CN110563829B

CN110563829B - Reflective protein system for regulating the behavior and function of liposome vesicles and its application

Info

Publication number: CN110563829B
Application number: CN201910875566.1A
Authority: CN
Inventors: 宋俊祎; 吴文健; 胡碧茹
Original assignee: National University of Defense Technology
Current assignee: National University of Defense Technology
Priority date: 2019-09-17
Filing date: 2019-09-17
Publication date: 2021-03-26
Anticipated expiration: 2039-09-17
Also published as: CN110563829A

Abstract

The present invention relates to a light-reflecting protein system for regulating the behavior and function of liposome vesicles and its application. RA1 and liposome vesicles have good and controllable aggregation characteristics based on the quantitative ratio. The special biochemical properties of RA1 can greatly change the spatial morphology of protein-liposome complex aggregates, including The controllable phospholipid membrane fusion process and the special spatial structure (tubular) formation process provide a method for constructing liposome vesicle aggregation, membrane fusion promotion and its spatial tubular morphology based on the biochemical properties of RA1.

Description

Light-reflecting protein system for regulating and controlling liposome vesicle behavior and function and application thereof

Technical Field

The invention belongs to the technical fields of biological pharmacy, drug target positioning and tumor treatment in biotechnology, and particularly relates to a light-reflecting protein system for regulating and controlling the behaviors and functions of liposome vesicles and application thereof.

Background

In the field of biomedicine, nanomaterials play an important role as carriers of bioactive molecules. Through the combination with bioactive molecules, the nano material effectively improves the solubility of the bioactive molecules and the stability of the bioactive molecules in organisms, supports passive and active target processes, enhances biocompatibility, reduces side effects and obtains outstanding treatment effects. Since the nano-carrier has a high surface area/volume ratio, it can carry a high dose of target molecules. Meanwhile, the cargo molecules are protected by the nano-carrier in the process of carrying, so that the cargo molecules are prevented from being influenced by the physiological environment, the dosage loss is greatly reduced, and the treatment effect is enhanced. Based on the above advantages, the nano material will bring revolutionary subversion in the treatment of cancer, cardiovascular disease, type I diabetes, fungal infection, lymphoma, leukemia and other diseases.

Liposomes (liposomes) are vesicular structures consisting of phospholipid molecules with an aqueous inner core. Liposomes can encapsulate hydrophobic components between bilayers, and can also place hydrophilic substrates in their internal aqueous core (aquouos core), thus having great potential for in vivo delivery, targeting of biopharmaceuticals. In clinical research, it has been applied to the field of biological medicine as a drug carrier. The potential for medical applications of lipid vesicles derives from their biocompatibility, tunability of composition and size, ease of modification of membrane surface characteristics, and their amphiphilicity, thus enabling the encapsulation of hydrophilic or hydrophobic materials.

The surface of nanoparticles that are injected intravenously into plasma can adsorb many biomolecules (proteins, liposomes, glycans, and other metabolites). These biomolecules adsorbed on the surface of the nanoparticles can exchange substances with the surrounding environment, and thus a dynamic shell called "biomolecule corona" is formed. Since most studies focus on the protein component of this layer of the coat, it is also known as "protein corona". This layer of covering imparts numerous pharmacological and biological functions to the nanoparticles: reducing nanoparticle accumulation within target tissues by blocking surface ligand action; modifying the surface characteristics of the carrier, improving the target specificity and promoting the internalization and absorption of cells; improve the immune response of the nano-particles in vivo, and the like.

It is presently believed that the results of the study of liposome-protein corona interactions will lead to subversive therapies for a variety of diseases. Meanwhile, the process of liposome-protein interaction and protein crown formation under physiological conditions also requires more deep systematic research.

Disclosure of Invention

The invention aims to provide a light reflecting protein system for regulating the behavior and the function of liposome vesicles.

