CN103694357A - Preparation and application of novel soluble fusion protein DT390-triTMTP1 - Google Patents

Abstract

The invention discloses a novel recombinant fusion protein and a preparation method of the novel recombinant fusion protein, wherein the fusion protein is based on a diphtheria toxin (DT) molecular effect group DT390 and three repetitive sequences of a tumor metastasis targeting peptide TMTP1 and can target to highly-metastatic tumor. The novel recombinant fusion protein comprises the diphtheria toxin molecular effect group, the three repetitive tandem sequences of the tumor metastasis targeting peptide TMTP1, and a fused His expressed sequence tag, wherein the DT390 is the No.1 to No. 390 amino acids of the diphtheria toxin. The pure soluble fusion protein DT390-triTMTP1 capable of targeting to the highly-metastatic tumor is prepared by soluble expression in escherichia coli BL21 through utilizing an inducing method at 37 DEG C, purification through a nickel metal chelating column aiming at the His tag, and protein lysis. The fusion protein can achieve a curative objective of targeting killing tumor cells further. The fusion protein is free of obvious influence on normal cells, but has significant proliferation suppressing and apoptosis-inducing effects on tumor cells with high metastasis potential, wherein the tumor cells comprise prostatic cancer cells and gastric cancer cells. The fusion protein has significant curative effects for all the tested tumor animal models, and can effectively inhibit tumor metastasis. The fusion protein is free of obvious curative-related toxicity, and provides a novel design mode for subsequent molecular treatment.

Description

The preparation and application of a kind of novel soluble fusion rotein DT390-triTMTP1

Technical field

The present invention relates to the preparation and application of a kind of novel soluble fusion rotein DT390-triTMTP1, its technical characterictic is: it (is GG that structure diphtheria toxin molecule (DT) effect group DT390 connects with three tumor-necrosis factor glycoproteinss of metastases targeted polypeptide TMTP1 nVVRQgGGGS nVVRQgGGGS nVVRQ) recombinant prokaryotic expression vector, and carry out preparation, expression, purifying and the renaturation of recombinant protein.Through experiment in vivo and vitro, confirm that this novel soluble fusion rotein DT390-triTMTP1 can be used as antitumor drug, energy selective killing tumour cell, effectively suppresses growth and the transfer of tumour, and normal cell and tissue is not had to obvious toxic side effect.Content of the present invention belongs to medical biotechnology field.

Background technology

Health and the life of malignant tumour serious threat numerous people, the annual cancer new cases in the whole world approximately have 1,000 ten thousand examples, because cancer causes dead case approximately 7,000,000.And shift, being the early stage event of Tumor cell dissemination, is to cause malignant tumour to be difficult to the major reason of radical cure and high mortality.Effectively control metastases and just mean remarkable lifetime of improving tumour patient.The ordinary method for the treatment of clinically malignant tumour mainly comprises operation, chemotherapy, radiotherapy etc.Operation is the first-selected treatment means of early stage malignant tumor patient, and chemotherapy is the most frequently used auxiliary treatment means, can bring into play the effect of removing the remaining focus of operation or metastasis.Chemotherapy and operation coupling, can control broad variety malignant tumour preferably.But chemotherapeutics can cause obvious general toxic side effect and patient's untoward reaction, and the repetition chemotherapy of many courses for the treatment of easily causes the resistance of tumour cell, the effect of opposing chemotherapeutics.Along with the arrival of global aging society and the continuous rising of tumor incidence, reduce the traditional methods for the treatment of recurrence rate of malignant tumour, improving the efficient of oncotherapy is an urgent demand to treatment malignant tumour at present.Clinically in the urgent need to researching and developing some medicines for the undesirable intractable tumour of conventional treatment effect (high metastatic tumo(u)r, resistant tumors).Target therapeutic agent arises at the historic moment, and its objective is the drug level that improves focus part, reduces medicine toxic side effects, reduces dosage, and medicine can be played a role safely and effectively.

Molecular targeted therapy, as an emerging technology in recent years, causes the very big concern of tumour circle.The auxiliary tumour of the routines such as current radiation and chemotherapy also has damage in various degree to normal cell in killing tumor cell, thereby cause oncotherapy toxic side effect, severe patient can affect treatment and carry out, even jeopardizes patient's life, therefore applies polypeptide and carries out the direction that tumor-targeting biotherapy is current tumor biotherapy development.Clinically, most of chemotherapeutics are not preferentially dense poly-at tumor focus.There are some researches show, the total amount that arrives the chemotherapeutics of tumour kitchen range only accounts for 5%～10% of healthy tissues, and the toxic side effect of chemotherapeutics has usually limited the optimum quantum of utilization of chemotherapeutics simultaneously, finally causes chemotherapy to recur not exclusively, in early days and chemotherapy resistance.So far, existing many optimization methods are used for improving the selection toxicity of chemotherapeutics, as being coated with of chemotherapeutics, guiding of monoclonal antibody or tumour-specific acceptor/peptide part etc.The means of current molecular targeted diagnosis and treatment mainly contain two classes: monoclonal antibody and targeted polypeptide (Homing peptide).Targeted polypeptide (homing peptide) is a kind of diagnosing tumor of thinking at present more satisfactory and the carrier of magnetic target therapy, and compared with monoclonal antibody, its advantage is:

plasma clearance speed is fast, high-affinity, high specific;

good tissue penetration, can be absorbed by tumour cell;

be easy to the feature of chemosynthesis and reduced immunogenicity.Nearly more than ten years, domestic and international investigator applies peptide storehouse technology and carries out different cancer target peptide screenings for different target spots, such as Arap W etc., mammary cancer tumor-bearing mice is carried out obtaining after peptide library selection a series of and small peptide tumor vessel specific binding, RGD sequence is wherein proved and can be combined with integrin, can selectively targeted kinds of tumors.Under the promotion of huge medical requirement, utilize targeted peptide for the preparation of contrast medium, the medicine of diagnosing tumor, treatment and prepare gene therapy vector preparation will be likely within the several years official listing become clinical treatment medicine, become an indispensable integral part of the existing therapy system of tumour, for the final solution of tumour problem has brought new hope.Arap W is applied to tumor-bearing mice after utilizing RGD sequence and Zorubicin crosslinked, and the antitumor curative effect of finding medicine significantly increases and toxic action obviously reduces.Recently the research that is applied to liver cancer severe severe combined immunodeficiency tumor-bearing mice after crosslinked to novel polypeptide SP94 and liposomal doxorubicin finds, optimizing drug delivery system increases the apoptosis of tumour cell and reduce tumor-blood-vessel growth, thereby significantly increases anti-knurl effect.

