CN1891718A - Fusion immunotoxin ML-L-SEC2 and gene and its preparation - Google Patents

Fusion immunotoxin ML-L-SEC2 and gene and its preparation Download PDF

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CN1891718A
CN1891718A CN 200510046819 CN200510046819A CN1891718A CN 1891718 A CN1891718 A CN 1891718A CN 200510046819 CN200510046819 CN 200510046819 CN 200510046819 A CN200510046819 A CN 200510046819A CN 1891718 A CN1891718 A CN 1891718A
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徐明恺
张成刚
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Institute of Applied Ecology of CAS
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Institute of Applied Ecology of CAS
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Abstract

The invention relates to a new type of anti-human epidermal growth factor receptor HER-2- Staphylococcus aureus enterotoxin C2 coalesce gene ml-l-sec2 of immunotoxin ML-L-SEC2 and its preparation, in which, there is a base sequence in the table SEQ ID No:1 and protein sequence in the table SEQ ID No:2. The invention provides a method for constructing the confluens immunotoxin B-L-SEC2 with single-chain antibody of anti - HER-2 elements and SEC2 via the link of short peptide, and expresses the confluence immunotoxin protein in the coliform bacterium via expression vector pET-32a.

Description

Fusion immunotoxin ML-L-SEC 2 and gene and preparation thereof
Technical field
The present invention relates to genetic engineering pharmaceutical, a kind of specifically novel anti-human epidermal growth factor acceptor HER-2-staphylococcus aureus enterotoxin C 2 fusion immunotoxin ML-L-SEC 2 gene ml-l-sec2 and preparation thereof.
Background technology
Immunotoxin is a kind of new therapeutic strategy that proposes on the basis of modern medicine biology development, now has been widely used in oncotherapy.Fusion immunotoxin is the product by targeted molecular and cytotoxicity molecule be combined into, antibody or genetic engineering antibody variable region fragment Chang Zuowei targeted molecular, (Single Chain Variable Fragment, scFv) strong, the blood of, tumour penetration power little because of its molecular weight is removed the research that characteristics such as fast and transformation period is short are widely used in the drug targeting carrier to single chain antibody fragments in recent years.
Human epidermal growth factor receptor 2 (Human Epidermal Growth Factor Receptor-2, HER-2) be a kind of tumor associated antigen, cross expressing of 25-30% arranged in mammary cancer and ovarian cancer patients, and its cross express relevant with patient's poor prognosis.The HER-2 expression of receptor is the suitable target spot of neoplasm targeted therapy in tumor cell surface.At present, the tumour of as oriented carrier HER-2 being crossed expression with anti-HER-2 single-chain antibody is carried out magnetic target therapy has become the research focus.
Staphylococcus aureus enterotoxin C 2 (SEC2) belongs to second hypotype in the C class enterotoxin family.As a kind of superantigen, when extremely low concentration, can combine with major histocompatibility complex two quasi-molecules (MHCII), stimulate major part that the T cell proliferation of TXi Baoshouti V β (TCRV β) sequence is arranged, make it to discharge the various kinds of cell factor such as IL-2, IFN-γ and TNF etc. produce extremely strong tumor inhibition effect in vitro and in vivo.At home, SEC2 has been applied to clinical cancer therapy and has obtained good therapeutic action.Yet the mhc class ii molecule mainly is expressed in normal human body cell, and in the tumor tissues expression amount seldom, this makes the oncotherapy of SEC2 lack specificity and produces side reaction.Therefore seek a kind of suitable can the transport vehicle of SEC2 target guiding tumour cell is significant for the oncotherapy of SEC2.
Summary of the invention
The purpose of this invention is to provide a kind of fusion immunotoxin ML-L-SEC 2 and gene and preparation thereof, the present invention with anti-HER-2 single-chain antibody as targeted molecular, with SEC2 action effect molecule, be built into fusion immunotoxin B-L-SEC2 through the small peptide connection, and in intestinal bacteria, efficiently express fusion immunotoxin albumen with expression vector pET-32a.But this fusion immunotoxin specific recognition is crossed the cancer cells of expression in conjunction with the HER-2 acceptor, and it is produced the specific cell restraining effect.
For achieving the above object, the technical solution used in the present invention is:
Fusion immunotoxin ML-L-SEC 2 has protein sequence among the sequence table SEQ ID No:2.
