CN106906230A

CN106906230A - Reconstituted drug carrier peptide gene and preparation method and application

Info

Publication number: CN106906230A
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: 2017-06-30
Anticipated expiration: 2037-03-22
Also published as: CN106906230B

Abstract

Reconstituted drug carrier peptide gene and preparation method and application, is related to a kind of gene.Using novel tumor is targetted, pH is sensitive, with self assembly characteristic recombination hepatitis B core protein virus-like particle be used for oncotherapy as pharmaceutical carrier, including：A kind of protein gene sequence of the recombination hepatitis B core protein virus-like particle of target tumor, a kind of preparation method of the recombination hepatitis B core protein virus-like particle of target tumor, a method that medicine is loaded using HBcAg virus-like particle, including the controllable self assembly behavior using HBcAg virus-like particle, and inner hydrophobic microenvironment loads medicine, the characteristic of medicine is discharged under acid condition.The shortcomings such as overcome Conventional nano vector tumors targeting, poor biocompatibility, immunogenicity strong.The problems such as also solving big classic chemotherapy drug medication dosage, strong toxicity, patient tolerability difference simultaneously.

Description

Reconstituted drug carrier peptide gene and preparation method and application

Technical field

The present invention relates to a kind of gene, more particularly, to a kind of target tumor restructuring of HBcAg virus-like particle Pharmaceutical carrier albumen (Hepatitis B virus like particles, HBc VLPs) gene and preparation method thereof and should With.

Background technology

HBcAg virus-like particle general introduction virus-like particle is free from the hollow shell structure of viral nucleic acid, many viruses Structural proteins all have the ability for being assembled into VLPs automatically, similar to natural virion on morphosis, with very strong Immunogenicity and BA.Because VLPs does not contain viral genetic, therefore without infectivity, some of which is Clinic is successfully applied to through as vaccine.VLPs allows the insertion of foreign gene or genetic fragment and forms mosaic type in structure Exogenous antigen is simultaneously illustrated in its surface by VLPs.

HBcAg (HBcAg) was used as exogenous antigen virus-like particle carrier for the first time in 1987^[1,2]。 C genes in HBV gene group include two initiation codon ATG, are translated since first initiation codon ATG and are then compared Many 29 HBeAg of amino acid of HBcAg；And if translate what is obtained since second initiation codon, be HBcAg.HBeAg It is identical with HBcAg Most amino-acids, but because overall amino acid number species is different, two kinds of conformations of albumen are not Together, antigen site is different.Expression system for HBcAg expression is thin with variation, including prokaryotic expression system, mammal Cellular expression system, Expressed in Transgenic Plant system, yeast expression system, insect cell expression system etc.^[3~5], and can reach To high yield, native granular shape structure is formed.

The HBcAg produced by escherichia expression system expression mainly has two kinds of particles size, and one kind includes 180 subunits (T=3, T=Triangulation number), another includes 240 subunits (T=4), large intestine is wrapped up in partial particulate Bacillus nucleic acid.Each subunit for forming particle is the dimer formed by HBcAg monomers, constitutes the furcella of extra-granular.Two By one subunit of disulfide formation, each HBcAg monomer has 4 Cys residues to individual HBcAg monomers, including Cys48, Cys61, Cys107、Cys183.Wherein Cys107 is wrapped in inside particle；Disulfide formation between Cys48 subparticipation monomers, it is remaining residual Base is free in particle surface；And disulfide formation between Cys61 and Cys183 then all participate in monomer or between dimer, it is and wild The HBcAg of type has same combination.For 183 HBcAg of amino acid of total length, preceding 144 amino acid belongs to Particle assembly section, major function and virion shape form relevant.And the 145th~183 amino acid belongs to nucleic acid land richness Containing arginine, with three SPRRR structures of repetition, Main Function is to combine viral nucleic acid and wrapped up to virion Protection is played to viral nucleic acid and the function of replicating place is provided in portion.In HBcAg amino acid sequences the 78th~82 amino acid it Between be principal immune region (Major Immunodominant Region, MIR), this region is presented on particle surface furcella Top；127th~133 amino acid forms a small furcella beside main furcella, and this two parts is HBcAg surfaces Major B-cell recognition site.

MIR regions due to being distributed in the furcella top of particle surface, with being easy to that external source piece is combined and do not influenceed with acceptor The advantage of Duan Ziran conformations^[4].Need to consider whether to present for the N-terminal and C-terminal of HBcAg monomers, during Insert Fragment In particle surface, whether influence that grain structure is assembled and Insert Fragment is enough native conformation this three aspects problems of keeping.C-terminal Small fragment sequence is inserted after 144 amino acid and does not interfere with particles self assemble, but the size of Insert Fragment to insertion piece Whether section can correctly fold and be that enough particle surfaces that can be presented on have a significant impact.N latter ends insertion exogenous array will keep sequence Correct conformation and particle surface are presented relatively difficult, so having strict limitation to Insert Fragment size.It is more open with structure Truncated-type be preceding 144 amino acids formed particles, the possibility that N-terminal Insert Fragment is presented on particle surface can be improved Property^[6]。

Referring to document：

[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.

