Recombinant ovalbumin with self-assembly performance and application thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a recombinant ovalbumin with self-assembly performance and its application. The first 100 positions of the amino acid sequence of the recombinant ovalbumin undergo systematic hydrophobic mutations, and the systematic hydrophobic mutations are continuous and discontinuous hydrophobic mutations in the first 100 amino acids, specifically the 30th, 43rd, 50th, 52nd, 53rd, 73rd, 76th and 90th hydrophilic amino acids mutate to hydrophobicity.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a recombinant ovalbumin with self-assembly performance and an application thereof. Background Art

[0002] In recent decades, humans have developed a variety of drug delivery carriers, common types include nanocarriers and antibody-drug conjugates (ADCs). However, traditional nanocarriers often have poor compatibility with organisms, unsatisfactory delivery efficiency, and high toxicity, while antibody-drug conjugates have certain disadvantages in terms of drug carrying capacity and stability. Therefore, researchers are more eager to find a drug carrier that comes from naturally occurring proteins in organisms, is non-toxic, easy to degrade, and not prone to rejection. It can contain the drug inside, so that it can have a larger drug loading capacity than ADC, and is more stable and safe; it is also unlike traditional nanocarriers that require an additional "positioning system" to identify target cells, thereby greatly reducing the difficulty and cost of production.

[0003] Ovalbumin accounts for about 54%-69% of the total amount of egg white protein. The naturally occurring ovalbumin has a spherical high-level structure, a molecular weight of about 42.881 kDa, and is composed of 386 amino acids. Due to its wide availability, low price, good biocompatibility, and ability to enhance immunity, ovalbumin is widely used in the fields of medicine, biochemistry, and drug carrier development.

[0004] The patent with publication number CN108125928A (20180608) discloses an ovalbumin nanoparticle loaded with epigallocatechin gallate (EGCG). The patent successfully prepared ovalbumin nanoparticles loaded with EGCG by heating self-assembly, and characterized a number of physical and chemical properties. The cumulative drug release of the ovalbumin nanoparticles reached 50% before 24 hours, and the drug release rate gradually slowed down after 24 hours. Therefore, although the self-assembled ovalbumin nanoparticles prepared by the patented method have a good drug loading capacity, the drug controlled release effect cannot meet the demand for drug sustained release.

[0005] In summary, it is necessary to propose new methods and strategies to supplement the shortcomings of existing technologies. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a recombinant ovalbumin with self-assembly performance and the application of the recombinant ovalbumin, and the specific technical scheme is as follows.

[0007] A recombinant ovalbumin with self-assembly performance, wherein the first 100 positions of the amino acid sequence of the recombinant ovalbumin undergo systematic mutations, wherein the systematic mutations include hydrophobic mutations at continuous amino acid sites and discontinuous amino acid sites; the amino acid sequence of the recombinant ovalbumin is shown in SEQ ID NO.1.

[0008] Furthermore, the systematic mutation is a hydrophobic mutation occurring at consecutive amino acid sites and discontinuous amino acid sites at positions 30-90 of the amino acid sequence of natural ovalbumin.

[0009] Preferably, the site of the systematic mutation is position 8, and the site of the systematic mutation is the hydrophilic amino acids at positions 30, 43, 50, 52, 53, 73, 76 and 90 of the complete structural fragment of natural ovalbumin are mutated to hydrophobic ones, specifically, tyrosine (Y), tyrosine (Y), threonine (T), threonine (T), glutamine (Q), glutamine (Q), threonine (T) and glutamine (Q) are mutated to phenylalanine (F), phenylalanine (F), valine (V), valine (V), leucine (L), leucine (L), valine (V) and leucine (L), respectively.

[0010] Furthermore, the nucleotide sequence of the recombinant ovalbumin is shown in SEQ ID NO.2.

[0011] A recombinant expression vector comprises the gene of the recombinant ovalbumin.

[0012] Furthermore, the types of expression vectors include plasmid vectors, bacteriophage vectors, adenovirus vectors, baculovirus vectors, and the like.

[0013] A recombinant bacterium expressing the recombinant ovalbumin.

[0014] Furthermore, the types of bacteria include Escherichia coli, Bacillus subtilis, Pichia pastoris, etc.

[0015] A nano micelle for delivering hydrophobic drugs is prepared from the recombinant ovalbumin.

