CN101337992A - Fusion protein of urokinase type plasminogen activator a chain and melittin and preparation thereof - Google Patents

Abstract

The invention provides a fusion protein formed by the combination of urokinase-type plasminogen activator a chain and melittin, the preparation method thereof, and the application of the fusion protein in tumor treatment.

Description

The fusion rotein of urokinase type plasminogen activator a chain and melittin and preparation thereof

Invention field

The present invention relates to fused protein and preparation thereof, particularly relate to the fusion rotein that forms that combines of human urokinase type plasminogen activator a chain (uPAa) and melittin, and preparation method thereof and the application in oncotherapy.

Background of invention

Before more than 100 year, " magic bullet " (" magic bullet ") notion that Paul Ehrlish proposes provides initial conception for the targeted therapy of tumour.His theory is that later researchist makes up monoclonal antibody-toxin binding substances, and promptly immunotoxin and other chimeric toxins have been established theoretical basis.

The recombinant immunotoxin treatment of tumour is a kind of novel method of treatment that occurs along with the neoplasm targeted therapy development in recent years.Recombinant immunotoxin (RIT) is the toxin protein with some bacterium and plant generation, be connected to form by modes such as gene recombination or small peptide connection and antibody or some cytokine etc., have cell-targeting identification and target cell and kill and wound two kinds of functions, in order to kill and wound the novel chimeric molecule of the target cell that the surface has specific antigen or acceptor.At present, the biotherapy method of tumour has become the 4th kind of oncotherapy pattern after operation, radiation and chemotherapy.Along with increasing tumor cell surface molecule is found and as the target molecule of neoplasm targeted therapy, the status of recombinant immunotoxin in neoplasm targeted therapy is outstanding day by day, become the important means in the biotherapy of tumour.The targeted therapy of tumour is to utilize product that tumor cell surface is rich in the sudden change or the oncogene of overexpression as pharmaceutically-active target spot, therefore can not damage normal tissue or cell in oncotherapy.Targeted molecular can be the specific antibody of tumour antigen or the acceptor of tumor cell surface overexpression.And effector molecule can be radio isotope, cell toxicity medicament, small molecules cytotoxin or toxin macromole.

Urokinase type plasminogen activator (uPA) system is one of important composition of substrate degradation enzyme system.The degradation of cell epimatrix needs the participation of a series of proteolytic enzyme, and plasmin (Pm) is except that direct degradation of cell epimatrix composition, but also activator metal proteolytic enzyme participates in the extracellular protein hydrolytic action, and the uPA of tumor cell secretion is the startup thing that plasmin forms.UPA participates in the adjusting of multiple physiology and pathologic process as important physical regulatory molecule in the body, and the expression of itself also is subjected to the multiple factor to comprise the influence of hormone, somatomedin and cytokine etc.The high expression level of uPA system in tumor tissues transforms relevant with the carinogenicity of cell.Cytologic experiment shows that the activation of some cellular oncogene can directly cause the high expression level of uPA system component.Experimental study and clinical data show that all uPA in the urokinase system and uPA acceptor (uPAR) are tangible positive correlation with metastases.Generally believe that the uPA system may become the target spot that a kind of new anti metastasis is treated.Suppress the tumor cell invasion transfer by interaction and the inhibition uPA activity of disturbing uPA/uPAR, become a kind of new approaches that antineoplastic invasion shifts treatment.

UPA is with the form secretion of the precursor uPA (pro-uPA) of its strand, and the double-stranded uPA of its fibrinolytic hangs down hundred times at least.By the peptide bond K158-I159 that ruptures in the catalysis of Iys158 place, pro-uPA is converted into the uPA of double chain form.The a chain contains the growth factor-like structural domain, and the b chain contains catalyst structure domain.Fibrinolytic significantly strengthens, but has lost fibrinous specificity.This activation can be through plasmin, cathepsin B and L, Hageman factor a, nerve growth factor, and catalysis such as prostate specific antigen are finished.UPA makes both reach combination efficiently by the identification of its somatomedin structural domain and in conjunction with the structural domain of uPAR.

Melittin is the essential substance that plays a role in the bee venom, is made up of 26 amino-acid residues.The constitutional features of melittin molecule has determined that it both can be soluble in water, again can with film natural combination, and then dissolved cell, and need not through cell internalizing.Therefore can substitute b chain (uPAb) performance antitumor action with melittin, the main mechanism of action is that the permeability of cytolemma is increased, and causes entocyte to be revealed and lysis.The melittin molecular weight is little, and is low as Ricin, toxin immunity originality that the diphtheria toxin equimolecular quantity is bigger compared with other some, is difficult for producing anaphylaxis, and these advantages make it have more research and using value.The present invention prepares first and a kind of fusion rotein, its preparation method and application of being made up of human urokinase type plasminogen activator a chain (uPAa) and melittin in oncotherapy thereof is provided.

Summary of the invention

An object of the present invention is to provide that a kind of (uPA a) combines the fusion rotein that forms with melittin (melittin), and wherein said uPA a chain and melittin have 85% sequence homology at least with natural human uPA and natural bee toxin respectively by urokinase type plasminogen activator a chain.

In fusion rotein of the present invention, can be the N-end that is positioned at fusion rotein with people uPA a chain homologous zone, the C-that is positioned at fusion rotein with natural bee toxin homologous zone holds; Also can be the C-end that is positioned at fusion rotein with people uPAa chain homologous zone, the N-that is positioned at fusion rotein with natural bee toxin homologous zone holds.That is to say, in the fusion protein molecule, uPAa and melittin be connected order or the position can exchange.

