CN117720671A - Recombinant proteins IIB-IIAIB having self-assembling repeat units rQTY Application and application thereof - Google Patents
Recombinant proteins IIB-IIAIB having self-assembling repeat units rQTY Application and application thereof Download PDFInfo
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- CN117720671A CN117720671A CN202410131162.2A CN202410131162A CN117720671A CN 117720671 A CN117720671 A CN 117720671A CN 202410131162 A CN202410131162 A CN 202410131162A CN 117720671 A CN117720671 A CN 117720671A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of biological medicine, and in particular relates to a repetitive structural unit recombinant protein IA I B-IA I B with self-assembly performance rQTY And (3) application. The recombinant protein comprises a group I-IB repeat fragment of 2 human serum albumin type I domains, wherein the amino group in the group 2 group I-IB repeat fragmentThe acid has mutation, specifically the hydrophilic amino acids at positions 125, 133, 138, 140, 150, 161, 166 and 170 in the 2 nd IIB structural fragment are mutated to be hydrophobic.
Description
Technical Field
The invention belongs to biological medicineTechnical field, in particular to a recombinant protein of repeated structural units IA IB-IA IB with self-assembly performance rQTY And (3) application.
Background
The drug carrier material plays an important role in the research of improving the water solubility of the hydrophobic drug, the bioavailability of the drug, the controlled release and the slow release of the drug, the targeted delivery of the drug, the penetration of the drug through biological barriers (such as endothelial cell barriers, blood brain barriers and the like) and the like. The drug carrier constructed based on protein can effectively improve the bioavailability, water solubility, safety and the like of the drug, and can also effectively reduce the administration frequency, thereby reducing the pain of patients caused by frequent administration. In the preparation process of the traditional protein drug carrier, the aim of self-assembling into a stable drug carrier is achieved by adding a chemical induction reagent, adjusting the pH of a solution or modifying self-assembled polypeptide and the like. However, the self-assembled drug carrier constructed based on protein is easy to denature and inactivate after contacting with factors such as organic chemical reagent, pH change, high temperature and high pressure due to the nature of the protein, so that the protein loses the original biological activity, and the prepared protein self-assembled drug carrier has larger immunogenicity. When the protein self-assembled drug carrier enters the body in the modes of injection and the like, stronger immune response can be caused, and the safety of the drug is affected.
Human Serum Albumin (HSA) is the most abundant protein in human plasma and consists of 585 amino acid residues with a molecular weight of about 67KDa, and a single molecule conformation is in a "heart-shaped" structure, and is mainly divided into three domains i, ii, iii, each of which is in turn divided into two subdomains i.e. ia and ib, iia and iib, iiia and iiib. Because of the wide source of HSA
Has the characteristics of good biocompatibility and the like, so that the method is widely applied to the fields of medicine, biochemistry, drug carrier development and the like.
Chinese patent publication No. CN114249813A (20220329), entitled "self-assembled albumin HSA-hydro-carboxylic-IB and its use", discloses a hydrophobic recombinant HSA protein having a partial IB subdomain fragment. The average particle diameter of the nanoparticle formed by self-assembly of the recombinant protein is 151.9nm, and the drug loading rate reaches 23.31%. However, the pursuit of higher drug loading and stability in complex body fluid environments is a continuing goal of those skilled in the art.
In view of the foregoing, there is a need for new methods and strategies that complement the deficiencies of the prior art.
Disclosure of Invention
The object of the present invention is therefore to provide a novel optical fiber having an IIB-IIA-IB structure rQTY The recombinant protein of the repeated structural unit and the application thereof are provided in the following specific technical scheme.
Recombinant protein of repeated structural unit IA IB-IA IB rQTY The recombinant proteins IB-IB rQTY The amino acid sequence of (2) is shown as SEQ ID NO. 1.
Further, the nucleotide sequence is shown as SEQ ID NO. 2.
Further, the recombinant proteins IB-IB rQTY Comprising 2 human serum albumin type I domains of IIB repeat structural fragments, wherein the amino acid in the 2 nd IIB structural fragment is mutated.
