CN107286249A - A kind of restructuring apoferritin nanocages of oligomerization polylysine modification and its preparation - Google Patents
A kind of restructuring apoferritin nanocages of oligomerization polylysine modification and its preparation Download PDFInfo
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- CN107286249A CN107286249A CN201710435975.0A CN201710435975A CN107286249A CN 107286249 A CN107286249 A CN 107286249A CN 201710435975 A CN201710435975 A CN 201710435975A CN 107286249 A CN107286249 A CN 107286249A
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Abstract
The invention discloses a kind of restructuring apoferritin nanocages of oligomerization polylysine modification and its preparation.The nanocages are the hollow spheres albumen being self-assembly of by protein protomer, and the N-terminal using DNA recombinant expression technology in protein protomer modifies different number of lysine, to obtain the restructuring apoferritin cage of surface modification oligomerization lysine.The protein nano cage, can realize protein protomer depolymerization and restructuring by changing pH value of solution, so as to realize that medicine is loaded；Biocompatibility, internal stability are good；Can occur specific recognition with the TfR of tumor cell surface height expression, realize active targeting；Mediated by encytosis after born of the same parents, in the presence of polylysine residue, in lysosomal acid environment, realize that lysosome is escaped by proton sponge effect, and then the medicine in protected protein cage is not degraded in lysosome.The recombinant protein nanocages are expected to have a good application prospect in terms of genomic medicine delivering, drug cell device targeted delivery.
Description
Technical field
The invention belongs to biomedicine technical field, and in particular to a kind of restructuring apoferritin nanometer of oligomerization polylysine modification
Cage and preparation method thereof.
Background technology
Chemotherapy is common treating malignant tumor method, but most of antineoplastics are due to a lack of selectivity, in production
Crude drug can also produce serious toxic side effect while effect to body.Therefore, the exploitation of antineoplastic targeted delivery systems has
Extremely important clinical value.Apoferritin not only has good biocompatibility as a kind of native protein
And stability, while can also be specifically bound with the TfR 1 (TfR1) of tumor cell surface height expression, and
Realized under the mediation of this receptor and effectively enter born of the same parents, so as to reach the effect of active targeting.Apoferritin is generally by 24 subunits
Self assembly and form hollow sphere cage structure, particle diameter is 12-13nm., can be by changing solution using its sensitiveness to pH
PH makes protein cage occur depolymerization with gathering again, so as to carry out containing for medicine；Simultaneously can be a variety of by genetic engineering, chemical method etc.
Means are modified the surfaces externally and internally of the albumen so that the albumen can effectively build novel multifunctional nano material.These
Superior performance causes apoferritin to become a kind of preferable multifunctional nano pharmaceutical carrier.
However, unmodified apoferritin nanocages realize across cell in the case where TfR1 is receptor-mediated by encytosis
After transhipment, it would generally be stopped in lysosome, and a large amount of enzyme systems existed in lysosome can cause protein cage structural damage,
Occur degraded and destruction so as to cause its medicine contained to be also exposed in lysosome environment, to needing the certain detail in endochylema
For the medicine played a role in born of the same parents' device or nucleus, a kind of apoferritin nanometer with lysosome escape function need to be designed and passed
Medicine system.Protonation can occur under the conditions of lysosomal acid, cause chlorion as a kind of basic amino acid for lysine
Largely enter lysosome with water, cause osmotic pressure in lysosome to raise, final rupture.Therefore utilize this " the proton sea of lysine
Continuous effect ", the N-terminal using genetic engineering recombination and expression techniques in protein protomer modifies different number of lysine, to obtain table
The restructuring apoferritin cage of oligomerization lysine is modified in face, it is possessed lysosome escape property.
The content of the invention
The invention aims to overcome natural human apoferritin cage to enter as drug delivery vehicle after born of the same parents easily by lyase
The deficiency of body degraded, and a kind of restructuring apoferritin nanocages of the oligomerization polylysine modification with lysosome escape function are provided
And preparation method thereof.The recombinant protein cage can not only realize that medicine, to the targeted delivery of tumour cell, is reduced to body normal group
The toxic side effect knitted, while the medicine in cage can also be protected not destroyed by lysosome, so as to realize the specific cells device in endochylema
Or endonuclear delivering, reach more preferable targeted therapy effect.
In order to solve the above technical problems, the present invention provides following technical scheme.