[0016] Application of the above nano micelles in the preparation of drug delivery carriers.

[0017] Furthermore, the nano-micelles are used to carry hydrophobic drugs, and the hydrophobic drugs include hydrophobic anti-tumor drugs, hydrophobic antibiotics, hydrophobic polypeptides or proteins.

[0018] Furthermore, the dosage form of the drug delivery carrier is an injection.

[0019] Furthermore, the injection includes intravenous injection, intradermal injection, subcutaneous injection or powder for injection.

[0020] Beneficial technical effects

[0021] 1. Compared with naturally occurring ovalbumin, the recombinant ovalbumin provided by the present invention has the ability of self-assembly, and thus can be assembled into nanoparticles with small particle sizes, while naturally occurring ovalbumin does not have the ability of self-assembly, and therefore cannot form nanoparticles.

[0022] 2. When the recombinant ovalbumin provided by the present invention is used to prepare a drug delivery carrier, it has a longer drug sustained release time compared to the prior art (CN108125928A) using ovalbumin to carry drugs. The cumulative drug release is about 50% at 72 hours and reaches more than 90% after 168 hours, with an outstanding drug sustained release effect.

[0023] 3. The recombinant ovalbumin provided by the present invention is a protein with "amphiphilic" properties, that is, the first half of the protein amino acid sequence (also known as one end) is hydrophilic, and the second half (also known as the other end) is hydrophobic. It can remain unchanged in a neutral pH solution and self-assemble into nanoparticles of a certain size without relying on inducing agents and other inducing means (such as heating in the prior art). The "hydrophobic core" structure formed in the middle of the nanoparticle can carry hydrophobic drugs through hydrophobic interactions. However, natural proteins that have not undergone amino acid mutations at key sites cannot achieve self-assembly.

[0024] 4. The recombinant ovalbumin provided by the present invention has low cytotoxicity, high safety and good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0026] Figure 1 The crystal structure of recombinant ovalbumin predicted by Alphafold 3;

[0027] Figure 2 The hydrophilicity analysis results of recombinant ovalbumin;

[0028] Figure 3 The SDS-PAGE image of the recombinant ovalbumin purified in the example and the natural ovalbumin without systematic mutation;

[0029] Figure 4 This is the transmission electron microscopy image of the recombinant ovalbumin self-assembled nanomicelles after freeze-drying (scale bar: 100 nm);

[0030] Figure 5 This is a transmission electron microscopy image of natural ovalbumin nanomicelles after freeze-drying without systematic mutation (scale bar: 100 nm);

[0031] Figure 6 The results of cytotoxicity analysis of recombinant ovalbumin self-assembled nanoparticles on human embryonic kidney cells (293T);

[0032] Figure 7 This is an experimental diagram of drug release rate from recombinant ovalbumin self-assembled drug-loaded nanomicelles;

[0033] Figure 8 This is a transmission electron microscopy image of a control mutant protein nanomicelle after freeze-drying in one of the embodiments of the present invention (scale bar: 100 nm);

[0034] Fig. 9 This is a transmission electron micrograph of another control mutant protein nanomicelle after freeze-drying in one of the embodiments of the present invention (scale bar: 100 nm). DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0036] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0037] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0038] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0039] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0040] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have 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 this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0041] Glossary:

[0042] The "self-assembly" mentioned in the present invention refers to the recombinant ovalbumin that has been systematically mutated and transformed by specific means of the present invention. From the perspective of the amino acid sequence, it has the characteristics of one end being hydrophilic and the other end being hydrophobic. Therefore, it can be aggregated together through hydrophobic interactions to form nanoparticles, which is called self-assembly.

[0043] The "hydrophobic mutation" mentioned in the present invention refers to the mutation of a hydrophilic amino acid into a hydrophobic amino acid.

[0044] Materials and reagents

[0045] Human embryonic kidney cells 293T were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-502, cell name: human embryonic kidney cells, animal species: human, tissue source: fetus, embryonic kidney.

[0046] Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Solebow Technology Co., Ltd.

[0047] The pET-22b(+) plasmid is a commercially available E. coli expression vector purchased from Beijing Qingke Biotechnology Co., Ltd. The vector tags are N-pelB and C-His, and the vector resistance is Ampicillin.

