterium for-80 ℃ and store and called after BL21-hCRY1-b.

Wherein preferred, the step of above-mentioned purification of recombinant proteins comprises, by the bacterial strain fragmentation of abduction delivering, centrifugal, obtains inclusion body, then by renaturing inclusion bodies, the activated recombinant protein of purified rear acquisition.Wherein, the step of renaturing inclusion bodies comprises: washing inclusion body, dissolves inclusion body, gradient dialysis, renaturation.Be specially: with the inclusion body washings (solution A) of the new configuration of 20ml, inclusion body is resuspended, centrifugal reservation precipitation; In precipitation after washing, add the solubilization of inclusion bodies liquid (solution B) of the new configuration of 20ml, first precipitation is blown and beaten into fritter, carry out again ultrasonication hydrotropy, after fragmentation, be positioned over 24 ℃ of shaking tables, 100rpm dissolves 2h, centrifugation retain supernatant after dissolving, precipitation repeats above-mentioned steps with appropriate lysate again and dissolves, until precipitation volume no longer obviously reduces; Protein solution is packed in dialysis tubing, the dialyzate dialysis containing 6M urea, 50mMTris-base, 50mM NaCl with 2L in 5L beaker, and gradually urea concentration in dialyzate is down to 3M; The dialysis tubing that protein solution is housed is moved to 4L protein renaturation liquid (solution C) from dialyzate before, in 4 ℃ of environmental chambers, stir dialysis 24h; After this dialysis tubing that protein solution is housed is moved into again and in 2L sample-loading buffer (solution D), in 4 ℃ of environmental chambers, stir dialysis, until have in dialysis tubing, obvious white is cotton-shaped to be separated out and quantity no longer increases (about 24-48h).Wherein, the proportioning of inclusion body washings (solution A) is: 50mM Tris-base, 0.5mM EDTA, 50mM NaCl, 1%Triton X-100,2M urea, with NaOH adjusting pH to 8.0; The proportioning of solubilization of inclusion bodies liquid (solution B) is: 50mM Tris-base, 0.5mM EDTA, 50mM NaCl, 8M urea, 1mM PMSF, with NaOH adjusting pH to 8.0, the proportioning of protein renaturation liquid (solution C) is: 100mM Tris-base, 400mM arginine, 2M urea, 5mM EDTA, 5mM GSH, 0.5mM GSSG, with NaOH adjusting pH to 8.0; The proportioning of sample-loading buffer (solution D) is 50mM Tris-base, 50mM NaCl, with NaOH adjusting pH to 8.0.Wherein, the step of purifying comprises: after ni-sepharose purification, and ultrafiltration displacement.The operating process of ni-sepharose purification is specially: by the centrifugal reservation supernatant of protein liquid after dialysis, with 10KD super filter tube protein concentrate volume, in 4 ℃ of environmental chambers, be splined in advance and purify in nickel post with the good 1ml of solution D balance, collect that stream is worn liquid and by its loading again, repeatedly to guarantee target protein and the abundant combination of nickel post, wash post with sample-loading buffer afterwards, residual sample on purification column sidewall is cleaned, wash away the foreign protein of combination on purification column with the sample-loading buffer that 10ml contains 40mM imidazoles after, the sample-loading buffer wash-out target protein that contains 200mM imidazoles with 10ml again, repeatedly repeat wash-out, to guarantee elution efficiency.The operating process of ultrafiltration displacement is specially: by after the protein sample dilution of upper step gained, then carry out ultrafiltration displacement with 10kD super filter tube is concentrated, repeatedly, to remove imidazoles in elutriant, finally protein solution is concentrated into 5ml.

