CN102558359A - Recombinant fusion toxin VEGF165-melittin and preparation method and application thereof - Google Patents

Abstract

The invention discloses recombinant fusion toxin VEGF165-melittin and a preparation method and application thereof, belonging to the field of a genetic engineering drug. VEGF165 and small-molecule toxin melittin are integrated via connecting peptides to construct the novel recombinant toxin VEGF165-melittin, and the solubility expression can be performed via pichia pastoris. Tests show that the VEGF165-melittin can simultaneously achieve the effects of combination of VEGFR and EGFR of tumor cells and release of melittin to kill tumor cells. Moreover, when the VEGF165-melittin is combined with the VEGFR and EGFR, tyrosine kinase activation of VEGF/VEGFR signals can be blocked, strong signals of multiple VEGFs and EGFs can be blocked, the proliferation and migration of tumors can be inhibited finally, the generation of neovascularization can be inhibited particularly, and new design concept and path are provided for tumor treatment.

Description

Reorganization fusion toxin VEGF 165-melittin

Technical field

The present invention relates to the genetically engineered drug field, be specifically related to a kind of reorganization fusion toxin VEGF ₁₆₅-melittin.

Background technology

The prevention of malignant tumour is a world-famous puzzle with treatment always, and vasculogenesis has crucial effects to growth of tumor and transfer.Tumour can promote the angiogenesis factor secretion when taking place, wherein most importantly vascular endothelial growth factor (Vascular endothelial growth factor, VEGF).The experimental results shows that VEGF passes through propagation and diffusion that its effect of quickening neovascularity growth promotes tumour cell, and is closely related with transfer with multiple solid tumor such as liver cancer, mammary cancer, lung cancer, colorectal carcinoma, ovarian cancer, cancer of the stomach, the generation of carcinoma of the pancreas, development.And with the directed targeted drug of blood vessel; Especially be that the fusion toxin of target has concurrently with vegf receptor and combines with the vegf receptor; Blocking VEGF/VEGFR signal path and the dual function of killing and wounding vascular endothelial cell except the inhibition tumor neogenetic blood vessels forms, can also be destroyed the tumor vessel that has generated; Be more promising treatment means, at aspects such as retinal diseases, AIDS the potential using value also arranged simultaneously.The mRNA of VEGF can form four kinds of different isomer, VEGF through shearing ₁₂₁, VEGF ₁₆₅, VEGF ₁₈₉And VEGF ₂₀₆, their key distinction is different with the bonding force of heparin and extracellular matrix.Existing research is reported in the isomer of these four kinds of VEGF, with VEGF ₁₂₁And VEGF ₁₆₅Be targeting vector, can produce stronger cytotoxic effect, this maybe with VEGF ₁₂₁, VEGF ₁₆₅There is more specific receptors relevant.

External existing scholar is with VEGF ₁₆₅Combine to form macromole fusion rotein target with diphtheria toxin and suppress tumor vascular generation, Hotz etc. are also with VEGF ₁₂₁Merge and carried out the anti-tumor activity detection with will He Xiang verticillium toxin, but these lps molecule amounts commonly used are big, and must at first will could get into cell through the cell internalizing effect and play a role, these have all influenced the performance of its antitumor action.

The small molecules toxin that mellitin (melittin) is made up of 26 amino acid; Has the advantage that molecular weight is little, be easy to go deep into solid tumor inside; Its dominant mechanism that plays a role is that the permeability of cytolemma is increased, and entocyte is revealed, lysis; Need not through cell internalizing and directly playing a role, this makes melittin be suitable as very much " bullet " and is used for making fusion toxin and carries out the antineoplastic targeting treatment.But the report that does not also have related fields at present.

Summary of the invention

The objective of the invention is to overcome prior art and only suppress the problem that tumor neogenetic blood vessels is the targeted therapy method existence of purpose with blocking VEGF/VEGFR signal path; Provide a kind of and have blood-vessels target concurrently and destroy living tumor vessel, and the novel reorganization fusion toxin VEGF of the dual function of ability kill tumor cell ₁₆₅-melittin is for the research and development of antineoplastic target treatment technology provides the basis.