[0048] Restriction endonucleases NdeI and XhoI were purchased from NEB (Beijing) Co., Ltd. His-tag protein purification resin (nickel column) was purchased from Shanghai Lianmai Bioengineering Co., Ltd., catalog number LM-616. IPTG, ampicillin, and DMSO (dimethyl sulfoxide) were all purchased from Beijing Solebow Technology Co., Ltd. DMEM culture medium was purchased from GIBCO. CCK8 reagent was purchased from Chongqing Baoguang Technology Co., Ltd. and used according to the reagent instructions. Adriamycin (English name adriamicin) is an antibiotic drug with a molecular formula of C 27 H 29 NO 11 , CAS registration number 23214-92-8, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0049] Culture medium

[0050] Each liter of LB medium contains: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and the pH is adjusted to 7.2-7.4.

[0051] Preparation method: Dissolve 5g yeast extract, 10g tryptone, and 10g sodium chloride in 950ml double distilled water, adjust the pH to 7.2-7.4 with sodium hydroxide solution, and dilute to 1L with double distilled water. If preparing solid culture medium, add agar at 1.5g / 100ml. Sterilize at 121℃ high pressure steam for 30min.

[0052] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, and can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of relevant reagent manufacturers.

[0053] Example 1

[0054] This embodiment provides an example of recombinant ovalbumin gene design and protein expression.

[0055] 1. Recombinant gene design

[0056] A recombinant gene is designed, and the recombinant gene is modified on the natural ovalbumin amino acid sequence. The principles of the modification are: 1) amino acids located in the first 100 positions and located on the α-helical structure; 2) the modified sites avoid the amino acid sites known to regulate protein functions; 3) the modification method is to mutate hydrophilic amino acids into hydrophobic amino acids. The protein expressed by the modified recombinant gene has 396 amino acids, which contains systematic mutations, and its sequence is shown in SEQ ID NO.1; the nucleotide sequence encoding the protein is shown in SEQ ID NO.2, and the total length is 1188bp. Further, NdeI restriction site (CATATG) and XhoI restriction site (CTCGAG) are added to the 5' end and 3' end of the gene respectively, and the nucleotide sequence of the gene with restriction sites is shown in SEQ ID NO.3, and the total length is 1197bp. The sequences involved in this embodiment are shown in Table 1.

[0057] Table 1

[0058]

[0059]

[0060] 2. Vector Construction

[0061] The 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 restriction digestion reaction with restriction endonucleases NdeI and XhoI, respectively, and then the target gene was connected to the pET-22b(+) vector by ligation reaction to obtain a ligation product. It is understood that the present embodiment can also use a bacteriophage, adenovirus vector or baculovirus vector as an expression vector to connect the target gene.

[0062] 3. Conversion

[0063] (1) Take out the competent E. coli BL21 (DE3) cells from the -80°C freezer and place them on ice for 5 min.

[0064] (2) After the glycerol containing the BL21(DE3) competent cells has melted, add the competent cells to the ligation product, pipette and release the pipette tip 3-4 times to mix, and place on ice for 30 min.

[0065] (3) Quickly wipe the tube wall dry with absorbent paper, then heat shock at 42°C for 90 seconds, and immediately place on ice for 2 minutes.

[0066] (4) Add 800 μl of LB liquid culture medium under sterile conditions and culture at 37°C, 150 rpm for 45 min.

[0067] (5) Collect the cells by centrifugation at 8000 rpm for 5 min, discard part of the supernatant, and use the remaining 100 μl of the supernatant to resuspend the E. coli. Then, spread it evenly on LB solid culture medium containing 100 μg / ml ampicillin, place it in a 37°C incubator, and invert and culture for 14-18 h.

[0068] (6) Pick a single clone and inoculate it in a liquid LB medium containing 100 μg / ml ampicillin. After culturing at 37° C. for 14-18 hours, identify the positive clones to obtain the positive clones containing the target recombinant gene plasmid. It is understood that in this embodiment, Bacillus subtilis cells or Pichia pastoris strain cells can also be used to prepare positive clones containing the target recombinant gene plasmid for the following experiments.