The fusion immunotoxin ML-L-SEC 2 gene has base sequence among the sequence table SEQ ID No:1.
The preparation of described fusion immunotoxin ML-L-SEC 2 gene, be the single-chain antibody gene ml3.9 (having base sequence among the sequence table SEQ ID No:3) of the anti-HER-2 of humanized and staphylococcus aureus enterotoxin C 2 (SEC2) gene sec2 are connected small peptide by coding DNA Linker with the connection of restriction endonuclease interconnection technique, resulting fusion immunotoxin gene has base sequence among the sequence table SEQ IDNo:1;
The base sequence of described connection small peptide DNA Linker is,
GGCCGCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGAATTC；
Described staphylococcus aureus enterotoxin C 2 has base sequence among the sequence table SEQ ID No:5.
The present invention has the following advantages:
1. the present invention has made up novel fusion immunotoxin gene, and it is the new gene of artificial preparation, belongs to the fusion gene that makes first; The catenation sequence of its introducing guarantees antibody fragment and enterotoxin molecule biologic activity separately.
2. quote the present invention, can further provide to be directly used in the engineering strain of producing the ML-L-SEC2 fusion immunotoxin.
3. this fusion immunotoxin can activate the human body immune response, and the tumour cell of crossing expression at the HER-2 acceptor produces lethal effect to this tumour on every side; Eliminate the side reaction that the whole body administration causes, for the immunotherapy of tumour provides new approach.
4. the fusion immunotoxin bulk of molecule is 1/3 of an antibody molecule, has overcome the deficiency that monoclonal antibody is difficult for being penetrated into tumor tissues, and antineoplastic action is more obvious.
Description of drawings
Fig. 1 is the pcr amplification agarose gel electrophoresis figure of the single-chain antibody gene ml3.9 of the anti-HER-2 of humanized, and wherein: 1 is the pcr amplification product of ml3.9, and 2 is the DL2000DNA molecular weight standard;
Fig. 2 is the pcr amplification agarose gel electrophoresis figure of SEC2 gene sec2, and wherein: 1 is the DL2000DNA molecular weight standard, and 2 is the pcr amplification product of sec2;
Fig. 3 is that the double digestion of fusion immunotoxin expression vector pET-32a-ml-l-sec2 is identified agarose gel electrophoresis figure, wherein 1 is the DL15000DNA molecular weight standard, 2 is NcoI and the checking of XhoI double digestion of pET-32a-ml-l-sec2,3 is NcoI and the checking of NotI double digestion of pET-32a-ml-l-sec2,4 is EcoRI and the checking of XhoI double digestion of pET-32a-ml-l-sec2, and 5 is DL 2000DNA molecular weight standard;
Fig. 4 is the intestinal bacteria heterogenous expression SDS-PAGE electrophorogram of fusion immunotoxin, wherein: 1 is the full cell expressing of inductive fusion immunotoxin, and 2 is the solubility expression of inductive fusion immunotoxin, and 3 is the inclusion body expression of inductive fusion immunotoxin, 4 (are respectively 116 from top to bottom for the molecular weight of albumen standard, 66,45,35,25,18,14KD), 5 is inductive pET-32a empty carrier;
Fig. 5 detects figure for the protein immunization marking of the fusion immunotoxin of expression, and wherein: 1 is the molecular weight of albumen standard of dying in advance, and 2 is the protein immunization marking detected result of fusion immunotoxin.
Embodiment
Embodiment 1
The single-chain antibody gene ml3.9 of the anti-HER-2 of a kind of humanized has base sequence among the sequence table SEQ ID No:3.
