CN100352838C - Interfusion protein of antineoplastic genetic engineering composed of target short peptide of receptor of epidermal growth factor and Lidamycin - Google Patents

Abstract

The present invention relates to a fusion protein SG-LDP-AE in strengthened genetic engineering, which is prepared by two technical lines: genetic engineering construction and molecule strengthening of the fusion protein. The fusion protein has strong effect on damaging tumor cells. The fusion protein can carry out specific combination with the tumor cells for expressing an epidermal growth factor receptor (EGFR) in vitro. An animal experiment shows that the fusion protein has obvious therapeutic effect on the transplanted liver cancer H22 of a mouse. When tolerable dose of the fusion protein is used, the inhibition ratio can reach to 95.8% (p<0.001). The result shows the characteristics of miniaturization and high efficiency of targeted drugs. The present invention is expected to be a novel targeted drug used for clinical tumor treatment.

Description

The antineoplastic genetic engineering fusion rotein that target short peptide of receptor of epidermal growth factor and lidamycin (LDM) constitute

Technical field:

The present invention relates to a kind of genetically engineered recombination fusion protein and preparation method thereof and the application in the antineoplastic target medicine of reinforcement.

Background technology:

Malignant tumour is a kind of serious threat human life and healthy disease.Wherein, alimentary tract cancer and respiratory cancer are the highest at China's sickness rate, mainly contain cancer of the stomach, liver cancer, lung cancer, the esophageal carcinoma, large bowel cancer etc., and along with the going from bad to worse of environment for human survival, the incidence of this class tumour is ascendant trend year by year.

At above-mentioned tumour, the treatment plan of present clinical employing mainly contains: surgical operation therapy, radiotherapy, chemotherapy, interventional radiology treatment, immunological therapy and targeted therapy etc.Said targeted therapy is to utilize antibody or the compound that tumour is had specificity avidity; Perhaps utilize the magnetic that the physics guide effect is arranged; The lipiodol that perhaps passes through tumor vessel specificity guiding is made conjugate, the purpose of less infringement healthy tissues to reach more killing tumor cells as carrier with " bullet " (radionuclide, chemotherapeutics, toxalbumin etc.) of tool killing tumor cells effect.Monoclonal antibody is of long duration as " carrier ", but the complete antibody molecular weight is approximately 150kDa, and is limited to the penetrating power of tumor tissues, causes the difficult deep of passing ECS arrival solid tumor of monoclonal antibody conjugate.Therefore, the molecule miniaturization has become the inexorable trend of monoclonal antibody targeted drug development.

(epidermal growth factor EGF) is made up of 53 amino acid Urogastron, and molecular weight is 6045KDa.The biological activity of EGF is very extensive, though its Urogastron by name, afterwards studies show that it all has short proliferation activity to the epithelial cell in multiple source, and relevant with wound healing.EGF also can be used as tumour and promotes factor enhanced virus or carcinogenic carcinogenesis.Many transformant secretion TGF-α also express Urogastron (EGFR) formation autocrine mechanism.The activation of EGFR has shown the process that helps strengthen growth of tumor and development, comprises the promotion hyperplasia, vasculogenesis, the invasion and attack/transfer of tumour and inhibition apoptosis.The development of the expression of EGFR and advancing of disease in the tumour cell, low survival rate, low result of treatment and pair cell poison, chemotherapeutics tolerance all has relation.Comprise prostate cancer in many kinds of tumours, mammary cancer, cancer of the stomach is observed the high level expression of EGFR in colorectal carcinoma and the uterus carcinoma.The related pathways that suppresses EGFR and EGFR comprises that monoclonal antibody that uses the outer ligand binding domain of anti-EGFR born of the same parents and the therapeutic strategies such as inhibitor that are directed to the tyrosine kinase activity of EGFR have entered clinical experiment.Tyrosine kinase inhibitor ZD1839 and OSI-774 are just carrying out III phase clinical study at present.On February 12nd, 2004, drugs approved by FDA anti-EGFR monoclonal antibody Erbitux ^TM(cetuximab is called IMC-C225 again) is used for the treatment of colorectal carcinoma patient in late period.Erbitux is one and contains people and the segmental genetically engineered chimeric antibody of musculus cdna simultaneously, and it is that EGFR with tumor cell surface is that target spot works, and disturbs the growth of tumour cell.