[0069] 4. Protein Expression and Purification

[0070] (1) Inoculation: Prepare liquid LB medium and sterilize it. Place the sterilized liquid LB medium in a clean bench and cool it to room temperature. Add ampicillin to the LB medium in the clean bench and mix it to a final concentration of 100 μg / mL. Then, inoculate 200 μl / L of Escherichia coli (positive clone) containing the target gene plasmid into the LB medium. Place the LB medium in a shaker and culture it at a speed of 170 rpm and a temperature of 37°C for 8-10 h.

[0071] (2) Induction: After culturing on a shaking table for 8-10 h, take out 2 ml of the bacterial solution and measure its OD600 value using a spectrophotometer. When the OD600 value of the bacterial solution reaches 0.6-0.8, add IPTG at a final concentration of 200 μl / mL to the bacterial solution, and then culture on a shaking table at 37°C and 170 rpm for 8 h.

[0072] (3) Purification: After adding IPTG and culturing on a shaking platform for 8 h, the bacterial solution was removed and centrifuged at 8000 rpm for 5 min at 4°C. After centrifugation, the supernatant was removed and the precipitate was retained (the precipitate is Escherichia coli).

[0073] (4) Ultrasonic disruption: E. coli was ultrasonically disrupted and then centrifuged. The precipitate obtained by centrifugation contained the target protein. The precipitate obtained by centrifugation was first washed with washing solution I (50mmol / L Tris-HCl, 1mol / L urea, 10mL / L Triton X-100), then washed with washing solution II (50mmol / L Tris-HCl, 2mol / L urea, 5ml / L Triton X-100), and then dissolved with inclusion body dissolution solution (50mmol / L Tris-HCl, 8mol / L urea, 100mmol / L NaCl).

[0074] (5) Finally, the inclusion body solution is subjected to gradient renaturation as follows: the inclusion body solution is placed in a dialysis bag, and the dialysis bag is then placed in 6M, 4M, 2M, 1M, and 0.5M urea solutions in sequence for gradient renaturation. The renaturation is performed for 4-6 hours at each urea concentration, and the renaturation must be performed at a low temperature of 4°C. The renaturation solution is purified by His-tag protein purification resin (nickel column) to obtain the recombinant protein recombinant ovalbumin (a histidine tag is added to the gene sequence of the target recombinant protein during design to facilitate protein purification). After purification, polyacrylamide gel electrophoresis (SDS-PAGE) is used to identify whether the purified target protein is successfully obtained. The results are as follows. Figure 3 As shown, recombinant ovalbumin with a size of about 44.169 kDa was obtained. Figure 3 The right side is the electrophoresis diagram of natural ovalbumin with a size of about 44.253 kDa that has not been systematically mutated (i.e., natural ovalbumin that has not been mutated at the relevant hydrophilic sites). Figure 3 In the figure, the left lane is a standard protein marker, and the right lane is purified recombinant ovalbumin or natural ovalbumin without systematic mutation.

[0075] The amino acid and nucleotide sequences of natural ovalbumin without systematic mutation are shown in Table 2. Similarly, NdeI restriction site (CATATG) and XhoI restriction site (CTCGAG) were added to the 5' end and 3' end of its coding gene, respectively.

[0076] Table 2

[0077]

[0078]

[0079] 5. Preparation of recombinant ovalbumin nanomicelles

[0080] (1) Take phosphate buffer saline dry powder in a beaker and add deionized water to dilute it, finally obtaining 1× PBS with a pH of 7.4 to prepare a dispersion solution for later use.

[0081] (2) Weigh 50 mg of the purified recombinant ovalbumin and dissolve it in 10 ml of deionized water under magnetic stirring to prepare a recombinant protein aqueous solution with a mass concentration of 5 mg / mL.

[0082] (3) 10 ml of the dispersion solution prepared in step (1) is measured and placed in a 25 ml beaker, and ultrasonically dispersed at a power of 400 W under ice bath conditions. At the same time, 10 ml of the recombinant protein solution prepared in step (2) is measured and dripped into the dispersion solution at a rate of 0.4 ml / min using a constant flow pump. After the dripping of the recombinant protein solution is completed, the ultrasound can be stopped to obtain a recombinant ovalbumin nanomicelle solution.

