CN106906230B

CN106906230B - Recombinant drug carrier protein gene and preparation method and application thereof

Info

Publication number: CN106906230B
Application number: CN201710171868.1A
Authority: CN
Inventors: 任磊; 单文俊
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2017-03-22
Filing date: 2017-03-22
Publication date: 2021-02-19
Anticipated expiration: 2037-03-22
Also published as: CN106906230A

Abstract

A recombinant drug carrier protein gene and a preparation method and application thereof relate to a gene. The novel tumor-targeted, pH-sensitive and self-assembly-characteristic recombinant hepatitis B core protein virus-like particle is adopted as a drug carrier for tumor treatment, and comprises the following steps: a protein gene sequence of a recombinant hepatitis B core protein virus-like particle targeting a tumor, a preparation method of the recombinant hepatitis B core protein virus-like particle targeting the tumor and a method for loading a drug by using the recombinant hepatitis B core protein virus-like particle include the controllable self-assembly behavior of the hepatitis B core protein virus-like particle and the characteristic that the drug is loaded by an internal hydrophobic microenvironment and is released under an acidic condition. Overcomes the defects of poor tumor targeting property, poor biocompatibility, strong immunogenicity and the like of the traditional nano-carrier. And simultaneously solves the problems of large dosage, strong toxicity, poor tolerance of patients and the like of the traditional chemotherapeutic drugs.

Description

Recombinant drug carrier protein gene and preparation method and application thereof

Technical Field

The invention relates to a gene, in particular to a targeted tumor recombinant drug carrier protein (HBc VLPs) gene of Hepatitis B core protein virus-like particles, and a preparation method and application thereof.

Background

Viral particles of hepatitis B core protein viral-like particles overview viral-like particles are empty-shell structures that do not contain viral nucleic acids, and many viral structural proteins have the ability to self-assemble into VLPs, are morphologically similar to natural viral particles, and have strong immunogenicity and biological activity. Since VLPs do not contain viral genetic material and are therefore not infectious, some of them have been successfully used clinically as vaccines. VLPs structurally allow insertion of foreign genes or gene fragments to form chimeric VLPs and display of foreign antigens on their surfaces.

Hepatitis B virus core antigen (HBcAg) was first used as a foreign antigen virus-like particle vector in 1987^[1，2]. The C gene in HBV genome contains two initiation codons ATG, and translation from the first initiation codon ATG obtains HBeAg with 29 amino acids more than HBcAg; and HBcAg when translated from the second start codon. Most of the amino acids of HBeAg and HBcAg are identical, but the two proteins differ in conformation and antigenic site due to the difference in the number and type of the total amino acids. The expression system for HBcAg expression has diversity, and comprises a prokaryotic expression system, a mammalian cell expression system, a transgenic plant expression system, a yeast expression system, an insect cell expression system and the like^[3～5]And can achieve higher yield and form a natural granular structure.

Hbcags produced by expression in e.coli expression systems are predominantly of two particle sizes, one comprising 180 subunits (T ═ 3, T ═ 4) and the other 240 subunits (T ═ 4), with part of the particles encapsulating e.coli nucleic acids. Each particle-forming subunit is a dimer formed from HBcAg monomers, constituting spikes on the outside of the particle. Two HBcAg monomers form a subunit through disulfide bonds, each HBcAg monomer having 4 Cys residues including Cys48, Cys61, Cys107, Cys 183. Wherein Cys107 is encapsulated within the granule; the Cys48 part is involved in the formation of inter-monomer disulfide bonds, and the remaining residues are free on the particle surface; cys61 and Cys183 are all involved in the formation of disulfide bonds between monomers or dimers, and have the same binding pattern as wild-type HBcAg. For a full length of 183 amino acids of HBcAg, the first 144 amino acids belong to the particle assembly region, with major functions associated with viral particle formation. And the 145-183 amino acids belong to nucleic acid binding regions rich in arginine, have three repeated SPRRR structures and mainly have the functions of binding virus nucleic acid and wrapping the virus nucleic acid into virus particles to protect the virus nucleic acid and provide replication sites. Between 78-82 amino acids in the HBcAg amino acid sequence is a Main Immune Region (MIR), and the Region is presented on the top of a spike on the surface of a particle; amino acids 127-133 form a small spike next to the main spike, which are the major B cell recognition sites on the HBcAg surface.

The MIR region has the advantages of easy binding with the receptor and no influence on the natural conformation of the exogenous fragment due to the distribution of the MIR region on the top of the spikes on the surface of the particle^[4]. For the N-terminal and C-terminal of the HBcAg monomer, the insertion fragment needs to be considered in terms of whether it is present on the surface of the particle, whether it affects the assembly of the particle structure, and whether the insertion fragment is capable of maintaining the native conformation. The insertion of a small fragment sequence after 144 amino acids at the C-terminus does not affect particle self-assembly, but the size of the insert has a large impact on whether the insert is correctly folded and can be presented on the particle surface. N-terminal insertion of foreign sequence to maintain correct sequence conformation and particle surface appearance is difficult, so the insertion fragment is difficultThere are severe limitations to the size. Using a particle with a more loosely constructed truncated form, i.e., the first 144 amino acids, increases the likelihood that the N-terminal insert will be present on the surface of the particle^[6]。

See literature:

[1]Gilbert R J C,Beales L,Blond D,Simon M N,Lin B Y,Chisari F V,Stuart D I,Rowlands D J.Hepatitis B small surface antigen particles are octahedral[J].Proceedings of the National Acad emy of Sciences of the United States of America,2005,102(41):14783.