The invention also aims to provide application of a light-reflecting protein system for regulating and controlling the behaviors and functions of liposome vesicles, and the liposome vesicles have good controllable aggregation characteristics based on quantity ratio, and the special biochemical characteristics of the protein can enable the spatial morphology of a protein-liposome complex to be greatly changed, including a controllable phospholipid membrane fusion process and a special spatial tubular structure forming process, so that a liposome vesicle aggregation and membrane fusion promotion method based on RA1 biochemical characteristics and a spatial morphology construction method thereof are provided.

The light reflecting protein system for regulating and controlling the behavior and the function of the liposome vesicle is prepared by the following steps:

1) dissolving dioleoyl lecithin (DOPC) and dioleoyl phosphatidylglycerol (DOPG) in chloroform at a ratio of 60:40, filtering with 0.22 μm filter membrane, and drying with clean nitrogen to form liposome membrane;

2) putting the liposome film obtained in the step into a vacuum drier for vacuum overnight;

3) the next day, 1ml of working buffer solution is added into each 100mg of liposome film, vortex and shake for 1 minute, and the process is repeated twice;

4) repeatedly freezing and thawing for 10 times by using liquid nitrogen and a water bath at 80 ℃, wherein each freezing and thawing time is 1 minute, and the total time is 20 minutes;

5) homogenization treatment of liposome vesicles: repeatedly treating liposome with a liposome extruder and a 100nm filter membrane, and transferring the homogenized liposome vesicles into a glass vial cleaned by ethanol and acetone for storage to obtain naked and stable 100 nm-scale liposome vesicles;

6) synthesizing the full length of RA1 gene by chemical synthesis;

RA1 is derived from membrane protein-reflectrin A1 (reflecin A1, RA1 for short) of iris cells of cuttlefish (Doryteuthis opalescens);

7) introducing RA1 gene into recipient cell E.coli Rosetta for protein expression;

8) after induction expression, carrying out inclusion body purification;

9) performing primary purification by adopting ion affinity chromatography and constantly changing the concentration of guanidine salt, performing hydrophobic chromatography by utilizing a trifluoroacetic acid and acetonitrile system, and freezing and drying the obtained purified protein and then freezing and storing at-80 ℃;

10) the protein crown is composed of RA1, RA1 covers the liposome vesicle obtained by the preparation method, and RA1 forms protein self-assemblies with the sizes of 8 +/-0.96 nm, 15.6 +/-3.4 nm and 34.4 +/-8.2 nm in pH4.5 acetic acid, pH 6.55mM propanesulfonic acid and pH 7.55mM propanesulfonic acid buffer solutions respectively to obtain the light-reflecting protein system for regulating and controlling the behaviors and functions of the liposome vesicle.

The liposome film formed in the step 1) of the invention is further subjected to fluorescence observation, and BODIPY is added during liposome dissolution and mixing^TM558/568C 12 fluorescent dye.

The working buffer solution in the step 3) comprises but is not limited to acetic acid with pH4.5, propanesulfonic acid with pH 6.55mM and propanesulfonic acid with pH 7.55mM.

Coli Rosetta in step 7), is e^TM2(DE 3); the protein expression is carried out by using LB culture medium and 50ng/ml kanamycin resistance.

After the induced expression in the step 8), carrying out inclusion body purification, namely carrying out induced expression for 3-4 hours at 37 ℃ by using 1mM IPTG, and then adopting

Inclusion body purification was performed.

The primary purification in the step 9) adopts a purification column as Hitrap^TM5mL SP XL and Mono

5/50GL (SIGMA).

Further, the protein self-assembly body in the step 10) comprises RA1 protein molecule aggregates formed under various conditions of pH value and salt ion concentration.