Immunotoxin is the fusion toxin that proteotoxin molecule (plant poison, bacteriotoxin, mycotoxins, zootoxin etc.) is connected by chemistry with from immune targeted molecular (monoclonal antibody, immunoglobulin (Ig), somatomedin etc.) or genetic engineering technique coupling forms.In recent years, along with the development of genetic engineering technique, be all generally the fusion gene that adopts this technique construction lps molecule and targeted molecular, and then expression and purified fusion protein.Conventional lps molecule is conventionally from bacteriotoxic diphtheria toxin (DT) or Pseudomonas exotoxin (PE).Targeting fusion toxin provides a kind of challenging brand-new methods for the treatment of for malignant tumor patient.More especially to radiotherapy, the undesirable patients of traditional treatment effect such as chemotherapy, the meaning of application targeting fusion toxin treatment is more far-reaching.Targeting fusion toxin therapeutic strategy is very simple: the targeted molecular that has carried toxin protein can be identified and in conjunction with tumour cell, then enter in cell, by toxin protein performance toxicity cell killing.Multiple different targeted molecular can be for the preparation of targeting fusion toxin, if the antibody molecule of tumor cell surface specific epi-position, or can identify and in conjunction with ligand molecular of tumor cell surface specific receptor etc.The lps molecule of performance cytotoxic effect derives from bacterium (as diphtheria toxin DT or Pseudomonas exotoxin) or plant (as Ricin or abrin) conventionally.It is synthetic that these toxin suppress cell protein by different mechanism of action, causes necrocytosis.Ricin is by cut-out rrna, and diphtheria toxin and Pseudomonas exotoxin are synthetic by the active interior white matter of cell egg that suppresses of performance ADP-ribosylation, finally cause apoptosis.Because fusion toxin molecule can only enter the cell that surface has guide molecule identification and bind receptor.So in theory this targeted fusion toxin can be optionally killing tumor cell at utmost, reduce the side reaction for the treatment of simultaneously.

Diphtheria toxin is the cytotoxin that a kind of toxicity is very powerful.Natural diphtheria toxin molecular energy wide spectrum kills and wounds eukaryotic cell, by its natural receptor, in conjunction with territory (Zone R), be combined with the Heparin-binding EGF-R ELISA on mammalian cell surface, through endocytosis, be transported to acid phagocytic vesicle, carry out the dependent configuration of pH value and change, enzymic activity territory (C district) is sheared and is discharged in born of the same parents.Enzymic activity territory in born of the same parents is by the ADP ribosylation of catalysis elongation factor 2, causes that albumen is synthetic to be blocked apoptosis.In recent years, the development and application of targeting fusion toxin all develops rapidly.The ligand substituting diphtheria toxin natural receptor that energy selectivity is targeted to malignant cell surface by genetic engineering technique, in conjunction with territory (Zone R), produces the diphtheria toxin fusion rotein of a large amount of brachymemmas or restructuring.These fusion toxins represent the cytotoxic drug that a class is novel.Different from chemotherapeutics, diphtheria toxin class fusion toxin causes receptor-mediated endocytosis by targeted molecular, optionally enters in toxin sensitive cells, causes target cell apoptosis.Up to now, the fusion toxin based on diphtheria toxin mainly comprises: DT508-MSF, DT486-IL-2, DT390-GM-CSF, DT390-IL3, DT388-GM-CSF, DT388-IL-3, DT385-VEGF, and the fusion of DT388 and μ PA.In these fusion toxins, nT388-IL3 has obtained some reasonable effects in clinical trial, yet only has DT389-IL2 restructuring fusion toxin (denileukin diftitox) by U.S. FDA approval listing, is used for the treatment of adult skin t cell lymphoma.

The strategy of employing genetic engineering technique linking ligand and toxin has not only overcome the shortcoming of chemical connection process, has also shown that it is as pinpoint accuracy, the stronger advantages such as stability.The diphtheria toxin class fusion toxin of producing has been used to tumour, transmissible disease, and hemopathic treatment, some drugs has been obtained good clinical efficacy.Between toxin and part, increase a connector, by better acceptor, ligand-ligand interaction, impel fusion toxin to bring into play better effect, can make the Increased Plasma Half-life of small molecules amount molecule with the long ligand binding of serum half-life, the immunotoxin side effect prepared according to the strategy of selectivity target tumor cell surface high expression level molecule is little is even free from side effects, and normal surrounding tissue is shown to better security.

In the decades in past, scholars pass through cultured cells, animal model, and clinical patient has been researched and developed the immunotoxins of a large amount of anti-various malignant tumours.The effect of the small molecules fusion toxin wherein, consisting of as guide molecule somatomedin or Fv fragment is best.Immunotoxin has all shown good biological effect in testing in vivo and in vitro, but in fact, up to the present, is only only applicable to experimental study and external application clinically.The anti-human T cellular immunization of bivalent toxin A-dmDT390-bisFv (Μ CHT1) prepared by the people such as Woo JH connects diphtheria toxin single-chain antibody sFv amalgamation and expression with two, result shows that the bonding force of divalence recombinant toxin is seven times of monovalence, and lethality is enhanced to ten times.Can be by the targeted molecular coupling of immunotoxin and multivalence, to overcome the low tumour perviousness of immunotoxin, the problems such as non-specific toxicity and immunogenicity in clinical application from now on.The antitumor novel biotechnological formulation of targeting of developing, can effectively make up the deficiency of current gene therapy targeting; Alleviate the toxic side effect of normal tissue simultaneously, fundamentally improve the safety issue of gene therapy, the research and development of efficient, safe magnetic target therapy medicine are had to directive significance, be expected to promote development and the process of malignant tumour molecular targeted therapy.

Summary of the invention

Based on above technical background and great medical requirement, the present invention will disclose a kind of soluble fusion protein of connecting with three tumor-necrosis factor glycoproteinss of metastases targeted polypeptide TMTP1 based on diphtheria toxin molecule (DT) effect group DT390 and preparation method thereof, the soluble fusion protein obtaining by this scheme has efficient target and suppresses high metastatic potential tumor growth and propagation, can effectively suppress its transfer and wait clear superiority, and normal cell or tissue is not had to obvious toxic side effect, can make up the weak point of current therapeutic field of tumor, for future, the biological targeting treatment of metastases provides new selection.

Technical scheme of the present invention is: a kind of target high metastatic potential soluble fusion protein DT390-triTMTP1, is characterized in that: it is expressed label by DT effect group, three tumor-necrosis factor glycoproteinss of targeted peptide TMTP1 with the high metastatic tumour of target and the His of fusion and forms; Wherein DT390 is 1-390 amino acid of diphtheria toxin.

The catalytic domain that described DT390 comprises diphtheria toxin (enzyme activation district, 1-193 amino acids), transmembrane transport district (205-379 amino acid); The described targeted peptide TMTP1 with the high metastatic tumour of target is the C-terminal of this soluble fusion protein; It is the N-terminal of this soluble fusion protein that the His of described fusion expresses label.

The soluble fusion protein DT390-triTMTP1 of the high metastatic tumour of described target realizes solubility expression with 37 ℃ of induction methods in e. coli bl21 (DE3) plysS, and use the nickel metal-chelating column purification for His label, after protein cleavage, obtain pure DT390-triTMTP1 albumen.

