CN101003568A

CN101003568A - Recombined diphtheria toxin, preparation method, and application

Info

Publication number: CN101003568A
Application number: CN 200610163285
Authority: CN
Inventors: 李泽鸿; 张国利; 岳玉环; 朱平; 陈萍; 张林波; 赵福广; 吴广谋; 郑艳军; 张玲
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2006-12-20
Filing date: 2006-12-20
Publication date: 2007-07-25
Anticipated expiration: 2026-12-20
Also published as: CN100519577C

Abstract

This invention discloses a method for preparing recombinant diphtherin and its application. The recombinant diphtherin comprises diphtherin DAB389, a linker peptide (Gly4Ser) 2, and alpha-melanocyte stimulating hormone. The method comprises: (1) preparing a gene fragment of DAB389(Gly4Ser)2-alpha-MSH and a leading peptide, constructing an expression plasmid containing the gene fragment, and transforming Escherichia coli; (2) inducing with IPTG to obtain the target protein, purifying, digesting with Xa factor, and separating to obtain DAB389(Gly4Ser)2-alpha-MSH fusion protein. The method has such advantages as easy purification, and high yield. The recombinant diphtherin has high activity, and can attack and kill malignant melanoma without injuring normal cells, thus can be used to treat malignant melanoma.

Description

Recombined diphtheria toxin and its production and application

Technical field

The present invention relates to a kind of immune recombinant toxin and its production and application, more specifically say so a kind of recombined diphtheria toxin and preparation method and application.

Background technology

Malignant tumour is one of principal disease of serious threat human health and existence, has 12% to die from cancer in world's death toll at present.Whole world malignant tumour sickness rate is in rising tendency generally.According to data, to the end of the nineties, cancer mortality becomes first killer of human health.In the town dweller, cancer 11.2% rises to now 18.14% by what account for the cause of the death seventies, and the first place that has occupied the cause of the death constitutes increasing threat to human health and life.

Traditional oncotherapy means mainly are operation, radiotherapy, chemotherapy at present, but also far unsatisfactory.Various countries actively be devoted to explore always a kind of new, safer and more effective, specificity better and the littler tumor therapeuticing method of toxic side effect, caused gram cancer new approaches---neoplasm targeted therapy method.Neoplasm targeted therapy is directed to attack tumour cell but not Normocellular methods of treatment, also is one of popular topic of current medical science, field of biology.

As far back as 1906, Germanization scholar Erhlich just proposed the notion of targeted therapy.With the antibody of tumour-specific and bacterial exotoxin coupling prepare can the specific killing tumour " magic bullet ".Up to the seventies in 20th century, along with the development of biotechnology, and some can be used as the screening and the purifying of the toxin of " warhead section ", and targeted drug has just had bigger development, thereby has been born first-generation immunotoxin.

Recombinant toxin also claims immunotoxin, and the immunotoxin of the first-generation prepares by the chemical coupling method.Guiding materials such as tumor specific antibody, cytokine and toxicity molecule such as animals and plants toxin, radioelement are made with the method for chemical coupling.But, easily be degraded problems such as the transformation period is short, and immunogenicity is strong, complicated process of preparation and its clinical application is very limited because the connecting key instability.The immune recombinant toxin of the s-generation is the new bio guided missile after the monoclonal antibody targeted drug occurs.Utilize gene recombination technology fusion vector portion gene (comprising monoclonal antibody, antibody fragment, cytokine etc.) and toxin moiety gene, through the immune recombinant toxin of expressing, purifying prepares.Toxic component in the targeted drug (recombinant toxin) adopts bacteriotoxin more, and is owing to their structure and the singularity of function make them very easily penetrate solid tumor, stronger to the specificity toxic action of tumour cell.

With respect to traditional tumor therapeuticing method, targeted therapy has unarguable advantage: 1, it has overcome the defective that radiotherapy, chemotherapy cause serious harm to normal cell also indiscriminately in killing tumor cell; 2, it all has lethal effect to the tumour cell in any period; 3, because it has catalyst mechanism efficiently, so very a spot of molecule just is enough to impel death of neoplastic cells.

Now,, be the DAB of target spot abroad with the IL-2 acceptor to the investigative technique strength richness of recombinant toxin ₃₈₉-IL2 (trade(brand)name ONTAK) sells DAB by the drugs approved by FDA official listing ₃₈₉-TGF α, DAB ₃₈₉-EGF etc. have entered the second phase, three phases are clinical, and have received good effect.Also there are domestic recent years a large amount of engineered biological products to emerge, as Interferon, rabbit, interleukin etc.Domestic diphtheria toxin recombinant toxin, Pseudomonas aeruginosa extracellular toxin recombinant toxin also have patent application and product to occur, as: LHRH-PE40 is remarkable to the treatment for cancer effect, it can overcome the defective of first-generation immunotoxin effectively, has demonstrated wide application prospect at present.To have good social effect and economic benefit.