A liposome vesicle aggregation, membrane promotion fusion and spatial morphology construction method based on RA1 biochemical characteristics comprises the following steps:

a. the processing method of the light-reflecting protein system for regulating the behavior and the function of the liposome vesicle comprises the following steps: in a buffer solution, RA1 with a proper concentration and vesicles firstly form large-scale RA 1-liposome aggregates, and the scale reaches hundreds of nanometers to the micron scale; then, treating the protein and vesicle aggregates for 50 minutes by using proteinase K with the working concentration of 0.15mg/ml in a propane sulfonic acid system with the pH value of 7.55mM to promote the large-scale membrane fusion behavior of the 100nm vesicles, so as to form the large vesicle monomers with the micron-sized dimensions;

b. the structure of the space tubular shape of the giant vesicle monomer formed after the liposome coacervate membrane is fused: in a system of pH7.55mM propanesulfonic acid, 0.15mg/ml proteinase K is used for treating protein and vesicle aggregates for 30 minutes, a proteinase activity inhibitor PMSF with the final concentration of 1mM is added to block the degradation process of RA1, and RA1 and RA1 self-assemblies in a semi-degradation state restrict the space morphology of a large vesicle monomer with micron-scale dimension formed after membrane promotion fusion, so that the large vesicle monomer has special tubular stacking characteristics.

In the buffer solution of step a of the present invention, the buffer solution refers to pH4.5 acetic acid, pH6.55mM propanesulfonic acid, pH7.55mM propanesulfonic acid; the appropriate concentration is 0.625. mu.M at pH4.5, 4.5. mu.M at pH6.5, and 13.5. mu.M at pH 7.5.

Compared with the prior art, the invention has the following advantages:

1. currently, academic reports of similar membrane functions are mainly focused on the pathological function research of natural proteins and protein families. For example, SV40 virus protein, BAR protein family, etc., wherein the mechanism of membrane upgoing of clathrin (clathrarin) is most clear: clathrin forms a hemispherical capsule pit on the membrane with the help of other chaperones (AP-2, etc.); subsequently, under the cohesion of actin, the capsular pit gradually invaginates; finally, the invaginated neck is severed and separated from the mother vesicle membrane with the aid of another population of sheared proteins. In the invention, RA1 can complete the capabilities of promoting membrane coagulation, promoting membrane fusion and maintaining the space tubular shape of the fused liposome without the assistance of other molecular chaperones and auxiliary proteins. It is sufficient to achieve the powerful function. In addition, the size of the RA1 protein self-assembly is controllable, i.e., the degree of polymerization of the protein molecule is controllable; meanwhile, the degradation treatment of proteinase K at a proper temperature is required in the process of promoting membrane fusion, and the formation of membrane fusion promoting and large vesicle monomers is controllable; finally, the special spatial morphology (tubular) of the fused large vesicle can be realized by adding PMSF, and the switching of the spatial morphology (spherical → tubular) is controllable.

2. By means of strong physiological and biochemical properties of RA1, the liposome based on the gliadin A1 has rich maneuverability and has potential of being applied to the fields of biological pharmacy, drug target positioning, tumor treatment technology and the like.

Gene sequence of RA 1:

ATGAATCGATATCTGAATCGACAGCGCCTGTACAACATGTACAGAAACAAGTACCGAGGTGTGATGGAACCGATGTCCAGAATGACCATGGACTTCCAAGGAAGATACATGGACTCCCAGGGCAGAATGGTCGACCCCCGATACTACGACCACTACGGAAGAATGCACGACTATGACCGATACTACGGAAGGTCCATGTTCAACCAGGGACACAGCATGGACAGTCAACGCTACGGCGGCTGGATGGACAACCCCGAGAGGTACATGGACATGTCTGGCTACCAGATGGACATGCAGGGACGCTGGATGGACGCCCAGGGGCGCTACAACAACCCATTTAGTCAAATGTGGCACAGCAGGCAAGGCCACTACCCTGGTTACATGTCACATCACTCCATGTATGGTAGAAATATGCACTACCCCTACCACAGCCATTCCGCCAGCCGGCATTTCGATTCCCCTGAAAGATGGATGGACATGTCCGGGTATCAGATGGACATGCAGGGACGCTGGATGGATAACTACGGCCGCTACGTGAACCCGTTCCACCACCACATGTATGGCAGAAACATGTTTTATCCTTACGGCAGCCATTGCAACAATCGGCACATGGAGCACCCCGAGAGGTACATGGACATGTCCGGCTATCAGATGGACATGCAGGGACGCTGGATGGACACACATGGACGTCACTGCAACCCGCTCGGTCAGATGTGGCACAACAGGCACGGTTACTATCCAGGACACCCACATGGTCGCAACATGTTCCAGCCCGAAAGATGGATGGATATGTCCAGCTATCAGATGGACATGCAAGGGCGTTGGATGGATAACTACGGCCGTTATGTGAACCCGTTCAGTCATAACTACGGCAGGCATATGAATTACCCTGGAGGTCACTACAACTACCACCACGGTCGCTACATGAATCACCCCGAGAGACAGATGGACATGTCCGGCTATCAGATGGACATGCACGGACGCTGGATGGACAACCAGGGCCGTTATATTGACAATTTCGATAGAAATTATTACGATTATCACATGTATTAA

