CN1394956A - Human epidermal growth factor receptor specific fusion protein and its application - Google Patents
Human epidermal growth factor receptor specific fusion protein and its application Download PDFInfo
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- CN1394956A CN1394956A CN01120100A CN01120100A CN1394956A CN 1394956 A CN1394956 A CN 1394956A CN 01120100 A CN01120100 A CN 01120100A CN 01120100 A CN01120100 A CN 01120100A CN 1394956 A CN1394956 A CN 1394956A
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- fusion protein
- fusion rotein
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Abstract
The present invention discloses a fusion protein formed by means of fusion of human epidermal growth factor and StxA45-247, nucleotide sequence of said fusion protein coding gene, antitumor medicine using said fusion protein as active component and application of said fusion protein in preparation of antitumor medicine. Said invented fusion protein, under the condition of low concentration, can possess obvious action for killing several human epidermal growth factor receptor positive tumor cell in vitro, and also has the obvious effect for inhibiting in vivo transplanted solid tumor.
Description
Technical field
The present invention relates to a kind of fusion rotein of antitumor action, be medicine and this proteic application of activeconstituents, particularly relate to a kind of fusion rotein with human epidermal growth factor receptor specific, be the medicine of activeconstituents and in the application of pharmaceutical industry with this albumen with this albumen.
Background technology
20th century, the seventies began to propose the conception of targeted drug, and the research of targeted drug has so far had very great development.The applying gene integration technology makes up targeted toxin in the world in recent years, can kill and wound target cell high selectivity, and the targeted toxin tool of passing by to make up has an enormous advantage.
On the surface of solid tumor cells such as human breast carcinoma, lung cancer, exist unusual a large amount of Urogastron (SEQID NO:1) acceptor (EGFR).According to this characteristic, can utilize part or antibody and the toxin protein fusion of EGFR, the antitumor drug of development targeted toxin formula.Human epidermal growth factor-the diphtheria toxin of external development has entered the clinical II phase, studies show that it has shown good tumor killing cell activity.But it is bigger that this fusion rotein exists molecular weight, influences immunotoxin and efficiently enter deficiencies such as target cell.
The Shigellae toxin subunit of StxA45-247 (its amino acid residue sequence the is seen SEQ ID NO:2) brachymemma that suddenlyd change, it is except having the lethal effect to target cell, it is low to have more molecular weight, is easy to enter target cell by pinocytosis, realizes the characteristics of lethal effect.
Summary of the invention
The purpose of this invention is to provide a kind of fusion rotein with human epidermal growth factor receptor specific, this albumen can enter target cell specifically, and target cell is killed.
Fusion rotein of the present invention is by human epidermal growth factor and StxA
45-247Fusion forms, and the encoding gene of this fusion rotein has the nucleotide sequence of SEQ ID NO:3.
Fusion rotein of the present invention has the amino acid residue sequence of SEQ ID NO:4.
It is the antitumor drug of activeconstituents that another object of the present invention provides with fusion rotein of the present invention.Particularly treat the medicine of mammary cancer, lung cancer, liver cancer, oral carcinoma and cell carcinoma.
Another purpose of the present invention provides the purposes of fusion rotein of the present invention aspect the preparation antitumor drug.
Medicine of the present invention is an activeconstituents with fusion rotein of the present invention, and this medicine is used for the treatment of tumour.
In needs, in said medicine, can also contain 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 tablet, pulvis, granula, capsule, oral liquid and injection liquid.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The survival dose of The compounds of this invention can be adjusted according to the severity of patient's age, body weight, disease, and per daily dose is generally 20-100 μ g/kg body weight.
The present invention utilizes low-molecular-weight StxA dexterously
45-247Form fusion rotein with the human epidermal growth factor in people source, the immunogenicity that both can avoid the external source toxin to produce reduces side effect, is easy to again enter target cell by pinocytosis, improves the specificity of killing tumor cell.Fusion rotein of the present invention has very high clinical value, is a kind of antitumor drug that leads efficiently.