According to the preferred embodiments of the invention, said fusion rotein produces with the DNA recombinant technology.

According to the preferred embodiments of the invention, wherein employed recombinant expression system is a yeast expression system, more preferably pichia spp, most preferably pichia spp X-33.

The present invention is with people uPA a chain molecule, or it has the analogue of at least 85% sequence homology to have the analogue of at least 85% sequence homology to merge mutually with melittin or its, formed fusion rotein had both kept the activity that combines with uPAR, had kept the activity of melittin dissolved cell again.Therefore, fusion rotein of the present invention both can combine with the tumour cell of expressing uPAR by people uPA a chain, the rhuPAa-melittin of enrichment high density around tumour cell, activity by the dissolved cell of melittin, reach the purpose of target killing tumor cell, simultaneously, when the uPAa-melittin with after uPAR combines, can also compete the binding site of uPAR with the uPA of double chain activity, thereby weakened the effect of uPA degradation of cell epimatrix, finally reached the effect of the adhesion, migration and the invasion and attack that suppress tumour cell.Experimental study confirms that rhuPAa-melittin fusion rotein of the present invention has the activity of efficient combination, target killing tumor cell.

Method well known in the art be can use, the polynucleotide of coding people uPA a chain and the polynucleotide of coding melittin obtained as methods such as PCR, RT-PCR method, artificial synthesis and structure screening cDNA libraries.Can derive from any tissue, cell and library etc. of containing corresponding mRNA or cDNA as pcr template with the mRNA or the cDNA that are used for the construction cDNA library, as obtaining, also can obtain with artificial synthetic method from human ovarian cancer tissue and worker bee poison gland cDNA library.The codon that can select for use the host to have a preference for during synthetic is so as to improving the expression efficiency of product.In case of necessity, can adopt that the known method in this area is suddenlyd change, lacks, inserts and is connected with other polynucleotide polynucleotide etc.Can use the known the whole bag of tricks in this area, as method by PCR, read separately under the constant prerequisite of frame in maintenance, suitable restriction endonuclease sites is introduced in both sides at encoding sequence, cut the generation sticky end by enzyme, and under the dna ligase effect, realize that the sticky end of the polynucleotide of coding people uPA a chain and coding melittin is connected to each other, obtain the gene of code book invention fusion rotein.If desired, can introduce suitable restriction enzyme enzyme recognition site in antigen-4 fusion protein gene of the present invention both sides.The gene order that available method well known in the art will contain the encoding fusion protein sequence is cloned in the various suitable expression.The molecular cloning method of used standard is referring to " molecular cloning test guide " third editions such as J. Sa nurse Brooker, Science Press, 2002.

Many expression vectors and its corresponding host such as Yeast expression carrier pPICZa, pPIC9, (Invitrogen Corp.San Diego, California USA.) all is commercially available pHIL-s1.The nucleotide sequence of code book invention fusion rotein or polypeptide can be cloned in the Yeast expression carrier.The preferred pichia pastoris phaff expression system of the present invention can be to express in the born of the same parents, also can be secreting, expressing.These carriers all comprise one by 5 ' alcohol oxidase operon (AOX1) sequence of about 0.9kb and approximately 0.3kb the Transcription Termination gene 3 '--the expression cassette that-sequence is formed, therefore can be so that goal gene is integrated in yeast chromosomal and expression.

The host of expressed fusion protein can be yeast, bacterium, zooblast, vegetable cell etc., but the present invention's yeast preferably.Fusion rotein or polypeptide may reside in the host cell, also can be that secretion is come out from the host, but preferably come out from the host cell internal secretion under the help of signal peptide.The preferred signal peptide of the present invention is alpha factor (α MF).The alpha factor signal sequence is made up of 87 amino acid, comes from the α sexual maturity factor leader sequence of cereuisiae fermentum (S.cerevsia).The nucleotide sequence of encoding fusion protein can be inserted into host chromosome, or exists with the free plasmid form.

After obtaining carrying the recombinant expression vector of antigen-4 fusion protein gene, can use usual method, as method transformed host cells such as lithium salts method, PEG method, spheroplast method and electroporations.Wherein, the preferred method for transformation of the present invention is an electroporation.Can be identified by the technology that people know,, be extracted DNA, be identified by successful cell transformed with PCR method then, promptly be contained the cell of DNA construct of the present invention as collecting and lysing cell.Perhaps, the antibody test cells and supernatant of available anti-people uPA a chain or anti-melittin or the albumen in the cytoclasis liquid.

Can use and shake bottle or bio-reactor, cultivate the host cell that contains DNA construct of the present invention, to produce fusion rotein of the present invention.Employed substratum should be able to provide thalline (or cell) growth and product to express required material, comprises nitrogenous source, carbon source, pH regulator and becomes to grade, and culture medium prescription should be according to different Objects of Development, by the test acquisition.Cultivation can be divided into two stages, and the fs is mainly used in thalline (or cell) growth, and subordinate phase is mainly used in the synthetic of product.

Can in bacterium that contain DNA construct of the present invention or cell culture, separate with the method for various albumen sepn, purified fusion protein, as saltout, the combination of technology such as organic solvent deposit, ultrafiltration, liquid chromatography (LC) and these technology.Wherein liquid chromatography (LC) can be used molecular sieve, affine, ion-exchange, chromatographic technique such as hydrophobic, anti-phase.

