CN117720670B

CN117720670B - Recombinant protein IIIAIIB-IIIAIIB with self-assembly performancerQTYApplication and application thereof

Info

Publication number: CN117720670B
Application number: CN202410130630.4A
Authority: CN
Inventors: 孟润; 周晓荣; 王磊; 张瑞雅
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2024-01-30
Filing date: 2024-01-30
Publication date: 2024-06-11
Anticipated expiration: 2044-01-30
Also published as: CN117720670A

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a recombinant protein III A III B-III A III B ^rQTY with self-assembly performance and application thereof. The recombinant protein comprises a IIIAIIB repeating structural fragment in a type III structural domain of 2 human serum albumin, wherein the amino acid in the type 2 IIIAIIB structural fragment has mutation, specifically, the hydrophilic amino acid at positions 132, 139, 143, 144, 160, 183 and 197 is mutated to be hydrophobic.

Description

Recombinant protein IIIAIIB-IIIAIIB rQTY with self-assembly performance and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a recombinant protein III A III B-III A III B ^rQTY with self-assembly performance and application thereof.

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 HSA sources are wide and have the characteristics of good biocompatibility and the like, the HSA is widely applied to the fields of medicine, biochemistry, drug carrier development and the like.

Chinese patent publication No. CN114249814A (20220329), entitled "albumin HSA-Hydrophoboic-IIIB with self-assembly properties" discloses a hydrophobic recombinant HSA protein with partial IIIB subdomain fragment. The average particle diameter of the nanoparticle formed by self-assembly of the recombinant protein is 86.7nm, and the drug loading rate reaches 19.26%. 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

In view of the above, the present invention aims to provide a recombinant protein having a repeated structural unit of IIIAIIB-IIIAIIB ^rQTY and its application, and the specific technical scheme is as follows.

A recombinant protein IIIAIIB-IIIAIIB ^rQTY with repeated structural units is provided, and the amino acid sequence of the recombinant protein IIIAIIB-IIIAIIB ^rQTY is shown as SEQ ID NO. 1.

Further, the nucleotide sequence is shown as SEQ ID NO. 2.

Further, the recombinant protein IIIAIIB-IIIAIIB ^rQTY comprises a IIIAIIB repeat structural fragment in the 2 human serum albumin type III domain, wherein the amino acid in the 2 nd IIIAIIB structural fragment is mutated.

Further, the mutation is a mutation of hydrophilic amino acids 132, 139, 143, 144, 160, 183 and 197 in the 2 nd IIIA IIIB structural fragment to hydrophobic, specifically a mutation of threonine (T), glutamine (Q), threonine (T) and glutamine (Q) to valine (V), leucine (L), valine (V) and leucine (L), respectively.

A recombinant expression vector comprising the gene of the recombinant protein iiiaib-iiiaiib ^rQTY.

Preferably, the expression vector is a plasmid vector.

A recombinant bacterium expressing the recombinant protein IIIAIIB-IIIAIIB ^rQTY.

A nano micelle for delivering hydrophobic drugs is prepared from the recombinant protein IIIAIIB-IIIAIIB ^rQTY.

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. Compared with the prior art, the recombinant protein IIIAIIB-IIIAIIB ^rQTY provided by the invention has smaller particle size and higher drug loading capacity.

2. The recombinant protein IIIAIIB-IIIAIIB ^rQTY provided by the invention is an amphiphilic protein, namely, one end of the protein presents hydrophilic characteristic and the other end presents hydrophobic characteristic, and can be self-assembled 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. And the recombinant protein IIIAIIB-IIIAIIB which is not subjected to key site amino acid mutation cannot realize self-assembly.

3. The recombinant protein IIIAIIB-IIIAIIB ^rQTY provided by the invention is taken as a carrier to carry the medicine, so that the sustained-release time is longer, the accumulated release amount of the medicine reaches more than 90% after 168 hours, and the accumulated release amount of the medicine carried by natural Human Serum Albumin (HSA) nano-micelle reaches 90% after 96 hours.