ATGGCCCAGG?TGCAGCTGGT?GCAGTCTGGG?GCAGAGGTGA?AAAAGCCCGG?50
GGAGTCTCTG?AAGATCTCCT?GTAAGGGTTC?TGGATACAGC?TTTACCAGCT?100
ACTGGATCGC?CTGGGTGCGC?CAGATGCCCG?GGAAAGGCCT?GGAGTACATG?150
GGGCTCATCT?ATCCTGGTGA?CTCTGACACC?AAATACAGCC?CGTCCTTCCA?200
AGGCCAGGTC?ACCATCTCAG?TCGACAAGTC?CGTCAGCACT?GCCTACTTGC?250
AATGGAGCAG?TCTGAAGCCC?TCGGACAGCG?CCGTGTATTT?TTGTGCGAGA?300
CATGACGTGG?GATATTGCAG?TAGTTCCAAC?TGCGCAAAGT?GGCCTGAATA?350
CTTCCAGCAT?TGGGGCCAGG?GCACCCTGGT?CACCGTCTCC?TCAGGTGGAG?400
GCGGTTCAGG?CGGAGGTGGC?TCTGGCGGTG?GCGGATCGCA?GTCTGTGTTG?450
ACGCAGCCGC?CCTCAGTGTC?TGCGGCCCCA?GGACAGAAGG?TCACCATCTC?500
CTGCTCTGGA?AGCAGCTCCA?ACATTGGGAA?TAATTATGTA?TCCTGGTACC?550
AGCAGCTCCC?AGGAACAGCC?CCCAAACTCC?TCATCTATGA?TCACACCAAT?600
CGGCCCGCAG?GGGTCCCTGA?CCGATTCTCT?GGCTCCAAGT?CTGGCACCTC?650
AGCCTCCCTG?GCCATCAGTG?GGTTCCGGTC?CGAGGATGAG?GCTGATTATT?700
ACTGTGCCTC?CTGGGACTAC?ACCCTCTCGG?GCTGGGTGTT?CGGCGGAGGA?750
ACCAAGCTGA?CCGTCCTAGG?TGCG 774
(1) information of SEQ ID No:3 (referring to sequence table)
(a) sequence signature
* length: 774 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: the single-chain antibody gene ml3.9 of the anti-HER-2 of humanized.
(2) preparation upstream primer V-F:5 '-TTA T of the single-chain antibody gene ml3.9 of the anti-HER-2 of humanized CC ATG GCC CAG GTG CAG CTG GTG CAGTCT-3 ';
Downstream primer V-R:5 '-TTC T GC GGC CGCACC TAG GAC GGT GAC CTTGGTC-3 ',
Horizontal line is depicted as the restriction enzyme site of interpolation, and upstream and downstream is respectively NcoI and NotI.
The PCR reaction system is: 10 * Pyrobest buffer, 5 μ l, dNTP 250 μ mol, 0.02%BSA 2 μ l, each 25pmol of upstream and downstream primer, template contain plasmid DNA 0.1 μ g, the Pyrobest archaeal dna polymerase 2U of scFv-ml, aseptic ultrapure water polishing volume to 50 μ l.
The PCR reaction conditions is:
95 ℃ of fs, 5 minutes;
94 ℃ of subordinate phase, 50 seconds; 55 ℃, 50 seconds; 72 ℃, 90 seconds; Totally 30 circulations;
72 ℃ of phase IIIs, 10 minutes;
(3) recovery of PCR product: pcr amplification product is through 1.2% agarose gel electrophoresis analysis and separation (shown in accompanying drawing 1), downcutting size is the purpose band of 770bp, reclaims the test kit working instructions by the clean biochemical technology of Hangzhou Wei Te company limited glue and carries out the glue recovery;
(4) double digestion of recovery after product: the PCR product after the recovery fully digests through restriction endonuclease NcoI and NotI, and digestion product is through 1.2% agarose gel electrophoresis analysis, and glue reclaims once more.
Embodiment 2
Staphylococcus aureus enterotoxin C 2 (SEC2) gene sec2 comes from streptococcus aureus, has base sequence among the sequence table SEQ ID No:5.
GAGAGTCAACCAGACCCTACGCCAGATGAGTTGCACAAATCAAGTGAGTTTACTGGTACGATGGGTAATATGAAATATTTATATGATGATCATTATGTATCAGCAACTAAAGTTATGTCTGTAGATAAATTTTTGGCACATGATTTAATTTATAACATTAGTGATAAAAAACTAAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGAAGATTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGTAAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGTAAAACTTGTATGTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGTACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAAGAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAAAAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAATAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGAAGTCCACCTTACAACAAAGAATGGA
(1) information of SEQ ID No:5 (referring to sequence table)
(A) sequence signature
* length: 717 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: streptococcus aureus
(2) preparation of SEC2 gene sec2
Upstream primer sec2-F:5 '-TCT GAA TTCGAG AGT CAA CCA GAC CCT A-3 '
Downstream primer sec2-R:5 '-ATA CTC GAGTTA TCC ATT CTT TGT TGT A-3 '
Horizontal line is depicted as the restriction enzyme site of interpolation, and upstream and downstream is respectively EcoRI and XhoI.
