CN110819651A

CN110819651A - Preparation method and application of heat-resistant ferritin

Info

Publication number: CN110819651A
Application number: CN201911184182.1A
Authority: CN
Inventors: 余佳成; 曹长乾; 张同伟; 潘永信
Original assignee: Institute of Geology and Geophysics of CAS
Current assignee: Institute of Geology and Geophysics of CAS
Priority date: 2019-11-27
Filing date: 2019-11-27
Publication date: 2020-02-21

Abstract

The invention provides a preparation method of a high heat-resistant ferritin cage-like structure and a method for preparing heat-resistant magnetic ferritin by using the same. Specifically, the protein is expressed and purified by gene engineering and protein engineering technology, and can be self-assembled to form a high-heat-resistant ferritin cage-shaped structure with the inner diameter of about 8nm and the outer diameter of about 12 nm. The ferritin cage-like structure and the magnetic ferritin prepared by the method can endure the high temperature condition of more than 110 ℃ for more than 30min, and have good pressure resistance.

Description

Preparation method and application of heat-resistant ferritin

Technical Field

The invention relates to the crossing technical field of genetic engineering, protein engineering and biomimetic synthesis of magnetic nano materials, in particular to a method for synthesizing heat-resistant ferritin in vitro by means of genetic engineering and protein engineering, synthesizing a heat-resistant magnetic ferritin material by biomimetic mineralization and application thereof.

Background

Ferritin is widely distributed inFerritin in stores in animals, plants and microorganisms play an important role in the regulation of iron metabolism and ferrous ions in the organism. Ferritin self-assembles in water phase and polar solution to form a 24-polymer nano cage-shaped structure (inner diameter 8nm and outer diameter 12nm), and ferritin is used as a nano reactor to synthesize ferritin-coated superparamagnetic nano magnetite (Fe)₃O₄) Or maghemite (gamma-Fe)₂O₃) The magnetic nano-particles have good dispersibility, particle size uniformity and higher saturation magnetization, and are widely applied to the biomedical fields of magnetic resonance contrast enhancers, magnetic targeting drugs and the like.

Currently, most of ferritin studied belongs to psychrophilic ferritin, such as equine ferritin, escherichia coli ferritin, human H subunit ferritin, etc., for example, patent document CN102115746A discloses a preparation method of monodispersion magnetic human ferritin, which comprises one-step biomimetic synthesis of monodispersion magnetic human ferritin, wherein the ferritin prepared by the method has the characteristics of uniform size, similar shape and good dispersibility, but the tolerance temperature is not more than 80 ℃. The materials synthesized based on the material need a relatively stable low-temperature environment during long-time transportation and storage. However, the maintenance of the cold chain is expensive, adding to the economic cost of material development. And the relatively low stability also limits its application in the fields of magnetic resonance imaging and high temperature catalysis in underground reservoirs (whose temperature is 80-120 ℃). Therefore, the improvement of the heat stability of the ferritin or the development of a new heat-resistant ferritin has higher economic and application values.

At present, few methods are available for improving the heat stability of ferritin, and the heat stability of ferritin is mainly improved by chemically modifying a stabilizing group. The chemical modification mainly refers to covalent or non-covalent chemical modification of ferritin by using a modifying agent, so as to change the dissolution behavior of ferritin, thereby enhancing the chemical and physical stability of ferritin. Such as PEG modification, chitosan glycosylation modification and the like, which are not obvious for improving the heat stability of ferritin on one hand, and the chemical modification has the well-known problems of complex modification process, relatively low success rate and expensive chemical modifier cost. The development of new thermostable ferritin is therefore the most efficient approach. Currently, ferritin AfFn and PfFn, which are expressed from the genes of Archaeoglobus fulgidus and Pyrococcus furiosus, have extremely high thermal stability, and the latter can tolerate a high temperature of 120 ℃ at a relatively low concentration (Jana et al, (2005) extreme, 10: 139-. However, since the number of thermotolerant ferritin is still small, it is critical to continue to develop a wider variety of thermotolerant ferritin.

In order to develop a novel ferritin with high thermal stability, the invention provides a method for constructing a prokaryotic expression vector for expressing and purifying a Pyrococcusayanoisi CH1 ferritin gene PcFn by a genetic engineering and protein engineering technology, so that the ferritin PcFn which can endure the high temperature of more than 110 ℃ is obtained in vitro, and the ferritin can be stored for a long time under the normal temperature condition without refrigeration. In addition, the invention also provides a magnetic nano material coated by the PcFn protein shell, which is synthesized by utilizing PcFn biomimetic mineralization and also has ultrahigh thermal stability capable of enduring more than 110 ℃.

