CN104211815A - Ferritin heavy chain subunit nano medicament carrying system as well as preparation method and application thereof - Google Patents
Ferritin heavy chain subunit nano medicament carrying system as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses derived protein of an epidermal growth factor-ferritin heavy chain subunit protein, namely epidermal growth factor-5 cysteine-ferritin heavy chain subunit protein (EGF-5Cys-FTH1), wherein the amino acid sequence of EGF-5Cys-FTH1 is shown by SEQ ID NO. 1 in a sequence table. The invention also discloses a ferritin heavy chain subunit nano medicament carrying system DOX/EGF-5Cys-FTH1. The ferritin heavy chain subunit nano medicament carrying system DOX/EGF-5Cys-FTH1 adopts EGF-5Cys-FTH1 as a medicament carrier, is connected with adriamycin by virtue of a crosslinking agent, namely trifluoroacetic acid salt containing maleimide hydrazide, and can be specifically enriched on the surfaces of EGFR-enriched tumor cells. The nano medicament carrying system disclosed by the invention improves the medicament carrying capacity, and is expected to be applied in manufacturing anti-tumor medicaments, particularly anti-breast-cancer medicaments.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to a kind of ferritin heavy chain subunit nano medicament carrying system and preparation method thereof and application.
Background technology
The characteristic of nano material makes it in diagnosing tumor analysis, have many advantages, one of research field of becoming with the fastest developing speed in field of nanometer technology of nanotechnology being combined with biomedical sector.
Ferritin as native protein have height biological safety, and ferritin as caged albumen can be assembled into voluntarily native conformation for its as nano drug-carrying carrier provide basis.Research in recent years shows, undersized nanoparticle can more in depth enter in the middle of solid tumor tissue, and this makes ferritin nanoparticle cause people more to pay close attention to.
In addition, result of study (Xu Li, Lihui Qiu, Xuni Cao et al., Epidermal Growth Factor-Ferritin H-Chain Protein Nanoparticles for Tumor Active Targeting " small2012; 8; No.16; 2505-2514) also show that Urogastron (EGF)-ferritin heavy chain subunit (FTH1) nanoparticle (EGF-FTH1) can be combined with the EGFR of many tumor cell surface process LAN effectively due to the EGF on its surface, make it can be enriched in tumor tissues in vivo.
But nanoparticle EGF-FTH1 forms drug-loading system owing to cannot directly be connected with medicine and directly acts on tumor cell surface, at present still not about nanoparticle EGF-FTH1 as the application of drug-loading system or report.
Summary of the invention
It is less that the present invention overcomes existing nanoparticle EGF-FTH1 particle diameter, lack the drug delivery technologies of suitable high medicine carrying thing and be difficult to the defect being applied directly to pharmaceutical carrier, the derived protein of a kind of Urogastron (EGF)-ferritin heavy chain subunit (FTH1) albumen is provided, i.e. Urogastron-5 halfcystines-ferritin heavy chain protein subunit (EGF-5Cys-FTH1 albumen), and it is connected medicine by linking agent, form ferritin heavy chain subunit nano medicament carrying system.
Existing EGF-FTH1 can be combined with the EGFR of many tumor cell surface process LAN effectively due to the EGF on its surface, can with tumour cell, particularly combine with the tumour cell of EGFR high expression level, illustrate that EGF-FTH1 has the prospect being applied to drug-loading system, but because its particle diameter is less, lack the drug delivery technologies of suitable high medicine carrying thing, therefore contriver considers that application linking agent is attempted connecting.
The cancer with the tumour cell of EGFR high expression level has mammary cancer, lung cancer, liver cancer, cancer of the stomach etc., at present, the medicine that can suppress wide spectrum or treat these cancers has cis-platinum, taxol, Zorubicin etc., consider the prospect of the pharmacokinetic properties of constructed pharmaceutical carrier, toxic side effect and reversing tumor cellular drug resistance, have chosen classical drug adriamycin as in drug-loading system by medicine carrying thing.