[2]Clarke B,Newton S,Carroll A,Francis M,Appleyard G,Syred A,Highfield P,Rowlands D,Brown F.Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein[J].Nature,1987,330(6146):381-384.

[3]Tan W S,Dyson M R,Murray K.Hepatitis B virus core antigen:enhancement of its produ ction in Escherichia coli,and interaction of the core particles with the viral surface antigen[J].Biol ogical Chemistry,2003,384(3):363-371.

[4]Hirschman S Z,Price P,Garfinkel E,Christman J,Acs G.Expression of cloned hepatitis B virus DNA in human cell cultures[J].Proceedings of the National Academy of Sciences,1980,77(9):5507.

[5]Beesley K,Francis M,Clarke B,Beesley J,Dopping-Hepenstal P,Clare J,Brown F,Roma nos M.Expression in yeast of amino-terminal peptide fusions to hepatitis B core antigen and their i mmunological properties[J].Nature Biotechnology,1990,8(7):644-649.

[6]Zlotnick A,Cheng N,Stahl S,Conway J,Steven A,Wingfield P.Localization of the C ter minus of the assembly domain of hepatitis B virus capsid protein:implications for morphogenesis a nd organization of encapsidated RNA[J].Proceedings of the National Academy of Sciences,1997,94(18):9556.

disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a recombinant drug carrier protein gene which is based on hepatitis B core protein virus-like particles, is tumor-targeted, pH-sensitive and has self-assembly characteristics, and a preparation method and application thereof.

The recombinant drug carrier protein gene based on the hepatitis B core protein virus-like particles and having tumor targeting, pH sensitivity and self-assembly characteristics is prepared and purified through gene engineering, wherein the expression gene of the carrier protein comprises an amino acid encoding a hepatitis B core protein site, a targeted tumor-encoding polypeptide sequence, a linked peptide, an amphipathic alpha helix encoding hydrophobic polypeptide NS5A (1-31 aa) and a gene sequence encoding pH-sensitive polyhistidine polypeptide.

The gene sequence of the recombinant drug carrier protein with tumor targeting, pH sensitivity and self-assembly characteristics based on the hepatitis B core protein virus-like particle is characterized in that a polypeptide sequence of a gene coding targeting tumor is positioned between 72 th to 92 th amino acids of the hepatitis B core protein virus-like particle; the amphipathic alpha helix of the gene coding hydrophobic polypeptide NS5A (1-31 aa) is positioned between 140-183 amino acids of the C end of the hepatitis B virus core protein; the polypeptide of polyhistidine encoded by the gene is positioned at the tail end of the hydrophobic polypeptide NS5A (1-31 aa) protein.

Any at least two of the gene sequences of the coding targeted tumor polypeptide, the coding hydrophobic polypeptide and the coding pH sensitive polyhistidine polypeptide molecule are combined with the hepatitis B virus core protein gene sequence.

The recombinant drug carrier protein based on the hepatitis B core protein virus-like particle with tumor targeting, pH sensitivity and self-assembly characteristics is used for completing the expression of soluble chimeric protein by escherichia coli BL21(DE 3).

The recombinant drug carrier protein based on the hepatitis B core protein virus-like particle with tumor targeting, pH sensitivity and self-assembly characteristics comprises the following conditions:

1) culturing in LB broth medium at 10-37 deg.C for 0.5-4 h;

2) inducing protein expression by IPTG (isopropyl thiogalactoside) with the concentration of 0.1-1 mM, the culture time of 4-16 h and the culture temperature of 16-37 ℃;

3) the rotating speed of the shaking table during the culture period is 120-300 r/min.

The purification method of the recombinant drug carrier protein drug based on the hepatitis B core protein virus-like particle with tumor targeting, pH sensitivity and self-assembly characteristics comprises the following steps:

1) ultrasonically crushing thalli, wherein the ultrasonic duration time of 250W and 50Hz is 1-10 s, and the interval is 1-10 s;

2) ammonium sulfate precipitation of the primary pure fusion protein, and salting out of the protein with 10-50% saturated ammonium sulfate solution;

3) purifying by ion exchange column chromatography, adopting DEAE resin column, and mobile phase is 10mM Tris-Cl (pH8.0) buffer solution;

4) purifying by using a molecular exclusion chromatography method to obtain the recombinant drug carrier protein drug which is based on the hepatitis B core protein virus-like particles and has tumor targeting, pH sensitivity and self-assembly characteristics.

In step 4), the size exclusion chromatography purification can be performed by Sepharose CL 4B molecular sieve column chromatography, and the mobile phase is 10mM Tris-Cl, 150mM NaCl (pH8.0) buffer.

The prepared recombinant hepatitis B virus core protein virus-like particles have uniform aggregate particle size, uniform appearance height and self-assembly characteristic, and the particle size range of the virus-like particles is 30-40 nm.

