CN105968182A

CN105968182A - Production process of recombinant human cryptochrome protein I (hCRY1) and composition thereof

Info

Publication number: CN105968182A
Application number: CN201610392874.5A
Authority: CN
Inventors: 黄文林; 吴全德
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-06-03
Filing date: 2016-06-03
Publication date: 2016-09-28
Also published as: WO2017205996A1

Abstract

The invention relates to a production process of a recombinant human cryptochrome protein I (hCRY1) and a composition thereof. The production process comprises the steps that the hCRY1 gene is cloned to pET-28 a to obtain a recombinant carrier pET-28 a (+)-hCRY1; the recombinant carrier is led to E. coli BL21 (DE3), and screening is performed to obtain a gene engineering bacterium BL21-hCRY1-b; the BL21-hCRY1-b is cultured in a fermentation tank and recombinant protein induced expression is performed; an inclusion body is collected and washed; the inclusion body undergoes degeneration dissolving; protein affinity chromatography purification is performed; protein gradient dialysis renaturation is performed; ultrafiltration protein concentration and solution displacement are performed to obtain the recombinant hCRY1 protein. The hCRY1 yield of the production process is 200-300 mg/L and is far higher than those of other existing known hCRY1 production methods.

Description

The production technology of a kind of recombined human cryptochrome protein I (hCRY1) and combinations thereof thing

Technical field

The present invention relates to bio-pharmaceuticals technology field, production technology being specifically related to a kind of recombined human cryptochrome protein I (hCRY1) and combinations thereof thing.

Background technology

Escherichia coli are engineering bacterias the most frequently used in current prokaryotic expression system, and it is clear that it has genetic background, genetic safety；Expressing quantity is high, and foreign gene expression levels is far above other gene expression systems；Breeding is rapidly, easy and simple to handle, can produce target protein with rapid scale；Advantage that toxigenic capacity is cheap etc.；It is approved by the FDA in the United States the genetic engineering receptor biological into safety.During building recombination engineering, it will usually use operon to control the controlled expression of exogenous gene.Isopropylthiogalactoside (IPTG) is efficient derivant the most frequently used in prokaryotic expression system, it is possible to stably start lac operon, makes genes of interest obtain the most efficiently and expresses.Control except expression vector, on expression vector, other auxiliary sequencels and genes of interest composition amalgamation and expression is often inserted in order to improve activity and the simplification downstream purification operation etc. of protein product, as inserted purification tag 6-His, GST, CAT etc., fusion protein entered a step affinity chromatograph and i.e. can get the product that homogeneity is higher.Prokaryotic expression system defect there is also certain limitation, such as destination gene expression, often forms insoluble, inactive inclusion bodies；As a kind of prokaryotic expression system, when expressing eukaryote exogenous gene, also tending to be to lack post translational modification, protein active is greatly affected.Although gene engineering system expands yeast, insecticide, plant and mammalian cell to from escherichia coli, and also occurs in that the most novel eukaryotic expression system, but escherichia coli remain the important tool of gene expression.

Up to now, the cryptochrome having been found that in animal body mainly has CRY1 and CRY2 two kinds, they regulate organism to ultraviolet and the adaptation response of blue light light exposure jointly with photodestruciton enzyme family, participate in the function such as DNA reparation, and know with the growth of animal, growth, magnetic strength and physiological rhythm etc. has close ties.Studies have found that, CRY1 participates in multiple smooth dependent form Transcription inhibition in animal body, it is possible to directly act on the rhythm and pace of moving things associated protein such as BmaL/CLOCK albumen dimer, Per1, Per2；HCRY1 participates in the various physiological processes such as transcriptional control, DNA damage reparation in human body；cry1/cry2Gene knockoutP53 ^KOCells in vitro test shows, the sensitivity of radiation, radiomimetic drug is improved by cell, and apoptosis ratio significantly rises；The rise of CRYs, downward etc. also develop with the generation of cell carcinogenesis, tumor close relationship.

ü r and Song etc. constructed respectively in 2007 and derive fromD. Plexippus,A. Gambiae, andA. PernyiMaltose-binding Protein-CRY fusion protein expression vector,E.coli BL21Middle abduction delivering, volume of culture 12 L about gathers in the crops 2 mg fusion protein.ü r etc. constructed insect cell expression system in 2009, expressed the plant such as arabidopsis, fruit bat and nonmammalian CRYs, through FLAG affinitive layer purification, it is thus achieved that obtained the yield of 2 mg/L.For the abduction delivering aspect research of mammal CRYs, particularly hCRYs, the most do not obtain greater advance.Owing in mammal, the cryptochrome content of native state is low, separate a large amount of natural products of acquisition the most difficult.And Peptide systhesis high cost, lack the application prospect as drug manufacture.ü r etc. built hCRY2-Flag fusion protein in 2003 and express in Hela cell, volume of culture 10 L, after FLAG M2 affinitive layer purification, and only results 5-15 μ g fusion protein；Wang Xiaomings etc. construct HsCRY1 insect expression system, by transfection insecticide sf9 cell, express HsCRY1, obtain 4.8 mg albumen HsCRY1 in 50 mL cultivation scales, and after extrapolating amplification culture, yield is about 96 mg/L.