Because Zorubicin contains carbonyl, therefore contriver wishes that linking agent can be connected with the carbonyl of medicine on the one hand, can also be connected on the one hand with EGF-FTH1.Linking agent is containing trifluoroacetate (N-[ε-Maleimidocaproic acid) hydrazide, the trifluoroacetic acid salt of maleimido hydrazide) one end of (EMCH) is that hydrazides can form stable hydrazone key with carbonyl reaction; And the other end is maleimide, can reacts with free sulfhydryl groups and form stable thioether bond.Hydrazone key can rupture under the condition of low pH (pH < 5), and the lysosome of tumour cell just can meet this pH condition, this also just means that use linking agent EMCH is connected with Zorubicin and can ruptures by hydrazone key when medicine enters tumour cell, thus drug controllable release can be reached, carry out the object of target location for target tumor, therefore select linking agent EMCH.
Thus, contriver considers to need to modify nanoparticle EGF-FTH1, makes its derived protein contain sulfydryl and be connected with linking agent EMCH.Contriver finds after repetition test screening, can insert the natural amino acid containing sulfydryl, preferably as halfcystine between EDF and FTH1 subunit.And the selection that halfcystine inserts number also needs screening: the minimizing of halfcystine number can cause the minimizing of Sulfhydryl Groups number and reduce drug loading; And halfcystine number is too much, as 9, EGF-Cys-FTH1 active site of protein then can be made to form disulfide linkage, destroy its sterie configuration, it is made to be difficult to keep stability, therefore contriver finally selects the EGF-5Cys-FTH1 albumen of insertion 5 halfcystines, each like this EGF-5Cys-FTH nanoparticle can provide 120 Sulfhydryl Groups, to realize carrying the height of medicine.
Thus, the invention provides the derived protein of a kind of Urogastron-ferritin heavy chain protein subunit, described derived protein is be inserted with 5 halfcystines in the middle of Urogastron-ferritin heavy chain protein subunit, be Urogastron-5 halfcystines-ferritin heavy chain protein subunit (EGF-5Cys-FTH1), the aminoacid sequence of described EGF-5Cys-FTH1 albumen is as shown in SEQ ID NO.1 in sequence table.
Preferably, carry out transforming and being applicable to the present invention to build plasmid expression vector better, the nucleotide sequence of the EGF-5Cys-FTH1 albumen described in coding is as shown in SEQ ID NO.2 in sequence table.
The present invention also provides a kind of DNA molecular of EGF-5Cys-FTH1 albumen of encoding described, the aminoacid sequence of described DNA molecule encode as shown in SEQ ID NO.1 in sequence table.
Preferably, carry out transforming and being applicable to the present invention to build plasmid expression vector better, the nucleotide sequence of described DNA molecular is as shown in SEQ ID NO.2 in sequence table.Further, those skilled in the art is clear, and the Nucleotide of encoding in the DNA molecular of the aminoacid sequence as shown in SEQ ID NO.1 in sequence table can carry out replacing or modifying to adapt to different expression vectors or transformant etc.
The present invention also provides a kind of expression vector containing above-mentioned nucleotide sequence as shown in SEQ ID NO.2 in sequence table.
The empty plasmid of described expression vector can be the empty plasmid of the expression vector of this area routine, preferably, owing to being convenient to obtain and test operation, described empty plasmid is pET-28a (+), and the expression vector obtained thus is pET-28a (+)/EGF-5Cys-FTH1.
The present invention also provides a kind of genetic engineering bacterium of nucleotide sequence of the DNA molecular containing the EGF-5Cys-FTH1 albumen described in coding.
Host cell in described genetic engineering bacterium can select the host cell of this area routine; Preferably, owing to being convenient to obtain and test operation, the host cell of described genetic engineering bacterium is intestinal bacteria; Be more preferably colon bacillus (E.coli) BL21 (DE3).
Preferably, described genetic engineering bacterium contains the expression vector of the nucleotide sequence of the DNA molecular of the EGF-5Cys-FTH1 albumen described in coding, pET-28a (+)/EGF-5Cys-FTH1 as escribed above, the genetic engineering bacterium obtained is E.coli BL21 (DE3)/pET-28a (+)/EGF-5Cys-FTH 1.
The inoculum size of described genetic engineering bacterium is the inoculum size of this area routine; Preferably, the inoculum size of described genetic engineering bacterium and the volume ratio of substratum are 1: 100.
The invention provides a kind of preparation method of described EGF-5Cys-FTH1 albumen, described preparation method comprises: the transformant of the expression vector of the nucleotide sequence of the DNA molecular containing the EGF-5Cys-FTH1 albumen described in coding described in cultivation, isolation and purification culture thing obtains described EGF-5Cys-FTH1 albumen.
