Coding sequence of recombinant protein, recombinant protein, recombinant protein-liposome nanoparticle, preparation method and application

Abstract

The invention relates to the technical field of gene engineering, and particularly discloses a coding sequence of a recombinant protein, the recombinant protein, a recombinant protein-lipidosome nanoparticle, a preparation method and application, and the coding sequence of the recombinant protein comprises a coding sequence shown as SEQ ID NO.01. The invention also discloses a preparation method of the recombinant protein-lipidosome nanoparticle. After the product is used, the number of blood vessels can be obviously increased, and the good effect of promoting blood vessel formation is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and in particular to a coding sequence of a recombinant protein, the recombinant protein, a recombinant protein-liposome nanoparticle, a preparation method and application. BACKGROUND

[0002] Vascular system injury is a key limiting factor for the prognosis of various major diseases. For example, in the process of diabetes, microangiopathy induced by hyperglycemia can cause delayed healing of wounds and gangrene of limbs; local vascular network damage after spinal cord injury exacerbates ischemia and hypoxia of nerve tissue, hindering functional recovery. According to statistics of the World Health Organization, more than 25% of the approximately 537 million diabetic patients worldwide are at risk of chronic wounds, and the number of new cases of spinal cord injury is 250-500 thousand cases per year. Such diseases highlight the urgent need to promote angiogenesis and repair. The fibroblast growth factor (FGF) family, as a core signal protein regulating endothelial cell proliferation and migration, is a key target for tissue regeneration and vascular reconstruction. However, natural FGF has problems such as short half-life, poor stability and easy degradation by proteases, which seriously restrict its application. SUMMARY

[0003] In order to promote the development of angiogenic natural drugs and overcome the problem of protein stability, the present application provides a recombinant protein which can significantly increase the number of blood vessels after use and has a good effect of promoting angiogenesis.

[0004] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: A coding sequence of a recombinant protein, comprising: a coding sequence as shown in SEQ ID NO. 01.

[0005] The present application also discloses a recombinant protein made from the coding sequence as shown in SEQ ID NO. 01.

[0006] The present application also discloses a preparation method of a recombinant protein, comprising the following steps: a. obtaining the coding sequence as shown in SEQ ID NO. 01 by gene synthesis of the nucleotide sequence of mouse FGF11a; b. recombining to a prokaryotic expression vector by using the homologous arm to obtain the corresponding recombinant plasmid vector; c. after transforming the engineering bacteria, picking a single colony and inoculating into an automatic induction complex culture medium for culture, and centrifuging to collect the bacterial bodies; d. placing the purified protein in a dialysis bag and soaking in a gradient dialysis buffer solution for dialysis.

[0007] Optionally, the specific centrifugal operation in step c comprises: adding bacterial lysis solution into the bacterial body, then ultrasonic lysis to obtain a precipitate; washing the precipitate, then adding urea solution, and dissolving overnight under shaking, centrifuging to collect supernatant; loading the supernatant onto a column, washing off impure proteins, eluting and collecting the purified proteins.

[0008] Optionally, the gradient dialysis buffer solution comprises a first gradient buffer, a second gradient buffer, a third gradient buffer and a fourth gradient buffer; the first gradient buffer comprises 25 mM Tris, 250 mM NaCl, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, 250 mM imidazole, 4 mM urea and 10% glycerol; the second gradient buffer comprises 25 mM Tris, 100 mM NaCl, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, 100 mM imidazole, 2 mM urea and 10% glycerol; the third gradient buffer comprises 25 mM Tris, 50 mM NaCl, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, 1 mM urea and 10% glycerol; and the fourth gradient buffer comprises 25 mM Tris, 25 mM NaCl, 2 mM reduced glutathione, 0.2 mM oxidized glutathione and 10% glycerol.

[0009] Optionally, the culturing operation in step c is specifically: culturing at 36-38℃ for 18-20h.

[0010] Optionally, the centrifugal operation in step c is specifically: centrifuging at 4-6℃ for 15-20min to collect the bacterial body.

[0011] Optionally, the urea solution comprises 20 mM Tris-HCl, 5 mM imidazole, 0.5 M sodium chloride and 8 M urea.

[0012] The application further discloses a recombinant protein-liposome nanoparticle, wherein the recombinant protein is prepared by the method.

