CN110200995A - A kind of preparation method and applications of the anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin - Google Patents
A kind of preparation method and applications of the anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin Download PDFInfo
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- CN110200995A CN110200995A CN201910375110.9A CN201910375110A CN110200995A CN 110200995 A CN110200995 A CN 110200995A CN 201910375110 A CN201910375110 A CN 201910375110A CN 110200995 A CN110200995 A CN 110200995A
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Abstract
The preparation method and applications of the invention discloses a kind of anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin, feature are the following steps are included: (1) passes sequentially through gene magnification, the building of prokaryotic expression carrier and prokaryotic expression and protein purification is obtained based on invertebrate Recombinant Ferritin；(2) it will be placed in bag filter based on invertebrate Recombinant Ferritin, it dialyses in 5 mM sodium arsenite solution, working fluid is simulated using magnetic stirring apparatus in entire enrichment process, it dialyses after 12 h, bag filter is placed in albumen buffer 12 h that dialyse, period replaces an albumen buffer every 4 h, take out bag filter in protein solution it is concentrated after, the anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin is obtained, advantage is that have to significantly inhibit effect to the proliferation of chronic granulocyte white blood corpuscle K562 cell.
Description
Technical field
Heavy metal nanosphere is prepared using Recombinant Ferritin the present invention relates to a kind of, more particularly, to one kind based on no vertebra
The preparation method and applications of the anti-tumor nano arsenic ball of animal Recombinant Ferritin.
Background technique
Siphon-worm, ribbon wirm, mud blood clam, razor clam of hanging, imitative stichopus japonicus and Haiti melon are the representatives of economic invertebrate.They are typical
Benthon is resistant to the heavy metal of higher concentration in environment, this characteristic is with its internal ferritin to the enrichment function of heavy metal
It can be inseparable.Therefore, the biological function of these invertebrate enriching heavy metals is studied, in biomedical and materials chemistry
And the application in the fields such as bio-nanotechnology provides possibility.In recent years, have and carry out target administration using ferritin.
Chronic myelocytic leukemia is a kind of malignant tumour originating from candidate stem cell, is often accompanied by leukocyte disorder hyperplasia
And differentiation and maturation obstacle, morbidity and mortality occupy first place in the malignant tumour of Children and teenager.The life of the disease
It is big that object is characterized in that hematopoietic cell proliferation is out of control, differentiation and maturation is obstructed, obstacle, the cell of abnormal differentiation occur for normal apoptotic process
Amount proliferation.Current clinically common leukaemia cell's differentiating inducer has arsenical, vitamin A acid and its derivative etc..Arsenical is people
Class is used for one of the drug of oncotherapy earliest in history, but since its toxic side effect is obvious, arsenical answering on antitumor
With also gradually being abandoned by people.In the 1970s, Chinese scholar Zhang Tingdong team uses the administration mode of modern science, with arsenic
Frost (As2O3) it is that " cancer spirit " injection is made in main component, initiative achievement is obtained to the treatment of acute granulocytic leukemia.
Sodium arsenite can inhibit its proliferation by inducing chronic granulocytic leukemia Apoptosis as a kind of inorganic arsenical.But
Since the arsenic agent treatment effect of low dosage is not ideal enough, and higher dosage has larger toxic side effect to normal cell, can cause
It is dead.Toxic side effect is reduced in order to play the effect of sodium arsenite treatment leukaemia, the present invention is based on invertebrates to recombinate iron
Sodium arsenite is made nano arsenic ball and is used to treat leukaemia by the characteristic of protein enrichment heavy metal.
Summary of the invention
Technical problem to be solved by the invention is to provide the proliferation of a kind of pair of chronic granulocyte white blood corpuscle K562 cell
The preparation method and applications of the anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin with the effect that significantly inhibits.
The technical scheme of the invention to solve the technical problem is: a kind of be based on invertebrate Recombinant Ferritin
Anti-tumor nano arsenic ball preparation method, comprising the following steps:
(1) preparation based on invertebrate Recombinant Ferritin
A. the clone of ferritin gene sequence
A. the gene magnification of more a arsenic binding sites
Using Fer147 gene order as template, which is cDNA sequence shown in SEQID NO.1, and design upstream is expanded
Increase primers F ER-F:5 '-CCGCTCGAGAATTAGGAGGAAGTCCAAGA-3 '；With downstream amplification primer FER-R:5 '-
CGCCATATGTCGGACTCAGAAGTCAATCA-3 ', PCR amplification Fer147 target gene, using plastic recovery kit to PCR
Product is recycled and is purified, and Fer147 target gene is obtained；
B. the building of prokaryotic expression carrier
Using two kinds of restriction enzymes of Nde I and Xho I respectively to Fer147 target gene and pET-28a (+) expression vector
Digestion is carried out, recovery purifying then is carried out to digestion products respectively using plastic recovery kit；It will be after digestion using DNA ligase
Fer147 target gene fragment and after pET-28a (+) empty plasmid is attached, be transformed into competent cell DH5 α, pass through
After plate screening PCR amplification and sequencing compare, the positive colony daughter colony containing target gene is obtained, after expanding culture, is utilized
Plasmid extraction kit extracts plasmid, obtains the recombinant expression carrier containing Fer147；Gained recombinant expression carrier is transformed into greatly
In enterobacteria BL21 competent cell, through plate screening PCR amplification, the e. coli bl21 expression bacterium containing target gene is obtained
Strain；