RA1 tag protein amino acid sequence:

MNRYLNRQRLYNMYRNKYRGVMEPMSRMTMDFQGRYMDSQGRMVDPRYYDHYGRMHDYDRYYGRSMFNQGHSMDSQRYGGWMDNPERYMDMSGYQMDMQGRWMDAQGRYNNPFSQMWHSRQGHYPGYMSHHSMYGRNMHYPYHSHSASRHFDSPERWMDMSGYQMDMQGRWMDNYGRYVNPFHHHMYGRNMFYPYGSHCNNRHMEHPERYMDMSGYQMDMQGRWMDTHGRHCNPLGQMWHNRHGYYPGHPHGRNMFQPERWMDMSSYQMDMQGRWMDNYGRYVNPFSHNYGRHMNYPGGHYNYHHGRYMNHPERQMDMSGYQMDMHGRWMDNQGRYIDNFDRNYYDYHMY

drawings

Fig. 1 is a graph showing experimental conditions including complexation and aggregation of RA1 with liposome vesicles at various pH values.

FIG. 2 is a graph showing the membrane fusion promoting process of glistening protein under three different fields of view (white scale: 10 μ M, protein concentration: 13.5 μ M, pH7.5) A-C) as a result of membrane fusion under a confocal microscope field of view.

FIG. 3 is a graph showing the results of membrane fusion + tube formation under a confocal microscope (white bar: 10 μ M, protein concentration: 13.5 μ M, pH 7.5).

Detailed Description

The present invention is described in further detail below by way of examples, which should not be construed as limiting the invention thereto.

Example (b):

example I: RA1+100nm liposome vesicle

FIG. 1:

1) the square point curve is experimental data under a pH4.5 system, the size of the naked vesicle is about 100nm when no protein exists, and no agglomeration occurs. After the protein is gradually added, the particle size of the detectable particles in the system rapidly rises, and the peak value reaches about 734.8nm when the protein concentration is 0.56 mu M. With the further increase of the protein concentration, the particle size of the particles in the system is rapidly reduced to below 200 nm.

2) The dot curve is experimental data under the condition of pH6.5, and the protein-free is that the size of the naked vesicle is about 100nm, and no agglomeration occurs. With the addition of protein, the particle size in the system increased and reached a maximum peak (1193nm) at a protein concentration of 4.5. mu.M, followed by a gradual decrease.

3) Under the condition of pH7.5 (triangle point curve), the size of the naked vesicle is about 100nm, the protein concentration at the maximum peak value is 13.5 mu M, the peak height is 1345 +/-95.78 nm, and then the protein concentration is reduced.

