CN1464058A - Tumor-repressed fusion protein, its encoding gene and use thereof - Google Patents
Tumor-repressed fusion protein, its encoding gene and use thereof Download PDFInfo
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Abstract
The present invention relates to bioengineering, and is one kind of fusion protein and its encoding gene and application. The present invention aims at providing one kind of fusion protein with tumor suppressing effect. The fusion protein with tumor suppressing effect has the DNA sequence of the sequence 1 in the sequence list, and the fusion protein with tumor suppressing effect has the amino acid residue sequence of the sequence 2. The other one aim of the present invention is to provide one kind of tumor treating medicine with the said fusion protein as its active component. The recombinant protein of the present invention has relatively strong tumor cell targeting and killing effect on various malignant tumors, such as mammary cancer, lung cancer, gastric cancer, etc.
Description
Technical field
The present invention relates to a kind of fusion rotein and the encoding gene and the application of bioengineering field, particularly relate to a kind of fusion rotein and encoding gene and application with tumor-inhibiting action.
Background technology
Utilize cytokine to combine the transmission recombinant cytokine-extracellular toxin fusion rotein that leads with the specificity of its acceptor, to reach the cancer cells that kills and wounds the corresponding cytokine receptor of high expression level with a definite target in view, be one of the important topic that cancer target kills and wounds (biological missile) research.
Targeting toxins comprises two portions: a part be can killer cell toxin, another part is for mediating the sign carrier that toxin arrives target cell.At present, made up the targeting toxins that many kinds are used to kill and wound cancer cells, comprising: plant poison (as: ricin), bacteriotoxin (as: diphtheria toxin (DT), Pseudomonas aeruginosa extracellular toxin (APE)) and mycotoxins etc., these toxin all have very strong cytotoxicity to mammalian cell.The sign carrier comprises multiple protein, as: transforming growth factor (TGF), Urogastron (EGF), interleukin-(IL-2, IL-4), granulocyte-macrophage colony stimutaing factor (GM-CSF), EGF receptor antibody, HER2 receptor antibody, Transferrins,iron complexes or the like.Toxin moiety can obtain fusion rotein by the chemical method coupling or by engineered method with the connection of carrier.Have report to show, the targeting toxins that genetically engineered makes up is more effective than the chemical coupling method structure.
Studies have shown that the EGF acceptor gene of many cancer cells has been exaggerated, and, comprising: mammary cancer, lung cancer, cancer of the stomach, bladder cancer, prostate cancer, carcinoma of the colon and rectum, cervical cancer and brain tumor at cell surface overexpression EGF acceptor.At present existing several targeting toxins are fabricated the cancer cells that kills overexpression EGF acceptor.As: 425.3-PE utilizes the immunotoxin (Engebraaten et al, 2002) of the monoclonal antibody of EGF with the chemistry connection structure of toxin PE.TP40 is the fusion rotein (Siegall et al, 1989) that the PE of a kind of TGF and deletion variation makes up.
Summary of the invention
The purpose of this invention is to provide a kind of fusion rotein and encoding gene thereof with tumor-inhibiting action.
A kind of encoding gene with tumor-inhibiting action fusion rotein, it is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
The dna sequence dna of sequence 1 is by 1401 based compositions in the sequence table of the present invention.
A kind of fusion rotein with tumor-inhibiting action, it have the amino acid residue sequence of sequence 2 in the sequence table or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2.
The protein of sequence 2 is made up of 466 amino-acid residues in the sequence table of the present invention, has the effect that suppresses tumour.
Another object of the present invention provides a kind of medicine for the treatment of tumour.
A kind of medicine for the treatment of tumour, its activeconstituents be have the amino acid residue sequence of sequence 2 in the sequence table or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2.
When needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.
Medicine of the present invention can be made various ways such as injection liquid, tablet, pulvis, granula, capsule, oral liquid, paste, creme.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The consumption of said medicine is generally 100ug/kg body weight every day.