The present invention will be further described below in conjunction with drawings and the specific embodiments.
Description of drawings
Fig. 1 is the lethal effect curve of the fusion rotein of different concns to human breast carcinoma MCF-7 cell strain
Fig. 2 is the lethal effect curve of the fusion rotein of different concns to human epithelium's like cell cancer A431 cell strain
Fig. 3 is the lethal effect curve of the fusion rotein of different concns to the strain of people's lung cancer A549 cell
Fig. 4 is the lethal effect curve of the fusion rotein of different concns to human oral squamous cell carcinoma KB cell strain
Fig. 5 is the lethal effect curve of the fusion rotein of different concns to human breast carcinoma MDA cell strain
Fig. 6 is the lethal effect curve of the fusion rotein of different concns to people's liver cancer HEPG2 cell strain
Fig. 7 is the photo of different treatment nude mice
Embodiment
Embodiment 1: the acquisition of fusion rotein of the present invention
1, gene fusion construct
According to known StxA
45-247With the EGF gene order, design primer respectively, go out StxA with pcr amplification
45-247, EGF fragment, be connected to after enzyme is cut: pET22b-StxA
45-247-EGF, through the enzyme detection of cutting and check order, sequence is correct.
2, the expression of fusion gene in intestinal bacteria
Constructed fusion gene Transformed E .coli JM109 (DE3) can also be that E.coli BL21 (DE3) waits other to have the intestinal bacteria of DE3 as the intestinal bacteria that transform.
Induce through IPTG, the SDS-PAGE electrophoretic analysis confirms that above-mentioned fusion gene obtains to efficiently express in E.coli.JM109 (DE3).
3, the sex change of fusion rotein, renaturation and purifying
With the inclusion body formal representation, through ultrasonication, after the inclusion body sex change, renaturation in renaturation solution utilizes metal chelating and chromatography to obtain the pure product albumen of purity more than 95% to fusion gene in intestinal bacteria.Through this proteic amino acid sequencing is shown, resulting albumen is fusion rotein of the present invention really.
Embodiment 2, fusion rotein StxA
45-247The cell in vitro toxic action of-EGF
In order to observe fusion rotein StxA
45-247-EGF is to the growth-inhibiting effect of different tumour cells, and the contriver uses fusion rotein StxA
45-247-EGF has done the dose-effect relationship test to six strain tumour cells respectively.With EGF is contrast, has compared fusion rotein StxA
45-247To six strain tumour cell cell in vitro toxic actions, the result was as shown in table 1 when-EGF was 1000nm/ml concentration:
The various tumour cell survival of table 1. per-cent
MDA A431 A549 MCF-7 KB HEPG2StxA
45-247-EGF?24.5 22.8 40.4 31.1 27.1 33.3
EGF 81.8 100.0 102.0 81.9 102 101.0 is wherein:
From the table data as can be seen, fusion rotein StxA
45-247-EGF is to human breast carcinoma MDA, MCF-7, human oral squamous cell carcinoma KB, and human epithelium's like cell cancer A431, people's lung cancer A549, people's liver cancer HEPG2 six strain tumor cell lines have tangible lethal effect.
Embodiment 3, fusion rotein StxA
45-247The dose-effect relationship of-EGF specific killing effect
Compare fusion rotein StxA respectively
45-247-EGF is 0,0.1,1,10,100, and to the cell in vitro toxic action of the different tumour cells of six strains, the result is shown in Fig. 1-6 during six concentration of 1000ng/ml, and X-coordinate is fusion rotein StxA among the figure
45-247The concentration of-EGF, ordinate zou are the maximum keep alive rate of cancer cells.
As can be seen from the figure, fusion rotein of the present invention is crossed the tumor cell line of expression to six strain EGF acceptors, along with the increase of immune fusion protein concentration, cell survival per-cent reduces, the cell toxicant lethal effect strengthens, and has shown tangible dose-effect relationship, promptly has dose-dependently.