The content of the invention

For the above-mentioned deficiency of prior art, HBcAg virus-like is based on it is an object of the invention to provide one kind Cancer target, pH sensitivities, the reconstituted drug carrier peptide gene with self assembly characteristic of particle and preparation method and application.

The cancer target based on HBcAg virus-like particle, pH are sensitive, the restructuring with self assembly characteristic Pharmaceutical carrier GFP, the expressing gene of its carrier protein includes coding hepatitis B virus core protein amino acids, coding target To oncopeptide sequence and link peptide, the α spirals of coding hydrophobic peptides NS5A (1~31aa) amphiphilic, encode the poly- of pH sensitivities The gene order of histidine polypeptide, and by genetic engineering, prepare purifying and obtain.

The cancer target based on HBcAg virus-like particle, pH are sensitive, the restructuring with self assembly characteristic The gene order of pharmaceutical carrier albumen, the polypeptide sequence of its gene code target tumor is located at hepatitis B virus core protein virus-like Position between 72nd~92 amino acids of particle；The α spiral shells of hydrophobic peptides NS5A (1~31aa) amphiphilic are encoded in its gene Rotation is between 140~183 amino acid of hepatitis B virus core protein C-terminal；The polypeptide of the polyhistidyl of its gene code is located at Hydrophobic peptides NS5A (1~31aa) albumen least significant end.

The base of the coding target tumor polypeptide, coding hydrophobic peptides, coding pH sensitive polyhistidyl peptide molecule In because of sequence any at least two are combined with hepatitis B virus core protein gene order.

The cancer target based on HBcAg virus-like particle, pH are sensitive, recombinate medicine with self assembly characteristic Thing carrier protein, the expression soluble chimeric albumen of recombinant protein is completed by e. coli bl21 (DE3).

The cancer target based on HBcAg virus-like particle, pH are sensitive, recombinate medicine with self assembly characteristic Thing carrier protein, it includes following condition in the method for Bacillus coli expression：

1) 10~37 DEG C of 0.5~4h of culture in LB broth bouillons；

2) using the expression of IPTG inducible proteins, IPTG concentration is 0.1~1mM, and incubation time is 4~16h, and cultivation temperature is 16~37 DEG C；

3) shaking speed is 120~300r/min during cultivating.

The cancer target based on HBcAg virus-like particle, pH are sensitive, recombinate medicine with self assembly characteristic The purification process of thing carrier protein medicine is comprised the following steps：

1) ultrasonication thalline, 250W, 50Hz ultrasound 1~10s of duration, is spaced 1~10s；

2) the first pure fusion protein of ammonium sulfate precipitation, 10%~50% saturated ammonium sulfate solution carries out albumen and saltouts；

3) ion exchange column chromatography purification, using DEAE resin columns, mobile phase is the buffering of 10mM Tris-Cl (pH8.0) Liquid；

4) molecular exclusion chromatography purifying, obtains the cancer target based on HBcAg virus-like particle, pH sensitivities, tool There is self assembly characteristic reconstituted drug carrier protein medicine.

In step 4) in, the molecular exclusion chromatography purifying can be pure using Sepharose CL 4B molecular sieve column chromatographies Change, mobile phase is the buffer solution of 10mM Tris-Cl, 150mM NaCL (pH8.0).

The poly- uniform particle sizes of recombinant hepatitis B virus core protein virus-like particle of preparation, topographical height are homogeneous, and depolymerization is from group Dress characteristic, virus-like particle particle size range is 30~40nm.

The cancer target based on HBcAg virus-like particle, pH are sensitive, recombinate medicine with self assembly characteristic The depolymerisation conditions of thing carrier protein are as follows：

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

The cancer target based on HBcAg virus-like particle, pH are sensitive, recombinate medicine with self assembly characteristic The restructuring condition of thing carrier protein is as follows：

Reassembly buffer liquid 1：50mM Tris-HCl, pH8.0,150mM NaCl, 10% glycerine, 1% glycine；

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

Described by described virus-like particle depolymerization, regrouping process carries out medicine loading again, the virus-like of medicine and depolymerization Particle is mixed, and virus-like particle and medicine mol ratio are 1 ︰ (50~10000), adjustment solution ph to medicine in itself PKa, incubation time is 0.5~4h.

The cancer target based on HBcAg virus-like particle, pH are sensitive, recombinate medicine with self assembly characteristic Thing carrier protein can be applied in antineoplastic polypeptide Nano medication and tumor photo-thermal/optical dynamic therapy medicine is prepared.