The diphtheria toxin that the present invention is used to make up recombination fusion protein is a kind of proteotoxin that contains two subunits, N-terminal subunit (1-388 residue) can cause protein synthesising disorder by the ADP-ribosylation of catalysis elongation factor 2, thereby cause apoptosis, the terminal subunit of C-is used to connect acceptor.Diphtheria toxin has high cell toxicity to mammalian cell, and the several DT molecules that enter cell interior will be enough to kill a cell.
The constructed recombinant protein of the present invention is to be made a variation by the diphtheria toxin of having deleted the receptors bind structural domain and people that EGF is recombinant expressed to be formed, its feature is that diphtheria toxin inherent toxicity reduces greatly by deletion receptors bind structural domain, so toxin will only kill and wound the cell with carrier acceptor specifically, other normal human tissue cells there is not injury, the EGF of the variation that the present invention transforms has improved the adhesion avidity to Receptor EGFR and HER2, also showed simultaneously adhesion avidity to HER3 and HER4, enlarge the scope of killing tumor cell, can effectively kill and wound expression EGFR, HER2, the cell of HER3 or HER4.
Experimental study shows in the external body, recombined diphtheria toxin human epidermal growth factor fusion rotein of the present invention to multiple malignant tumour as: mammary cancer, lung cancer, cancer of the stomach, bladder cancer, prostate cancer, carcinoma of the colon and rectum, cervical cancer and brain tumor have stronger tumour cell targeting killing effect.
The present invention will be further described below in conjunction with specific embodiment.
Embodiment
Embodiment 1, Recombinant Protein Expression of the present invention
1, diphtheria toxin residue 1-388 (DT388) clone is advanced carrier pet28b (Novagen)
Obtain the DT388PCR product from Corynebacterium diphtheriae virus genome.The PCR product is pressed the described method digestion of New England BioLabs with restriction endonuclease NheI and Nde I.Insert connects into the pet28b carrier of digestion with the T4 ligase enzyme.Synthetic carrier called after 28bDT388.Nucleotide sequence is proved conclusively through dna sequencing.
2, human epidermal growth factor gene obtains through PCR from people's spleen cDNA library, and the sequence of a pair of primer is as follows:
1：GGAGTTCATATGTCTCACCTGGTTAAATGCCCGCTGTCCCACG
2：GGGCTCGAGTTAACGCAGTTCCCACCATTTCAGATCACGG
The first five of this Urogastron amino-acid residue has been mutated into SHLVK.
The PCR product is pressed the described method digestion of New England BioLabs with restriction endonuclease NheI and XhoI.This insert connects into the 28bDT388 carrier of digestion with the T4 ligase enzyme.The plasmid called after 28bDT388mEGF that generates.
3, plasmid 28bDT388mEGF is transformed into e. coli bl21 (DE3) and carries out protein expression.The intestinal bacteria that transform are inoculated in the 10ml LB substratum that contains 20 μ g/ml kantlex, and 37 ℃ of shaking tables growths 12 hours are got the above-mentioned LB substratum of 10ml transferred species and contained the LB substratum of 20 μ g/ml kantlex in 1L, as substratum OD
600Value reaches at 0.6 o'clock, adds 0.5ml 1M IPTG inducible protein in the 1L substratum and expresses, and induces collecting cell after 3 hours.
4, be harvested from the Bacillus coli cells centrifugation in the 1L substratum and be resuspended in (pH7.9) 150mM NaCl in the 100ml 100mM Tris damping fluid.4 ℃ of ultrasonic treatment cells, cell debris and insolubles because expressing protein is a histidine mark, therefore can pass through the metal-chelating column purification with the centrifugation under the 15000rpm condition of Beckman JA20 rotor with comparalive ease.
5, the supernatant liquor after centrifugal pumps into the Ni post of 10ml resin, the Ni post fully washes pillar with 50mM Tris damping fluid (pH7.9), 0.5M NaCl and 50mM imidazole and removes foreigh protein removing, will be by pillar bonded recombinant protein by 50mM Tris damping fluid (pH7.9), 0.5M NaCl and 200mM imidazole wash-out.