[0013] The application further discloses a preparation method of a recombinant protein-liposome nanoparticle. 1) hydrogenated soybean lecithin, cholesterol and distearoyl phosphatidyl ethanolamine-methoxy polyethylene glycol (DSPE-mPEG) are respectively dissolved in an organic solvent, and after dissolution, a part of each component is taken to a centrifugal tube to mix, to obtain an oil phase; 2) an equal amount of recombinant protein solution is taken as an aqueous phase as the oil phase; 3) the oil phase and the aqueous phase are subjected to a microfluidic device to prepare an oil phase-aqueous phase mixture; 4) The oil phase-water phase mixture product obtained by microfluidization is placed in a dialysis bag for dialysis to obtain recombinant protein-liposome nanoparticles.

[0014] Optionally, the organic solvent comprises methanol.

[0015] The application further discloses an application of the recombinant protein in preparation of a medicine for promoting blood vessel formation.

[0016] The application has at least the following beneficial effects: the recombinant protein can obviously increase the number of blood vessels and has a good effect of promoting blood vessel formation. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is an SDS-PAGE detection protein expression detection diagram of example 1 of the application; Figure 2 is a recombinant protein target protein expression detection diagram using different label antibodies of example 1 of the application; Figure 3 is a transmission electron microscope diagram of liposomes and liposome-GFP nanoparticles of example 1 of the application; Figure 4 is a liposome-GFP nanoparticle potential and particle size detection diagram of example 1 of the application; Figure 5 is a transfection efficiency diagram of liposomes of example 1 of the application; Figure 6 is a blood vessel formation diagram of HUVEC cells after being treated with the recombinant protein for 8h of example 1 of the application; Figure 7 is a statistical analysis diagram of blood vessel formation ability of HUVEC cells after being treated with the recombinant protein for 8h of example 1 of the application.

[0018] APPENDIX Figure 1 and APPENDIX Figure 2 In the figure: M, marker; 1, culture medium; 2, bacterial body; 3, supernatant after bacterial body crushing; 4, precipitate after bacterial body crushing; 5, protein after nickel column purification. Figure 5 In the figure: DAPI (4', 6-diamidino-2-phenylindole) is a blue fluorescent nucleic acid dye widely used in biological research. Merge is a double staining technique, which uses fluorescent dye DAPI and another fluorescent dye (such as Rhodamine or FITC) to simultaneously stain the cell nucleus and cytoplasm. DETAILED DESCRIPTION

[0019] The present application is further described below in conjunction with the accompanying drawings and specific examples. The raw materials and equipment used in the following specific examples of the present application are all known products, which are obtained by purchasing commercially available products. The engineering bacteria in the present application use Transetta (DE3) Chemically Competent Cell of Beijing Quanshi Jinsheng Technology Co., Ltd. Transetta (DE3) is an imported bacterial strain made by special process and can be used for chemical transformation of DNA. The cell has chloramphenicol (Camr) resistance. Using pUC19 plasmid DNA detection, the transformation efficiency can reach 107 cfu / μg DNA. Control Plasmid I (Amp+) is used to detect whether the cell has expression function, and the expression protein size is 25 kDa.

[0020] In the present application, SDS-PAGE is the abbreviation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is mainly used for separating and analyzing components in protein mixtures. In the present application, Tris-HCl, i.e. tris(hydroxymethyl)aminomethane hydrochloride, is a commonly used buffer, which can use the product of Solabio Technology Co., Ltd. In the present application, mM refers to mmol / L (millimoles per liter). M refers to mol / L (moles per liter). In the present application, GFP (green fluorescent protein) is a protein composed of about 238 amino acids. In the present application, the method of GFP (green fluorescent protein) is the same as that of expressing recombinant protein, and the carrier used is the carrier of recombinant protein. The concentration of recombinant protein solution is 100 ng / ul. In the present application, recombinant protein solution can use the buffer recommended in the instruction: commonly used PBS, 20 mM Tris + 150 mM NaCl (pH 8.0) or sterilized ultrapure water. In the present application, His tag (also known as histidine tag) is a short peptide tag widely used in recombinant protein expression and purification.

[0021] In the present application, micro-control flow device can use micro-control flow chip of Shanghai Pengzan Biotechnology Co., Ltd.