Diphtheria toxin (DT) is the extracellular toxin that is produced by the diphtheria corynebacterium that infects corynephage.Complete DT molecule is made up of 535 amino acid, and molecular weight is 58kDa.According to its 26S Proteasome Structure and Function characteristics, be divided into three structural areas to the C end from the N end: catalytic domain (C district), form by 1～193 amino-acid residue; Stride film district (T district), form by 205～378 amino-acid residues; Receptor binding domain (Zone R) is made up of 386～535 amino-acid residues.Zone R can with eukaryotic cell surface DT receptors bind, the T district inserts in the cytolemma after Zone R and receptors bind, and the C district is entered in the cell.C contains in the district rrna transferase active that ADP relies on, can catalyzing N AD ⁺The ADP ribosyl shift to elongation factor 2 (EF-2), generate niacinamide, make the EF-2 inactivation, blocking-up cell protein synthetic.Utilize 3 characteristics that functional zone are spatially separate of DT molecule, can Zone R be deleted by gene recombination, replace and be aglucon such as cytokine, the monoclonal antibody etc. of energy specific recognition tumor cell surface acceptor, this kind structure is recombinant toxin, it is combined in surface of tumor cells by receptor-ligand binding, utilize diphtheria toxin to have extremely strong cytotoxicity again, arrestin matter is synthetic, finally causes necrocytosis.The characteristics of recombinant toxin are to normal cell toxigenicity not, but can reach the purpose of treatment tumour by the specific killing tumour cell with the tumor surface receptors bind.

Application number is that 01138705.X and 01138706.8 Chinese patent application have made up receptor-directed sex-mosaicism toxin and the Diphtherin-gonadoliberin chimeric protein that has joint sequence, test shows that above-mentioned two kinds of recombinant toxins all have good targeting specific, certain cancers there is good therapeutic action, but do not have effect for malignant melanoma, be necessary to construct the recombinant toxin that targeting specific is arranged at melanoma for this reason.

Summary of the invention

Purpose of the present invention provides a kind of recombined diphtheria toxin, and it comprises: lps molecule diphtheria toxin DAB ₃₈₉, connection peptides (Gly ₄Ser) ₂, guide molecule melanotropin α MSH, SEQ ID NO.1 in its base sequence such as the sequence table.

Another object of the present invention provides a kind of recombined diphtheria toxin gene fragment, by HisTag+Ser+Gly+Xafactor+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH constitutes, SEQ ID NO.2 in its base sequence such as the sequence table, HisTag among the HisTag+Ser+Gly+Xa factor is an affinity labelling, Ser+Gly connects little peptide, Xa factor is the restriction endonuclease recognition sequence of Xa factor, and this HisTag+Ser+Gly+Xa factor complex body is referred to as the prefix body.

Another object of the present invention provides a kind of recombined diphtheria toxin preparation method, and this method comprises:

1, chemosynthesis contains prefix body+DAB ₃₈₉+ (Gly ₄Ser) ₂Upstream primer (SEQ ID NO.3), contain the downstream primer (SEQ ID NO.4) of α MSH reverse sequence, utilize pET28a/DAB ₃₈₉(Gly ₄Ser) ₂-EGF is a template, amplifies to contain prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The gene fragment of-α MSH, purifying reclaims, and must reclaim fragment;

2, fetch the section of taking up and add among the pMD-18T vector, use T ₄Dna ligase connects, and connects product and is converted in the intestinal bacteria, screens positive recombinant plasmid, obtains to contain prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The segmental recombinant plasmid of-α MSH, this recombinant plasmid of double digestion and expression vector pET28a (+) reclaim both endonuclease bamhis, with prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH gene fragment is mixed, is connected with pET28a plasmid fragment, will connect product and be converted in the intestinal bacteria, screens positive bacterium colony and cultivates, and extracts plasmid DNA;

3, with recombinant plasmid pET28a/ prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH is converted into and expresses in the bacterium, and abduction delivering is collected thalline;

4, adopt ultrasonic treatment, make the somatic cells fragmentation, centrifugal, collect supernatant, ammonium sulfate precipitation, affinitive layer purification and Xa factor cutting recombinant toxin expressing protein, recovery, obtain to remove the DA of prefix body ₃₈₉(Gly ₄Ser) ₂-α MSH fusion rotein.

As a kind of improvement, the described recombinant plasmid pET28a/ of recombined diphtheria toxin preparation method step 3 prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The optimum expression bacterial classification of-α MSH plasmid is BL21 (λ DE3); Optimum substratum is M ₉B ₂

Prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The best abduction delivering temperature and time of-α MSH be 30 ℃ 6 hours, the optimum amount of inductor IPTG is 1.4mmol/L.