Preferably, described transformant is the genetic engineering bacterium of the nucleotide sequence of the described DNA molecular containing the EGF-5Cys-FTH1 albumen described in coding, above-mentioned E.coli BL21 (DE3)/pET-28a (+)/EGF-5Cys-FTH1 of such as the present invention.
The invention provides a kind of ferritin heavy chain subunit nano medicament carrying system, it adopts EGF-5Cys-FTH1 albumen of the present invention as pharmaceutical carrier.
Described ferritin heavy chain subunit nano medicament carrying system also comprises the medicine containing carbonyl; Described EGF-5Cys-FTH1 albumen is connected by linking agent with the described medicine containing carbonyl.Preferably, described linking agent can one end with containing the medicine of carbonyl and the other end be connected with described EGF-5Cys-FTH1 albumen.
Preferably, the described medicine containing carbonyl is Zorubicin (DOX).
More preferably, described linking agent is the trifluoroacetate (EMCH) containing maleimido hydrazide.Certainly, based on inventive concept of the present invention, other meet one end and can be connected with the medicine containing carbonyl, and the bi-functional cross-linking agent that the other end is connected with the sulfydryl of described derived protein also can apply to the preparation of drug-loading system.
More preferably, described ferritin heavy chain subunit nano medicament carrying system is that Zorubicin is connected by linking agent EMCH with described EGF-5Cys-FTH1 albumen, obtains DOX/EGF-5Cys-FTH1 nano medicament carrying system.
The invention provides a kind of preparation method of ferritin heavy chain subunit nano medicament carrying system, comprise the following steps: that described linking agent EMCH and described DOX is connected to form compd A, described compd A is EMCH-DOX; Described compd A is connected with described EGF-5Cys-FTH1 albumen and get final product.The preparation method of described compd A can see Willner, D et al., (6-Maleimidocaproyl) hydrazone of doxorubicin--a new derivative for the preparation of immunoconjugates of doxorubicin.Bioconjug.Chem.1993,4,521-527.
Preferably, the pH condition of described connection is 6.5 ~ 7.5; In described connection, the mol ratio of the consumption of described compd A and described EGF-5Cys-FTH1 albumen is (1 ~ 240): 1, is preferably 120: 1.The solvent of described connection is conventional buffered soln, as PBS etc.; The temperature of described connection is 4 ~ 65 DEG C; The time of described connection is 1 ~ 24 hour.
Described EGF-5Cys-FTH1 albumen due to thermostability fabulous, still can carry out ligation at 65 DEG C.
Contriver finds through experiment, when the mol ratio of the consumption of described compd A and described EGF-5Cys-FTH1 albumen is for being less than 1: 1, compd A quantity is very few, joint efficiency is too low, and the mol ratio of the consumption of described compd A and described EGF-5Cys-FTH1 albumen is when being greater than 240: 1, greatly preparation cost can be increased.When both mol ratios are 120: 1, can reach and preferably connect effect, cost compare is reasonable simultaneously.
Contriver finds through experiment, and during pH < 6.5 or > 7.5, ligation side reaction is relatively more serious, and the efficiency that described compd A is connected with sulfydryl is lower.
The invention provides described ferritin heavy chain subunit nano medicament carrying system and prepare the application in anti-tumor drug.
Preferably, described medicine is the medicine of anti-breast cancer.
Preferably, the application in overriding resistance strain tumour cell medicine is being prepared.
More preferably, described persister tumour cell is mammary cancer MCF-7/ADR cell.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: build targeted drug for carrier by linking agent EMCH with nanoparticle EGF-FTH1, can make the tissue that drug-rich acts at medicine; The hydrazone key formed due to the hydrazides of EMCH and the carbonyl of medicine can rupture at low ph conditions, thus release medicine, so apply the controllable release that pharmaceutical carrier of the present invention can realize medicine; In EGF-FTH1 subunit, insert 5 halfcystines containing sulfydryl, each like this nanoparticle will provide 120 Sulfhydryl Groups, to realize the high drug load to medicine; Drug-loading system of the present invention is obvious to persister cyto-inhibition, is expected to apply to some extent in the medicine manufacturing artitumor multi-medicine-resistant.
Accompanying drawing explanation
Fig. 1 is the EGF-5cys-FTH1 albumen transmission electron microscope picture of preparation.