Embodiment 4, StxA
45-247Tumor-inhibiting action in the body of-EGF
Employing nude mice in six ages in week makes up the nude mice tumor model with oral cavity squamous epithelial cell (KB), and cell inoculation density is 10
5Cells/ only, it is subcutaneous to be injected in the back, the inoculation back observe tumor growth to naked eyes as seen, random packet, wherein, three test group, StxA
45-247-EGF use respectively height (80ug/ml), in (40ug/ml), low (20ug/ml) three kinds of dosage, positive controls is used ring mebenil (CTX), negative control group is used PBS, and abdominal injection was injected 5 days continuously, observe the tumor growth situation, the results are shown in Table 2 and Fig. 7, among Fig. 7, No. 1 is the PBS group, be the CTX group No. 2, No. 4 is StxA
45-247-EGF group.
The interior restraining effect group dosage of table 2 fusion rotein body (ug/ is only) heavy (mg) tumour inhibiting rate (%) PPBS of number of animals knurl placebo 5 916 ± 166.4 to human oral squamous cell carcinoma (KB) //CTX 400 * 1d 5 34.5 ± 6.4 96.2＜0.01StxA
45-247-80 * 5d 5 42.33 ± 3.51 95.4＜0.01 EGF
40×5d 5 121.4±8.59 86.7 ＜0.01
20×5d 5 217.8±10.8 76.2 ＜0.01
As can be seen from the table, the fusion rotein high dose group is suitable with the tumour inhibiting rate of ring mebenil (CTX), and low dose group also reaches 76.2%, compares difference extremely significantly (P＜0.01) with PBS group (contrast).
Sequence table (1) general information: (I) applicant: Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A (II) denomination of invention: the information of human epidermal growth factor receptor specific fusion protein and application thereof (2) SEQ ID NO:1
(i) sequence signature
(A) length: 53 amino-acid residues
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: protein
(xi) sequence description: the information of SEQ ID NO:1NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWEL R 53 (2) SEQ ID NO:2
(i) sequence signature
(A) length: 180 amino-acid residues
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: protein
(xi) sequence description: the information of SEQ ID NO:2TGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFS HVTFPGTT 60AVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAM LRFVTV 120TAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQD SVRVGRI 180 (2) SEQ ID NO:3
(i) sequence signature
(A) length: 870 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: DNA
( xi ) ：SEQ ID NO：3NTACNAAACNCTTNCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGA 63GATATACATATGACAGGGGATAATTTGTTTGCAGTTGATGTCAGAGGGATAGATCCAGAGGAA 126GGGCGGTTTAATAATCTACGGCTTATTGTTGAACGAAATAATTTATATGTGACAGGATTTGTTA 190ACAGGACAAATAATGTTTTTTATCGCTTTGCTGATTTTTCACATGTTACCTTTCCAGGTACAAC 254AGCGGTTACATTGTCTGGTGACAGTAGCTATACCACGTTACAGCGTGTTGCAGGGATCAGTCG 317TACGGGGATGCAGATAAATCGCCATTCGTTGACTACTTCTTATCTGGATTTAATGTCGCATAAGT 381GGAACCTCACTGACGCAGTCTGTGGCAAGAGCGATGTTACGGTTTGTTACTGTGACAGCTGA 443AGCTTTACGTTTTCGGCAAATACAGAGGGGATTTCGTACAACACTGGATGATCTCAGTGGGC 505GTTCTTATGTAATGACTGCTGAAGATGTTGATCTTACATTGAACTGGGGAAGGGTGAGTAGN 567GTCCTGCCTGACTATCATGGACAAGACTCTGNTCGTGTNGGAAGAANTTCTTTTGGAAGCAT 629TAATGCAATTCTGGGAAGCGTGGCATTAATATTGTTATCGCATCATCATGCAThe information of AATTCCGAC 693TCTGAATGCCCGCTGTCTCACGACGGTTACTGCCTGCACGATGGTGTTTGCAT GTATATCGAA 756GCTCTGGACAAATACGCGTGCGGCTGTGTTGTTGGTTACATCGGTGAACGTTG CCAGTACCG 818TGACCTGAAATCGTGGGAACTGCGTCTCGAGCACCACCACCACCACCACTGA 870 (2) SEQ ID NO:4
(i) sequence signature
(A) length: 233 amino-acid residues
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: protein
(xi) sequence description: SEQ ID N0:4TGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFAD 50FSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMS 100HSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTA 150EDVDLTLNWGRLSSVLPDYHGQDSVRVGRINSDSECPLSHDGYCLHDGVC 200MYIEALDKYACNCVVGYIGERCQYRDLKWWELR 233
Claims (9)
1, the encoding gene of fusion rotein has the nucleotide sequence of SEQ ID NO:3.