Further object of the present invention recombined diphtheria toxin or a kind of recombined diphtheria toxin gene fragment are in the application of producing the melanoma medicine.

The present invention has adopted the diphtheria toxin DAB of brachymemma ₃₈₉As the effect part, it is made up of preceding 386 amino acid of diphtheria toxin and the 484th～486 3 amino acid, DT is much smaller for the molecular weight ratio diphtheria toxin, molecular weight is very large for the curative effect influence of solid tumor, this mainly is because press very high in the solid tumor, the lps molecule amount is big more, and it is inner just difficult more to enter solid tumor, and the molecular weight that reduces immunotoxin has not only guaranteed activity but also improve the solid tumor tumour inhibiting rate;

Selecting α MSH for use is targeting part, alpha-Melanocyte stimulating hormone (Alpha-melanocyte stimulatinghormone, α MSH) is a kind of of melanotropin, and it is made up of 13 amino acid, molecular weight 1 430Da derives from its prohormone proopiomelanocortin (POMC).Except that hypophysis, keratinocyte (KC) in the epidermis, melanophore (MC) and bright rare cell all can produce α MSH and related peptides class.α MSH sequence is silk-junket-Si-egg-paddy-paddy-phenylpropyl alcohol-essence-Se-Gan-Lai-dried meat-figured silk fabrics (α MSH (1-13)); Be a kind of neuro-endocrinology hormone, except that promoting that melanophore propagation, differentiation and melanocyte form, bringing into play many physiological actions in vivo.This hormone also has strong anti-inflammatory, regulates the effect of immunity; Under the mankind's pathological state, α MSH reaches inflammation part in blood plasma concentration significantly increases, it may be a kind of natural means of body to anti-inflammatory that prompting α MSH level raises, α MSH and its receptors bind, the performance anti-inflammatory action, also have significantly bring down a fever, anti-inflammatory and immunoregulation effect, be one of key material during nerve immunity is regulated; α MSH be meant can with the natural MSH of MSH receptor-specific bonded and analogue or the derivative on the cell surface, its acceptor extensively is present in a lot of surface of tumor cells, especially the surface of malignant melanoma, the overexpression of α MSH acceptor on cancer cells becomes the target spot of control cancer cell, α MSH can specific combination at the acceptor on cancer cells surface, therefore α MSH is well suited for the carrier as immunotoxin, carries toxin, the special cancer cells that kills and wounds.Animal and human research confirm, α-MSH and derivative toxicity is low, side effect is slight, and easily tolerance, anti-inflammatory action is strong and extensively, no obvious adverse reaction has the potential potential applicability in clinical practice; Therefore α MSH can be used for tumour, the especially preparation of malignant melanoma targeted drug;

The present invention is at diphtheria toxin DAB ₃₈₉Added with (Gly between the two with alpha-Melanocyte stimulating hormone ₄Ser) ₂The little connection peptides of forming, fusion gene is expressed the new shank that inserts in back and is not produced new antigenicity, simultaneously can not influence the original antigenicity of its both sides peptide chain, the existence of flexible peptide linker can make correct the folding in the protein function district at its two ends, thereby produce correct biological structure and space structure, make the extensibility of recombinant protein better, the wetting ability height.

For the convenience of recombinant toxin purifying, prefix body in the interpolation amplifies prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH gene fragment makes up pET28a/ prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH expression plasmid is transformed into expression in escherichia coli, induces through IPTG, obtains to contain prefix body+DAB ₃₈₉(Gly ₄Ser) ₂Acquisition DAB is cut, separated to the expressing protein of-α MSH through purifying, Xa factor enzyme again ₃₈₉(Gly ₄Ser) ₂The fusion rotein of-α MSH.The present invention proves by experiment: it is convenient that the toxin of reorganization has purifying, reduces cost, and the yield height improves the active advantage of recombinant toxin; Simultaneously malignant melanoma is had the characteristic that specificity is attacked and killed and wounded, and normal cell is not had injury, reach the purpose of effective treatment malignant melanoma disease.

Description of drawings

Accompanying drawing 1 shows that the present invention is used for the recombinant expression plasmid pET28a/ prefix body+DAB of expressed fusion protein ₃₈₉(Gly ₄Ser) ₂The construction strategy of-α MSH.

Accompanying drawing 2 shows the present invention prefix body+DAB that encodes ₃₈₉(Gly ₄Ser) ₂1% agarose gel electrophoretogram of the pcr amplification product of the segmental dna sequence dna of-α MSH, wherein swimming lane 1 is a dna molecular amount mark, swimming lane the 2, the 3rd, coding prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The segmental dna fragmentation of-α MSH.