SDS-PAGE (A) fluoroscopic examination figure (B) the coomassie brilliant blue staining figure of Fig. 2 DOX/EGF-5Cys-FTH1.Swimming lane 1, EGF-5Cys-FTH1 nanoparticle (8 μ g); Swimming lane 2, DOX/EGF-5Cys-FTH1 nanoparticle (20 μ g).
Fig. 3 is the broken line graph of DOX/EGF-5cys-FTH1 administration nano-drug administration system for MCF-7/ADR cell survival rate after 48 hours of preparation.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Embodiment 1 builds pET-28a (+)/EGF-5Cys-FTH1 engineering and expresses bacterium
1, by between the Nco I on nucleotide sequence insertion pET-28a (+) plasmid of coding EGF-FTH1 albumen and Not I multiple clone site, obtain pET-28a (+)/EGF-FTH1, specifically can see (Xu Li, Lihui Qiu, Xuni Cao et al., Epidermal Growth Factor-Ferritin H-Chain Protein Nanoparticles for Tumor Active Targeting, small 2012,8, No.16,2505-2514).Then design a pair reverse primer, between EGF-FTH1, insert 5 halfcystines, (see table 1).
Table 1
| Primer | Sequence (5 '-3 ') |
| Primer f | tgcggttcttgcggttcttgcggttcttgcggttcttgcgttaacatgacgaccgcgtccacctcgc |
| Primer r | gaattcgcgcagttcccaccactt |
The nucleotide sequence of the EGF-5Cys-FTH1 albumen described in coding is as shown in SEQ ID NO.2 in sequence table; The nucleotide sequence of described primer is as shown in SEQ ID No.3 (primer f) and SEQ ID No.4 (primer r) in sequence table;
Primer (primer f and primer r) described in table 1 is diluted to 10pmol, and template pET-28a (+)/EGF-FTH1 plasmid concentration is adjusted to 50ng/ μ L, carry out iPCR (KOD-Plus mutagenesis kit, TOYOBO Co., Ltd.), iPCR product pET-28a (+)/EGF-5Cys-FTH 1 is obtained.
2, with DPn I enzyme, PCR primer is digested, and make PCR primer recirculation.Take out DH5 α competent cell (purchased from Tian Gen biochemical corp) 100 μ L, ice bath dissolves; Get above-mentioned recirculation product 10 μ L, add in competent cell, mix gently, ice bath places 30min; 42 DEG C, 30s heat-shocked, ice bath places 5min.Add SOC substratum 900 μ L, 37 DEG C of shaking culture 2h.Get the LB that 100 μ L coat containing kantlex (Kanamycin) dull and stereotyped, room temperature just puts 0.5h; Be inverted in the CO of 37 DEG C
2more than 16h cultivated by incubator.
3, random choose list bacterium colony 5 on each solid LB flat board, joins respectively in the fresh LB 10mL containing Kanamycin, and marks.Put 37 DEG C of thermostat container shaken overnight; Get 8mL overnight shaking cultivate the centrifugal 20min of LB substratum, speed 5500rpm, obtain thalline.
4, middle amount test kit extraction plasmid is carried with the high purity of Tian Gen biochemical corp is little: Hinc II enzyme digestion verification is carried out to the plasmid extracted: the plasmid getting 5 μ L extractions adds (purchased from Tian Gen biochemical corp) in E.coli BL21 (DE3) competent cell, gently after mixing, place 30min on ice.42 DEG C of water-bath 90s, then leave standstill 5min on ice.Add the aseptic SOC substratum of 900 μ L, mixing placement 37 DEG C of shaking table 2h.The supernatant liquor of the centrifugal 5min of 1000rpm, sucking-off 800 μ L.Resuspended remaining bacterium liquid is also spread evenly across on the LB solid plate containing 50ng/ μ L Kanamycin.Room temperature places 0.5h, treats that whole bacterium liquid has been absorbed, flat board is inverted 37 DEG C of incubators and spends the night.
Namely obtain and transform pET-28a (+)/EGF-5Cys-FTH1 engineering expression bacterium E.coli BL21 (DE3)/pET-28a (+)/EGF-5Cys-FTH1, described engineering expresses the aminoacid sequence of the EGF-5cys-FTH1 albumen that bacterium is expressed as shown in SEQ ID No.1 in sequence table.