Further, the mutation is a mutation of hydrophilic amino acids at positions 125, 133, 138, 140, 150, 161, 166 and 170 in the 2 nd IIB structural fragment to hydrophobic, specifically, threonine (T), tyrosine (Y), threonine (T) and glutamine (Q) are mutated into valine (V) respectively valine (V), phenylalanine (F), valine (V), and leucine (L).
A recombinant expression vector comprising the recombinant protein IB-IB rQTY Is a gene of (a).
Preferably, the expression vector is a plasmid vector.
Recombinant bacteria expressing the recombinant proteins IIB-IIIB rQTY 。
A nanomicelle for hydrophobic drug delivery is prepared from the recombinant protein IB-IB rQTY Is prepared.
The application of the nano micelle in preparing a drug delivery carrier.
Further, the nanomicelle is used for carrying a hydrophobic drug, wherein the hydrophobic drug comprises a hydrophobic antitumor drug, a hydrophobic antibiotic, a hydrophobic polypeptide or a protein.
Further, the drug delivery carrier is in the form of injection.
Beneficial technical effects
1. The recombinant protein IB-IB provided by the invention rQTY Compared with the prior art, the composition has smaller particle size and higher drug loading.
2. The recombinant protein IB-IB provided by the invention rQTY The protein with 'amphipathic' performance is protein, namely one end of the protein presents hydrophilic characteristic and one end presents hydrophobic characteristic, and can self-assemble into nano particles with a certain size under the condition that neutral pH solution is not denatured and no induction reagent is relied on, and a 'hydrophobic core' structure formed in the middle of the nano particles can carry hydrophobic drugs through hydrophobic interaction. In contrast, recombinant proteins IIB-IIB, which have not undergone amino acid mutation at critical sites, are unable to achieve self-assembly.
3. The recombinant protein IB-IB provided by the invention rQTY The drug carried by the carrier has longer slow release time, the accumulated release amount of the drug reaches more than 90% after 168 hours, and the accumulated release amount of the drug carried by the natural Human Serum Albumin (HSA) nano-micelle reaches 90% after 96 hours.
4. The recombinant protein IB-IB provided by the invention rQTY Has small cytotoxicity, high safety and good biocompatibility.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.
FIG. 1 shows recombinant proteins IIB-IIIB predicted by alpha fold 2 rQTY A crystal structure diagram;
FIG. 2 shows recombinant proteins IB-IB purified in the examples rQTY And SDS-PAGE of recombinant proteins IB-IB (A is IB-IB rQTY B is unmutated IB-IB;
FIG. 3 is a diagram of recombinant proteins IB-IB rQTY A transmission electron microscope image (scale: 100 nm) of the self-assembled nano micelle after freeze drying;
FIG. 4 is a transmission electron microscope image (scale: 100 nm) of recombinant proteins IB-IB nano-micelle after lyophilization;
FIG. 5 is a recombinant protein IB-IB rQTY Experimental graphs of the effect of self-assembled nanoparticles on human embryonic kidney cell (293T) viability;
FIG. 6 is a recombinant protein IB-IB rQTY And a drug release rate experimental graph of the self-assembled drug-loaded nano-micelle and the natural human serum albumin drug-loaded nano-micelle prepared by an anti-solvent precipitation method.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Herein, "and/or" includes any and all combinations of one or more of the associated listed items.
Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.
In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, a rangeThe description of (c) should be taken as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
Noun interpretation
The invention relates to a recombinant protein of a repeated structural unit IA IB-IA IB rQTY "means that the structural region I (IIB) of human serum albumin is linked to the mutated structural region I (IIB) by flexible linker (GGGGSGGGGSGGGGS) rQTY ) The recombinant protein obtained by the method. Wherein rQTY represents a protein design method to convert a specific hydrophilic alpha helix into a hydrophobic alpha helix.
Materials and reagents
Human embryonic kidney cells 293T were purchased from the China academy of sciences typical culture Collection, catalog number SCSP-502, cell name: human embryonic kidney cells, animal species: human, tissue origin: fetal, embryonic kidney.
Coli BL21 (DE 3) competent cells were purchased from Beijing Soy Bao technology Co.