Accompanying drawing 3 shows that the present invention contains prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The SDS-PAGE of-α MSH recombinant toxin expression product analyzes, and wherein swimming lane 1 is the protein molecular weight mark; Swimming lane 2 is full bacterium of host cell e. coli bl21 (λ DE3); Swimming lane 3 is to comprise prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The full bacterium ultrasonic degradation supernatant of-α MSH recombinant toxin; Swimming lane 4 is to comprise prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The full bacterium ultrasonic degradation precipitation of-α MSH recombinant toxin; Swimming lane 5 is to comprise prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The whole bacterial protein of-α MSH recombinant toxin.

What accompanying drawing 4 showed purifying comprises prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The SDS-PAGE of the expression product of-α MSH recombinant toxin analyzes, and swimming lane 1 is the protein molecular weight mark; Swimming lane 2 is the expressing proteins after the purifying desalination.

The DAB that obtains after accompanying drawing 5 demonstrations are cut with the Xa factor enzyme ₃₈₉(Gly ₄Ser) ₂The SDS-PAGE analytical results of-α MSH fusion rotein, swimming lane 1 are the protein molecular weight marks, and swimming lane 2 is prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH expressing protein, swimming lane 3 are the DAB that obtain after cutting with the Xa factor enzyme ₃₈₉(Gly ₄Ser) ₂-α MSH fusion rotein.

Accompanying drawing 6 shows DAB of the present invention ₃₈₉(Gly ₄Ser) ₂-α MSH fusion rotein is to people's malignant melanoma cell A375, human lung cancer cell A549's specific killing activity, and wherein 6.1 is DAB ₃₈₉(Gly ₄Ser) ₂-α MSH fusion rotein is to the cytotoxic effect of A375 cell, 6.1.1 control group; 6.2 be DAB ₃₈₉(Gly ₄Ser) ₂-α MSH fusion rotein is to the cytotoxic effect of A549 cell, and 6.2.1 is a control group.

Accompanying drawing 7 shows DAB of the present invention ₃₈₉(Gly ₄Ser) ₂-α MSH fusion rotein is to the 503nhibiting concentration (IC of malignant melanoma cell A375, lung cell A549 ₅₀).

Embodiment

Embodiment 1: recombinant plasmid pET28a/ prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The structure of-α MSH.

Present embodiment is described the recombinant expression plasmid pET28a/ prefix body+DAB that is used to express recombinant toxin of the present invention ₃₈₉(Gly ₄Ser) ₂The construction strategy of-α MSH and basic skills see also accompanying drawing 1;

In order to finish the present invention, can use gene fusion technology well known by persons skilled in the art to prepare recombinant toxin protein of the present invention.For this reason, can finish various DNA operations according to known DNA recombinant technology (as referring to Sambrook etal, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, 1989).PET28a/DAB ₃₈₉(Gly ₄Ser) ₂EGF is provided by Zhang Guoli, and plasmid pET28a (+) (Novagen), recipient bacterium JM109, BL21 (DE3) be all available from Novagen company.PCR test kit, DNA Marker DL-2000, DNA reclaim test kit, restriction enzyme, T4 dna ligase, Ex Taq archaeal dna polymerase, IPTG, pMD18-T carrier, protein molecular weight mark available from TakaRa company, and primer synthesizes and sequential analysis is finished by the precious biotech firm in Dalian; Horse diphtheria toxin positive serum is provided by Zhang Guoli, and the mouse-anti Ma Erkang of HRP mark is provided by Changchun Jin Kelong company;

1) primer design is with synthetic

With reference to Collier RJ (Mol Microbiol.1988,2 (2): 293-296) Bao Dao DAB ₃₈₉The nucleotide sequence of gene and Zhang Guoli etc. (Chinese Journal of Immunology .2004,20 (3): 171-172) Bao Dao sequence, design and synthesize 2 primers, chemosynthesis contains prefix body+DAB ₃₈₉+ (Gly ₄Ser) ₂Upstream primer-Primer 1 (SEQ ID NO.3), contain the downstream primer-Primer 2 (SEQ ID NO.4) (Dalian precious biotech firm) of α MSH antisense sequences, use pET28a/DA ₃₈₉(Gly ₄Ser) ₂EGF is a template, pcr amplification prefix body+DAB ₃₈₉(Gly ₄Ser) ₂α MSH gene fragment, for the benefit of the clone introduces Nco I and EcoR I restriction enzyme site respectively among Primer 1 and the Primer 2;

Primer 1：5’catg ccatgg gcagc catcatcatcatcatcac agcggc attgagggtcgc ggcgctgatgatgttgttg 3’

Nco I His.Tag Ser Gly Xa factor

Primer 2：5’cg gaattcttataccggtttaccccagcggaagtgttccatgctgtagctggatccacctccgcctga 3’