The preparation method of antineoplastic polypeptide Nano medication of the invention is comprised the following steps：

1) being obtained by gene synthesis has cancer target of the coding based on HBcAg virus-like particle, pH quick The gene order of the reconstituted drug carrier protein of sense, expression vector plasmid is building up to by Xho I/Nde I restriction enzyme sites In pEt43.1 (a)；

2) step 1 is made) vector plasmid that obtains is in expression in escherichia coli destination protein；

3) by step 2) vector plasmid for obtaining is collected after expression in escherichia coli destination protein is isolated and purified, by adjusting Its self assembling process is controlled, chemotherapeutics is loaded, antineoplastic polypeptide Nano medication is made.

The present invention provide a kind of cancer target based on HBcAg virus-like particle, pH it is sensitive, with self assembly Characteristic reconstituted drug carrier peptide gene and its expression product, the virus-like particle Nano medication comprising cancer target polypeptide, The hydrophobic peptides inserted to improve its drugloading rate, the polyhistidyl acid response functional polypeptide with pH sensitivities.

The present invention is transformed hepatitis B virus core protein virus-like particle, and cancer target polypeptide fragment is illustrated in Hydrophobic peptides are inserted inside it to strengthen it to hydrophobicity to assign the ability of its targets neoplastic cells and tissue in grain surface The delivered payload capability of medicine, improves package-contained hydrophobic drug stability in vivo, reduces the damage of medicine normal tissue cell Wound；Polyhistidyl polypeptide fragment is introduced simultaneously, and acid response sexual function is obtained inside particle.It is constructed based on hepatitis B core The cancer target of prion sample particle, pH be sensitive, with self assembly characteristic reconstituted drug carrier protein good biocompatibility, surely It is fixed, improve to the targeting of tumor locus and its bioavilability.Further, since containing acid response sexual function in the medicine Molecule causes that the virus-like particle has sour response, and in slightly acidic environment, internal polyhistidine is protonated, proton Interior stream, the positive charge repulsive force between hydrophobic side makes self-assembly depolymerization, under the environment that tumor tissues pH value is acid, quickly Release medicine, reaches preferable oncotherapy effect.

Cancer target polypeptide of the invention is the tripeptide sequence of RGD (arginine-glycine-aspartic acid), its two ends point The connection peptide of 19 rich serine glycine not being connected with is constituted.Positioned at the principal immune area position of hepatitis B virus core protein, This region is an exposure to the surface of virus-like particle, is thus advantageous to cancer target rgd peptide molecule and tumor cell surface mistake Amount expression integrin receptor is combined, so as to play special cancer target effect.

Hydrophobic peptides NS5A (1-33aa) of the present invention is inserted into the C-terminal of particle, and its C sections is mainly embedded in particle Portion, therefore the cavity structure of inner hydrophobic can be formed, then pass through HBcAg the poly- restructuring of solution process by therapeutic Medicine be loaded into inside particle.

The acid ph value of acid response of the present invention is that for the pH value in normal structure in human body, human body is just Often the pH value of tissue is 7.4, and the pH value of tumour cell is 5~7.2, therefore for human normal tissue, tumor group The pH environment for knitting position is faintly acid.

Sour response functional molecular can be protonated when pH value is 5~6, cause many particle inside polyhistidyl matter Sonization, between produce positive charge repulsive force, make self-assembling nanoparticles form be destroyed.

The cancer target of HBcAg virus-like particle of the present invention, pH are sensitive, the weight with self assembly characteristic It is any one in the polypeptide with cancer target, hydrophobic peptides, polyhistidyl peptide molecule in group pharmaceutical carrier albumen Plant or at least two and hepatitis B virus core protein fragment combination.

A kind of cancer target based on HBcAg virus-like particle of the present invention, pH sensitive reconstituted drug is carried The particle diameter of body protein is 30~40nm.Nanoparticles stable in the range of this, with tumour enrichment, can improve medicine The bioavilability of targeting and medicine.

Coding target tumor polypeptide two ends connecting peptides are to contain 5~30 serines or glycine.

A kind of table of the cancer target based on HBcAg virus-like particle, pH sensitive reconstituted drug carrier protein Realized by synthesizing up to genetic fragment.

Cancer target based on HBcAg virus-like particle of the present invention, pH sensitive reconstituted drug carrier Albumen overcomes virus-like particle target tumor difference and carries, the relatively low shortcoming of dose, there is provided one kind has tumor locus weakly acidic condition Response, good biocompatibility, stability are strong, tumor-targeting is good, safe be easy to large-scale production, low cost and biology Availability polypeptide nano pharmaceutical carrier high.