The pET-22b (+) plasmid is a commercially available E.coli expression vector, purchased from Beijing, biotech Co., ltd. The vector tags were N-pelB and C-His, and the vector resistance was Ampicillin (Ampicillin).
Restriction enzymes NdeI and XhoI were purchased from NEB (Beijing) Inc. His-tag protein purification resin (Nickel column), purchased from Shanghai-Haimai bioengineering Co., ltd., cat# LM-616.IPTG, ampicillin, DMSO (dimethyl sulfoxide) were all purchased from beijing solebao technologies. DMEM medium was purchased from GIBCO. CCK8 reagent is purchased from Chongqing Bao optical technology Co., ltd and is used according to the operation of the reagent instruction. Doxorubicin (english name adriamycin) is an antibiotic drug, and has the molecular formula of C 27 H 29 NO 11 CAS registry number 23214-92-8, purchased from Shanghai microphone Biochemical technologies Co., ltd.
Culture medium
Each liter of LB medium contains: 5g of yeast extract, 10g of tryptone and 10g of sodium chloride, and the pH was adjusted to 7.0.
The preparation method comprises the following steps: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride were dissolved in 950ml of double distilled water, the pH was adjusted to 7.0 with sodium hydroxide solution, and the volume was adjusted to 1L with double distilled water. If a solid medium is formulated, agar is added at 1.5g/100 ml. Sterilizing with steam at 121deg.C for 30min.
The experimental reagents not specifically described in the present invention are all conventional reagents in the art and can be formulated according to conventional methods in the art or purchased from related reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and reference may be made to the relevant experimental manuals, for example, molecular cloning experimental manuals or instructions of the relevant reagent manufacturers.
Example 1
This embodiment provides a type of IB-IB rQTY Gene design and protein expression examples
1. Gene design
A gene was designed comprising 2 repeat structural fragments of the type I domain of the HSA protein (IIB-IIA IB) in which the amino acid sequence of the 2 nd stage IIB comprises a plurality of hydrophilic amino acid mutations (IIB-IIB rQTY ) The protein expressed by the gene has 416 amino acids, and the sequence of the protein is shown as SEQ ID NO. 1; the nucleotide sequence of the coded protein is shown as SEQ ID NO.2, and the total length is 1248bp. Further, ndeI restriction enzyme site (CATATG) and XhoI restriction enzyme site (CTCGAG) are respectively added at the 5 'end and the 3' end of the gene, the nucleotide sequence of the gene with the restriction enzyme site is shown as SEQ ID NO.3, and the full length is 1257bp. The sequences involved in this example are shown in Table 1.
TABLE 1
2. Vector construction
pET-22b (+) plasmid was used as an expression vector. The pET-22b (+) plasmid and the target gene (SEQ ID No. 1) were subjected to double digestion reaction with restriction enzymes NdeI and XhoI, respectively, and then the target gene was ligated into the pET-22b (+) vector by ligation reaction, to obtain a ligation product.
3. Transformation
(1) Competent cells of E.coli BL21 (DE 3) were removed from the freezer at-80℃and ice-bathed for 5min.
(2) After glycerol of BL21 (DE 3) competent cells is preserved and melted, the competent cells are added into the connection product, and the gun head is sucked and put for 3-4 times and is uniformly mixed, and the ice bath is kept stand for 30min.
(3) The water on the pipe wall is quickly wiped by the absorbent paper, and then the pipe wall is thermally shocked for 90s at 42 ℃, and the pipe wall is immediately ice-bathed for 2min.
(4) 800. Mu.l of LB liquid medium was added under aseptic conditions, and the culture was continued at 37℃for 45min at 150 rpm.
(5) The cells were collected by centrifugation at 8000rpm for 5min, part of the supernatant was discarded, E.coli was resuspended in about 100. Mu.l of the remaining supernatant, and then uniformly spread on LB solid medium containing 100. Mu.g/ml ampicillin, placed in a 37℃incubator, and cultured upside down for 10-16h.
(6) The monoclonal is selected and inoculated into liquid LB culture medium containing 100 mug/ml ampicillin, and positive clone identification is carried out after the culture for 10 to 16 hours at 37 ℃ so as to obtain positive clone containing target gene plasmid.