EcoR I

2) pcr amplified fragment

With pET28a/DAB ₃₈₉(Gly ₄Ser) ₂EGF is a template, pcr amplification prefix body+DAB ₃₈₉(Gly ₄Ser) ₂α MSH gene fragment, reaction conditions are 94 ℃, 60s, 58 ℃, 70s, 72 ℃, 80s, 30 circulations; 72 ℃, 600s, 4 ℃, 300s, 1% agarose gel electrophoresis detects result's (seeing accompanying drawing 2) of pcr amplification, with the recovery of PCR product, purifying;

The PCR reaction system

Composition	Volume (μ L)
Composition	Volume (μ L)	dd H ₂O 10×Ex Taq Buffer DNTPs Mixture Primer 1(15μmol/L) Primer 2(15μmol/L) pET28a/DAB ₃₈₉(Gly ₄Ser) ₂-EGF Ex Taq Total	36 5 5 1 1 1 1 50

3) pMD18T/ prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The structure of-α MSH plasmid

Reclaim dna fragmentation and be cloned on the pMD18-T carrier, obtain pMD18T/ prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH plasmid transforms in the competence e. coli jm109, extracts plasmid and identifies with endonuclease NcoI and EcoR I double digestion, filters out and contains prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The segmental recombinant plasmid of α MSH;

The ligation system

Composition	Volume (μ L)
Composition	Volume (μ L)	PMD18-T carrier prefix body+DAB ₃₈₉(Gly ₄Ser) ₂α MSH fragment Ligation Solution I Total	0.5 4.5 5 10

4) recombinant plasmid pET28a/ prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The structure of-α MSH

PET28a (+) and cloned plasmids pMD18T/ prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH plasmid is used Nco I and EcoR I double digestion respectively, and purifying reclaims the purpose fragment respectively then, connects with the T4 dna ligase, makes up recombinant expression plasmid pET28a/ prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH plasmid transforms in competence JM109, makes up recombinant bacterial strain, extracts plasmid, and enzyme is cut evaluation, with the accuracy that guarantees that the purpose fragment is inserted;

The ligation system

Composition	Volume (μ L)
Composition	Volume (μ L)	10 * T4DNA connects the purpose fragment pET28a/EcoR I+Nco I fragment T4 dna ligase Total that the damping fluid enzyme cuts back to close	1.2 8.2 1.8 0.8 12

5) dna sequence analysis

A large amount of pET28a/ prefix body+DAB that extract ₃₈₉(Gly ₄Ser) ₂-α MSH plasmid carries out nucleotide sequencing to the insertion fragment in the recombinant plasmid, guarantees to insert segmental exactness; Sequencing result shows, contained base sequence is as described in the SEQ ID NO.2 in the recombinant plasmid.

Embodiment 2: expression of recombinant plasmid protein expression and evaluation

Present embodiment is described the abduction delivering of recombinant toxin of the present invention in e. coli host cell.

With the recombinant plasmid pET28a/ prefix body+DAB that makes among the embodiment 1 ₃₈₉(Gly ₄Ser) ₂-α MSH transformed competence colibacillus e. coli bl21 (λ DE3) bacterial strain (Novagen), the positive bacterium colony of picking, 37 ℃ of cultivations according to a conventional method, when OD600nm=0.6, the reduction culture temperature adds the IPTG inductor to 30 ℃ of best inducing temperatures and in culture and carries out abduction delivering, the optimum amount of inductor IPTG is 1.4mmol/L, induces 6 hours; Collect BL21 (λ DE3) somatic cells and be suspended in 10 times of volumes and contain in the 20mmol/LTris-HCL solution, get cleer and peaceful throw out behind the ultrasonic degradation respectively and carry out the SDS-PAGE electrophoretic analysis; Analytical results shows, has the band of an about 46.17KDa of molecular weight in the expression product, and recombinant toxin is expressed (referring to accompanying drawing 3) simultaneously with soluble form in the born of the same parents and inclusion body form;

By identifying expression product with the immunoblotting assay method, the result shows that the specific reaction band appears in expression product and horse diphtheria toxin.

Embodiment 3: the purifying of fused protein

Method purifying such as use is saltoutd, Cu Chelating Sepharose Fast Flow affinity chromatography, desalination, Xa factor enzyme are cut obtain fusion rotein;

In bacterium cracking supernatant liquor, add ammonium sulfate and make its concentration reach 55%, 4 ℃ to place behind the 1h centrifugal, (contain 2mmol/LEDTA with 20mmol/LTris-Cl, pH8.0) dissolving target protein precipitation, centrifugal collection supernatant sample carries out analyzing and testing with SDS-PAGE to sample.With Cu Chelating Sepharose Fast Flow chromatography purification albumen, sample on the sample, with A (20mmol/L Tris-Cl, 0.5mol/L NaCl, pH7.5) → B (A liquid+50mmol/L imidazoles) carries out gradient elution, and (flow velocity is 3mL/min, time 90min), collect each component peaks composition and carry out the SDS-PAGE analysis; Target protein solution is crossed the desalination of Superdex G-25 post, collect solution, ultraviolet spectrophotometer analysis and efficient liquid phase chromatographic analysis, obtaining purity is 90.53%, concentration is the expressing protein (seeing accompanying drawing 4) of 1.5347mg/mL; Cut expressing protein with Xa factor enzyme, separate, obtain purity and reach 93.26% DAB with G-25 ₃₈₉(Gly ₄Ser) ₂-α MSH fusion rotein (seeing accompanying drawing 5) carries out the cytoactive analysis with this fusion rotein then.