C. prokaryotic expression
By the e. coli bl21 expression bacterial strain containing target gene, the ratio of 1:50 is inoculated in 10 mL respectively and contains by volume
30 μ g/mL kanamycins LB culture mediums, 37 DEG C, 120 rpm are cultivated to OD600=0.6, add final concentration of 0.5 mM's
IPTG(isopropylthiogalactoside) in culture medium, shaking speed is 125 rpm, respectively under the conditions of temperature is 20 DEG C
It induces 18 h and temperature is 37 DEG C of 5 h of induction；Gained bacterium solution is centrifuged 15 min in 6500 rpm and collects thallus, abandons supernatant culture
Base, thallus are suspended with 2 mL disruption buffers, and the solution after ultrasonication is centrifuged 10 in 12000 rpm refrigerated centrifuges
Min collects supernatant, is separately added into 40 μ L Tris-HCl and 10 μ L 5 × albumen sample-loading buffers, mixes well, be put into 100
DEG C metal bath in heat 10 min, then 30 s of low-speed centrifugal, collecting albumen supernatant is to contain target gene albumen
The sample solution of Fer147；
B. protein purification
Nickel column equipped with filler is connected on protein purification system, nickel column is rinsed with albumen buffer；It will be centrifuged
Albumen supernatant solution flow through nickel column after, the foreign protein in nickel column is rinsed with albumen washing buffer I, until albumen is pure
Change the signal that instrument detects and drops to steady state；Then destination protein is eluted with albumen washing buffer II, works as egg
When white purifying instrument detects that signal is on the rise, the solution eluted is the protein solution for needing to collect, and collects eluent,
Until signal drops to baseline steady state；The eluent containing destination protein of collection is placed in the super filter tube concentration of 30 kD,
Revolving speed control is repeatedly exchanged in 3000-3500 rpm, centrifugal process with albumen buffer, to remove remaining high concentration
Imidazoles, until finally protein solution is concentrated in Melon yellow color；Suitable SUMO enzyme is added in the albumen of concentration, as 4
Digestion is stayed overnight in DEG C environment, and the mixed solution after digestion is purified again by nickel column, take and flow through liquid, i.e., not tape label after digestion
Destination protein is based on invertebrate Recombinant Ferritin；
(2) enrichment of arsenic
It will be placed in bag filter, be dialysed in 5 mM sodium arsenite solution, entirely based on invertebrate Recombinant Ferritin
Working fluid is simulated using magnetic stirring apparatus in enrichment process, dialyses after 12 h, bag filter is placed in albumen buffer and is dialysed
During which 12 h replace an albumen buffer every 4 h, to remove the heavy metal ion that do not adsorbed by recombinant protein, take out saturating
Analyse bag in protein solution it is concentrated after, obtain the anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin.
The formula of the disruption buffer is 25 mM Tris, 150 mM NaCL, pH 8.0,0.5% triton X-
100；The formula of the albumen buffer is 25 mM Tris, 150 mM NaCl, pH 8.0；The albumen washing buffer
The formula of liquid I is 25 mM Tris, 150 mM NaCl, 50/70/100 mM imidazoles, pH 8.0；The albumen washing buffer
The formula of liquid II is 25 mM Tris, 150 mM NaCl, 500 mM imidazoles, pH 8.0.
It is above-mentioned that chronic granulocyte white blood corpuscle is being prepared based on the anti-tumor nano arsenic ball of invertebrate Recombinant Ferritin
Application in terms of K562 inhibitor.
Compared with the prior art, the advantages of the present invention are as follows: it is a kind of based on the antitumor of invertebrate Recombinant Ferritin
The preparation method and applications of nano arsenic ball, by having constructed the ferritin prokaryotic expression system of invertebrate, through inducing table
Large batch of soluble iron protein solution is obtained after reaching, and determines it after being prepared into albumin crystal, parsing by the arsenic of enriched
Structure, and this ferritin-sodium arsenite nanosphere is verified to chronic granulocyte white blood corpuscle K562's by cell experiment
Inhibiting effect lays the foundation for it in the clinical treatment of leukaemia.
In conclusion a kind of novel siphon-worm ferritin Fer147 joint sodium arsenite mixed processing K562 cell of the present invention
Method.It is shown experimentally that, Fer147, which combines sodium arsenite, has significant inhibiting effect to the proliferation of K562 cell, has and uses
In the potentiality for administering chronic grain leukaemia.
Detailed description of the invention
Fig. 1 is Fer147 gene amplification product detected through gel electrophoresis；
Fig. 2 is the SDS-PAGE protein electrophoresis figure of Fer147；1 swimming lane standard protein, 2-4 are the total protein for not adding inducer, swimming
Road 5-7 is the total protein being added after inducer induction 18h, and swimming lane 8 is the albumen after ni-sepharose purification, and 9 be the albumen after digestion；
Fig. 3 is that Fer147 is enriched with As3+Circular dichroism figure；
Fig. 4 is Fer147 crystallographic structural analysis；The three-phase channel Surface electrostatic potential of A.Fer147, including three-phase channel design section
Surface electrostatic potential, three-phase passway Surface electrostatic potential and from three-phase channel show entire ferritin nanocages surface
Electrostatic potential；The four phase channel Surface electrostatic potentials of B.Fer147, Surface electrostatic potential, four Xiang Tongdao including four phase channel structural profiles
The Surface electrostatic potential of the Surface electrostatic potential of mouth and the entire ferritin nanocages shown from four phase channels；C.Fer147 subunit knot
Structure is shown, including Fer147 subunit helical structure annotation, the Surface electrostatic potential and Fer147 subunit three of Fer147 subunit communicate
Road and four phase channel Surface electrostatic potential distribution situations；D.Fer147 is enriched with the structure chart of As, including the entire ferritin of Fer147 is received
Rice basket structure shows, the distribution situation of not homoatomic and molecule on Fer147 subunit and three TAS at three-phase passway