6, the recombinant protein of wash-out is dialysed with 2L PBS damping fluid, and is further purified with Superdex 200 gel-filtration columns that are installed on AKTA HPLC system of Pharmacia company, obtains finished product.
Embodiment 2, recombinant protein of the present invention are to the effect of cancer cells
With DT388 is contrast, with recombinant protein of the present invention respectively to mammary cancer (MCF-7), lung cancer (H125, H358), the rectum cancer (SW480), cervical cancer (A431) and brain tumor (U251, U87, U373) eight strain tumour cells are done the dose-effect relationship test, and the IC50 of fusion rotein and DT388 (ng/ml) result is as shown in table 1:
Table 1. recombinant protein and DT388 handle various tumour cell IC50 (ng/ml)
2.5 17.5 15.0 2.5 2 15 20 25.0 contrasts＞1000＞1000＞1000＞1000＞1000＞1000＞1000＞1000 of MCF-7 H125 H358 SW480 A431 U251 U87 U373 recombinant protein are annotated: IC50 is the protein concentration of the cell half death of handling.
From the table data as can be seen, recombinant protein of the present invention all has tangible lethal effect to various tumor cell strains such as mammary cancer, lung cancer, the rectum cancer, cervical cancer, brain tumors.
＜160〉2＜210〉1＜211〉1401＜212〉DNA＜213〉＜220〉＜223〉＜400〉1atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atggctagcg gcgctgatga tgttgttgat tcttctaaat cttttgtgat ggaaaacttt 120tcttcgtacc acgggactaa acctggttat gtagattcca ttcaaaaagg tatacaaaag 180ccaaaatctg gtacacaagg aaattatgac gatgattgga aagggtttta tagtaccgac 240aataaatacg acgctgcggg atactctgta gataatgaaa acccgctctc tggaaaagct 300ggaggcgtgg tcaaagtgac gtatccagga ctgacgaagg ttctcgcact aaaagtggat 360aatgccgaaa ctattaagaa agagttaggt ttaagtctca ctgaaccgtt gatggagcaa 420gtcggaacgg aagagtttat caaaaggttc ggtgatggtg cttcgcgtgt agtgctcagc 480cttcccttcg ctgaggggag ttctagcgtt gaatatatta ataactggga acaggcgaaa 540gcgttaagcg tagaacttga gattaatttt gaaacccgtg gaaaacgtgg ccaagatgcg 600atgtatgagt atatggctca agcctgtgca ggaaatcgtg tcaggcgatc agtaggtagc 660tcattgtcat gcataaatct tgattgggat gtcataaggg ataaaactaa gacaaagata 720gagtctttga aagagcatgg ccctatcaaa aataaaatga gcgaaagtcc caataaaaca 780gtatctgagg aaaaagctaa acaataccta gaagaatttc atcaaacggc attagagcat 840cctgaattgt cagaacttaa aaccgttact gggaccaatc ctgtattcgc tggggctaac 900tatgcggcgt gggcagtaaa cgttgcgcaa gttatcgata gcgaaacagc tgataatttg 960gaaaagacaa ctgctgctct ttcgatactt cctggtatcg gtagcgtaat gggcattgca 1020gacggtgccg ttcaccacaa tacagaagag atagtggcac aatcaatagc tttatcgtct 1080ttaatggttg ctcaagctat tccattggta ggagagctag ttgatattgg tttcgctgca 1140tataattttg tagagagtat tatcaattta tttcaagtag ttcataattc gtataatcgt 1200cccgcgtatt ctccggggca taaaacgcaa ccacatatgt ctcacctggt taaatgcccg 1260ctgtcccacg acggttattg tctgcacgac ggtgtttgca tgtatatcga agctctggac 1320aaatacgctt gcaactgcgt agttggttac atcggtgaac gttgccagta ccgtgatctg 1380aaatggtggg aactgcgtta a 1401＜210〉2＜211〉466＜212〉PRT＜213〉＜220〉＜223〉＜400〉2Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val 1 5 10 15Pro Arg Gly Ser His Met Ala Ser Gly Ala Asp Asp Val Val Asp