4. Protein expression and purification
(1) Inoculating: preparing a liquid LB culture medium, sterilizing, placing the sterilized liquid LB culture medium in a super clean workbench, cooling to room temperature, adding ampicillin into the LB culture medium in the super clean workbench, uniformly mixing to ensure that the final concentration of the ampicillin is 100 mug/mL, inoculating escherichia coli (positive clone) containing target gene plasmid into the LB culture medium according to the amount of 200 mug/L, placing the LB culture medium in a shaking table, and culturing for 8-10h according to the conditions that the rotating speed is 170rpm and the temperature is 37 ℃.
(2) Induction: after shaking culture for 8-10h, 2mL of bacterial liquid is taken out, the OD600 value of the bacterial liquid is measured by a spectrophotometer, when the OD600 value of the bacterial liquid reaches 0.6-0.8, IPTG with the final concentration of 200 mu L/mL is added into the bacterial liquid, and then shaking culture is carried out for 8h according to the conditions of 37 ℃ and 170 rpm.
(3) Purifying: adding IPTG and shake culturing for 8 hr, taking out bacterial liquid, centrifuging at 4deg.C and 8000rpm for 5min, removing supernatant after centrifuging, and retaining precipitate (the precipitate is Escherichia coli).
(4) Ultrasonic crushing: the escherichia coli is ultrasonically crushed and then centrifuged, the sediment obtained by centrifugation contains target protein, the sediment obtained by centrifugation is washed by washing liquid I (50 mmol/L Tris-HCl,1mol/L urea and 10mL/L Triton X-100), then washed by washing liquid II (50 mmol/L Tris-HCl,2mol/L urea and 5mL/L Triton X-100), and then dissolved by inclusion body dissolving liquid (50 mmol/L Tris-HCl,8mol/L urea and 100mmol/L NaCl).
(5) Finally, carrying out gradient renaturation on the inclusion body dissolution liquid, wherein the method comprises the following steps: the inclusion body solution is filled in a dialysis bag, and then the dialysis bag is sequentially placed in urea solutions of 6M, 4M, 2M, 1M and 0.5M for gradient renaturation, and renaturation is carried out at the low temperature of 4 ℃ for 2-4 hours under each urea concentration. Purifying the solution after renaturation by His-tag protein purification resin (nickel column) to obtain novel recombinant protein IB-IB rQTY (the gene sequence of the target protein is added with a histidine tag when designing, so as to facilitate protein purification). After purification, it was identified whether the purified target protein was successfully obtained by running polyacrylamide-gel electrophoresis (SDS-PAGE). As a result, as shown in FIG. 2A, a recombinant protein IIB-IIB having a size of about 47.08kDa was obtained rQTY . FIG. 2B is an electrophoretogram of recombinant proteins IIB-IIB (without hydrophilic site mutation) of approximately 47.16 kDa. In FIG. 2, the left lane is labeled with protein molecules and the right lane is purified to obtain AIB-AIB rQTY Proteins or IB-IB proteins.
The amino acid and nucleotide sequences of the recombinant proteins IIB-IIB without hydrophilic site mutations are shown in Table 2. Similarly, ndeI cleavage site (CATATG) and XhoI cleavage site (CTCGAG) were added to the 5 'and 3' ends of the coding gene, respectively.
TABLE 2
5. Recombinant protein IIBⅠAⅠB rQTY Preparation of nanomicelle
(1) And taking phosphate buffer solution dry powder in a beaker, adding deionized water for dilution, and finally obtaining 1 XPBS with pH of 7.4, so as to prepare a dispersion solution for later use.
(2) Weighing 50mg of the novel recombinant protein obtained after purification, and dissolving the novel recombinant protein in 10mL of deionized water under the condition of magnetic stirring to prepare a novel recombinant protein aqueous solution with the mass concentration of 5 mg/mL.