[0022] The BCA kit of this invention can be purchased from Beyotime Biotechnology Co., Ltd. The PBS buffer solution can be purchased from Solarbio Science & Technology Co., Ltd. In this invention, Western blotting refers to immunoblotting, also known as protein blotting, which is a method for detecting a specific protein in a complex sample based on the specific binding of antigen and antibody. The PC12 cells in this invention are a commonly used neural cell line. The PC12 cell line is derived from a transplantable mouse pheochromocytoma, which exhibits a reversible neuronal phenotypic response to nerve growth factor (NGF) and does not synthesize adrenaline. BSA (bovine serum albumin) can be purchased from Thermo Fisher Scientific Co., Ltd. PFA (paraformaldehyde) can be purchased from Solarbio Science & Technology Co., Ltd. Distearatel phosphatidylethanolamine-methoxy polyethylene glycol can be purchased from Shanghai Kaiwei Chemical Technology Co., Ltd. Hydrogenated soybean lecithin can be purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0023] This invention discloses a coding sequence for a recombinant protein, comprising: the coding sequence shown in SEQ ID NO.01. SEQ ID NO.01 of this invention is listed in the sequence listing. The nucleotide sequence of SEQ ID NO.01 corresponds to the mouse FGF11a nucleotide sequence NM_001362623.1 (this nucleotide sequence is published on NCBI and was synthesized by the inventors using a gene).

[0024] A recombinant protein, made using the coding sequence shown in SEQ ID NO.01.

[0025] This invention discloses a method for preparing a recombinant protein, comprising the following steps: a. The coding sequence shown in SEQ ID NO. 01 was obtained by gene synthesis from the mouse FGF11a nucleotide sequence; b. Recombining homologous arms into a prokaryotic expression vector to obtain the corresponding recombinant plasmid vector; c. After transforming the engineered bacteria, select single clones and inoculate them into an automated induction complex medium for culture. Collect the bacterial cells by centrifugation. The specific centrifugation operation includes: adding bacterial lysis buffer to the bacterial cells, followed by sonication to obtain a precipitate; washing the precipitate, adding urea solution, and centrifuging overnight with shaking to dissolve the precipitate. Centrifuge at 4-6℃ for 15-20 minutes and collect the supernatant; load the supernatant onto a column, wash away impurities, and elute and collect the purified protein. d. Place the purified protein in a dialysis bag and immerse it in a gradient dialysis buffer solution for dialysis.

[0026] The application further discloses a preparation method of the recombinant protein-liposome nanoparticle. 1) Hydrogenated soybean lecithin, cholesterol and distearoyl phosphatidyl ethanolamine-methoxy polyethylene glycol (DSPE-mPEG) are respectively dissolved in methanol, and after dissolution, one part of each component is taken to a centrifugal tube to obtain an oil phase; 2) An equal amount of a recombinant protein solution is taken as the water phase; 3) The oil phase and the water phase are subjected to a microfluidic device to obtain an oil phase-water phase mixture; 4) The oil phase-water phase mixture is subjected to a microfluidic device to obtain a product, and the product is placed in a dialysis bag to dialysis to obtain the recombinant protein-liposome nanoparticle.

[0027] Example 1: A preparation method of a recombinant protein, comprising the following steps: a. According to the mouse FGF11a nucleotide sequence NM_001362623.1 published in the NCBI database, a FGF11a coding sequence is obtained by gene synthesis; b. The homologous arm is recombined to a prokaryotic expression vector pET30(a)-His-GFP to obtain a pET30(a)-His-GFP-FGF11a recombinant plasmid vector; c. The recombinant plasmid is transformed into a commercial Transetta (DE3) competent (full type gold), and a single colony is picked and inoculated into 250 mL of an automatic induction complex culture medium containing 0.1% kanamycin, and cultured at 37°C and 200 rpm for 18 h. The bacteria are collected by centrifugation at 4000 g and 4°C for 20 min. 30 mL of bacterial lysis solution (containing 1% protease inhibitor mixture) is added to the bacteria, and then the bacteria are lysed by ultrasonic lysis (30% power, 3 s of ultrasonic lysis and 3 s of stop) for 30 min. The precipitate is obtained by centrifugation at 10000 g and 4°C for 15 min. After the precipitate is washed with PBS, 30 mL of a high-concentration urea solution (20 mM Tris-HCl, 5 mM imidazole, 0.5 M sodium chloride and 8 M urea) is added, and the solution is dissolved by oscillation at 4°C overnight. The supernatant is collected by centrifugation at 10000 g for 20 min. The supernatant is loaded onto a His-tag protein purification nickel column, and the flow rate is 10 times the column volume per hour. The column is washed with 15 times the column volume of a high-concentration urea solution to wash away impurities. The high-concentration imidazole and urea solution is used for elution, and the purified protein solution is collected; d. The purified protein is placed in a dialysis bag with a Mw of 50000, and is soaked in different gradient refolding solutions, and each solution is dialyzed for 6 h.