Before this, we report the correlational study utilizing escherichia coli expression hCRY1.Wherein, we use shake-flask culture recombination bacillus coli, after carrying out ultrasonic bacteria breaking, it is thus achieved that the destination protein expressed with inclusion bodies, then use the resuspended inclusion body of high concentration urea, and ultrasonic dissolution.After obtaining solubilization of inclusion bodies liquid, first by protein renaturation, after carry out affinitive layer purification again, then be concentrated by ultrafiltration with 10 kDa super filter tubes and solution replacement obtains the hCRY1 yield of 10-15 mg/L.

Summary of the invention

On the basis of research before, the present invention is directed to the problem that in prior art, hCRY1 yield is relatively low, it is provided that production technology of a kind of recombined human cryptochrome protein I (hCRY1) and combinations thereof thing.

The present invention is achieved in that

The production technology of a kind of recombined human cryptochrome protein I (hCRY1), comprises the following steps

(1) recombinant expression plasmid is built: willhCRY1Genetic fragment restructuring to prokaryotic expression carrier pET28a (+), it is thus achieved that recombinant expression plasmid pET28a (+)-hCRY1.Particularly as follows: obtained by PCR amplificationhCRY1Gene, then with carrier pET28a (+) be connected with T4 DNA ligase after restricted enzyme Xho I and Baml I enzyme action respectively, final obtain recombinant expression plasmid pET28a (+)-hCRY1.DescribedhCRY1The hCRY1 protein amino acid sequence that gene translation obtains is as shown in SEQ ID NO:1.

(2) build engineering bacteria: by recombinant expression plasmid pET28a (+)-hCRY1 proceeds to competence host cellE.coliBL21 (DE3), builds containing recombinant expression carrier pET-28a(+)-hCRY1 genetic engineering bacterium, then screens, by SDS-PAGE, the clone strain that hCRY1 expression is highBL21-hCRY1-b。

(3) recombinant and recombiant protein abduction delivering: strainBL21-hCRY1-bIncubated overnight, next day, picking list bacterium colony accessed in the LB fluid medium containing kanamycin, be transferred in the fresh LB culture medium containing kanamycin carry out secondary amplification culture with 1:100 after shaking bacterium 6 h, shake bacterium overnight after, ferment to NBS series of fermentation tank with 1:100 inoculum concentration, cultivation temperature 36.5-37.5 DEG C, dissolved oxygen 20-40%, pH6.8-7.4, mixing speed 200-350 rpm, after cultivating 5-8 hOD ₆₀₀During=7-8, add final concentration 0.5-3.0 mM IPTG inducing culture 3-4 h.

null(4) inclusion body is collected and washing: 3000-5000 rpm is centrifugal collects thalline，With power 200-250 W、Working time 3-8 s、The condition ultrasonication thalline 60-90 min of intermittent time 5-10 s，3000-5000 rpm is centrifuged 50-80min，Collection inclusion body precipitates，With component mM Tris Han 10-100、0.1-1.0 mM EDTA、0-100 mM NaCl、1-10% Triton X-100、1-2 M Urea，The resuspended inclusion body of lavation buffer solution of pH7.5-8.5，With power 100-150 W、Working time 3-8 s、The condition supersound washing 60-90 min of intermittent time 5-10 s，12000-14000 rpm is centrifuged 60-90 min and collects precipitation.

(5) inclusion body degeneration is dissolved: by component containing the inclusion body precipitation after 4-8 M guanidine hydrochloride, the resuspended washing of pH7.5-8.5 phosphate buffer, with power 200-250 W, working time 3-8 s, the condition ultrasonic dissolution 60-90 min of intermittent time 5-10 s；Dilute 6-10 times with containing 6-8 M Urea, pH7.5-8.5 phosphate buffer again, with power 200-250 W, working time 3-8 s, the condition ultrasonic dissolution 60-90 min of intermittent time 5-10 s, obtain albuminous degeneration solvent soln.

(6) albumen affinitive layer purification: by albuminous degeneration solvent soln 12000-14000 rpm high speed centrifugation 40-60 min, collect supernatant, through 0.22-0.45 μm membrane filtration, add imidazoles to the final concentration of 5-30 mM of imidazoles in filtrate, HIS label affinity chromatograph is carried out, chromatograph: AKT Purifier 100 with loading volume 1-1.5 CV, loading flow velocity 5-10 ml/min, elution flow rate 10-20 ml/min；Chromatographic stuffing: in ProteinIso Ni-NTA Resin, Ni-NTA His Bind Resin any one；Balance: balance 2-4 CV with buffer 0-500 mM NaCl, 6-8 M Urea, 0-60 mM imidazoles, pH7.5-8.5, washes away impurity and puts down to baseline；Eluting: with buffer 0-500 mM NaCl, 6-8 M Urea, 200-300 mM imidazoles, pH7.5-8.5 eluting 2-2.5 CV, collect eluted protein peak.