[0083] The method for preparing natural ovalbumin nanoparticles without systematic mutation is the same as that for preparing recombinant ovalbumin nanoparticles. The purpose of preparing natural ovalbumin nanoparticles without systematic mutation is to verify whether they can self-assemble like recombinant ovalbumin, so as to further highlight that recombinant ovalbumin can self-assemble without adding inducing agents (such as ethanol, isopropanol, dimethyl sulfoxide, etc.).

[0084] Example 2

[0085] This example provides a cytotoxicity evaluation of recombinant ovalbumin

[0086] The toxicity of recombinant ovalbumin to human embryonic kidney 293T cells (cell bank of the Chinese Academy of Sciences Type Culture Collection Committee) was studied by CCK8 method to evaluate the biosafety of recombinant ovalbumin. 4 Human embryonic kidney cells 293T were added to DMEM culture medium, and 5 experimental groups and 1 control group were set up, with 3 replicate wells in each group. After culturing the cells at 37°C for 24 hours, recombinant ovalbumin was added to the wells of the experimental group (experimental wells), so that the final concentrations of recombinant ovalbumin in the 5 experimental groups were 10μg / ml, 50μg / ml, 100μg / ml, 200μg / ml, and 400μg / ml, respectively. No recombinant ovalbumin was added to the wells of the control group (control wells). Continue to culture at 37°C for 24 hours, and then use the CCK8 method to detect the number of live cells. The steps are as follows: add 10μl CCK8 reagent to each well and incubate at 37°C for 3 hours. Then use an enzyme reader to measure the absorbance of each well at 450nm.

[0087] The cell viability rate was calculated as follows: [absorbance of experimental wells (culture medium containing cells, CCK8 reagent, protein sample) - absorbance of blank wells (culture medium without cells and protein sample, CCK8 reagent)] / [absorbance of control wells (culture medium containing cells, CCK8 reagent, no protein sample) - absorbance of blank wells (culture medium without cells and protein sample, CCK8 reagent)] × 100%.

[0088] The cell survival rate of each group is the average of the cell survival rates of the three replicate wells in the group. The cell survival rate of the control group is 100%, and the results are shown in Figure 2. Figure 6As shown, the horizontal axis is the concentration of recombinant ovalbumin self-assembled nanoparticles in the culture medium (μg / ml), and the vertical axis is the survival rate (%) of human embryonic kidney cells (293T). The survival rate of the control group cells without recombinant ovalbumin self-assembled nanoparticles in the culture medium is 100%. Figure 6 It can be seen that the cells showed a higher survival rate after adding recombinant ovalbumin at a concentration of 10-400 μg / ml, proving that recombinant ovalbumin has low cytotoxicity and good biocompatibility.

[0089] Example 3

[0090] This example provides a performance test of recombinant ovalbumin

[0091] 1. Transmission electron microscopy observation of recombinant ovalbumin nanomicelles

[0092] Sample preparation: Use a pipette to take 10 μl of the recombinant ovalbumin nanomicelle solution prepared in Example 1 and drop it onto a copper mesh (300 mesh), then use a pipette to take 4 μl of uranyl acetate and drop it onto the copper mesh to stain it, and then naturally dry it for morphological observation. Continue to use the natural ovalbumin nanomicelle solution prepared in Example 1 without systematic mutation, and prepare the electron microscopy observation sample of natural ovalbumin without systematic mutation on the copper mesh according to the above steps.

[0093] Sample observation: The morphology of the recombinant ovalbumin and the natural ovalbumin without systematic mutation on the copper mesh was observed using a transmission electron microscope. The results showed that the recombinant ovalbumin prepared by the present invention can self-assemble into nanoparticles without adding an inducing agent ( Figure 4 ), while natural ovalbumin without systematic mutation cannot self-assemble into nanoparticles without the addition of inducing agents ( Figure 5 ).

[0094] 2. Determination of particle size, encapsulation efficiency and drug loading of recombinant ovalbumin nanomicelles

[0095] Taking doxorubicin as an example, recombinant ovalbumin was used to encapsulate doxorubicin, and the particle size, encapsulation efficiency and drug loading were measured.