Compare with existing immunotoxin, the present invention has reached following effect:

1 soluble fusion protein DT390-triTMTP1 builds three tumor-necrosis factor glycoproteinss of the 1-390 amino acids that comprises diphtheria toxin and the targeted peptide TMTP1 with the high metastatic tumour of target, in theory both retain the lethal effect of diphtheria toxin, brought into play again metastases targeted peptide TMTP1 target effect simultaneously.Due to this brand-new design, make this new construct can realize to a greater extent targeting killing off tumor cells, reach inhibition tumor cell growth and shift object.In addition, the present invention also will provide a kind of new Design Mode for follow-up other molecular therapies.

2 experiment in vitro results show, this soluble fusion protein, for the tumour cell that has high metastatic potential, comprises prostate cancer and cancer of the stomach, have significant inhibition propagation and apoptosis-induced effect, and normal cell is had no significant effect.

The body inner model of 3 pairs of animal models for tumour studies confirm that, by the route of administration of abdominal injection, this soluble fusion protein is to the tested significant therapeutic action of the equal tool of all animal models for tumour, and can effectively suppress metastases, and to animal without significantly treatment is xicity related.

4 prepare the fusion rotein of tumor targeting peptide and diphtheria toxin, biotherapy preparation as a kind of effective antitumour, because it had both had diphtheria toxin high efficiency cell toxicity, again can be in high metastatic tumour cell place's enrichment, therefore this novel soluble fusion rotein possesses the prospect of clinical development application, such as patients such as the advanced ovarian cancer shifting at height and cancer of the stomach, as a kind of new biotherapy strategy.

Accompanying drawing explanation:

The structure schematic diagram of Fig. 1, fusion rotein DT390-triTMTP1;

The three-dimensional simulation figure of Fig. 2, fusion rotein DT390-triTMTP1;

The electrophorogram of the purifying of Fig. 3, fusion rotein DT390-triTMTP1;

The propagation of Fig. 4, fusion rotein DT390-triTMTP1 inhibition tumor cell;

Fig. 5, fusion rotein DT390-triTMTP1 inducing apoptosis of tumour cell;

Fig. 6, fusion rotein DT390-triTMTP1 anti-tumor in vivo growth;

Restraining effect to metastases in Fig. 7, fusion rotein DT390-triTMTP1 body;

The distribution of Fig. 8, fusion rotein DT390-triTMTP1 each internal organs in nude mouse

Embodiment:

Below in conjunction with drawings and Examples, the present invention is described in further detail.

Tumor targeting peptide TMTP1 and the DT effect group DT390 fusion rotein with target high metastatic potential tendency of three tumor-necrosis factor glycoproteinss preparing according to technical scheme of the present invention, applying gene clone technology, build the tumor targeting peptide TMTP1 with target high metastatic potential tendency of three tumor-necrosis factor glycoproteinss and the fusion gene of DT effect group DT390, and in intestinal bacteria, obtained high efficient expression.

Realize the tumor targeting peptide TMTP1 with target high metastatic potential tendency of three tumor-necrosis factor glycoproteinss of the present invention and the preparation method of DT effect group DT390 fusion rotein, according to the following steps preparation:

(1) acquisition of DT390-triTMTP1 gene fragment:

At DT390-triTMTP1 primer two ends, restriction enzyme site Ndel and Xhol have been designed respectively, so that directed cloning.And on downstream primer, designed the sequence of three TMTP1 of one section of coding.With above-mentioned primer pcr amplification from the plasmid that contains diphtheria toxin full length sequence, obtain the gene fragment that DT390 and TMTP1 merge, be called for short DT390-triTMTP1 fragment

1. pcr amplification goal gene:

According to diphtheria toxin 1-390 amino acids sequence, adopt primer-design software Oligo 6.0, design specific amplification TMTP1-triDT390 primer, holds the restriction enzyme site sequence that adds respectively Nde I and Xho I at 5', and primer sequence is:

Upstream: 5'-CAT ATG GGC GCT GAT GAT GTT GTT G-3'

Downstream: 5'-CTC GAG ATT ATT GAC GCA CCA CGT TAG AAC CAC CAC CAC CTT GAC GCA CCA CGT TTG AAC CAC CAC CAC CTT GAC GCA CCA CGT TAC CAC C-3'

Usining my chamber preserves containing the plasmid of diphtheria toxin as template:

PCR reaction system (50 μ l)

Taq enzyme 10 * b μ ffer 5 μ l

MgCl ₂ (25mM) 3μl

dNTP (10mM) 1μl

Goal gene upstream primer 0.5 μ l

Goal gene downstream primer 0.5 μ l

GAPDH upstream primer 0.5 μ l

GAPDH downstream primer 0.5 μ l

Plasmid 1 μ l containing diphtheria toxin

Taq enzyme 2 μ l

ddH ₂O 38μl

Mix, simply centrifugal.The amplification condition of gene is: 95 ℃ of denaturation 5min; 94 ℃ of 30s, 60 ℃ of 1min, 5 circulations; 94 ℃ of 30s, 68 ℃ of 1min, 30 circulations, 72 ℃ of 10min.

PCR product is separated through 1.5% agarose gel electrophoresis, observes and take pictures under ultraviolet lamp.Utilize PCR product purification test kit purified pcr product.

(2) goal gene and T easy cloning vector is connected

After 1.PCR product purification, be connected and spend the night with T easy carrier.

2×bμffer 5μl

PCR product 3 μ l

T carrier 1 μ l

T4DNA ligase enzyme 1 μ l

Final volume 10μl

2. the competent preparation of intestinal bacteria E.coli DH5 α:

1. from flat board, the single colony inoculation of picking E.coli DH5 α is in 5m1 LB nutrient solution, and 37 ℃ of jolting 225rpm spend the night.

2. by 1:100, be inoculated in 150 ml LB nutrient solutions and continue jolting 2-3h, to OD value be 0.4-0.6.

3. by bacterium liquid ice bath 10min, 4 ℃ centrifugal, and 4000g * 10min collects bacterium.

4. 0.1 %CaC12 that adds 20 ml precoolings, slow suspension cell, ice bath 5min.

5. 4 ℃ centrifugal, 4000g * 10min, collects bacterium.

6. 0.1 %CaCL2 that adds 60 ml precoolings, the bacterium that slowly suspends, ice bath 30min.

7. competence bacterium packing 200 μ l are a, adding final concentration is that 15% glycerine is preserved liquid, and-80 ℃ save backup.

3. connect product transformed competence colibacillus bacterium E coli.DH5 α.

1. get competence bacterium, after thawing on ice, add and connect product 5 μ l, ice bath 30min after mixing gently.

2. 42 ℃ of heat-shocked 90s, rapidly ice bath 5min.

3. add 800 μ l LB nutrient solutions, 37 ℃ of jolting 225rpm, 60min.

4. mix bacterium liquid, get 100 μ l and coat on the LB culture plate containing IPTG, X-gal and Amp 100 μ g/ μ l, 37 ℃ of incubator overnight incubation.