The present invention realizes above-mentioned purpose through following technical scheme:

A kind of reorganization fusion toxin VEGF ₁₆₅-melittin is to connect VEGF through connection peptides at the aminoterminal of melittin small peptide melittin ₁₆₅Obtain, or pass through connection peptides at VEGF ₁₆₅Aminoterminal connect melittin small peptide melittin and obtain; The aminoacid sequence of said connection peptides is shown in SEQ ID NO:1.That is to say, in the fusion toxin molecule, VEGF ₁₆₅With melittin be connected order or the position can exchange.

Wherein said VEGF ₁₆₅With melittin small peptide melittin respectively with natural human VEGF ₁₆₅Has 90% sequence homology at least with the natural bee toxin.

The aminoacid sequence of said reorganization fusion toxin is shown in SEQ ID NO:2.Or for the amino acid residue sequence of SEQ ID NO:2 through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of function of selective killing endothelial cells in tumor neogenetic blood vessels.

The present invention also protects above-mentioned reorganization fusion toxin VEGF ₁₆₅The encoding sox of-melittin.The nucleotides sequence of this encoding sox is classified the gene of protein sequence shown in the coding SEQ ID NO:2 as; Concrete nucleotide sequence perhaps has 90% above homology with sequence shown in the SEQ ID NO:3 and has the nucleotide sequence of function of selective killing endothelial cells in tumor neogenetic blood vessels shown in SEQ ID NO:3.Perhaps under the rigorous condition of height can with the nucleotide sequence of sequence hybridization shown in the SEQ ID NO:3.

A kind of reorganization fusion toxin VEGF ₁₆₅The expression vector of-melittin is by VEGF ₁₆₅The MCS that the encoding sox of-melittin is inserted into the carrier that sets out constitutes, and the said carrier that sets out is preferably pPICZa, pPIC9 or pHIL-sl.

The transgenic cell line or the host bacterium that contain above-mentioned expression vector.

Reorganization fusion toxin VEGF according to the invention ₁₆₅-melittin can be through the preparation of DNA recombinant technology, and employed expression system is a yeast expression system, preferred pichia spp, and pichia spp X-33 is best.

Can use methods such as method well known in the art such as PCR, RT-PCR method, artificial synthesis and structure screening cDNA library to obtain coding human VEGF ₁₆₅Polynucleotide and the polynucleotide of coding melittin melittin.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,, also can use artificial synthetic method to obtain as obtaining from people's liver 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,, read separately under the constant prerequisite of frame in maintenance like method through PCR; Suitable restriction endonuclease sites is introduced in both sides at encoding sequence; Cut the generation sticky end through enzyme, and under the dna ligase effect, realize coding human VEGF ₁₆₅Be connected to each other with the sticky end of polynucleotide of coding melittin, obtain the gene of code book invention fusion toxin.If desired, can introduce suitable restriction enzyme enzyme recognition site in fusion toxin gene of the present invention both sides.The gene order that available method well known in the art will contain coding fusion toxin 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, (Invitrogerl Corp.San Diego, California USA.) all is commercially available pHIL-sl.Can the nucleotide sequence that code book is invented fusion toxin or polypeptide 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 an expression cassette of being made up of 5 ' alcohol oxidase operon (AOXl) sequence of about 0.9kb and about 3 ' sequence of the Transcription Termination gene of 0.3kb, therefore can be so that goal gene is integrated in yeast chromosomal and expression.

The host who expresses fusion toxin can be yeast, bacterium, zooblast, vegetable cell etc., but the present invention's yeast preferably.Fusion toxin or polypeptide may reside in the host cell, also can be that secretion is come out from the host, but preferably under the help of signal peptide, come out from the host cell internal secretion.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 fusion toxin gene, can use usual method, like 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 identify through the technology that people know,, extract DNA, identify by successful cell transformed with PCR method then, promptly contain the cell of DNA construct of the present invention as collecting and lysing cell.Perhaps, available anti-people VEGF ₁₆₅Or the antibody test cells and supernatant of 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 toxin 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, through 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, technology and these technological combinations such as organic solvent deposit, ultrafiltration, LC.Wherein LC can be used chromatographic techniques such as molecular sieve, affine, IX, hydrophobic, anti-phase.