Our experimental result shows, the fusion rotein rhuPAa-melittin that the present invention obtains has the characteristics of " target economic benefits and social benefits ", and one to imitate be the rhuPAa-melittin with after the uPAR of tumour cell combines, melittin release killing tumor cell; Two imitate be when the rhuPAa-melittin with after uPAR combines, thereby weakened the effect of uPA degradation of cell epimatrix with binding site that the uPA of double chain activity competes uPAR, suppressed adhesion, migration and the invasion and attack effect of tumour cell.The present invention is with the target spot of uPA/uPAR system as the treatment tumour, and construction of fusion protein uPAa-melittin is realized specificity target spot, efficient combination, target killing tumor cell, for tumor treatment is opened up a new design idea and approach.

Further biological experiment shows, can combine and can suppress the growth of ovarian cancer cell with the receptor target on ovarian cancer cell surface according to the fusion rotein of the inventive method preparation.The rhuPAa-melittin suppresses the mechanism of ovarian cancer cell growth and may can induce ovarian cellular apoptosis relevant with it, and the apoptosis pathway of ovarian cancer cell may be relevant with the plastosome approach.

Fusion rotein among the present invention can have various derivatives, and these derivatives can be but be not limited to its multi-form salt, modification after product etc.As on the amino of polypeptide, carboxyl, hydroxyl, sulfydryl, modifying again.Used modifier can be but be not limited to polyoxyethylene glycol, dextran etc.

Fusion rotein or derivatives thereof among the present invention can use separately, preferably forms pharmaceutical preparation with one or more pharmaceutically acceptable carriers.Pharmaceutical carrier generally should be compatible with fusion rotein and can not be harmful to receptor autophosphorylation, and typical carrier is water, salt solution, carbohydrate, alcohols or amino acid, and their must be aseptic and have a pyrogeneous substance.Pharmaceutical preparation can prepare by methods known in the art.These methods comprise fusion rotein and one or more ancillary component blended steps.Preferred drug substances comprises that aqueous liquid preparation and water content are lower than 10% or water-free freeze-dried preparation.These preparations can contain buffer reagent, salt, small molecules carbohydrate etc.

Fusion rotein among the present invention and derivative thereof or its pharmaceutical composition can comprise method administrations such as injection (as subcutaneous or muscle), venoclysis, transdermal, suction by any known method.Preferable methods is venoclysis or drug administration by injection.Treatment is included in and uses single dose or compound dosage in for some time.

The invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 shows that the PCR of recombinant plasmid rhuPAa-melittin-pPICZ α transformed yeast bacterium DNA identifies electrophoretogram: wherein swimming lane 1,3～5,7～8 is the PCR product of different yeast transformant genomic dnas; Swimming lane 2 is DNAmarker; Swimming lane 6 is the PCR product of the yeast genomic dna of empty plasmid conversion.

Fig. 2 shows the proteinic sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rhuPAa-melittin (SDS-PAGE) result (coomassie brilliant blue staining) of purifying.Wherein swimming lane 1 is the rhuPAa-melittin behind the purifying, and M is wide molecular weight albumen marker.

Fig. 3 shows the Western immunoblotting assay result of fusion rotein rhuPAa-melittin.Wherein swimming lane 1,2,3 and 4 is rhuPAa-melittin samples that the present invention prepares.

Fig. 4 shows the result of uPAR immunofluorescence dyeing in the SKOV3 cell.

Fig. 5 shows that the rhuPAa-melittin of observing different concns under the transmission electron microscope is the influence of SKOV3 cell ultrastructure to Proliferation of Human Ovarian Cell.Wherein Fig. 5 A is for using the variation of 16.0 μ g/mlrhuPAa-melittin group cell ultrastructures, and Fig. 5 B is for using the variation of 32.0 μ g/ml rhuPAa-melittin group cell ultrastructures.

Fig. 6 shows that different concns rhuPAa-melittin is the proliferating cycle of SKOV3 cell and the influence of apoptosis to Proliferation of Human Ovarian Cell.Wherein Fig. 6 A is the proliferating cycle of use 0 μ g/ml rhuPAa-melittin group cell and the image of apoptosis, Fig. 6 B is the proliferating cycle of use 2.0 μ g/ml rhuPAa-melittin group cells and the image of apoptosis, Fig. 6 C is the proliferating cycle of use 4.0 μ g/ml rhuPAa-melittin group cells and the image of apoptosis, Fig. 6 D is the proliferating cycle of use 8.0 μ g/ml rhuPAa-melittin group cells and the image of apoptosis, and Fig. 6 E is the proliferating cycle of use 16.0 μ g/ml rhuPAa-melittin group cells and the image of apoptosis.

Embodiment

Below by embodiment preferred forms of the present invention is described.These embodiment are intended to further illustrate for example the present invention, rather than limit the await the reply scope of claim of the present invention by any way.

Embodiment 1: the clone and the amplification of people uPA a chain gene

1, the preparation of .RNA:

The ovarian cancer tissue of getting in the surgical procedure through rapid pathology turns out to be serous cystadenocarcinoma puts into the liquid nitrogen quick-frozen.(Invitrogen Corp.San Diego, California USA.), extract the RNA of ovarian cancer tissue with single stage method to use Trizol reagent.Concrete steps are as follows:

Specifically, at first freezing fritter ovarian cancer tissue being put into the mortar that fills liquid nitrogen is ground to Powdered.Then, the tissue that will pulverize moves into 1.5ml not to be had in the RNA enzyme EP pipe, adds about 1ml Trizol.Add 200 μ l chloroforms again, thermal agitation shakes up, and places 30 seconds under the room temperature.4 ℃ centrifugal (12 000r/min) carefully transferred to supernatant liquor in the EP pipe of another no RNA enzyme after 5 minutes.To wherein adding the equal-volume Virahol, room temperature was placed 5 minutes, and in 4 ℃ centrifugal (12 000r/min) 5 minutes, supernatant discarded.Add 70% ethanol 1ml washing precipitation twice, 4 ℃ centrifugal (12 000r/min) 5 minutes.Behind the remaining ethanol of air evaporation, precipitation is dissolved in the 50 μ l DEPC water under the room temperature, the analysis of carrying out RNA is got 1 100 times of μ l diluted samples after ultraviolet spectrophotometer is measured OD with quantitative ₂₆₀And OD ₂₈₀, calculate its concentration and purity by following formula.Rna content: RNA (μ g/ml)=40 * OD ₂₆₀* extension rate, OD ₂₆₀/ OD ₂₈₀=1.8～2.0 expression purity are qualified.With agarose gel electrophoresis method, observe the integrity of RNA.

2, the clone of people uPA a chain gene (RT-PCR method)

Get the human ovarian cancer total tissue RNA 1 μ g that as above extracts, set up reverse transcription (RT) reaction system in following ratio:

Human ovarian cancer total tissue RNA 1 μ g; 10 * RNA PCR damping fluid, 2 μ l; MgCl ₂(25mmol/L) 4 μ l; RNA enzyme inhibitors (40U/ μ l) 0.5 μ l; DNTPs (each 10mmol/L) 2 μ l; AMV reversed transcriptive enzyme (200U/ μ l) 1 μ l; Oligo dT (20pmol/ μ l) 1 μ l; No RNA enzyme aqua sterilisa is added to final volume 20 μ l.42 ℃ of reactions are 60 minutes on the PCR instrument, and 99 ℃ made the reversed transcriptive enzyme deactivation in 5 minutes then.Behind the synthetic cDNA, set up the PCR reaction system according to following ratio:

Above-mentioned cDNA reaction solution 20 μ l; MgCl ₂(25mmol/L) 6 μ l; 10 * LA PCR damping fluid, 8 μ l; Upstream primer: 5 '-GTT CCA TCG AAC TGT GAC TG-3 ' (SEQ ID NO:1), 1 μ l (20pmol/ μ l); Downstream primer: 5 '-GGT TCT CGA TGG TGG TGA AT-3 ' (SEQ ID NO:2), 1 μ l (20pmol/ μ l); TaKaRa LA Taq (5U/ μ l) 1 μ l; The sterilization ultrapure water is to final volume 100 μ l.The PCR reaction conditions is: 94 ℃ 4 minutes; 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 1 minute, totally 30 circulations, then 72 ℃ 8 minutes.Amplified production is carried out 0.8% agarose gel electrophoresis analysis, observe clip size, determine whether to obtain correct goal gene.

3, the structure of cloning vector, conversion and amplification

Reclaim the people uPA a chain gene fragment of RT-PCR amplification and carry out 0.8% agarose gel electrophoresis analysis.After the recovery, people uPA a chain gene fragment is connected 30 minutes for 16 ℃ with the T carrier.The ligation system comprises: people uPA a chain RT-PCR product 0.1～0.3pmol; T carrier 0.03pmol; Solution I (contain dna ligase and be connected damping fluid, see Takara company's T support agent box) 5 μ l; The sterilization ultrapure water is to final volume 10 μ l.

According to ordinary method, from 37 ℃ of XL that cultivate 16 hours ₁The last single bacterium colony of picking of the negative culture plate of-Blue (not containing antibiotic LB agar plate) (diameter 2～3mm) is inoculated in the 1L culturing bottle that contains 100ml LB substratum, with preparation competence Bacillus coli cells, and frozen standby in-80 ℃.

Get a frozen competent cell, melt the back and add above-mentioned connection product, carry out routine and transform.Get competent cell that 200 μ l have transformed then and coat on the LB agar plate that contains X-Gal, IPTG (the two can be before being coated with bacterium half an hour be applied to LB agar plate surface) and penbritin (100 μ g/ml), cultivated 12～16 hours in 37 ℃ of incubators.

4, the evaluation of recombinant vectors

White single bacterium colony (" indigo plant is screened in vain ") after picking transforms at random is inoculated in the LB substratum that contains penbritin (100 μ g/ml), 37 ℃ of strong concussion overnight incubation, the conventional then plasmid DNA of extracting.Get that 1 μ l dissolved DNA throw out carries out the agarose gel electrophoresis quantitative analysis and enzyme is cut evaluation.Identify digestion 2 hours with SalI, BglII double digestion down, be accredited as correct clone according to the inscribe zymogram behind 1% agarose gel electrophoresis for 37 ℃.Select enzyme and cut the correct clone of evaluation, extract plasmid, be used for the dna sequencing analysis.

The structure of embodiment 2:huPAa-melittin pichia spp secreted expression carrier huPAa-melittin-pPICZ alpha expression carrier

1, makes up the huPAa gene eukaryotic expression vector skeleton huPAa-X-pPICZ α that can replace the uPAb chain arbitrarily

Get above-mentioned order-checking and identify the cloned plasmids 100ng that correctly contains huPAa, in 0.2ml EP pipe, set up the PCR reaction system in following ratio:

HuPAa recombinant plasmid 100ng; Contain Mg ²⁺10 * LA PCR damping fluid, 10 μ l; DNTPs (each 2.5mmol/L) 8 μ l; Upstream primer: 5 '-CTG CTC GAG AAG AGA TCT AAC GAG CAC CAA GTTCCA TCG AAC-3 ' (SEQ ID NO:3), 1 μ l (20pmol/ μ l); Downstream primer: 5 '-CAG GAATTC TCA AAT CTT AAA CCG CGG GCC TCA GAG TCT TTT-3 ' (SEQ ID NO:4), 1 μ l (20pmol/ μ l); TaKaRa LA Taq (5U/ μ l) 1 μ l; The sterilization ultrapure water is to final volume 100 μ l.On the PCR instrument, carry out cyclic amplification.Amplification program is: 94 ℃ 4 minutes; 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 1 minute, totally 30 circulations, then 72 ℃ 8 minutes.