Disclosure of Invention

The invention aims to provide a novel high heat-resistant and high pressure-resistant ferritin and an in-vitro preparation method of a ferritin cage structure thereof, wherein the PcFn ferritin obtained by genetic engineering and protein engineering can be self-assembled in a water phase and a polar solvent to form a particle size of about 12 nm. And a preparation method for biomimetically synthesizing high heat-resistant and high pressure-resistant magnetic nanoparticles by using the ferritin cage-like structure as a template.

To achieve the above objects, the present invention provides a method for preparing a ferritin cage structure, which comprises transforming a vector comprising a gene sequence encoding Pyrococcusayanosii CH1 ferritin (PcFn) into a bacterium, and inducing expression; after the expression is finished, extracting and purifying to obtain the ferritin cage-like structure.

Preferably, the coding gene sequence of the ferritin Pyrococcusyayanosii CH1 can be chemically synthesized or directly cloned from the genome of the thermophilic bacterium Pyrococcusyayanosii CH 1.

Preferably, the coding gene sequence codes an amino acid sequence shown as SEQ ID NO. 2. Further preferably, the coding gene sequence is identical to the sequence shown in SEQ ID NO:1 or SEQ ID NO:3 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In one embodiment of the present invention, the coding gene sequence is as shown in SEQ ID NO: 1. SEQ ID NO:3 or their degenerate gene sequences. Wherein, SEQ ID NO:1 or SEQ ID NO:3 also encodes the degenerate gene sequence of SEQ ID NO: 2.

Wherein, in order to improve the expression quantity of PcFn in bacteria, the expression vector adopts SEQ ID NO:3 as a coding gene sequence, SEQ ID NO:3 is obtained by performing prokaryotic codon preference optimization on the gene sequence SEQ ID NO. 1.

Preferably, the vector is a prokaryotic expression vector, and the prokaryotic expression vector is any vector in the prior art which can carry an inserted exogenous nucleic acid sequence into a prokaryotic cell for expression. Further preferably, the prokaryotic expression vector is selected from pET-30a, pET-31b, pET-34b, pET-35b, pET22b or pET-43.1.

In one embodiment of the invention, the prokaryotic expression vector is pET22 b.

Preferably, the bacterium is Escherichia coli, and preferably, the bacterium is Escherichia coli BL21(DE3) or Rosetta series strain.

In a particular embodiment of the invention, the bacterium is Escherichia coli BL21(DE 3).

Preferably, said induced expression comprises the step of adding IPTG (isopropyl- β -D-thiogalactoside).

In one embodiment of the invention, the IPTG is added under conditions such that the bacteria grow to an OD of 0.55-0.65, under induction conditions of 25-35 deg.C, most preferably 30 deg.C, for an induction time of 8-10 hours.

Preferably, said extraction comprises disrupting the cells to release the protein, and the applicant of the present invention has unexpectedly found that better separation can be achieved by adding water at 80-100 ℃ to the mixture, agitating the mixture, centrifuging the mixture to collect the supernatant. Further preferably, the heating and stirring time is 20 to 30 min.

Further preferably, the method for disrupting cells may employ one or a combination of two or more of conventional biochemical, physical or mechanical methods. Wherein the biochemical method is selected from the group consisting of a chemical reagent method (e.g., toluene, acetone, chloroform or Triton), an autolysis method, and an enzymatic hydrolysis method. The physical method is selected from a temperature difference method, a pressure difference method and an ultrasonic method. The mechanical method is selected from a mashing method, a grinding method or a homogenizing method.

In one embodiment of the invention, disrupting the cell to release the protein comprises adding lysozyme and sonicating. Wherein, in order to completely break the bacterial cells and completely flow out cell sap, lysozyme is added for treatment for 2-3h, and then ultrasonic treatment is carried out for 5-10 min.

Further preferably, the centrifugation temperature is 4-5 ℃, the rotation speed is 15000-.

Preferably, the purification comprises the steps of filtering and desalting the obtained supernatant.

In one embodiment of the present invention, the purification further comprises a step of desalting and then performing electrophoresis to determine purity.

In one embodiment of the present invention, the preparation method further comprises the steps of filtering, sterilizing and storing the purified protein.

In one embodiment of the present invention, the preparation method comprises the following steps:

1) encoding the polypeptide of SEQ ID NO:2 is cloned into plasmid to prepare prokaryotic expression vector; preferably, the nucleic acid encoding SEQ ID NO:2 is the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3, the prokaryotic expression vector is selected from pET-30a, pET-31b, pET-34b, pET-35b, pET22b or pET-43.1;

2) transforming the prokaryotic expression vector obtained in the step 1) into escherichia coli, adding IPTG (isopropyl-beta-thiogalactoside) to activate a promoter, and performing inducible expression; preferably, the Escherichia coli is Escherichia coli BL21(DE3) or Rosetta series strain; preferably, the promoter is T7 promoter;

3) after the expression is finished, breaking the cell to release protein, adding water with the temperature of 80-100 ℃, stirring, centrifuging and collecting supernate; preferably, the water is added and stirred for 20-30min, the centrifugation temperature is 4-5 ℃, the rotation speed is 15000-20000g, and the time is 25-35 min;

4) filtering and desalting the supernatant obtained in the step 3);

obtaining the ferritin cage-like structure.