Relative to prior art, the invention has the advantages that：

Recombinant virus sample granular biological compatibility of the invention is good, and toxic and side effect is low, with acid pH response with from group Dress characteristic, nano material is protide in itself, it is easy to biodegradable in vivo.It is of the invention based on hepatitis B virus core protein Virus-like particle target tumor polypeptide nano pharmaceutical carrier can be enriched in tumor locus by active targeting, in tumor locus In weak acid environment, the nanoparticle morphology depolymerization of Nano medication loads medicine and is effectively discharged, to reach antineoplaston Effect, improves the bioavilability of antineoplastic.Antineoplastic polypeptide nano-medicament carrier of the invention can be in large intestine bar Largely produced in bacterium, simple production process is with low cost, has broad application prospects.

Brief description of the drawings

Fig. 1 is the SDS-PAGE electrophoresis point of embodiment 1 and recombinant hepatitis B virus core protein virus-like particle in embodiment 2 Analysis figure；

Fig. 2 is the TEM result figures of the recombinant hepatitis B virus core protein virus-like particle of embodiment 1；

Fig. 3 is the TEM result figures of recombinant hepatitis B virus core protein virus-like particle in embodiment 2；

Fig. 4 is the hydration radius result of embodiment 1 and recombinant hepatitis B virus core protein virus-like particle in embodiment 2 Figure；

Fig. 5 is the liquid chromatogram of recombinant hepatitis B virus core protein virus-like particle loading drug adriamycin in embodiment 1 Figure；

Fig. 6 is the liquid chromatogram of recombinant hepatitis B virus core protein virus-like particle loading drug adriamycin in embodiment 2 Figure；

Fig. 7 is the recombinant hepatitis B virus core protein virus-like in embodiment 3 to being prepared in embodiment 1 and embodiment 2 Grain carries out the result figure of acid response measure；

Fig. 8 is the recombinant hepatitis B virus core protein virus-like in embodiment 3 to being prepared in embodiment 1 and embodiment 2 Grain particle diameter in PBS changes over time figure；

Fig. 9 is the recombinant hepatitis B virus core egg of the embodiment 1 of embodiment 1 with preparation in embodiment 2 of measure in embodiment 4 Leukovirus sample particle targets neoplastic cells design sketch in vitro；

Figure 10 is the restructuring that is loaded with adriamycin of the embodiment 1 of embodiment 1 with preparation in embodiment 2 of measure in embodiment 5 The design sketch of the extracorporeal suppression tumor cell growth of hepatitis B virus core protein virus-like particle；

Figure 11 is the recombinant hepatitis B virus core protein being mounted with the embodiment 2 of CG of measure in embodiment 6 The external 808 laser irradiation heating curve of virus-like particle.

Specific embodiment

Technical scheme is further illustrated below by specific embodiment.

Embodiment 1

It is an object of the invention to provide a kind of target tumor medicine based on hepatitis B virus core protein virus-like particle The design and preparation method of carrier, by genetic modification, insert after the amino acids of HBcAg C-terminal 144 blocked Hepatitis C virus non-structural protein NS5A (1~31aa) is come from as hydrophobic peptides, while insert 6 histidines in its afterbody making Be acid-sensitive polypeptide, in Bacillus coli expression and be finally recovered purifying, obtain with pattern rule, single point of 30~40nm of particle diameter Dissipate virus-like particle.Loading chemotherapeutics by the self assembling process of its own is used for neoplasm targeted therapy.

Genes of interest fragment is obtained by gene chemical synthesis first, being inserted into expression using Xho I/Nde I restriction enzyme sites carries In constitution grain, it is transformed into afterwards in e. coli bl21 (DE3), the induced expression of destination protein is carried out in LB culture mediums.

1. the induced expression of fusion protein described in

1) BL21 (DE3) single bacterium colony of picking containing expression vector, in LB culture mediums (containing 100 μ g/mL ampicillins) 37 DEG C, 255rpm is cultivated to logarithmic phase；

2) bacterium solution presses 1 with culture medium:1000 dilution proportion, 37 DEG C, 255rpm overnight incubations reach its OD600 value 0.5~0.6；Strain is diluted to fresh culture, 37 DEG C, 255rpm cultures 2h according to 1 ︰ 1；

3) induce at a temperature of 37 DEG C, the final concentration of 1mM of IPTG；

4) after induction 4h, 1mL bacterium solutions are taken out, supernatant is removed after 12,000rpm centrifugation 1min, add the 100 μ L resuspended bacterium of PBS Body, sample 12%SDS-PAGE gel electrophoresis analysis.

2. ammonium sulfate precipitation just pure fusion protein

1) bacterium solution for terminating will be induced with collects thalline after 4000rpm centrifugations 10min, with 10mM Tris, 0.5% Triton pH8.0 cushioning liquid blows afloat thalline.