(AsO2 -) with periphery hydrone and amino acid residue form covalent bond；
Fig. 5 is the metamorphosis situation that normal cell, sodium arsenite and Fer147 combine that sodium arsenite handles K562 cell；
Fig. 6 is that Annexin V-FITC/PI double-staining observes K562 cell；A. the K562 cell of control group；B. arsenous
The K562 cell of sour sodium processing；
Fig. 7 is influence of the sodium arsenite processing to Apoptosis；Q1 indicates mechanical loss cell；Q2 indicates non-viable apoptotic cell；
Q3 indicates viable apoptotic cell；Q4 indicates living cells；Point in figure represents the K562 cell branch situation under different disposal；Cell
Apoptosis rate is presented as the ratio (cell including early apoptosis and late apoptic) of apoptotic cell；
Fig. 8 is that sodium arsenite combines influence of the sodium arsenite processing to Apoptosis with Fer147；Q1 indicates mechanical loss cell；
Q2 indicates non-viable apoptotic cell；Q3 indicates viable apoptotic cell；Q4 indicates living cells；Point in figure represents under different disposal
K562 cell branch situation；Apoptosis rate is presented as that the ratio of apoptotic cell is (thin including early apoptosis and late apoptic
Born of the same parents)；
Fig. 9 is sodium arsenite, sodium arsenite combines the influence that sodium arsenite handles cell cycle with Fer147.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
Specific embodiment one
Preparation based on invertebrate Recombinant Ferritin
1. the clone of ferritin gene sequence
1.1 gene magnification
Using Fer147 sequence (SUMO+ferritin gene) as template, the 5.0 software design upstream Primer Primer is utilized
Expression primer FER-F:5 '-CCGCTCGAGAATTAGGAGGAAGTCCAAGA-3 '；With downstream expression primer FER-R:5 '-
CGCCATATGTCGGACTCAGAAGTCAATCA-3 ', PCR amplification Fer147 target gene, using plastic recovery kit to PCR
Product is recycled and is purified, and after sequencing compares confirmation, obtains Fer147 target gene.(Fer147 is that seminar utilizes ferment
A kind of new albumen-Fer147 that can be interacted with Phascolosoma ferritin that female two-hybrid system filters out.Pass through sequence
Column analysis finds that Fer147 is ferritin family member, and DNA sequence dna overall length includes 916 bases, by 174 amino acid groups
At.The gene order of Fer147 not yet uploads to GenBank, encodes original gene sequence (multiple arsenic knots of this 174 amino acid
The gene order of coincidence point) as follows: ATGTCTCTTTCGAGACCACGTCAAAACTACCACACGGAATCGGAATCAGCGGT
CAACAAGCAAATCAACTTGGAGCTTTATGCCAGCTATGTCTATCAGTCTATGTCGAGATATTTCAACCGAGATGAT
GTGGCGCTGAAAGGATTTCATGAATACTTCAAGGAAGCGAGTGAGGAGGAGCGCCAACACGCGGAGGAACTAATGG
AATACCAGAGCACGCGTGGCGGTCGCATTATGCTCTCGGACATTAAGCGCCCTGAAAATGATGAGTGGGGAACTGG
TCTGGAGGCCATGGAAACTGCTCTGAATCTAGAAAAGAACGTCAACCAATCGCTACTGGACCTGCACAAGACGGCA
GAGAAACACGTCGACGCGCAGATGCAAGATTTCATCGAGGAAAACTTTCTAAGGGAGCAGGTGGAGTCTATCGAGG
AAATATCTGATCACATTACAAACCTGAAGCGTGTTGGACCAGGTGAAGGCGAGTACATGTTCGACGAGAACCTAAG
TGAAGAACTTCAATGA。
The building of 1.2 prokaryotic expression carriers
(1) double enzyme digestion reaction: using two kinds of restriction enzymes of Nde I and Xho I respectively to Fer147 target gene and pET-
28a (+) expression vector carries out digestion, then carries out recovery purifying to digestion products respectively using plastic recovery kit；
(2) the Fer147 target gene fragment after digestion is attached with pET-28a (+) empty plasmid using DNA ligase；
(3) connection product is transformed into competent cell DH5 α, is coated on the LB plate containing 30 μ g/mL kanamycins,
It being incubated overnight, picking single bacterium falls within 10 mL and contains 30 μ g/mL kanamycins LB liquid mediums (yeast extract 0.5%(w/v),
Tryptose powder 1.0%(w/v), sodium chloride 1.0%(w/v)) in 37 DEG C be incubated overnight, collect thallus, PCR amplification, through sequencing compare
Afterwards, it obtains the positive colony daughter colony containing target gene and (after plate screening PCR amplification and sequencing compare, confirms purpose base
Cause and expression vector successful connection), the positive colony daughter colony containing target gene is expanded into culture, plasmid is utilized to extract reagent
Box extracts plasmid, obtains the recombinant expression carrier containing Fer147.Gained recombinant expression carrier is transformed into e. coli bl21 sense
By in state cell, (Fig. 1) is verified through plate screening PCR amplification, and using agarose gel electrophoresis, then to PCR product
After carrying out sequencing comparison, the expression bacterial strain containing target gene is obtained, after expanding culture, after being mixed with 20% glycerol (v/v), point
In -80 DEG C of cryo-conservations after dress.
1.3 prokaryotic expression
(1) e. coli bl21 containing target gene is taken out from -80 DEG C of refrigerators express bacterial strain (DE3), 37 DEG C of recoveries；
(2) ratio of the bacterium solution of recovery 1:50 by volume is inoculated in 10 mL respectively and contains 30 μ g/mL kanamycins LB training
Base is supported, 37 DEG C, 120 rpm are cultivated to OD600=0.6, the IPTG(isopropylthiogalactoside of final concentration of 0.5 mM is added,
Isopropyl β-D-Thiogalactoside) in culture medium, shaking speed is 125 rpm, is respectively 20 DEG C in temperature
Under the conditions of induce 18 h and the temperature to be 37 DEG C of 5 h of induction, and the culture bacterium to be not added with IPTG is negative right as what is tested
According to；
(3) gained bacterium solution is centrifuged 15 min in 6500 rpm and collects thallus, abandons supernatant culture medium, the thallus broken buffering of 50 mL
Liquid (25 mM Tris, 150 mM NaCl, pH 8.0,0.5% triton X-100) suspends, cell ultrasonication parameter setting
Are as follows: 6 min of total time surpass 2 s and stop 3 s, the solution after ultrasonication collects supernatant in 12000 rpm refrigerated centrifuge, 10 min
Liquid is detected with SDS-PAGE.
(4) the SDS-PAGE electrophoresis of inducing expression product: take 40 μ L supernatants that 10 μ L 5 × albumen loadings are added slow
Fliud flushing mixes well, 100 DEG C of 10 min of metal bath, then 30 s of low-speed centrifugal, takes 10 μ L for SDS-PAGE electrophoretic analysis.
SDS-PAGE electrophoretic analysis result is shown in Fig. 2, and as seen from the figure, Fer147 has single band at 30 kDa or so place.