Through the optimum combination of kinds of schemes, finally determine most effective a kind of reorganization fusion toxin VEGF ₁₆₅The preparation method of-melittin, step is following:

(1) be primer with SEQ ID NO:4 ~ 5, amplification VEGF ₁₆₅Gene;

(2) synthetic melittin small peptide melittin gene, two ends connect restriction enzyme site respectively EcoRI with XbaI is connected to the MCS of carrier pPICZ α, obtains recombinant vectors;

(3) use respectively EcoRI with XhoI is to VEGF ₁₆₅Gene and recombinant vectors carry out double digestion, and after the connection, electroporation transforms host's Pichia yeast, makes up VEGF ₁₆₅-Melittin genetic engineering bacterium;

(4) cultivate VEGF ₁₆₅-Melittin genetic engineering bacterium, substratum are the BMGY substratum, cultivate down for 28 ℃, induce it to produce solubility recombination fusion protein VEGF ₁₆₅-Melittin;

(5) culture of step (4) is centrifugal, collection supernatant carries out affinity column chromatography purifying VEGF ₁₆₅-Melittin albumen, the said filler of column chromatography is the Ni-NTA resin, and adsorption conditions is between the pH 5.5-8.5, and elution requirement is for reducing pH, progressively improving the concentration of imidazoles or add EDTA or EGTA, and pH is between the 4-6, imidazole concentration is 0.02-0.5M;

(6) adopt molecular weight 1000 daltonian ultra-filtration membrane ultrafiltration, adopt semi-permeable membranes dialysis (dialyzate: 10mM TrisHCl, 150mM NaCl), freeze-drying obtains VEGF ₁₆₅-Melittin albumen.

Biological experiment shows that fusion toxin of the present invention can combine and can suppress the growth of liver cancer cell with the receptor target on liver cancer cell surface, can be used for preparing the targeting anti-tumor medicine.

Reorganization fusion toxin VEGF among the present invention ₁₆₅-melittin can have various verivates, and these verivates 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, VISOSE etc.

Reorganization fusion toxin VEGF among the present invention ₁₆₅-melittin or derivatives thereof can use separately, preferably forms pharmaceutical prepn with one or more pharmaceutically acceptable carriers.Pharmaceutical carrier generally should be compatible with fusion toxin 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 prepn can be through the method preparation of oneself knowledge of this area.These methods comprise fusion toxin and one or more ancillary component blended steps.Preferred drug substances comprises that aqueous liquid preparation and water cut are lower than 10% or water-free freeze-dried prepn.These preparations can contain buffer reagent, salt, small molecules carbohydrate etc.

Reorganization fusion toxin VEGF among the present invention ₁₆₅-melittin and verivate thereof or its pharmaceutical composition can comprise method administrations such as injection (like subcutaneous or muscle), venoclysis, transdermal, suction through 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.

Compared with prior art, the present invention has following beneficial effect:

The present invention is with people VEGF ₁₆₅Molecule; Or it has the analogue of at least 90% sequence homology to have the analogue of at least 90% sequence homology to merge mutually with melittin or its; Formed fusion toxin had both kept the activity that combines with VEGFR, EGFR, had kept the activity of melittin dissolved cell again.Therefore fusion toxin of the present invention both can pass through people VEGF ₁₆₅Combine with the tumour cell of VEGF expression R, EGFR, the VEGF165-melittin of enrichment high density around the tumour cell by the activity of the dissolved cell of melittin, reaches the purpose of target killing tumor cell, simultaneously, works as VEGF ₁₆₅-melittin is with after VEGFR, EGFR combine; Can also compete the binding site of VEGFR, EGFR with endogenic VEGF; Thereby the Tyrosylprotein kinase activation of blocking VEGF/VEGFR signal; Block multiple VEGFs, EGFs crosses strong signal, finally reaches the propagation, the migration that suppress tumour, especially suppresses the effect of the generation of new vessel.Experimental study confirms that VEGF165-melittin fusion toxin of the present invention has the activity of efficient combination, target killing tumor cell.

Compared with prior art, the present invention has following beneficial effect:

The present invention is with VEGF ₁₆₅Merge with small molecules toxin melittin, make up novel recombinant toxin VEGF ₁₆₅-melittin; In the eukaryotic expression system pichia pastoris, obtain solubility expression; And the lethal effect of its tumour cell detected; Filter out and have stronger target property and the active novel recombinant toxin of tumor cytotoxicity, the antineoplastic target treatment technology that will help further research and development to be correlated with has market outlook preferably.