Embodiment 4:DAB ₃₈₉(Gly ₄Ser) ₂The vitro cytotoxicity test of-α MSH fusion rotein

Present embodiment is intended to test the human cancer cell toxicity of recombinant toxin fusion rotein of the present invention to various cultivations.

People's colon-cancer cell is that Lovo, human cervical carcinoma cell are HeLa, human leukemia cell line JurKet, HTL Hut102, human lung cancer cell line A549, SGC-7901 BGC823, human skin malignant melanoma A375 and Madin-Darby canine kidney(cell line) (MDCK) DK, hamster kidney cell BHK totally nine kinds of clones, all can buy from China typical culture collection center (CCTCC) or Nat'l Pharmaceutical ﹠ Biological Products Control Institute; Culturing cell is carried out om observation;

Fusion rotein DAB with purifying ₃₈₉(Gly ₄Ser) ₂-α MSH, quantitatively filtration sterilization adds respectively in the different cell holes, first hole adds target protein 10 μ L (parallel 3 holes), below each hole add 20 μ L (parallel 3 holes) successively, add the substratum of different amounts then, making it cumulative volume is 100 μ L, and each gradient is established three parallel holes; Blank (adding isopyknic empty mycoprotein and substratum) is established in experiment.37 ℃ of 5%CO ₂Cultivate 36h under the condition, take out and be put in observations under the inverted microscope; Found that DAB ₃₈₉(Gly ₄Ser) ₂-α MSH fusion rotein has the obvious suppression effect to human skin malignant melanoma A375 cell line growth, lethal effect to human lung cancer cell line A549 take second place (seeing accompanying drawing 6), increase along with protein concentration, the mortality of cell also increases gradually, and the most of change circle of experimental group cell, look are secretly, refractivity is poor even cracking death; To people's colon-cancer cell be Lovo, human cervical carcinoma cell be HeLa, human leukemia cell line JK, SGC-7901 BGC823, HTL Hut102 to no effect; These two kinds of normal cells of Madin-Darby canine kidney(cell line) (MDCK) DK, hamster kidney cell BHK are not then had injury, and the cell growth does not normally have obvious variation; Demonstrated fully fusion rotein specific tumor cell toxic action.

Recombinant toxin is to tumour cell 503nhibiting concentration IC ₅₀Determine, regulate cell count to 2 * 10 ₄Individual/hole is inoculated in 96 porocyte culture plates, and 37 ℃ according to a conventional method, 5%CO ₂2h makes it adherent in the incubator, adds in each cell hole by the pure protein sample that waits times dilution method with different concns, and making it cumulative volume is 100 μ L, 5%CO ₂, cultivate 72h under 37 ℃ of conditions, observations under the inverted microscope adds 100 μ LXTT staining reagents respectively in each hole, and 37 ℃ of effect 4min under the 490nm wavelength, use microplate reader to measure light absorption value and calculate 503nhibiting concentration IC ₅₀The result shows, fusion toxin of the present invention can surface in conjunction with human skin malignant melanoma A375 cell on, lethal effect is fairly obvious; Blank group experimental cell does not all have influence.Fusion rotein is to human skin malignant melanoma A375, human lung cancer cell A549's IC ₅₀Be respectively: 4.672 μ g/mL and 7.243 μ g/mL (seeing accompanying drawing 7).

Embodiment 5:DAB ₃₈₉(Gly ₄Ser) ₂-α MSH is to the effect experiment of animal

This enforcement is intended to further observe the tumor killing effect to the solid tumor of tumor bearing nude mice, provides foundation for determining pharmacology, toxicological experiment dosage.

20 of nude mices (female) are given every nude mice back subcutaneous injection 1 * 10 available from Military Medical Science Institute's Experimental Animal Center ⁶Individual A375 cell; Inoculate after 15 days, when finding that there is the above projection of 0.5 * 0.5cm at the back, nude mice is divided into two groups at random, 10 is one group; One group is experimental group, and one group is control group.Every of experimental group is tail vein injection DAB every other day ₃₈₉(Gly ₄Ser) ₂The pure product 0.2mL of-α MSH (being equivalent to pure product 2 μ g), every of control group be tail vein injection saline 0.2mL every other day; Inject 5 times; Drug withdrawal after 5 days is put to death two groups of laboratory animal, and the weighing knurl is heavy, and calculates tumour inhibiting rate; The result shows that tumour inhibiting rate is 64%, and tumor killing effect is obvious.