1% agarose gel electrophoresis is analyzed the PCR product, carries out 0.8% agarose gel electrophoresis then, separates and reclaim the PCR product of suitable size.

2, the structure of huPAa-X-pPICZ alpha expression carrier

With the PCR product huPAa and the carrier pPICZ α of XhoI and EcoRI digestion as above-mentioned recovery, after agarose gel electrophoresis was quantitative, 16 ℃ of connections were spent the night.The ligation system is as follows: goal gene 0.1～0.3pmol; Carrier pPICZ α 0.03pmol after enzyme is cut; 10 * connection damping fluid, 1 μ l; T4 dna ligase (350U/ μ l) 1 μ l; The sterilization ultrapure water is to final volume 10 μ l.

To connect product and competent cell XL1-blue mixing gently, ordinary method transforms.Get the Bacillus coli cells that 200 μ l have transformed and coat on the less salt LB agar plate that contains Zeocin (25 μ g/ml), 37 ℃ of incubators were cultivated 12～16 hours.The clone that picking is different, overnight incubation in the LB liquid nutrient medium is extracted plasmid then, and carries out XhoI and the evaluation of EcoRI double digestion.

Choose enzyme and cut the correct clone of evaluation, extract plasmid with plasmid rapid extraction test kit.Carry out determined dna sequence.

3, the structure of huPAa-melittin-pPICZ alpha expression carrier

According to gene bank retrieve sequence, synthetic has one group of complementary oligonucleotide of melittin of specificity restriction enzyme site, 5 '-AA TTC TCA TTG TTG TCT CTT TCT CTT AAT CCA AGA AAT CAA AGCTGG CAA ACC AGT AGT CAA AAC CTT CAA AAC AGC ACC AAT CTT GAA CCG C-3 ' (SEQID NO:5); 5 '-GG TTC AAG ATT GGT GCT GTT TTG AAG GTT TTG ACT ACT GGT TTGCCA GCT TTG ATT TCT TGG ATT AAG AGA AAG AGA CAA CAA TGA G-3 ' (SEQ IDNO:6)

Article two, chain is respectively got 25umol and 0.5mol NaCl 6ul sets up the 20ul system, and 95 ℃ were boiled 2 minutes, and 80 ℃ were boiled 3 minutes, and slowly cooled to room temperature.Form the joint that has SacII and EcoRI sticky end two strands after the complementary strand annealing respectively.

Cut huPAa-X-pPICZ α carrier with SacII and EcoRI enzyme, reclaim go forward side by side row agarose gel electrophoresis quantitatively after, 16 ℃ of connections are spent the night.The ligation system is as follows: annealed melittin gene 0.1～0.3pmol; HuPAa-X-pPICZ α 0.03pmol; 10 * connection damping fluid, 1 μ l; T4 dna ligase (350U/ μ l) 1 μ l; The sterilization ultrapure water is to final volume 10 μ l.

To connect product and competent cell XL1-blue mixing gently, ordinary method transforms.Get the Bacillus coli cells that 200 μ l have transformed and coat on the less salt LB agar plate that contains Zeocin (25 μ g/ml), 37 ℃ of incubators were cultivated 12～16 hours.The clone that picking is different, overnight incubation in the LB liquid nutrient medium is extracted plasmid then and is carried out two enzymes (XhoI and SacII) and cut evaluation.

Choose enzyme and cut the correct clone of evaluation, extract plasmid with plasmid rapid extraction test kit.Carry out determined dna sequence.

The foundation and the screening of embodiment 3:rhuPAa-melittin Pichia anomala expression system

Get the correct cultivation bacterium liquid of order-checking, extract plasmid DNA and carry out quantitative analysis with agarose gel electrophoresis method by the plasmid extraction kit specification sheets.Get 20～25 μ g expression vector huPAa-melittin-pPICZ α, after SacI enzymic digestion (linearizing) with phenol/chloroform extracting and use ethanol sedimentation.Linearizing plasmid is standby on ice with 10 μ, 1 ultrapure water dissolving postposition.

(not containing antibiotic YPD Agar flat board) single bacterium colony of picking from the negative culture plate of the YPD of pichia spp X-33 is inoculated in the 5ml YPD substratum, 250rpm, and 30 ℃ of concussions were cultivated 8 hours, and ordinary method prepares the yeast competent cell.

Get the above-mentioned competent cell of 80 μ l then, mix, move in the 0.2cm electricity conversion cup and carry out the electricity conversion with the linearizing recombinant expression plasmid of 20～25 μ g.The bacterium liquid of getting after 50～100 μ l transform is coated on the YPD flat board that contains Zeocin (100 μ g/ml), and 30 ℃ of incubators were cultivated 2～3 days, observed the upgrowth situation of transformant.

Use PCR method (employed primer is a upstream primer: 5 '-GAC TGG TTC CAA TTGACA AGC-3 ' (SEQ ID NO:7) and downstream primer: 5 '-ATC GAT CTC ACA GTG TTGACC-3 ' (SEQ ID NO:8), reaction conditions is the same) screening transformed yeast bacterium then.Behind the centrifugal recovery thalline, extract the pastoris genomic dna performing PCR of going forward side by side with glass bead method and identify that amplified production carries out 1.0% agarose gel electrophoresis, observe whether obtaining expecting the gene fragment of size.Qualification result as shown in Figure 1.