(7) protein ladder dialysis renaturation: the eluted protein peak sample collected is loaded in 30-50 kDa bag filter, comprises 6 M Urea in 8-10 times of volume, in 0-100 mM Tris, 0-100 mM NaCl, pH7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 4 M Urea, in 0-100 mM Tris, 0-100 mM NaCl, pH 7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 2 M Urea, in 0-100 mM Tris, 0-400 mM Argine, 0-10 mM EDTA, 0-10 mM GSH, 0-5 mM GSSG, pH 7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12h；Sample is transferred to comprise 1 M Urea, 0-100 mM Tris, 0-100 mM NaCl, 0-2% glycerol, in pH7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 0-100 mM Tris, 0-500 mM NaCl, 0-10% glycerol, in pH7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；After having dialysed, collect sample in bag filter.

(8) albumen is concentrated by ultrafiltration and solution replacement: use ultrafiltration instrument Millipore, with 30-50 kDa ultrafilter membrane bag, dialysis gained sample is concentrated by ultrafiltration: after sample concentration to original volume about 1/4-1/5, progressively add and comprise 0-100 mM Tris, 0-200 mM NaCl, 0-20% glycerol, the displacement buffer of pH7.5-8.5 is diluted, finally sample volume is concentrated into the 1/5 of original volume, obtain solvable hCRY1 protein solution, again after 0.22 μm membrane filtration, i.e. obtain solubility hCRY1 protein solution finished product.

Compositions, the restructuring hCRY1 albumen prepared containing above-mentioned production technology and pharmaceutically acceptable carrier or adjuvant.

Compared with research before, production technology is optimized by the present invention: abduction delivering in fermentor cultivation system；Two kind different denaturation liquid are successively used in combination and dissolve inclusion body.After obtaining the hCRY1 protein dissolution liquid of degeneration, first carrying out degeneration affinitive layer purification, after obtaining the hCRY1 eluting peak of higher degree, then carry out dialysis renaturation, be finally concentrated by ultrafiltration with 30 ~ 50kD ultrafilter membrane bag and obtain hCRY1, yield has reached 200-300 mg/L.HCRY1 yield of the present invention studies described method and our the disclosedest production method far above other; produced, by the present invention, the protein product empirical tests obtained and there is intended radiation absorption function and Protective agent on radiation injury activity, be the activated protein having and being developed into radiotherapy protection class medicine potential quality.

The invention has the beneficial effects as follows:

(1) the hCRY1 productivity of this production technology is at 200-300 mg/L, far above other hCRY1 production methods in prior art, is that we disclose 10-15 times of technique in early days.

(2) this technique chromatography purification phase, uses purification after degeneration, and the hCRY1 protein purification response rate is close to 100%, and purity is more than 95%；Overcoming before this after first renaturation in purifying process, renaturation stage albumen separates out with foreign protein, hanging column weak effect, the deficiency of protein degradation.

(3) this technique uses fermentor cultivation abduction delivering hCRY1 albumen, fermentation ends, and thalline weight in wet base improves to about 120 g from about 1 g of former shake-flask culture technique.

(4) this technique solubilization of inclusion bodies part, have employed 4-8M guanidine hydrochloride the most molten, then dissolves with 6-8 M carbamide secondary, improve the dissolved efficiency of inclusion body protein.

(5) in this technique protein renaturation stage, initially with hyperfiltration technique, sample is concentrated by ultrafiltration, reduces sample volume, thus it is required long-pending to dialysis buffer liquid to decrease dialysis renaturation, save the material input of about 5 times.

Accompanying drawing explanation

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

Fig. 2: left figure is the agarose gel electrophoresis testing result of PCR primer, and right figure is the enzyme action testing result of prokaryotic expression plasmid, and 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 be corresponding band gray value and target protein and foreign protein ratiometric result, and wherein M is protein Marker；

Fig. 4: during fermentation tank abduction delivering hCRY1, growth curve of bacteria；

Fig. 5:BL21-hCRY1-bAbduction delivering result, upper figure is SDS-PAGE result, and figure below is Western blot result；

Fig. 6: inclusion body degeneration course of dissolution SDS-PAGE detection；

Fig. 7: upper figure is Mass Spectrometric Identification target protein slice result, and underscore part is the part matched with hCRY1 aminoacid sequence, and figure below is above sequence comparison result in ncbi database, wherein hCRY1 highest scoring；

Fig. 8: during affinitive layer purification, to loading sample (Sample), loading penetrates (Flowthrough), and eluting reclaims peak (Elution) and carries out SDS-PAGE electrophoretic analysis result；

Fig. 9: use AKT purifier 100 chromatograph to carry out affinitive layer purification hCRY1 and chromatograph collection of illustrative plates；

Figure 10: before and after ultrafiltration concentration, sampling carries out SDS-PAGE electrophoretic analysis result；

Figure 11: left figure is the matched group (cBSA) after X-ray irradiates and experimental group (hCRY1) intracellular FOCI fluorescence intensity comparison result, and right figure is above two groups of cell fluorescence intensity quantized result (* * * P < 0.001)；

Figure 12: the picture left above is the HaCaT cell adding variable concentrations gradient hCRY1 intracellular FOCI fluorescence intensity results when irradiating without X-ray, lower-left figure is the HaCaT cell adding variable concentrations gradient hCRY1 intracellular FOCI fluorescence intensity results after X-ray irradiates, right figure is often to organize cell fluorescence intensity quantized result (* * P < 0.05, * * * P < 0.001)；

Figure 13: hCRY1 and process after BSA affect comparison result to apoptotic.