4. the positive bacterium colony of picking, adds in the LB nutrient solution 5m1 containing Amp 100 μ g/ml, and 37 ℃ of 225rpm jolt and spend the night, and by plasmid extraction kit explanation, extracts plasmid.

5.Nde I and Xho I enzyme cut and identify after row 1% agarose gel electrophoresis, after size conforms to, serve the order-checking of Hai Ying fine horse bio-engineering corporation and with Genbank in sequence alignment.

(3) structure of pET-28a-DT390-triTMTP1 prokaryotic expression carrier

1. the plasmid of the correct T carrier containing DT390-triTMTP1 gene of order-checking, and carrier pET-28a is all used Nde I and Xho I double digestion, row 1% agarose gel electrophoresis, and object fragment and carrier are reclaimed respectively.

2. object fragment is connected, transforms, chosen clone with carrier, shake bacterium, to extract plasmid method the same, recombinant plasmid is the prokaryotic expression carrier containing complete DT390-triTMTP1 gene, called after pET-28a-DT390-triTMTP1.With Nde I and Xho I double digestion, row 1% agarose gel electrophoresis is identified.

By above experimental procedure, obtain recombination fusion protein pET-28a-DT390-triTMTP1 ,this fusion rotein is built and is formed by three tumor-necrosis factor glycoproteinss of TMTP1 and DT effect group DT390, and its building process is shown in Fig. 1.

(4) expression of DT390-triTMTP1 fusion rotein

1. recombinant plasmid transformed is entered to prokaryotic expression Host Strains BL21(DE3) plysS, method for transformation is the same.Picking list bacterium colony, is seeded in the LB substratum of 1ml containing kantlex (50 μ g/ml), paraxin (34 μ g/ml) and shakes bacterium, jolting 6h～8h, and PCR identifies.In the ratio of 1:50,1:100,1:100, be seeded in the SOB substratum of 5ml containing kantlex (50 μ g/ml), paraxin (34 μ g/ml) respectively; 37 ℃ of violent joltings, until OD value is 0.6; Add IPTG to final concentration 1mM, 2mM; 37 ℃ are continued to cultivate, and every 1h gets bacterium liquid 1ml, cultivates 4h; 4,000g 10min collects thalline.

2. get the thalline under different inductive conditions, get portion and add ultrasonic damping fluid ^*, fully mix in rear liquid nitrogen and freeze 10min, in frozen water, melt; Brief impulse ultrasound (5W, 30s * 6 time); The centrifugal 20min of 10,000g, its supernatant is partly soluble proteins in born of the same parents, and precipitation is soluble albumen in born of the same parents.Separately get portion and add 20ml 30mMTris/HCL, 20% sucrose (pH8.0), adds EDTA to 1mM after fully mixing, and concussion shakes up rear room temperature and places 5-10min; 8,000 4 ℃, 10min, removes supernatant, is resuspended in the 5mM MgSO4 of 20ml precooling, shakes 10min in ice bath; 8,000 4 ℃, 10min, collects supernatant, i.e. periplasm protein.

* ultrasonic damping fluid (50Mm sodium phosphate; 300mM NaCL pH 7.8)

3.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) detects protein expression situation.

1. the preparation of reagent:

30% polyacrylamide: 29g acrylamide is dissolved in 80ml tri-distilled water (heating to 35～37 ℃), more slowly add the two methene acrylamides of 1g, limit edged stirs, and adds water to 100ml, and filter paper filtering, is placed in brown bottle, room temperature preservation.

1.5mol/L TrisCl(pH8.8), 1mol/L TrisCl(pH6.8), 1mol/L TrisCl(pH7.4): take respectively 18.2g, 12.1g, 12.1g Tris alkali, add water to 80ml, with HCl, be adjusted to after corresponding pH value, add water to 100ml.

Tris-glycine electrophoretic buffer (pH8.3): 25 mmol/L Tris alkali, 192 mmol/L glycine, 0.1%SDS, prepares with tri-distilled water.

Transfering buffering liquid (pH8.3): 25mmol/L Tris alkali, 192 mmol/L glycine, 20% methyl alcohol, prepares with tri-distilled water.

2 * SDS gel loading buffer: 50mmol/L TrisCl(pH6.8), 20% glycerine, 4% SDS, 0.001% bromjophenol blue, faces the used time and adds 5% 2 mercapto ethanol.

2. glue

Separation gel and spacer gel be mixed with submeter

Composition 10% separation gel (10 ml) 5% spacer gel (5ml)

1.5mol/L Tris·Cl（pH8.8） 2.5ml

1mol/L Tris·Cl（pH6.8） 630μl

30% polyacrylamide 3.3ml 830 μ l

Tri-distilled water 4ml 3.4ml

10% SDS 100μl 50μl

10% ammonium persulphate 100 μ l 50 μ l

TEMED 4μl 5μl

3. loading and electrophoresis

Get and respectively organize different components protein 20 μ l, add 20 μ l 2 * SDS gel loading buffers, mix in rearmounted 100 ℃ of boiling water and boil 5～10min, of short duration centrifugal, get 20 μ l loadings.Positive source connects lower groove, negative pole connects groove, with 60V voltage to separation gel, then use 120V voltage to bromjophenol blue apart from bottom line 1cm left and right.

4. coomassie brilliant blue staining: (0.25g coomassie brilliant blue R250 is dissolved in 100ml destainer to be dipped in staining fluid after gel is fixing, with filter paper, remove insoluble particles) in 30min at least, washing, proceeds to 45-60 ℃ of decolouring extremely completely of destainer (water: formaldehyde: Glacial acetic acid is 45:45:10).

4.Western Blot detects the expression with the fusion rotein of HIS label.

1. SDS-polyacrylamide gel electrophoresis is the same.

2. electrotransfer

After electrophoresis completes, take off gel, cut spacer gel and contain bromjophenol blue part, and the mark upper left corner.System " sandwich " is sandwich in the following order: porose poly (methyl methacrylate) plate, sponge, 3～5 metafiltration paper, gel, PVDF film or nitrocellulose (NC) filter membrane, 3～5 metafiltration paper, sponge, poly (methyl methacrylate) plate.In system " sandwich " sandwich process, drain bubble as far as possible, after making, put into the transfer groove that fills transfering buffering liquid, NC film, near anodal, shifts 1h with 350mA.

3. immunoblotting

5% the skimmed milk room temperature sealing 1h that A joins with TBST, or 4 ℃ spent the night;

B discards confining liquid, adds the primary antibodie with confining liquid 1:250 dilution, and 1h is hatched in room temperature jolting, or after room temperature jolting 30min, 4 ℃ are spent the night;

C washes film 10min with TBST, 3 times;

D adds HRP mark two anti-of 1:200 dilution, room temperature jolting 1h;

E TBST washes film 10min, 3 times;

F adds ECL chromogenic substrate;

After G 5min, develop (can successively decrease according to the result time), photographic fixing, and flowing water flushing, kept dry, takes a picture.