The preparation of embodiment 2 EGF-5Cys-FTH1 target protein
1, from single bacterium colony that random choose the LB solid medium having transformed plasmid is full, add in the fresh LB liquid nutrient medium containing 50ng/ μ L kantlex, 37 DEG C are shaken bacterium and spend the night and activate.Then add in 50mL fresh LB spread cultivation with the volume ratio of 1: 100.When the OD value to 0.6 of bacterium liquid, add the inductor IPTG of 1mM, overnight induction, collected by centrifugation thalline.
2, the re-suspension liquid A (re-suspension liquid A: containing 50mM PBS (phosphate buffered saline buffer), 150mM NaCl, pH 7.9) that past thalline adds 10-15ml carries out cell ultrasonication.Broken condition is: by the reiteration circulation 30min of Ultrasonic Cell Disruptor work 1s, interval ls, power 300w.Centrifugal 10min under 12000rpm after fragmentation, collects bacterial sediment.Add washings (washings: 50mMTris, 50mM NaCl, 1mM EDTA (ethylenediamine tetraacetic acid (EDTA)), 1%Triton-100 (C
34h
62o
11)), pH 7.9 washs the inclusion body 4 times of gained, namely obtains purer inclusion body.
3, after washing, grind inclusion body, add sex change liquid (sex change liquid: 50mM Tris, 1mM EDTA, 8M urea, 10mM DTT (dithiothreitol (DTT)), pH 7.9) inclusion body after resuspended washing, the shaking table being placed in 28 DEG C spends the night and inclusion body sex change is dissolved.It is 0.1mg/mL that the inclusion body sex change liquid that sex change is good is diluted to final concentration, and volume is 50mL.With being placed in dialysis tubing, being immersed in renaturation solution 1-6 and progressively dialysing at 4 DEG C, changed a renaturation solution every 6 hours, each renaturation solution formula changed is in table 2.After treating that renaturation terminates, the protein solution after collecting by filtration renaturation, with the super filter tube ultrafiltration and concentration protein solution of 50KD, namely obtain EGF-5Cys-FTH1 target protein, the transmission electron microscope of described target protein as shown in Figure 1.
The formula of table 2 renaturation solution
The connection of embodiment 3 linking agent (EMCH) and Zorubicin (DOX)
1, get DOX and the 19mg of 12mg EMCH powder mixing after be dissolved in the methanol solution of 3.5mL, after the powder of DOX and EMCH dissolves completely, in mixing solutions, add the trifluoroacetic acid of 1 μ L, with magnetic stirring apparatus at ambient temperature lucifuge stir 24 hours.
2, to reaction solution decompression batch distillation, lower water temperature is 31 DEG C, and underpressure distillation adds the acetonitrile of 2.5mL in the solution and puts into 4 DEG C of refrigerators and make it crystallization 48 hours when overall solution volume is 150 μ L.The centrifugal 2min of 8000rpm rotating speed after crystallisation process completes, removes supernatant liquor, with the mixing solutions washing precipitation twice containing 0.5mL methyl alcohol and 5mL acetonitrile.Last vacuum-drying at ambient temperature becomes the Powdered of drying to moistening product, namely obtains EMCH-DOX compound.Identify EMCH-DOX compound, appraising datum is as follows: detected from proton nmr spectra, NMR (CD3OD) 7.94 (bd, 1H), 7.82 (t, 1H), 7.55 (d, 1H), 6.78 (s, 2H), 5.48 (s, 1H), 5.07 (t, 1H), 4.59 (d, 1H), 4.21 (m, 1H), 4.02 (s, 3H), 3.63-3.30 (m, 5H), 2.55-2.26 (m, 4H), 2.19-1.88 (m, 3H), 1.69-1.18 (m, 12H); MS (M+H)+751, illustrates and has really synthesized EMCH-DOX compound.
The molecular structural formula of described EMCH-DOX compound is:
The connection of embodiment 4 EMCH-DOX and EGF-5Cys-FTH1
1, dissolving EMCH-DOX powder compounds with DMSO makes concentration be 10 μ g/ μ L; Configuration PBS buffer A: containing 0.1M sodium phosphate, 0.15M NaCl, regulates pH to be 7.2.
