CN108424924A - Fusion protein HFBI-RGD genes and albumen - Google Patents

Fusion protein HFBI-RGD genes and albumen Download PDF

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CN108424924A
CN108424924A CN201810148650.9A CN201810148650A CN108424924A CN 108424924 A CN108424924 A CN 108424924A CN 201810148650 A CN201810148650 A CN 201810148650A CN 108424924 A CN108424924 A CN 108424924A
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rgd
hfbi
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CN108424924B (en
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王泽方
肖云杰
王斌
杨海涛
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Tianjin University
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Abstract

The invention discloses fusion protein HFBI RGD genes and albumen, the nucleotide sequence of fusion protein HFBI RGD genes is shown in SEQ ID NO.1.The fusion protein HFBI RGD of the present invention have the characteristics that dissolubility is good, with short production cycle and yield is high.The HFBI RGD fusion proteins of the present invention had not only had the structure and function of HFBI, but also active with cell adhesion, and this fusion protein can navigate to the tumor locus containing integrin and have preferable targeting.HFBI RGD fusion proteins, which can still act as emulsifier, makes small oil droplet be stabilized in water, dispersant dispersed graphite alkene, carbon nanotube and hydrophobic fluorescence probe can be used as, solve hydrophobic material scattering problem, and since the fusion protein includes small peptide RGD, this assigns the fusion protein integrin targeting again, can be used for the fields such as diagnosing tumor, drug delivery.

Description

Fusion protein HFBI-RGD genes and albumen
Technical field
The invention belongs to the genetic engineering fields of albumen, more particularly to a kind of fusion protein HFBI-RGD genes and its system Preparation Method.
Background technology
Epiphyte hydrophobic protein (hydrophobins) is to contain abundant hydrophobic amino acid by what filamentous fungi secretion generated Molecular weight be about 10kDa Amphiphilic proteins, have special physics and chemical property, risen in contact of the mycelia with air To important function.Epiphyte hydrophobic protein has hydrophobic and hydrophilic two parts, can be formed about in interface by self assembly The Amphiphilic proteins film of 10nm, these films can make hydrophilic and hydrophobic surface take a turn for the worse so that hydrophilic substance has hydrophobic Property, lyophobic dust has hydrophily.According to the dissolubility for the protein film that hydrophobin is formed, epiphyte hydrophobic protein is divided into I types With II types.Wherein, II types hydrophobin includes HFBI.Epiphyte hydrophobic protein HFBI is considered as that known surface-active is highest One of albumen.Epiphyte hydrophobic protein was widely used in the world in recent years, and in different field, there are many different reasons Value and application value, such as personal care product and lotion, isolation technics, biosensor and electrode, biomaterial etc..
Cancer has become one of the great illness for threatening human life, therefore studies the method for diagnosing and treating cancer As the research focus of many researchers.In order to preferably detect tumour, in order to improve the utilization rate of drug, tumor target The research of tropism becomes main research direction.RGD is three containing arginine-glycine-aspartic acid (Arg-GlyAsp) Peptide sequence.Integrin is the membrane receptor protein family of heterodimer composition, can specific recognition RGD.RGD peptide is deposited extensively It is in organism there is that small molecular mass, shearing force dispersion and organic solvent influence are smaller.Integrin is sub- by α and β Base forms, and is located at cell surface.So far it has been found that be made of 18 kinds of different α subunits and 8 kinds of different β subunits 24 Kind heterodimer.Wherein αvβ3It studies relatively broad, mainly works in Tumor Angiongesis and Nasopharyngeal neoplasms. The recognition site that RGD is integrin with the interaction of its ligandin, mediated cell and extracellular matrix and intercellular sticks work With, while having the function of signal transduction, to mediate many important vital movements.The integrin of RGD and tumor cell surface αVβ3There is strong compatibility, be used to transmit drug, the targeting vector etc. of gene to tumour cell.
Currently, there has been no the reports that a kind of epiphyte hydrophobic protein HFBI and RGD sequence are prepared into fusion protein.Although right For hydrophobin have many important theories and application value, but to be applied to molecule diagnosis, drug delivery and There are still deficiencies for targeting vector etc..
Invention content
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of fusion protein HFBI-RGD genes.
Second object of the present invention is to provide a kind of albumen of fusion protein HFBI-RGD gene expressions.
Third object of the present invention is to provide a kind of plasmids containing fusion protein HFBI-RGD genes.
Fourth object of the present invention is to provide a kind of recombinant bacterial strain containing fusion protein HFBI-RGD genes.
Technical scheme of the present invention is summarized as follows：
The nucleotide sequence of fusion protein HFBI-RGD genes, the gene is shown in SEQ ID NO.1.
The amino acid sequence of the albumen of fusion protein HFBI-RGD gene expressions, the albumen is shown in SEQ ID NO.2.