A kind of protein nano cage, formula is：N-Lys-HFn, wherein, Lys represents lysine, and n is lysine in single egg
The modification number of Bai Yaji N-terminals, n=4,8, HFn refer to the protein cage as formed by 24 people ferritin H subunits (FTH) self assemblies.
The ferritin heavy chain subunit nanocages of above-mentioned oligomerization polylysine modification, its preparation method is mainly included the following steps that：
(1) recombinant protein cage expresses the structure of engineering bacteria：Based on FTH encoding genes, in its 5 ' terminal modified different number
Purpose AAG or AAA (Lys encoding gene), and obtained gene order is subcloned after double digestion to pET-30a (+) matter
In grain carrier, that is, obtain plasmid template to be expressed.The template is converted to competent escherichia coli cell through heat shock, and led to
Cross the means such as Kanamycin resistances and gene sequencing and filter out positive monoclonal, mass propgation is carried out to positive monoclonal, and protect
Deposit its glycerol stock.
(2) expression of fusion protein and receipts bacterium：The positive engineering bacteria that step (1) is obtained is inoculated in LB-Kan+ culture mediums
In, culture to bacterium solution OD600When reaching 0.3-0.5, IPTG induced expressions are added, thalline is collected by centrifugation.
(3) fusion protein is isolated and purified：Ultrasonication step (2) obtains thalline, and supernatant is collected by centrifugation, supernatant is put
In after heating water bath, centrifugation again obtains the supernatant containing purposeful fusion protein.By supernatant loading to Ni+Post, uses gradient
Imidazole buffer successively elutes foreign protein and purpose fusion protein, collects eluent and simultaneously carries out SDS-PAGE analyses, selection contains mesh
Fusion protein at most and the minimum eluent of foreign protein, be placed in 100kDa super filter tubes and centrifuge desalination, obtain mesh after purification
Fusion protein.
(4) enterokinase of fusion protein is cut：Recombinant enterokinase is added in the purpose fusion protein obtained to step (3), is made
It is histidine-tagged to cut off for the enterokinase site in fusion protein, then reaction product is placed in 100kDa super filter tubes
Centrifugation, finally gives the destination protein (n-Lys-HFn).
Pass through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscope (TEM)
Product is characterized.
The ferritin heavy chain subunit nanocages (n-Lys-HFn) for the oligomerization polylysine modification that the present invention is obtained can be with height expression
Specific recognition occurs for TfR1 tumour cell, and realizes effectively to enter born of the same parents and enter lysosome in the presence of receptor-mediated.
In lysosome, n-Lys-HFn occurs lysosome by proton sponge effect and escaped, and protects the medicine in cage from degraded, increase
Medicine specific cells device or endonuclear concentration in endochylema, improve the therapeutic effect of medicine.The recombinant protein passs medicine system
System not only has good biocompatibility and stability, can also realize medicine to the active targeting of tumor locus, reduction is aligned
The toxic side effect often organized, provides and a kind of preferably multi-functional receives for the targeted delivery of genomic medicine and other antineoplastics
Rice pharmaceutical carrier.
Brief description of the drawings
Fig. 1：PET-30a (+)/4-Lys-FTH (A) and pET-30a (+)/8-Lys-FTH (B) exist in embodiment 2
ArcticExpressTMThe soluble analysis result of middle expression product.
Fig. 2：4-Lys-HFn (A) and 8-Lys-HFn (B) respectively flow in affinity chromatography method purge process in embodiment 4
Go out the SDS-PAGE figures of liquid.
Fig. 3：The SDS-PAGE of product schemes before and after 4-Lys-HFn and 8-Lys-HFn digestions in embodiment 5, wherein with digestion
Front and rear HFn is used as control, A) be product, B before digestion) it is product after digestion.
Fig. 4：In embodiment 5 after digestion end-product 4-Lys-HFn (B) and 8-Lys-HFn (C) transmission electron microscope picture, wherein
Control is used as using the HFn (A) after digestion.
Embodiment
The present invention is further explained and illustrated with reference to specific embodiment, it should be appreciated that given embodiment
It is only used as example, it does not constitute any limitation to the scope of the present invention in any way.