The PCR reaction system is: 10 * Pyrobest buffer, 5 μ l, dNTP 250 μ mol, 0.02%BSA 2 μ l, each 25pmol of upstream and downstream primer, template contain plasmid DNA 0.1 μ g, the PyrobestDNA polysaccharase 2U of sec2, aseptic ultrapure water polishing volume to 50 μ l.
The PCR reaction conditions is:
95 ℃ of fs, 5 minutes;
94 ℃ of subordinate phase, 45 seconds; 55 ℃, 45 seconds; 72 ℃, 90 seconds; Totally 30 circulations;
72 ℃ of phase IIIs, 10 minutes;
(3) recovery of PCR product: pcr amplification product is through 1.2% agarose gel electrophoresis analysis and separation (shown in accompanying drawing 2), downcutting size is the purpose band of 720bp, reclaims the test kit working instructions by the clean biochemical technology of Hangzhou Wei Te company limited glue and carries out the glue recovery;
(4) double digestion of recovery after product: the PCR product after the recovery fully digests through restriction endonuclease EcoRI and XhoI, and digestion product is through 1.2% agarose gel electrophoresis analysis, and glue reclaims once more.
Embodiment 3
Coding connects the DNA Linker sequence of small peptide, has following base sequence (oligonucleotide chain):
GGCCGCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGAATTC
(1) preparation of DNA Linker sequence gene
1) design of DNA Linker sequence gene is synthetic:
Design and synthesize two oligonucleotide chains:
Forward chain L-F:5 '- G GCC GCA AGC GGC TCA GGA TCT GGA TCA GGA TCTGGC G-3 ';
Reverse strand L-R:5 '- AA TTCGCC AGA TCC TGA TCC AGA TCC TGA GCCGCT TGC-3 '
Upstream and downstream adds NotI respectively and EcoRI is sticking terminal, and coded glycine Serine small peptide is (GS) 2Sequence.
2) annealing of oligonucleotide chain:
10 * annealing hybridization buffer (SSC) composition: 1.5mol/L NaCl, 0.3M trisodium citrate 2H 2O transfers pH to 7.0 with HCl;
Annealing reaction system: 10 * SSC buffer, 1 μ l, positive and negative each 5pmol of oligonucleotide chain, aseptic ultrapure water polishing volume to 10 μ l;
Annealing reaction condition: 94 ℃ of 10min; 0 ℃ of 10min; 72 ℃ of 20min
Embodiment 4 fusion immunotoxin genes have the base sequence of sequence table SEQ ID No:1.
ATGGCCCAGG?TGCAGCTGGT?GCAGTCTGGG?GCAGAGGTGA?AAAAGCCCGG?50
GGAGTCTCTG?AAGATCTCCT?GTAAGGGTTC?TGGATACAGC?TTTACCAGCT?100
ACTGGATCGC?CTGGGTGCGC?CAGATGCCCG?GGAAAGGCCT?GGAGTACATG?150
GGGCTCATCT?ATCCTGGTGA?CTCTGACACC?AAATACAGCC?CGTCCTTCCA?200
AGGCCAGGTC?ACCATCTCAG?TCGACAAGTC?CGTCAGCACT?GCCTACTTGC?250
AATGGAGCAG?TCTGAAGCCC?TCGGACAGCG?CCGTGTATTT?TTGTGCGAGA?300
CATGACGTGG?GATATTGCAG?TAGTTCCAAC?TGCGCAAAGT?GGCCTGAATA?350
CTTCCAGCAT?TGGGGCCAGG?GCACCCTGGT?CACCGTCTCC?TCAGGTGGAG?400
GCGGTTCAGG?CGGAGGTGGC?TCTGGCGGTG?GCGGATCGCA?GTCTGTGTTG?450
ACGCAGCCGC?CCTCAGTGTC?TGCGGCCCCA?GGACAGAAGG?TCACCATCTC?500
GTGGTGTGGA?AGGAGGTCCA?AGATTGGGAA?TAATTATGTA?TGGTGGTACG?550
AGCAGCTCCC?AGGAACAGCC?CCCAAACTCC?TCATCTATGA?TCACACCAAT?600
CGGCCCGCAG?GGGTCCCTGA?CCGATTCTCT?GGCTCCAAGT?CTGGCACCTC?650
AGCCTCCCTG?GCCATCAGTG?GGTTCCGGTC?CGAGGATGAG?GCTGATTATT?700
ACTGTGCCTC?CTGGGACTAC?ACCCTCTCGG?GCTGGGTGTT?CGGCGGAGGA?750
ACCAAGCTGA?CCGTCCTAGG?TGCGGCCGCA?AGCGGCTCAG?GATCTGGATC?800
AGGATCTGGC?GAATTCGAGA?GTCAACCAGA?CCCTACGCCA?GATGAGTTGC?850
ACAAATCAAG?TGAGTTTACT?GGTACGATGG?GTAATATGAA?ATATTTATAT?900
GATGATCATT?ATGTATCAGC?AACTAAAGTT?ATGTCTGTAG?ATAAATTTTT?950