The invention also provides a ferritin cage-like structure obtained by the preparation method.

The invention further provides a preparation method of the magnetic ferritin, which comprises the step of adding metal salts and an oxidant into the prepared ferritin cage structure for reaction.

Preferably, the oxidant is H₂O₂The pH value of the reaction is 7-11, the reaction temperature is 20-85 ℃, wherein the molar ratio of the metal ions to the oxidant is 1:0.33-0.5, and the molar ratio of the ferritin cage-like structure to the metal ions is 1: 100-40000.

Further preferably, the reaction temperature is 65-85 ℃.

In one embodiment of the invention, the method of preparation comprises mixing a ferrous salt or a combination of a ferrous salt and a metal oxide, and H₂O₂Adding into purified ferritin cage-like structure solution for reaction, controlling pH to 7-11 with NaOH concentration of 500mM or less, controlling temperature to 20-85 deg.C, wherein metal oxide core is formed inside ferritin cage-like structure, the liquid adding speed of metal ions is 10-200 metal ions per ferritin cage-like structure per minute, the number of metal ions and H₂O₂The ratio of the number of molecules is 2:1 or 3:1, the concentration of the ferritin cage-like structure is 0.25-2mg/mL, wherein the chemical formula of the metal oxide is Fe_(3-x)M_xO₄(x is more than or equal to 0 and less than or equal to 3) or Fe_(2-x)M_xO₃(x is more than or equal to 0 and less than or equal to 2) and whichWherein M is selected from Mn, Cu, Zn, Ni, Gd or Co.

Preferably, after the reaction is finished, sodium citrate is added to chelate ferric ions and ferrous ions which do not enter the ferritin shell, and finally, the synthesized magnetic nanoparticles (magnetic ferritin) are desalted and subjected to exclusion chromatography for purification.

The invention further provides the magnetic ferritin obtained by the preparation method.

The invention also provides the application of the ferritin cage-like structure or the magnetic ferritin in drug loading, drug delivery, antibody screening, magnetic resonance imaging, disease diagnosis reagents or cell labeling.

Preferably, the drug-loading step can be carried out by connecting the drug to a ferritin cage structure or a shell of magnetic ferritin, or directly filling the drug into the ferritin cage structure for drug loading.

Preferably, the antibody screening may be to link a protein that specifically binds or recognizes the antibody to a ferritin cage structure or the shell of a magnetoferritin.

Preferably, the disease diagnostic reagent may be a structure in which a ferritin cage structure or a shell of magnetoferritin is modified or engineered to specifically recognize a disease tissue or cell.

Preferably, the cell marker can be realized by fusing a marker molecule to a ferritin cage structure or a ferritin magnetic shell, and utilizing the natural cell targeting property of the ferritin shell to be combined with a ferritin receptor highly expressed on the surface of a highly proliferative cell.

The "ferritin cage structure" described in the present invention represents an enucleated ferritin, i.e., an empty ferritin shell without a core structure. The inner diameter is about 8nm and the outer diameter is about 12 nm.

The term "about" as used herein means 20% of the original number and the above or below error, for example, about 8nm means 8. + -. 1.6 nm.

The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that specify the presence of the stated elements or steps, and not substantially affect the presence of other stated elements or steps.

The ferritin cage-like structure and the magnetic ferritin obtained by the preparation method have pressure resistance and higher thermal stability, and can endure temperature higher than 110 ℃ for at least 30min at high concentration (1 mg/mL). Meanwhile, the protein solution can be stored at room temperature for a long time without refrigeration storage, and the protein solution still keeps stable and clear. Furthermore, the prepared ferritin has a cage-like structure and good magnetic ferritin dispersibility, and the magnetic ferritin has no magnetic interaction, so that the problem of aggregation of nano magnetic particles is avoided, and the magnetic ferritin can exist stably for a long time and has a larger surface area.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: a is a SDS polyacrylamide gel electrophoresis pattern of the PcFn protein expressed before and after induction by Escherichia coli BL21(DE3) and purified by heating at 100 ℃ in example 1 of the present invention. Wherein M is marker (kD), and the molecular weights of the bands from top to bottom are respectively 116.0, 66.2, 45.0, 35.0, 25.0, 18.4 and 14.4; band 1 is the bacterial protein band before addition of inducer, Escherichia coli BL21(DE 3); lane 2 is Escherichia coli BL21(DE3) mycoprotein after 8 hours of induction with inducer; band 3 is the post-purification PcFn protein band. B is a negative staining photograph of the PcFn protein purified in example 1 of the present invention by Transmission Electron Microscopy (TEM).