Detailed description of the invention

Describing the present invention below in conjunction with drawings and Examples, following example explain with invention optimal effectiveness.

Embodiment 1:

(1) recombinant expression plasmid is built: the hCRY1 eukaryon expression plasmid pcDNA3.1-hCRY1 preserved with laboratory, as masterplate, designs upstream and downstream specific primer: hCRY1FW according to the coding region sequence of hCRY1 (5'-ATACTCGAGGCTAAGCCTTCC-3'), hCRY1RV (5'-CGCGGATCCTACGTTTATACT-3') (upstream and downstream primer is synthesized by the prosperous bio tech ltd of Beijing AudioCodes).PCR amplifies required purpose fragment (total system 25 uL, 94 DEG C of denaturation 3 min, 32 circulations: 94 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 2 min, last 72 DEG C extend 10 minutes).With restricted enzyme XhoI and BamlI(TAKARA) enzyme action purpose fragment and prokaryotic expression carrier pET28a (+), then the carrier with sticky end and purpose fragment are connected with T4 DNA ligase, final acquisition prokaryotic expression plasmid pET28a (+)-hCRY1, as shown in Figure 1.With agarose gel electrophoresis, amplified production is detected, obtain the band that size is about 1900 bp, shown in figure as left in Fig. 2, it was demonstrated that purpose fragment Successful amplification.Use electrophoresis detection digestion products, available size to be about the purpose fragment band of 1900 bp and size is about the carrier ribbon of 5300 bp, shown in figure as right in Fig. 2, it was demonstrated that prokaryotic expression plasmid successfully constructs.DescribedhCRY1The hCRY1 protein amino acid sequence that gene translation obtains is as shown in SEQ ID NO:1.

(2) build engineering bacteria: by above-mentioned recombiant plasmid pET28a (+)-hCRY1 convert to competent cellE.coliIn BL21 (DE3), coated plate is cultivated, next day, 5 different single colony clones of picking, were respectively connected in the LB culture medium containing 50 mg/L kanamycin, shook bacterium and were transferred in the fresh LB culture medium containing 50 mg/L kanamycin be amplified cultivating with 1:100 after overnight.Bacterium is shaken to bacterium solution in conical flaskOD ₆₀₀After value 0.6, add 1 mM derivant IPTG, 24 DEG C, 200 rpm are centrifugal after inducing 6 h receives bacterium.Received thalline PBS is washed and is resuspended in 30 ml PBS, after adding 1 mM protease inhibitor PMSF, carries out ultrasonication.From every part of disrupted sample, take 20 μ l samples carry out SDS-PAGE analysis, shown in electrophoresis result figure as upper in Fig. 3, measure target protein and foreign protein band gray value respectively and calculate ratio, select b clone (clone that i.e. target protein band is brighter, foreign protein band is more weak) (Fig. 3 figure below) that target protein ratio is the highest, protect bacterium for-80 DEG C and store and namedBL21-hCRY1-b.All experiments afterwards all use this bacterial strain to complete.

(3) recombinant and recombiant protein abduction delivering: willBL21-hCRY1-bIt is seeded in 5 ml LB culture medium containing kanamycin, inoculates 100 ml LB culture medium containing kanamycin with 1:100 after shaking bacterium 6 h and carry out expanding species, after shaking bacterium 12 h, be seeded in the NBS series of fermentation tank containing 9 L culture medium ferment with 1:100.Culture medium prescription yeast extract 24 g/L, tryptone 12 g/L, glycerol 4 mL/L, KH₂PO₄ 2.3 g/L, K₂HPO₄·3H₂O 12.5 g/L, defoamer 0.2 mL/L.Set cultivation temperature 37 DEG C, dissolved oxygen 25%, pH 7.0, mixing speed 200-350 rpm, cultivate 5.5hOD ₆₀₀After value reaches 7.0, add IPTG to final concentration in tank about 1.0 mM, after induction 3h, complete fermentation.To fermenting, the sampling of each time period is carried outOD ₆₀₀Measure, draw growth curve, see accompanying drawing 4；And before taking induction, each time period thalline sample after induction, carry out SDS-PAGE testing goal protein expression situation, see accompanying drawing 4.After fermentation ends, 3500 rpm are centrifuged 10 min and collect thalline, thalline weight in wet base about 130 g.

Showing from accompanying drawing 4 growth curve, being seeded to fermentation tank 4 to 7 h, escherichia coli are in exponential phase, are initially added into 1.0 mM inductions when logarithmic growth middle and late stage (6 h), and bacterial multiplication starts to ease up.From Fig. 4 SDS-PAGE electrophoresis showed, after starting to induce 1 h, have been able to detect that hCRY1 expresses.