The expression and the purifying of embodiment 4:rhuPAa-melittin

1, the expression of rhuPAa-melittin

Get above-mentioned qualification result male clone and be inoculated in 10ml BMGY (pH6.0) substratum, 30 ℃ of concussions were cultivated 24 hours, to OD ₆₀₀Reach 2.0～6.0 o'clock collecting cells.With equal-volume (10ml) BMMY (pH6.0) re-suspended cell precipitation, abduction delivering is cultivated in 30 ℃ of concussions.Induce in the process, replenished a methyl alcohol to final concentration 0.5% in per 24 hours, replenish the sterilization ultrapure water simultaneously, the fermented liquid cumulative volume is remained unchanged.Respectively get the 0.5ml fermented liquid at the 0th, 24,48,72,96,120, the 144 and 168 hour equi-time point of cultivating, centrifugal collection supernatant is used for protein analysis (SDS-PAGE, Western Blot and n terminal amino acid sequential analysis).

2, the purifying of rhuPAa-melittin

(1) solution and reagent: solution A: HAc-NaAc damping fluid 200mmol/L (pH 4.0); Solution B: 20mmol/L HAc-NaAc damping fluid (pH 4.0); Solution C: solution B contains 2mol/L NaCl; Solution D: 1% (v/v) TFA; Solution E: 0.1% (v/v) TFA; Solution F: solution E contains 100% acetonitrile.

(2) 1 Mono S cation seperation column chromatographies

With 10 times of column volume solution B balance Mono S positively charged ion chromatography posts (Sigma company).Adjusting fermented supernatant fluid pH value with 0.5mol/LNaOH is 4.0, and centrifugal (12000rpm) 10 minutes removes particulate matter, and then supernatant adds 1/10 volume solution A damping fluid.With the speed application of sample of 0.5ml/min to the MonoS resin cation (R.C.), wavelength 280nm, the monitoring of 260nm on-line ultraviolet.After application of sample finishes, wash to the OD280 value with solution B and to reduce to baseline.Use solution B and solution C gradient elution then, and the fraction collection protein peak.The SDS-PAGE method is determined the elution peak of huPAa-melittin.

(3) the anti-phase hydrophobic chromatography of Source 30 RPC

With 10 times of anti-phase hydrophobic chromatography posts of column volume solution E balance source 30 RPC (Sigma company).Protein sample with above-mentioned collection adds 1/10 volume solution D then.With the speed application of sample of 0.5ml/min, wavelength 280nm, the monitoring of 260nm on-line ultraviolet.Application of sample washes to OD with solution E after finishing ₂₈₀Value is reduced to baseline.Carry out gradient elution with solution E and solution F, and the fraction collection protein peak.The SDS-PAGE method is determined the elution peak of huPAa-melittin.

The 15%SDS-PAGE analytical results shows, can reach about 95% (referring to accompanying drawing 2) through the huPAa-of this method purifying melittin purity.

The physico-chemical property of embodiment 5:rhuPAa-melittin fusion rotein is identified

1.SDS polyacrylamide gel electrophoresis (SDS-PAGE)

Carry out protein molecule flow measurement and preliminary quantitative analysis with the SDS-PAGE method.Method is as follows:

Prepare 15% separation gel 5% and concentrate glue.Get per 24 hours culture supernatants respectively and add 5 * SDS sample buffer, behind the mixing, boiled 3～5 minutes in 4: 1 ratios.Get above-mentioned sample, be cooled to room temperature after, centrifugal (10000rpm) 30 seconds gets the every hole of supernatant application of sample 20 μ l.The 40V electrophoresis is adjusted voltage to 100V to concentrating glue and separation gel intersection, continues the constant voltage electrophoretic separation.After coomassie brilliant blue staining and the decolouring, the observation analysis result.

2. the Western engram analysis of expression product

According to ordinary method with fermented liquid supernatant behind SDS-PAGE, be transferred on nitrocellulose (NC) film drying at room temperature 30～60 minutes.Above-mentioned NC film is placed plate, add 20ml confining liquid (TTBS that contains 0.2%BSA), room temperature was vibrated 2～3 hours gently or 4 ℃ of sealings are spent the night.Then, the NC film is put into hybridization bag by 0.1ml antibody-solutions/cm ²Film adds the anti-people uPA of rabbit antibody, room temperature vibration 1～4 hour.After the TTBS rinsing 3～5 times, will dilute the goat anti-rabbit antibody (1: 200～1: 1000) of horseradish peroxidase-labeled, add in the hybridization bag, with NC film room temperature vibration 2～4 hours with antibody diluent.Add 1ml0.3% (W/V) NiCl or CoCl ₂And 10 μ l 30%H ₂0 ₂Solution.After washing film, film is moved in the colour developing liquid, shake and observe color reaction under the room temperature gently.The result as shown in Figure 3.

3) amino acid sequence analysis of expression product

Proteinic primary structure is the basis of its higher structure, during with the gene recombination technology expression secreted protein, signal peptide mistake cutting phenomenon appears in the heterologous protein of expressing easily in the processing ripening process, influence the higher structure and the biologic activity of expression product then.Therefore, determine under the correct situation of molecular weight, tackle expressed recombinant protein and carry out protein N-terminal amino acids sequence mensuration, to determine the exactness of its primary structure at SDS-PAGE.Our sequencing result shows to have and the aminoacid sequence of expecting according to rhuPAa-melittin fusion rotein of the present invention.