FIG. 2: is a PcFn protein heat stability analysis. A: the PcFn protein solution is stored for 106 days at normal temperature and is compared before and after the protein solution is stored; b: the value of PcFn concentration varies with increasing temperature; c: heating PcFn with the same concentration at different temperatures for 30min to obtain a CD spectrogram; d: differential Scanning Calorimetry (DSC) result of PcFn.

FIG. 3: is the analysis of the iron feeding rate of the PcFn protein before and after being heated for 30min at 110 ℃.

FIG. 4: the magnetic ferritin M-PcFn is synthesized by using PcFn protein as a template₅₀₀₀And (4) microscopic characterization and analysis. A, negative staining transmission electron microscope 100KV picture; b, M-PcFn₅₀₀₀Counting the particle size of the kernel; c, a transmission electron microscope 200KV picture; and D, high resolution image of transmission electron microscope.

FIG. 5: is M-PcFn₅₀₀₀And (5) analyzing thermal stability. A, same concentration of M-PcFn₅₀₀₀CD spectrogram after heating for 30min at different temperatures; b, M-PcFn₅₀₀₀Differential Scanning Calorimetry (DSC) of (1).

FIG. 6: purified PcFn protein and M-PcFn₅₀₀₀Transmission Electron Microscopy (TEM) negative staining transmission electron microscopy 100KV image.

FIG. 7: and comparing SDS polyacrylamide gel electrophoresis images of the PcFn ferritin under different purification conditions, wherein the middle strip is Marker, the left strip is PcFn ferritin obtained after heating and stirring at 100 ℃ for 30min, and the right strip is PcFn ferritin obtained after heating and stirring at 75 ℃ for 30 min.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The reagents and other materials used in this example were all commercially available products unless otherwise specified, and the methods used were conventional methods known to those skilled in the art unless otherwise specified. The experimental procedures not described in the examples of the present invention, such as digestion and ligation of a target gene, transformation of a plasmid, preparation of an LB medium and a PBS buffer, were carried out by referring to the procedures described in molecular cloning, A.C. (J. SammBruke, D.W. Lassel, Huang Peyer, scientific Press, 2002). The preferred embodiments of the present invention will be described more fully hereinafter with reference to the accompanying drawings, which are provided in the examples of the invention.

Example 1

1. Construction of expression vector for PcFn-encoding Gene

The gene sequence of Pyrococcusayanosii CH1 encoding ferritin PcFn was found in GenBank under accession number 10837266, and NdeI and EcoRI cleavage sites were added to the head and tail ends as shown in SEQ ID NO: 1. The SEQ ID NO. 1 sequence is subjected to codon preference modification as shown in SEQ ID NO. 3, so that the gene can be expressed in an Escherichia coli expression system more efficiently. The cDNA chain of the sequence shown in SEQ ID NO. 3 was synthesized by Beijing Liu-Hei-Hua DageneCo, and the synthesized product was pUC57-PcFn plasmid and stored in puncturing bacteria. The puncturing bacterium is amplified, purified and cultured, pUC57-PcFn plasmid is extracted by a kit, the pUC57-PcFn plasmid and pET22b plasmid are subjected to double enzyme digestion by restriction enzymes NdeI and EcoRI at the same time, and after agarose electrophoresis detection, the plasmid is recovered and purified by a DNA gel recovery kit. Then, the target genes PcFn and pET22b were ligated by using T4DNAligase (NEB Biolabs), and the ligated plasmid was named pET22 b-PcFn.

2. pET22b-PcFn plasmid was transformed into Escherichia coli BL21(DE3) engineering bacteria

pET22b-PcFn vector was transferred into competent cells of Escherichia coli BL21(DE3) strain. And (3) screening positive clones on a solid LB culture medium containing ampicillin to obtain successfully transformed strains, and performing strain preservation and next protein expression.