4. The recombinant protein IIIAIIB-IIIAIIB ^rQTY provided by the invention has the advantages of low 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 is a diagram of the crystal structure of recombinant protein IIIAIIB-IIIAIIB ^rQTY predicted by Alphafold;

FIG. 2 is a SDS-PAGE of recombinant proteins IIIAIIB-IIIAIIB ^rQTY and recombinant proteins IIIAIIB-IIIAIIB purified in the examples (A is IIIAIIB-IIIAIIB ^rQTY, B is unmutated IIIAIIB-IIIAIIB);

FIG. 3 is a transmission electron microscope image (scale: 100 nm) of a self-assembled nanomicelle of recombinant proteins IIIAIIB-IIIAIIB ^rQTY after lyophilization;

FIG. 4 is a transmission electron microscope image (scale: 100 nm) of a recombinant protein IIIAIIB-IIIAIIB nano-micelle after lyophilization;

FIG. 5 is a graph showing the effect of self-assembled nanoparticles of recombinant proteins IIIAIIB-IIIAIIB ^rQTY on human embryonic kidney (293T) viability;

FIG. 6 is a graph showing the drug release rate of recombinant protein IIIAIIB-IIIAIIB ^rQTY self-assembled drug-loaded nanomicelles and natural human serum albumin drug-loaded nanomicelles 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 III (IIIAIIB) -IIIAIIB ^rQTY which is obtained by connecting a structural region III (IIIAIIB) of human serum albumin with a mutated structural region III (IIIAIIB ^rQTY) through flexibility linker (GGGGSGGGGSGGGGS). 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 of the formula C ₂₇H₂₉NO₁₁, CAS accession number 23214-92-8, purchased from shanghai microphone biochemistry technologies, inc.

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

The implementation provides a IIIAIIB-IIIAIIB ^rQTY gene design and a protein expression example

1. Gene design

Designing a gene, comprising 2 repeated structural fragments (IIIAIIB-IIIAIIB) of an I type structural domain in an HSA protein, wherein the amino acid sequence of the 2 nd section IIIAIIB comprises a plurality of hydrophilic amino acid mutations (IIIAIIB-IIIAIIB ^rQTY), and the protein expressed by the gene has 424 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 1272bp. 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 total length is 1281bp. 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 IIIAIIB-IIIAIIB ^rQTY (histidine tag is added in the design of the gene sequence of target protein 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 IIIAIIB-IIIAIIB ^rQTY having a size of about 47.28kDa was obtained. FIG. 2B is an electrophoretogram of recombinant protein IIIAIIB-IIIAIIB (without hydrophilic site mutation) of about 47.35 kDa. In FIG. 2, the left lane is a protein molecular marker, and the right lane is a purified IIIAIIB-IIIAIIB ^rQTY protein or IIIAIIB-IIIAIIB protein.

The amino acid and nucleotide sequences of the recombinant proteins IIIAIIB-IIIAIIB 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. Preparation of recombinant protein IIIAIIB-IIIAIIB ^rQTY nano-micelle

(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) And (3) measuring 10mL of the dispersion solution prepared in the step (1) in a 25mL beaker, performing ultrasonic dispersion with 400W of power 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 IIIAIIIB-IIIAIIIB ^rQTY nano micelle solution.

The preparation method of the recombinant protein IIIAIIB-IIIAIIB protein nano-micelle is consistent with that of the recombinant protein IIIAIIB-IIIAIIB ^rQTY nano-micelle. The purpose of the recombinant protein IIIAB-IIIAIIB nano-micelles was to verify whether they self-assemble like recombinant protein IIIAIIB-IIIAIIB ^rQTY to further highlight that IIIAIIB-IIIAIIB ^rQTY self-assembles without the addition of inducing agents (e.g., ethanol, isopropanol, dimethyl sulfoxide, etc.).