[0028] The gradient dialysis buffer solution comprises a first gradient buffer, a second gradient buffer, a third gradient buffer and a fourth gradient buffer; the first gradient buffer comprises 25 mM of Tris, 250 mM of NaCl, 2 mM of reduced glutathione, 0.2 mM of oxidized glutathione, 250 mM of imidazole, 4 mM of urea and 10% of glycerol; the second gradient buffer comprises 25 mM of Tris, 100 mM of NaCl, 2 mM of reduced glutathione, 0.2 mM of oxidized glutathione, 100 mM of imidazole, 2 mM of urea and 10% of glycerol. The third gradient buffer comprises 25 mM of Tris, 50 mM of NaCl, 2 mM of reduced glutathione, 0.2 mM of oxidized glutathione, 1 mM of urea and 10% of glycerol; the fourth gradient buffer comprises 25 mM of Tris, 25 mM of NaCl, 2 mM of reduced glutathione, 0.2 mM of oxidized glutathione and 10% of glycerol.

[0029] A preparation method of a recombinant protein-liposome nanoparticle, comprising the following steps: hydrogenated soybean lecithin, cholesterol and distearoyl phosphatidyl ethanolamine-methoxy polyethylene glycol (DSPE-mPEG) are respectively dissolved in methanol, and after dissolution, 2 mL of each is taken to a centrifuge tube to mix, to obtain an oil phase. Then, an equal amount of recombinant protein solution to the oil phase is taken as an aqueous phase. The oil phase and the aqueous phase are each taken 5 mL by a syringe and operated by a microfluidic device, the flow rate is controlled to be 0.1 mL / min, and the product is collected. The product obtained by microfluidics is placed in a 50 kDa dialysis bag for dialysis for 24 h.

[0030] Embodiment 2: A preparation method of a recombinant protein, comprising the following steps: a. According to the mouse FGF11a nucleotide sequence NM_001362623.1 published in the NCBI database, the FGF11a coding sequence is obtained by gene synthesis; b. The homologous arm is recombined to the prokaryotic expression vector pET30(a)-His-GFP to obtain the pET30(a)-His-GFP-FGF11a recombinant plasmid vector; c. Transform the recombinant plasmid into Transetta (DE3) competent (full form gold) commercially. Pick a single colony and inoculate into 250 mL of auto-induction complex medium containing 0.1% kanamycin, and incubate at 37°C, 200 rpm for 18 h. Centrifuge at 4000g, 6°C for 15 min to collect the bacteria. Add 30 mL of bacterial lysis solution (containing 1% protease inhibitor cocktail) to the bacteria, and then ultrasonic lysis (30% power, ultrasonic 3 s, stop 3 s) for 30 min. Centrifuge at 10000g, 4°C for 15 min to obtain the precipitate. After washing the precipitate with PBS, add 30 mL of high-concentration urea solution (20 mM Tris-HCl, 5 mM imidazole, 0.5 M sodium chloride and 8 M urea), and shake to dissolve at 4°C overnight. Centrifuge at 10000g for 20 min to collect the supernatant. Load the supernatant into a His-tag protein purification nickel column, and the flow rate is 10 column volumes / hour. Use 15 column volumes of high-concentration urea solution to wash the column to remove impurities. Use high-concentration imidazole and urea solution for elution, and collect the purified protein solution; d. Place the purified protein in a Mw50000 dialysis bag, and soak it in different gradient renaturation solutions, each for 6 h.

[0031] The gradient dialysis buffer solution includes a first gradient buffer, a second gradient buffer, a third gradient buffer and a fourth gradient buffer; the first gradient buffer includes 25 mM Tris, 250 mM NaCl, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, 250 mM imidazole, 4 mM urea and 10% glycerol; the second gradient buffer includes 25 mM Tris, 100 mM NaCl, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, 100 mM imidazole, 2 mM urea and 10% glycerol. The third gradient buffer includes 25 mM Tris, 50 mM NaCl, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, 1 mM urea and 10% glycerol; and the fourth gradient buffer includes 25 mM Tris, 25 mM NaCl, 2 mM reduced glutathione, 0.2 mM oxidized glutathione and 10% glycerol.