Embodiment 1.pET-30a (+)/n-Lys-FTH expresses the structure of engineering bacteria
According to the literature treat fully synthetic FTH gene coded sequences, in order that FTH subunits N-terminal can be modified with not
With the Lys of number, 5 ' ends of the gene coded sequence plus AAGAAAAAGAAA (4-Lys) and
AAGAAAAAGAAAAAGAAAAAGAAA (8-Lys), then 5 ' ends of the gene coded sequence after the modification and 3 ' ends are distinguished
Plus CCATGGCT and GCGGCCGC to obtain Nco I and Not I digestion recognition sites, finally gene order is optimized,
The sequence is set to realize correct expression in E. coli system.Two sequences length is respectively 580bp and 592bp, coding
The nucleotide sequence of described 4-Lys-HFn and 8-Lys-HFn protein protomers is respectively such as SEQ ID NO.1, SEQ ID NO.2 institutes
Show.Double digestion is carried out to the sequence of synthesis using Nco I and Not I, and same pair is carried out to plasmid vector pET-30a (+)
Digestion, by T4DNA ligases, 4 DEG C connect two kinds of digestion products overnight, and the product is synthesized by Nanjing Jin Sirui companies.While root
According to《Molecular Cloning:A Laboratory guide》Use CaCl2Method prepares Host Strains ArcticExpressTMThe competent cell of (AE bacterium), and lead to
Heat shock is crossed by connection product in 42 DEG C of conversions to the competent cell.The LB culture mediums without resistance are used to train at 37 DEG C
Product after conversion is supported, is incubated after 1h, is coated to Kan+- LB flat boards, are placed after 30min at 37 DEG C, are inverted flat board, are cultivated
Night.Picking positive monoclonal transformant is cultivated and preserves strain.Obtain conversion pET-30a (+)/4-Lys-FTH respectively
Bacterium ArcticExpress is reached with pET-30a (+)/8-Lys-FTH quantity sheetTM。
The analysis of the fusion protein solubility expression form of embodiment 2.n-Lys-HFn mesh
Positive restructuring bacterium is added to fresh Kan+In-LB culture mediums, at 37 DEG C, 220r/min, shaken overnight is with work
Change.5mL Kan are forwarded to 1% inoculum concentration+- LB culture mediums, at 37 DEG C, 220r/min, culture to bacterium solution OD600Reach
0.5.Induced expression about 6h under final concentration of 0.1mM IPTG, similarity condition is added into test tube.Take 4 DEG C of 1mL bacterium solutions, 8000
× g, centrifuges 3min.After supernatant discarding, 200 μ L combination buffers (20mM Tris, 5mM imidazoles, 0.5M NaCl, pH are added
7.9) it is resuspended, is centrifuged again under the same terms after resuspension, abandoned supernatant and collect thalline, 200 μ L is resuspended in again and combine buffering
Liquid, -20 DEG C freeze overnight.Carrying out ultrasonic bacteria breaking after thalline melts is taken out, sample is taken out and is used as total bacterium；Collected after centrifugation supernatant, takes out
Sample is used as supernatant.Using supernatant after the bacterial protein of Host Strains and broken bacterium as control, the parallel selection of every kind of positive restructuring bacterium
Four samples, through SDS-PAGE electrophoretic analysis solubility expression of protein situations and expression quantity.SDS-PAGE results as shown in figure 1,
As a result show, total bacterium of two kinds of recombination engineerings and bacteria break supernatant are it is observed that the band that molecular weight is about 29kDa, with restructuring
The single molecular weight subunits of albumen 4-Lys-HFn (26.8kDa) and the basic phase of the single molecular weight subunits of 8-Lys-HFn (27.32kDa)
When, with the oneself expression albumen of space-time Host Strains without this band, therefore can determine whether the band at 29kDa be respectively two kinds restructuring
The subunit of albumen.In addition, destination protein band area quite, shows recombinant protein in total bacterium of positive restructuring bacterium and bacteria break supernatant
Mainly expressed with soluble form.
The great expression of the fusion protein of embodiment 3.n-Lys-HFn mesh is with receiving bacterium
The positive restructuring bacterium of recovery overnight is forwarded to 100mLKan with 1% inoculum concentration+- LB culture mediums, at 37 DEG C,
220r/min, culture to bacterium solution OD600Reach 0.5.Add under final concentration of 0.1mM IPTG, similarity condition and induce into test tube
Express about 6h.4 DEG C, 8000 × g, centrifugation 3min adds 30mL combination buffers and is resuspended, after resuspension again to collect thalline
Centrifuged under the same terms, abandon supernatant and collect thalline, 30mL combination buffers are resuspended in again, add final concentration of 100 μ g/mL's
Lysozyme, -20 DEG C freeze.