The tumor targeting, pH sensitive and self-assembly characteristic depolymerization conditions of the recombinant drug carrier protein based on the hepatitis B core protein virus-like particle are as follows:

depolymerizing buffer: 50mM Tris-HCl, pH8.0, 0-10M urea; depolymerization time: 0.5-4 h.

The recombination conditions of the recombinant drug carrier protein based on the hepatitis B core protein virus-like particle with tumor targeting, pH sensitivity and self-assembly property are as follows:

recombination buffer 1: 50mM Tris-HCl, pH8.0, 150mM NaCl, 10% glycerol, 1% glycine;

recombination buffer 2: 50mM Tris-HCl, pH8.0, 150mM NaCl, 1% glycine.

Drug loading is carried out through the depolymerization and recombination processes of the virus-like particles, the drug and the depolymerized virus-like particles are mixed together, the molar ratio of the virus-like particles to the drug is 1: 50-10000, the pH value of the solution is adjusted to the pKa of the drug, and the incubation time is 0.5-4 h.

The recombinant drug carrier protein based on the hepatitis B core protein virus-like particles with tumor targeting, pH sensitivity and self-assembly characteristics can be applied to preparation of anti-tumor polypeptide nano-drugs and tumor photothermal/photodynamic therapy drugs.

The preparation method of the anti-tumor polypeptide nano-medicament comprises the following steps:

1) obtaining a gene sequence of a recombinant drug carrier protein which is based on hepatitis B virus core protein virus-like particle and has tumor targeting and pH sensitivity through a gene synthesis method, and constructing the gene sequence into an expression vector plasmid pEt43.1(a) through an Xho I/Nde I enzyme cutting site;

2) expressing the target protein in the escherichia coli by using the vector plasmid obtained in the step 1);

3) separating and purifying the target protein expressed by the vector plasmid collected in the step 2) in escherichia coli, and loading chemotherapeutic drugs by regulating and controlling the self-assembly process to prepare the antitumor polypeptide nano-drug.

The invention provides a recombinant drug carrier protein gene with tumor targeting, pH sensitivity and self-assembly characteristics based on hepatitis B core protein virus-like particles and an expression product thereof, wherein the virus-like particle nano-drug comprises tumor targeting polypeptide, hydrophobic polypeptide inserted for improving drug-loading capacity of the tumor targeting polypeptide and pH-sensitive polyhistidine acid-responsive functional polypeptide.

The invention modifies hepatitis B virus core protein virus-like particles, displays tumor targeting polypeptide fragments on the surfaces of the particles to endow the particles with the ability of targeting tumor cells and tissues, inserts hydrophobic polypeptide in the particles to enhance the loading ability of the particles to hydrophobic drugs, improves the stability of the encapsulated hydrophobic drugs in vivo, and reduces the damage of the drugs to normal tissue cells; meanwhile, a polyhistidine polypeptide fragment is introduced into the particle to obtain acid-responsive function. The constructed recombinant drug carrier protein based on the hepatitis B core protein virus-like particles has good biocompatibility and stability, is targeted to tumors, is pH sensitive, has a self-assembly characteristic, and improves the targeting property and the bioavailability of the tumor parts. In addition, the virus-like particles have acid responsiveness due to the fact that the medicine contains acid-responsive functional molecules, in a slightly acidic environment, internal polyhistidine is protonated, proton flows in, positive charge repulsive force between hydrophobic ends depolymerizes self-assemblies, and the medicine is quickly released in an environment that the pH value of tumor tissues is acidic, so that a good tumor treatment effect is achieved.

The tumor targeting polypeptide is a tripeptide sequence of RGD (arginine-glycine-aspartic acid), and the two ends of the tripeptide sequence are respectively connected with 19 connecting peptides rich in serine and glycine. Is positioned in the main immune area of hepatitis B virus core protein, and the area is exposed on the surface of virus-like particles, thereby being beneficial to the combination of tumor-targeted RGD polypeptide molecules and tumor cell surface overexpression integrin receptors, and further playing the specific tumor-targeted role.

The hydrophobic polypeptide NS5A (1-33aa) of the invention is inserted into the C-terminal of the particle, the C section of the hydrophobic polypeptide is mainly embedded in the particle, so that an internal hydrophobic cavity structure can be formed, and then a therapeutic drug is loaded in the particle through the depolymerization-recombination process of hepatitis B core protein.

The pH value of the acid response acidity is relative to the pH value in normal tissues in a human body, the pH value of the normal tissues of the human body is 7.4, and the pH value of tumor cells is 5-7.2, so that the pH environment of the tumor tissue parts is weakly acidic relative to the normal tissues of the human body.

When the pH value of the acid-responsive functional molecule is 5-6, the acid-responsive functional molecule can be protonated, so that polyhistidine in the multi-particle is protonated, and positive charge repulsive force is generated between the acid-responsive functional molecule and the polyhistidine, so that the self-assembled nano-particle form is damaged.

In the recombinant drug carrier protein with tumor targeting, pH sensitivity and self-assembly characteristics of the hepatitis B core protein virus-like particle, any one or at least two of the polypeptide with tumor targeting, hydrophobic polypeptide and polyhistidine polypeptide molecules is combined with a hepatitis B core protein fragment.