SEQUENCE LISTING

＜110〉Jilin Agriculture University

＜120〉recombined diphtheria toxin and its production and application

<160>4

<210>1

<211>1233

<212>DNA

＜213〉artificial

<400>1

GGCGCTGATG ATGTTGTTGA TTCTTCTAAA TCTTTTGTGA TGGAAAACTT TTCTTCGTAC 60

CACGGGACTA AACCTGGTTA TGTAGATTCC ATTCAAAAAG GTATACAAAA GCCAAAATCT 120

GGTACACAAG GAAATTATGA CGATGATTGG AAAGGGTTTT ATAGTACCGA CAATAAATAC 180

GACGCTGCGG GATACTCTGT AGATAATGAA AACCCGCTCT CTGGAAAAGC TGGAGGCGTG 240

GTCAAAGTGA CGTATCCAGG ACTGACGAAG GTTCTCGCAC TAAAAGTGGA TAATGCCGAA 300

ACTATTAAGA AAGAGTTAGG TTTAAGTCTC ACTGAACCGT TGATGGAGCA AGTCGGAACG 360

GAAGAGTTTA TCAAAAGGTT CGGTGATGGT GCTTCGCGTG TAGTGCTCAG CCTTCCCTTC 420

GCTGAGGGGA GTTCTAGCGT TGAATATATT AATAACTGGG AACAGGCGAA AGCGTTAAGC 480

GTAGAACTTG AGATTAATTT TGAAACCCGT GGAAAACGTG GCCAAGATAC GATGTATGAG 540

TATATGGCTC AAGCCTGTGC AGGAAATCGT GTCAGGCGAT CAGTAGGTAG CTCATTGTCA 600

TGCATAAATC TTGATTGGGA TGTCATAAGG GATAAAACTA AGACAAAGAT AGAGTCTTTG 660

AAAGAGCATG GCCCTATCAA AAATAAAATG AGCGAAAGTC CCAATAAAAC AGTATCTGAG 720

GAAAAAGCTA AACAATACCT AGAAGAATTT CATCAAACGG CATTAGAGCA TCCTGAATTG 780

TCAGAACTTA AAACCGTTAC TGGGACCAAT CCTGTATTCG CTGGGGCTAA CTATGCGGCG 840

TGGGCAGTAA ACGTTGCGCA AGTTATCGAT AGCGAAACAG CTGATAATTT GGAAAAGACA 900

ACTGCTGCTC TTTCGATACT TCCTGGTATC GGTAGCGTAA TGGGCATTGC AGACGGTGCC 960

GTTCACCACA ATACAGAAGA GATAGTGGCA CAATCAATAG CTTTATCGTC TTTAATGGTT 1020

GCTCAAGCTA TTCCATTGGT AGGAGAGCTA GTTGATATTG GTTTCGCTGC ATATAATTAT 1080

GTAGAGAGTA TTATCAATTT ATTTCAAGTA GTTCATAATT CGTATAATCG TCCCGCGTAT 1140

TCTCCGGGGC ATAAAACGCA TGCGGGTTTA GGCGGTTCAG GCGGAGGTGG ATCCAGTTAT 1200

AGTATGGAGC ACTTCAGGTG GGGAAAGCCA GTA 1233

<210>2

<211>1275

<212>DNA

＜213〉artificial

<400>2

GGCAGCCATC ATCATCATCA TCACAGCGGC ATTGAGGGTC GCGGCGCTGA TGATGTTGTT 60

GATTCTTCTA AATCTTTTGT GATGGAAAAC TTTTCTTCGT ACCACGGGAC TAAACCTGGT 120

TATGTAGATT CCATTCAAAA AGGTATACAA AAGCCAAAAT CTGGTACACA AGGAAATTAT 180

GACGATGATT GGAAAGGGTT TTATAGTACC GACAATAAAT ACGACGCTGC GGGATACTCT 240

GTAGATAATG AAAACCCGCT CTCTGGAAAA GCTGGAGGCG TGGTCAAAGT GACGTATCCA 300

GGACTGACGA AGGTTCTCGC ACTAAAAGTG GATAATGCCG AAACTATTAA GAAAGAGTTA 360

GGTTTAAGTC TCACTGAACC GTTGATGGAG CAAGTCGGAA CGGAAGAGTT TATCAAAAGG 420

TTCGGTGATG GTGCTTCGCG TGTAGTGCTC AGCCTTCCCT TCGCTGAGGG GAGTTCTAGC 480

GTTGAATATA TTAATAACTG GGAACAGGCG AAAGCGTTAA GCGTAGAACT TGAGATTAAT 540

TTTGAAACCC GTGGAAAACG TGGCCAAGAT ACGATGTATG AGTATATGGC TCAAGCCTGT 600

GCAGGAAATC GTGTCAGGCG ATCAGTAGGT AGCTCATTGT CATGCATAAA TCTTGATTGG 660

GATGTCATAA GGGATAAAAC TAAGACAAAG ATAGAGTCTT TGAAAGAGCA TGGCCCTATC 720

AAAAATAAAA TGAGCGAAAG TCCCAATAAA ACAGTATCTG AGGAAAAAGC TAAACAATAC 780

CTAGAAGAAT TTCATCAAAC GGCATTAGAG CATCCTGAAT TGTCAGAACT TAAAACCGTT 840

ACTGGGACCA ATCCTGTATT CGCTGGGGCT AACTATGCGG CGTGGGCAGT AAACGTTGCG 900

CAAGTTATCG ATAGCGAAAC AGCTGATAAT TTGGAAAAGA CAACTGCTGC TCTTTCGATA 960

CTTCCTGGTA TCGGTAGCGT AATGGGCATT GCAGACGGTG CCGTTCACCA CAATACAGAA 1020

GAGATAGTGG CACAATCAAT AGCTTTATCG TCTTTAATGG TTGCTCAAGC TATTCCATTG 1080

GTAGGAGAGC TAGTTGATAT TGGTTTCGCT GCATATAATT ATGTAGAGAG TATTATCAAT 1140