Taking the red curve (pH6.5, 5mM propanesulfonic acid) in FIG. 1 as an example: when the protein concentration is 0, the particle size of the naked liposome vesicle is about 100 nm; when the protein concentration in the solution is 0.625 mu M, the DLS detection result shows that the particle size in the solution is 180 nm; when the protein concentration in the solution gradually increases from 1.25 μ M to 4.5 μ M, the size of the particles in the solution transits from 200nm to 1200nm, indicating that a large-scale aggregation process occurs between the reflectrin and the liposome vesicle. When the protein concentration was further increased to 9, 13.5 or 18 μ M, the coacervation process was terminated without massive coacervate formation due to saturation of RA1 self-assemblies covering the liposome vesicle surface.

Similar results were obtained with the 100nm vesicle system and the protein system in two other buffers. In the acetic acid solution with pH4.5 (black line in FIG. 1), the size of the naked vesicle is still around 100nm when the protein concentration is 0. Under the condition, the hydration diameter of the protein self-assembly is 8 +/-0.96 nm, and compared with the condition of pH6.5, the number of protein molecules contained in a single protein self-assembly is greatly reduced. Thus, at equivalent protein concentrations, the volume of protein self-assemblies in solution is reduced and the number is greatly increased. The maximum peak of vesicle aggregation occurred earlier at a protein concentration of 0.625 μ M (. about.750 nm). Subsequently, the coagulation status decays rapidly with increasing protein concentration; when the protein concentration exceeds 2.25. mu.M, the particle size in the solution is kept below 200 nm.

In contrast, in pH7.5 propanesulfonic acid (blue line in FIG. 3.5), the hydrated diameter of the protein self-assemblies increased to 34.4. + -. 8.2nm, and higher concentrations were required to achieve equivalent levels of protein self-assemblies in solution at low pH. The particle agglomeration peak in solution was delayed to the right until significant aggregation (-800 nm) did not occur until the protein concentration reached 4.5. mu.M and peaked (-1400 nm) at a protein concentration of 13.5. mu.M. Subsequently, when the protein concentration was further increased to 18. mu.M, the aggregation of vesicles on a large scale rapidly disappeared, and the particle size of the substance in the reaction solution rapidly decreased to about 200 nm.

Example II: RA1+ Liposomal vesicles (pH7.5) + proteinase K (37 ℃ C.)

In example I, the RA1 concentration reached 13.5. mu.M with propane sulfonic acid at pH7.5, giving the largest size of RA 1-liposome aggregates formed. The RA 1-liposome aggregate formed under these conditions was selected as a sample, and treated with proteinase K at a final concentration of 0.15mg/ml at 37 ℃ for 50 minutes, whereby the results shown in FIG. 2 were obtained. In fig. 2, after proteinase K degrades RA1 and its formed protein self-assembly, RA 1-liposome aggregate is not, but not, dissociated, but triggers a large-scale, vigorous vesicle fusion process: the 100nm vesicles used in the beginning of the experiment are rapidly fused to form a huge and new vesicle monomer with the size of micron.

FIG. 2 shows the membrane fusion promoting process of the glistening protein under three different fields of view (white scale is 10 μ M, protein concentration is 13.5 μ M, pH is 7.5) A-C) based on the membrane fusion results under the field of view of confocal microscope.

Considering the amino acid sequence characteristics of the glistening protein, the hydrophobic fragment is likely to be embedded in the self-assembly body during the formation process, and the part exposed in the solution is weaker in hydrophobicity; meanwhile, the functional domain of the membrane protein generally spans the phospholipid bilayer of the plasma membrane and should have strong hydrophobicity. In the process of treating the reflective protein by using protease K, the protein self-assembly is gradually untied, and the amino acid sequence which is wrapped by hydrophobicity and has the function on a specific membrane is released; the specific amino acid functional sequences perform the function on the membrane within a certain time range, and the process of promoting liposome vesicles with the particle size of about 100nm to fuse into micron-sized giant vesicles is completed.

Example III: RA1+ liposome vesicles (pH7.5) + proteinase K (37 ℃) + PMSF

Notably, proteinase K randomly cleaves up to 22 serine sites in the amino acid sequence of the reflectrin. At the end of the enzymatic hydrolysis, the entire gliadin will be thoroughly minced. In this process, various functional sequences including the membrane-promoting fusion sequence may be sequentially released or destroyed.