The particle size of the recombinant drug carrier protein with tumor targeting and pH sensitivity based on the hepatitis B core protein virus-like particles is 30-40 nm. The nanoparticles in the range are stable, have the effect of tumor enrichment, and can improve the targeting property and bioavailability of the medicine.

The polypeptide connected with the two ends of the coding targeted tumor polypeptide contains 5-30 serines or glycines.

The expression gene segment of the recombinant drug carrier protein with tumor targeting and pH sensitivity based on the hepatitis B core protein virus-like particle is realized by synthesis.

The recombinant drug carrier protein based on the hepatitis B core protein virus-like particle with tumor targeting and pH sensitivity overcomes the defects of poor tumor targeting and low drug dosage of the virus-like particle, and provides a polypeptide nano-drug carrier which has weak acid environment responsiveness at a tumor part, good biocompatibility, strong stability, good tumor targeting property, high safety, easiness for large-scale production, low cost and high bioavailability.

Compared with the prior art, the invention has the following beneficial effects:

the recombinant virus-like particles have good biocompatibility and low toxic and side effects, have acidic pH responsiveness and self-assembly characteristics, and are easily biodegraded in vivo because the nano materials are proteins. The tumor-targeted polypeptide nano-drug carrier based on the hepatitis B virus core protein virus-like particles can be enriched on a tumor part through active targeting, the nano-particle form of the nano-drug is depolymerized in a weak acid environment of the tumor part, and the loaded drug is effectively released, so that the effect of anti-tumor treatment is achieved, and the bioavailability of the anti-tumor drug is improved. The anti-tumor polypeptide nano-drug carrier can be produced in large quantities in escherichia coli, has simple production process and low cost, and has wide application prospect.

Drawings

FIG. 1 is an SDS-PAGE electrophoretic analysis of recombinant hepatitis B virus core protein virus-like particles of examples 1 and 2;

FIG. 2 is a TEM result of recombinant hepatitis B virus core protein virus-like particle in example 1;

FIG. 3 is a TEM result of recombinant hepatitis B virus core protein virus-like particle in example 2;

FIG. 4 is a graph showing the results of the radius of hydration of the recombinant hepatitis B virus core protein virus-like particles in examples 1 and 2;

FIG. 5 is a liquid chromatogram of recombinant hepatitis B virus core protein virus-like particle loaded with drug doxorubicin in example 1;

FIG. 6 is a liquid chromatogram of recombinant hepatitis B virus core protein virus-like particle loaded with drug doxorubicin in example 2;

FIG. 7 is a graph showing the results of an acid responsiveness assay in example 3 on recombinant hepatitis B virus core protein virus-like particles prepared in examples 1 and 2;

FIG. 8 is a graph showing the change of the particle size of the recombinant hepatitis B virus core protein virus-like particles prepared in example 1 and example 2 with time in PBS buffer in example 3;

FIG. 9 is a graph showing the effect of the recombinant hepatitis B virus core protein virus-like particles prepared in example 1 and example 2 in targeting tumor cells in vitro, as determined in example 4;

FIG. 10 is a graph showing the effect of doxorubicin-loaded recombinant hepatitis B virus core protein virus-like particle prepared in example 1 and example 2 on inhibiting tumor cell growth in vitro, as determined in example 5;

fig. 11 is an in vitro 808 laser irradiation temperature ramp profile of recombinant hepatitis b virus core protein virus-like particles of example 2 loaded with indocyanine green as determined in example 6.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments.

Example 1

The invention aims to provide a design and preparation method of a targeted tumor drug carrier based on hepatitis B virus core protein virus-like particles, which is characterized in that through gene modification, 144 th amino acid at the C end of truncated hepatitis B core protein is inserted into non-structural protein NS5A (1-31 aa) derived from hepatitis C virus as hydrophobic polypeptide, and simultaneously 6 histidine is inserted into the tail of the protein as acid-sensitive polypeptide, and the protein is expressed in escherichia coli, finally separated and purified to obtain monodisperse virus-like particles with regular appearance and 30-40 nm particle size. The chemotherapeutic drug is loaded for tumor targeted therapy through the self-assembly process of the chemotherapeutic drug.

First, a target gene fragment is obtained by gene synthesis, inserted into an expression vector plasmid using an Xho I/Nde I cleavage site, transformed into Escherichia coli BL21(DE3), and subjected to inducible expression of a target protein in LB medium.

1. Inducible expression of the fusion protein

1) BL21(DE3) single colonies containing the expression vector were picked and cultured in LB medium (containing 100. mu.g/mL ampicillin) at 37 ℃ and 255rpm to log phase;

2) diluting the bacterial liquid and the culture medium according to the proportion of 1:1000, culturing overnight at 37 ℃ and 255rpm to enable the OD600 value to reach 0.5-0.6; diluting the strain to fresh culture medium at a ratio of 1:1, and culturing at 37 deg.C and 255rpm for 2 h;

3) inducing at 37 ℃ with a final concentration of 1mM IPTG;

4) after 4h of induction, 1mL of the bacterial solution was removed, centrifuged at 12,000rpm for 1min, the supernatant was removed, 100. mu.L of PBS was added to resuspend the cells, and the samples were analyzed by 12% SDS-PAGE gel electrophoresis.