GGCACATGAT?TTAATTTATA?ACATTAGTGA?TAAAAAACTA?AAAAATTATG?1000
ACAAAGTGAA?AACAGAGTTA?TTAAATGAAG?ATTTAGCAAA?GAAGTACAAA?1050
GATGAAGTAG?TTGATGTGTA?TGGATCAAAT?TACTATGTAA?ACTGCTATTT?1100
TTCATCCAAA?GATAATGTAG?GTAAAGTTAC?AGGTGGTAAA?ACTTGTATGT?1150
ATGGAGGAAT?AACAAAACAT?GAAGGAAACC?ACTTTGATAA?TGGGAACTTA?1200
CAAAATGTAC?TTATAAGAGT?TTATGAAAAT?AAAAGAAACA?CAATTTCTTT?1250
TGAAGTGCAA?ACTGATAAGA?AAAGTGTAAC?AGCTCAAGAA?CTAGACATAA?1300
AAGCTAGGAA?TTTTTTAATT?AATAAAAAAA?ATTTGTATGA?GTTTAACAGT?1350
TCACCATATG?AAACAGGATA?TATAAAATTT?ATTGAAAATA?ACGGCAATAC?1400
TTTTTGGTAT?GATATGATGC?CTGCACCAGG?CGATAAGTTT?GACCAATCTA?1450
AATATTTAAT?GATGTACAAC?GACAATAAAA?CGGTTGATTC?TAAAAGTGTG?1500
AAGATAGAAG?TCCACCTTAC?AACAAAGAAT?GGA 1533
(1) information of SEQ ID No:1 (referring to sequence table)
(A) sequence signature
* length: 1533 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: CDNA
(C) suppose: not
(d) antisense: not
(e) initial source: artificial sequence
(2) preparation of fusion gene
With the linker fragment after the annealing, through the scFv-ml fragment of NcoI and NotI double digestion, through the sec2 fragment of EcoRI and XhoI double digestion and through the pET-32a fragment of NcoI and XhoI double digestion in molar ratio example be 3: 3: 3: 1 mixed, with 16 ℃ of connections of T4DNA ligase enzyme, make up fusion immunotoxin expression vector pET-32a-ml-l-sec2.(conversion operation is pressed F. Ao Sibai, R. Brunt, R.E. James Kingston to connect product Transformed E .coli AD494 (DE3) competent cell, D.D. Moore, J.G. Sai Deman, J.A. Smith, K. Si Telaer " fine works molecular biology experiment guide " USA New York JohnWiley; The nineteen ninety-five third edition P39-40 of Sons press), with the acillin of 100 μ g/ml and the kalamycin resistance screening transformant of 15 μ g/ml.
(3) checking of fusion gene
The positive transformant of selecting is spread cultivation, (the plasmid DNA extracting method is pressed J. Sa nurse Brooker to extract plasmid DNA, E.F. Flitch, T. Manny A Disi " molecular cloning experiment guide " cold spring port, the USA New York calendar year 2001 third edition P27-30 of press), identify (shown in accompanying drawing 3) through double digestion, and to identify that correct recombinant clone plasmid is a template, be primer with T7 promotor and terminator, confirm with the terminal cessation method order-checking of the two deoxidations of Sanger.
The expression and the activity identification of embodiment 5 fusion immunotoxins
(1) expression of fusion immunotoxin: inoculation has transformed the single bacterium colony of AD494 (DE3) of pET-32a-ml-l-sec2 plasmid in the liquid LB of the kantlex of acillin that contains 100 μ g/ml and 15 μ g/ml substratum, the activation culture of spending the night, with 1% inoculum size switching next stage, 37 ℃ of shaking tables are cultured to OD 600Be 0.6, add the IPTG of final concentration 1.0mmol/L, 30 ℃ of abduction deliverings 6 hours.Centrifugal collection thalline, (buffer formulation and treatment process are pressed Wang Jiazheng with the processing of 5 * sample-loading buffer, model bright " protein technical manual " 2002 first version P81 and the P87 of Science Press), the 12%SDS-PAGE electrophoresis, coomassie brilliant blue staining is analyzed expression product.