[0032] A method for preparing recombinant protein-liposome nanoparticles includes the following steps: hydrogenated soybean lecithin, cholesterol, and distearate-phosphatidylethanolamine-methoxy polyethylene glycol (DSPE-mPEG) are dissolved in methanol, and 2 mL of each is taken into a centrifuge tube and mixed to obtain an oil phase. Then, an equal volume of recombinant protein solution is taken as the aqueous phase. 5 mL of each phase is drawn using a syringe and collected using a microfluidic device at a flow rate of 0.1 mL / min. The microfluidic-obtained product is then dialyzed in a 50 kDa dialysis bag for 24 h.

[0033] In Examples 1 and 2, during the expression and purification of the protein, 200 μL each of the culture medium, bacterial cells, supernatant after bacterial cell lysis, precipitate after bacterial cell lysis, and purified protein were collected. 50 μL of 5X protein loading buffer was added, and the mixture was boiled at 100°C for 10 min. After cooling, the protein was detected by SDS-PAGE electrophoresis and Western blot. The experimental results of Example 1 are as follows: Figure 1 and Figure 2 As shown, after protein purification, the bands of impurity proteins were significantly reduced. The protein precipitation during the refolding process of the recombinant protein in Example 1 is shown in Table 1 below.

[0034] Table 1. Protein precipitation during refolding of recombinant proteins .

[0035] A recombinant protein-liposome nanoparticle was used to determine the liposome size and potential using a particle size analyzer; simultaneously, the liposome structure was examined using transmission electron microscopy. Specific transmission electron micrographs of the liposomes and liposome-GFP nanoparticles are shown below. Figure 3 Transmission electron microscopy imaging results showed that both the liposomes and the prepared liposome-GFP nanoparticles were uniform spherical structures.

[0036] The potential and particle size of liposome-GFP nanoparticles are shown in the figure. Figure 4 ,analyze Figure 4 The potential of the prepared liposome-GFP nanoparticles was found to be -18.64 mV. Particle size analysis showed that the particle size of the prepared liposome-GFP nanoparticles was 107.64 ± 1.88 nm.

[0037] For the detection of liposome nanoparticle transfection efficiency: Commercial PC12 cells were cultured in vitro, and the lipid nanoparticles encapsulating GFP protein were added into the cell culture medium. After 24 h of culture, the cells were fixed, and the expression of the target protein in the cells was detected by immunofluorescence. 30,000 PC12 cells were seeded in a 24-well plate, and 250 μL of culture medium was added for incubation for 24 hours. 20 μL of the protein-lipid nanoparticle solution was added into the culture medium. After 48 hours, the cells were fixed with 4% PFA, and blocked with 1% bovine serum albumin (BSA) in PBS. After washing with PBS for three times, the cells were incubated with a rabbit monoclonal anti-His antibody (1:5000; Zenbio, China) to detect the expression of the recombinant protein in the cells. The transfection efficiency of the liposome is shown in Figure 5 .

[0038] Figure 5 The transfection efficiency detection results of the liposome show that the prepared liposome has high transfection efficiency, and the transfection efficiency is 98.83±1.12%.

[0039] The application discloses an application of a recombinant protein-liposome nanoparticle in preparation of a drug for promoting blood vessel formation.

[0040] For identification of the function of the recombinant protein-liposome nanoparticle in promoting blood vessels: HUVEC cells were plated in a 48-well plate and cultured at 37°C and 5% CO2 for 24 hours. After the GFP-His or MFGF11a-GFP-His recombinant protein was prepared into a protein-liposome nanoparticle solution with the liposome, 2 μg of each was added into the culture medium, and the cells were further cultured for 12 hours. Then, the cells were collected by digestion and plated in a 48-well plate containing Matrigel (Corning) for culture. After 8 hours of culture, the blood vessel formation was observed under an optical microscope and photographed. The specific photograph of the product of Example 1 is shown in Figure 6 . The blood vessel formation of Example 1 was statistically analyzed by using software ImageJ, and the specific analysis is shown in Figure 7 , the expression of the two groups p <0.01.