The biologic activity of embodiment 6:rhuPAa-melittin

Present embodiment is by observing the influence of rhuPAa-melittin to the form of Proliferation of Human Ovarian Cell SKOV3 cell, growth curve, cell generation cycle, apoptosis, cell death related protein etc., judges and the biologic activity of the rhuPAa-melittin that definite the present invention produces.

1.RT-PCR method detects the expression of SKOV3 cell strain uPAR

Collect SKOV3 1.5 * 10 ⁶Individual cell adds 1mLTrizol piping and druming back room temperature and placed 5 minutes.Extract the RNA (referring to embodiment 1) of SKOV3 cell strain then, and (upstream primer of use is 5 '-CAG TGT AAG ACC AAC GGG GAT-3 ' downstream primer: 5 '-TCA GGT TTA GGT CCA GAG GAG-3 ') to detect the expression of uPAR with the RT-PCR method.The result shows that in the SKOV3 cell strain, the uPAR expression amount is higher.

To clean in advance, handle high pressure disinfectant cover glass through poly-lysine and put into 24 well culture plates.To digest then, the SKOV3 cell (10% foetal calf serum H-DMEM nutrient solution is resuspended with containing) of centrifuge washing adds 24 orifice plates (0.5ml/ hole).Culture plate is placed 37 ℃, 5%CO ₂Incubator continue to be cultivated, and is cultured to cell log vegetative period after about 24 hours.Experimental group adds the huPAa-melittin makes its final concentration reach 4.0 μ g/ml, 8.0 μ g/ml, and control group adds nutrient solution to equal-volume.Take out 24 orifice plates after 24 hours, the sucking-off nutrient solution adds twice of PBS flushing.37 ℃, take out cell climbing sheet after fixing 30 minutes with 4% Paraformaldehyde 96, with distilled water flushing 2 times.Drying at room temperature ,-20 ℃ frozen, uses in two weeks.

Cell climbing sheet added the distilled water aquation 30 minutes.Drip 0.1%TritonX-100, handled 10 minutes.

After time (5 minutes/time), use 0.3%H 0.01MPBS give a baby a bath on the third day after its birth ₂O ₂Solution blocking-up endogenous peroxydase was washed 3 times with PBS once more and was sealed 30 minutes with serum after 15 minutes.The serum deprivation that inclines then adds the anti-people uPAR of rabbit polyclonal antibody, 37 ℃ 1 hour.Replace one to resist with PBS as negative control.PBS washes the goat anti-rabbit antibody that adds the FITC mark after 3 times, and the room temperature lucifuge was hatched 30 minutes.PBS washes 30 minutes (lucifuge) back glycerine mounting.The laser confocal microscope observations shows that the uPAR of SKOV3 cell surface expresses and is strong positive (seeing accompanying drawing 4).

2. fusion rotein huPAa-melittin of the present invention is to the effect of Proliferation of Human Ovarian Cell SKOV3

(1), mtt assay is measured the restraining effect of huPAa-melittin to the growth of SKOV3 cell

Get the SKOV3 cell (1 * 10 of exponential phase of growth ⁴) 2 96 orifice plates of inoculation.Wherein 1 96 orifice plate is normal dosing group, and other 1 is that uPAR antibody antagonism is handled back dosing group.Each concentration is established 3 parallel holes.Normal dosing group huPAa-melittin concentration is respectively 0.1 μ g/ml, 0.2 μ g/ml, 0.4 μ g/ml, 0.8 μ g/ml, 1.6 μ g/ml, 3.2 μ g/ml, 6.4 μ g/ml, 12.8 μ g/ml, control group adds H-DMEM nutrient solution to the equal-volume of 10% foetal calf serum and puts into 37 ℃, 5%CO ₂Incubator is cultivated.The dosing group adds uPAR antibody 20 μ g/ml, 37 ℃, 5%CO in advance behind the uPAR antibody antagonism in every hole ₂Cultivate and press above-mentioned concentration adding huPAa-melittin after 2 hours more respectively, control group adds the H-DMEM nutrient solution of 10% foetal calf serum and put into 37 ℃, 5%CO to equal-volume ₂Incubator is cultivated.Take out 96 orifice plates after 24 hours, in each hole, add MTT solution 20 μ l (5mg/ml), 37 ℃ of 5%CO ₂Hatch and stop after 4 hours cultivating.The careful suction abandoned supernatant liquor in the hole, and every hole adds 100 μ l DMSO, vibrates 10 minutes.Treat on Bio-red 550 microplate reader, to measure absorbance (OD value) after precipitation is dissolved fully.Calculate two groups of inhibitory rate of cell growth (three multiple mean value) after the effect of different concns huPAa-melittin respectively by following formula.With inhibitory rate of cell growth drug level is done curve, obtain IC ₅₀

Shown in the following tabulation 1 of result.As seen the cell of surviving in the antagonism group is obviously more than rhuPAa-melittin group, prompting is owing to add the uPAR polyclonal antibody in advance in the antagonism group, this antibody can combine with the uPAR in the SKOV3 cell, can reduce for the acceptor quantity of rhuPAa-melittin bonded uPAR thereby make in the antagonism group, and then discharge the ability attenuating of melittin killing tumor cell.The result shows that rhuPAa-melittin of the present invention has the ability of target bind receptor uPAR, also proves the nontoxic or hypotoxicity of rhuPAa-melittin under the fusion state, fusion rotein have only with its receptors bind after just can bring into play toxic action.