Wherein, SEQ ID NO 1

1 ATGCTGAGCGAAAGGATGCTTAAGGCACTCAATGAGCAGATAAACAAGGAGCTATTTTCC

61 GCATATTTCTACCTTGGGATAGCTGCCTACTTCAAGGACAAGGGGCTTGAGGGCTTCGCC

121 AAATGGATGGAGGCCCAGGCTGAGGAGGAGCTTGGCCACGCGATGAGGATATACGACTAT

181 GTCTTCAACCGCGGAGGCAAGGTCGAGCTTTATGAGATTGAAAAGCCAAAGCAGGACTTC

241 GAGAGCCCGCTCAAGGCCTTCGAGGCCGTTTATCTCCATGAGGTAGGGGTCACACAGTCC

301 ATATTCAAGCTCGTCGAGCTGGCTCAGGAGGAAAATGACCACGCAACATACAACTTCCTC

361 CAGTGGTTCGTCGAAGAGCAGGTAGAGGAGGAGGCCTCGACAAAGGCGATACTCGACAAG

421 CTCAAGATAATCGGCGACAATCCCCAAGCCTTATTCATGCTCGACAGAGAGCTTGGCCAG

481 AGACAGGCAAAGCTCGGGACCTTGATCCAAGATAAGGAGTGA

SEQ ID NO:2

1 MLSERMLKAL NEQINKELFS AYFYLGIAAY FKDKGLEGFA

41 KWMEAQAEEE LGHAMRIYDY VFNRGGKVEL YEIEKPKQDF

81 ESPLKAFEAV YLHEVGVTQS IFKLVELAQE ENDHATYNFL

121 QWFVEEQVEE EASTKAILDK LKIIGDNPQA LFMLDRELGQ

161 RQAKLGTLIQ DKE

SEQ ID NO:3

1 CATATGCTGTCTGAACGTATGCTGAAAGCTCTGAACGAACAGATCAACAAAGAACTGTTC

61 TCTGCTTACTTCTACCTGGGTATCGCTGCTTACTTCAAAGACAAAGGTCTGGAAGGTTTC

121 GCTAAATGGATGGAAGCTCAGGCTGAAGAAGAACTGGGTCACGCTATGCGTATCTACGAC

181 TACGTTTTCAACCGTGGTGGTAAAGTTGAACTGTACGAAATCGAAAAACCGAAACAGGAC

241 TTCGAATCTCCGCTGAAAGCTTTCGAAGCTGTTTACCTGCACGAAGTTGGTGTTACCCAG

301 TCTATCTTCAAACTGGTTGAACTGGCTCAGGAAGAAAACGACCACGCTACCTACAACTTC

361 CTGCAGTGGTTCGTTGAAGAACAGGTTGAAGAAGAAGCTTCTACCAAAGCTATCCTGGAC

421 AAACTGAAAATCATCGGTGACAACCCGCAGGCTCTGTTCATGCTGGACCGTGAACTGGGT

481 CAGCGTCAGGCTAAACTGGGTACCCTGATCCAGGACAAAGAATA

3. PcFn protein expression and purification

And (3) inoculating the positive clone screened in the step 2 into a liquid LB culture medium, culturing at 37 ℃ until the OD value is 0.6, adding an IPTG (isopropyl-beta-thiogalactoside) inducer to activate a T7 promoter, and culturing at 30 ℃ for 8 h. The thalli is collected by centrifugation, resuspended by PBS buffer solution, and treated for 2.5h by adding lysozyme at 37 ℃. The resuspended solution was then placed on ice for sonication for 8min, the cell disruption solution was centrifuged at 20,000g for 30min at 4 ℃ and the protein supernatant was collected. The supernatant was heated in boiling water at 100 ℃ for 30min and centrifuged at 20,000g at 4 ℃ for 30min to obtain purified PcFn protein. FIG. 1A shows SDS-acrylamide gel electrophoresis of PcFn protein before, after and after induction, showing that the PcFn protein is successfully highly expressed in E.coli and that the purified ferritin has high purity. FIG. 1B is a transmission electron microscope negative staining pattern of PcFn ferritin, from which it is clear that PcFn is capable of self-assembly and forms nanocage structures with good dispersibility in aqueous phase.

4. Protein thermal stability analysis

FIG. 2A shows that the PcFn protein solution after purification (1 XPBS buffer, pH8.5) is stored directly at room temperature, and after 106 days, the solution state is still clear and transparent, which indicates that refrigeration is not needed for the storage and transportation of the PcFn protein solution.