[0096] Preparation of recombinant ovalbumin drug-loaded nanomicelles: Take recombinant ovalbumin, dissolve it in deionized water, and prepare a protein aqueous solution with a mass concentration of 5 mg / ml. Then prepare a doxorubicin-PBS solution with a concentration of 0.4 mg / ml (the pH value of the PBS solution is 7.4). The doxorubicin-PBS solution needs to be stirred continuously, otherwise the hydrophobicity of doxorubicin will cause the drug to precipitate and cannot be evenly dispersed in the PBS solution. Finally, under ice bath conditions, an equal volume of recombinant ovalbumin aqueous solution is dripped into a doxorubicin-PBS solution with a concentration of 0.4 mg / ml at a rate of 0.4 ml / min. Ultrasonic dispersion is performed during the dripping process. After the dripping is completed, ultrasonic dispersion is stopped to obtain a recombinant ovalbumin drug-loaded nanomicelle solution.

[0097] The particle size, encapsulation efficiency and drug loading capacity of the prepared recombinant ovalbumin drug-loaded nanomicelles were measured according to the following method.

[0098] 1) Particle size determination method:

[0099] Use a pipette to draw 2 ml of the prepared recombinant ovalbumin drug-loaded nanomicelle solution and place it in a 4.5 ml transparent cuvette. Place the transparent cuvette containing the drug-loaded nanomicelle solution in the sample analysis hole of a nanoparticle size and ZETA potential analyzer (Malvern, UK, model: Nano ZS90), and determine the particle size of the nanoparticles according to the instructions for use of the Nano ZS90 nanoparticle size and ZETA potential analyzer.

[0100] 2) Method for determination of encapsulation efficiency:

[0101] The prepared recombinant ovalbumin drug-loaded nanomicelle solution was centrifuged at a speed of 10,000 r / min for 15 min, and then the supernatant was taken and the absorbance of the supernatant at 480 nm was measured by ultraviolet spectrophotometer. The free drug content was calculated according to the formula: A=0.0184C+0.0216 (A is absorbance, C is doxorubicin concentration).

[0102] Encapsulation efficiency = (W 总 -W 游 ) / W 总 *100%

[0103] W 总 is the initial dosage, W 游 is the amount of free drug not encapsulated in the nanoparticles.

[0104] 3) Method for determining drug loading:

[0105] The prepared recombinant ovalbumin drug-loaded nanomicelle solution was centrifuged at a speed of 10,000 r / min for 15 min, and then the supernatant was taken and the absorbance of the supernatant at 480 nm was measured by ultraviolet spectrophotometer. The free drug content was calculated according to the formula: A=0.0184C+0.0216 (A is absorbance, C is doxorubicin concentration).

[0106] Drug loading = (W 总 -W 游 ) / W 总重 *100%

[0107] W 总 is the initial dosage, W 游 is the amount of free drug not encapsulated in the nanoparticles, W 总重 is the total weight of recombinant ovalbumin nanoparticles after drug loading.

[0108] W 总重 The determination method is as follows: recombinant ovalbumin loaded nanoparticles are centrifuged at 12000r / min, and the supernatant is discarded after centrifugation. The weight of the precipitate is the total weight of the recombinant ovalbumin nanoparticles loaded with drugs. 总重 ) = total weight of centrifuge tube containing precipitate - weight of blank centrifuge tube.

[0109] The results are shown in Table 3. The particle size of the recombinant ovalbumin drug-loaded nanomicelles is small and uniform, and the drug loading capacity reaches 20.53%.

[0110] Table 3 Particle size, encapsulation efficiency and drug loading of recombinant ovalbumin nanomicelles (taking adriamycin as an example)

[0111]

[0112] 3. Drug release experiment of recombinant ovalbumin loaded nanomicelles

[0113] The prepared recombinant ovalbumin drug-loaded nanomicelles were subjected to drug release experiments.

[0114] Drug release experimental method: 5 ml of recombinant ovalbumin drug-loaded nanomicelle solution was transferred into a dialysis bag, placed in 20 ml of release medium containing 1 g / L Tween 80 in phosphate buffer (pH 7.4), placed on a constant temperature shaker, and the drug release experiment was carried out at 37°C and 170 r / min. 1 ml of the solution was taken out at 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h and 168 h (and 1 ml of the release medium was added), and the absorbance of the 1 ml solution taken out at 480 nm was measured by ultraviolet spectrophotometer, and the doxorubicin content was calculated according to the formula: A = 0.0184C + 0.0216 (A is absorbance, C is doxorubicin concentration). The experiment was repeated 3 times and the results were averaged. The cumulative drug release (%) at 3h, 6h, 12h, 24h, 48h, 72h, 96h, 120h, 144h and 168h was calculated and the drug release curve was plotted.