Lidamycin (LDM) (lidamycin, LDM, claim C1027 again) be to separate the strain actinomycetes obtain from Qianjiang county, China Hubei soil (Streptomyces globisporu, the s culture presevation number is: what CGMCC No.0704) produce has the large-molecular peptides antitumor antibiotics of strong lethal effect to tumour cell.Animal experiment shows in the body, and LDM is to mouse junction cancer 26, and transplanting in kinds of tumors such as the people's liver cancer Bel-7402 of nude mice and carcinoma of cecum Hce-8693 all has significant curative effect (Chinese microbiotic magazine 1994,19 (2): 164-168).LDM comprises two parts: (activeenediyne AE), has extremely strong cytotoxicity to a part, but unstable for the chromophoric group of enediyne structure; Another part is that (lidaprotein's apoprotein of forming of 110 amino-acid residues LDP), shields to chromophoric stability.Chromophoric group combines by non covalent bond with apoprotein, and both combinations are special, and can rebuild (Chinese Academy of Medical Sciences's journal 2001,23 (6): 563-567) after splitting.Lidamycin (LDM) has the molecular structure characteristics of unique detachable reconstruction, is convenient to DNA reorganization and molecule and strengthens; On the other hand, LDM apoprotein LDP molecular weight has only 10.5kDa, and when the fusion rotein that the somatomedin of LDP and different sources prepares, its molecular weight only is 15-20kDa, will be significantly less than known other immunotoxin fusion protein at present.What the present invention made up be " bullet " medicine with lidamycin (LDM), with EGFR targeting specific bound short peptide SG be " carrier " efficiently, the energized fusion protein SG-LDP-AE of miniaturization, not seeing as yet so far has relevant report.

The object of the present invention is to provide a kind of antitumor drug energized fusion protein SG-LDP-AE.

Another object of the present invention is to provide said energized fusion protein SG-LDP-AE preparation method.

Another purpose of the present invention is to provide a kind of energized fusion protein SG-LDP-AE application in neoplasm targeted therapy.

Summary of the invention:

In order to improve warm proteic effect, this laboratory is studied " carrier " and " bullet " and is selected, used " bullet " medicine is a lidamycin (LDM): the EGFR targeting specific bound short peptide SG that selects for use is when construction of expression vector, to add one section intestinal bacteria secreting, expressing signal peptide gene fragment with the upstream of the small peptide GE7 gene order of EGFR specific combination, between this gene fragment and small peptide fragment, introduce a restriction enzyme site NcoI.Wherein said small peptide GE7 is the principle that mutually combines according to part and acceptor, adopts computer image board design synthetic, it can with tumour cell specific combination (" Chinese science (C collects) " 1998,28 (6): 554-562) of expressing EGFR.When expression vector carries out secreting, expressing, stay two amino acid Met and Ala at the N of small peptide GE7 end behind the excision signal peptide, thereby obtain small peptide SG.This small peptide is that first is used for the EGFR targeting specific bound short peptide that merges with lidamycin (LDM), can make contributions for exploring new species target spot specificity binding fragment application facet.

The said energized fusion protein SG-LDP-AE of the present invention is made of the fusion rotein SG-LDP (molecular weight is about 13.9kDa) of the Histidine six aggressiveness urogenesis of EGFR target bonded small peptide SG, Lidamycin agon albumen LDP and carboxyl terminal and activated form enediyne chromophoric group AE (molecular weight is 843 Da), the full length gene 492bp of coding SG-LDP, 141 amino acid of encoding.SG full length gene 54bp wherein, 18 amino acid (the signal peptide gene sequence that 66bp is arranged before the SG gene) of encoding; LDP full length gene 330bp, 110 amino acid of encoding; Flexible peptide gene 15bp between the two, 5 amino acid of encoding; The Histidine six aggressiveness coda genes of carboxyl terminal are 18bp, 6 amino acid of encoding; XhoI restriction enzyme site gene between the Histidine six aggressiveness coda genes of SG-LDP gene and carboxyl terminal is 6bp, 2 amino acid of encoding, and terminator codon is 3bp.

Technology of preparing route of the present invention is:

Concrete steps are as follows:

The fusion gene of A.EGFR target bonded small peptide SG and Lidamycin agon albumen LDP makes up:

The structure that contains the expression vector pET30SGLDP of EGFR target bonded small peptide SG and Lidamycin agon albumen LDP fusion gene, it is the utilization genetic engineering technique, the clone obtains EGFR target bonded small peptide SG gene and Lidamycin agon albumen LDP gene respectively, the dna sequence dna of the two and one section little peptide of flexibility of coding is linked to each other and is built into the fusion gene of SG-spacer-LDP form, before the SG gene, introduce a segment signal peptide sequence simultaneously, introduce the specificity restriction enzyme site after this signal peptide sequence.

B. the structure of fusion rotein intestinal bacteria recombinant expression plasmid pET30SGLDP:

Fusion gene cloning to colibacillus expression plasmid pET-30a (+), is made up recombinant expression plasmid pET30SGLDP.

C. fusion rotein SG-LDP intestinal bacteria pET30SGLDP (deposit number: the abduction delivering CGMCC No.1320):

Fusion rotein SG-LDP is at e. coli bl21 (DE3) star ^TMIn abduction delivering comprise pET30SGLDP be transformed in the e. coli host bacteria, obtain the active fusion rotein SG-LDP of secretor type through the IPTG abduction delivering.