1.4 protein purification
(1) will be connected on protein purification system (AKTA) equipped with the nickel column of filler, with albumen buffer (25 mM Tris, 150
MM NaCl, pH 8.0), 15-20 min is rinsed to nickel column with the flow velocity of 2 mL/min；
(2) the albumen supernatant solution being centrifuged is flowed through into nickel column with the flow velocity of 2 mL/min；
(3) after supernatant solution all flows through nickel column, with albumen washing buffer I (25 mM Tris, 150 mM NaCl, 50/70/
100 mM imidazoles, pH 8.0) foreign protein in nickel column is rinsed with the flow velocity of 2 mL/min, until protein purification instrument detects
To signal drop to steady state；
(4) it according to above-mentioned elution profile, determines the best imidazole concentration of elution foreign protein, then uses albumen washing buffer II
(25 mM Tris, 150 mM NaCl, 500 mM imidazoles, pH 8.0) elutes destination protein with the flow velocity of 2 mL/min,
When protein purification instrument detects that signal is on the rise, the solution eluted is target protein solution, collects albumen, until
Signal drops to baseline steady state；
(5) destination protein of above-mentioned collection is placed in the super filter tube of 30 KD and is concentrated by ultrafiltration, revolving speed is controlled in 3000-3500
Rpm is repeatedly exchanged with albumen buffer (25 mM Tris, 150 mM NaCl, pH 8.0) in centrifugal process, to remove
Remaining high concentration imidazoles, until finally protein solution is concentrated in Melon yellow color；
(6) suitable SUMO enzyme is added in the albumen of concentration, is stayed overnight as digestion in 4 DEG C of environment, the mixed solution after digestion is logical
It crosses nickel column to purify again, takes and flow through liquid, i.e., the not destination protein of tape label after digestion recombinates iron egg based on invertebrate
It is white；
(7) destination protein for removing label is concentrated again, it is spare is placed in -80 DEG C of freezer storages.In order to determine the albumen after digestion
In SUMO label completely removed, verified using SDS-PAGE electrophoresis, electrophoresis result is as shown in Fig. 2, You Tuke
Know, the band for removing the Fer147 of SUMO label is distributed in 20 kDa or so.
Specific embodiment two
The enrichment of heavy metal
1. being based on preliminary result, after the metallic solution concentration range for determining ferritin enriching heavy metal ion, 5 mM gold is chosen
Belong to solution as enriched concentration.The ferritin of removal SUMO enzyme label after purification is placed in bag filter, in 5 mM arsenious acid
Sodium (NaAsO2) dialyse in solution, working fluid is simulated using magnetic stirring apparatus in entire enrichment process, after 12 h that dialyse,
Bag filter is placed in albumen buffer (25 mM Tris, 150 mM NaCl, pH 8.0) 12 h that dialyse, during which more every 4 h
An albumen buffer is changed, to remove the heavy metal ion that do not adsorbed by recombinant protein.The protein solution taken out in bag filter is set
It in super filter tube, is required according to subsequent experimental, protein concentrate, is placed in -80 DEG C and stores for future use.
2.Fer147 being enriched with the measurement of arsenic content
1 mg/mL of liquor ferri albuminati of enrichment arsenic is taken, the dust technology of 0.1 mL, 5 wt% is added, shake makes sample dispersion, then
Be fitted into microwave digestion system and carry out microwave digestion, finally using icp ms to contained metal into
Row quantitative analysis.
1 Fer147 of table is enriched with the measurement of metal ion content after arsenic
As shown in Table 1, the iron atom that each Fer147 ferritin molecules contain is 157, and each Fer147 molecule combines 17
Arsenic atom.
3, Fer147 is enriched with the secondary structure analysis of arsenic
2 Fer147 of table is enriched with the content of secondary structure before and after arsenic
Table 2 is the measurement that Fer147 is enriched with Protein secondary structure before and after arsenic, is that Fer147 is enriched with As by Fig. 33+Circular dichroism
Figure and table 2 it is found that Fer147 enrichment arsenic before and after,αHelical content is kept approximately constant,βCorner and random coil contain
There is a certain range of fluctuation in amount, after this may be enriched with arsenic with Fer147, certain amino in the arsenic ion and ferritin of trivalent
It is related that sour residue forms ionic bond.
Specific embodiment three
The preparation and parsing of crystal
1, the preparation of protein sample
(1) it is placed in defrosting (fresh sample is then directly centrifuged) on ice from -80 DEG C of refrigerators by the ferritin sample frozen, will thawed
Protein sample afterwards with 4 DEG C of high speed freezing centrifuge, 12000 rpm, be centrifuged 15 min, remove denaturation protein precipitation and
Impurity, and guarantee the uniform of sample, the supernatant solution after centrifugation is transferred in the new centrifuge tube being pre-chilled, in transfer process
If occurring more foam in solution, it can be centrifuged 5 min again except defoaming；
(2) it measures protein concentration: utilizing BCA determination of protein concentration kit measurement protein concentration, determine residual protein after centrifugation
Concentration, provide reference for the most suitable protein crystal concentration of subsequent determination.
2, the primary dcreening operation of crystallization condition
Using the crystallization condition of seat drop benefit of vapor diffusion method screening albumen, albumen primary dcreening operation concentration is 20 mg/mL, uses Crystal
Albumin crystal plate of the preliminary screening agents such as the Screen 1&2 box in 48 holes carries out primary dcreening operation to albumin crystal growth conditions, and every hole contains 150
μ L albumin crystal conditioned, two sides little Chi respectively contain 1 μ L albumin crystal conditioned, the two each other one it is parallel, mutually compare, often
After the mixing of 1 μ L protein solution is added in a little Chi, is sealed with special adhesive tape, form closed cultivating system.Crystal slab is transferred to
Crystal culture is carried out under 18 DEG C of cryogenic conditions.Albumin crystal growing state in several days observation crystal slabs, to growing crystalline substance
The conditioned of body is further optimized.