[0041] The specific embodiments described in the present application are only illustrative of the spirit of the present application. Those skilled in the art to which the present application belongs can make various modifications or supplements to the described specific embodiments or use similar ways to replace, but will not deviate from the spirit of the present application or exceed the scope defined by the appended claims.

[0042] Although detailed descriptions have been made for the present application and some specific embodiments have been cited, it is obvious for those skilled in the art to make various changes or modifications without departing from the spirit and scope of the present application.

Claims

1. A coding sequence for a recombinant protein, characterized in that, The coding sequence shown as SEQ ID NO.

01.

2. A recombinant protein, characterized in that, The coding sequence shown as SEQ ID NO. 01 is prepared by using the coding sequence shown as SEQ ID NO.

01.

3. A method for producing a recombinant protein, characterized by, The method comprises the following steps: a. obtaining the coding sequence shown as SEQ ID NO. 01 by gene synthesis of the mouse FGF11a nucleotide sequence; b. recombining the coding sequence shown as SEQ ID NO. 01 to a prokaryotic expression vector by using the homologous arms to obtain a corresponding recombinant plasmid vector; c. after transforming the engineering bacteria, picking a single colony to inoculate into an automatic induction complex culture medium for culture, centrifuging to collect the bacterial bodies; d. placing the purified protein into a dialysis bag and soaking in a gradient dialysis buffer solution for dialysis.

4. The method for preparing a recombinant protein according to claim 3, characterized in that, The specific centrifugation operation in step c comprises: adding a bacterial lysis solution to the bacterial bodies, then performing ultrasonic lysis to obtain a precipitate; after washing the precipitate, adding a urea solution and dissolving under oscillation overnight, centrifuging to collect the supernatant; loading the supernatant onto a column, washing away the impure proteins, eluting and collecting the purified protein.

5. A method for preparing a recombinant protein according to claim 3 or 4, characterized in that, The gradient dialysis buffer solution comprises a first gradient buffer solution, a second gradient buffer solution, a third gradient buffer solution and a fourth gradient buffer solution; the first gradient buffer solution comprises 25 mM of Tris, 250 mM of NaCl, 2 mM of reduced glutathione, 0.2 mM of oxidized glutathione, 250 mM of imidazole, 4 mM of urea and 10% of glycerol; the second gradient buffer solution comprises 25 mM of Tris, 100 mM of NaCl, 2 mM of reduced glutathione, 0.2 mM of oxidized glutathione, 100 mM of imidazole, 2 mM of urea and 10% of glycerol; the third gradient buffer solution comprises 25 mM of Tris, 50 mM of NaCl, 2 mM of reduced glutathione, 0.2 mM of oxidized glutathione, 1 mM of urea and 10% of glycerol; and the fourth gradient buffer solution comprises 25 mM of Tris, 25 mM of NaCl, 2 mM of reduced glutathione, 0.2 mM of oxidized glutathione and 10% of glycerol.

6. A method for preparing a recombinant protein according to claim 3 or 4, characterized in that, The culture operation in step c is specifically: culturing at 36-38℃ for 18-20h.

7. A method for preparing a recombinant protein according to claim 3 or 4, characterized in that, The centrifugation operation in step c is specifically: centrifuging at 4-6℃ for 15-20 minutes to collect the bacterial bodies.

8. A recombinant protein-liposome nanoparticle characterized by, The recombinant protein is prepared by the method for preparing the recombinant protein of any one of claims 3 to 7; and the recombinant protein is wrapped in a nanoscale liposome.

9. A method for preparing a recombinant protein-liposome nanoparticle, characterized by, The method for preparing the recombinant protein-liposome nanoparticle of claim 8 comprises the following steps: 1) dissolving hydrogenated soybean lecithin, cholesterol and distearoyl phosphatidyl ethanolamine-methoxy polyethylene glycol in organic solvents respectively, after dissolving, taking one part of each component to a centrifuge tube to mix, to obtain an oil phase; 2) taking the same amount of recombinant protein solution as the oil phase as an aqueous phase; 3) passing the oil phase and the aqueous phase through a microfluidic device to prepare an oil phase-aqueous phase mixture; 4) placing the product obtained by the microfluidic device into a dialysis bag for dialysis to prepare the recombinant protein-liposome nanoparticle.

10. Use of a recombinant protein-liposome nanoparticle for the preparation of a medicament for promoting angiogenesis, characterized in that, The recombinant protein-liposome nanoparticle is the recombinant protein-liposome nanoparticle of claim 8.

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