TTATTTCAAG TAGTTCATAA TTCGTATAAT CGTCCCGCGT ATTCTCCGGG GCATAAAACG 1200

CATGCGGGTT TAGGCGGTTC AGGCGGAGGT GGATCCAGTT ATAGTATGGA GCACTTCAGG 1260

TGGGGAAAGC CAGTA 1275

<210>3

<211>70

<212>DNA

＜213〉artificial

<400>3

catgccatgg gcagccatca tcatcatcat cacagcggca ttgagggtcg cggcgctgat 60

gatgttgttg 70

<210>4

<211>68

<212>DNA

＜213〉artificial

<400>4

cggaattctt ataccggttt accccagcgg aagtgttcca tgctgtagct ggatccacct 60

ccgcctga 68

Claims

1, recombined diphtheria toxin, it comprises: diphtheria toxin DAB ₃₈₉, connection peptides (Gly ₄Ser) ₂, melanotropin α MSH.

2, a kind of recombined diphtheria toxin gene fragment is characterized in that: the contained gene of the described recombined diphtheria toxin of claim 1 has a prefix body, and its base sequence is as described in the SEQ ID NO.2 in the sequence table.

3, recombined diphtheria toxin preparation method according to claim 1, this method comprises successively:

(1), chemosynthesis contains prefix body+DAB ₃₈₉+ (Gly ₄Ser) ₂Upstream primer, contain the downstream primer of α MSH reverse sequence, utilize pET28a/DAB ₃₈₉(Gly ₄Ser) ₂-EGF is a template, amplifies to contain prefix body+DAB ₃₈₉(Gly ₁Ser) ₂The gene fragment of-α MSH, purifying reclaims, and must reclaim fragment;

(2), fetch the section of taking up and add among the pMD-18T vector, use T ₄Dna ligase connects, and connects product and is converted in the intestinal bacteria, screens positive recombinant plasmid, obtains to contain prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The segmental recombinant plasmid of-α MSH, this recombinant plasmid of double digestion and expression vector pET28a (+) reclaim both endonuclease bamhis, with prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH gene fragment is mixed, is connected with pET28a plasmid fragment, will connect product and be converted in the intestinal bacteria, screens positive bacterium colony and cultivates, and extracts plasmid DNA;

(3), with recombinant plasmid pET28a/ prefix body+DAB ₃₈₉(Gly ₄Ser) ₂-α MSH is converted into and expresses in the bacterium, and abduction delivering is collected thalline;

(4), adopt ultrasonic treatment, make the somatic cells fragmentation, centrifugal, collect supernatant, ammonium sulfate precipitation, affinitive layer purification and Xa factor cutting recombinant toxin expressing protein, recovery, obtain to remove the DAB of prefix body ₃₈₉(Gly ₄Ser) ₂-α MSH fusion rotein.

4, recombined diphtheria toxin preparation method according to claim 3 is characterized in that: the described recombinant plasmid pET28a/ of step (3) prefix body+DAB ₃₈₉(Gly ₄Ser) ₂The expression bacterial classification of-α MSH plasmid is BL21 (λ DE3), and substratum is M ₉B ₂

5, according to claim 3,4 described recombined diphtheria toxin preparation methods, it is characterized in that: expressing 30 ℃ of temperature and time is 6 hours, and inductor IPTG consumption is 1.4mmol/L.

6, according to of the application of the described recombined diphtheria toxin of claim 1 at production melanoma medicine.