(5) a large amount of abduction deliverings of fusion rotein, purifying, renaturation and concentrated.

1. with a small amount of expression method, gather in the crops 1000ml bacterium liquid.

2. the extraction and purification of inclusion body

The expression bacterium slag that aforesaid method is collected is put-80 ℃ of refrigerators frozen 10 minutes, then takes out and puts into 37 ℃ of water-baths 10 minutes rapidly, then put frozen 10 minutes of-80 ℃ of refrigerators into, and so multigelation is 3 times.Add lysis buffer 50ml (lysing buffer) ultrasonic degradation on ice, 30HZ continues 30 seconds, 30 seconds, interval, and repetitive operation is until detect cell crashing ratio >=95% under microscope.Centrifugal 15 minutes of 4 ℃ of 12000rpm, abandon supernatant, lysis buffer for precipitation (containing Triton-X1OO) supersound washing on ice, fully resuspended rear recentrifuge.By this, operate repeated washing inclusion body 3 times, finally in 50ml centrifuge tube, collect inclusion body precipitation.Add 40ml and dissolve damping fluid (50mMGlycine, 100mM Tris buffer, pH8.0) ultrasonic resuspended inclusion body on ice, abandon supernatant, with sex change damping fluid (8M urea; 0.IM NaH2PO4; 0.01M Tris-cl, PH8.0) ultrasonic resuspended inclusion body on ice, then inclusion body suspension is added in the chromatographic column containing 6ml50%Ni-NTA HisBind resin.4 ℃ are reacted 1 hour.Successively through inclusion body lavation buffer solution C (8M urea; 0.1MNaH2PO4; 0.01M Tris-CL pH6.3) and damping fluid D (8M urea; 0.INaH2PO4; 0.01M Tris-CL pH5.9) cross post washing.Finally use elution buffer E (8M urea; 0.1MNaH2PO4; 0.01M Tris-CL pH4.5) wash-out.

3. extract amount and the purity detecting of inclusion body

Get the inclusion body sample 10 μ l of wash-out, add 2.5 μ l5x sample-loading buffers to mix, microwave oven boils 10 minutes.Albumen marker and ready sample are carried out to SDS-PAGE electrophoresis (5% concentrated glue, 10% separation gel).After gel electrophoresis finishes, gel is steeped into SDS-PAGE silver ammonia dyeing gel stationary liquid (guaranteeing that stationary liquid fully covers gel), horizontal shaking table, room temperature is fixed 1 hour.Abandon stationary liquid, add appropriate coomassie brilliant blue staining liquid (R250), horizontal shaking table slowly shakes, and room temperature lucifuge is dyed glue and spent the night.Next day, reclaim coomassie brilliant blue staining liquid, add distilled water, horizontal shaking table slowly shakes decolouring, during repeatedly to change water transparent to gel clean background.

Experimental result shows: get and carry the recombinant expression vector bacterial classification that sequencing result confirmation successfully constructs, a large amount of induction expression of recombinant plasmid, through collecting bacterium, ultrasonic degradation discharges inclusion body on ice, then uses 50%Ni-NTA HisBind resin purification, finally with elutriant, the inclusion body protein gradation in chromatographic column is eluted, collect respectively each eluate, thermally denature is by SDS-PAGE gel electrophoresis, and coomassie brilliant blue staining detects amount and the purity of the fusion rotein at every turn eluting.As shown in Figure 3, just comprise for the first time target protein, and 3 times reach wash-out peak value in elutriant, the inclusion body protein of wash-out reduces gradually afterwards, and the inclusion body protein concentration in the 9th elutriant is extremely low.The foreign protein that in front 2 elutriants, total white egg contains a little non-target protein, after the 3rd wash-out, foreign protein disappears, and has obtained the target protein (about 55kd) of very high purity.

4. renaturing inclusion bodies is with concentrated

The inclusion body solution of purifying mixes in the ratio of l:10 and dilution refolding liquid (1mM GSH, 0.2mM GSSG), and 4 ℃ are stirred 48 hours.Finally use 4 ℃ of super filter tubes, the fusion rotein after 4000rpm centrifugal concentrating renaturation.Get the fusion rotein after 20 μ l renaturation, separately get 2 μ g, 4 μ g, 6 μ g, 1O μ g BSA, after adding the sample-loading buffer of respective volume to mix, microwave oven boils 10 minutes.Albumen marker and ready sample are carried out to SDS-PAGE electrophoresis (5% concentrated glue, 10% separation gel).By the method for introducing, gel is fixed and coomassie brilliant blue staining again to rinsing, concentration and the purity of observing renaturation rear fusion protein above.

(6) in vitro study to multiple high metastatic cancer cell target killing effect of recombination fusion protein DT390-triTMTP1

1. cell cultures

L) with mitten from taking out the PC-3M-1E8 of conservation in liquid nitrogen, MKN-45, HEK293 cell, puts into rapidly 37 ℃ of water-baths and shakes, quick-thawing.

2) after cell thaws for suspension completely, 800rpm, centrifugal 5 minutes of room temperature, with 75% ethanol spray disinfectant, in ultra-clean cell platform, with careful suction of 1ml sample loading gun, abandon frozen storing liquid, add fresh RPMI1640 substratum re-suspended cell precipitation, proceed in T25 Tissue Culture Flask, mend RPMI1640 substratum to the about 6ml of cumulative volume.Put 37 ℃, 5%CO2 incubator is cultivated.

3) under inverted microscope, observe next day, visible cell is adherent good, only has a little dead cell to be suspended in nutrient solution, inhales and abandons outmoded substratum, changes first liquid, puts incubator and continues to cultivate.

4) after 48 hours, again observe, visible recovery cytogamy has reached 80%-90%, prepares to go down to posterity.Old substratum is abandoned in suction, with the PBS crossing through autoclaving, softly wash 2 times, add 37 ℃ of preheatings 0.25% pancreatin, at the bottom of covering culturing bottle, tighten lid and examine under a microscope, it is clear that cell boundary becomes, cell starts to become bowlder, inhales and abandons pancreatin in culturing bottle, twists the lid of good culturing bottle, place after 1 minute, with suction pipe, blow and beat gently cell suspension the cell digesting is fully disperseed.And go down to posterity by 1:3, supply nutrient solution, blow even rearmounted 37 ℃ of incubators and cultivate.

5) within 2-3 days, change nutrient solution once.

6) select to carry out vitro effect experiment in logarithmic growth cell.

2. MTT (tetrazolium bromide) experimental implementation step:

1) digest above-mentioned logarithmic phase cell, the concentration of cell counting count board counting cells suspension, adjusts cell concn, is inoculated into 96 orifice plates, 8000/hole.

2) put 37 ℃, 5%C02 incubator is cultivated overnight incubation.

3) add aseptic fusion toxin DT390-triTMTPI:l, 2,3,4,5, the 6 μ g/ml of following concentration, each concentration is established 3 multiple holes, respectively establish 3 holes and do not add the blank group of fusion rotein and the hole (acellular, to only have isopyknic substratum) of returning to zero simultaneously.Put 37 ℃, 5%CO2 incubator overnight incubation.