3. crystallization condition optimizes
Optimize crystallization condition by changing the concentration of the protein concentration in conditioned, pH, precipitating reagent and inorganic salt solution, it is right
PH and precipitant concentration carry out gradient and increase or decrease 0.2 ratio to adjust, and 9 gradients are arranged in precipitating reagent, with original condition
Centered on, pH is arranged 8 gradients and forms the new condition liquid kit of 9*8 equally centered on original pH.It is dripped using seat
Crystal culture is carried out with the cultural method of hanging drop benefit of vapor diffusion.
4, the X-ray diffraction of albumin crystal
(1) screening and processing of crystal
The higher crystal of crystal quality is filtered out from crystal slab, screening criteria is that crystal head is big, surface-brightening and shape rule
Then.Prepare corresponding crystal anti-icing fluid (0.1 M HEPES pH 7.7,22% v/v Jeffamine M-600,25% glycerol).
Crystal is pulled out in pond with Loop ring, is gently stained with and is touched in anti-icing fluid, is immersed in liquid nitrogen and is rapidly frozen preservation.
(2) collection of crystal data
Crystal is transferred in the Puck of automatic sampling, illustrates to carry out crystal loading according to line station loading.Log in manipulator operation
Software, illustratively manipulator carries out crystal diffraction, and in Shanghai, it is brilliant to collect protein for synchrotron radiation light source (BL17U1 line station)
The diffraction data of body.
(3) preliminary treatment of initial data
Initial data is integrated using HKL2000 program, is merged and normalized, determines the cell parameter of protein structure
And space group, according in original data processing result Rmerge value and the parameters such as signal-to-noise ratio determine that structural model is effectively usable.
Finally obtain the .sca file of later period solution structure.Use the ray diffraction data of HKL2000 program bag processing ferritin crystal, side
Method is as follows:
A. HKL2000 program is opened, corresponding detector is selected, creates the storing path of data after processing, checks program parameter
It is whether correct, it is set for the diffraction resolution range and intensity limit of integration of space group judgement；
B. selected part diffraction picture carries out indexing and determines the space lattice type of crystal；
C. intensity integral is carried out to a whole set of data after carrying out explication de texte to structure cell；
D. the space group of crystal is determined；
E. the final diffraction resolution of crystal is determined.
5, albumin crystal structure elucidation
Crystal structure amendment mainly includes phase determination and structural modifications.During wherein phase determination is crystallographic structural analysis
Difficult point, current main solution have molecular replacement, Single wavelength anomalous scattering and multi-wavelength anomalous scattering.We attempt to use
Molecular replacement solves the phase of crystal structure.
(1) molecular replacement
The amino acid sequence of ferritin is compared in the website PDB and finds the highest several structures of sequence similarity therewith
The homology of 5wpn, 5jkl, 3ajio and 3ajp, they and ferritin are respectively 70.00%, 63.47%, 61.85% and 61.27%；
The intensity value of diffraction data is converted into structure factor amplitude using the program of Import Merged Data in CCP4 program bag；
Possible molecular number in an asymmetry unit is calculated using the Matthews program in program bag；Using in program bag
Model sequence is replaced with the sequence of ferritin by Chainsaw；It is obtained using the Phaser in program bag by molecular replacement technique trial
The phase obtained, uses and similarly spends highest several structures in PDB as model；
(2) refine of structure
Obtained PDB file and mtz file are opened with Wincoot, according in Validate Ramachandran Plot and
Density fit analysis carries out refine to structure.Using the Refmac5 program in CCP4 software package to modified manually
Conformation is automatically corrected；
(3) crystal structure finally obtained the explanation of structure: is subjected to structural analysis by PyMOL software.
Experiment uses Crystal Screen 2TMKit sessile drop method culture albumin crystal, albumin crystal conditioned is through screening
After optimization, determine that optimal crystallization condition is: protein concentration 20 mg/mL, Fer147 are enriched with As3+The best item of crystal growth
Part is No. 39 conditioneds (0.2 M magnesium chloride hexahydrate, 0.1 8.5,3.4 M 1 of M Tris, pH, 6- hexylene glycol).It is same through Shanghai
After walking radiating light source diffraction, 1.25 crystal data of resolution ratio, space group I432 are obtained, cell parameter isa = b = c =
151.930, α=β=γ=90, Rmerge=7.40%.Matthews constant experience range (1.7 ~ 103.Da),
Using Matthews Coeff analysis it is found that the Ma Xiusi constant of Fer147-As albumin crystal is 3.583.Da-1, solvent contains
Amount about 65.67%, thus it is speculated that each structure cell asymmetry unit is made of a protein molecular.Divided using CCP4i suite
Son displacement, PBD template is human heavy chain ferritin (PDB:3AJO chain A), due to the 1st, 2 and 172,173,174 amino acids
Residue lacks enough cloud densities, and the amino acid structure in the region not can determine that.The successful protein structure warp of molecular replacement
REFMAC5 determines the structure of the albumin crystal repeatedly after refine repeatedly.