20 25 30Ser?Ser?Lys?Ser?Phe?Val?Met?Glu?Asn?Phe?Ser?Ser?Tyr?His?Gly
35 40 45Thr?Lys?Pro?Gly?Tyr?Val?Asp?Ser?Ile?Gln?Lys?Gly?Ile?Gln?Lys
50 55 60Pro?Lys?Ser?Gly?Thr?Gln?Gly?Asn?Tyr?Asp?Asp?Asp?Trp?Lys?Gly
65 70 75Phe?Tyr?Ser?Thr?Asp?Asn?Lys?Tyr?Asp?Ala?Ala?Gly?Tyr?Ser?Val
80 85 90Asp?Asn?Glu?Asn?Pro?Leu?Ser?Gly?Lys?Ala?Gly?Gly?Val?Val?Lys
95 100 105Val?Thr?Tyr?Pro?Gly?Leu?Thr?Lys?Val?Leu?Ala?Leu?Lys?Val?Asp
110 115 120Asn?Ala?Glu?Thr?Ile?Lys?Lys?Glu?Leu?Gly?Leu?Ser?Leu?Thr?Glu
125 130 135Pro?Leu?Met?Glu?Gln?Val?Gly?Thr?Glu?Glu?Phe?Ile?Lys?Arg?Phe
140 145 150 Gly?Asp?Gly?Ala?Ser?Arg?Val?Val?Leu?Ser?Leu?Pro?Phe?Ala?Glu
155 160 165Gly?Ser?Ser?Ser?Val?Glu?Tyr?Ile?Asn?Asn?Trp?Glu?Gln?Ala?Lys
170 175 180Ala?Leu?Ser?Val?Glu?Leu?Glu?Ile?Asn?Phe?Glu?Thr?Arg?Gly?Lys
185 190 195Arg?Gly?Gln?Asp?Ala?Met?Tyr?Glu?Tyr?Met?Ala?Gln?Ala?Cys?Ala
200 205 210Gly?Asn?Arg?Val?Arg?Arg?Ser?Val?Gly?Ser?Ser?Leu?Ser?Cys?Ile
215 220 225Asn?Leu?Asp?Trp?Asp?Val?Ile?Arg?Asp?Lys?Thr?Lys?Thr?Lys?Ile
230 235 240Glu?Ser?Leu?Lys?Glu?His?Gly?Pro?Ile?Lys?Asn?Lys?Met?Ser?Glu
245 250 255Ser?Pro?Asn?Lys?Thr?Val?Ser?Glu?Glu?Lys?Ala?Lys?Gln?Tyr?Leu
260 265 270Glu?Glu?Phe?His?Gln?Thr?Ala?Leu?Glu?His?Pro?Glu?Leu?Ser?Glu
275 280 285Leu?Lys?Thr?Val?Thr?Gly?Thr?Asn?Pro?Val?Phe?Ala?Gly?Ala?Asn
290 295 300Tyr?Ala?Ala?Trp?Ala?Val?Asn?Val?Ala?Gln?Val?Ile?Asp?Ser?Glu
305 310 315Thr?Ala?Asp?Asn?Leu?Glu?Lys?Thr?Thr?Ala?Ala?Leu?Ser?Ile?Leu
320 325 330Pro?Gly?Ile?Gly?Ser?Val?Met?Gly?Ile?Ala?Asp?Gly?Ala?Val?His
335 340 345His?Asn?Thr?Glu?Glu?Ile?Val?Ala?Gln?Ser?Ile?Ala?Leu?Ser?Ser
350 355 360Leu?Met?Val?Ala?Gln?Ala?Ile?Pro?Leu?Val?Gly?Glu?Leu?Val?Asp
365 370 375Ile?Gly?Phe?Ala?Ala?Tyr?Asn?Phe?Val?Glu?Ser?Ile?Ile?Asn?Leu
380 385 390Phe?Gln?Val?Val?His?Asn?Ser?Tyr?Asn?Arg?Pro?Ala?Tyr?Ser?Pro
395 400 405Gly?His?Lys?Thr?Gln?Pro?His?Met?Ser?His?Leu?Val?Lys?Cys?Pro
410 415 420Leu?Ser?His?Asp?Gly?Tyr?Cys?Leu?His?Asp?Gly?Val?Cys?Met?Tyr
425 430 435Ile?Glu?Ala?Leu?Asp?Lys?Tyr?Ala?Cys?Asn?Cys?Val?Val?Gly?Tyr
440 445 450Ile?Gly?Glu?Arg?Cys?Gln?Tyr?Arg?Asp?Leu?Lys?Trp?Trp?Glu?Leu
455 460 465Arg466
Claims (9)
1, a kind of encoding gene with tumor-inhibiting action fusion rotein, it is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
2, gene according to claim 1 is characterized in that: described encoding gene with tumor-inhibiting action fusion rotein is the dna sequence dna of sequence 1 in the sequence table.