The purified PcFn was desalted with a 0.1M sodium chloride solution, and the protein concentration was measured with a BSA bovine serum albumin kit and then diluted to 0.5 mg/mL. Respectively taking 1mL of sample, and heat treating at 25 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, 100 deg.C, 110 deg.C, and 120 deg.C for 30 min. The concentration after heating was determined by BSA kit after centrifugation at 20000g for 10 min. FIG. 2B shows the result of concentration measurement of PcFn after heating for 30min at different temperatures, and it can be seen from the graph that the PcFn concentration can be maintained above 0.4mg/mL between 25 deg.C and 110 deg.C, and the protein concentration is significantly reduced when the temperature reaches 120 deg.C. The heated PcFn was diluted to 0.1mg/mL with 0.1M NaCl solution and analyzed by CD spectrometer (Chirascan)^TMPlus CDspectrometer) scanning wavelength range of 200-260nm to detect the change of protein secondary structure with treatment temperature, FIG. 2C is CD spectrum result, which shows that the PcFn protein secondary structure is a typical α helix, the secondary structure does not change significantly at the treatment temperature of 25-110 ℃, the temperature reaches 120 ℃, the α helix begins to change, thus showing that the PcFn protein secondary structure can endure at least 110 ℃ for 30min, another 1mg/mL sample is measured by a differential scanning calorimeter (Nano DSC Microcalorimeter), FIG. 2D is scanning result, which shows that the PcFn temperature T is thermal denaturation temperature T_m＝112℃。

5. Functional verification of ferritin thermal stability

The functional verification of ferritin adopts the measurement comparison of iron feeding rate before and after heat treatment. 10mM ferrous ammonium sulfate is prepared as Fe²⁺Donor, HEPES (hydroxyethylpiperazine ethanethiosulfonic acid, molecular formula: C)₈H₁₈N₂O₄S) buffer (0.1M, pH7.0) as a reaction solution. The experiment is carried out in three groups, and the reaction system is 20uL Fe²⁺+ sample (final concentration 0.025mg/mL) + HEPES buffer, total volume 2 mL. The first group was blank control without ferritin; the second group added ferritin before heat treatment to a final concentration of 0.025 mg/mL; and the third group is added with ferritin with final concentration of 0.025mg/mL and heat-treated at 110 ℃ for 30 min. Mixing the protein immediately after adding, and monitoring wavelength at 315nm with spectrophotometer for 20 min. FIG. 3 shows the results of iron feeding rate, which shows that ferritin after heating at 110 deg.C for 30min still has good iron feeding rate, and the maximum iron feeding rate of 87.4% is maintained by calculation analysis of derivation. Thus PcFn ferritin has ultra-high thermal stability that can tolerate at least 110 ℃.

6. Analysis of protein pressure resistance

The pressure resistance analysis of the prepared PcFn ferritin shows that the PcFn ferritin prepared in the embodiment can bear the pressure of at least 50.5MPa, and has a significant difference with the pressure resistance of the ferritin prepared in the prior art.

Example 2

1. Magnetoferritin M-PcFn₅₀₀₀Biomimetic synthesis of

The PcFn ferritin cage structure purified in example 1 was desalted with 0.1M sodium chloride solution, and the concentration was measured and diluted to 0.5mg/mL, and 50mL of the solution was taken out and placed in a reaction vessel for further use. And (3) evacuating deionized water by using a vacuum pump to remove oxygen for 30min, and then filling the deionized water by using high-purity argon. Preparing 0.1M NaOH, 50mM ammonium ferrous sulfate and 16.67mM hydrogen peroxide solution in an anaerobic box by using deoxygenated water, connecting a reaction device, starting heating and stirring, setting the reaction temperature to 65 ℃, adjusting the pH of the protein solution to 8.5 by using sodium hydroxide, starting a reaction program when the temperature reaches 65 ℃, controlling the reaction rate to be that each ferritin shell enters 80 ferrous ions per minute, ensuring the molar ratio of the ferrous ions added into a reaction system to the hydrogen peroxide to be 3:1, namely ensuring the adding rate of the ammonium ferrous sulfate to the hydrogen peroxide to be 1:1, and theoretically synthesizing Fe nanoparticles in a ferritin cage-like structure under the condition of the stoichiometric ratio₃O₄. Stopping the reaction liquid pumping procedure when the volume of the added ammonium ferrous sulfate reaches the stoichiometric amount of 5000 iron atoms entering each ferritin,stopping heating, maintaining stirring for 10min, adding 300 μ L of prepared 1M sodium citrate solution to chelate ferrous ions and ferric ions which do not enter the ferritin cage structure, and finishing the reaction. The reaction solution is well proportioned and put into a centrifuge for 20000g to be centrifuged for 30min, the precipitate is removed, and the supernatant is the synthesized magnetic ferritin M-PcFn₅₀₀₀. FIG. 4A is M-PcFn₅₀₀₀The negative staining pattern of transmission electron microscope (M-PcFn) was found₅₀₀₀Has an intact ferritin cage structure, has a particle size of about 12nm, and is highly dispersed (wherein, purified PcFn protein and M-PcFn₅₀₀₀A comparison of Transmission Electron Microscopy (TEM) negative staining transmission electron microscopy 100KV is shown in FIG. 6). FIG. 4B shows magnetoferritin M-PcFn₅₀₀₀Particle size distribution of the core, particle size was 4.7 ± 1.3nm in all statistical 790 data. FIG. 4C is M-PcFn₅₀₀₀The electron microscope image of the core, FIG. 4D is a high resolution image of the core, the atomic arrangement of the core can be clearly seen, and the (222) and (220) planes are measured to show that the core crystal is Fe₃O₄Or gamma-Fe₂O₃。