Wherein preferred, the step of above-mentioned recombinant protein activity and Function detection comprises, utilizes FOCL fluorescence intensity in born of the same parents to detect the functional of hCRY1 albumen.Be specially: hCRY1 and treated BSA(reference protein that purifying is obtained) add in the substratum that contains Hela cell with same concentrations respectively, after X-ray irradiates, successively with after antibody Phospho-Histone H2A.X, goat anti-rabbit igg/FITC incubated cell, film-making is observed, under identical observation condition, the FOCI fluorescence intensity difference forming in the visible two groups of cells of naked eyes is very remarkable.Wherein being treated to of BSA configured to certain density BSA with imidazoles, flow out and during with hCRY1 sample preparation after identical ultrafiltration displacement, be diluted to identical with hCRY1 concentration (cBSA) in contrast through nickel post loading.

In addition, for the minimizing that further confirms FOCI in cell is the radiation protection effect due to hCRY1, in HaCaT cell, carry out the experiment of hCRY1 concentration gradient and set up the control group irradiating without X-ray, result shows, in the time that the hCRY1 concentration in cell culture medium reaches 50 μ g/ml, the fluorescence intensity of FOCI obviously reduces.In addition, hCRY1 and contrast BSA are also detected respectively to apoptotic impact, process two groups of same cells with hCRY1 and the BSA after treatment of same concentrations respectively, together with not adding the cell of any albumen with another group after 1h, detect apoptosis situation, with cell apoptosis detection kit, cell is carried out to two dying and analyze with flow cytometer afterwards; Within result shows 1h, no matter be that hCRY1 or BSA can not cause apoptosis, this result further proves that the reduction of FOCI fluorescence intensity is acellular apoptosis because of the minimizing of DNA institute damaged in cell.

At present, escherichia expression system is widely used in the production of recombinant protein, and its advantage is that expression level is higher, simple to operate.This research selects pET28a (+) as expression vector, by above-mentioned Prokaryotic expression, purification method, average every liter of bacterium liquid can be gathered in the crops the about 20-25mg of hCRY1, productive rate is far above current known additive method, and due to the needed substratum of this Prokaryotic expression, purification method and subsequent purification raw material relatively inexpensive, cost is as calculated far below eukaryotic expression purification process and chemically synthesized polypeptide method.In large-scale production and application, the possibility that the method provides renaturation raw material to reuse, this also will greatly reduce costs.This method operating process is simple in addition, and serious forgiveness is high, and operator do not need a large amount of expertise can carry out production work.And the protein product empirical tests being obtained by present method purifying has gamma absorption function and the Protective agent on radiation injury activity of expection, be to there is exploitation to become the activated protein of radiotherapy protection class medicine potential quality.

Owing to having verified that by embodiment the cell that is added with hCRY1 produces H2AX FOCI and obviously reduces in the time being subject to RADI, and this minimizing has not caused damage caused by hCRY1 to cell, therefore can confirm that hCRY1 has the effect of ultraviolet injury protection to cell.Therefore the restructuring hCRY1 that prepared by the present invention can be used as radiation therapy protective reagents, as is prepared into radiation therapy protective reagents class medicine, particularly skin radiation therapy protective reagents.The present invention also provides a kind of composition, and said composition contains restructuring hCRY1 and pharmaceutically acceptable carrier or auxiliary material prepared by the present invention, and said composition can be used for radiotherapy protection related fields.

Accompanying drawing explanation

Now relevant the present invention accompanying drawing is described, wherein:

Fig. 1: the structure schema of prokaryotic expression plasmid pET28a (+)-hCRY1;

Fig. 2: left figure is the agarose gel electrophoresis detected result of PCR product, and the enzyme that right figure is prokaryotic expression plasmid is cut detected result, wherein M is DNA Marker;

Fig. 3: the selection result of high efficient expression hCRY1 bacterial strain, upper figure is different single bacterium colony abduction delivering effect detection results, and figure below is corresponding band gray-scale value and target protein and foreign protein ratio result, and wherein M is protein Marker;

The abduction delivering result of Fig. 4: BL21-hCRY1-b, upper figure is SDS-PAGE result, figure below is Western blot result;