2) with the method for ultrasonication by bacterial cell disruption, power 300W altogether ultrasound 30min by bacterial cell disruption, now bacterium solution into Viscous semi-transparent shape, 12000rpm centrifugation 10min, collects supernatant (destination protein is soluble protein in supernatant).

3) supernatant is divided in 1.5ml centrifuge tubes, often the μ L of pipe 500, respectively with 10%, 20%, 30%, 40%, 50% saturated ammonium sulfate solution carries out albumen and saltouts, and saltout 30min at 4 DEG C, 12,000rpm centrifugation 10min, collects heavy Form sediment, the precipitation of each concentration takes sample carries out the distribution of SDS-PAGE detection fusions albumen.

3.DEAE ion-exchange chromatogram purifications

1) base fluid balance pillar：Pillar is rinsed with 10 times of base fluids of column volume Tris-Cl containing 10mM (pH8.0), to outflow The pH of liquid is consistent with base fluid pH.

2) loading：Albumen CE after the sample that will be collected into i.e. ammonium sulfate precipitation dialysis is with the flow velocity of 3mL/min Loading.

3) loading wash-out：Chromatographic column is rinsed with 5 base fluids of column volume, the registration to Ultraviolet Detector comes back to base Line.

4) eluent I is eluted：Rushed with 5 times of eluents of column volume I (Tris-Cl containing 10mM (pH8.0), 50mM NaCl) Pillar is washed, eluting peak is collected.

5) eluent II is eluted：With 5 times of eluents of column volume II (Tris-Cl containing 10mM (pH8.0), 100mM NaCl pillar) is rinsed, eluting peak is collected.

6) eluent III is eluted：With 5 times of eluents of column volume III (Tris-Cl containing 10mM (pH8.0), 200mM NaCl pillar) is rinsed, eluting peak is collected.

7) eluent III is eluted：With 5 times of eluents of column volume IV (Tris-Cl containing 10mM (pH8.0), 300mM NaCl pillar) is rinsed, eluting peak is collected.

8) eluent III is eluted：With 5 times of eluents of column volume IV (Tris-Cl containing 10mM (pH8.0), 400mM NaCl pillar) is rinsed, eluting peak is collected.

4. fusion protein molecule sieve Sepharose CL 4B purifying

The liquid in pillar is slowly declined, when liquid level drops to gel interface, sample is added drop-wise in glue surface, can not make Liquid level is stirred, and treats that it is deposited to below glue surface, adds the deionized water of degassing, is slowly added to, until pillar is filled, is connected Inlet adapter；Post is crossed with constant flow velocity 1.5mL/min.2mL/ pipes collect efflux.

5. albumen dialysis and concentration

1) bag filter that will fill protein solution is put into the good dialyzate of precooling, is placed on 4 DEG C of refrigerators and is dialysed；

2) whole dialysis procedure changes 4 dialyzates, until dialysis is complete；

3) the complete protein solution that will dialyse is put into new large beaker, plus polyethylene glycol powder is covered on bag filter, Bag internal solvent is oozed out and is sucked rapidly by polyethylene glycol, and beaker is placed in into 4 DEG C of refrigerators is concentrated；

4) in concentration process, dry polyethylene glycol powder is repeatedly added on a small quantity, to increase concentration speed, polyethylene glycol By more renewing after water saturation until reaching required concentration volume；

5) after concentration terminates, bag filter is rinsed well with distilled water, the protein solution that will be concentrated takes out, legal with BCA Protein concentration is determined in measurement.

6. medicine is loaded

1) dissociation of virus-like particle

Dissociation solution is prepared：The urea (urea) of 50mM Tris-HCl, pH 8.0,150mM NaCl, 8.0M.Take 10 μ L diseases Malicious sample particle (~10mg/mL) is incubated 3h with the above-mentioned dissociation solution for preparing at 4 DEG C.

2) restructuring of virus-like particle

Reassembly buffer liquid 1 ︰ 50mM Tris-HCl, pH 8.0,150mM NaCl, 10% glycerine, 1% glycine；Restructuring is slow Fliud flushing 2 ︰ 50mM Tris-HCl, pH 8.0,150mM NaCl, 1% glycine.Virion solution after dissociation is moved into and is divided It is placed in 100mL reassembly buffers liquid 1 and the dialyzed overnight at 4 DEG C in the sub bag filter measured as 8000~14000Da, after 12h It is replaced by reassembly buffer liquid 2.Dialysis time is total up to 48h, and period changes buffer solution 2 times.