Plasmid containing fusion protein HFBI-RGD genes, is built with following methods：With Filamentous fungi (Trichoderma Reesei HFBI nucleotides sequences shown in SEQ ID NO.3 are classified as template in), are reversely to draw with R1 using F1 as forward primer Object carries out first round PCR, obtains the first PCR product；Using the first PCR product as template, using F1 as forward primer, with R2 For reverse primer, carry out the second wheel PCR, obtain the first sequence for containing fusion protein HFBI-RGD genes, through digestion and T4DNA ligases connect, and fusion protein HFBI-RGD is gene constructed on PET-28a carriers, obtain PET-28a-HFBI- RGD plasmids；Using PET-28a-HFBI-RGD plasmids as template, using F2 as forward primer, using R3 as reverse primer, PCR is carried out, is obtained To second of sequence containing fusion protein HFBI-RGD genes, connected through digestion and T4DNA ligases, by fusion protein HFBI-RGD is gene constructed on pPIC9k carriers, obtains pPIC9k-HFBI-RGD plasmids；
The nucleotide sequence of F1 is shown in SEQ ID NO.6；
The nucleotide sequence of R1 is shown in SEQ ID NO.7；
The nucleotide sequence of R2 is shown in SEQ ID NO.8；
The nucleotide sequence of F2 is shown in SEQ ID NO.9；
The nucleotide sequence of R3 is shown in SEQ ID NO.10；
The nucleotide sequence of fusion protein HFBI-RGD genes is shown in SEQ ID NO.1.
Recombinant bacterial strain containing fusion protein HFBI-RGD genes, is built with following methods：By pPIC9k-HFBI-RGD matter In grain electrotransformation to Pichia pastoris GS115 bacterial strain, the recombinant bacterial strain containing fusion protein HFBI-RGD genes is obtained.
Advantages of the present invention：
The present invention uses the expression system of Pichia pastoris, is changed to original HFBI genes using the method for genetic engineering It makes, the fusion protein HFBI-RGD of acquisition has the characteristics that dissolubility is good, with short production cycle and yield is high.Experiment shows 1L BMM culture mediums can obtain 15mg destination proteins HFBI-RGD.The HFBI-RGD fusion proteins of the present invention both have HFBI Structure and function, and with cell adhesion activity, this fusion protein can navigate to containing the tumor locus of integrin and have There is preferable targeting.HFBI-RGD fusion proteins, which can still act as emulsifier, makes small oil droplet be stabilized in water, can be with As dispersant dispersed graphite alkene, carbon nanotube and hydrophobic fluorescence probe, hydrophobic material scattering problem is solved, and by Include small peptide RGD in the fusion protein, this assigns the fusion protein integrin targeting again, can be used for diagnosing tumor, drug passes Defeated equal fields.
Description of the drawings
Fig. 1 is BCA Tot Prot Analysis and Screening positive colonies.
Fig. 2 is HFBI-RGD albumen high performance liquid chromatography chromatography detection figure.
Fig. 3 is HFBI-RGD protein SDS-PAGE gel detection figures.
Fig. 4 is that HFBI-RGD albumen disperses fluorescence probe BODIPY.
Fig. 5 is that the BODIPY of HFBI-RGD albumen dispersion is enriched in mouse U-87MG and HeLa tumor locus figure.
Fig. 6 is fusion protein HFBI-RGD modifying multiwall carbon nano-tubes.
Fig. 7 is that HFBI-RGD/BODIPY enters U-87MG and HeLa cell co-focusing imagings.
Specific implementation mode
Below by specific embodiment, the present invention is further illustrated.
PPIC9k and PET-28a is commercially available.Pichia pastoris GS115 bacterial strain is commercially available.
Experiment material：
(1) LB culture mediums：It prepares per 1000mL culture mediums, peptone 10g, ferment is added in the first water of 1000mL Female powder 5g, NaCl 10g, after dissolving, 121 DEG C, 0.1MPa sterilizings 20min.It is added into culture medium when configuring solid LB media 1.5% agar powder.
(2) YPD culture mediums：It prepares per 1000mL culture mediums, 20 g of peptone is added in the first water of 1000mL, Yeast powder 10g, NaCl 8g, glucose 20g.
(3) MD solid mediums：It prepares per 100mL culture mediums, is added without amino acid ferment in the first water of 100mL Female nitrogen source 1.34g, glucose 2.0g, agar 2.2g.
(4) MM solid mediums：It prepares per 100mL culture mediums, is added without amino acid ferment in the first water of 100mL Female nitrogen source 1.34g, agar 2.2g.After sterilizing, before pouring into tablet, 0.75mL absolute methanols are added.
(5) BMG culture mediums：It prepares per 1000mL culture mediums, is added without amino acid leaven in the first water of 890mL The phosphate buffer of nitrogen source 14.4g, 100mL 1M pH 6.0,10mL glycerine.
(6) BMM culture mediums：It prepares per 1000mL culture mediums, is added without amino acid leaven in the first water of 890mL The phosphate buffer of nitrogen source 14.4g, 100mL 1M pH 6.0,6mL absolute methanols.