Example 2

This example provides cytotoxicity assessment of recombinant proteins IIIAIIB-IIIAIIB ^rQTY

Toxicity of recombinant protein IIIAIIB-IIIAIIB ^rQTY to human embryonic kidney cells 293T (China academy of sciences typical culture Collection Committee cell bank) was studied by CCK8 method to evaluate biosafety of recombinant protein IIIAIIB-IIIAIIB ^rQTY. In 96-well plates, 5 experimental groups and 1 control group were set with 3 duplicate wells per well, with 1×10 ⁴ human embryonic kidney cells 293T per well inoculated into DMEM medium. After culturing the cells at 37℃for 24 hours, recombinant protein IIIAIIB-IIIAIIB ^rQTY was added to the wells of the experimental groups (experimental wells) so that the final concentrations of recombinant protein IIIAIIB-IIIAIIB ^rQTY were 10. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, 200. Mu.g/ml, 400. Mu.g/ml, respectively, for 5 experimental groups. The control wells (control wells) did not exacerbate the histone IIIAIIB-IIIAIIB ^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 plotted with 100% cell viability of the control group, as shown in FIG. 5, where the abscissa is the concentration of IIIAIIB-IIIAIIB ^rQTY self-assembled nanoparticles in the medium (μg/mL) and the ordinate is the viability (%) of human embryonic kidney cells (293T), with 100% cell viability of the control group without IIIAIIB-IIIAIIB ^rQTY self-assembled nanoparticles in the medium. FIG. 5 shows that the cells show higher survival rate after the recombinant protein IIIAIIB-IIIAIIB ^rQTY with the concentration of 10-400 mug/ml is added, and the recombinant protein IIIAIIB-IIIAIIB ^rQTY is proved to have low cytotoxicity and good biocompatibility.

Example 3

This example provides performance testing of recombinant proteins IIIAIIB-IIIAIIB ^rQTY

1. Transmission electron microscope observation of recombinant protein IIIAIIB-IIIAIIB ^rQTY nano-micelle

Sample preparation: 10 μl of the recombinant protein IIIAIIB-IIIAIIB ^rQTY nano-micelle solution prepared in example 1 was pipetted by a pipette and added dropwise onto a copper mesh (300 mesh), and then 4 μl of uranyl acetate was pipetted by a pipette and added dropwise onto the copper mesh to dye it, and then naturally dried for morphology observation. The preparation of the recombinant protein IIIAIIB-IIIAIIB nano-micelle solution with unmutated hydrophilic sites prepared in example 1 was continued, and a sample was prepared on a copper mesh for electron microscopy observation according to the above procedure.

Sample observation: and respectively observing the morphology of the recombinant protein IIIAIIB-IIIAIIB and the recombinant protein IIIAIIB-IIIAIIB ^rQTY on the copper net by using a transmission electron microscope. The results show that the recombinant protein IIIAIIB-IIIAIIB ^rQTY prepared by the method can self-assemble to form nanoparticles under the condition of no addition of an induction reagent (figure 3), while the recombinant protein IIIAIIB-IIIAIIB without mutation of hydrophilic amino acid can not self-assemble to form nanoparticles under the condition of no addition of an induction reagent (figure 4).

2. Determination of particle size, encapsulation efficiency and drug loading capacity of recombinant protein IIIAIIB-IIIAIIB ^rQTY nano-micelle

Taking doxorubicin as an example, the recombinant protein IIIAIIB-IIIAIIB ^rQTY was used to encapsulate doxorubicin and the particle size, encapsulation efficiency and drug loading were determined.

Preparation of recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded nano-micelle: and (3) dissolving the recombinant protein IIIAIIB-IIIAIIB ^rQTY in deionized water to prepare a protein aqueous 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. And finally, under the ice bath condition, instilling an equal volume of recombinant protein IIIAIIB-IIIAIIB ^rQTY aqueous solution into an doxorubicin-PBS solution with the concentration of 0.4mg/mL at the rate of 0.4mL/min, performing ultrasonic dispersion in the instilling process, and stopping ultrasonic dispersion after instilling is finished to obtain the recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded nano micelle solution.

The particle size, the encapsulation efficiency and the drug loading capacity of the prepared recombinant protein IIIAIIB-IIIAIIB ^rQTY drug loading nano-micelle are measured according to the following method.