2. Magnetoferritin M-PcFn₅₀₀₀Analysis of thermal stability

The synthesized magnetoferritin M-PcFn₅₀₀₀Protein concentration was determined using the BSA kit. Using 0.1M NaCl solution to mix the magnetic ferritin M-PcFn₅₀₀₀Diluting to 0.1mg/mL, heat treating 1mL diluted magnetic ferritin solution at 25 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, 100 deg.C, 110 deg.C, 120 deg.C for 30min, centrifuging for 10min at 20000g, and separating with CD spectrometer (Chirascan)^TMPlus CD spectrometer) scanning wavelength range 200-260nm to detect the change of the magnetoferritin secondary structure with the treatment temperature. The measured concentration of M-PcFn₅₀₀₀Diluted to 1mg/mL with 0.1M NaCl, and measured by Differential Scanning Calorimetry (DSC), FIG. 5A shows the same concentration of M-PcFn₅₀₀₀The CD spectrum measured after heating for 30min at different temperatures shows that the CD spectrum does not change obviously in the heating temperature range of 25-110 ℃, and does not change obviously until the heating temperature reaches 120 ℃. FIG. 5B is M-PcFn₅₀₀₀The DSC result of (2) shows that M-PcFn₅₀₀₀The determined denaturation temperature T_m＝99.7℃。

3. Magnetoferritin M-PcFn₅₀₀₀Analysis of pressure resistance

For the prepared magnetic ferritin M-PcFn₅₀₀₀Analysis of pressure resistance was conducted, and the results showed that the magnetic ferritin M-PcFn prepared in this example₅₀₀₀Can bear the pressure of at least 51MPa, and has obvious pressure resistance difference with the ferritin prepared in the prior art.

Example 3

The PcFn-encoding gene-containing engineering bacteria were obtained by the procedure of constructing expression vectors for the PcFn-encoding genes, transforming pET22b-PcFn plasmids into Escherichia coli BL21(DE3) engineering bacteria in example 1, and then inoculating the selected positive clones into liquid LB medium and culturing at 37 ℃ until OD was 0.6, adding IPTG inducer to activate T7 promoter, and culturing at 30 ℃ for 8 hours. The thalli is collected by centrifugation, resuspended by PBS buffer solution, and treated for 2.5h by adding lysozyme at 37 ℃. The resuspended solution was then placed on ice for sonication for 8min, the cell disruption solution was centrifuged at 20,000g for 30min at 4 ℃ and the protein supernatant was collected.

The supernatant was heated in 80 ℃ water for 30min and centrifuged at 20,000g at 4 ℃ for 30min to obtain purified PcFn protein. The results showed that the PcFn protein was successfully highly expressed in E.coli and that the purified ferritin protein was substantially free of contaminating proteins.

The heat resistance and pressure resistance of the prepared PcFn ferritin were analyzed according to the procedures described in example 1, and the results show that the PcFn ferritin prepared in this example can be stored at room temperature for 107 days, and the solution state is still clear and transparent, which indicates that the PcFn ferritin solution does not need to be refrigerated during storage and transportation. Meanwhile, the concentration of PcFn can be kept above 0.4mg/mL between 25 ℃ and 110 ℃, the secondary structure is not obviously changed, the protein concentration is obviously reduced when the temperature reaches 115 ℃, the secondary structure is changed, and the differential scanning calorimeter shows that the thermal denaturation temperature T of the PcFn_m111 deg.c. That is, the secondary structure of the PcFn protein prepared in this example can withstand a high temperature of at least 110 ℃ for up to 30 min. Comparing the iron feeding rate before and after heat treatment, the ferritin after being heated at 110 ℃ for 30min still has good iron feeding rate, and is measured by a derivative meterThe maximum iron feed rate of 86.9% was maintained by calculation analysis. Further, the pressure resistance of the iron protein material is at least 50MPa, and the difference of the pressure resistance of the iron protein material and the pressure resistance of the iron protein material prepared in the prior art is significant.

Example 4

The magnetoferritin M-PcFn in example 2 was used₅₀₀₀The step of biomimetic synthesis of (2) preparing the magnetoferritin M-PcFn₅₀₀₀The reaction temperature was adjusted to 85 ℃ only. The results show that the prepared M-PcFn₅₀₀₀Has an intact ferritin cage structure, has a particle size of about 12nm, and is highly dispersed. Meanwhile, the temperature is increased to synthesize the magnetic ferritin, so that the heat resistance, pressure resistance and stability of the magnetic ferritin have more excellent effects.