Table 1. antagonism group and rhuPA _a-melittin group is to the growth inhibition ratio of SKOV3

( ^＊P＜0.05； ^＊＊P＜0.01)

(2), transmission electron microscope observation

With the SKOV3 cell of trysinization 80% fusion state, cell counting is with 1 * 10 ⁷Be inoculated in the 250ml culturing bottle.It is 4.0 μ g/ml, 8.0 μ g/ml that experimental group adds final concentration, huPAa-melittin effect 24h.Control group is added to and the isopyknic nutrient solution of experimental group.After PBS washes 2-3 time, with 0.25% trypsinase cell dissociation, with the nutrient solution termination reaction that contains serum.Centrifugal 5 minutes of 800rpm also washes 2-3 time with PBS.After for the last time centrifugal, the cell mass of formation gently adds the glutaraldehyde of 1-2ml, places for 4 ℃ and also fixes 1 hour with 1% goose acid at 4 ℃ in 2 hours.Discard the goose acid solution, PBS washes 2 times, each 20 minutes.0.5% uranyl acetate dyes in advance, room temperature, and shaking table spends the night.With 30%, 50%, 70%, 90%, 100% each 10-15 of gradient ethanol dehydration minute, use 100% ethanol dehydration 2 times each 10 minutes at last.With the embedding of Epon812 Resins, epoxy, LKB-III type ultramicrotome semithin section is done ultrathin section(ing) behind the location.1% (w/v) uranyl acetate and lead citrate double staining.The JEM-1200EX transmission electron microscope observation.

Result shown in the accompanying drawing 5 shows that in the finite concentration scope, apoptosis may be the mechanism of action that the rhuPAa-melittin suppresses the ovarian cancer cell growth, and its effect to ovarian cancer cell may be direct when excessive concentration.

(3), Flow cytometry different concns huPAa-melittin is to the influence of SKOV3 cell cycle and apoptosis rate

Collect respectively and respectively to organize SKOV3 cell 5 * 10 through 0 μ g/ml, 2.0 μ g/ml, 4.0 μ g/ml, 8.0 μ g/ml, 16.0 μ g/mlhuPAa-melittin effects 24 hours ⁶Individual; With 0.25% trypsinase cell dissociation, make cell suspension; 5 minutes centrifugal back PBS of 1000rpm clean 2-3 time, and 70% ice ethanol is fixed, and preserves down at 4 ℃.The first detecting centrifugal 5 minutes, add 20 μ g RNaseA after abandoning supernatant with 1000rpm; Insulation added 800 μ l PI dye liquor mixings after 30 minutes in 37 ℃ of water-baths.After keeping in Dark Place 30 minutes under 4 ℃, on flow cytometer, detect.

Result shown in the accompanying drawing 5 shows that the rhuPAa-melittin can make it blocking-up in the S phase by influencing the period profile of SKOV3 cell, induce the SKOV3 apoptosis simultaneously, and then the division growth of inhibition ovarian cancer cell reaches the purpose that suppresses its growth.

Sequence table

＜110〉the holy first Science and Technology Ltd. in Jilin

＜120〉fusion rotein of urokinase type plasminogen activator a chain and melittin and preparation thereof

<140>

<141>

<160>8

<210>1

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as the structure cloning vector.

<400>1

GTTCCATCGA?ACTGTGACTG

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as the structure cloning vector.

<400>2

GGTTCTCGAT?GGTGGTGAAT

<210>3

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as construction of expression vector.

<400>3

CTGCTCGAGA?AGAGATCTAA?CGAGCACCAA?GTTCCATCGA?AC

<210>4

<211>42

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as construction of expression vector.

<400>4

CAGGAATTCT?CAAATCTTAA?ACCGCGGGCC?TCAGAGTCTT?TT

<210>5

<211>93

<212>DNA

＜213〉artificial sequence

<220>

＜223〉encoding sequence of the melittin of chemosynthesis.

<400>5

AATTCTCATT?GTTGTCTCTT?TCTCTTAATC?CAAGAAATCA?AAGCTGGCAA?ACCAGTAGTC?AAAACCTTCA

AAACAGCACC?AATCTTGAAC?CGC

<210>6

<211>87

<212>DNA

＜213〉artificial sequence

<220>

＜223〉encoding sequence of the melittin of chemosynthesis.

<400>6

GGTTCAAGAT?TGGTGCTGTT?TTGAAGGTTT?TGACTACTGG?TTTGCCAGCT?TTGATTTCTT?GGATTAAGAG

AAAGAGACAA?CAATGAG

<210>7

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the primer that designs according to specific nucleotide sequence as the PCR evaluation.

<400>7

GACTGGTTCC?AATTGACAAG?C

<210>8

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the primer that designs according to specific nucleotide sequence as the PCR evaluation.

<400>8

ATCGATCTCA?CAGTGTTGAC?C

Claims (5)

1. It is 1, a kind of that (uPA a) combines the fusion rotein that forms with melittin, wherein said uPA a chain and melittin have 85% sequence homology at least with natural human uPA and natural bee toxin respectively by urokinase type plasminogen activator a chain.
2. 2,, be characterised in that said fusion rotein produces with the DNA recombinant technology according to the fusion rotein of claim 1.
3. 3, according to the fusion rotein of claim 1, be characterised in that said fusion rotein be the pichia pastoris phaff system expression that uses produce.
4. 4,, be characterised in that in the said fusion protein molecule being connected order and can exchanging of uPA a and melittin according to the fusion rotein of claim 1.
5. 5, the fusion rotein of claim 1 is to produce the application in the anti-tumor medicine.

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