FIG. 3 results of membrane fusion promotion and tube formation under confocal microscope (white scale: 10 μ M, protein concentration: 13.5 μ M, pH7.5)

In view of this, the present invention preferably selects a processing method: using benzyl sulfuryl fluoride (PMSF), a common protease inhibitor, the enzymatic hydrolysis was terminated after 30 minutes of reaction (0.15mg/ml proteinase K, 37 ℃ C.), and after standing for ten minutes, the experimental results shown in FIG. 3 were obtained. In the test process, RA1 firstly facilitates the membrane fusion behavior among liposome vesicles of 100nm in the gradual degradation process, and large liposomes are formed; subsequently, since PMSF prevents further degradation of RA1, so that it is in a semi-degraded state, a part of functional amino acid sequence is preserved, thus leading to that the fused liposome vesicle monomer can maintain a special spatial tubular structure. In the present embodiment, RA1 has three functions of liposome large-scale agglomeration, membrane fusion promotion and maintenance of liposome membrane structure space tube-packaging characteristics.

Sequence listing

<110> China national defense science and technology university of people liberation army

<120> light-reflecting protein system for regulating and controlling liposome vesicle behavior and function and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1053

<212> DNA

<213> Membrane protein of rainbow cell of cuttlefish (logo pealei)

<400> 1

atgaatcgat atctgaatcg acagcgcctg tacaacatgt acagaaacaa gtaccgaggt 60

gtgatggaac cgatgtccag aatgaccatg gacttccaag gaagatacat ggactcccag 120

ggcagaatgg tcgacccccg atactacgac cactacggaa gaatgcacga ctatgaccga 180

tactacggaa ggtccatgtt caaccaggga cacagcatgg acagtcaacg ctacggcggc 240

tggatggaca accccgagag gtacatggac atgtctggct accagatgga catgcaggga 300

cgctggatgg acgcccaggg gcgctacaac aacccattta gtcaaatgtg gcacagcagg 360

caaggccact accctggtta catgtcacat cactccatgt atggtagaaa tatgcactac 420

ccctaccaca gccattccgc cagccggcat ttcgattccc ctgaaagatg gatggacatg 480

tccgggtatc agatggacat gcagggacgc tggatggata actacggccg ctacgtgaac 540

ccgttccacc accacatgta tggcagaaac atgttttatc cttacggcag ccattgcaac 600

aatcggcaca tggagcaccc cgagaggtac atggacatgt ccggctatca gatggacatg 660

cagggacgct ggatggacac acatggacgt cactgcaacc cgctcggtca gatgtggcac 720

aacaggcacg gttactatcc aggacaccca catggtcgca acatgttcca gcccgaaaga 780

tggatggata tgtccagcta tcagatggac atgcaagggc gttggatgga taactacggc 840

cgttatgtga acccgttcag tcataactac ggcaggcata tgaattaccc tggaggtcac 900

tacaactacc accacggtcg ctacatgaat caccccgaga gacagatgga catgtccggc 960

tatcagatgg acatgcacgg acgctggatg gacaaccagg gccgttatat tgacaatttc 1020

gatagaaatt attacgatta tcacatgtat taa 1053

<210> 2

<211> 350

<212> PRT

<213> Membrane protein of rainbow cell of cuttlefish (logo pealei)