[0115] Cumulative drug release (%) = released drug weight / total encapsulated drug weight.

[0116] The results are as follows Figure 6 As shown, the cumulative drug release of the recombinant ovalbumin drug-loaded nanomicelles is about 50% at 72 hours, and reaches more than 90% after 168 hours. Therefore, the recombinant ovalbumin nanomicelles have a significant drug sustained release effect. Preferably, the recombinant protein recombinant ovalbumin drug-loaded nanomicelles take effect by being injected into the body. Injections include intravenous injections, intradermal injections, subcutaneous injections or powders for injection.

[0117] It is to be understood that the present embodiment only takes loading of doxorubicin as an example and not a limitation, and other hydrophobic drugs, including hydrophobic anti-tumor drugs or hydrophobic polypeptides, are also applicable to the present invention.

[0118] Example 4

[0119] This example provides control validation of other mutations.

[0120] Control 1: The following 7 positions of natural ovalbumin were subjected to hydrophobic mutation: position 30, position 43, position 50, position 52, position 53, position 73 and position 76. Specifically, tyrosine (Y), tyrosine (Y), threonine (T), threonine (T), glutamine (Q), glutamine (Q) and threonine (T) were mutated to phenylalanine (F), phenylalanine (F), valine (V), valine (V), leucine (L), leucine (L) and valine (V), respectively.

[0121] Nano-micelles of mutant protein were prepared according to the method of Example 1, and then the nano-micelle solution was dripped onto a copper mesh (300 mesh) according to the method of Example 3. Then 4 μl of uranyl acetate was taken by a pipette and dripped onto the copper mesh to stain it. Then it was naturally dried and the morphology was observed under an electron microscope. The results are shown in FIG. Figure 8 The results showed that ovalbumin with 7 amino acid mutations could not self-assemble.

[0122] Control 2: The following 6 positions of natural ovalbumin were subjected to hydrophobic mutation: position 30, position 43, position 50, position 52, position 53 and position 73. Specifically, tyrosine (Y), tyrosine (Y), threonine (T), threonine (T), glutamine (Q) and glutamine (Q) were mutated to phenylalanine (F), phenylalanine (F), valine (V), valine (V), leucine (L) and leucine (L), respectively.

[0123] Nano-micelles of mutant protein were prepared according to the method of Example 1, and then the nano-micelle solution was dripped onto a copper mesh (300 mesh) according to the method of Example 3. Then 4 μl of uranyl acetate was taken by a pipette and dripped onto the copper mesh to stain it. Then it was naturally dried and the morphology was observed under an electron microscope. The results are shown in FIG. Fig. 9 The results showed that ovalbumin with 6 amino acid mutations could not self-assemble.

[0124] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A recombinant ovalbumin with self-assembly performance, characterized in that: The first 100 positions of the amino acid sequence of the recombinant ovalbumin undergo systematic mutations, and the systematic mutations include hydrophobic mutations at consecutive amino acid sites and discontinuous amino acid sites; the amino acid sequence of the recombinant ovalbumin is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: A gene comprising the recombinant ovalbumin according to claim 1.

3. The recombinant expression vector according to claim 2, characterized in that The types of the vector include plasmid vector, bacteriophage, adenovirus vector or baculovirus vector.

4. A recombinant bacterium, characterized in that Expressing the recombinant ovalbumin according to claim 1.

5. The recombinant bacterium according to claim 4, characterized in that The types of bacteria include strains of Escherichia coli, Bacillus subtilis or Pichia pastoris.

6. A nanomicelle for hydrophobic drug delivery, characterized in that: Prepared from the recombinant ovalbumin described in claim 1.

7. Use of the nanomicelles according to claim 6 in preparing drug delivery carriers.

8. The use according to claim 7, characterized in that The nano micelles are used to carry hydrophobic drugs, hydrophobic polypeptides or proteins; the hydrophobic drugs include hydrophobic anti-tumor drugs or hydrophobic antibiotics.

9. The use according to claim 7, characterized in that The dosage form of the drug delivery carrier is injection.

10. The use according to claim 9, characterized in that The injection includes intravenous injection, intradermal injection or subcutaneous injection.

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