(2) biologic activity of fusion immunotoxin: the immunogenicity of identifying the fusion immunotoxin of expressing with protein immunization marking method: the fusion immunotoxin albumen of heterogenous expression is separated with SDS-PAGE, half-dried commentaries on classics film instrument goes to nitrocellulose membrane, the sealing of 2% skimmed milk, goat anti-rabbit antibody with the anti-SEC2 antibody of rabbit, alkali phosphatase enzyme mark is handled successively, the colour developing of NBT/BCIP colouring reagents box.(used damping fluid and concrete operation method are pressed Wang Jiazheng, model bright " protein technical manual " the 2002 first version P166 of Science Press).
The result: in the cell whole protein electrophoresis of engineering bacteria an obvious expression band is arranged, the inducible protein molecular weight is about 72kD (shown in accompanying drawing 4).This fusion immunotoxin can keep the proteic immunogenicity of former SEC2 by anti-SEC2 antibodies specific identification in conjunction with (shown in accompanying drawing 5).
SEQUENCE?LISTING
＜110〉Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences
＜120〉fusion immunotoxin ML-L-SEC 2 and gene and preparation thereof
<130>
<160>6
<170>PatentIn?version?3.1
<210>1
<211>1533
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1533)
<223>
<400>1
atg?gcc?cag?gtg?cag?ctg?gtg?cag?tct?ggg?gca?gag?gtg?aaa?aag?ccc 48
Met?Ala?Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro
1 5 10 15
ggg?gag?tct?ctg?aag?atc?tcc?tgt?aag?ggt?tct?gga?tac?agc?ttt?acc 96
Gly?Glu?Ser?Leu?Lys?Ile?Ser?Cys?Lys?Gly?Ser?Gly?Tyr?Ser?Phe?Thr
20 25 30
agc?tac?tgg?atc?gcc?tgg?gtg?cgc?cag?atg?ccc?ggg?aaa?ggc?ctg?gag 144
Ser?Tyr?Trp?Ile?Ala?Trp?Val?Arg?Gln?Met?Pro?Gly?Lys?Gly?Leu?Glu
35 40 45
tac?atg?ggg?ctc?atc?tat?cct?ggt?gac?tct?gac?acc?aaa?tac?agc?ccg 192
Tyr?Met?Gly?Leu?Ile?Tyr?Pro?Gly?Asp?Ser?Asp?Thr?Lys?Tyr?Ser?Pro
50 55 60
tcc?ttc?caa?ggc?cag?gtc?acc?atc?tca?gtc?gac?aag?tcc?gtc?agc?act 240
Ser?Phe?Gln?Gly?Gln?Val?Thr?Ile?Ser?Val?Asp?Lys?Ser?Val?Ser?Thr
65 70 75 80
gcc?tac?ttg?caa?tgg?agc?agt?ctg?aag?ccc?tcg?gac?agc?gcc?gtg?tat 288
Ala?Tyr?Leu?Gln?Trp?Ser?Ser?Leu?Lys?Pro?Ser?Asp?Ser?Ala?Val?Tyr
85 90 95
ttt?tgt?gcg?aga?cat?gac?gtg?gga?tat?tgc?agt?agt?tcc?aac?tgc?gca 336
Phe?Cys?Ala?Arg?His?Asp?Val?Gly?Tyr?Cys?Ser?Ser?Ser?Asn?Cys?Ala
100 105 110