<400> 2

Met Asn Arg Tyr Leu Asn Arg Gln Arg Leu Tyr Asn Met Tyr Arg Asn

1 5 10 15

Lys Tyr Arg Gly Val Met Glu Pro Met Ser Arg Met Thr Met Asp Phe

20 25 30

Gln Gly Arg Tyr Met Asp Ser Gln Gly Arg Met Val Asp Pro Arg Tyr

35 40 45

Tyr Asp His Tyr Gly Arg Met His Asp Tyr Asp Arg Tyr Tyr Gly Arg

50 55 60

Ser Met Phe Asn Gln Gly His Ser Met Asp Ser Gln Arg Tyr Gly Gly

65 70 75 80

Trp Met Asp Asn Pro Glu Arg Tyr Met Asp Met Ser Gly Tyr Gln Met

85 90 95

Asp Met Gln Gly Arg Trp Met Asp Ala Gln Gly Arg Tyr Asn Asn Pro

100 105 110

Phe Ser Gln Met Trp His Ser Arg Gln Gly His Tyr Pro Gly Tyr Met

115 120 125

Ser His His Ser Met Tyr Gly Arg Asn Met His Tyr Pro Tyr His Ser

130 135 140

His Ser Ala Ser Arg His Phe Asp Ser Pro Glu Arg Trp Met Asp Met

145 150 155 160

Ser Gly Tyr Gln Met Asp Met Gln Gly Arg Trp Met Asp Asn Tyr Gly

165 170 175

Arg Tyr Val Asn Pro Phe His His His Met Tyr Gly Arg Asn Met Phe

180 185 190

Tyr Pro Tyr Gly Ser His Cys Asn Asn Arg His Met Glu His Pro Glu

195 200 205

Arg Tyr Met Asp Met Ser Gly Tyr Gln Met Asp Met Gln Gly Arg Trp

210 215 220

Met Asp Thr His Gly Arg His Cys Asn Pro Leu Gly Gln Met Trp His

225 230 235 240

Asn Arg His Gly Tyr Tyr Pro Gly His Pro His Gly Arg Asn Met Phe

245 250 255

Gln Pro Glu Arg Trp Met Asp Met Ser Ser Tyr Gln Met Asp Met Gln

260 265 270

Gly Arg Trp Met Asp Asn Tyr Gly Arg Tyr Val Asn Pro Phe Ser His

275 280 285

Asn Tyr Gly Arg His Met Asn Tyr Pro Gly Gly His Tyr Asn Tyr His

290 295 300

His Gly Arg Tyr Met Asn His Pro Glu Arg Gln Met Asp Met Ser Gly

305 310 315 320

Tyr Gln Met Asp Met His Gly Arg Trp Met Asp Asn Gln Gly Arg Tyr

325 330 335

Ile Asp Asn Phe Asp Arg Asn Tyr Tyr Asp Tyr His Met Tyr

340 345 350

Claims

1. a light-reflecting protein system for regulating liposome vesicle behavior and function, characterized in that: prepared by the following steps:

1) dissolving dioleoyl lecithin and dioleoyl phosphatidyl glycerol in chloroform according to a ratio of 60:40 of the amount of substance, and drying with clean nitrogen after filtration with a 0.22 μm filter membrane to form a liposome film;

2) vacuum the liposome film obtained in the previous step in a vacuum desiccator overnight;

3) The next day, add 1 ml of working buffer to every 100 mg of liposome film, vortex for 1 minute, and repeat twice;

4) Use liquid nitrogen and an 80°C water bath to freeze and thaw 10 times, each time for 1 minute, for a total time of 20 minutes;

5) Homogenization treatment of liposome vesicles: use a liposome extruder and a 100 nm filter membrane to repeatedly process liposomes. After homogenization, the liposome vesicles are transferred to a glass vial washed with ethanol and acetone for preservation. Obtain naked and stable liposome vesicles with a size of 100 nm;

6) Synthesize the full length of the RA1 gene by chemical synthesis;

RA1 is derived from the membrane protein of squid iridocytic--Reflectin A1, Reflectin A1, referred to as RA1;

7) The RA1 gene was introduced into the recipient cell E. coli Rosetta for protein expression;

8) After inducing expression, carry out inclusion body purification;

9) After the initial purification by ion affinity chromatography and continuously changing the guanidine salt concentration, hydrophobic chromatography was performed using trifluoroacetic acid and acetonitrile system, and the purified protein obtained was freeze-dried and stored at -80°C;

10) The protein corona is composed of RA1, and RA1 covers the liposome vesicles obtained by the above preparation method, in a buffer solution of one of pH4.5 acetic acid, pH6.55mM propanesulfonic acid, and pH7.55mM propanesulfonic acid, RA1 forms protein self-assemblies of 8±0.96nm, 15.6±3.4nm, and 34.4±8.2nm, respectively, and RA1 and vesicles form large-scale RA1-liposome aggregates with scales ranging from hundreds of nanometers to micrometers. A light-reflecting protein system for regulating the behavior and function of liposome vesicles.