Fig. 5: upper figure is Mass Spectrometric Identification target protein slice result, underscore part is the part matching with hCRY1 aminoacid sequence, and figure below is above sequence comparison result in ncbi database, and wherein hCRY1 score is the highest;

Fig. 6: left figure is the measuring result of protein concentration before and after ni-sepharose purification, and right figure is in ni-sepharose purification process, before loading, loading stream wears liquid, 50mM imidazoles and wash the SDS-PAGE analytical results of assorted liquid and 300mM imidazoles elutriant, wherein M is protein Marker;

Fig. 7: left figure is the measuring result of protein concentration before and after ultrafiltration displacement, and right figure is the SDS-PAGE analytical results before and after ultrafiltration displacement, and wherein M is protein Marker;

Fig. 8: left figure is FOCI fluorescence intensity comparison result in the postradiation control group of X-ray (cBSA) and experimental group (hCRY1) cell, and right figure is above two groups of cell fluorescence intensity quantized result (* * * P<0.001);

Fig. 9: the picture left above is intracellular FOCI fluorescence intensity result while adding the HaCaT cell of different concns gradient hCRY1 to irradiate without X-ray, lower-left figure is the HaCaT cell intracellular FOCI fluorescence intensity result after X-ray irradiates that adds different concns gradient hCRY1, right figure is every group of cell fluorescence intensity quantized result (* * P<0.05, * * * P<0.001);

BSA after Figure 10: hCRY1 and processing is on the apoptotic comparison result that affects.

Embodiment

embodiment

Described in below the present invention, be that the present invention is further described with embodiment, the present invention is not limited to following examples.Embodiment for the method for the invention is as follows:

1, the structure of prokaryotic expression plasmid pET28a (+)-hCRY1:

The hCRY1 eukaryon expression plasmid pcDNA3.1-hCRY1 preserving take laboratory is as masterplate, coding region sequence design Auele Specific Primer according to hCRY1: upstream primer is hCRY1FW (5'-ATACTCGAGGCTAAGCCTTCC-3'), downstream primer is hCRY1RV (5'-CGCGGATCCTACGTTTATACT-3') (upstream and downstream primer is synthetic by the prosperous bio tech ltd of Beijing AudioCodes).Pcr amplification goes out required object fragment (total system 25uL, 94 ℃ of denaturation 3min, 32 circulations: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 2min, last 72 ℃ of extensions 10 minutes).With restriction enzyme XhoI and BamlI(TAKARA) enzyme cuts object fragment and prokaryotic expression carrier pET28a (+), then use carrier and the object fragment of T4DNA ligase enzyme connecting band toughness end, final prokaryotic expression plasmid pET28a (+)-hCRY1 that obtains, as shown in Figure 1.

Amplified production is detected with agarose gel electrophoresis, obtain the band that size is about 1900bp, as shown in figure as left in Fig. 2, confirm that object fragment successfully increases.Cut product with electrophoresis detection enzyme, can obtain object fragment band and the big or small carrier strap that is about 5300bp that size is about 1900bp, as shown in figure as right in Fig. 2, confirm that prokaryotic expression plasmid successfully constructs.

2, induction expression of recombinant proteins

2.1 outstanding clones' screening

Above-mentioned recombinant plasmid pET28a (+)-hCRY1 is converted in competent cell E.coli BL21 (DE3), coated plate is cultivated, 5 different single colony clones of picking next day, access is containing in the LB substratum of 50mg/L kantlex respectively, shakes after bacterium spends the night and is transferred to and fresh carries out amplification culture containing in the LB substratum of 50mg/L kantlex with 1:100.In Erlenmeyer flask, shake bacterium after (being preferably about 0.6) between bacterium liquid OD600 value 0.6-0.8, add 1mM inductor IPTG, centrifugal receipts bacterium after 24 ℃, 200rpm induction 6h.Received thalline is washed and is resuspended in 30ml PBS with PBS, carry out ultrasonication after adding 1mM proteinase inhibitor PMSF.From every part of broken sample, get 20 μ l samples and carry out SDS-PAGE analysis, as shown in electrophoresis result figure as upper in Fig. 3, measurement target albumen and foreign protein band gray-scale value calculate ratio respectively, select the b clone that target protein ratio is the highest (being the clone that target protein band is brighter, foreign protein band is weak) (Fig. 3 figure below), protect bacterium for-80 ℃ and store and called after BL21-hCRY1-b.All experiments afterwards are all used this bacterial strain to complete.