3) medicine is loaded

Take 1mL virus-like particles (~1mg/mL) and 2.5h, overall solution are incubated at 4 DEG C with the dissociation solution for preparing in advance Product is 10mL.Now to 500mg adriamycins are added in above-mentioned dissociation solution, with slight concussion, gained mixed solution continues training altogether Support 30min.Afterwards, in moving it to the bag filter that molecular weight is 8000~14000Da, it is initially positioned in reassembly buffer liquid 1 simultaneously The dialyzed overnight at 4 DEG C, is replaced by reassembly buffer liquid 2 after 12h.Dialysis time is total up to 48h, and period changes buffer solution 2 times.Institute Must be mounted with the virus-like particle of medicine is stored in -20 DEG C and protect.

Demonstrated by SDS-PAGE electrophoresis, protein monomer molecular weight (Fig. 1) is obtained.Transmission electron microscope demonstrates institute The virus-like particle of structure can effectively be assembled into the homogeneous monodisperse particles of pattern, such as Fig. 2.Laser particle analyzer is further demonstrated Its particle diameter is the grain structure of 30~40nm, such as Fig. 4.High-efficient phase chromatogram analysis (Fig. 5) confirms that the present embodiment obtains virus-like Particle can efficient loading chemotherapeutic drugs Doxorubicin.

Its nucleotides sequence is classified as：

atgGACATCGACCACTACAAAGAATTCGGTGCTTCCGTTGAACTGCTGTCCTTCCTGCCGTCCGACTTCTTCCCGTC CATCCGTGACCTGCTGGACACTGCTTCCGCTCTGTACCGTGAAGCTCTGGAATCCCCGGAACACTGCTCCCCGCACC ACACTGCTCTGCGTCAGGCTATCCTGTGCTGGGGTGAACTGATGAACCTGGCTACTTGGGTTGGTTCCAACCTGGAA GACCCGGCTTCCCGTGAACTGGTTGTTGGTTACGTTAACGTTAACATGGGTCTGAAAATCCGTCAGATCCTGTGGTT CCACATCTCCTGCCTGACTTTCGGTCGTGAAACTGTTCTGGAATACCTGGTTTCCTTCGGTGTTTGGATTCGTACTC CGCCGGCTTACCGTCCGCCGAACGCTCCGATCCTGTCCACTCTGCCGGCCGGTTCCTGGCTAAGGGACATCTGGGAC TGGATATGCGAGGTGCTGAGCGATTTTAAGACCTGGCTGAAGGCCAAGCTCATGCCAACCATGCACCACCACCACCA CCAC

Its coding amino acid sequence be：

MDIDHYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLA TWVGSNLEDPASRELVVGYVNVNMGLKIRQILWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPAG SWLRDIWDWICEVLSDFKTWLKAKLMPTMHHHHHH

Embodiment 2

In the present embodiment, on the basis of original embodiment 1, inserted between 72~82, its principal immune area Cancer target polypeptide sequence.By expression and purification method same as Example 1 and step, realize for tumour cell table The tumor-targeting that the integrin receptor high-affinity of face overexpression is combined.Through self assembly same as Example 1 Journey loads antineoplastic adriamycin, obtains antineoplastic polypeptide Nano medication system.

Confirm that the virus-like particle that the present embodiment is obtained is expected molecular weight (Fig. 1) by SDS-PAGE electrophoresis means.

Form and diameter characterization (Fig. 3) are carried out to the virus-like particle for obtaining using transmission electron microscope and laser particle analyzer, Result shows that the virus-like particle for preparing is spherical in shape, and granular size is more uniform, and hydration particle diameter distribution is 30~40nm (figures 4), average grain diameter is about 36.5 ± 2.1nm.

Fig. 6 illustrates the high performance liquid chromatography result of embodiment 2 after loading adriamycin.

Its nucleotides sequence is classified as：

atgGACATCGACCACTACAAAGAATTCGGTGCTTCCGTTGAACTGCTGTCCTTCCTGCCGTCCGACTTCTTCCCGTC CATCCGTGACCTGCTGGACACTGCTTCCGCTCTGTACCGTGAAGCTCTGGAATCCCCGGAACACTGCTCCCCGCACC ACACTGCTCTGCGTCAGGCTATCCTGTGCTGGGGTGAACTGATGAACCTGGCTACTTGGGTTGGTTCCAACCTGGAA GACGGTACCTCCGGTTCCTCCGGTTCCGGTTCCGGTGGTTCCGGTTCCGGTGGTGGTGGTCGAGGTGACGGTGGTGG TGGTTCCGGTTCCGGTGGTTCCGGTTCCGGTTCCTCCGGTTCCACCGGTTCCCGTGAACTGGTTGTTGGTTACGTTA ACGTTAACATGGGTCTGAAAATCCGTCAGATCCTGTGGTTCCACATCTCCTGCCTGACTTTCGGTCGTGAAACTGTT CTGGAATACCTGGTTTCCTTCGGTGTTTGGATTCGTACTCCGCCGGCTTACCGTCCGCCGAACGCTCCGATCCTGTC CACTCTGCCGGCCGGTTCCTGGCTAAGGGACATCTGGGACTGGATATGCGAGGTGCTGAGCGATTTTAAGACCTGGC TGAAGGCCAAGCTCATGCCAACCATGCACCACCACCACCACCAC*