(7) SDS-PAGE (polyacrylamide gel electrophoresis)
30% acrylamide solution (100mL)：The methylene diacrylamide of 30g acrylamides and 0.8g is dissolved in It is positioned in the distilled water of 100mL, after dissolving in 4 DEG C of brown bottles.
1.5M Tris-HCl pH=8.8 buffer solutions (1L)：It weighs 181.71g Tris to be dissolved in 800mL pure water, adjust PH to 8.8 is settled to 1L, room temperature preservation.
0.5M Tris-HCl pH=6.8 buffer solutions (500mL)：60.57g Tris are weighed to be dissolved in 400mL pure water, PH to 6.8 is adjusted, 500mL, room temperature preservation are settled to.
10% lauryl sodium sulfate (10%SDS) solution (50mL)：5g SDS are weighed, add pure water to 50mL, room temperature is protected It deposits.
10% ammonium persulfate (10%APS) solution (20mL)：2g ammonium persulfates are weighed, add pure water to 20mL, often 500 μ L of pipe Packing, -20 DEG C save backup.
1 12%SDS-PAGE separation gels of table prepare (10mL)
Table 2 SDS-PAGE concentration glue prepares (5mL)
(8) 5 × Tris- glycine running buffers (5L)：75.5g Tris, 470g glycine are weighed, 25g SDS add Pure water dissolves, and is settled to 5L.
(9) 6 × albumen sample-loading buffers：0.35M pH=6.8Tris-HCl, 10.28%W/V SDS, 36% glycerine, 5% β-mercaptoethanols, 0.012g/mL bromophenol blues, often pipe dispenses 1mL, and -20 DEG C save backup.
The structure of 1 pET-28a-HFBI-RGD of embodiment：
It is classified as mould with HFBI nucleotides sequences shown in SEQ ID NO.3 in Filamentous fungi (Trichoderma reesei) Plate, using R1 as reverse primer, carries out first round PCR, obtains the first PCR product using F1 as forward primer；With the first PCR Product is template, using F1 as forward primer, using R2 as reverse primer, carries out the second wheel PCR, obtains the first and contain fusion protein The sequence of HFBI-RGD genes is connected through digestion and T4DNA ligases, and fusion protein HFBI-RGD is gene constructed to PET- On 28a carriers, PET-28a-HFBI-RGD plasmids are obtained.It is as follows：
A) digestion system
B) condition
I. target gene digestion 1h, 37 DEG C.
Ii. plasmid enzyme restriction 1h, 37 DEG C.
Iii. 80 DEG C of inactivation 5min after digestion.
C) mould is classified as with HFBI nucleotides sequences shown in SEQ ID NO.3 in Filamentous fungi (Trichoderma reesei) Plate, the linker and RGD sequence of additional design carry out PCR to obtain HFBI-RGD sequences.Wherein linker gene orders are such as Shown in SEQ ID NO.4, RGD sequence is as shown in SEQ ID NO.5.It is as follows：
According to the gene order of Filamentous fungi (Trichoderma reesei) HFBI, RGD and Linker, pass through primer Software designs sense primer and downstream primer, is respectively designated as F1, R1, R2.During first time PCR, sense primer is F1, downstream primer R1, template are the plasmid containing HFBI gene orders.Second of PCR system is as first time system, no With downstream primer R2, template is the PCR product of first time.Pass through twice PCR in PCR instrument, obtains target gene sequence Row.PCR system and program setting are as shown in the table：
3 PCR system of table
4 PCR instrument setting program of table
PCR terminates, and the FD Green Buffer of 1 μ L are added in the PCR product of 10 μ L, is blown and beaten uniformly with pipettor, Then sample is added in 2% Ago-Gel prepared and is separated by electrophoresis, target fragment is cut under ultraviolet lamp, used DNA Ago-Gels QIAquick Gel Extraction Kit (Tiangeng) is recycled.
Target gene fragment and carrier PET-28a after above-mentioned PCR is used respectively FastDigest EcoRI and FastDigest XhoI carry out double digestion, 37 DEG C of water-bath digestion 30min, digestion products into row agarose gel electrophoresis, then into Row glue recycles, and obtains the target gene fragment containing cohesive end and PET-28a carriers.50 μ L of digestion system, each component and content As shown in table 5：
5 digestion system of table
Above-mentioned product is subjected to glue recycling with plastic recovery kit, obtain target gene fragment containing cohesive end and PET-28a carriers, and by it according to 5:The ratio of 1 molar ratio is added in linked system, and 22 DEG C of connection 2h obtain PET- 28a-HFBI-RGD plasmids, 20 μ L of linked system, each component and content are as shown in table 6：
6 linked system of table
The structure of 2 pPIC9k-HFBI-RGD of embodiment：
1) select carrier for pPIC9k, restriction enzyme site is NotI and EcoRI, and design primer carries out PCR, PCR system such as table 3 It is shown.Design of primers is F2, R3.The plasmid PET-28a-HFBI-RGD that above-described embodiment 1 is built can be obtained as template, PCR To second of sequence containing fusion protein HFBI-RGD genes that restriction enzyme site is NotI and EcoRI.