(4) inclusion body is collected and washing: take wherein 10 g thalline, PBS is added resuspended by 4 ml/g wet thallus, with power 225 W, working time 5 s, condition ultrasonication thalline 80 min of intermittent time 8 s, take the different suspension of crushing stage, upper cleer and peaceful precipitation carries out SDS-PAGE electrophoresis detection, and result is shown in accompanying drawing 5.From accompanying drawing 5 it is observed that after carrying out ultrasonic bacteria breaking, hCRY1 all exists with precipitation form, showing that hCRY1 inclusion body in an insoluble form is expressed, during broken bacterium, hCRY1 is insoluble in PBS, contributes to the enrichment of target protein.4000 rpm are centrifuged 60 min and collect precipitation, the component newly prepared with 40 ml contains 50 mM Tris, 0.5 mM EDTA, 50 mM NaCl, 1% Triton X-100,2 M Urea, the resuspended inclusion body of lavation buffer solution of pH 8.0, with power 125 W, working time 5 s, condition supersound washing 60 min of intermittent time 8 s, 14000 rpm are centrifuged 80 min and collect precipitation, and repeated washing is once；During supersound washing, sampling carries out SDS-PAGE, detects clean result, sees accompanying drawing 6.Can observe from front 5 swimming lanes of accompanying drawing 6 and obtain, in two stages of inclusion body washing, a large amount of foreign proteins are dissolved in supernatant and being removed, and hCRY1 exists with insoluble inclusion bodies, the enrichment of beneficially hCRY1 and primary purification.Target stripe being cut, carry out Mass Spectrometric Identification, the aminoacid sequence of albumen is as shown in SEQ ID NO:1.With ncbi database comparing result as shown in Figure 7, this inclusion body protein official confirmation is hCRY1.

(5) inclusion body degeneration is dissolved: add component that 40 ml newly prepare containing the inclusion body precipitation after 6 M guanidine hydrochlorides, the resuspended washing of pH8.0 phosphate buffer, with power 225 W, working time 5 s, condition ultrasonic dissolution 90 min of intermittent time 8 s；Diluting 8 times with containing 8 M Urea, pH8.0 phosphate buffers again, with power 225 W, working time 5 s, program ultrasonic dissolution 90 min of intermittent time 8 s, sampling carries out SDS-PAGE detection inclusion body protein and dissolves situation, sees accompanying drawing 6.From accompanying drawing 6 the 6th, 7,8 swimming lanes are it is observed that under ultrasound condition, hCRY1 inclusion body starts to be dissolved in supernatant.

(6) albumen affinitive layer purification: by albuminous degeneration solvent soln 14000 rpm high speed centrifugation 45min, collect supernatant, through 0.45 μm membrane filtration, add imidazoles to the final concentration of 20mM of imidazoles in sample in filtrate, HIS label affinity chromatograph is carried out, chromatograph: AKT Purifier 100 with loading volume 1 CV, loading flow velocity 8 ml/min, elution flow rate 15 ml/min；Chromatographic stuffing: ProteinIso Ni-NTA Resin 100 ml；Balance: balance 2 CV with buffer 500 mM NaCl, 8 M Urea, 20 mM imidazoles, pH 8.0；Eluting: with buffer 500 mM NaCl, 8 M Urea, 200 mM imidazoles, pH 8.0 eluting 2CV, collect eluted protein peak.To loading sample, penetrating peak and eluting peak sampling, carry out SDS-PAGE electrophoresis detection, as shown in Figure 8, chromatography collection of illustrative plates is as shown in Figure 9 for result.

(7) protein ladder dialysis renaturation: the eluted protein peak sample that will collect loads in 30-50 kDa bag filter, be placed in 8 times of volumes containing 6 M Urea, 50 mM Tris, in 50 mM NaCl, pH8.0 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Again sample is transferred to containing 4 M Urea, 50 mM Tris, in 50 mM NaCl, pH 8.0 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 2 M Urea, 100 mM Tris, 400 mM Argine, 5 mM EDTA, 5 mM GSH, in 0.5 mM GSSG, pH8.0 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 1 M Urea, 50 mM Tris, 50 mM NaCl, 10% glycerol, in pH8.0 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 50 mM Tris, 500 mM NaCl, 10% glycerol, in pH8.0 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；After having dialysed, collect sample in bag filter.

(8) albumen is concentrated by ultrafiltration and solution replacement: use ultrafiltration instrument Millipore, with 30 kDa ultrafilter membrane bags, dialysis gained sample is concentrated by ultrafiltration: after sample concentration to original volume about 1/4, progressively add and comprise 50 mM Tris, 100 mM NaCl, 10% glycerol, the displacement buffer of pH8.0 is diluted, finally sample volume is concentrated into the 1/5 of original volume, obtain hCRY1 protein solution, then after 0.22 μm membrane filtration, i.e. obtain aseptic solubility hCRY1 protein solution.Before and after taking concentration, sample carries out SDS-PAGE electrophoretic analysis, and result is as shown in Figure 10.By Bradford protein quantification detection kit, sample being carried out protein quantification, protein concentration is about 0.52 mg/ml, calculates that protein yield is about 300mg/L.