(3) Measuring 10mL of the dispersion solution prepared in the step (1) in a 25mL beaker, performing ultrasonic dispersion at 400W under the ice bath condition, simultaneously measuring 10mL of the novel recombinant protein solution prepared in the step (2), instilling the novel recombinant protein solution into the dispersion solution at the rate of 0.4mL/min by a constant flow pump, and stopping ultrasonic treatment after instilling the novel recombinant protein solution to obtain the recombinant protein IIB-IIB rQTY A nano-micelle solution.
Preparation method of recombinant protein IB-IB protein nano-micelle and recombinant protein IB-IB rQTY The nano-micelle is consistent. The purpose of preparing recombinant proteins IB-IB nanomicelles is to verify whether they resemble recombinant proteins IB-IB nanomicelles rQTY Self-assembly is then performed to further highlight the IIB-IIAIB rQTY Self-assembly can be performed without the addition of an inducing agent (e.g., ethanol, isopropanol, dimethyl sulfoxide, etc.).
Example 2
This example provides recombinant proteins IB-IB rQTY Cytotoxicity evaluation of (2)
Investigation of recombinant proteins IB-AIB by CCK8 method rQTY Toxicity to human embryonic kidney cells 293T (China academy of sciences typical culture Collection Committee cell Bank) for evaluation of recombinant proteins IIB-IIIB rQTY Is a biological safety feature of (1). 1X 10 of each well was seeded in 96-well plates 4 Personal embryonic kidney cells 293T into DMEM medium, 5 experimental groups and 1 control group were set with 3 duplicate wells each. After culturing the cells at 37℃for 24 hours, the recombinant proteins IB-IB were added to wells of the experimental group (experimental wells) rQTY So that 5Recombinant proteins IB-IB of each experimental group rQTY The final concentrations of (C) were 10. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, 200. Mu.g/ml, 400. Mu.g/ml, respectively. The control wells (control wells) did not exacerbate the histones IIB-IIIB rQTY . The incubation was continued for 24h at 37℃and then the number of viable cells was detected using the CCK8 method as follows: mu.l of CCK8 reagent was added to each well and incubated at 37℃for 3h. The absorbance of each well at 450nm was then measured using a microplate reader.
The cell viability was calculated as follows: [ experimental well absorbance (cell-containing medium, CCK8 reagent, protein sample) -blank well absorbance (cell and protein sample-free medium, CCK8 reagent) ]/[ control well absorbance (cell-containing medium, CCK8 reagent, protein sample-free) -blank well absorbance (cell and protein sample-free medium, CCK8 reagent) ]. Times.100%.
The cell viability of each group is the average of the cell viability of the three duplicate wells of the group. The results are shown in FIG. 5, which plots 100% cell viability for the control group, with the abscissa representing IB-IB in culture rQTY Concentration of self-assembled nanoparticles (. Mu.g/mL), and survival rate (%) of human embryonic kidney cells (293T) on the ordinate, without the addition of IB-IB to the medium rQTY The survival rate of the control cells of the self-assembled nanoparticle was 100%. FIG. 5 shows that cells were supplemented with recombinant protein IB-IB at a concentration of 10-400 μg/ml rQTY The recombinant proteins IA-IB show higher survival rate after that, and the recombinant proteins IA-IB-IA-IB are proved rQTY Low cytotoxicity and good biocompatibility.
Example 3
This example provides recombinant proteins IB-IB rQTY Performance testing of (C)
1. Recombinant proteins IIB-IIAIB rQTY Transmission electron microscope observation of nano micelle
Sample preparation: mu.l of the recombinant protein IB-IB A IB prepared in example 1 was pipetted using a pipette rQTY The nano micelle solution is dripped on a copper mesh (300 meshes), 4 mu l of uranyl acetate is sucked by a liquid-transfering gun and is dripped on the copper mesh to dye the copper mesh, and then the copper mesh is naturally dried for shapingAnd (5) appearance observation. A sample of the recombinant proteins IB-IB was prepared on a copper mesh by following the procedure described above using the unmutated hydrophilic site recombinant protein IB-IB nano-micelle solution prepared in example 1.
Sample observation: using transmission electron microscopy to observe recombinant proteins IB-IB and IB-IB on copper mesh, respectively rQTY Morphology. The results show that the recombinant protein IB-IB prepared by the present invention rQTY The recombinant proteins IIB-IIB, which do not mutate hydrophilic amino acids, are not self-assembled to form nanoparticles without addition of an inducing agent (FIG. 4).