D. the affinitive layer purification of fusion rotein SG-LDP and separation:

The affinitive layer purification of fusion rotein SG-LDP and separation preparation thereof are by affinitive layer purification fusion rotein SG-LDP, dialyse again, concentrate, and prepare functional fusion rotein.

E. the preparation of energized fusion protein SG-LDP-AE and separation:

The preparation of energized fusion protein SG-LDP-AE separates, be with purifies and separates fusion rotein SG-LDP that obtains and the chromophoric active form enediyne of the lidamycin (LDM) AE for preparing through methanol extraction, with molecular ratio is that 50: 1 and enhanced time are 12 hours, carry out molecule under the room temperature and strengthen, obtain energized fusion protein SG-LDP-AE.

The immunologic competence of different tumour cells is detected and energized fusion protein SG-LDP-AE shows the tumour cell of energized fusion protein SG-LDP-AE preferred therapeutic EGFR high expression level in the application of treatment tumour or the tumour cell of Her2 high expression level to the cytotoxicity detected result of tumour cell by fusion rotein SG-LDP.

The tumour cell of EGFR high expression level comprises people's liver cancer Bel-7402, human oral tesselated epithelium KB cell, human colon carcinoma HT-29 cell, cells such as cervical cancer HeLa.

Advantage of the present invention and positively effect are, said energized fusion protein SG-LDP-AE good stability, and difficult drop-off can the using gene engineering means carry out fermentative production, and is with low cost.With EGFR target bonded small peptide is carrier, molecular weight is little, a little less than the immunogenicity, be difficult for causing the immune response of human body to exogenous antibodies, side effect is little, and the while small peptide is as the aglucon of target acceptor, and is strong to the seepage force of solid tumor, help to arrive the target tumour cell rapidly, give full play to the strong lethal effect of lidamycin (LDM) tumour cell.Energized fusion protein SG-LDP-AE of the present invention is a kind of targeted drug of new and effective miniaturization, being first is used for the antineoplastic target medicine that merges with lidamycin (LDM) with EGFR target bonded small peptide SG, the tumor-targeting of this fusion rotein is good, has good potential applicability in clinical practice.

Description of drawings:

Fig. 1: the restriction endonuclease analysis of recombinant plasmid pET-GLDP

Wherein: 1.DNA Marker DL15000 2.pET-30a (+)

3.pET-GLDP 4.pET-30a(+)/NdeI+XhoI

5.pET-GLDP/NdeI+XhoI the GLDPgene of 6.PCR amplification

7.DNA?Marker?DL2000

Fig. 2: the restriction endonuclease analysis of recombinant plasmid pET-SGLDP

Wherein: the SGLDP gene of 1.MarkerDL15000+2000 2.PCR amplification

3.pET-30a(+)/NdeI+XhoI 4.pET-30a(+)

5.pET-SGLDP/NdeI+XhoI 6.pET-SGLDP

Fig. 3: expression vector pET-SGELDP is at e. coli bl21 (DE3) star ^TMThe SDS-PAGE of middle abduction delivering analyzes

Wherein: 1. whole bacterial protein component before empty carrier is induced

2. empty carrier is induced back whole bacterial protein component

3.37 a ℃ expression strain is induced back whole bacterial protein component

4.28 a ℃ expression strain is induced back whole bacterial protein component

Albumen Marker (molecular weight is respectively 97.4kDa, 66.2kDa, 43kDa, 31kDa, 20kDa, 14.4kDa)

6. protein ingredient in the substratum before empty carrier is induced

7. empty carrier is induced protein ingredient in the substratum of back

8.37 a ℃ expression strain is induced protein ingredient in the substratum of back

9.28 a ℃ expression strain is induced protein ingredient in the substratum of back

Fig. 4: the SDS-PAGE of expression product and Western-blot analyze

Wherein: 1. albumen Marker (molecular weight is respectively 97.4kDa, 66.2kDa, 43kDa, 31kDa, 20kDa, 14.4kDa)

2. cell whole bacterial protein component before inducing

3. protein ingredient in the substratum before inducing

4. induce back cell whole bacterial protein component

5. induce protein ingredient in the substratum of back

6. periplasmic space protein ingredient

7. soluble protein component

8. soluble protein ingredient

Fig. 5: the SDS-PAGE that metal is integrated chromatographic separation purified fusion protein SG-LDP analyzes

Wherein: 1. albumen Marker (molecular weight is respectively 97.4kDa, 66.2kDa, 43kDa, 31kDa, 20kDa, 14.4kDa)