The abduction delivering of 2.2 fusion roteins and evaluation

Using b clone superinduce to express tests and amplifies induction system to 1L, after bacterial cell disruption, supernatant is analyzed with SDS-PAGE respectively with precipitation, as shown in electrophoresis result figure as upper in Fig. 4, carry out Western Blot with His antibody, result, as shown in Fig. 4 figure below, proves the form expression of most of target protein with inclusion body.Target stripe is cut, transfer to Institute of Micro-biology of Chinese Academy of Sciences centre to carry out Mass Spectrometric Identification.As shown in Mass Spectrometric Identification result figure as upper in Fig. 5, the aminoacid sequence of hCRY1 albumen is as shown in SEQ ID NO:1.As shown in Fig. 5 figure below, confirm that this albumen is hCRY1 really with ncbi database comparison result.

3, purification of recombinant proteins

The washing of 3.1 inclusion bodys

With the inclusion body washings of the new configuration of 20ml (solution A, filling a prescription is: 50mM Tris-base, 0.5mM EDTA, 50mM NaCl, 1%Triton X-100,2M urea, regulate pH to 8.0 with NaOH), inclusion body is resuspended, ultrasonication, centrifugal reservation precipitation.

The dissolving of 3.2 inclusion bodys

In precipitation after washing, add the solubilization of inclusion bodies liquid (solution B of the new configuration of 20ml, formula is 50mM Tris-base, 0.5mM EDTA, 50mM NaCl, 8M urea, 1mM PMSF, with NaOH adjusting pH to 8.0), first precipitation is blown and beaten into fritter, then carry out ultrasonication hydrotropy.After fragmentation, be positioned over 24 ℃ of shaking tables, 100rpm dissolves 2h.Centrifugation retain supernatant after dissolving, precipitation repeats above-mentioned steps dissolving with appropriate lysate again, until precipitation volume no longer obviously reduces.Finally will repeatedly dissolve rear supernatant and be collected together, remove precipitation.

3.3 gradient dialysis renaturations

Dialysis is omnidistance to be carried out and keeps magnetic agitation in 4 ℃ of environmental chambers.Upper step gained protein solution is packed in 10kD dialysis tubing, the dialyzate dialysis containing 6M urea, 50mM Tris-base, 50mM NaCl with 2L in 5L beaker, and gradually urea concentration in dialyzate is down to 3M.The dialysis tubing that protein solution is housed is moved to 4L protein renaturation liquid (solution C from dialyzate before, formula is 100mM Tris-base, 400mM arginine, 2M urea, 5mM EDTA, 5mM GSH, 0.5mM GSSG, with NaOH adjusting pH to 8.0) middle dialysis 24h.After this again the dialysis tubing that protein solution is housed is moved into 2L sample-loading buffer (solution D, formula is 50mM Tris-base, 50mM NaCl, regulate pH to 8.0 with NaOH) in dialyse, until have in dialysis tubing, obvious white is cotton-shaped to be separated out and quantity no longer increases (about 24-48h).