Our experimental result shows, the fusion toxin VEGF that the present invention obtains ₁₆₅-melittin possesses simultaneously with VEGFR, the EGFR of tumour cell and combines, and discharges the effect of melittin killing tumor cell, and works as VEGF ₁₆₅-melittin is with after VEGFR, EGFR combine, and the Tyrosylprotein kinase activation of blocking VEGF/VEGFR signal is blocked multiple VEGFs, EGFs crosses strong signal, finally reaches the propagation, the migration that suppress tumour, especially suppresses the effect of the generation of new vessel.The present invention makes up fusion toxin VEGF with the target spot of VEGF/VEGFR system as the treatment tumour ₁₆₅-melittin realizes specificity target spot, efficient combination, target killing tumor cell, for tumor treatment is opened up a new design idea and approach.

Description of drawings

Fig. 1. the RT-PCR VEGF that increases ₁₆₅Gene mapping, wherein, swimming lane 1 is VEGF ₁₆₅PCR product, swimming lane 2 are dna molecular amount standard substance.

Fig. 2. pPICZ α/VEGF ₁₆₅The enzyme of-Melittin expression plasmid is cut evaluation figure, and swimming lane 1 is the recombinant plasmid pPICZ alpha/VEGF of Xho I and Xba I double digestion ₁₆₅-Melittin; Swimming lane 2 is the dna molecular mark; The recombinant plasmid that swimming lane 3 is cut for enzyme not.

Fig. 3. VEGF ₁₆₅Figure is identified in the expression of-melittin, wherein, swimming lane 1 for empty carrier conversion bacterial strain inducing after; Swimming lane 3 is the protein molecular weight standard article; Swimming lane 2,4,5 is recombinant plasmid pPICZ α/VEGF ₁₆₅-melittin transforms the band behind the bacterial strain inducing.

Fig. 4. VEGF ₁₆₅The purifying of-melittin and western-blotting identify figure.Wherein, figure A is the VEGF behind the purifying ₁₆₅-melittin identifies collection of illustrative plates, and swimming lane 1 is the VEGF of purifying ₁₆₅-melittin, swimming lane 2 is the protein molecular weight standard article; Figure B is western-blotting evaluation figure, swimming lane 1 for empty carrier conversion bacterial strain inducing after Protein Detection, swimming lane 2,3 is purifying VEGF ₁₆₅-melittin Protein Detection.

Fig. 5. RT-PCR detects the expression of VEGF, VEGFR1, VEGFR2 among tumor cell line AsPC-1, MHCC97-H and the HepG-2.

Fig. 6. mtt assay is measured the restraining effect of VEGF-Melittin fusion toxin to growth of tumour cell, and wherein for A liver cancer cell HepG-2, B are pancreatic cancer cell AsPC-1, C is liver cancer cell MHCC97-H.

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

(1) get the liver cancer cell HepG2 of VEGF high expression level, use Trizol reagent (Invitrogen Corp.San Diego, Ca] ifornia, USA.), extract total RNA with single stage method.Concrete steps are following: specifically, after the HepG2 cell counting that cultivation conditions is good, about 10 ⁶～10 ⁷Individual cell adds 1mL Trizol, and moving into 1.5mL does not then have in the RNA enzyme EP pipe.Add 200 μ L chloroforms again, thermal agitation shakes up, room temperature held 30 seconds.4 ℃ centrifugal (12 000 r/min) carefully transferred to supernatant 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 (12 000 r/min) 5 minutes, supernatant discarded.Add 70% (volume) ethanol lmL washing precipitation twice, 4 ℃ centrifugal (12 000 r/min) 5 minutes.Under the room temperature behind the remaining ethanol of air evaporation; Deposition is dissolved in the 50 μ L DEPC water; The analysis of carrying out RNA is with quantitative; Get 1 100 times of μ L diluted samples after ultraviolet spectrophotometer is measured OD260 and OD280, calculate its concentration and purity by following formula: rna content: RNA (μ g/mL)=40 * OD260 * extension rate, OD260/OD280=1.8～2.0 expression purity are qualified.With agarose gel electrophoresis method, observe the integrity of RNA.

(2) RT-PCR method human cloning VEGF ₁₆₅Gene: get the total RNA 1 μ g that as above extracts, add 1 μ L Oligo dT, 70 ℃ of sex change 10min, ice bath 1min; Add 10 * RNA PCR damping fluid, 2 μ L then, MgCl ₂(25 mmol/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; O1igo dT (20pmol/ μ L) 1 μ L, no RNA enzyme aqua sterilisa is added to final volume 20 μ L, sets up rt (RT) reaction system.42 ℃ of reactions are 60 minutes on the PCR appearance, and 99 ℃ made the reversed transcriptive enzyme deactivation in 5 minutes then, synthetic cDNA.