2. induce protein ingredient in the substratum of back

3. sample after dialysing behind the ammonium sulfate precipitation

4. not in conjunction with component

5. scrubbed component for the first time

6. scrubbed component for the second time

7. elution fraction for the first time

8. elution fraction for the second time

9. elution fraction for the third time

Fig. 6 A:SG-LDP to the immunity of different tumour cells in conjunction with activation analysis

Wherein: ◆ Bel-7402 ■ MCF-7

△MCF-7/Her2 ×KB

*Hela ●HT-29

Fig. 6 B:LDP to the immunity of different tumour cells in conjunction with activation analysis

Wherein: ◆ Bel-7402 ■ MCF-7

△MCF-7/Her2 ×KB

*Hela ●HT-29

Fig. 7: energized fusion protein is to Bel-7402 cells in vitro cytotoxicity

Wherein: ■ LDM

◆SG-LDP-AE

◆

Fig. 8 A:LDM is to MCF-7 cells in vitro cytotoxicity

Wherein: ■ MCF-7

◆MCF-7/Her2

Fig. 8 B: energized fusion protein is to MCF-7/Her2 cells in vitro cytotoxicity

Wherein: ■ MCF-7

◆MCF-7/Her2

◆

Fig. 9: energized fusion protein SG-LDP-AE is to the growth-inhibiting effect of rat liver cancer H22

Wherein: ◆ contrast ■ SG-LDP-AE 0.2mg/kg

△SG-LDP-AE?0.4mg/kg ×SG-LDP-AE?0.8mg/kg

*LDM?0.05mg/kg ●SG-LDP?1.6mg/kg

|MMC?1mg/kg

Embodiment:

Following illustrated embodiment, just illustrative for the present invention, and nonrestrictive.

Embodiment 1. contains the structure of EGFR target bonded small peptide SG/ Lidamycin agon albumen LDP expression carrier pET30SGLDP

L1 upstream 5 ' GA CAT ATGAAC CCC GTG GTG GGC TAC ATC GGT GAA CGT CCT CAG

Nde?I

TAT?CGT?GAC?CTG?GGT?GGA?GGC?GGT?TCA?GCG?CCC?GCC?TTC?TCC?GTC3’

R1 downstream 5 ' GTTA CTC GAGGCC GAA GGT CAG AGC CAC GTG3 '

Xho?I

L2 upstream 5 ' GAATCCA CAT ATGAAA TAC CTG CTG CCG ACC GCT GCT GCT GGT CTG

Nde?I

CTG?CTC?CTC?GCT?GCC?CAG?CCG?GCG?ATG?GCC?ATG?GCC?AAC?CCC?GTG

GTG?GGC?TAC3’

R2 downstream 5 ' GTTA CTC GAGGCC GAA GGT CAG AGC CAC GTG3 '

Xho?I

(contain the LDP gene with plasmid pIJ1027GRGDS, make up by this laboratory, this laboratory can provide and provide relevant proof to the public) be template, with primer L1 and R1 (Shanghai bio-engineering corporation is synthetic) routinely the PCR reaction conditions increase, obtain being about the GLDP gene fragment of 0.4kb.GLDP carries out 1% agarose gel electrophoresis with the pcr amplification reaction product, and the GLDP gene fragment is reclaimed with the glass milk test kit of BioDev company in dyed back.This gene fragment is connected with the T4DNA ligase enzyme with through the plasmid pET-30a (+) of Nde I/Xho I double digestion with Nde I/XhoI double digestion obtains plasmid pET30GLDP, to connect product Transformed E .coli DH5 α competent cell, picking mono-clonal after 37 ℃ of cultivations is cultivated back extraction plasmid in a small amount and is carried out Nde I/Xho I double digestion evaluation (Fig. 1).The recombinant expression plasmid that will downcut about 0.4kb gene fragment changes host bacterium BL21 (DE3) star over to ^TMIn the competent cell of (Invitrogen company product), screening obtains recombinant conversion and extracts transformant.With T7 universal primer order-checking, the result shows, this fusion gene conform to expection (the learned Beijing Company in Shanghai is carried out sequencing).

With plasmid pET30GLDP is template, with primer L2 and R2 (Shanghai bio-engineering corporation is synthetic) routinely the PCR reaction conditions increase, obtain being about the SG-LDP gene fragment of 0.5kb.All the other operations are the same, obtain expression plasmid pET30SGLDP, carry out enzyme and cut evaluation (Fig. 2).The recombinant expression plasmid that will downcut about 0.5kb gene fragment changes host bacterium BL21 (DE3) star over to ^TMIn the competent cell of (Invitrogen company product), screening obtains recombinant conversion and extracts transformant.With T7 universal primer order-checking, the result shows, this fusion gene conform to expection (the learned Beijing Company in Shanghai is carried out sequencing).SG full length gene 54bp, 18 amino acid (the signal peptide gene sequence that 66bp is arranged before the SG gene) of encoding; LDP full length gene 330bp, 110 amino acid of encoding; Flexible peptide gene 15bp between the two, 5 amino acid of encoding; The Histidine six aggressiveness coda genes of carboxyl terminal are 18bp, 6 amino acid of encoding; XhoI restriction enzyme site gene between the Histidine six aggressiveness coda genes of SG-LDP gene and carboxyl terminal is 6bp, 2 amino acid of encoding.The expression plasmid pET-30a (+) that the present invention uses, merge one section encoding histidine, six poly-tails arranged the (gene order of His * 6-Tag) at 3 ' end of its multiple clone site, behind accurate translation, His * 6-Tag is convenient to Expression of Fusion Protein and is identified and separation and purification.