2) double digestion is carried out respectively to above-mentioned PCR product and carrier pPIC9k with restriction enzyme NotI and EcoRI, made There is cohesive end in target gene fragment and carrier.Specific digestion system and method are as follows：
3) target gene fragment of toughness end and carrier pPIC9k are attached using T4DNA ligases, are connected System is as shown in upper table 6.
4) it is verified using conversion and double digestion, obtained positive findings is subjected to sequence verification.Finally obtain pPIC9k- HFBI-RGD builds successfully plasmid.
3 fusion protein HFBI-RGD positive colonies of embodiment screen and expression and purification：
1) recombinant expression carrier will be inserted into higher copy in Yeast genome in yeast cell, need be transferred to ferment It is linearized before mother cell.The restriction enzyme site analytic function pair provided in primer-design software Primer 5.0 is provided PPIC9k and HFBI-RGD genes carry out restriction enzyme site analysis, are grasped in pichia yeast expression system in conjunction with Ivitrogen companies The restriction enzyme site information provided in handbook is provided, linearization for enzyme restriction is finally carried out to carrier with restriction enzyme SalI.Digestion line The reaction system of property is as shown in table 7.This system is in 37 DEG C of water-bath digestion 2h.Take 5 μ L products into row agarose gel electrophoresis, inspection Survey whether digestion is complete, then the band of product is subjected to glue recycling.
Table 7 linearizes system
2) preparation of Pichia pastoris electrotransformation competence
It draws 15 μ L Pichia pastoris GS115 bacterium solutions to be forwarded in 5mL YPD Tube propagation bases, 30 DEG C, 250rpm was cultivated Night.0.1-0.5mL overnight cultures are taken from tube culture within second day, the 2L for being seeded to the fresh YPD medium containing 500mL shakes Bottle, overnight growth to OD600About 1.3-1.5.Yeast culture is taken from shaking table, 4 DEG C, it is thin to collect that 1500g centrifuges 5min Born of the same parents (are added the HEPES buffer solution of 20mL pH 8.0, add in 100mL YPD-HEPES-DTT later in 77.5mL YPD The DTT of the 1M of 2.5mL Fresh) suspension cell again in culture medium, 30 DEG C are cultivated 15min on shaking table；It is taken from shaking table Lower cell, ice bath half an hour；4 DEG C, 1500g centrifuges 5min to collect cell, then the aqua sterilisa suspension cell being pre-chilled with 250mL； As above centrifugation, then the 1M sorbitol aqueous solution suspension cells with 20mL precoolings；As above centrifugation, the 1M sorbierite water being pre-chilled with 1mL Solution suspension cell, until final volume about 1.5mL；As above centrifugation, it is standby in ice bath with 500 μ L precooling ultra-pure waters again suspension cell With.
3) electrotransformation of Pichia pastoris
The pPIC9k-HFBI-RGD of 5-20 μ g linearisations is added in 80 μ L competent cells, the 0.2 of precooling is transferred to In cm electricity revolving cups, 5min is placed on ice；After 1.5kV electric shocks, immediately by the 1M sorbitol aqueous solutions of 1mL precoolings to converting cup In, then transfer them in sterile centrifugation tube, recovery 1h is stood in 30 DEG C of incubators, is divided into 200-600 μ L not equal portions, is applied In on MD tablets.Culture generates (about needing 3 days) to clone in 30 DEG C of incubators, screens Mut+/Muts phenotypes.
4) screening of Pichia pastoris positive transformant
Square grids are drawn on new MD tablets, and have marked serial number.The His grown is selected from MD tablets with sterile toothpick On+transformant to new MD tablets, a clone is chosen in each small lattice.Allow its overnight growth, second day, by 40 differences Monoclonal choose in 40 different 5mL YPD Tube propagation bases, 250rpm, 30 DEG C of overnight incubations, then by 40 Dan Ke Grand bacterium solution is inhaled 600 μ L and is added in the EP pipes equipped with 400 μ L, 50% glycerine, and glycerol stock is preserved.Remaining carry out yeast genes Group extraction and transformant PCR identifications.PCR system is as shown in table 8.
8 PCR system of table
Amplification condition is as follows：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 1min, 60 DEG C of annealing 45s, 72 DEG C of 1.5 min of extension, Totally 30 cycles, last 72 DEG C of extensions 10min.The product of PCR can be detected into row agarose gel electrophoresis by running cementing fruit Go out whether to have HFBI-RGD genes to be inserted into genome, finds positive colony.Above-mentioned primer AOX1-3 ' and AOX1-5 ' is respectively Shown in SEQ ID NO.11 and SEQ ID NO.12.