The prepared magnetoferritin M-PcFn was coupled according to the procedure described in example 2₅₀₀₀The results of the analyses of the heat resistance and the pressure resistance showed that the same concentration of M-PcFn was obtained₅₀₀₀After being heated for 30min at different temperatures, the secondary structure is not obviously changed between 25 ℃ and 110 ℃, the protein concentration is not obviously reduced until the temperature reaches 120 ℃, the secondary structure begins to change, and a differential scanning calorimeter shows that M-PcFn₅₀₀₀Temperature T of thermal denaturation_mAt 100 ℃. Further, the pressure resistance of the iron protein material is at least 50MPa, and the difference of the pressure resistance of the iron protein material and the pressure resistance of the iron protein material prepared in the prior art is significant.

Example 5

The supernatant was heated in 75 ℃ water for 30min and centrifuged at 20,000g at 4 ℃ for 30min to obtain purified PcFn protein. As shown in FIG. 7, the PcFn protein was successfully expressed in E.coli, but the purified ferritin contained a little impure protein, whereas in example 1, the supernatant was heated in 100 ℃ water bath for 30min, and almost no impure band was observed. The purification method of the invention has the advantages of high purity of the purified ferritin, simple and easily obtained purification reagent, low price, no impurity, no pollution and suitability for industrial large-scale production, and the purification temperature is optimal at 80-100 ℃.

Meanwhile, the stability, heat resistance and pressure resistance of the purified PcFn ferritin were analyzed according to the procedures described in example 1, and the results show that the PcFn ferritin prepared in this example starts to be turbid when the solution state is observed after being stored at room temperature for less than 100 days, although the storage time is still long, the storage time is much shorter than that of the PcFn ferritin prepared in examples 1 and 3 due to the influence of impure protein; meanwhile, the heat resistance and the pressure resistance are also influenced, and the pressure resistance is reduced to about 48 MPa.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Sequence listing

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Claims

1. A method for preparing a ferritin cage structure, which comprises transforming a vector comprising a gene sequence encoding Pyrococcusayanosii CH1 ferritin into a bacterium, and inducing expression; after the expression is finished, extracting and purifying to obtain the ferritin cage-like structure.

2. The method according to claim 1, wherein the coding gene sequence encodes the amino acid sequence shown in SEQ ID NO. 2; preferably, the coding gene sequence is identical to the sequence shown in SEQ ID NO:1 or SEQ ID NO:3 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

3. The method of claim 1 or 2, wherein the vector is a prokaryotic expression vector, preferably the prokaryotic expression vector is selected from the group consisting of pET-30a, pET-31b, pET-34b, pET-35b, pET22b, and pET-43.1; the bacteria are Escherichia coli, and preferably the bacteria are Escherichia coli BL21(DE3) or Rosetta series strains; the induced expression comprises the step of adding IPTG.

4. The method according to any one of claims 1 to 3, wherein the extraction comprises disrupting the cell-released protein, adding water at 80-100 ℃ under stirring, centrifuging, and collecting the supernatant; the purification comprises the steps of filtering and desalting the obtained supernatant.

5. The process according to any one of claims 1 to 4, wherein the process comprises the steps of:

2) transforming the prokaryotic expression vector obtained in the step 1) into escherichia coli, adding IPTG (isopropyl-beta-thiogalactoside) to activate a promoter, and performing inducible expression; preferably, the Escherichia coli is Escherichia coli BL21(DE3) or Rosetta series strain;

3) after the expression is finished, breaking the cell to release protein, adding water with the temperature of 80-100 ℃, stirring, centrifuging and collecting supernate;

4) filtering and desalting the supernatant obtained in the step 3);

obtaining the ferritin cage-like structure.

6. A ferritin cage structure obtainable by the process according to any one of claims 1 to 5.

7. A method for preparing a ferritin magnetic material, comprising adding a metal salt and an oxidizing agent to the ferritin cage structure of claim 6 to react.

8. The method of claim 7, wherein the oxidizing agent is H₂O₂The pH value of the reaction is 7-11, the reaction temperature is 20-85 ℃, wherein the molar ratio of the metal ions to the oxidant is 1:0.33-0.5, and the molar ratio of the ferritin cage-like structure to the metal ions is 1: 100-40000.

9. A magnetoferritin obtained by the preparation method according to claim 7 or 8.

10. Use of the ferritin cage structure of claim 6 or the magnetic ferritin of claim 9 in drug loading, drug delivery, antibody screening, magnetic resonance imaging, disease diagnostic agents or cell labelling.