Embodiment 2. fusion rotein SG-LDP are at e. coli bl21 (DE3) star ^TMIn abduction delivering

The expression vector that the present invention uses is coli expression system E.coli BL21 (DE3) star ^TM, be Invitrogen company product.With recombinant plasmid transformed e. coli bl21 (DE3) star that builds ^TMObtain the recombinant conversion bacterium, picking mono-clonal colony inoculation is to the LB substratum of the kantlex that contains 50 μ g/ml from the LB flat board, and 37 ℃, the 180rpm shaking culture is spent the night.Press 2-3% inoculum size transferred species next day, 37 ℃, shaking culture 2-3 hour to OD ₆₀₀About 0.8-1.0, adding final concentration in culture is the isopropyl-(IPTG) of 0.05mmol/L, induces 8 hours.15% SDS-PAGE analytical results (Fig. 3) shows, in nutrient solution, tangible exogenous protein expression band is arranged through the about 14kDa of inductive intestinal bacteria place, expression amount accounts for more than 70% of nutrient media components, then do not have this band without the inductive thalline in the nutrient solution, the expression of a spot of foreign protein is also arranged in the cytoplasm.Illustrate that expressed proteins mainly is present in the nutrient solution supernatant.The expression of foreign protein is all arranged under the inducing temperature of 37 ℃ and 28 ℃, and under the inducing temperature of expression amount than 28 ℃ under 37 ℃ the inducing temperature expression amount height.From whole bacterial protein, nutrient solution supernatant, cell pericentral siphon, endochylema, prepare sample respectively then, simultaneously with the sample of the bacterial strain of the pET-30a of band empty carrier with do not carry out the recombinant expressed thalline sample of inductive as negative control.Carry out SDS-PAGE and Western-blot and detect (Fig. 4) (half-dried electricity changes, and electric commentaries on classics condition is: continuous current 0.85mA/cm2, about 2 hours of time).One anti-is anti-His-Tag monoclonal antibody (Novagen company product), two anti-are the sheep anti-mouse igg of HRP mark (Santa Cruz company product), carry out color reaction (Western Blotting Luminol Reagent is a Santa Cruz company product), also confirm to have the expression of the foreign protein of band his-tag tail, and foreign protein is expressed in the nutrient solution supernatant mainly at the 14kDa place.Conversion bacterial strain with the optimal expression fusion rotein finally picked out, the plasmid pET30SGLDP that wherein contains energy expressed fusion protein SG-LDP, called after pET30SGLDP, deliver on March 3rd, 2005 and to be positioned at the common micro-organisms center preservation of Pekinese China Committee for Culture Collection of Microorganisms, deposit number: 1320.

The affinitive layer purification of embodiment 3. fusion rotein SG-LDP and separation preparation thereof

Collect the thalline fermented liquid supernatant of abduction delivering, at 4 ℃, slowly add ammonium sulfate under the stirring condition with the concentration of 113g/L.4 ℃ of static placements 1 hour, 4 ℃, centrifugal 20 minutes of 10000g collects supernatant.Supernatant at 4 ℃, is slowly added ammonium sulfate under the stirring condition, and making its final concentration is 390g/L.4 ℃ of static placement half an hour, 4 ℃, centrifugal 20 minutes of 10000g, collecting precipitation.Every 100ml fermented liquid gained precipitation 2ml 1 * Ni-NTA Binding Buffer (50mMNaH ₂PO ₄, 300mM NaCl, 10mM imidazole, pH8.0) dialyse with same solution in the dissolving back.