3.4 ni-sepharose purification

By the centrifugal reservation supernatant of protein liquid after dialysis.Protein solution cumulative volume is concentrated into 20ml with 10KD super filter tube.10 times of the total protein diluted samples taking a morsel after renaturation, with Bradford protein content detection kit (Kai Ji biological substance Development Co., Ltd buys by Nanjing) measurement protein concentration.In 4 ℃ of environmental chambers, be splined in advance and purify in nickel post (Qi Hai Fu Tai bio tech ltd buys by Shanghai) with the good 1ml of solution D balance, collect that stream is worn liquid and by its loading again, repeatedly (at least 5 times) are to guarantee target protein and the abundant combination of nickel post.Use afterwards sample-loading buffer (solution D) to wash post, the residual sample on purification column sidewall is cleaned.Wash away the foreign protein of combination on purification column with the sample-loading buffer that 10ml contains 40mM imidazoles after, then the sample-loading buffer wash-out target protein that contains 200mM imidazoles with 10ml, wash-out repeatedly repeated to guarantee elution efficiency.Albumen, after ni-sepharose purification, is again measured protein concentration (as shown in figure as left in Fig. 6) after 10 times of the diluted samples of taking a morsel 10ml elutriant, and the total protein rate of recovery that can obtain after ni-sepharose purification is about 22.2%.The stream of collecting in protein solution sample before ni-sepharose purification and ni-sepharose purification process is worn to liquid, washed assorted liquid, elutriant and jointly carry out SDS-PAGE electrophoresis, after coomassie brilliant blue staining as shown in figure as right in Fig. 6, visible foreign protein is significantly removed, and purity of protein has reached more than 95%.

3.5 ultrafiltration displacements

By 10 times of sample-loading buffer dilutions for the protein sample of upper step gained, be concentrated into original volume with 10KD super filter tube again, repeatedly (at least 3 times), to remove imidazoles in elutriant, finally protein solution is concentrated into 5ml, obtains solubility hCRY1 protein solution finished product.Before and after displacement, take a morsel respectively after 10 times of diluted samples and use Bradford protein content detection kit (Kai Ji biological substance Development Co., Ltd buys by Nanjing) to measure protein concentration, as shown in figure as left in Fig. 7, protein recovery is about 93.8%.Respectively sample before and after ultrafiltration displacement is carried out to SDS-PAGE electrophoresis and coomassie brilliant blue staining, as shown in figure as right in Fig. 7.

4, recombinant protein activity and Function detection

Getting protein solution after purifying, to be diluted to 500 μ g/ml for subsequent use to do, simultaneously with 300mM imidazoles configuration 1mg/ml BSA albumen, through nickel post loading flow out and with the identical ultrafiltration displacement of hCRY1 sample after, be diluted to 500 μ g/ml (cBSA) in contrast.When being cultured to about 80%-90% cell density, changes by Hela cell climbing sheet bed board liquid, after three hours, respectively by the hCRY1 getting ready and reference protein according to adding cell culture fluid with the ratio of substratum 1:1, making total liquid volume in each hole is 2ml, adding final concentration of protein is 250 μ g/ml.At once two groups of cells are placed in X-ray irradiating machine and are irradiated afterwards, per minute dosage is 1.132/Gy, irradiation time 530s, and total dose is 10Gy.Substratum (DMEM) the continuation cultivation at once cell culture fluid being all changed to after irradiating before experiment was received cell climbing sheet after 45 minutes.Develop a film 3 times with PBS, 100% methyl alcohol-20 ℃ fixed cell 5min, wash 3 times with PBS again, after 5%BSA sealing is spent the night,, then wash 3 times with PBS in 37 ℃ of incubated cell 1h with antibody Phospho-Histone H2A.X, use again antibody goat anti-rabbit igg/FITC in 37 ℃ of incubated cell 1h, wash rear mounting for PBS3 time, the brightness of two groups of endocellular phosphorus light bodies of confocal laser scanning microscope is got respectively 3 different visual field statistical average fluorescence intensities for every under identical shooting condition.Under identical observation condition, the FOCI fluorescence intensity difference forming in the visible two groups of cells of naked eyes is (the left figure of Fig. 8) very significantly, choose at random 3 visuals field and fluorescence intensity is quantized, extremely significantly (P<0.001) (right figure of Fig. 8) of difference between two groups.Hence one can see that, and hCRY1 prepared by present patent application has reduced the fluorescence intensity of FOCI in cell, and cell has been played to certain provide protection, has reduced the infringement of X-ray.