aag?tgg?cct?gaa?tac?ttc?cag?cat?tgg?ggc?cag?ggc?acc?ctg?gtc?acc 384
Lys?Trp?Pro?Glu?Tyr?Phe?Gln?His?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
115 120 125
gtc?tcc?tca?ggt?gga?ggc?ggt?tca?ggc?gga?ggt?ggc?tct?ggc?ggt?ggc 432
Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly
130 135 140
gga?tcg?cag?tct?gtg?ttg?acg?cag?ccg?ccc?tca?gtg?tct?gcg?gcc?cca 480
Gly?Ser?Gln?Ser?Val?Leu?Thr?Gln?Pro?Pro?Ser?Val?Ser?Ala?Ala?Pro
145 150 155 160
gga?cag?aag?gtc?acc?atc?tcc?tgc?tct?gga?agc?agc?tcc?aac?att?ggg 528
Gly?Gln?Lys?Val?Thr?Ile?Ser?Cys?Ser?Gly?Ser?Ser?Ser?Asn?Ile?Gly
165 170 175
aat?aat?tat?gta?tcc?tgg?tac?cag?cag?ctc?cca?gga?aca?gcc?ccc?aaa 576
Asn?Asn?Tyr?Val?Ser?Trp?Tyr?Gln?Gln?Leu?Pro?Gly?Thr?Ala?Pro?Lys
180 185 190
ctc?ctc?atc?tat?gat?cac?acc?aat?cgg?ccc?gca?ggg?gtc?cct?gac?cga 624
Leu?Leu?Ile?Tyr?Asp?His?Thr?Asn?Arg?Pro?Ala?Gly?Val?Pr0?Asp?Arg
195 200 205
ttc?tct?ggc?tcc?aag?tct?ggc?acc?tca?gcc?tcc?ctg?gcc?atc?agt?ggg 672
Phe?Ser?Gly?Ser?Lys?Ser?Gly?Thr?Ser?Ala?Ser?Leu?Ala?Ile?Ser?Gly
210 215 220
ttc?cgg?tcc?gag?gat?gag?gct?gat?tat?tac?tgt?gcc?tcc?tgg?gac?tac 720
Phe?Arg?Ser?Glu?Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Ala?Ser?Trp?Asp?Tyr
225 230 235 240
acc?ctc?tcg?ggc?tgg?gtg?ttc?ggc?gga?gga?acc?aag?ctg?acc?gtc?cta 768
Thr?Leu?Ser?Gly?Trp?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu
245 250 255
ggt?gcg?gcc?gca?agc?ggc?tca?gga?tct?gga?tca?gga?tct?ggc?gaa?ttc 816
Gly?Ala?Ala?Ala?Ser?Gly?Ser?Gly?Ser?Gly?Ser?Gly?Ser?Gly?Glu?Phe
260 265 270
gag?agt?caa?cca?gac?cct?acg?cca?gat?gag?ttg?cac?aaa?tca?agt?gag 864
Glu?Ser?Gln?Pro?Asp?Pro?Thr?Pro?Asp?Glu?Leu?His?Lys?Ser?Ser?Glu
275 280 285
ttt?act?ggt?acg?atg?ggt?aat?atg?aaa?tat?tta?tat?gat?gat?cat?tat 912
Phe?Thr?Gly?Thr?Met?Gly?Asn?Met?Lys?Tyr?Leu?Tyr?Asp?Asp?His?Tyr
290 295 300
gta?tca?gca?act?aaa?gtt?atg?tct?gta?gat?aaa?ttt?ttg?gca?cat?gat 960
Val?Ser?Ala?Thr?Lys?Val?Met?Ser?Val?Asp?Lys?Phe?Leu?Ala?His?Asp
305 310 315 320
tta?att?tat?aac?att?agt?gat?aaa?aaa?cta?aaa?aat?tat?gac?aaa?gtg 1008
Leu?Ile?Tyr?Asn?Ile?Ser?Asp?Lys?Lys?Leu?Lys?Asn?Tyr?Asp?Lys?Val
325 330 335
aaa?aca?gag?tta?tta?aat?gaa?gat?tta?gca?aag?aag?tac?aaa?gat?gaa 1056
Lys?Thr?Glu?Leu?Leu?Asn?Glu?Asp?Leu?Ala?Lys?Lys?Tyr?Lys?Asp?Glu
340 345 350
gta?gtt?gat?gtg?tat?gga?tca?aat?tac?tat?gta?aac?tgc?tat?ttt?tca 1104
Val?Val?Asp?Val?Tyr?Gly?Ser?Asn?Tyr?Tyr?Val?Asn?Cys?Tyr?Phe?Ser