The amino acid sequence of its coded polypeptide is：

MDIDHYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLA TWVGSNLEDGTSGSSGSGSGGSGSGGGGRGDGGGGSGSGGSGSGSSGSTGSRELVVGYVNVNMGLKIRQILWFHISC LTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPAGSWLRDIWDWICEVLSDFKTWLKAKLMPTMHHHHHH

Embodiment 3

The present embodiment purpose is to determine based on grain of the hepatitis B virus core protein virus-like particle in slightly acidic solution Footpath.

By embodiment 1 and the virus-like particle obtained in embodiment 2 and different pH value (8.0,7.4,6.4,5.8,5,4) PBS is incubated 12h altogether, and its hydration particle diameter is determined using laser particle analyzer.As shown in figure 3, being less than or equal to 5 in pH When, prepared virus-like particle particle diameter occurs substantially to become big, more than 100nm (Fig. 7).And under physiological ph conditions, virus-like There is no significant change (Fig. 8) in the particle diameter of grain.In an acidic solution, prepared virus-like particle is no longer nanometer for this explanation It is spherical, but due to its internal protonation so that nanometer spherical structure produces repulsive force, causes it that irreversible depolymerization occurs.

Embodiment 4

The present embodiment purpose is to determine the cancer target based on HBcAg virus-like particle, pH sensitivities in vitro Reconstituted drug carrier protein in vitro to the targeting of tumour cell.

By U87MG cells with 5 × 10⁵The density in cells/ holes is inoculated into 6 well culture plates, is incubated 24h and is treated cell attachment After growth, old nutrient solution is discarded, give the adriamycin that is loaded with diluted through serum-free medium in embodiment 1 and embodiment 2 Virus-like particle.Doxorubicin concentration is 5 μ g/mL, and 0.5h, 1h and 2h are incubated respectively.After incubation terminates, old nutrient solution is discarded, used Ice-cold PBS is rinsed 3 times.100 μ L pancreatin are added per hole, cell is collected after terminating digestion, be centrifuged, discard upper liquid, add 500 μ L After PBS cell dispersions, determined with flow cytometer.As shown in figure 9, the recombinant virus sample particle of embodiment 2 be easier it is thin by tumour Born of the same parents (U87Mg) absorb.

Embodiment 5

The U87MG cells in exponential phase are taken, with 7 × 10³Individual/hole is inoculated in 96 orifice plates, after 37 DEG C of culture 24h, It is separately added into load medicine recombinant virus sample particle, 3 parallel holes of each concentration, while setting in the embodiment 1 of 100 μ L and embodiment 2 Blank control group.Put after being incubated 48h in incubator, suck pastille nutrient solution, the MTT solution (0.5mg/ of 100 μ L is added per hole ML), continue to be incubated 4h, suck MTT solution, the DMSO of 100 μ L is added per hole, 2min is to dissolve bluish violet crystal for vibration, most The absorbance (OD) per hole is determined under 492nm wavelength with ELIASA afterwards, and cell survival rate is calculated using following formula (such as Figure 10).

Embodiment 6

In the present embodiment, sensitising agent CG is contained for tumor photo-thermal/optical dynamic therapy by embodiment 2.Implement Virus-like particle in example 2 makes its depolymerization under the conditions of 2.5M urea, after being incubated 4~8h altogether with sensitising agent CG afterwards, In reassembly buffer liquid dialysis 48h, period changes dialyzate twice.Finally give the recombinant hepatitis B virus core for being loaded with CG Albumen.In power for 808 laser of 1W irradiate 0~300s, the embodiment 1 for being loaded with CG can be increased to 45 DEG C or so, With excellent light thermal property (such as Figure 11), tumor photo-thermal and optical dynamic therapy are can be applied to.

The present invention shortcomings such as overcome Conventional nano vector tumors targeting, poor biocompatibility, immunogenicity strong.This The problems such as invention also solves big classic chemotherapy drug medication dosage, strong toxicity, patient tolerability difference simultaneously.Preparation side of the invention Method is simple, suitable for mass production, have selectivity to tumour cell, and tumour can be effectively suppressed after being loaded with chemotherapeutics, Sensitising agent can be loaded carries out tumor photo-thermal dynamic therapy altogether.