Ni-NTA His-Bind resin (Novagen) is packed in the chromatography column, with 1 * Ni-NTA Binding Buffer balance with dialyzed sample with slowly adding in the chromatography column behind the membrane filtration of 0.45um.Collect effluent liquid, flow velocity is 10-15ml/ hour; With 1 * Ni-NTA Binding Buffer washing chromatography column of 8 times of column volumes, collect effluent liquid, flow velocity is 20-30ml/ hour; 1 * Ni-NTA Elution Buffer (50mM NaH with 4 times of column volumes ₂PO ₄, 300mMNaCl, 200mM imidazole, pH8.0) target protein of elution of bound to the chromatography column collected effluent liquid, and flow velocity is 10-15ml/ hour; The each several part effluent liquid is respectively got 50ul and 50ul 2 * go up sample buffered soln (100mM Tris-HCl, pH6.8,4%SDS, 5%2-ME, 20% glycerine, 0.2% bromjophenol blue) mix boiling water bath 5 minutes with metaprotein ,-20 ℃ of preservations are in order to electrophoretic analysis.Carry out SDS-PAGE electrophoretic analysis (Fig. 5).Collection contains the elutriant of target protein, will contain elutriant PBS (137mM NaCl, 2.7mM KCl, the 10mM Na of target protein ₂HPO ₄, 2mM KH ₂PO ₄PH7.4) solution is dialysed, and obtains the target protein of high density again through ultrafiltration and concentration.

The preparation of embodiment 4. energized fusion protein SG-LDP-AE separates

Get the LDM dried frozen aquatic products (number of patent application: 001215272, publication number: 1284566) 10mg, add 5ml cold methanol jolting 5 minutes, placed 1 hour for-20 ℃, middle jolting 1 time, at 0 ℃, 12000rpm/min, centrifugal 20 minutes, supernatant liquor is rich in AE, and sediment is a peptide chain, repeats to extract 2 times.The spontaneous evaporation concentrated methanol solution, aforesaid operations need carry out under 4 ℃, lucifuge condition.The SG-LDP that gets certain volume and concentration is dissolved in the 0.01mol/L phosphoric acid buffer (pH7.0), the AE methanol solution (volume ratio 50: 1) that adds 5 times of molecular weight, mixed jolting, room temperature was placed 12 hours, to mix liquid with PD-10 post (business-like Sephadex G-25 post, the Pharmacia product) chromatographic separation is abandoned excessive unconjugated AE behind the A280nm ultraviolet monitoring, collect energized fusion protein SG-LDP-AE.

The immunologic competence of embodiment 5. fusion rotein SG-LDP detects

The tumour cell of taking the logarithm vegetative period is according to 2 * 10 ⁴The density in individual/hole is spread 96 orifice plates, cultivated 24 hours, through 4 ℃ of precooling 0.05% glutaraldehyde fix 15 minutes, after 1% the BSA sealing, every hole adds the fusion rotein of doubling dilution, 37 ℃ hatch 1-2 hour after, be an anti-(Novagen company product successively with anti-His antibody, 1: 2000 times of dilution), the goat anti-mouse igg antibody of HRP mark is two anti-(Santa Cruz company products, 1: 2500 times of dilution) 37 ℃ hatch half an hour respectively after, every hole adds 100 μ l OPD substrate reactions liquid (O-Phenylene Diamines: substrate buffer solution: H ₂O ₂=4mg: 10ml: 15ul) developed the color 10 minutes in the dark place, and microplate reader is measured 492nm place light absorption value.The result shows, tumour cell such as the KB of targent fused protein SG-LDP and different high level expression EGFR, 7402, HeLa, HT-29 etc. have stronger immunity in conjunction with activity, and Lidamycin agon albumen LDP to above-mentioned tumour cell almost do not have immunity in conjunction with active (Fig. 6 A, 6B).

Embodiment 6. energized fusion protein SG-LDP-AE detect the MTT of the cytotoxicity of tumour cell

The cell dissociation of taking the logarithm vegetative period, counting, 3 * 10 ³Individual/hole is laid on 96 well plates, contains 5%CO at 37 ℃ ₂Incubator in cultivate to inhale after 24 hours and abandon supernatant, add medicine with the different concns of RPMI1640 dilution, 200ul/ well, each drug level are established 3 parallel wells.Continue to cultivate after 48-72 hour, every well adds with PBS dissolved MTT (2mg/ml) 50ul, and 37 ℃ were continued to cultivate after 4 hours, inhaled and abandoned supernatant, added 150ul DMSO, and the shaking table jolting is 15 minutes under the room temperature, measures the absorbance value of 560nm on the microplate reader.No medicine contrast well and each 3 well of acellular blank well are all established in each experiment.Calculate the survival rate of cell and the IC of sample by following formula ₅₀Value.

The result shows, SG-LDP-AE is to the KB of high expression level EGFR, 7402, and HeLa, tumour cells such as HT-29 have the intensive lethal effect, IC ₅₀Value is respectively 8.9 * 10 ^-11M, 4.04 * 10 ^-11M, 1.14 * 10 ^-9M, 1.19 * 10 ^-9M (Fig. 7), and the IC of LDM ₅₀Be respectively 3.66 * 10 ^-11M, 6.98 * 10 ^-12M, 1.74 * 10 ^-10M, 1.92 * 10 ^-10M compares with LDM, its IC ₅₀Slightly high, it is former because the chromophoric group packaging efficiency that SG-LDP-AE is external is poorer than the chromophoric group packaging efficiency of natural LDM.