2, fusion rotein has the amino acid residue sequence of SEQ ID NO:4.
3, be the antitumor drug of activeconstituents with the described fusion rotein of claim 2.
4, antitumor drug according to claim 3 is characterized in that: described medicine is the medicine of treatment mammary cancer.
5, antitumor drug according to claim 3 is characterized in that: described medicine is the treatment lung cancer drugs.
6, antitumor drug according to claim 3 is characterized in that: described medicine is the medicine of treatment liver cancer.
7, antitumor drug according to claim 3 is characterized in that: described medicine is the medicine of treatment oral carcinoma.
8, antitumor drug according to claim 3 is characterized in that: described medicine is the medicine of treatment cell carcinoma.
9, the application of the fusion rotein of claim 1 in the preparation antitumor drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011201002A CN1161464C (en) | 2001-07-09 | 2001-07-09 | Human epidermal growth factor receptor specific fusion protein and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011201002A CN1161464C (en) | 2001-07-09 | 2001-07-09 | Human epidermal growth factor receptor specific fusion protein and its application |
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| Publication Number | Publication Date |
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| CN1394956A true CN1394956A (en) | 2003-02-05 |
| CN1161464C CN1161464C (en) | 2004-08-11 |
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|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100436481C (en) * | 2003-08-18 | 2008-11-26 | 中国医学科学院基础医学研究所 | Targeted anti-tumour fused protein containing adenovirus E40rf4 protein |
| CN1673388B (en) * | 2004-03-24 | 2010-12-01 | 董强刚 | Reagent kit for real-time quantitative detecting epidermal growth factor receptor gene expression level |
| CN1924021B (en) * | 2006-09-08 | 2012-08-29 | 张力建 | Gene recombination humanized pigment epithelium derivative factor and application therof |
| CN103290018A (en) * | 2013-05-07 | 2013-09-11 | 南方医科大学 | Nucleic acid aptamer specifically bound with human epidermal growth factor receptor III-type mutant and application of nucleic acid aptamer |
-
2001
- 2001-07-09 CN CNB011201002A patent/CN1161464C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100436481C (en) * | 2003-08-18 | 2008-11-26 | 中国医学科学院基础医学研究所 | Targeted anti-tumour fused protein containing adenovirus E40rf4 protein |
| CN1673388B (en) * | 2004-03-24 | 2010-12-01 | 董强刚 | Reagent kit for real-time quantitative detecting epidermal growth factor receptor gene expression level |
| CN1924021B (en) * | 2006-09-08 | 2012-08-29 | 张力建 | Gene recombination humanized pigment epithelium derivative factor and application therof |
| CN103290018A (en) * | 2013-05-07 | 2013-09-11 | 南方医科大学 | Nucleic acid aptamer specifically bound with human epidermal growth factor receptor III-type mutant and application of nucleic acid aptamer |
| CN103290018B (en) * | 2013-05-07 | 2015-07-29 | 南方医科大学 | A kind of nucleic acid aptamer of being combined with Human epidermal growth factor receptor type III mutant specific and application thereof |
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| Publication number | Publication date |
|---|---|
| CN1161464C (en) | 2004-08-11 |
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Granted publication date: 20040811 Termination date: 20130709 |