The interaction of metal ion and albumen with combined with protein surface amino acid residue Surface electrostatic potential distribution
There is close relationship, and the ion channel on ferritin surface is the important way that metal ion enters albumen caged cavity inside
Diameter, therefore surface electrostatic potential analysis is carried out to ferritin using the APBS kit in PyMOL software.Fig. 4 A is Fer147 three-phase
The Surface electrostatic potential of channel design section, the Surface electrostatic potential of three-phase passway and the entire ferritin shown from three-phase channel
The distribution situation of the Surface electrostatic potential of nanocages.In figure it is found that from three-phase channel design section to ferritin it has been observed that caged
Molecular surface in cavity is dispersed with around a large amount of negative electrical charges, especially three-phase passway, implies the acidic amino acid in molecule
And the negative potential generated plays a decisive role to the entrance and combination of itself and metal ion.Fig. 4 B is tetra- phase channel design of Fer147
The Surface electrostatic potential of section, the Surface electrostatic potential of four phase passways and the entire ferritin nanocages that are shown from four phase channels
The distribution situation of Surface electrostatic potential, the passway inlet integrated distribution of the caged cavity inside positive potential mixed with zero potential
The region of intersection, passway centre is strong positive potential area, and four phase channel surface entrances are zero potential region, outer profile packet
Round strong positive potential area, this is incorporated in four phase passways for some metal ions and provides possibility.Fig. 4 C is Fer147 subunit
Structure chart, the subunit are to terminate since the 3rd leucine (Leu) of N-terminal to the 171st glutamic acid (Glu), which is by α
Spiral A, B, C and D connect in 60 jiaos with α spiral E through AB Loop, BC Loop and CD Loop, then through DE Loop and four-helix bundle
It connects.By the subunit surface electrostatic potential analysis it is found that three-phase channel is made of the amino acid residue for being dispersed with a large amount of negative potentials, this
Ferroxidase activity neutrality is smoothly entered for metal ion and ferritin caged cavity inside provides possibility.Fig. 4 D
The structure chart of As is enriched with for Fer147, it includes an arsenous acid molecule (TAS) which is made of 169 amino acid residues,
Two Fe atoms, two Cl atoms, five Mg atoms and four 1,6- hexylene glycol molecules.The ferritin nanocages are by 24 Asias
Base, 8 three-phase channels and 6 four phase channels form, and include 24 TAS molecules, 48 Fe atoms, 48 Cl in ferritin cage
Atom, 120 Mg atoms and 96 1,6- hexylene glycol molecules.Each three-phase passway arsenous acid molecule in conjunction with there are three, each
Arsenious acid respectively with the 120th glutamic acid (Glu120) and a H2O molecule forms covalent bond.
Specific embodiment four
The application of anti-tumor nano arsenic ball
1, cell experiment
K562 cell culture is placed in 37 DEG C, the CO containing 5 vt% in the IMDM culture medium containing 10 wt% fetal calf serums2Constant temperature
It is cultivated in incubator, according to cell culture situation, carries out cell passage or other experiments in the logarithmic phase of cell growth.
2, cellular morphology is observed
0 μM of NaAsO of experimental setup2、2 µM NaAsO2、4 µM NaAsO2、6 µM NaAsO2、8 µM NaAsO2、10 µM
NaAsO2、12 µM NaAsO2、14 µM NaAsO2、16 µM NaAsO2With 20 μM of NaAsO2Single treatment group and 0 μM
NaAsO2To 30 μM of NaAsO2Group is mixed with 0.5 mg/mL Fer147, after handling K562 cell respectively for 24 hours, its form is become
Change is observed.
By the metamorphosis situation of Fig. 5 normal cell, sodium arsenite and Fer147 joint sodium arsenite group cell it is found that just
Normal K562 cell growth state is good, and cellular morphology is complete, is in regular circle, and the cell of single sodium arsenite processing group
Apparent clustering phenomena is presented, with the increase of sodium arsenite concentration, the cell of aggregation occurs significantly collapsing the phenomena of mortality.This
Outside, in sodium arsenite concentration from 6 μM to 12 μM during variation, there is the abnormal phenomenon of strip in cell.And arsenious acid
The cell of sodium and 0.5 mg/mL Fer147 mixed processing group, cell distribution are comparatively dispersed, and are 6 μ in sodium arsenite concentration
In the case where M, the abnormal phenomenon that strip occurs in cell is obvious, and sodium arsenite concentration is since 8 μM, Apoptosis
Speed is accelerated.
3, cell viability detects
The cell inoculation of logarithmic growth phase is in 96 orifice plates, every hole 1 × 105A cell, if 4 multiple holes, by no Fer147 and
Fer147 group is added to be separately added into 2 μM of NaAsO2、4 µM NaAsO2、6 µM NaAsO2With 8 μM of NaAsO2Handle K562 cell
24 h.The CCK-8 solution (5 g/L) of 10 μ L is added in every hole, and 37 DEG C are continued after being incubated for 2 h, is to measure at 450 nm in wavelength
Its light absorption value.
The influence of 3 sodium arsenite group of table, Fer147 joint sodium arsenite to K562 cell Proliferation
Note: compared with the control group, the statistically significant (* of differenceP< 0.05, * *P< 0.01).
The result shows that sodium arsenite has significant growth inhibition effect, sodium arsenite and 0.5 mg/mL to K562 cell
After Fer147 mixed processing, inhibiting effect is become apparent.
Fig. 6 is that Annexin V-FITC/PI double-staining observes K562 cell, and A is the K562 cell of control group, and B is
The K562 cell of sodium arsenite processing；Show that the K562 of control group is thin by the amphophilic K562 cell of Annexin V-FITC/PI
Born of the same parents, fluorescence distribution is uniform, and cell is relatively regular, and 12 μM of NaAsO2Treated, and there is apparent apoptosis, cell in cell
Volume shrinkage is reduced, and cytoplasm concentration is fine and close, and cell edges are irregular.
4, using Apoptosis by Flow Cytometry
According to 2 × 105A/mL final concentration of cells is inoculated in 6 orifice plates, 3 parallel laboratory tests of every group of carry out.0 μM of experimental setup
NaAsO2、2 µM NaAsO2、4 µM NaAsO2、6 µM NaAsO2、8 µM NaAsO2、10 µM NaAsO2、12 µM
NaAsO2、14 µM NaAsO2、16 µM NaAsO2With 20 μM of NaAsO2Single treatment group and 0 μM of NaAsO2To 20 μM
NaAsO2Group is mixed with 0.5 mg/mL Ferritin, after handling 24 h of K562 cell respectively, cell is collected, with the PBS of pre-cooling
Buffer washes twice cell, according to apoptosis kit specification, is separately added into Annexin V-FITC and PI, room temperature
It is thin using flow cytometer (Gallios, Beckman coulter, the U.S.) detection apoptosis in 1 h after being protected from light 15 min
Born of the same parents calculate the apoptosis rate of cell.