2, every 1mg EGF-5Cys-FTH1 albumen and 130 μ g EMCH-DOX react, the mol ratio of compd E MCH-DOX and EGF-5Cys-FTH1 albumen be 120: 1 (method of calculation:
Mol[EMCH-DOX]：(130μg)÷(751g/mol)＝1.7×10
-7mol，
Mol[EGF-5Cys-FTH1]：(1mg)÷(703KD)＝1.4×10
-9mol，
Mol[EMCH-DOX]/Mol[EGF-5Cys-FTH1]＝120∶1)。
After being added by EMCH-DOX solution in albumen, lucifuge 4 DEG C of reactions are spent the night and are obtained by reacting reaction solution; Reaction solution is poured in the super filter tube of 15mL, ultrafiltration and concentration 6 ~ 8min under 3500rpm, by the removing of free small molecules EMCH-DOX compound.Repeat this step repeatedly until effluent liquid clarification is without color.
3, the product obtained is carried out SDS-PAGE gel electrophoresis, electrophoresis takes fluorescent bands after running through at once under ultraviolet, with Xylene Brilliant Cyanine G, glue is dyeed again, observe whether have protein band in fluorescent bands position after decolouring, its result as shown in Figure 2, because protein band and fluorescent bands position consistency, the successful connection of DOX and EGF-5Cys-FTH1 albumen can be judged.Namely DOX/EGF-5cys-FTH1 drug-loading system has built.Then Coomassie Brilliant Blue is adopted to measure the concentration of the Zorubicin medicine that protein concentration is connected with Their Determination by Spectrophotometry respectively, the drug loading that can calculate this drug-loading system is 72mol/mol nano medicament carrying system, and namely every 1molDOX/EGF-5cys-FTH1 drug-loading system can carry the DOX of 72mol.
The Toxicity Analysis of embodiment 5 DOX/EGF-5Cys-FTH1 nano medicament carrying system
1, with 1 × 10 after MCF-7/ADR cancer cells (purchased from Nanjing KeyGen Biotech.Co., the Ltd) tryptic digestion growing to desired number being counted
4the concentration of individual cell per well is inoculated in 96 orifice plates, every hole 100 μ L; Add aseptic PBS damping fluid to ensure the temperature and humidity in hole, at 37 DEG C, 5%CO
2in incubator, overnight incubation makes cell attachment.
2, by the sucking-off of 10%FBS 1640 substratum, change to the fresh substratum containing different concns medicine, each concentration need establish the repetition in 3 holes; Free Zorubicin and DOX/EGF-5Cys-FTH1 nano medicament carrying system are configured to respectively the concentration of 0.01 μ g/mnL, 0.1 μ g/mL, 0.2 μ g/mL, 0.5 μ g/mL, 1 μ g/mnL, 2 μ g/mL, 5 μ g/mL, 10 μ g/mL and 20 μ g/mL, every hole 100 μ L adds 96 orifice plates; In addition arrange 3 blank well as zeroising, 3 pure cell holes as negative control, all medicines add latter 37 DEG C, 5%CO
2cultivate 48 hours in incubator.
3, in every hole, add MTT (3-(4,5-dimethylthiazole-2)-2, the 5-diphenyltetrazolium bromide bromine salt) working fluid of 20 μ L 5mg/mL after 48 hours, then continue 37 DEG C and hatch 4 hours.
4, hatched and carefully sucked all supernatant liquors in hole afterwards; Then add 150 μ L dimethyl sulfoxide (DMSO) liquid, room temperature condition is positioned on horizontal shaker and shakes 10min slowly, ties brilliant formazan resolution of precipitate by hatching the bluish voilet formed at the bottom of metapore.
5, use microplate reader 490nm place to detect each hole light absorption value, record data, and with the concentration of DOX for X-coordinate, cell survival rate is ordinate zou drawing standard curve, and result as shown in Figure 3.Adopt free Zorubicin to compare with DOX/EGF-5Cys-FTH1 nano medicament carrying system, on cell survival rate, there is significant difference (p < 0.05).
Result display DOX/EGF-5Cys-FTH1 drug-loading system has stronger killing effect compared with DOX to MCF-7/ADR cell.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. the derived protein of Urogastron-ferritin heavy chain protein subunit, it is characterized in that, described derived protein is be inserted with 5 halfcystines in the middle of Urogastron-ferritin heavy chain protein subunit, be Urogastron-5 halfcystines-ferritin heavy chain protein subunit, the aminoacid sequence of described derived protein is as shown in SEQ ID NO.1 in sequence table; Preferably, the nucleotide sequence of the derived protein described in coding is as shown in SEQ ID NO.2 in sequence table.