2. Ammonium sulfate precipitation of primary pure fusion protein

1) After the induction, the bacterial suspension was centrifuged at 4000rpm for 10min, and the cells were collected and blown up with 10mM Tris, 0.5% Triton pH8.0 buffer.

2) The bacterial cells are crushed by an ultrasonic crushing method, the power is 300W, the ultrasonic treatment is carried out for 30min, the bacterial liquid is viscous and semitransparent, centrifugation is carried out at 12000rpm for 10min, and supernatant (target protein in the supernatant is soluble protein) is collected.

3) The supernatant was dispensed into 1.5ml centrifuge tubes, 500. mu.L each, and protein salting-out was performed with 10%, 20%, 30%, 40%, 50% saturated ammonium sulfate solutions, respectively, and the precipitates were collected by salting-out at 4 ℃ for 30min, centrifuged at 12,000rpm for 10min, and the precipitate samples at each concentration were subjected to SDS-PAGE to detect the distribution of fusion proteins.

DEAE ion exchange chromatography purification

1) Column equilibration with base solution: the column was washed with 10 column volumes of a base solution containing 10mM Tris-Cl (pH8.0) until the effluent pH was consistent with the base solution pH.

2) Loading: the collected sample, i.e., the crude protein extract after dialysis by ammonium sulfate precipitation, was loaded at a flow rate of 3 mL/min.

3) Loading and eluting: the column was washed with 5 column volumes of base solution until the UV detector reading returned to baseline.

4) And (3) eluting by eluent I: the column was washed with 5 column volumes of eluent I (containing 10mM Tris-Cl (pH8.0), 50mM NaCl) and the eluted peak was collected.

5) And (3) eluting with an eluent II: the column was washed with 5 column volumes of eluent II (containing 10mM Tris-Cl (pH8.0), 100mM NaCl) and the eluted peak was collected.

6) And (3) eluting with eluent III: the column was washed with 5 column volumes of eluent III (containing 10mM Tris-Cl (pH8.0), 200mM NaCl) and the eluted peak was collected.

7) And (3) eluting with eluent III: the column was washed with 5 column volumes of eluent IV (containing 10mM Tris-Cl (pH8.0), 300mM NaCl) and the eluted peak was collected.

8) And (3) eluting with eluent III: the column was washed with 5 column volumes of eluent IV (containing 10mM Tris-Cl (pH8.0), 400mM NaCl) and the eluted peak was collected.

4. Purification of fusion protein molecular sieve Sepharose CL 4B

Slowly lowering the liquid in the column, dripping the sample on the gel surface when the liquid surface is lowered to the gel interface, adding degassed deionized water slowly until the liquid surface is below the gel surface, and connecting with an inlet adapter; the column was run at a constant flow rate of 1.5 mL/min. The effluent was collected at 2 mL/tube.

5. Protein dialysis and concentration

1) Putting the dialysis bag containing the protein solution into the precooled dialysate, and dialyzing in a refrigerator at 4 ℃;

2) the dialysis solution is changed for 4 times in the whole dialysis process until the dialysis is complete;

3) putting the completely dialyzed protein solution into a new big beaker, adding polyethylene glycol powder to cover the dialysis bag, quickly sucking out the solvent in the bag by polyethylene glycol when the solvent seeps out, and putting the beaker into a refrigerator at 4 ℃ for concentration;

4) during the concentration process, adding a small amount of dried polyethylene glycol powder for multiple times to increase the concentration speed, and replacing the saturated polyethylene glycol with new polyethylene glycol until the required concentration volume is reached;

5) after the concentration is finished, the dialysis bag is washed clean with distilled water, and the concentrated protein solution is taken out and the protein concentration is quantitatively determined by using a BCA method.

6. Drug loading

1) Dissociation of virus-like particles

Preparing dissociation liquid: 50mM Tris-HCl, pH8.0, 150mM NaCl, 8.0M urea (urea). mu.L of virus-like particles (. about.10 mg/mL) was incubated with the prepared dissociation solution at 4 ℃ for 3 h.

2) Recombination of virus-like particles

Recombinant buffer solution 1: 50mM Tris-HCl, pH8.0, 150mM NaCl, 10% glycerol, 1% glycine; recombinant buffer 2: 50mM Tris-HCl, pH8.0, 150mM NaCl, 1% glycine. Transferring the dissociated virus particle solution into a dialysis bag with the molecular weight of 8000-14000 Da, placing the dialysis bag into 100mL of recombinant buffer solution 1, performing overnight dialysis at 4 ℃, and replacing the solution with recombinant buffer solution 2 after 12 h. The dialysis time was 48h in total, during which the buffer was changed 2 times.

3) Drug loading

1mL of virus-like particles (about 1mg/mL) was incubated with the prepared dissociation solution at 4 ℃ for 2.5h, the total volume of the solution being 10 mL. At this time, 500mg of doxorubicin was added to the above dissociation solution, and the resulting mixed solution was further co-cultured for 30min with gentle shaking. Then, the sample was transferred to a dialysis bag with a molecular weight of 8000-14000 Da, and the sample was first placed in a recombinant buffer solution 1 and dialyzed overnight at 4 ℃ for 12 hours, and then replaced with a recombinant buffer solution 2. The dialysis time was 48h in total, during which the buffer was changed 2 times. The resulting drug-loaded virus-like particles were stored at-20 ℃.