Through on NCBI, searching VEGF ₁₆₅Sequence (sequence number: NM_001204384.1), the design upstream primer 5 '-AAT CTC GAGAAC TTT CTG CTG TCT TGG G-3 ' (SEQ ID NO:4), downstream primer 5 '-AAT GAA TTCCCG CCT CGG CTT GTC ACA T-3 ' (SEQ ID NO:5); Entrust Shanghai Ying Jun company synthetic.Again by following PCR system amplification VEGF ₁₆₅Gene: above-mentioned cDNA reaction solution 20 μ L; MgCl ₂(25 mmol/L) 6 μ L; 10 * LA PCR damping fluid, 8 μ L; Upstream primer 1 μ L (20pmol/ μ L); Downstream primer 1 μ L (20pmol/ μ L); TaKaRa LA Taq (5U/ μ L) 1 μ L; Sterilization ultrapure water to final volume 100 μ L.The PCR condition is 94 ℃, 5min, 94 ℃, 30s, 57 ℃, 1min, 72 ℃, 30s, 35 circulations, 72 ℃, 10min; Whether 1.0% agarose gel electrophoresis is observed clip size and is matched with estimating the product size, and amplification is seen Fig. 1, visible about 500bp band.

(3) structure of cloning vector and evaluation: the people VEGF that reclaims the RT-PCR amplification ₁₆₅After the gene fragment, be connected 30 minutes for 16 ℃ with the T carrier.The ligation system comprises: people VEGF ₁₆₅Gene 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; Sterilization ultrapure water to final volume 10 μ L.

According to ordinary method, preparation bacillus coli DH 5 alpha competent cell adds above-mentioned connection product, carries out ordinary method and transforms.Get the competent cell that 200 μ L have transformed then and coat on the LB agar plate that contains X-Gal, IPTG and penbritin (100 μ g/mL), cultivated 12～16 hours in 37 ℃ of incubators.

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 cultures, the conventional then DNA that extracts.Get that 1 μ L dissolved DNA throw out carries out the agarose gel electrophoresis quantitative analysis and enzyme is cut evaluation.Use down for 37 ℃ XhoI with EcoR I double digestion is identified digestion 2 hours, is accredited as correct clone according to the inscribe zymogram behind 1% agarose gel electrophoresis.Select enzyme and cut the correct clone of evaluation, extract plasmid, be used for the dna sequencing analysis.

(4) synthetic 26 peptide Melittin genes, the N end carries (GGGGS) ₂Flexible small peptide, restriction enzyme site is carried at two ends respectively EcoRI with XbaI be connected expression vector pPICZ α structure recombinant vectors pPICZ α/melittin for merging the back with VEGF, and we introduces restriction enzyme site between connection peptides and melittin ApaI is so that can be with the VEGF in the fusion toxin molecule ₁₆₅Exchange with the order that is connected of melittin. EcoRI with XbaCarrying out routine with the pPICZ α carrier of same double digestion behind the above-mentioned synthetic gene of I double digestion is connected; Transformed into escherichia coli DH5 α competent cell after 16 ℃ of connections are spent the night; Zeocin microbiotic (25 μ g/mL) resistance screening; The picking positive colony is inoculated in the 5mL LB substratum at random, 37 ℃ of concussion overnight cultures, the conventional then DNA that extracts.1% agarose gel electrophoresis quantitative analysis and enzyme are cut evaluation and (are used down for 37 ℃ XhoI with EcoR I double digestion is identified, was digested 2 hours).Select enzyme and cut the correct clone of evaluation, extract plasmid, be used for the dna sequencing analysis.

(5) cut glue and reclaim the VEGF behind pcr amplification ₁₆₅Gene is used EcoR I with XhoI double digestion VEGF ₁₆₅PPICZ α/melittin recombinant vectors with same double digestion behind the gene is connected according to ordinary method, and is transformed into DH5 α competent cell, the several clones of random choose on flat board, and the extracting plasmid is used XhoI with XbaThe I double digestion is identified recon pPICZ α/VEGF ₁₆₅-melittin, qualification result is seen Fig. 2, the recombinant plasmid that swimming lane 3 is cut for enzyme not, the band of visible about 750bp, recon is confirmed the sequence accuracy through the order-checking of Beijing six directions China major company simultaneously, sequencing result with design consistent.