Recombiant protein activity and Function detection:

Taking protein solution after purification, to be diluted to 500 μ g/ml standby to make, configure 1 mg/ml BSA albumen with 300 mM imidazoles simultaneously, flow out and after ultrafiltration identical with hCRY1 sample displacement through nickel post loading, be diluted to 500 μ g/ml as comparison (cBSA).Liquid is changed when cultivating Hela cell climbing sheet bed board to about 80-90% cell density, after three hours, respectively according to the ratio with culture medium 1:1, the hCRY1 that gets ready being added cell culture fluid with reference protein, making total liquid volume in each hole is 2 ml, and adding final concentration of protein is 250 μ g/ml.Being irradiated in the most at once two groups of cells being placed in X-ray irradiating machine, dosage per minute is 1.132/Gy, irradiation time 530 s, and accumulated dose is 10 Gy.Culture medium (DMEM) before at once cell culture fluid being all changed to experiment after irradiation receives cell climbing sheet after continuing to cultivate 45 minutes.Develop a film 3 times with PBS, 100% methanol-20 DEG C fixing cell 5 min, 3 times are washed again with PBS, after 5% BSA closes overnight, with antibody Phospho-Histone H2A.X in 37 DEG C of incubated cell 1 h, then wash 3 times with PBS, again with antibodies Goat anti-rabbit IgG/FITC in 37 DEG C of incubated cell 1 h, PBS washes rear mounting, two groups of endocellular phosphorus body of light brightness of confocal laser scanning microscope for 3 times, and under identical shooting condition, every takes 3 different visual field statistical average fluorescence intensities respectively.Under identical observation condition, the FOCI fluorescence intensity difference formed in two groups of cells seen from naked eyes is very notable (the left figure of Figure 11), randomly selects 3 visuals field and fluorescence intensity is quantified, and between two groups, difference is extremely notable (P < 0.001) (the right figure of Figure 11).It follows that the hCRY1 that we prepare reduces the fluorescence intensity of FOCI in cell, cell is served certain protective effect, decreases the infringement of X-ray.

It is the radiation protection effect due to hCRY1 for being further characterized by the minimizing of intracellular FOCI, we carry out hCRY1 Concentraton gradient experiment in HaCaT cell further and arrange the cellular control unit processed without X-ray, Concentraton gradient is set up with above-mentioned BSA solution dilution hCRY1 solution after treatment, processing cell with same method, film-making is also observed.Result shows, when the hCRY1 concentration in cell culture medium reaches 50 μ g/ml, the fluorescence intensity of FOCI substantially reduces (Figure 12).

We have detected the BSA after hCRY1 and process the most respectively on apoptotic impact.The BSA after 250 μ g/ml hCRY1 and 250 μ g/ml process it is separately added in two groups of cells, detect apoptosis situation together with the cell not adding any albumen with another group after 1h, be analyzed with flow cytometer after cell being carried out double dye with cell apoptosis detection kit (being bought by Nanjing Kai Ji biological substance Development Co., Ltd).Within result shows 1h, whether hCRY1 or BSA is all without causing apoptosis, and this result proves that the reduction of FOCI fluorescence intensity is because the minimizing of intracellular DNA institute damaged and acellular apoptosis (Figure 13) further.

Last it is noted that obviously, above-described embodiment is only used to example of the present invention is clearly described, and not to the restriction implemented.For the those of ordinary skill in described field, can also make other changes in different forms on the basis of the above description.Here without also cannot be to all of embodiment party's formula with exhaustive.And the obvious change thus amplified out or variation still in protection scope of the present invention among.

SEQUENCE LISTING

<110>yellow galaxy of literary talent

<120>production technology of a kind of recombined human cryptochrome protein I (hCRY1) and combinations thereof thing

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 586

<212> PRT

<213>recombined human cryptochrome protein I (hCRY1)