1) Particle size measurement method:

2mL of the prepared recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded Nano-micelle solution is sucked by a pipetting gun, 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 Nano-particle size of Nano ZS90 and the use instruction of the ZETA potential analyzer.

2) The method for measuring the encapsulation efficiency comprises the following steps:

Centrifuging the prepared recombinant protein IIIAIIB-IIIAIIB ^rQTY 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)} x 100%

W _{Total (S)} is the initial dose and W _{Swimming device} is the amount of unencapsulated free drug in the nanoparticle.

3) The method for measuring the drug loading comprises the following steps:

Drug loading= (W _{Total (S)}-W_{Swimming device})/W_{Total weight of} x 100%

W _{Total (S)} is the initial dosage, W _{Swimming device} is the non-encapsulated free drug amount in the nanoparticle, and W _{Total weight of} is the total weight of the recombinant protein IIIAIIB-IIIAIIB ^rQTY nanoparticle after drug loading.

Method for measuring W _{Total weight of}: centrifuging the recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded nano-micelle under the condition of 12000r/min, discarding supernatant after centrifugation, and obtaining the precipitated weight which is the total weight of the drug-loaded recombinant protein IIIAIIB-IIIAIIB ^rQTY nano-particle. I.e., total weight of recombinant protein iiiaiibb-iiiaiib ^rQTY nanoparticle after drug loading (W _{Total weight of}) =total weight of centrifuge tube containing pellet-weight of blank centrifuge tube.

The results are shown in Table 3, the recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded nano-micelle has smaller and uniform particle size, and the drug-loading rate reaches 25.56%.

TABLE 3 determination of particle size, encapsulation efficiency, drug loading of recombinant proteins IIIAIIB-IIIAIIB ^rQTY nanomicelle (Adriamycin loading is an example)

3. Drug release experiment of recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded nano-micelle

And carrying out a drug release experiment on the prepared recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded nano-micelle.

Drug release experimental method: transferring 5mL of recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded nano-micelle solution into a dialysis bag, placing the dialysis bag in 20mL of release medium containing phosphate buffer solution (pH 7.4) of 1g/L Tween 80, placing the dialysis bag on a constant temperature shaking table, performing drug release experiments according to the conditions of 37 ℃ and 170r/min, respectively taking out 1mL of solution (supplemented with 1mL of release medium) at 3h, 6h, 12h, 24h, 48h, 72h, 96h, 120h, 144h and 168h, measuring the absorbance of the taken 1mL of solution at 480nm by an ultraviolet spectrophotometer, and according to the formula: 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.

As shown in FIG. 6, the cumulative drug release of the recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded nano-micelle reaches more than 90% after 168 hours, and the cumulative drug release of the natural Human Serum Albumin (HSA) drug-loaded nano-micelle reaches 90% after 96 hours. Therefore, the recombinant protein IIIAIIB-IIIAIIB ^rQTY nano-micelle has obvious drug slow release effect. Preferably, the recombinant protein IIIAIIB-IIIAIIB ^rQTY drug-loaded nano-micelle is effective by injection into a 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

1. The recombinant protein IIIAIIB-IIIAIIB ^rQTY with the repeated structural units is characterized in that the amino acid sequence of the recombinant protein IIIAIIB-IIIAIIB ^rQTY is shown as SEQ ID NO. 1.

2. A recombinant expression vector comprising the gene for recombinant protein iiiaiib-iiiaiib ^rQTY of claim 1.

3. A recombinant bacterium expressing the recombinant protein iiiaiib-iiiaiiib ^rQTY of claim 1.

4. A nanomicelle for hydrophobic drug delivery prepared from the recombinant protein iiiaiib-iiiaiib ^rQTY of claim 1.

5. Use of the nanomicelle of claim 4 for the preparation of a drug delivery vehicle.

6. The use according to claim 5, wherein the nanomicelle is for carrying a hydrophobic drug comprising a hydrophobic anti-neoplastic drug, a hydrophobic antibiotic, a hydrophobic polypeptide or protein.

7. The use according to claim 5, wherein the drug delivery vehicle is in the form of an injection.