(6) get the correct culture bacteria liquid of order-checking, extract 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 pPICZ α/VEGF ₁₆₅-melittin, warp SacI enzymic digestion (linearizing) back is with phenol/chloroform extracting and use ethanol sedimentation.Linearizing plasmid is subsequent use on ice with 10 μ L ultrapure waters dissolving postposition.

(it is dull and stereotyped not contain antibiotic YPD Agr) 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 '-AAC TTT CTG CTG TCT TGG G-3 ' < SEQ ID NO:6>and downstream primer: 5 '-CCG CCT CGG CTT GTC ACA T-3 ' < SEQ ID NO:7 >, 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.

(7) fusion toxin VEGF ₁₆₅The expression of-melittin, evaluation: get above-mentioned qualification result male clone and be inoculated in 10mL BMGY (pH 6.0) substratum, 28 ℃ of concussions were cultivated 24 hours, to OD ₆₀₀Reach 2.0～6.0 o'clock collecting cells.With equal-volume (10mL) BMMY (pH 6.0) re-suspended cell deposition, abduction delivering is cultivated in 28 ℃ of concussions; The the 0th, 24,48,72,96,120,144 and 168 hour equi-time point cultivating respectively got the 0.5mL fermented liquid; Centrifugal collection supernatant adds sample buffer and prepares the electrophoresis sample, and SDS-PAGE, western-blotting identify the secreting, expressing of recombination fusion protein; Qualification result is seen Fig. 3, and Fig. 3 shows the about 28kD of fusion toxin protein expression band after inducing.Simultaneously, in inducing process, replenished methyl alcohol a to final concentration 0.5% in per 24 hours, replenish the sterilization ultrapure water simultaneously, the fermented liquid TV is remained unchanged.

(8) western-blotting of fusion toxin identifies: expression product is behind SDS-PAGE; Electrotransfer is to the PVDF film; Use confining liquid (TBS+1% polysorbas20+10% skim-milk) sealing 2h then; Then spend the night 4 ℃ of reactions with the anti-vegf protein antibody of rabbit, the anti-melittin antibody of rabbit (with the dilution proportion of confining liquid by 1:2500) respectively, (TBS+1% polysorbas20) washes film 5 times with elutriant; Add the goat-anti rabbit (1:6000) of confining liquid dilution then, room temperature reaction 1 h washes film 5 times with elutriant; Place the colour developing liquid that contains the horseradish peroxidase substrate to develop the color the PVDF film at last.

(9) the high recon of picking expression amount is equipped with its access in the culturing bottle of 5mL YPD substratum by 1% inoculum size, spends the night, and be forwarded in 500mL BMGY substratum by 1% next day, is cultured to OD ₆₀₀Reach 2.0～6.0, with equal-volume BMMY (pH 6.0) re-suspended cell deposition, 28 ℃ of concussions are cultivated, and abduction delivering is collected fermented liquid, centrifugal collection supernatant in the time of 96 hours.

(10) purifying of fusion toxin (Ni post affinity chromatography combines ultra-filtration membrane ultrafiltration, dialysis):

A, wash the post material with deionized water; Add 0.5 times of column volume 0.1M single nickel salt, fully the gantry post material; Add 5 times of volumes of deionized water and wash post, repeat 2 times after, the level pad balance, broken supernatant sample is gone up appearance with 0.5mL/min, is in charge of collection then; 1.5 the sample that do not adsorb of sample volume level pad flush away doubly, flow velocity 1-2mL/min is in charge of collection; With 1/2 times of volume elution buffer wash-out, flow velocity 1-2mL/min is in charge of the collection eluting peak.SDS-PAGE, western-blotting electrophoresis detection purifying protein VEGF ₁₆₅-melittin, qualification result is seen Fig. 4.

B, albumen adopt 10mM TrisHCl then through molecular weight 10000 daltonian ultra-filtration membrane ultrafiltration, after the 150mM NaCl dialysis, and freeze-drying.