2. a DNA molecular for the derived protein of the encoding epidermal growth factor-ferritin heavy chain protein subunit, is characterized in that, the aminoacid sequence of described DNA molecule encode as shown in SEQ ID NO.1 in sequence table; Preferably, the nucleotide sequence of described DNA molecular is as shown in SEQ ID NO.2 in sequence table.
3. the expression vector containing, for example the nucleotide sequence of DNA molecular according to claim 2; Preferably, the empty plasmid of described expression vector is pET-28a (+).
4. the genetic engineering bacterium containing, for example the nucleotide sequence of DNA molecular according to claim 2; Preferably, described genetic engineering bacterium is containing, for example expression vector according to claim 3; Preferably, the host cell of described genetic engineering bacterium is colon bacillus (E.coli) BL21 (DE3).
5. a preparation method for derived protein as claimed in claim 1, is characterized in that, described preparation method comprises: cultivate the transformant containing, for example expression vector according to claim 3, and isolation and purification culture thing obtains described derived protein; Preferably, described transformant is genetic engineering bacterium as claimed in claim 4.
6. a ferritin heavy chain subunit nano medicament carrying system, is characterized in that, described ferritin heavy chain subunit nano medicament carrying system adopts derived protein as claimed in claim 1 as pharmaceutical carrier.
7. ferritin heavy chain subunit nano medicament carrying system as claimed in claim 6, it is characterized in that, described ferritin heavy chain subunit nano medicament carrying system also comprises the medicine containing carbonyl, and described derived protein is connected by linking agent with the described medicine containing carbonyl; Preferably, the described medicine containing carbonyl is Zorubicin; More preferably, described linking agent is the trifluoroacetate containing maleimido hydrazide.
8. the preparation method of a ferritin heavy chain subunit nano medicament carrying system, it is characterized in that, described preparation method comprises the following steps: that linking agent contains the trifluoroacetate of maleimido hydrazide and Zorubicin and reacts and form compd A, and described compd A is connected with derived protein as claimed in claim 1.
9. preparation method as claimed in claim 8, it is characterized in that, the pH condition of described connection is 6.5 ~ 7.5; In described connection, the mol ratio of described compd A and described derived protein consumption is (1 ~ 240): 1, is preferably 120: 1.
10. the application of ferritin heavy chain subunit nano medicament carrying system in the medicine preparing antitumor cell as described in any one of claim 6-7; Preferably, described medicine is the medicine of anti-breast cancer; Preferably, the application in the medicine preparing overriding resistance strain tumour cell; More preferably, described persister tumour cell is MCF-7/ADR cell.
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| CN113476588A (en) * | 2021-07-06 | 2021-10-08 | 武汉大学 | Multifunctional breast cancer-targeted nanoparticle, and preparation method and application thereof |
| WO2022206451A1 (en) * | 2021-03-30 | 2022-10-06 | 南京纳么美科技有限公司 | Targeted ferritin cage nano-carrier co-loading hydrophilic/hydrophobic drugs and use thereof |
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| WO2007073932A2 (en) * | 2005-12-27 | 2007-07-05 | Lts Lohmann Therapie-Systeme Ag | Protein-based delivery system for overcoming resistance in tumour cells |
| CN101942023A (en) * | 2010-07-29 | 2011-01-12 | 华东理工大学 | Human epidermic growth factor ferritin heavy chain subunit fusion protein, constructing method and use thereof |
| CN104017089A (en) * | 2014-06-25 | 2014-09-03 | 华东理工大学 | Ferritin heavy-chain subunit nanoparticles, and preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022206451A1 (en) * | 2021-03-30 | 2022-10-06 | 南京纳么美科技有限公司 | Targeted ferritin cage nano-carrier co-loading hydrophilic/hydrophobic drugs and use thereof |
| CN113476588A (en) * | 2021-07-06 | 2021-10-08 | 武汉大学 | Multifunctional breast cancer-targeted nanoparticle, and preparation method and application thereof |
| CN113476588B (en) * | 2021-07-06 | 2022-11-22 | 武汉大学 | Multifunctional nanoparticles targeting breast cancer, preparation method and application thereof |
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