The fusion protein of embodiment 4.n-Lys-HFn mesh is isolated and purified
Taking-up freezes carrying out ultrasonic bacteria breaking after bacterium solution is melted, and ultrasound condition is：Power 600W, ultrasound opens 1s, stops 2s, altogether ultrasound
12min.4 DEG C, 10000 × g centrifuges 35min.Collect supernatant and be placed in 60 DEG C of water-baths, heat 20min to remove heat labile miscellaneous egg
In vain.4 DEG C again, 10000 × g centrifuges 35min, obtains the supernatant containing destination protein.Due to containing in purpose fusion protein
It is histidine-tagged, therefore destination protein can be purified using affinity chromatography method.2mL resins are added into chromatographic column, are used
NiSO is added after sterile water wash4Solution is combined, after resin fully becomes basket, cleans many to wash off with combination buffer
Remaining NiSO4.With slow flow velocity loading supernatant, albumen is set fully to be combined with nickel post.It is flat with combination buffer after end of the sample
Weigh chromatographic column, then successively elutes foreign protein and destination protein with gradient imidazole solution (30,50,70,90 and 300mM), and collects
Eluent is through SDS-PAGE methods analysis eluent composition.As a result SDS-PAGE results as shown in Fig. 2 show, two kinds of recombinant proteins
Maximum level is shown in 300mM imidazole elutions, and now foreign protein content is also minimum.Therefore 300mM imidazoles is selected
Eluent carries out ultrafiltration (100kDa, 4 DEG C, 12000 × g, 6min) desalination, and final product is stored in storage buffer solution (20mM
Tris, 0.15M NaCl, pH 8.0).
The enterokinase of the fusion protein of embodiment 5.n-Lys-HFn mesh is cut
Appropriate recombinant enterokinase is added into 1.5mg/mL destination protein solution, under 37 DEG C of water-baths, 16h is reacted, with
Excision is histidine-tagged.Reaction product is transferred to 100kDa super filter tubes, 4 DEG C, 12000 × g centrifuges 6min, to remove restructuring
Enterokinase and cutting it is histidine-tagged, finally give the destination protein of the end-product after digestion, i.e. high-purity.HFn before digestion,
4-Lys-HFn, 8-Lys-HFn molecular weight subunit are respectively 26.29kDa, 26.80kDa, 27.32kDa, and after digestion then
21.50kDa, 22.00kDa, 22.53kDa are reduced to respectively.Therefore use SDS-PAGE to analyze the destination protein before and after digestion, knot
Fruit is as shown in Figure 3.Test result indicates that, HFn, 4-Lys-HFn, 8-Lys-HFn molecular weight subunit are substantially reduced after digestion, and
Molecular weight increasing trend is presented between three kinds of albumen, is consistent with each protein protomer corresponding theory molecular weight.4-Lys- after digestion
The amino acid sequence of HFn, 8-Lys-HFn protein protomer is respectively as shown in SEQ ID NO.3, SEQ ID NO.4.In addition, from electricity
It can be seen that in swimming figure, the destination protein purity after digestion and ultrafiltration is higher, almost without other foreign protein bands.Therefore it can determine whether mesh
Albumen 4-Lys-HFn, 8-Lys-HFn successful expression and purified.Table is carried out to the form of destination protein finally by TEM
Levy, as a result as shown in Figure 4.As a result show, the Recombinant Ferritin subunit of oligomerization polylysine modification is in escherichia expression system
Self assembly still is able to successfully, with unmodified HFn forms without significant difference, the small and homogeneous hollow cage knot of particle diameter is all shown as
Structure.
Claims (2)
1. the restructuring apoferritin cage of a kind of oligomerization polylysine modification, it is characterised in that its amalgamation mode relies to be different number of
Propylhomoserin is blended in the N-terminal of people's ferritin H subunits (FTH), and formula is：N-Lys-HFn, wherein, Lys represents lysine, and n is to rely ammonia
The sour modification number in single protein protomer N-terminal, HFn refers to the protein cage as formed by 24 FTH self assemblies.
2. the preparation method of the restructuring apoferritin cage of the oligomerization polylysine modification described in claim 1, it is characterised in that including
Procedure below：
1) recombinant protein cage expresses the structure of engineering bacteria：
In 5 ' terminal modified Lys encoding genes of FTH gene coded sequences, it is subcloned into plasmid vector pET-30a (+), and turns
Change to E.coli ArcticExpressTMCompetent cell.By Kanamycin resistance screening positive monoclonal transformants,
To obtain purpose recombinant protein cage expression engineering bacteria.