For the minimizing that further confirms FOCI in cell is the radiation protection effect due to hCRY1, in present patent application, in HaCaT cell, further carry out the experiment of hCRY1 concentration gradient and the cellular control unit of processing without X-ray is set, set up concentration gradient with above-mentioned BSA solution dilution hCRY1 solution after treatment, process cell with same method, film-making is also observed.Result shows, in the time that the hCRY1 concentration in cell culture medium reaches 50 μ g/ml, the fluorescence intensity of FOCI obviously reduces (Fig. 9).

Present patent application has also detected respectively hCRY1 and has processed BSA afterwards to apoptotic impact.In two groups of cells, add respectively 250 μ g/ml hCRY1 and 250 μ g/ml BSA after treatment, together with not adding the cell of any albumen with another group after 1h, detect apoptosis situation, with cell apoptosis detection kit (Kai Ji biological substance Development Co., Ltd buys by Nanjing), cell is carried out pair dying and analyze with flow cytometer afterwards.Within result shows 1h, no matter be that hCRY1 or BSA can not cause apoptosis, this result further proves that the reduction of FOCI fluorescence intensity is acellular apoptosis (Figure 10) because of the minimizing of DNA institute damaged in cell.

Table 1: protein purification table

By statistics, every liter of bacterium liquid can obtain about 25mg hCRY1; The total hCRY1/ previous step of yield=each step total protein concentration

Claims (10)

1. a prokaryotic expression method for recombinant human cryptochrome protein I (hCRY1), is characterized in that, comprises the following steps: construction expression plasmid, induction expression of recombinant proteins, purification of recombinant proteins, recombinant protein activity and Function detection.

2. method as claimed in claim 1, is characterized in that, wherein the step of construction expression plasmid comprises: hCRY1 gene fragment is connected into prokaryotic expression carrier pET28a (+), obtain recombinant expression plasmid pET28a (+)-hCRY1.

3. method as claimed in claim 1, is characterized in that, wherein induces the step of expression of recombinant proteins to comprise: the expression plasmid building is proceeded to abduction delivering after host cell E.coli BL21 (DE3); Wherein preferably proceeding to after host cell, screening by SDS-PAGE the clone cell that expression amount is high.

4. method as claimed in claim 1, is characterized in that, wherein the step of purification of recombinant proteins comprises: by the bacterial strain fragmentation of abduction delivering, centrifugal, obtain inclusion body, then by renaturing inclusion bodies, the activated recombinant protein of purified rear acquisition.

5. method as claimed in claim 4, is characterized in that, wherein the step of renaturing inclusion bodies comprises: washing inclusion body, dissolves inclusion body, gradient dialysis renaturation; The step of purifying comprises: after ni-sepharose purification, carry out ultrafiltration displacement.

6. method as claimed in claim 1, is characterized in that, wherein the step of recombinant protein activity and Function detection comprises: utilize FOCL fluorescence intensity in born of the same parents to detect activity and the function of hCRY1 albumen.

7. the restructuring hCRY1 albumen that prepared by the arbitrary described method of claim 1-6 is in the application of preparing in radiation therapy protective reagents.

8. composition, contains restructuring hCRY1 albumen and pharmaceutically acceptable carrier or auxiliary material prepared by the arbitrary described method of claim 1-6.

Described in claim 8 composition in the application of preparing in radiation therapy protective reagents.

10. application as described in claim 7 or 9, is characterized in that, wherein radiation therapy protective reagents is skin radiation therapy protective reagents.

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