<400> 1

Met Gly Val Asn Ala Val His Trp Phe Arg Lys Gly Leu Arg Leu His

1 5 10 15

Asp Asn Pro Ala Leu Lys Glu Cys Ile Gln Gly Ala Asp Thr Ile Arg

20 25 30

Cys Val Tyr Ile Leu Asp Pro Trp Phe Ala Gly Ser Ser Asn Val Gly

35 40 45

Ile Asn Arg Trp Arg Phe Leu Leu Gln Cys Leu Glu Asp Leu Asp Ala

50 55 60

Asn Leu Arg Lys Leu Asn Ser Arg Leu Phe Val Ile Arg Gly Gln Pro

65 70 75 80

Ala Asp Val Phe Pro Arg Leu Phe Lys Glu Trp Asn Ile Thr Lys Leu

85 90 95

Ser Ile Glu Tyr Asp Ser Glu Pro Phe Gly Lys Glu Arg Asp Ala Ala

100 105 110

Ile Lys Lys Leu Ala Thr Glu Ala Gly Val Glu Val Ile Val Arg Ile

115 120 125

Ser His Thr Leu Tyr Asp Leu Asp Lys Ile Ile Glu Leu Asn Gly Gly

130 135 140

Gln Pro Pro Leu Thr Tyr Lys Arg Phe Gln Thr Leu Ile Ser Lys Met

145 150 155 160

Glu Pro Leu Glu Ile Pro Val Glu Thr Ile Thr Ser Glu Val Ile Glu

165 170 175

Lys Cys Thr Thr Pro Leu Ser Asp Asp His Asp Glu Lys Tyr Gly Val

180 185 190

Pro Ser Leu Glu Glu Leu Gly Phe Asp Thr Asp Gly Leu Ser Ser Ala

195 200 205

Val Trp Pro Gly Gly Glu Thr Glu Ala Leu Thr Arg Leu Glu Arg His

210 215 220

Leu Glu Arg Lys Ala Trp Val Ala Asn Phe Glu Arg Pro Arg Met Asn

225 230 235 240

Ala Asn Ser Leu Leu Ala Ser Pro Thr Gly Leu Ser Pro Tyr Leu Arg

245 250 255

Phe Gly Cys Leu Ser Cys Arg Leu Phe Tyr Phe Lys Leu Thr Asp Leu

260 265 270

Tyr Lys Lys Val Lys Lys Asn Ser Ser Pro Pro Leu Ser Leu Tyr Gly

275 280 285

Gln Leu Leu Trp Arg Glu Phe Phe Tyr Thr Ala Ala Thr Asn Asn Pro

290 295 300

Arg Phe Asp Lys Met Glu Gly Asn Pro Ile Cys Val Gln Ile Pro Trp

305 310 315 320

Asp Lys Asn Pro Glu Ala Leu Ala Lys Trp Ala Glu Gly Arg Thr Gly

325 330 335

Phe Pro Trp Ile Asp Ala Ile Met Thr Gln Leu Arg Gln Glu Gly Trp

340 345 350

Ile His His Leu Ala Arg His Ala Val Ala Cys Phe Leu Thr Arg Gly

355 360 365

Asp Leu Trp Ile Ser Trp Glu Glu Gly Met Lys Val Phe Glu Glu Leu

370 375 380

Leu Leu Asp Ala Asp Trp Ser Ile Asn Ala Gly Ser Trp Met Trp Leu

385 390 395 400

Ser Cys Ser Ser Phe Phe Gln Gln Phe Phe His Cys Tyr Cys Pro Val

405 410 415

Gly Phe Gly Arg Arg Thr Asp Pro Asn Gly Asp Tyr Ile Arg Arg Tyr

420 425 430

Leu Pro Val Leu Arg Gly Phe Pro Ala Lys Tyr Ile Tyr Asp Pro Trp

435 440 445

Asn Ala Pro Glu Gly Ile Gln Lys Val Ala Lys Cys Leu Ile Gly Val

450 455 460

Asn Tyr Pro Lys Pro Met Val Asn His Ala Glu Ala Ser Arg Leu Asn

465 470 475 480

Ile Glu Arg Met Lys Gln Ile Tyr Gln Gln Leu Ser Arg Tyr Arg Gly

485 490 495

Leu Gly Leu Leu Ala Ser Val Pro Ser Asn Pro Asn Gly Asn Gly Gly

500 505 510

Phe Met Gly Tyr Ser Ala Glu Asn Ile Pro Gly Cys Ser Ser Ser Gly

515 520 525

Ser Cys Ser Gln Gly Ser Gly Ile Leu His Tyr Ala His Gly Asp Ser

530 535 540

Gln Gln Thr His Leu Leu Lys Gln Gly Arg Ser Ser Met Gly Thr Gly

545 550 555 560

Leu Ser Gly Gly Lys Arg Pro Ser Gln Glu Glu Asp Thr Gln Ser Ile

565 570 575

Gly Pro Lys Val Gln Arg Gln Ser Thr Asn

580 585

Claims

1. the production technology of recombined human cryptochrome protein I (hCRY1), it is characterised in that comprise the following steps:

(1) recombinant expression plasmid is built: willhCRY1Genetic fragment restructuring to prokaryotic expression carrier pET28a (+), it is thus achieved that recombinant expression plasmid pET28a (+)-hCRY1；

(2) build engineering bacteria: by recombinant expression plasmid pET28a (+)-hCRY1 proceeds to competence host cellE.coliBL21 (DE3), then screens, by SDS-PAGE, the clone strain that hCRY1 expression is highBL21-hCRY1-b；