2) expression of fusion protein and receipts bacterium：
Expression engineering bacteria is inoculated in LB-Kan with 1% inoculative proportion+In culture medium, IPTG induced expressions are added, are collected by centrifugation
Thalline.
3) fusion protein is isolated and purified：
The thalline that destruction step (2) is obtained, is collected by centrifugation supernatant.Supernatant is placed in heating water bath, acquisition is centrifuged again and contains mesh
Fusion protein supernatant.Using affinity chromatography method the foreign protein and purpose fusion protein of supernatant are carried out separation with
Purifying.
4) enterokinase of fusion protein is cut：
Recombinant enterokinase is added in the purpose fusion protein obtained to step (3), it is histidine-tagged to cut off, finally give described
Destination protein (n-Lys-HFn).
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Cited By (8)
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| CN109486827A (en) * | 2018-12-04 | 2019-03-19 | 南京林业大学 | A kind of apoferritin nanocages and preparation method thereof of tumor-homing cell-penetrating peptide tLyP-1 modification |
| CN110075087A (en) * | 2019-04-10 | 2019-08-02 | 中国药科大学 | SiRNA- biology calcification recombinates apoferritin nanoparticle and preparation method thereof |
| CN110208236A (en) * | 2019-06-28 | 2019-09-06 | 郑州大学 | Ratio-type fluorescent pH nano-probe based on apoferritin, preparation method and applications |
| CN110327308A (en) * | 2019-07-02 | 2019-10-15 | 中国药科大学 | A kind of recombination apoferritin nanocages and preparation method thereof being loaded with siRNA |
| CN114452266A (en) * | 2022-02-09 | 2022-05-10 | 山东大学 | Nucleic acid drug delivery system based on recombinant ribosomal protein and preparation method and application thereof |
| CN116196398A (en) * | 2023-03-09 | 2023-06-02 | 中国人民解放军空军军医大学 | Hemoglobin crowned protein nanocage and construction method thereof |
| WO2023165467A1 (en) * | 2022-03-04 | 2023-09-07 | 南京纳么美科技有限公司 | Ferritin nanocage vector loaded with small nucleic acid drug in inner cavity and use |
| CN117599209A (en) * | 2024-01-23 | 2024-02-27 | 中山大学 | Self-assembled nano protein cage and preparation method and application thereof |
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| CN109486827A (en) * | 2018-12-04 | 2019-03-19 | 南京林业大学 | A kind of apoferritin nanocages and preparation method thereof of tumor-homing cell-penetrating peptide tLyP-1 modification |
| CN110075087A (en) * | 2019-04-10 | 2019-08-02 | 中国药科大学 | SiRNA- biology calcification recombinates apoferritin nanoparticle and preparation method thereof |
| CN110208236A (en) * | 2019-06-28 | 2019-09-06 | 郑州大学 | Ratio-type fluorescent pH nano-probe based on apoferritin, preparation method and applications |
| CN110327308A (en) * | 2019-07-02 | 2019-10-15 | 中国药科大学 | A kind of recombination apoferritin nanocages and preparation method thereof being loaded with siRNA |
| CN114452266A (en) * | 2022-02-09 | 2022-05-10 | 山东大学 | Nucleic acid drug delivery system based on recombinant ribosomal protein and preparation method and application thereof |
| WO2023165467A1 (en) * | 2022-03-04 | 2023-09-07 | 南京纳么美科技有限公司 | Ferritin nanocage vector loaded with small nucleic acid drug in inner cavity and use |
| CN116196398A (en) * | 2023-03-09 | 2023-06-02 | 中国人民解放军空军军医大学 | Hemoglobin crowned protein nanocage and construction method thereof |
| CN116196398B (en) * | 2023-03-09 | 2023-11-24 | 中国人民解放军空军军医大学 | Hemoglobin crowned protein nanocage and construction method thereof |
| CN117599209A (en) * | 2024-01-23 | 2024-02-27 | 中山大学 | Self-assembled nano protein cage and preparation method and application thereof |
| CN117599209B (en) * | 2024-01-23 | 2024-05-03 | 中山大学 | Self-assembled nano protein cage and preparation method and application thereof |
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