Fig. 7 is that sodium arsenite handles Fer147 joint sodium arsenite processing to the apoptosis situation of K562 cell；Fig. 8 is
Fer147 combines influence of the sodium arsenite processing to Apoptosis, and Q1 indicates mechanical loss cell；Q2 indicates non-viable apoptotic cell；
Q3 indicates viable apoptotic cell；Q4 indicates living cells；Point in figure represents the K562 cell branch situation under different disposal；Cell
Apoptosis rate is presented as the ratio (cell including early apoptosis and late apoptic) of apoptotic cell.It can be seen from the figure that with
Sodium arsenite concentration is continuously increased, and Apoptosis is also increasingly severe.Table 4 and table 5 be respectively single sodium arsenite group and
Fer147 combines sodium arsenite under the conditions of various concentration to the apoptotic effect of K562 cell.
Influence of 4 sodium arsenite of table to K562 Apoptosis
Note: compared with the control group, the statistically significant (* of differenceP< 0.05, * *P< 0.01).
5 Fer147 of table combines influence of the sodium arsenite to K562 Apoptosis
Note: compared with the control group, the statistically significant (* of differenceP< 0.05, * *P< 0.01).
Table 4 and table 5 are respectively single sodium arsenite group and sodium arsenite and Fer147 mixed processing group to K562 cell
Apoptosis is under the conditions of various concentration to the apoptotic effect of cell.From table 4, it can be seen that single sodium arsenite processing K562 is thin
Born of the same parents, apoptosis rate is followed successively by 2.23% ± 0.24%, 10.82% ± 3.19%, 16.06% ± 5.14%, 22.90% ±
5.24%, 37.37% ± 6.46%, 43.90% ± 6.86%, 46.11% ± 5.82%, 57.1% ± 4.06% and 61.3%
± 4.66%.As can be seen from Table 5, Fer147 combine sodium arsenite 12.00% is followed successively by K562 apoptosis rate ±
2.97%、11.61%± 2.02%、17.20% ± 4.25%、23.27% ± 3.51%、33.40% ± 4.83%、31.91%
± 3.46%, 72.50% ± 3.91%, 73.00% ± 3.29% and 81.60% ± 2.58%.The result shows that with arsenious acid
The apoptosis rate of the increase of na concn, K562 cell gradually increases, and Fer147 combines sodium arsenite to the apoptotic effect of cell more
Obviously.
5, the variation of flow cytomery cell cycle
The K562 cell cycle is grouped unanimously, collects cell after handling 24 h, is washed carefully with the PBS of pre-cooling by with Apoptosis grouping
Born of the same parents twice, be added pre-cooling 70% ethyl alcohol be resuspended cell, 24 h are fixed under the conditions of 4 DEG C, with PBS wash cell it is primary after, receipts
Collection cell is simultaneously transferred in fluidic cell pipe, and pre-configured 0.5 mL of dyed blended liquid containing RNase A and PI is added,
Cell slowly and is sufficiently resuspended, 37 DEG C are protected from light after 30 min of incubation in detecting the cell cycle on flow cytometer, and experiment repeats 3
It is secondary.
Influence of 6 sodium arsenite of table to the K562 cell cycle
Note: compared with the control group, the statistically significant (* of differenceP< 0.05, * *P< 0.01).
7 Fer147 of table combines influence of the sodium arsenite to the K562 cell cycle
Note: compared with the control group, the statistically significant (* of differenceP< 0.05, * *P< 0.01).
From flow cytomery result, (Fig. 9 is the shadow of sodium arsenite, Fer147 joint sodium arsenite cell cycle
Loud and table 6) it is found that after single sodium arsenite group processing 24 h of K562 cell, when the concentration of sodium arsenite increases to 12 from 0 μM
μM when, the cell proportion of S phase and G2/M phase gradually decrease, and the cell proportion of G0/G1 phase is in the trend that gradually increases, illustrates this
The sodium arsenite of concentration range is by K562 cell-cycle arrest in the G0/G1 phase.And when sodium arsenite increases to 20 μM from 16 μM
When, the cell proportion of G0/G1 phase is declined, and the cell proportion of S phase increases, and illustrates that the sodium arsenite of the two concentration will
K562 cell-cycle arrest is in G0/G1 phase and S phase.From Fig. 9 and table 7 it is found that when Fer147 joint sodium arsenite processing K562 is thin
When born of the same parents, the cell proportion of G0/G1 phase and S phase are higher, and when the concentration of sodium arsenite is less than 6 μM, the K562 cell cycle is hindered
It is stagnant to be concentrated mainly on the G0/G1 phase.When the sodium arsenite (being greater than 10 μM) of high concentration is with Fer147 mixed processing K562 cell, G0/
G1 phase cell proportion is substantially in the trend gradually decreased, and the cell proportion of S phase then gradually increases, and illustrates the arsenous of higher concentration
After sour sodium and Fer147 mixed processing K562 cell, cell-cycle arrest is concentrated mainly on G0/G1 phase and S phase.