4) after 24 hours, with careful suction of sample loading gun, abandon supernatant in hole, add 90 μ lRPMI1640 nutrient solutions, then add 10 μ g MTT solution (5mg/ml).37 ℃, 5%CO2 incubator is cultivated 4h.

5) 96 orifice plates are put in whizzer to centrifugal 5 minutes of normal temperature 800rpm.

6) the careful suction of sample loading gun abandoned liquid in hole.Add 150 μ l dimethyl Asias to sough (DMSO), put lucifuge on shaking table and softly sway skilful minute, abundant dissolving crystallized thing.

7) upper microplate reader detects the light absorption value in each hole, 490nm wavelength place.

8) processing of result, all light absorption values all represent with mean value scholar SD.The blank hole light absorption value of each strain cell of take is contrast, and the average cell survival rate of calculating under different fusion toxin mass actions (is treatment group mean value: blank cell mean).

Experimental result shows: as shown in Figure 4, DT390-triTMTP1 fusion toxin is (l-6 μ g/ml) under different activities, and the propagation of the remarkable inhibition tumor cell of dose-dependently increases apoptosis of tumor cells; 6 μ g/ml DT390-triTMTP1 cause almost 100% PE-3M-1E8 apoptosis, about 95%MKN-45 apoptosis, but normal HEK293 cell does not have obvious apoptosis (approximately 12% apoptosis).Be DT390-triTMTP1 to prostate cancer PE-3M-1E8 cell dosage dependency brought into play violent toxic action, cancer of the stomach MKN-45 cell has also been brought into play to significant lethal effect, and normal people's embryonic kidney epithelial cell HEK293 has not been had to obvious cytotoxicity.

3. flow cytometer detects apoptosis

Experimental procedure:

L) cell of digestion in logarithmic phase, is inoculated in 6 orifice plates, adjusts cell density, makes orifice plate inner cell degrees of fusion approximately 60%.

2) put 37 ℃, 5%C02 incubator is cultivated overnight incubation, makes cell attachment.

3) adding working concentration is the DT390-triTMTPI fusion toxin of 3 μ g/ml, establishes the blank hole that does not add toxin simultaneously.

4) for detecting the toxicity of TMTP1 micromolecule polypeptide to cell, add respectively in addition 3 μ g/mlTMTP1, establish equally the control wells that does not add processing factor.

5) effect, after 24 hours, carefully digests collecting cell with the pancreatin that does not contain EDTA.

6) 37 ℃, the cell of digestion being collected, centrifugal 5 minutes of 800rpm.Abandon supernatant, with phosphoric acid salt PBS re-suspended cell, adjust concentration, collect l * 10 ⁵individual cell, 800rpm is centrifugal 5 minutes again, with PBS, repeats to wash cell once.

7) inhale and abandon PBS, the Binding Buffer500 μ L re-suspended cell in Jia Kaiji apoptosis test kit.

8) add Annexinv-FITC 5 μ L, after softly mixing, add Propidium lodide 5 μ L, mix.

9) normal temperature, lucifuge reaction 10 minutes.

10) in 1 hour, upflowing cell instrument detects.

11) regulate fluidic cell instrument parameter, excitation wavelength is transferred to 488nm; Emission wavelength is transferred to 530nm.The AnnexinV-FITC of mark viable apoptotic cell detects with FLIC passage, and the PI of mark non-viable non-apoptotic cell detects with FL3 passage.And with the blank Cell regulate fluorescence compensation that does not add processing factor, set cross door, remove spectra overlapping.

Experimental result shows: as shown in Figure 5, compare with untreated control group, the TMTP1 peptide of 3 μ g/ml is not significantly increased the apoptosis rate of two kinds of tumour cells, and the DT390-triTMTP1 fusion toxin of 3 μ g/ml has significantly increased the apoptosis rate of MKN-45 and PC-3M-1E8 cell.

(7) research in the body of distribution and neoplasm targeted therapy in the DT390-triTMTPI fusion toxin body of recombination fusion protein

1. the foundation of primary nude mice by subcutaneous knurl model

1) get in the PC-3M-1E8 of logarithmic phase prostate cancer cell and MKN-45 stomach cancer cell, use 0.25% trysinization, centrifugal 5 minutes collecting cells of 800rpm, aseptic PBS re-suspended cell, adjusts cell concn after counting by tally.

2) after male nude mouse is fixing, take cotton swab to dip in iodophor disinfection nude mice left side thigh root skin, (concentration is 1.5 * l0 with 1ml syringe, to get 200 μ lPC-3M-1E8 prostate cancer cell suspensions ¹⁰/ L), be expelled to sterilized subcutaneous.Be every nude inoculation 3 * l0 ⁶individual cell.Inject altogether 3 of male nude mouses.

3) after female nude mice is fixing, take cotton swab to dip in iodophor disinfection nude mice left side thigh root skin, (concentration is l * l0 with 1ml syringe, to get 200 μ l MKN-45 stomach cancer cell suspensions ¹⁰/ L), be expelled to sterilized subcutaneous.Be every nude inoculation 2 * l0 ⁶/ L cell.Inject altogether 3 of female nude mices.

4) put into SPF environment and continue to raise, observe the tumour formational situation of injection site every day, after 10 days left and right Subcutaneous tumors have obviously formed, observe every other day tumor growth situation and by the value of vernier caliper measurement tumour maximum diameter and path.

2. the foundation of prostate cancer and cancer of the stomach subcutaneous tumors model

1) when primary prostate cancer/cancer of the stomach subcutaneous tumors grows to about 1cm * 1cm, get each 2 of prostate cancer that subcutaneous tumors growth conditions is good and gastric cancer in nude mices, cervical vertebra dislocation art is put to death tumor bearing nude mice, in Bechtop, with sharp sterile scissors and tweezers, peel off knurl body, put in stroke-physiological saline solution, remove reticular tissue, with scissors, knurl body is cut into about 2mm ³the knurl piece of size.

2) the fixedly nude mice to be transplanted of holding in Bechtop, iodophor disinfection left side thigh root, scalpel is carefully scratched one and is about the skin that 3mm sterilized, the careful blunt separation subcutis of tweezers, select a knurl piece shredding to insert wherein, No. 4.0 sutures skin sutures.Prostate cancer is transplanted 20 nude mices, and cancer of the stomach is transplanted 16 nude mices.

3) put back to SFP level Animal House and continue to raise, observe the tumour formational situation of transplantation site every day, after 7 days left and right Subcutaneous tumors have obviously formed, observe every other day tumor growth situation and by the value of vernier caliper measurement tumour maximum diameter and path.