5) positive colony, which is taken temperature, reaches
It is positive colony to have 13 results by PCR verifications, this 13 monoclonals take temperature reaching.It will be previously stored The glycerol stock of this 13 positive colonies is respectively inhaled 30 μ L and is gone to respectively in the YPD culture mediums of 5mL, 30 DEG C, 250rpm incubator overnights Culture.Then the bacterium solution for drawing certain volume is gone in the BMG culture mediums of 10mL so that OD values at this time are 0.025.Often cross A period of time surveys OD values, when OD values are 2 to 6, then draw a certain amount of bacterium solution and goes in BMM so that OD values at this time It is 1.After going to BMM, every the methanol that 100 μ L are added for 24 hours as derivant, 120h is induced.Bacterium is received after the completion of induction, by bacterium Supernatant is carried out BCA Tot Prot measurement, the highest positive colony 8 of expression quantity is selected, such as Fig. 1 by its supernatant of liquid centrifuging and taking It is shown.
6) the great expression purifying of Pichia pastoris HFBI-RGD
It is reached by taking temperature, filters out the highest positive colony of expression quantity 8.Then, expression and purification is carried out with the transformant.It inhales It takes in the glycerol stock to the YPD culture mediums of 5mL of 30 μ L, 30 DEG C, 250rpm is incubated overnight.Then a certain amount of bacterium solution point is drawn Not to 5 in the 1L culture bottles of the BMG culture mediums containing 200mL so that OD values at this time are 0.025.It is every to survey after a period of time OD values once when OD values are 2 to 6, then are drawn a certain amount of bacterium solution and are gone in BMM so that OD values at this time are 1.Go to BMM Afterwards, every the methanol that 2mL is added for 24 hours 120h is induced as derivant.After the completion of induction, then centrifuging and taking supernatant carries out dense Contracting, by volume concentration to 30mL.Sample after concentration is handled, pure destination protein HFBI-RGD is obtained with HPLC. Sample treatment is as follows：To acetonitrile is added in sample to acetonitrile final concentration of 40%, be dispensed into 16000g in EP pipes, 40min from Then the heart takes supernatant to filter.Column is prepared using HPLC to be purified.HPLC purifying is as shown in Figure 2.Obtained HFBI-RGD melts Hop protein carries out SDS-PAGE electrophoresis, from electrophoretogram (Fig. 3)：HPLC system after purification, between 10kDa-15kDa, goes out An existing pure specific protein band, molecular weight match with the molecular weight of fusion protein HFBI, show HFBI-RGD Hydrophobin purifies successfully.
Embodiment 4
The preparation of compound (I)
1) to the synthesis of two pyrroles's alkene fluorine boron compound (BOD) (VI) of methoxycarbonyl group phenyl -1,3,5,7- tetramethyls
100mL single necked round bottom flask is taken, it is new to methoxycarbonyl group benzaldehyde (III) and 32mL that 0.864g (5.2mmol) is added The dichloromethane of steaming；The 2,4- dimethyl pyrroles (II) that 1mL (10.0mmol) newly steams are added.Under light protected environment, nitrogen is bubbled 30min removes the oxygen in flask；Magnetic agitation under room temperature.After bubbling, under nitrogen protection, it is slowly added to syringe 0.08mL trifluoroacetic acids continue to be protected from light.Gap is reacted, is sampled every 30min and carries out TLC analyses, after about 4h, raw material point It disappears, obtains compound (IV).2,4- dimethyl pyrroles remove nitrogen protection, with 5mL dichloromethane and 5mL after the reaction was complete Tetrahydrofuran dissolves bis- chloro- 5,6- dicyan 1,4-benzoquinone (DDQ) of 1.2g (2.6mmol) 2,3-, dropwise with constant pressure funnel (20min or so) is added in above-mentioned reaction solution, and the reaction was continued under magnetic agitation 1 hour, obtains compound (V).Measure 8.6mL Triethylamine is added dropwise, and lasts about 10min；Reaction solution 5min is cooled down with ice-water bath later, it is borontrifluoride that 8.6mL is then added dropwise Borate ether, duration about 20min.Ice-water bath is removed in half an hour recession, and magnetic agitation is protected from light at room temperature.After reacting 3h, newly The material concentration of generation no longer changes, and stops reaction.Reaction solution is washed with water three times, anhydrous sulphur is added in dichloromethane extraction Sour sodium is dried overnight.By obtained material first with short silicagel column initial gross separation, the tar fraction of atropurpureus is removed completely；It is spin-dried for gained The darkviolet crude product solution arrived.Column chromatography for separation selects 300-400 mesh silica gel, dichloromethane：Petroleum ether=2:1 (v/v) drenches It washes, picks up the eluant, eluent of second orange-yellow colour band, be spin-dried for, recrystallized with dichloromethane and n-hexane, obtain red powder Last shape solid BOD (compound VI) 210mg (0.524mmol), yield 12%.1HNMR(400MHz,CDCl3):δ8.18(d, J=8.0Hz, 2H), 7.41 (d, J=8.0Hz, 2H), 5.99 (s, 2H), 3.97 (s, 3H), 2.56 (s, 6H), 1.36 (s, 6H).MALDI-TOF-MS:[M]+calcd for C21H21BF2N2O2, 382.1660,found 382.30.