In conclusion the present invention constructs soluble prokaryotic expression system, measured by icp ms
Fer147 is to As3+Accumulation ability, using circular dichroism technology to Fer147 be enriched with As3+Secondary structure afterwards is analyzed,
Utilize Fer147 and As3+It is found after acting on chronic myeloid leukemia cell K562 after mixing, with single As3+Processing group
It compares, mixing group can significantly improve the apoptosis rate of K562 cell.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Sequence table
<110>University Of Ningbo
<120>a kind of preparation method and applications of the anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 525
<212> DNA
<213>Fer147 gene order
<400> 1
ATGTCTCTTTCGAGACCACGTCAAAACTACCACACGGAATCGGAATCAGCGGTCAACAAGCAAATCAACTTG
GAGCTTTATGCCAGCTATGTCTATCAGTCTATGTCGAGATATTTCAACCGAGATGATGTGGCGCTGAAAGGATTTC
ATGAATACTTCAAGGAAGCGAGTGAGGAGGAGCGCCAACACGCGGAGGAACTAATGGAATACCAGAGCACGCGTGG
CGGTCGCATTATGCTCTCGGACATTAAGCGCCCTGAAAATGATGAGTGGGGAACTGGTCTGGAGGCCATGGAAACT
GCTCTGAATCTAGAAAAGAACGTCAACCAATCGCTACTGGACCTGCACAAGACGGCAGAGAAACACGTCGACGCGC
AGATGCAAGATTTCATCGAGGAAAACTTTCTAAGGGAGCAGGTGGAGTCTATCGAGGAAATATCTGATCACATTAC
AAACCTGAAGCGTGTTGGACCAGGTGAAGGCGAGTACATGTTCGACGAGAACCTAAGTGAAGAACTTCAATGA 525
<210> 2
<211> 29
<212> DNA
<213>upstream amplification primer FER-F
<400> 2
CCGCTCGAGAATTAGGAGGAAGTCCAAGA 29
<210> 3
<211> 29
<212> DNA
<213>downstream amplification primer FER-R
<400> 3
CGCCATATGTCGGACTCAGAAGTCAATCA 29
Claims (3)
1. a kind of preparation method of the anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin, it is characterised in that including with
Lower step:
(1) preparation based on invertebrate Recombinant Ferritin
A. the clone of ferritin gene sequence
A. gene magnification
Using Fer147 gene order as template, which is cDNA sequence shown in SEQID NO.1, and design upstream is expanded
Increase primers F ER-F:5 '-CCGCTCGAGAATTAGGAGGAAGTCCAAGA-3 '；With downstream amplification primer FER-R:5 '-
CGCCATATGTCGGACTCAGAAGTCAATCA-3 ', PCR amplification Fer147 target gene, using plastic recovery kit to PCR
Product is recycled and is purified, and Fer147 target gene is obtained；
B. the building of prokaryotic expression carrier
Using two kinds of restriction enzymes of Nde I and Xho I respectively to Fer147 target gene and pET-28a (+) expression vector
Digestion is carried out, recovery purifying then is carried out to digestion products respectively using plastic recovery kit；It will be after digestion using DNA ligase
Fer147 target gene fragment and after pET-28a (+) empty plasmid is attached, be transformed into competent cell DH5 α, pass through
After plate screening PCR amplification and sequencing compare, the positive colony daughter colony containing target gene is obtained, after expanding culture, is utilized
Plasmid extraction kit extracts plasmid, obtains the recombinant expression carrier containing Fer147；Gained recombinant expression carrier is transformed into greatly
In enterobacteria BL21 competent cell, through plate screening PCR amplification, the e. coli bl21 expression bacterium containing target gene is obtained
Strain；
C. prokaryotic expression
By the e. coli bl21 expression bacterial strain containing target gene, the ratio of 1:50 is inoculated in 10 mL respectively and contains by volume
30 μ g/mL kanamycins LB culture mediums, 37 DEG C, 120 rpm are cultivated to OD600=0.6, add final concentration of 0.5 mM's
IPTG(isopropylthiogalactoside) in culture medium, shaking speed is 125 rpm, respectively under the conditions of temperature is 20 DEG C
It induces 18 h and temperature is 37 DEG C of 5 h of induction；Gained bacterium solution is centrifuged 15 min in 6500 rpm and collects thallus, abandons supernatant culture
Base, thallus are suspended with 2 mL disruption buffers, and the solution after ultrasonication is centrifuged 10 in 12000 rpm refrigerated centrifuges
Min collects supernatant；It takes 40 μ L supernatants that 10 μ L 5 × albumen sample-loading buffers are added, mixes well, be put into 100 DEG C of gold
Belong to and heat 10 min, then 30 s of low-speed centrifugal in bath, collecting albumen supernatant is to contain target gene albumen Fer147
Sample solution；
B. protein purification
Nickel column equipped with filler is connected on protein purification system, nickel column is rinsed with albumen buffer；It will be centrifuged
Albumen supernatant solution flow through nickel column after, the foreign protein in nickel column is rinsed with albumen washing buffer I, until albumen is pure
Change the signal that instrument detects and drops to steady state；Then destination protein is eluted with albumen washing buffer II, works as egg
When white purifying instrument detects that signal is on the rise, the solution eluted is the protein solution for needing to collect, and collects eluent,
Until signal drops to baseline steady state；The eluent containing destination protein of collection is placed in the super filter tube concentration of 30 kD,
Revolving speed control is repeatedly exchanged in 3000-3500 rpm, centrifugal process with albumen buffer, to remove remaining high concentration
Imidazoles, until finally protein solution is concentrated in Melon yellow color；Suitable SUMO enzyme is added in the albumen of concentration, as 4
Digestion is stayed overnight in DEG C environment, and the mixed solution after digestion is purified again by nickel column, take and flow through liquid, i.e., not tape label after digestion
Destination protein is based on invertebrate Recombinant Ferritin；
(2) enrichment of heavy metal
It will be placed in bag filter, be dialysed in 5 mM sodium arsenite solution, entirely based on invertebrate Recombinant Ferritin
Working fluid is simulated using magnetic stirring apparatus in enrichment process, dialyses after 12 h, bag filter is placed in albumen buffer and is dialysed
During which 12 h replace an albumen buffer every 4 h, to remove the heavy metal ion that do not adsorbed by recombinant protein, take out saturating
Analyse bag in protein solution it is concentrated after, obtain the anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin.
2. a kind of preparation side of anti-tumor nano arsenic ball based on invertebrate Recombinant Ferritin according to claim 1
Method, it is characterised in that: the formula of the disruption buffer is 25 mM Tris, 150 mM NaCL, pH 8.0,0.5%
triton X-100；The formula of the albumen buffer is 25 mM Tris, 150 mM NaCl, pH 8.0；The albumen
The formula of washing buffer I is 25 mM Tris, 150 mM NaCl, 50/70/100 mM imidazoles, pH 8.0；The albumen
The formula of washing buffer II is 25 mM Tris, 150 mM NaCl, 500 mM imidazoles, pH 8.0.
3. prepared by a kind of claim 1 is preparing chronic grain based on the anti-tumor nano arsenic ball of invertebrate Recombinant Ferritin
Application in terms of cell white blood corpuscle K562 inhibitor.
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| CN106480076A (en) * | 2016-11-25 | 2017-03-08 | 淮海工学院 | A kind of method of protecting high efficiency recombinant expressed restriction enzyme of utilization methylase |
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| CN106480076A (en) * | 2016-11-25 | 2017-03-08 | 淮海工学院 | A kind of method of protecting high efficiency recombinant expressed restriction enzyme of utilization methylase |
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