(11) mtt assay is measured fusion toxin VEGF ₁₆₅-melittin is to the restraining effect of liver cancer cell HepG-2, pancreatic cancer cell AsPC-1 and liver cancer cell MHCC97-H growth

Get liver cancer cell HepG-2, pancreatic cancer cell AsPC-1 and the liver cancer cell MHCC97-H (1 * 10 of exponential phase of growth respectively ⁴) inoculation 96 orifice plates.Every kind of cell is divided into control group and experimental group, and experimental group adds VEGF ₁₆₅-melittin, concentration is respectively 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, and each concentration is established 5 multiple holes.Control group adds the H-DMEM nutrient solution of 10% foetal calf serum to equal-volume.Put into 37 ℃, 5%CO ₂Incubator is cultivated.Take out 96 orifice plates after 24 hours, in each hole, add MTT solution 20 μ L (5 mg/mL), 37 ℃ of 5%CO ₂Hatch and stop after 4 hours cultivating.The careful suction abandoned supernatant in the hole, and every hole adds 100 μ L DMSO, vibrates 10 minutes.Treat on Bio-red 550 ELIASAs, to measure absorbance (OD value) after deposition is dissolved fully.Calculate two groups respectively at different concns VEGF by following formula ₁₆₅Inhibitory rate of cell growth after the-melittin effect (three multiple MV).With inhibitory rate of cell growth drug level is done curve, obtain IC50.Inhibitory rate of cell growth (%)=(cellular control unit OD value-experimental group cell OD value)/cellular control unit OD value * 100%.

Fig. 5 is that the sxemiquantitative RT-PCR of VEGF, VEGFR1, VEGFR2 expression detects collection of illustrative plates.Detected the expression of VEGF, VEGFR1, VEGFR2 in the three strain tumor cell lines (human pancreatic cancer cell AsPC-1, Bel7402 MHCC97-H and HepG-2) respectively; The result shows the high expression level that all presents VEGF in the three strain clones; And the expression of VEGFR1, VEGFR2 is different: wherein the expression of VEGFR1 all is positive in AsPC-1 pancreatic cancer cell and the MHCC97-H liver cancer cell; The expression of VEGFR2 also is positive among the MHCC97-H simultaneously, and has only VEGFR2 to be positive in the HepG-2 liver cancer cell.

Purifying VEGF ₁₆₅After-melittin albumen and liver cancer cell HepG-2, pancreatic cancer cell AsPC-1 and liver cancer cell MHCC97-H were hatched altogether, mtt assay was measured VEGF ₁₆₅-melittin fusion toxin is as shown in Figure 6 to the restraining effect result of growth of tumour cell.It is thus clear that VEGF ₁₆₅-melittin is the strongest to the liver cancer cell HepG-2 growth-inhibiting effect of VEGFR2 high expression level; To the VEGFR1 high expression level and VEGFR2 expresses the growth-inhibiting effect of relatively low liver cancer cell MHCC97-H and takes second place; And the VEGFR1 high expression level, the growing state of the negative pancreatic cancer cell AsPC-1 that express of VEGFR 2 is compared with control group a little less than.The result shows, reorganization fusion toxin VEGF ₁₆₅-melittin has good tumor cytotoxicity effect, and the killing activity of tumour cell is demonstrated the characteristic of targeting in VEGFR2.

The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included within protection scope of the present invention.

SEQUENCE LISTING

< 110>Guangdong Pharmaceutical University

< 120>reorganization fusion toxin VEGF165-melittin

<130>

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 15

<212> PRT

< 213>artificial sequence

<400> 1

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr

1 5 10 15

<210> 2

<211> 218

<212> PRT

< 213>artificial sequence

<400> 2

Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu Tyr

1 5 10 15

Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly Gly

20 25 30

Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg

35 40 45

Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr

50 55 60

Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met

65 70 75 80

Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr

85 90 95

Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln

100 105 110

Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu

115 120 125

Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val Arg

130 135 140

Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr Lys

145 150 155 160

Ser Trp Ser Val Cys Asp Lys Pro Arg Arg Glu Phe Gly Ser Gly Gly

165 170 175

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr Asp Gly Pro

180 185 190

Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu

195 200 205

Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln

210 215

<210> 3

<211> 654

<212> DNA

< 213>artificial sequence

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aactttctgc tgtcttgggt gcattggagc cttgccttgc tgctctacct ccaccatgcc 60