To detecting cytotoxicity with mtt assay behind the low MCF-7 cell transfecting Her2 gene of expressing EGFR, with the MCF-7 cell of untransfected in contrast, find that LDM changes little (Fig. 8 A), its IC to the cytotoxicity of two strain oncocytes ₅₀Value is respectively 1.02 * 10 ^-10(before the transfection) and 6.6 * 10 ^-11(after the transfection); And the cytotoxicity of the MCF-7 cell of SG-LDP-AE after to transfection Her2 gene is than the cytotoxicity strong (Fig. 8 B) of the MCF-7 cell of untransfected, its IC ₅₀Value is respectively 3.42 * 10 ^-9(before the transfection) and 5.6 * 10 ^-11(after the transfection), prompting SG-LDP-AE may also have the potential synergy to the tumour cell of high expression level Her2, and makes its tumour cell to high expression level Her2 more responsive.

The animal experiment treatment plan of embodiment 7. energized fusion protein SG-LDP-AE

According to dosage primary dcreening operation result, the administering mode of designing animal test of cure and dosage.Get body weight and be 60 of the kunming mices of 18-22g, random packet, 10 every group.Get rat liver cancer H22 ascites, being diluted to cell count with physiological saline is 7.5 * 10 ⁶Individual/ml, 0.2ml/ only, it is subcutaneous to be inoculated in the kunming mice armpit.Plant knurl after one day, control group intravenous injection physiological saline, all the other each groups give SG-LDP-AE respectively, LDM, SG-LDP, mitomycin (MMC).Be tail vein injection, 0.2ml/ only.Experimental session, tumour major diameter a of measurement in per 3 days and tumour minor axis b, and record the weight of animals.With formula V=1/2ab ²Calculate the knurl volume, draw tumor growth curve, and calculate inhibiting rate.As Fig. 9, shown in the table 1.

Table 1. energized fusion protein SG-LDP-AE is to the growth-inhibiting effect (testing the 21st day) of rat liver cancer H22

Group

Dose (mg/kg)

No.of?mice Start/end

?Body?wt.(g) ?Start/end

Tumor-size(cm 3) X±s

Inhibition rate(％)

Control LDM SG-LDP-AE SG-LDP MMC

- 0.05 0.8 0.4 1.6 1

10/10 10/10 10/10 10/10 10/10 10/10

40.639 30.483 25.462 29.829 39.533 36.157

8.74±5.40 0.98±1.21 0.36±0.66 1.81±2.05 6.39±3.24 5.82±4.06

- 88.8** 95.8** 79.3* 26.9 33.4

*P＜0.05vs.control；**P＜0.01vs.control。

The 21st day result of test A shows (table 1), in the energized fusion protein SG-LDP-AE body significant curative effect is arranged, at 0.4mg/kg, 0.8mg/kg dosage, can obviously suppress the growth of H22 subcutaneous tumors, from (Fig. 9), can see, SG-LDP-AE curative effect when 0.8mg/kg dosage is especially remarkable, and its inhibiting rate of the 21st day reaches 95.8%.

＜110〉Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences

＜120〉the antineoplastic genetic engineering fusion rotein of target short peptide of receptor of epidermal growth factor and lidamycin (LDM) formation

<140>

<141>

<160>2

<170>

<210>1

<211>492

<212>DNA

＜213〉artificial sequence

<220>

<221>sig_peptide

<222>(1)…(66)