2. Recombinant proteins IIB-IIAIB rQTY Particle size, encapsulation efficiency and drug loading of nano-micelle
By way of example, the use of recombinant proteins IIB-IIIB rQTY Doxorubicin was encapsulated and the particle size, encapsulation efficiency and drug loading were determined.
Recombinant proteins IIB-IIAIB rQTY Preparation of drug-loaded nano-micelle: recombinant proteins IB-IB rQTY Dissolving in deionized water to prepare a protein water solution with the mass concentration of 5 mg/mL. And preparing the doxorubicin-PBS solution with the concentration of 0.4mg/mL (the pH value of the PBS solution is 7.4), wherein the doxorubicin-PBS solution needs to be continuously stirred, otherwise, the doxorubicin can be settled due to the hydrophobicity of the doxorubicin, and cannot be uniformly dispersed in the PBS solution. Finally, under ice bath conditions, an equal volume of recombinant protein IIB-IIAIB is prepared rQTY Dripping the aqueous solution into 0.4mg/mL doxorubicin-PBS solution at a rate of 0.4mL/min, performing ultrasonic dispersion during dripping, and stopping ultrasonic dispersion after dripping to obtain recombinant protein IB-IB rQTY Drug-loaded nano-micelle solution.
The recombinant proteins obtained were subjected to the following procedure rQTY The drug-loaded nano-micelle is used for measuring the particle size, the encapsulation efficiency and the drug-loaded amount.
1) Particle size measurement method:
by movingA liquid gun aspirates 2mL of the prepared recombinant protein IIB-IIAIB rQTY The drug-loaded Nano micelle solution is placed in a 4.5mL transparent cuvette, the transparent cuvette containing the drug-loaded Nano micelle solution is placed in a sample analysis hole of a Nano-particle size and ZETA potential analyzer (British Markov company, model: nano ZS 90), and the particle size of the Nano-particles is measured according to the use instruction of the Nano-particle size and the ZETA potential analyzer.
2) The method for measuring the encapsulation efficiency comprises the following steps:
the prepared recombinant protein IB-IB rQTY Centrifuging the drug-loaded nano micelle solution for 15min at a rotating speed of 10000r/min, taking supernatant, measuring absorbance of the supernatant at 480nm by an ultraviolet spectrophotometer, and according to the formula: a=0.0184c+0.0216 (a is absorbance and C is doxorubicin concentration) to calculate the free drug content.
Encapsulation efficiency= (W) Total (S) -W Swimming device )/W Total (S) *100％
W Total (S) For initial dose, W Swimming device Is the amount of free drug in the nanoparticle that is not encapsulated.
3) The method for measuring the drug loading comprises the following steps:
the prepared recombinant protein IB-IB rQTY Centrifuging the drug-loaded nano micelle solution for 15min at a rotating speed of 10000r/min, taking supernatant, measuring absorbance of the supernatant at 480nm by an ultraviolet spectrophotometer, and according to the formula: a=0.0184c+0.0216 (a is absorbance and C is doxorubicin concentration) to calculate the free drug content.
Drug loading= (W) Total (S) -W Swimming device )/W Total weight of *100％
W Total (S) For initial dose, W Swimming device W is the amount of unencapsulated free drug in the nanoparticle Total weight of Is recombinant protein IB-IB after drug loading rQTY The total weight of the nanoparticles.
W Total weight of Is determined by the following steps: recombinant proteins IB-IB rQTY Centrifuging the drug-loaded nano micelle under the condition of 12000r/min, discarding the supernatant after centrifuging, and obtaining the precipitated weight which is the recombinant protein after drug loadingⅠAⅠB-ⅠAⅠB rQTY The total weight of the nanoparticles. I.e., recombinant proteins IIB-IIIB after drug delivery rQTY Total weight of nanoparticles (W) Total weight of ) Total weight of centrifuge tube containing pellet-weight of blank centrifuge tube.
The results are shown in Table 3, recombinant proteins IB-IB rQTY The particle size of the drug-loaded nano micelle is small and uniform, and the drug-loaded rate reaches 24.12%.