2) synthesis of purpose BODIPY derivatives (compound I)
50mL single necked round bottom flask is taken, 20mg (0.0524mmol) compound BOD (compound VI) and 42.7 mg is added Two Bian (compound VII) of (0.157mmol) N- ethyl -3- formoxyls, is dissolved in 3mL solvent benzols, while with syringe to solution Middle addition catalyst piperidines and glacial acetic acid, each 0.1mL.The condensing reflux at 90 DEG C is protected from light lower magnetic agitation.In reaction process, TLC detects reaction process.After 1h, reaction solution becomes brick-red, and color is more and more deeper, and after 2.5h, reaction solution gradually becomes Green, the reaction was continued, if solvent is reduced, adds solvent and catalyst piperidines, glacial acetic acid, after reacting 11-12 h, raw material and production Object concentration no longer changes substantially, stops reaction, is protected from light natural cooling.Reaction solution, dichloromethane extraction, anhydrous slufuric acid is washed with water Sodium is dried, and product is spin-dried for, column chromatography for separation, eluant, eluent is dichloromethane:Petroleum ether=2:1 (v/v) receives blue color, It is recrystallized with dichloromethane and n-hexane, obtains dark green solid 18.7mg, products collection efficiency 50%.1HNMR(400MHz, DMSO):δ 9.37 (s, 8.37Hz, 2H), 8.23 (d, J=7.7Hz, 4H), 7.86 (4m, 7.86Hz, 4H), 7.52 (m, J= 13.8Hz, 6H), 7.46 (m, J=8.2Hz, 4H), 7.32 (t, J=7.4Hz, 2H), 6.74 (s, 2H), 4.34 (t, J= 7.1Hz,4H),4.02(s,3H),1.90(m,4H)；MALDI-TOF-MS:[M]+calcd for C55H53BF2N4O2 850.4230,found 848.0673.(compound I)
Embodiment 5
Fusion protein HFBI-RGD modifying multiwall carbon nano-tubes
(1) the accurate fusion protein HFBI-RGD (SEQ ID NO.2) for weighing 1mg adds distilled water to 500 μ L (fusion eggs The concentration 2mg/mL of white HFBI-RGD)；
(2) the fusion protein HFBI-RGD solution that 75 μ L steps (1) are prepared is drawn, PBS (pH=7.4) to 1mL is added；
(3) the fusion protein HFBI-RGD solution for using step (2) to obtain is mixed with 1mg multi-walled carbon nanotubes (MWCNTs), Ultrasound is carried out using probe ultrasound system, power 180W, ultrasonic 1h obtain the HFBI-RGD/MWCNTs of stable dispersion, see Fig. 6.
Embodiment 6
Fusion protein HFBI-RGD modification BODIPY derivatives (compound I)
(1) the accurate fusion protein HFBI-RGD (SEQ ID NO.2) for weighing 1mg adds distilled water to 500 μ L (fusion eggs The concentration 2mg/mL of white HFBI-RGD)；
(2) the fusion protein HFBI-RGD solution that 75 μ L steps (1) are prepared is drawn, PBS (pH=7.4) to 1mL is added；
(3) the fusion protein HFBI-RGD solution for using step (2) to obtain is mixed with 1mgBODIPY derivatives, uses probe Ultrasound Instrument carries out ultrasound, and power 180W, ultrasonic 1h obtain the HFBI-RGD/BODIPY of stable dispersion, such as Fig. 4.
Embodiment 7
The HFBI-RGD/BODIPY for the stable dispersion that embodiment 6 obtains is used for living imaging
1) cell fluorescence is imaged
Using DMEM medium culture Hela cells, using 1640 medium culture U-87MG cells of RPMI, wait for that cell is given birth to When length is to 60% or so plating efficiency, using trypsin digestion cell, it is added separately to training in 48 orifice plates that cell climbing sheet is added in advance Support cell.Plating cells density is respectively：Hela cells, per 10,000 cell of hole；U-87MG cells, per 8000 cell of hole.Add per hole Enter original 200 μ l culture cells of culture medium respectively used.After cell culture for 24 hours, original fluid is abandoned in suction, respectively with containing 1 μM The continuous culture of Cell relay is added in the respective culture medium of HFBI-RGD/BODIPY.4h is cultivated, cell climbing sheet is contaminated using DAPI Color, mounting.Specific mounting process is as follows：
(1) after cultivating 4h, the former culture medium containing HFBI-RGD/BODIPY is abandoned in suction, is cleaned using PBS (pH=7.4) thin Born of the same parents are three times；
(2) 100 μ l, 4% paraformaldehydes are added into cell and fix 4min；
(3) PBS (pH=7.4) is added to clean 3 times；
(4) DAPI is added into cell and dyes 5min, cleaned 3 times using PBS (pH=7.4)；
(5) anti-fluorescence quenching is added on glass slide, on it by the cell climbing sheet handled well back-off, use is transparent Nail oil seal, 4 DEG C of preservations；
(6) it is taken pictures using Laser Scanning Confocal Microscope, the results are shown in Figure 7.