The molecular weight of the prepared protein monomer is verified by SDS-PAGE electrophoresis (figure 1). Transmission electron microscopy demonstrated that the constructed virus-like particles can be efficiently assembled into monodisperse particles with uniform morphology, as shown in FIG. 2. The laser particle sizer further verifies the particle structure of the particle size of 30-40 nm, as shown in figure 4. High performance chromatography (FIG. 5) confirmed that the virus-like particles obtained in this example were effectively loaded with the chemotherapeutic drug doxorubicin.

The nucleotide sequence is as follows:

atgGACATCGACCACTACAAAGAATTCGGTGCTTCCGTTGAACTGCTGTCCTTCCTGCCGTCCGACTTCTTCCCGTCCATCCGTGACCTGCTGGACACTGCTTCCGCTCTGTACCGTGAAGCTCTGGAATCCCCGGAACACTGCTCCCCGCACCACACTGCTCTGCGTCAGGCTATCCTGTGCTGGGGTGAACTGATGAACCTGGCTACTTGGGTTGGTTCCAACCTGGAAGACCCGGCTTCCCGTGAACTGGTTGTTGGTTACGTTAACGTTAACATGGGTCTGAAAATCCGTCAGATCCTGTGGTTCCACATCTCCTGCCTGACTTTCGGTCGTGAAACTGTTCTGGAATACCTGGTTTCCTTCGGTGTTTGGATTCGTACTCCGCCGGCTTACCGTCCGCCGAACGCTCCGATCCTGTCCACTCTGCCGGCCGGTTCCTGGCTAAGGGACATCTGGGACTGGATATGCGAGGTGCTGAGCGATTTTAAGACCTGGCTGAAGGCCAAGCTCATGCCAACCATGCACCACCACCACCACCAC

the coded amino acid sequence is as follows:

MDIDHYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVVGYVNVNMGLKIRQILWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPAGSWLRDIWDWICEVLSDFKTWLKAKLMPTMHHHHHH

example 2

In this embodiment, based on the original embodiment 1, a tumor targeting polypeptide sequence is inserted between positions 72-82 of the main immune region. By the same expression purification method and steps as in example 1, tumor active targeting with high affinity binding to integrin receptors overexpressed on the surface of tumor cells is achieved. The antitumor drug adriamycin is loaded through the self-assembly process the same as the example 1, and the antitumor polypeptide nano-drug system is obtained.

The expected molecular weight of the virus-like particles obtained in this example was confirmed by SDS-PAGE electrophoresis (FIG. 1).

The morphology and the particle size characterization (figure 3) of the obtained virus-like particles are carried out by using a transmission electron microscope and a laser particle sizer, and the results show that the prepared virus-like particles are spherical, the particle sizes are uniform, the distribution of hydrated particle sizes is 30-40 nm (figure 4), and the average particle size is about 36.5 +/-2.1 nm.

Figure 6 shows the hplc results of example 2 after doxorubicin loading.

The nucleotide sequence is as follows:

atgGACATCGACCACTACAAAGAATTCGGTGCTTCCGTTGAACTGCTGTCCTTCCTGCCGTCCGACTTCTTCCCGTCCATCCGTGACCTGCTGGACACTGCTTCCGCTCTGTACCGTGAAGCTCTGGAATCCCCGGAACACTGCTCCCCGCACCACACTGCTCTGCGTCAGGCTATCCTGTGCTGGGGTGAACTGATGAACCTGGCTACTTGGGTTGGTTCCAACCTGGAAGACGGTACCTCCGGTTCCTCCGGTTCCGGTTCCGGTGGTTCCGGTTCCGGTGGTGGTGGTCGAGGTGACGGTGGTGGTGGTTCCGGTTCCGGTGGTTCCGGTTCCGGTTCCTCCGGTTCCACCGGTTCCCGTGAACTGGTTGTTGGTTACGTTAACGTTAACATGGGTCTGAAAATCCGTCAGATCCTGTGGTTCCACATCTCCTGCCTGACTTTCGGTCGTGAAACTGTTCTGGAATACCTGGTTTCCTTCGGTGTTTGGATTCGTACTCCGCCGGCTTACCGTCCGCCGAACGCTCCGATCCTGTCCACTCTGCCGGCCGGTTCCTGGCTAAGGGACATCTGGGACTGGATATGCGAGGTGCTGAGCGATTTTAAGACCTGGCTGAAGGCCAAGCTCATGCCAACCATGCACCACCACCACCACCAC*

the amino acid sequence of the coded polypeptide is as follows:

MDIDHYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDGTSGSSGSGSGGSGSGGGGRGDGGGGSGSGGSGSGSSGSTGSRELVVGYVNVNMGLKIRQILWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPAGSWLRDIWDWICEVLSDFKTWLKAKLMPTMHHHHHH

example 3

This example is directed to the determination of the particle size of virus-like particles based on the hepatitis B virus core protein in a slightly acidic solution.