(3) recombinant and recombiant protein abduction delivering: use fermentor cultivationBL21-hCRY1-b, cultivation temperature 36.5-37.5 DEG C, dissolved oxygen 20-40%, pH6.8-7.4, mixing speed 200-350 rpm, after cultivating 5-8 h, OD₆₀₀During=7-8, add IPTG inducing culture 3-4 h；

null(4) inclusion body is collected and washing: 3000-5000 rpm is centrifugal collects thalline，With power 200-250 W、Working time 3-8 s、The condition ultrasonication thalline 60-90 min of intermittent time 5-10 s，3000-5000 rpm is centrifuged 50-80min，Collection inclusion body precipitates，With component mM Tris Han 10-100、0.1-1.0 mM EDTA、0-100 mM NaCl、1-10% Triton X-100、1-2 M Urea，The resuspended inclusion body of lavation buffer solution of pH7.5-8.5，With power 100-150 W、Working time 3-8 s、The condition supersound washing 60-90 min of intermittent time 5-10 s，12000-14000 rpm is centrifuged 60-90 min and collects precipitation；

(5) inclusion body degeneration is dissolved: by component containing the inclusion body precipitation after 4-8 M guanidine hydrochloride, the resuspended washing of pH7.5-8.5 phosphate buffer, with power 200-250 W, working time 3-8 s, the condition ultrasonic dissolution 60-90 min of intermittent time 5-10 s；6-10 times is diluted again, with power 200-250 W, working time 3-8 s, the condition ultrasonic dissolution 60-90 min of intermittent time 5-10 s, it is thus achieved that albuminous degeneration solvent soln with containing 6-8 M Urea, pH7.5-8.5 phosphate buffer；

(6) albumen affinitive layer purification: by albuminous degeneration solvent soln 12000-14000 rpm high speed centrifugation 40-60 min, collect supernatant, through 0.22-0.45 μm membrane filtration, add imidazoles to the final concentration of 5-30 mM of imidazoles in filtrate, carry out HIS label affinity chromatograph with loading volume 1-1.5 CV, loading flow velocity 5-10 ml/min, elution flow rate 10-20 ml/min, use buffer 0-500 MM NaCl, 6-8 M Urea, 0-60 mM imidazoles, pH7.5-8.5 balance 2-4 CV, with buffer 0-500 mM NaCl, 6-8 M Urea, 200-300 mM imidazoles, pH7.5-8.5 eluting 2-2.5 CV, collect eluted protein peak；

(7) protein ladder dialysis renaturation: the albumen eluting peak sample of collection is carried out gradient dialysis renaturation, and buffer comprises 0-6 M Urea, 0-20% glycerol, 0-1 M NaCl, 0-100 mM Tris, 0-400mM Arginine, 0-10mM EDTA, 0-10mM GSH, 0-5mM GSSG, pH7.5-8.5；

(8) albumen is concentrated by ultrafiltration and solution replacement: use 30-50 KDa ultrafilter membrane bag that dialysis gained sample is concentrated by ultrafiltration: after sample concentration to original volume 1/4-1/5, progressively add and comprise 0-100 mM Tris, 0-200 mM NaCl, 0-20% glycerol, the displacement buffer of pH7.5-8.5 is diluted, and sample volume is finally concentrated into the 1/5 of original volume, obtains solvable hCRY1 protein solution, again after 0.22 μm membrane filtration, i.e. obtain solubility hCRY1 protein solution；

The aminoacid sequence of described hCRY1 is as shown in SEQID NO:1.

The production technology of recombined human cryptochrome protein I (hCRY1) the most according to claim 1, it is characterised in that described fermentation tank is NBS series of fermentation tank.

The production technology of recombined human cryptochrome protein I (hCRY1) the most according to claim 2, it is characterised in that the final concentration of 0.5-3.0 of described IPTG mM。

The production technology of recombined human cryptochrome protein I (hCRY1) the most according to claim 1, it is characterized in that: the filler of described affinity chromatograph is ProteinIso Ni-NTA Resin, in Ni-NTA His Bind Resin any one, chromatography instrument is AKT Purifier 100.

The production technology of recombined human cryptochrome protein I (hCRY1) the most according to claim 1, it is characterized in that: the program of described gradient dialysis renaturation is: the eluted protein peak sample collected is loaded in 30-50 kDa bag filter, 6 M Urea are comprised in 8-10 times of volume, 0-100 mM Tris, 0-100 mM NaCl, in pH7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 4 M Urea, in 0-100 mM Tris, 0-100 mM NaCl, pH 7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 2 M Urea, in 0-100 mM Tris, 0-400 mM Argine, 0-10 mM EDTA, 0-10 mM GSH, 0-5 mM GSSG, pH7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 1 M Urea, 0-100 mM Tris, 0-100 mM NaCl, 5-15% glycerol, in pH7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；Sample is transferred to comprise 0-100 mM Tris, 0-500 mM NaCl, 0-20% glycerol, in pH7.5-8.5 dialysis buffer liquid, 4 DEG C of dialysis 8-12 h；After having dialysed, collect sample in bag filter.

The production technology of recombined human cryptochrome protein I (hCRY1) the most according to claim 1, it is characterised in that: described ultrafiltration uses ultrafiltration instrument Millipore.

7. compositions, it is characterised in that: the restructuring hCRY1 albumen prepared containing production technology described in any one of claim 1-6 and pharmaceutically acceptable carrier or adjuvant.