TABLE 3 recombinant proteins IB-IB rQTY Particle size, encapsulation efficiency and drug loading rate of nano micelle (taking doxorubicin as an example)
3. Recombinant proteins IIB-IIAIB rQTY Drug release experiment of drug-loaded nano micelle
For the recombinant protein IB-IB obtained rQTY And carrying out a drug release experiment on the drug-loaded nano micelle.
Drug release experimental method: 5mL of recombinant protein IB-IB were removed rQTY The drug-loaded nano micelle solution is placed in a dialysis bag, placed in 20mL of a release medium containing 1g/L Tween 80 (pH 7.4), placed on a constant temperature shaking table, and subjected to drug release experiments according to the conditions of 37 ℃ and 170r/min, 1mL of the solution (and 1mL of the release medium are supplemented) are respectively taken out at 3h, 6h, 12h, 24h, 48h, 72h, 96h, 120h, 144h and 168h, the absorbance of the taken 1mL of the solution at 480nm is measured by an ultraviolet spectrophotometer, and the following formula is adopted: a=0.0184c+0.0216 (a is absorbance and C is doxorubicin concentration) the doxorubicin content is calculated, the experiment is repeated 3 times, and the result is averaged. The cumulative drug release (%) for 3h, 6h, 12h, 24h, 48h, 72h, 96h, 120h, 144h and 168h was calculated and a drug release curve was drawn.
Cumulative drug release (%) = weight of drug released/total weight of drug encapsulated.
The results are shown in FIG. 6, recombinant proteins IB-IB rQTY The accumulated release of the drug loaded nano-micelle reaches after 168 hoursMore than 90 percent, and the accumulated release amount of the natural Human Serum Albumin (HSA) drug-loaded nano micelle reaches 90 percent in about 96 hours. Thus, the recombinant proteins IB-IB rQTY The nano micelle has remarkable drug slow release effect. Preferably, the recombinant protein IB-IB rQTY The drug-loaded nano-micelle acts through injection into the body.
It will be appreciated that the present embodiment is described by way of example only and not limitation, and that other hydrophobic drugs, including hydrophobic antineoplastic agents or hydrophobic polypeptides and the like, are suitable for use in the present invention.
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.
Claims (10)
1. Recombinant protein of repeated structural unit IA IB-IA IB rQTY Characterized in that the recombinant protein IB-IB rQTY The amino acid sequence of (2) is shown as SEQ ID NO. 1.
2. The recombinant protein IIB-IIAIB of claim 1 rQTY The nucleotide sequence of the polypeptide is shown as SEQ ID NO. 2.
3. The recombinant protein IIB-IIAIB of claim 1 rQTY Characterized in that the recombinant protein IB-IB rQTY Comprising 2 human serum albumin type I domains of IIB repeat structural fragments, wherein the amino acid in the 2 nd IIB structural fragment is mutated.
4. The recombinant protein IIB-IIAIB of claim 3 rQTY Characterized in that the mutation is 2 ndThe hydrophilic amino acids at positions 125, 133, 138, 140, 150, 161, 166 and 170 in the IIB structural fragment are mutated to hydrophobic, specifically, threonine, tyrosine, threonine and glutamine are mutated into valine respectively valine, phenylalanine, valine and leucine.
5. A recombinant expression vector comprising the recombinant protein of any one of claims 1 or 2 rQTY Is a gene of (a).
6. A recombinant bacterium which expresses the recombinant protein IB-IB according to claims 1-4 rQTY 。
7. A nanomicelle for hydrophobic drug delivery, characterized in that it is composed of the recombinant proteins IIB-IIB according to any of claims 1-4 rQTY Is prepared.
8. Use of the nanomicelle of claim 7 in the preparation of a drug delivery vehicle.
9. The use according to claim 8, wherein the nanomicelle is for carrying a hydrophobic drug comprising a hydrophobic anti-neoplastic drug, a hydrophobic antibiotic, a hydrophobic polypeptide or protein.
10. The use according to claim 8, wherein the drug delivery vehicle is in the form of an injection.
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