3, a kind of fusion rotein with tumor-inhibiting action, it have the amino acid residue sequence of sequence 2 in the sequence table or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2.
4, protein according to claim 3 is characterized in that: described protein has the amino acid residue sequence of sequence 2 in the sequence table.
5, a kind of medicine for the treatment of tumour, its activeconstituents are the described fusion roteins of claim 3.
6, a kind of medicine for the treatment of tumour according to claim 5, the activeconstituents that it is characterized in that this medicine is the protein with amino acid residue sequence of sequence 2 in the sequence table.
7, according to claim 5 or 6 described a kind of medicines for the treatment of tumour, it is characterized in that: also contain the acceptable pharmaceutical carrier of human body in the described medicine.
8, the application of the described fusion rotein of claim 3 in the medicine of preparation treatment tumour.
9, the application of the described fusion rotein of claim 4 in the medicine of preparation treatment tumour.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102639555A (en) * | 2009-09-24 | 2012-08-15 | 加利福尼亚大学董事会 | Bladder cancer specific ligand peptides |
| CN106967743A (en) * | 2017-05-02 | 2017-07-21 | 通用生物系统（安徽）有限公司 | A kind of production method of restructuring NotI restriction enzymes in Escherichia coli |
| CN111499695A (en) * | 2020-04-28 | 2020-08-07 | 上海市农业科学院 | Functional protein MU20410, its coding sequence and application |
| CN112316115A (en) * | 2020-10-26 | 2021-02-05 | 济宁医学院 | Protein sequence capable of inhibiting malignant tumor and application thereof |
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2002
- 2002-06-28 CN CN02123449A patent/CN1464058A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102639555A (en) * | 2009-09-24 | 2012-08-15 | 加利福尼亚大学董事会 | Bladder cancer specific ligand peptides |
| US8946379B2 (en) | 2009-09-24 | 2015-02-03 | The Regents Of The University Of California | Bladder cancer specific ligand peptides |
| CN102639555B (en) * | 2009-09-24 | 2015-11-25 | 加利福尼亚大学董事会 | Bladder cancer specific ligand peptide |
| US9539340B2 (en) | 2009-09-24 | 2017-01-10 | The Regents Of The University Of California | Bladder cancer specific ligand peptides |
| US10335365B2 (en) | 2009-09-24 | 2019-07-02 | The Regents ot the Univershy of California | Bladder cancer specific ligand peptides |
| CN106967743A (en) * | 2017-05-02 | 2017-07-21 | 通用生物系统（安徽）有限公司 | A kind of production method of restructuring NotI restriction enzymes in Escherichia coli |
| CN111499695A (en) * | 2020-04-28 | 2020-08-07 | 上海市农业科学院 | Functional protein MU20410, its coding sequence and application |
| CN111499695B (en) * | 2020-04-28 | 2022-09-09 | 上海市农业科学院 | Functional protein MU20410, its coding sequence and application |
| CN112316115A (en) * | 2020-10-26 | 2021-02-05 | 济宁医学院 | Protein sequence capable of inhibiting malignant tumor and application thereof |
| CN112316115B (en) * | 2020-10-26 | 2022-07-01 | 济宁医学院 | Protein sequence capable of inhibiting malignant tumor and application thereof |
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