<223>

<400>1

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60

atggccatgg cgaaccccgt ggtgggctac atcggtgaac gtcctcagta tcgtgacctg 120

ggtggaggcg gttcagcgcc cgccttctcc gtcagtcccg cctcgggtct gagtgacgga 180

cagagcgtgt cggtgtcggt cagcggtgcc gccgccggcg agacctacta catcgcccag 240

tgcgctccgg tcggtggcca ggacgcgtgc aacccggcga ccgcgacgtc cttcaccacg 300

gacgcgtccg gagcggcgtc gttcagcttc gtcgtgcgca agtcgtacac gggctccacg 360

cccgaaggca cgccggtcgg cagcgtcgac tgcgccacgg ccgcctgtaa cctcggcgcc 420

ggcaactccg ggctcgacct cggccacgtg gctctgacct tcggcctcga gcaccaccac 480

caccaccact ga 492

<210>2

<211>141

<212>PRT

＜213〉artificial sequence

<220>

<221>misc_feature

<222>

<223>

<400>2

atg aaa tac ctg ctg ccg acc gct gct?gct ggt ctg ctg ctc ctc 45

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu

gct gcc cag ccg gcg atg gcc atg gcg?aac ccc gtg gtg ggc tac 90

Ala Ala Gln Pro Ala Met Ala Met Ala Asn Pro Val Val Gly Tyr

1 5

atc ggt gaa cgt cct cag tat cgt gac ctg ggt gga ggc ggt tca 135

Ile Gly Glu Arg Pro Gln Tyr Arg Asp Leu Gly Gly Gly Gly Ser

10 15 20

gcg ccc gcc ttc tcc gtc agt ccc gcc tcg ggt ctg agt gac gga 180

Ala Pro Ala Phe Ser Val Ser Pro Ala Ser Gly Leu Ser Asp Gly

25 30 35

cag agc gtg tcg gtg tcg gtc agc ggt gcc gcc gcc ggc gag acc 225

Gln Ser Val Ser Val Ser Val Ser Gly Ala Ala Ala Gly Glu Thr

40 45 50

tac tac atc gcc cag tgc gct ccg gtc ggt ggc cag gac gcg tgc 270

Tyr Tyr Ile Ala Gln Cys Ala Pro Val Gly Gly Gln Asp Ala Cys

55 60 65

aac ccg gcg acc gcg acg tcc ttc acc acg gac gcg tcc gga gcg 315

Asn Pro Ala Thr Ala Thr Ser Phe Thr Thr Asp Ala Ser Gly Ala

70 75 80

gcg tcg ttc agc ttc gtc gtg cgc aag tcg tac acg ggc tcc acg 360

Ala Ser Phe Ser Phe Val Val Arg Lys Ser Tyr Thr Gly Ser Thr

85 90 95

ccc gaa ggc acg ccg gtc ggc agc gtc gac tgc gcc acg gcc gcc 405

Pro Glu Gly Thr Pro Val Gly Ser Val Asp Cys Ala Thr Ala Ala

100 105 110

tgt aac ctc ggc gcc ggc aac tcc ggg ctc gac ctc ggc cac gtg 450

Cys Asn Leu Gly Ala Gly Asn Ser Gly Leu Asp Leu Gly His Val

115 120 125

gct ctg acc ttc ggc ctc gag cac cac cac cac cac cac tga 492

Ala Leu Thr Phe Gly Leu Glu His His His His His His

130 135 140

Claims (6)

1, a kind of energized fusion protein SG-LDP-AE, it is characterized in that it be by with EGF-R ELISA EGFR target bonded small peptide SG, the molecular weight of Lidamycin agon albumen LDP and the six aggressiveness urogenesis of carboxyl terminal Histidine is that the fusion rotein SG-LDP of 13.9kDa and activated form enediyne chromophoric group AE that molecular weight is 843Da constitute, SG-LDP full length gene 492bp, 141 amino acid of encoding, the signal peptide gene sequence that 66bp is arranged before the SG gene, fusion rotein SG-LDP is the active fusion rotein of secretor type that obtains through the IPTG abduction delivering, and its gene coded sequence is the sequence 1 in the sequence table.

2, prepare the method for energized fusion protein SG-LDP-AE according to claim 1, it is characterized in that said method mainly adopts DNA reorganization and molecule intensifying technology route, concrete steps are as follows:

A, utilization genetic engineering technique, the clone obtains EGFR target bonded small peptide SG gene and Lidamycin agon albumen LDP gene respectively, the dna sequence dna of one section little peptide of flexibility of the two and coding is linked to each other, be built into the fusion gene of SG-spacer-LDP form, introduce the specificity restriction enzyme site behind the signal peptide sequence before the SG gene simultaneously;

B, with resulting fusion gene cloning to colibacillus expression plasmid pET-30a (+), make up recombinant expression plasmid pET30SGLDP;

C. fusion rotein SG-LDP is a abduction delivering among the intestinal bacteria pET30SGLD of CGMCC No.1320 at deposit number;

D. the affinitive layer purification of fusion rotein SG-LDP and separation;

E. the preparation of energized fusion protein SG-LDP-AE and separation.

3, preparation method as claimed in claim 2 is characterized in that pET30SGLDP is transformed in the e. coli host bacteria, through the IPTG abduction delivering, obtains the active fusion rotein SG-LDP of secretor type.

4, preparation method as claimed in claim 2 is characterized in that dialysing by affinitive layer purification fusion rotein SG-LDP again, concentrates, and prepares functional fusion rotein.

5, preparation method as claimed in claim 2, it is characterized in that fusion rotein SG-LDP that purifies and separates is obtained and the chromophoric active form enediyne of the lidamycin (LDM) AE for preparing through methanol extraction, strengthen by carrying out molecule under 1: 5 molecular ratio, 12 hours, room temperature, obtain energized fusion protein SG-LDP-AE.

6, the application of the described energized fusion protein SG-LDP-AE of claim 1 in preparation new antitumoral targeted drug.

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