Sequence table

<110>Xiamen University

<120>Reconstituted drug carrier peptide gene and preparation method and application

<130> 2017

<160> 4

<170> PatentIn version 3.3

<210> 1（The DNA sequence dna of embodiment 1）

<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（The DNA sequence dna of embodiment 2）

<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（The protein sequence of embodiment 1）

<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（The protein sequence of embodiment 2）

<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. the cancer target based on HBcAg virus-like particle, pH sensitivities, the reconstituted drug with self assembly characteristic are carried Body protein gene, it is characterised in that the expressing gene of its carrier protein includes coding hepatitis B virus core protein amino acids, volume Code target tumor polypeptide sequence and link peptide, the α spirals of coding hydrophobic peptides NS5A (1~31aa) amphiphilic, coding pH are sensitive Polyhistidyl polypeptide gene order, and by genetic engineering, prepare purifying and obtain.

2. the cancer target based on HBcAg virus-like particle, pH sensitivities, the reconstituted drug with self assembly characteristic are carried The gene order of body protein, it is characterised in that the polypeptide sequence of its gene code target tumor is located at hepatitis B virus core protein disease Position between 72nd~92 amino acids of malicious sample particle；Hydrophobic peptides NS5A (1~31aa) amphiphilic is encoded in its gene α spirals be located at hepatitis B virus core protein C-terminal 140~183 amino acid between；The polypeptide of the polyhistidyl of its gene code Positioned at hydrophobic peptides NS5A (1~31aa) albumen least significant end.

3. the cancer target based on HBcAg virus-like particle, pH are sensitive, special with self assembly as claimed in claim 2 Property reconstituted drug carrier protein gene order, it is characterised in that the coding target tumor polypeptide, coding hydrophobic peptides, Any at least two and hepatitis B virus core protein gene in the gene order of coding pH sensitive polyhistidyl peptide molecule Sequence is combined.

4. based on the cancer target of HBcAg virus-like particle, pH it is sensitive, with self assembly characteristic reconstituted drug carrier Albumen, it is characterised in that the expression soluble chimeric albumen of recombinant protein is completed by e. coli bl21 (DE3).

5. the cancer target based on HBcAg virus-like particle, pH are sensitive, special with self assembly as claimed in claim 4 Property reconstituted drug carrier protein, it is characterised in that its Bacillus coli expression method include following condition：

1) 10~37 DEG C of 0.5~4h of culture in LB broth bouillons；

2) using the expression of IPTG inducible proteins, IPTG concentration is 0.1~1mM, and incubation time is 4~16h, cultivation temperature is 16~ 37℃；

3) shaking speed is 120~300r/min during cultivating.

6. based on the cancer target of HBcAg virus-like particle, pH it is sensitive, with self assembly characteristic reconstituted drug carrier The purification process of protein drug, it is characterised in that comprise the following steps：

3) ion exchange column chromatography purification, using DEAE resin columns, mobile phase is the buffer solution of 10mM Tris-Cl (pH8.0)；

4) molecular exclusion chromatography purifying, obtain the cancer target based on HBcAg virus-like particle, pH it is sensitive, with from Assembling characteristic reconstituted drug carrier protein medicine.

7. the cancer target based on HBcAg virus-like particle, pH are sensitive, special with self assembly as claimed in claim 6 Property reconstituted drug carrier protein medicine purification process, it is characterised in that in step 4) in, molecular exclusion chromatography purifying Purified using Sepharose CL 4B molecular sieve column chromatographies, mobile phase is slow for 10mM Tris-Cl, 150mM NaCl's, pH8.0 Fliud flushing.

8. the cancer target based on HBcAg virus-like particle, pH are sensitive, special with self assembly as claimed in claim 4 Property reconstituted drug carrier protein, it is characterised in that its depolymerisation conditions is as follows：

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

The cancer target based on HBcAg virus-like particle, pH are sensitive, carried with self assembly characteristic reconstituted drug The restructuring condition of body protein is as follows：

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

Described by described virus-like particle depolymerization, regrouping process carries out medicine loading again, the virus-like particle of medicine and depolymerization Mix, virus-like particle and medicine mol ratio are 1 ︰ (50~10000), adjustment solution ph to medicine pKa in itself is incubated The time is educated for 0.5~4h.

9. based on the cancer target of HBcAg virus-like particle, pH it is sensitive, with self assembly characteristic reconstituted drug carrier Albumen is applied in antineoplastic polypeptide Nano medication and tumor photo-thermal/optical dynamic therapy medicine is prepared.

10. application as claimed in claim 9, it is characterised in that the preparation method of the antineoplastic polypeptide Nano medication includes following Step：

1) obtain have cancer target of the coding based on HBcAg virus-like particle, pH sensitive by gene synthesis The gene order of reconstituted drug carrier protein, expression vector plasmid pEt43.1 is building up to by Xho I/Nde I restriction enzyme sites In (a)；

3) by step 2) vector plasmid for obtaining is collected after expression in escherichia coli destination protein is isolated and purified, by regulating and controlling it Self assembling process, loads chemotherapeutics, is made antineoplastic polypeptide Nano medication.