2) mouse living imaging
The Female nude mice (nude mouse) of 20 6 week old of purchase is raised, and then connects U-87MG and Hela cells Kind in nude mice right front leg oxter, it is allowed to grow tumour, then by HFBI-RGD/BODIPY by tail vein injection to mouse body, Carry out living imaging.It is as follows：
(1) recovery Hela and U-87MG cells, are cultivated, are passed on by cell, and sufficient amount of Hela and U- is obtained 87MG cells.
(2) cell suspending liquid being made with PBS (pH=7.4) in cultured cell, U-87MG cell volumes are 1mL, Hela Cell suspension volumes are 800 μ L.
(3) tumour cell is injected in nude mice oxter.Inject the tumour cell of 50 μ L, about 1x10 in every nude mice oxter7It is a.
(4) after being inoculated with tumour cell, the general 2-3 weeks time of nude mice is raised, waits for that its tumor size reaches 5-8mm progress Test.
(5) after tumour is grown well, BODIPY-HFBI-RGD samples are prepared to nude mice tail vein injection.Every rat tail It is injected intravenously the sample of 200 μ L (HFBI-RGD/BODIPY containing 17.7nmol).
(6) 2h, 4h, 6h, 8h respectively after injection, for 24 hours, 48h and 72h observe mouse with living imaging instrument, The results are shown in Figure 5.
The BODIPY fluorescence probes derivative fluorescence probe has a good near-infrared fluorescent characteristic, launch wavelength 732nm, It can be applied to carry out living imaging in cell or organism, reduce the background interference of cell or tissue.
Fusion protein HFBI-RGD gene order SEQ ID NO.1 (artificial synthesized)
AGCAACGGCAACGGCAATGTTTGCCCTCCCGGCCTCTTCAGCAACCCCCAGTGCTGTGC CACCCAAGTCCTTGGCCTCATCGGCCTTGACTGCAAAGTCCCCTCCCAGAACGTTTACG ACGGCACCGACTTCCGCAACGTCTGCGCCAAAACCGGCGCCCAGCCTCTCTGCTGCGT GGCCCCCGTTGCCGGCCAGGCTCTTCTGTGCCAGACCGCCGTCGGTGCTGGTGGAGGA GGTTC TGGCGGTGGAGGTTCTCGTGGTGATTGA
Fusion protein HFBI-RGD amino acid sequence SEQ ID NO.2 (artificial synthesized)
SNGNGNVCPPGLFSNPQCCATQVLGLIGLDCKVPSQNVYDGTDFRNVCAKTGAQPLCCVA PVAGQALLCQTAVGAGGGGSGGGGSRGD
HFBI nucleotide sequences are SEQ ID NO.3 Filamentous fungis (Trichoderma reesei)
ATGAAGTTCTTCGCCATCGCCGCTCTCTTTGCCGCCGCTGCCGTTGCCCAGCCTCTCGAG GACCGCAGCAACGGCAACGGCAATGTTTGCCCTCCCGGCCTCTTCAGCAACCCCCAGT GCTGTGCCACCCAAGTCCTTGGCCTCATCGGCCTTGACTGCAAAGTCCCCTCCCAGAAC GTTTACGACGGCACCGACTTCCGCAACGTCTGCGCCAAAACCGGCGCCCAGCCTCTCTG CTGCGTGGCCCCCGTTGCCGGCCAGGCTCTTCTGTGCCAGACCGCCGTCGGTGCTT GA Linker nucleotide sequences SEQ ID NO.4 (artificial synthesized)
GGTGGAGGAGGTTCTGGCGGTGGAGGTTCT
RGD nucleotide sequence SEQ ID NO.5 (artificial synthesized)
CGTGGTGAT
Forward primer F1 nucleotide sequence SEQ ID NO.6 (artificial synthesized)
GGAATTCAGCAACGGCAACGGCAATGTTTGCCCTCC
Reverse primer R1 nucleotide sequence SEQ ID NO.7 (artificial synthesized)
GAACCTCCTCCACCAGCACCGACGGCGGTCTG
Reverse primer R2 nucleotide sequence SEQ ID NO.8 (artificial synthesized)
CCGCTCGAGATCACCACGAGAACCTCCACCGCC
Forward primer F2 nucleotide sequence SEQ ID NO.9 (artificial synthesized)
GGAATTC AGCAACGGCAACGGCAATGT
Reverse primer R2 nucleotide sequence SEQ ID NO.10 (artificial synthesized)
TTGCGGCCGC ATCACCACGAGAACCTCCACCGCCAGA
AOX1-3 ' nucleotide sequence SEQ ID NO.11 (artificial synthesized)
GCAAATGGCATTCTGACATCC
AOX1-5 ' nucleotide sequence SEQ ID NO.12 (artificial synthesized)
GACTGGTTCCAATTGACAAG。
Sequence table
<110>University Of Tianjin
<120>Fusion protein HFBI-RGD genes and albumen
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 267
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
agcaacggca acggcaatgt ttgccctccc ggcctcttca gcaaccccca gtgctgtgcc 60
acccaagtcc ttggcctcat cggccttgac tgcaaagtcc cctcccagaa cgtttacgac 120
ggcaccgact tccgcaacgt ctgcgccaaa accggcgccc agcctctctg ctgcgtggcc 180
cccgttgccg gccaggctct tctgtgccag accgccgtcg gtgctggtgg aggaggttct 240
ggcggtggag gttctcgtgg tgattga 267
<210> 2
<211> 88
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