The virus-like particles obtained in example 1 and example 2 were incubated with phosphate buffered solutions at different pH values (8.0, 7.4, 6.4, 5.8, 5, 4) for 12h, and their hydrated particle sizes were determined using a laser particle sizer. As shown in FIG. 3, when the pH is 5 or less, the particle size of the prepared virus-like particles becomes significantly large and exceeds 100nm (FIG. 7). Whereas the particle size of the virus-like particles did not change significantly under physiological pH conditions (fig. 8). This indicates that in acidic solution, the prepared virus-like particles are no longer nanosphere-shaped, but rather, due to protonation inside the nanosphere, the nanosphere structure generates repulsive force, which causes irreversible depolymerization thereof.

Example 4

This example is directed to the determination of targeting of hepatitis b core protein virus-like particle-based tumor-targeted, pH-sensitive recombinant drug carrier proteins to tumor cells in vitro.

U87MG cells were cultured at 5X 10⁵cells/well were plated in 6-well plates at a density, incubated for 24h, and after adherent growth of cells, the old medium was discarded, and virus-like particles loaded with doxorubicin as in example 1 and example 2 were diluted with serum-free medium. The doxorubicin concentration was 5. mu.g/mL, and the incubations were 0.5h, 1h, and 2h, respectively. After the incubation was completed, the old culture medium was discarded and washed 3 times with ice-cold PBS. Add 100. mu.L of pancreatin to each well, collect cells after termination of digestion, centrifuge, discard supernatant, disperse cells by adding 500. mu.L of PBS, and measure with flow cytometer. As shown in FIG. 9, the recombinant virus-like particles of example 2 were more easily taken up by tumor cells (U87 Mg).

Example 5

Taking U87MG cells in logarithmic growth phase at 7X 10³Each well was inoculated into a 96-well plate, and after incubation at 37 ℃ for 24 hours, 100. mu.L of each of the drug-loaded recombinant virus-like particles of example 1 and example 2 was added thereto, and 3 wells were placed in parallel at each concentration, and a blank control was set. After 48h incubation in the incubator, the drug-containing medium was aspirated, 100. mu.L of MTT solution (0.5mg/mL) was added to each well, incubation was continued for 4h, MTT solution was aspirated, 100. mu.L of DMSO was added to each well, shaking was performed for 2min to dissolve the bluish violet crystals, and finally the absorbance value (OD) was measured at 492nm using a microplate reader, and the cell viability was calculated using the following formula (see FIG. 10).

Example 6

In this example, the photosensitizer indocyanine green encapsulated in example 2 was used for photothermal/photodynamic therapy of tumors. The virus-like particles in example 2 were depolymerized under 2.5M urea, incubated with the photosensitizer indocyanine green for 4-8 h, and dialyzed against the recombinant buffer for 48h, during which the dialysate was changed twice. Finally, the recombinant hepatitis B virus core protein carrying indocyanine green is obtained. The 808W laser irradiation power is 1W for 0-300 s, the temperature of the embodiment 1 carrying the indocyanine green can be raised to about 45 ℃, the indocyanine green has excellent photo-thermal performance (as shown in figure 11), and the indocyanine green can be applied to tumor photo-thermal and photodynamic therapy.

The invention overcomes the defects of poor tumor targeting property, poor biocompatibility, strong immunogenicity and the like of the traditional nano-carrier. The invention also solves the problems of large dosage, strong toxicity, poor tolerance of patients and the like of the traditional chemotherapeutic drugs. The preparation method is simple, is suitable for large-scale production, has selectivity on tumor cells, can effectively inhibit tumors after being loaded with chemotherapeutic drugs, and can also be loaded with photosensitizers for tumor photothermal co-kinetic treatment.

Sequence listing

<110> university of mansion

<120> recombinant drug carrier protein gene and preparation method and application thereof

<130> 2017

<160> 4

<170> PatentIn version 3.3

<210> 1 (example 1 DNA sequence)

<211> 543

<212> DNA

<213> Artificial sequence

<400> 1

atggacatcg accactacaa agaattcggt gcttccgttg aactgctgtc cttcctgccg 60

tccgacttct tcccgtccat ccgtgacctg ctggacactg cttccgctct gtaccgtgaa 120

gctctggaat ccccggaaca ctgctccccg caccacactg ctctgcgtca ggctatcctg 180

tgctggggtg aactgatgaa cctggctact tgggttggtt ccaacctgga agacccggct 240

tcccgtgaac tggttgttgg ttacgttaac gttaacatgg gtctgaaaat ccgtcagatc 300

ctgtggttcc acatctcctg cctgactttc ggtcgtgaaa ctgttctgga atacctggtt 360

tccttcggtg tttggattcg tactccgccg gcttaccgtc cgccgaacgc tccgatcctg 420

tccactctgc cggccggttc ctggctaagg gacatctggg actggatatg cgaggtgctg 480

agcgatttta agacctggct gaaggccaag ctcatgccaa ccatgcacca ccaccaccac 540

cac 543

<210> 2 (example 2 DNA sequence)