3. prostate cancer and cancer of the stomach subcutaneous tumors nude mice packet transaction

1) when prostate cancer/cancer of the stomach subcutaneous transplantation knurl grows to about 3mm * 3mm, 15 prostate cancer tumor bearing nude mices are divided into 3 groups at random, 5 every group, every group is a cage, carries out mark.12 human gastric cancer in nude mices are divided into 3 groups at random, and 4 every group, every group is a cage, carries out mark.

2) prostate cancer/cancer of the stomach subcutaneous transplantation knurl tumor bearing nude mice is accepted respectively following processing (being abdominal injection): first group: 50 μ lPBS (blank group), second group; 50 μ lPBS TMTPl (containing 10 μ g peptides), the 3rd group; 50 μ l DT390-triTMTPl (containing 10 μ g DT390-triTMTPl fusion roteins).

3. within every 3 days, process in a manner described once, and before each processing, first use the value of vernier caliper measurement tumour maximum diameter and path.

4. process after 21 days (treating 8 times), by each group prostate cancer subcutaneous transplantation knurl nude mice, with after etherization, camera is taken pictures.And press formula V=4/ π * (d/2) ²* (D/2) calculating each gross tumor volume of measuring, d represents the value of the path of tumour, D represents the value of tumour maximum diameter, draws gross tumor volume growth curve.After taking pictures, stop processing, continue to raise, observe every day, until process for the first time latter the 46th day, observes and respectively organize tumor bearing nude mice death condition, and record is observed dead every day, and nude mice survival curve is respectively organized in drafting.

5. process after 21 days (treating 8 times), each is organized to cancer of the stomach subcutaneous transplantation knurl nude mice with putting to death after etherization, peel off tumour, each group tumour is first taken pictures with camera, with PBS, cleaning rearmounted 3.7% paraformaldehyde of bloodstain again soaks, the volume ratio of stationary liquid and knurl body is 10:1, send Wuhan Bioisystech Co., Ltd of Google to carry out paraffin embedding, 4 μ m sections.And press formula V=4/ π * (d/2) ²* (D/2) calculate each gross tumor volume of measuring, d represents the value of the path of tumour, D represent tumour maximum diameter value [51, draw gross tumor volume growth curve.

6. the foundation of cancer of the stomach model in situ

1) when primary cancer of the stomach subcutaneous tumors grows to about 1cm * 1cm, get 2 of the nude mices that subcutaneous tumors growth conditions is good, cervical vertebra dislocation art is put to death tumor bearing nude mice, in Bechtop, with sharp sterile scissors and tweezers, peel off knurl body, put in stroke-physiological saline solution, remove reticular tissue, with scissors, knurl body is cut into about 1mm ³the knurl piece of size.

2) by the female nude mice fasting after 12 hours of 12 preparation modelings, abdominal injection speed is slept and is newly anaesthetized, Iodophor and 75% alcohol disinfecting nude mice left side and middle part skin of abdomen, tweezers pick up the skin of sterilizing, scissors is cut off stomach wall, in paramesial left side, open abdominal cavity, stomach is carefully pulled out with tweezers, with suture needle, carefully repeatedly gently sting placenta percreta 3-5 time of greater gastric curvature side antetheca, choose a fresh cancer of the stomach subcutaneous tumors knurl piece shredding and be placed in herein, then with 5-0 absorbable suture, knurl piece is sewn on the greater gastric curvature placenta percreta of stabbing.By normal anatomy position, stomach is put back to abdominal cavity, with 1-0 suture, sew up stomach wall and skin.Clean bloodstain, the cage of putting back to disinfection by ultraviolet light continues to raise at SPF level Animal House.

7. the processing of cancer of the stomach tumor in situ

1) cancer of the stomach orthotopic transplantation tumor is postoperative the 7th day, and 15 postoperative nude mices are divided into 3 groups at random, 5 every group, by cutting ear, carries out mark.

2) transplanted tumor nude mice is accepted respectively following processing (being abdominal injection): first group: 50 μ lPBS (blank group), second group; 50 μ lPBS TMTPl (containing 10 μ g peptides), the 3rd group; 50 μ l DT390-triTMTPl (containing 10 μ g DT390-triTMTPl fusion roteins).

3) within every 3 days, process once in a manner described.

4) process 10 times after etherization process nude mice, dissect nude mice, take out primary tumor, liver, spleen and Abdominal wall metastasis knurl, after photographic camera is taken pictures, get macroscopic metastatic tumor in primary tumor and each tissue, PBS cleans rearmounted 3.7% paraformaldehyde of bloodstain and soaks, the volume ratio of stationary liquid and knurl body is 10:1, carry out paraffin embedding, 4 μ m sections, paraffin section HE dyeing.

5) incidence of nude mice primary tumor and metastatic tumor, statistic data respectively organized in record.

Experimental result shows: as shown in Figure 6, result shows that DT390-triTMTP1 fusion toxin has significantly suppressed the growth of PC-3M-1E8 prostate cancer subcutaneous tumors and significant prolongation the lifetime of tumor bearing nude mice.After DT39O-triTMTP1 fusion toxin is processed, the gross tumor volume of tumor bearing nude mice is obviously than the gross tumor volume little (P<0.05) of TMTP1 processing and control group tumor bearing nude mice., compare with TMTP1 meanwhile, DT390-triTMTPI fusion toxin significant prolongation the lifetime of nude mice.Similar to the result for the treatment of of PC-3M-1E8 prostate cancer subcutaneous tumors, DT390-triTMTP1 fusion toxin has significantly suppressed the growth of MKN-45 cancer of the stomach subcutaneous tumors, and TMTPI has no significant effect the growth of MKN-45 cancer of the stomach subcutaneous tumors.

As shown in Figure 7: each organizes the visible huge tumor in situ of stomach-tissue of nude mice, except DT390-triTMTPI fusion rotein treatment group, also visible obvious white metastasis on liver or spleen, stomach wall also has larger metastatic tumor.Statistics is respectively organized the incidence of each internal organs original position of nude mice or metastatic tumor, as shown in Figure 7, there is tumour in PBS, TMTP1 group nude mice stomach original position equal 100%, PBS nude mice of control group 100% liver metastasis, TMTP1 organizes 66.7% liver metastasis, there is spleen and shift in PBS, TMTP1 group 33.3%, there is Abdominal wall metastasis in PBS group 100%, there is Abdominal wall metastasis in TMTP1 group 33.3%, and DT390-triTMTP1 fusion rotein treatment group nude mice is without the visible liver spleen of naked eyes metastasis, Abdominal wall metastasis kitchen range, and cancer of the stomach original position generation tumour rate is 33.3%.

Above data presentation, fusion rotein DT390-triTMTP1 has the generation development of remarkable inhibition tumour and suppresses the effect of metastases, is expected to become a kind of new antitumoral diversion medicaments.

8. distribution and the metabolism of DT390-triTMTPl in tumor bearing nude mice body

1) by 1.2.2 introduction method, set up prostate cancer PC-3M-1E8 subcutaneous tumors.