aagtggtccc aggctgcacc catggcagaa ggaggagggc agaatcatca cgaagtggtg 120

aagttcatgg atgtctatca gcgcagctac tgccatccaa tcgagaccct ggtggacatc 180

ttccaggagt accctgatga gatcgagtac atcttcaagc catcctgtgt gcccctgatg 240

cgatgcgggg gctgctgcaa tgacgagggc ctggagtgtg tgcccactga ggagtccaac 300

atcaccatgc agattatgcg gatcaaacct caccaaggcc agcacatagg agagatgagc 360

ttcctacagc acaacaaatg tgaatgcaga ccaaagaaag atagagcaag acaagaaaaa 420

aaatcagttc gaggaaaggg aaaggggcaa aaacgaaagc gcaagaaatc ccggtataag 480

tcctggagcg tatgtgacaa gccgaggcgg gaattcggtt ccggtggtgg tggttctggt 540

ggtggtggtt ctggtggtgg tggtacggat gggcccggta tcggtgctgt gctgaaagtt 600

ctgaccactg gtctgccggc actgatttct tggatcaaac gcaaacgtca gcag 654

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<212> DNA

< 213>artificial sequence

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aatctcgaga actttctgct gtcttggg 28

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<212> DNA

< 213>artificial sequence

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aatgaattcc cgcctcggct tgtcacat 28

<210> 6

<211> 19

<212> DNA

< 213>artificial sequence

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aactttctgc tgtcttggg 19

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<212> DNA

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ccgcctcggc ttgtcacat 19

Claims (10)

1. reorganization fusion toxin VEGF ₁₆₅-melittin is characterized in that it being to connect VEGF through connection peptides at the aminoterminal of melittin small peptide melittin ₁₆₅Obtain, or pass through connection peptides at VEGF ₁₆₅Aminoterminal connect melittin small peptide melittin and obtain; The aminoacid sequence of said connection peptides is shown in SEQ ID NO:1.

2. according to the said reorganization fusion toxin of claim 1 VEGF ₁₆₅-melittin is characterized in that its aminoacid sequence is shown in SEQ ID NO:2.

3. claim 1 or 2 said reorganization fusion toxin VEGF ₁₆₅The encoding sox of-melittin.

4. according to the said reorganization fusion toxin of claim 3 VEGF ₁₆₅The encoding sox of-melittin is characterized in that nucleotide sequence is shown in SEQ ID NO:3.

5. claim 1 or 2 said reorganization fusion toxin VEGF ₁₆₅The expression vector of-melittin.

6. expression vector according to claim 5 is characterized in that it being by the said reorganization fusion toxin of claim 3 VEGF ₁₆₅The MCS that the encoding sox of-melittin inserts the carrier that sets out obtains, and the said carrier that sets out is pPICZa, pPIC9 or pHIL-sl.

7. the transgenic cell line that contains the said expression vector of claim 5.

8. the host bacterium that contains the said expression vector of claim 5.

9. reorganization fusion toxin VEGF ₁₆₅The preparation method of-melittin is characterized in that step is following:

(1) be primer with SEQ ID NO:4 ~ 5, amplification VEGF ₁₆₅Gene;

(2) synthetic melittin small peptide melittin gene, two ends connect restriction enzyme site respectively EcoRI with XbaI is connected to the MCS of carrier pPICZ α, obtains recombinant vectors;

(3) use respectively EcoRI with XhoI is to VEGF ₁₆₅Gene and recombinant vectors carry out double digestion, and after enzyme was cut the product connection, electroporation transformed host's Pichia yeast, makes up VEGF ₁₆₅-Melittin genetic engineering bacterium;

(4) cultivate VEGF ₁₆₅-Melittin genetic engineering bacterium, substratum are the BMGY substratum, cultivate down for 28 ℃, induce it to produce solubility recombination fusion protein VEGF ₁₆₅-Melittin;

(5) culture of step (4) is centrifugal, collection supernatant carries out affinity column chromatography purifying VEGF ₁₆₅-Melittin albumen, the said filler of column chromatography is the Ni-NTA resin, and adsorption conditions is between the pH 5.5 ~ 8.5, and elution requirement is for reducing pH, progressively improving the concentration of imidazoles or add EDTA or EGTA, and pH is between 4 ~ 6, and imidazole concentration is 0.02 ~ 0.5M;

(6) adopt molecular weight 1000 daltonian ultra-filtration membrane ultrafiltration, adopt the semi-permeable membranes dialysis, dialyzate is that 10mM TrisHCl and 150mM NaCl, freeze-drying obtain VEGF ₁₆₅-Melittin albumen.

10. claim 1 or 2 said reorganization fusion toxin VEGF ₁₆₅The application of-melittin in preparation targeting anti-tumor medicine.

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