<211> 660

<212> DNA

<213> Artificial sequence

<400> 2

atggacatcg accactacaa agaattcggt gcttccgttg aactgctgtc cttcctgccg 60

tccgacttct tcccgtccat ccgtgacctg ctggacactg cttccgctct gtaccgtgaa 120

gctctggaat ccccggaaca ctgctccccg caccacactg ctctgcgtca ggctatcctg 180

tgctggggtg aactgatgaa cctggctact tgggttggtt ccaacctgga agacggtacc 240

tccggttcct ccggttccgg ttccggtggt tccggttccg gtggtggtgg tcgaggtgac 300

ggtggtggtg gttccggttc cggtggttcc ggttccggtt cctccggttc caccggttcc 360

cgtgaactgg ttgttggtta cgttaacgtt aacatgggtc tgaaaatccg tcagatcctg 420

tggttccaca tctcctgcct gactttcggt cgtgaaactg ttctggaata cctggtttcc 480

ttcggtgttt ggattcgtac tccgccggct taccgtccgc cgaacgctcc gatcctgtcc 540

actctgccgg ccggttcctg gctaagggac atctgggact ggatatgcga ggtgctgagc 600

gattttaaga cctggctgaa ggccaagctc atgccaacca tgcaccacca ccaccaccac 660

<210> 3 (example 1 protein sequence)

<211> 181

<212> PRT

<213> Artificial sequence

<400> 3

Met Asp Ile Asp His Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Glu Leu Val Val Gly Tyr Val Asn Val Asn Met Gly Leu Lys

85 90 95

Ile Arg Gln Ile Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Ala Gly Ser Trp Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu

145 150 155 160

Ser Asp Phe Lys Thr Trp Leu Lys Ala Lys Leu Met Pro Thr Met His

165 170 175

His His His His His

180

<210> 4 (example 2 protein sequence)

<211> 220

<212> PRT

<213> Artificial sequence

<400> 4

Met Asp Ile Asp His Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Gly Thr

65 70 75 80

Ser Gly Ser Ser Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly Gly Gly

85 90 95

Gly Arg Gly Asp Gly Gly Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser

100 105 110

Gly Ser Ser Gly Ser Thr Gly Ser Arg Glu Leu Val Val Gly Tyr Val

115 120 125

Asn Val Asn Met Gly Leu Lys Ile Arg Gln Ile Leu Trp Phe His Ile

130 135 140

Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser

145 150 155 160

Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala

165 170 175

Pro Ile Leu Ser Thr Leu Pro Ala Gly Ser Trp Leu Arg Asp Ile Trp

180 185 190

Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Leu Lys Ala

195 200 205

Lys Leu Met Pro Thr Met His His His His His His

210 215 220

Claims

1. A gene encoding a tumor-targeted, pH-sensitive, self-assembling recombinant drug carrier protein based on hepatitis B core protein virus-like particles, wherein the amino acid sequence of the recombinant drug carrier protein is shown in SEQ ID NO: 4.

2. A tumor-targeted, pH-sensitive, self-assembled recombinant drug carrier protein based on hepatitis B core protein virus-like particles, characterized in that the amino acid sequence of the recombinant drug carrier protein is shown in SEQ ID NO: 4.

3. the production method of recombinant drug carrier protein as claimed in claim 2 is characterized in that comprising the following steps:

1) introducing the plasmid vector containing the gene of claim 1 into Escherichia coli BL21, and culturing Escherichia coli BL21 in LB broth medium at 10 to 37°C for 0.5 to 4 hours;

2) Use IPTG to induce protein expression, the IPTG concentration is 0.1-1mM, the culture time is 4-16h, and the culture temperature is 16-37°C;

3) The rotating speed of the shaker is 120-300 r/min during the incubation.

4. the purification method of recombinant drug carrier protein as claimed in claim 2 is characterized in that comprising the following steps:

1) ultrasonication of Escherichia coli BL21 obtained by the method as claimed in claim 3, 250W, 50Hz ultrasonic duration 1～10s, interval 1～10s;

2) Precipitate the primary pure recombinant drug carrier protein with ammonium sulfate, and perform protein salting out with 10%-50% saturated ammonium sulfate solution;

3) Purification by ion exchange column chromatography, using DEAE resin column, the mobile phase is 10mM Tris-Cl buffer, pH 8.0;

4) Purification by molecular size exclusion chromatography to obtain a tumor-targeted, pH-sensitive, self-assembled recombinant drug carrier protein based on hepatitis B core protein virus-like particles.

5. the purification method of recombinant drug carrier protein as claimed in claim 4 is characterized in that in step 4), described size exclusion chromatography is purified by Sepharose CL 4B molecular sieve column chromatography, and mobile phase is 10mM Tris-Cl, 150 mM NaCl, pH 8.0 buffer.

6 . The tumor-targeted, pH-sensitive, self-assembled recombinant drug carrier protein based on hepatitis B core protein virus-like particles according to claim 2 is used in the preparation of anti-tumor polypeptide nano-drugs and tumor photothermal/photodynamic therapy drugs. 7 .

7. application as claimed in claim 6 is characterized in that the preparation method of described anti-tumor polypeptide nanomedicine comprises the following steps:

1) A gene sequence encoding a tumor-targeted, pH-sensitive recombinant drug carrier protein based on hepatitis B core protein virus-like particles was obtained by gene synthesis, and constructed into the expression vector plasmid pEt43.1 through the Xho I/Nde I restriction site (a);

2) make the vector plasmid obtained in step 1) express the target protein in Escherichia coli;

3) After separating and purifying the vector plasmid collected in step 2) to express the target protein in Escherichia coli, by regulating its self-assembly process, loading chemotherapeutic drugs to prepare anti-tumor polypeptide nano-drugs.