2) get 9 male tumor bearing nude mices, the about 0.5-1.0cm of subcutaneous tumors volume ³, abdominal injection 50 μ l PBS, other 8 abdominal injection 50 μ l DT390-triTMTPl (containing 10 μ g DT390-triTMTPl fusion roteins) all.After half an hour, etherization is put to death injection 50 μ l PBS tumor bearing nude mices and a tumor bearing nude mice of injecting 50 μ l DT390-triTMTPl, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours respectively anesthesia put to death one of tumor bearing nude mice having injected 50 μ l DT390-triTMTPl.

3) tumor bearing nude mice of execution is anaesthetized in fresh dissection at every turn, gets subcutaneous tumors, heart, liver, spleen, lung, kidney, cerebral tissue, PBS cleans the rearmounted 3.7% paraformaldehyde soaked overnight of bloodstain, the volume ratio of stationary liquid and tissue is 10:1, carries out paraffin embedding, 4 μ m sections, paraffin section HE dyeing.

Experimental result shows: as shown in Figure 8, compare with PBS control group, injected His protein positive expression in the subcutaneous tumors tissue of DT390-triTMTP1 fusion rotein and obviously strengthened.Adopt H-score scoring, inject latter 1 hour, DT390-triTMTP1 fusion rotein starts in tumor tissues significantly dense poly-, still than control group is remarkable, increases after continuing to 48 hours.Prompting DT390-triTMTP1 fusion rotein can be in the short period of time in tumor bearing nude mice body efficient targeting in tumor tissues.Liver and renal tissue occur after half an hour that at injection DT390-triTMTP1 fusion rotein one crosses property and assembles, and expression intensity declines gradually afterwards, after 48 hours in tissue without positive expression.

Above result shows that DT39O-triTMTP1 fusion rotein energy high density is targeted to tumor by local, and toxic side effect is little simultaneously, reaches the object of bringing into play safely and effectively antitumor action.

The complete sequence of three tumor-necrosis factor glycoproteins fusion genes of diphtheria toxin molecule (DT) effect group DT390 and metastases targeting peptides TMTP1:

ATGGGCGCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAACTATGACGATGATTGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTcACTGAACCGTTGATGGAGCAAGTCGGAACGGAAGaGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGGCGATCAGTAGGTAGCTCATTGTCATGCATAAATCTTGATTGGGATGTCATAAGGGATAAAACTAAGACAAAGATAGGGTCTTTGAAAGAGCATGGCCCTATCAAAAATAAAATGAGCGAAAGTCCCAATAAAACAGTATCTGAGGAAAAAGCTAAACAATACCTAGAAGAATTTCATCAAACGGCATTAGAGCATCCTGAATTGTCAGAACTTAAAACCGTTACTGGGACCAATCCTGTATTCGCTGGGGCTAACTATGCGGCGTGGGCAGTAAACGTTGCGCAAGTTATCgatAGCGAAACAGCTGATAATTTGGAAAAGACAACTGCTGCTCTTTCGATACTTCCTGGTATCGGTAGCGTAATGGGCATTGCAGATGGTGCCGTTCACCACAATACAGAAGAGATAGTGGCACAATCAATAGCTTTATCGTCTTTAATGGTTGCTCAAGCTATTCCATTGGTAGGAGAGCTAGTTGATATTGGTTTCGCTGCATATAATTTTGTAGAGAGTATTATCAATTTATTTCAAGTAGTTCATAATTCGTATAATCGTCCCGCGTATTCTCCGGGGCATAAAACGCAACCATTTCTTGGAGGAAACGTGGTGCGTCAAGGAGGAGGAGGATCAAACGTGGTGCGTCAAGGTGGTGGTGGTTCTAACGTGGTGCGTCAATAA

Aminoacid sequence

MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIGSLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFL GGNVVRQGGGGSNVVRQGGGGSNVVRQ*。

Claims (6)

1. preparation and application of the soluble fusion protein DT390-triTMTP1 of novel targeted high metastatic tumour, is characterized in that: it is expressed label by three tumor-necrosis factor glycoproteinss of DT effect group, the high metastatic tumour targeted peptide of target TMTP1 and the His of fusion and forms; Wherein DT390 is 1-390 amino acid of diphtheria toxin.

2. a kind of novel targeted high metastatic potential soluble fusion protein DT390-triTMTP1 according to claim 1, it is characterized in that: described DT390 comprises the catalytic domain of diphtheria toxin (enzyme activation district, 1-193 amino acids), transmembrane transport district (205-379 amino acid); Described can the high metastatic tumour targeted peptide of target TMTP1 be the C-terminal of this soluble fusion protein; It is the N-terminal of this soluble fusion protein that the His of described fusion expresses label.

3. described in, high metastatic tumour targeting soluble fusion protein DT390-triTMTP1 realizes solubility expression with 37 ℃ of induction methods in e. coli bl21 (DE3) plysS, and use the nickel metal-chelating column purification for His label, after protein cleavage, obtain pure DT390-triTMTP1 albumen.

4.DT390-triTMTP1 aminoacid sequence:

MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIGSLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFL GGNVVRQGGGGSNVVRQGGGGSNVVRQ

The nucleotide sequence of DT390-triTMTP1:

ATGGGCGCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAACTATGACGATGATTGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTcACTGAACCGTTGATGGAGCAAGTCGGAACGGAAGaGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGGCGATCAGTAGGTAGCTCATTGTCATGCATAAATCTTGATTGGGATGTCATAAGGGATAAAACTAAGACAAAGATAGGGTCTTTGAAAGAGCATGGCCCTATCAAAAATAAAATGAGCGAAAGTCCCAATAAAACAGTATCTGAGGAAAAAGCTAAACAATACCTAGAAGAATTTCATCAAACGGCATTAGAGCATCCTGAATTGTCAGAACTTAAAACCGTTACTGGGACCAATCCTGTATTCGCTGGGGCTAACTATGCGGCGTGGGCAGTAAACGTTGCGCAAGTTATCgatAGCGAAACAGCTGATAATTTGGAAAAGACAACTGCTGCTCTTTCGATACTTCCTGGTATCGGTAGCGTAATGGGCATTGCAGATGGTGCCGTTCACCACAATACAGAAGAGATAGTGGCACAATCAATAGCTTTATCGTCTTTAATGGTTGCTCAAGCTATTCCATTGGTAGGAGAGCTAGTTGATATTGGTTTCGCTGCATATAATTTTGTAGAGAGTATTATCAATTTATTTCAAGTAGTTCATAATTCGTATAATCGTCCCGCGTATTCTCCGGGGCATAAAACGCAACCATTTCTTGGAGGAAACGTGGTGCGTCAAGGAGGAGGAGGATCAAACGTGGTGCGTCAAGGTGGTGGTGGTTCTAACGTGGTGCGTCAATAA。

5. novel restructuring soluble fusion protein claimed in claim 1 suppresses the multiplication capacity of tumour and the application in inducing apoptosis of tumour cell in vitro.

6. novel restructuring soluble fusion protein claimed in claim 1 is at neoplasm growth and suppress the purposes in metastases.

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