2. The light-reflecting protein system for regulating liposome vesicle behavior and function according to claim 1, characterized in that: step 1) forming a liposome film, carrying out fluorescence observation, and dissolving in the liposome Add BODIPY ^™ 558/568 C12 Fluorescent Dye while mixing.

3. The reflectin system for regulating liposome vesicle behavior and function according to claim 1, characterized in that: the working buffer described in step 3) is selected from pH4.5 acetic acid, pH6.5-5mM One of propanesulfonic acid, pH7.5 5mM propanesulfonic acid.

4. the light-reflecting protein system for regulating liposome vesicle behavior, function according to claim 1, is characterized in that: E.coli Rosetta described in step 7) is E.coli Rosetta ^TM 2 DE3; The protein expression described above was performed using LB medium, 50ng/ml kanamycin resistance.

5. The reflectin system for regulating the behavior and function of liposome vesicles according to claim 1, characterized in that: after the induction and expression in step 8), inclusion body purification is performed using 1mM IPTG at 37°C 3-4 hours after induction of expression, use

Inclusion body purification was performed.

6. the reflectin system for regulating liposome vesicle behavior, function according to claim 1, is characterized in that: the described primary purification of step 9) adopts purification column to be Hitrap ^TM 5mL SP XL and Mono

5/50 GL SIGMA conducted.

7. The light-reflecting protein system for regulating the behavior and function of liposome vesicles according to claim 1, characterized in that: the protein self-assembly described in step 10) is a variety of pH values, salt ion concentration conditions One of the RA1 protein molecular aggregates formed under the

8. A method for constructing liposome vesicles based on RA1 biochemical properties, promoting membrane fusion and spatial morphology thereof, comprising the following steps:

a. The processing method of the light-reflecting protein system for regulating the behavior and function of liposome vesicles according to claim 1: in the buffer, RA1 and vesicles of appropriate concentration first form large-scale RA1-liposome aggregation Then, in the pH7.5 5mM propanesulfonic acid system, the protein and vesicle aggregates were treated with proteinase K at a working concentration of 0.15mg/ml for 50 minutes to promote the size of 100nm vesicles. Scale membrane fusion behavior to form giant vesicle monomers with a scale of microns;

or

b. The structure of the spatial tubular morphology of the giant vesicle monomers formed after the fusion of the liposome aggregate membrane: the processing method of the reflectin system for regulating the behavior and function of liposome vesicles according to claim 1 : In the buffer solution, RA1 and vesicles at an appropriate concentration first form large-scale RA1-liposome aggregates with a scale of several hundred nanometers to micrometers; mg/ml proteinase K treated protein and vesicle aggregates for 30 minutes, and then added PMSF, a protease activity inhibitor with a final concentration of 1 mM, to block the degradation process of RA1. The micron-scale giant vesicles formed after membrane fusion are bound by their spatial morphology, giving them special tubular stacking characteristics.

9. a kind of liposome vesicle aggregation based on RA1 biochemical property according to claim 8, promote membrane fusion and its spatial morphology construction method, it is characterized in that: step a described in buffer, buffer It refers to one of pH4.5 acetic acid, pH6.5 5mM propanesulfonic acid, pH7.5 5mM propanesulfonic acid; the suitable concentration refers to 0.625μM in pH4.5 acetic acid, pH6.55mM propanesulfonic acid 4.5 μM in acid, 13.5 μM in 5 mM propanesulfonic acid, pH 7.5.