Ser Asn Gly Asn Gly Asn Val Cys Pro Pro Gly Leu Phe Ser Asn Pro
1 5 10 15
Gln Cys Cys Ala Thr Gln Val Leu Gly Leu Ile Gly Leu Asp Cys Lys
20 25 30
Val Pro Ser Gln Asn Val Tyr Asp Gly Thr Asp Phe Arg Asn Val Cys
35 40 45
Ala Lys Thr Gly Ala Gln Pro Leu Cys Cys Val Ala Pro Val Ala Gly
50 55 60
Gln Ala Leu Leu Cys Gln Thr Ala Val Gly Ala Gly Gly Gly Gly Ser
65 70 75 80
Gly Gly Gly Gly Ser Arg Gly Asp
85
<210> 3
<211> 294
<212> DNA
<213>Filamentous fungi (Trichoderma reesei)
<400> 3
atgaagttct tcgccatcgc cgctctcttt gccgccgctg ccgttgccca gcctctcgag 60
gaccgcagca acggcaacgg caatgtttgc cctcccggcc tcttcagcaa cccccagtgc 120
tgtgccaccc aagtccttgg cctcatcggc cttgactgca aagtcccctc ccagaacgtt 180
tacgacggca ccgacttccg caacgtctgc gccaaaaccg gcgcccagcc tctctgctgc 240
gtggcccccg ttgccggcca ggctcttctg tgccagaccg ccgtcggtgc ttga 294
<210> 4
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ggtggaggag gttctggcgg tggaggttct 30
<210> 5
<211> 9
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
cgtggtgat 9
<210> 6
<211> 36
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
ggaattcagc aacggcaacg gcaatgtttg ccctcc 36
<210> 7
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gaacctcctc caccagcacc gacggcggtc tg 32
<210> 8
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
ccgctcgaga tcaccacgag aacctccacc gcc 33
<210> 9
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ggaattcagc aacggcaacg gcaatgt 27
<210> 10
<211> 37
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
ttgcggccgc atcaccacga gaacctccac cgccaga 37
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
gcaaatggca ttctgacatc c 21
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
gactggttcc aattgacaag 20

Claims (4)

1. fusion protein HFBI-RGD genes, it is characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO.1.
2. the albumen of the fusion protein HFBI-RGD gene expressions of claim 1, it is characterized in that the amino acid sequence of the albumen It is shown in SEQ ID NO.2.
3. the plasmid of the fusion protein HFBI-RGD genes containing claim 1, it is characterized in that being built with following methods：With Rui Shi HFBI nucleotides sequences shown in SEQ ID NO.3 are classified as template in trichoderma (Trichoderma reesei), draw by forward direction of F1 Object carries out first round PCR, obtains the first PCR product using R1 as reverse primer；Using the first PCR product as template, with F1 The second wheel PCR is carried out, the first is obtained and contains fusion protein HFBI-RGD genes using R2 as reverse primer for forward primer Sequence is connected through digestion and T4 DNA ligases, and fusion protein HFBI-RGD is gene constructed on PET-28a carriers, is obtained PET-28a-HFBI-RGD plasmids；It is reversely to draw with R3 using F2 as forward primer using PET-28a-HFBI-RGD plasmids as template Object carries out PCR, obtains second of sequence containing fusion protein HFBI-RGD genes, is connected through digestion and T4 DNA ligases, Fusion protein HFBI-RGD is gene constructed on pPIC9k carriers, obtain pPIC9k-HFBI-RGD plasmids；
The nucleotide sequence of F1 is shown in SEQ ID NO.6；
The nucleotide sequence of R1 is shown in SEQ ID NO.7；
The nucleotide sequence of R2 is shown in SEQ ID NO.8；
The nucleotide sequence of F2 is shown in SEQ ID NO.9；
The nucleotide sequence of R3 is shown in SEQ ID NO.10；
The nucleotide sequence of fusion protein HFBI-RGD genes is shown in SEQ ID NO.1.
4. the recombinant bacterial strain containing claim 1 fusion protein HFBI-RGD genes, it is characterized in that being built with following methods：It will power Profit requires in 3 pPIC9k-HFBI-RGD plasmids electrotransformation to Pichia pastoris GS115 bacterial strain, obtains containing fusion protein HFBI- The recombinant bacterial strain of RGD genes.
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