355 360 365
tcc?aaa?gat?aat?gta?ggt?aaa?gtt?aca?ggt?ggt?aaa?act?tgt?atg?tat 1152
Ser?Lys?Asp?Asn?Val?Gly?Lys?Val?Thr?Gly?Gly?Lys?Thr?Cys?Met?Tyr
370 375 380
gga?gga?ata?aca?aaa?cat?gaa?gga?aac?cac?ttt?gat?aat?ggg?aac?tta 1200
Gly?Gly?Ile?Thr?Lys?His?Glu?Gly?Asn?His?Phe?Asp?Asn?Gly?Asn?Leu
385 390 395 400
caa?aat?gta?ctt?ata?aga?gtt?tat?gaa?aat?aaa?aga?aac?aca?att?tct 1248
Gln?Asn?Val?Leu?Ile?Arg?Val?Tyr?Glu?Asn?Lys?Arg?Asn?Thr?Ile?Ser
405 410 415
ttt?gaa?gtg?caa?act?gat?aag?aaa?agt?gta?aca?gct?caa?gaa?cta?gac 1296
Phe?Glu?Val?Gln?Thr?Asp?Lys?Lys?Ser?Val?Thr?Ala?Gln?Glu?Leu?Asp
420 425 430
ata?aaa?gct?agg?aat?ttt?tta?att?aat?aaa?aaa?aat?ttg?tat?gag?ttt 1344
Ile?Lys?Ala?Arg?Asn?Phe?Leu?Ile?Asn?Lys?Lys?Asn?Leu?Tyr?Glu?Phe
aac?agt?tca?cca?tat?gaa?aca?gga?tat?ata?aaa?ttt?att?gaa?aat?aac 1392
Asn?Ser?Ser?Pro?Tyr?Glu?Thr?Gly?Tyr?Ile?Lys?Phe?Ile?Glu?Asn?Asn
450 455 460
ggc?aat?act?ttt?tgg?tat?gat?atg?atg?cct?gca?cca?ggc?gat?aag?ttt 1440
Gly?Asn?Thr?Phe?Trp?Tyr?Asp?Met?Met?Pro?Ala?Pro?Gly?Asp?Lys?Phe
465 470 475 480
gac?caa?tct?aaa?tat?tta?atg?atg?tac?aac?gac?aat?aaa?acg?gtt?gat 1488
Asp?Gln?Ser?Lys?Tyr?Leu?Met?Met?Tyr?Asn?Asp?Asn?Lys?Thr?Val?Asp
485 490 495
tct?aaa?agt?gtg?aag?ata?gaa?gtc?cac?ctt?aca?aca?aag?aat?gga 1533
Ser?Lys?Ser?Val?Lys?Ile?Glu?Val?His?Leu?Thr?Thr?Lys?Asn?Gly
500 505 510
<210>2
<211>511
<212>PRT
＜213〉artificial sequence
<400>2
Met?Ala?Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro
1 5 10 15
Gly?Glu?Ser?Leu?Lys?Ile?Ser?Cys?Lys?Gly?Ser?Gly?Tyr?Ser?Phe?Thr
20 25 30
Ser?Tyr?Trp?Ile?Ala?Trp?Val?Arg?Gln?Met?Pro?Gly?Lys?Gly?Leu?Glu
35 40 45
Tyr?Met?Gly?Leu?Ile?Tyr?Pro?Gly?Asp?Ser?Asp?Thr?Lys?Tyr?Ser?Pro
50 55 60
Ser?Phe?Gln?Gly?Gln?Val?Thr?Ile?Ser?Val?Asp?Lys?Ser?Val?Ser?Thr
65 70 75 80
Ala?Tyr?Leu?Gln?Trp?Ser?Ser?Leu?Lys?Pro?Ser?Asp?Ser?Ala?Val?Tyr
85 90 95
Phe?Cys?Ala?Arg?His?Asp?Val?Gly?Tyr?Cys?Ser?Ser?Ser?Asn?Cys?Ala
100 105 110
Lys?Trp?Pro?Glu?Tyr?Phe?Gln?His?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
115 120 125
Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly
130 135 140
Gly?Ser?Gln?Ser?Val?Leu?Thr?Gln?Pro?Pro?Ser?Val?Ser?Ala?Ala?Pro
145 150 155 160
Gly?Gln?Lys?Val?Thr?Ile?Ser?Cys?Ser?Gly?Ser?Ser?Ser?Asn?Ile?Gly
165 170 175
Ash?Asn?Tyr?Val?Ser?Trp?Tyr?Gln?Gln?Leu?Pro?Gly?Thr?Ala?Pro?Lys
180 185 190
Leu?Leu?Ile?Tyr?Asp?His?Thr?Asn?Arg?Pro?Ala?Gly?Val?Pro?Asp?Arg
195 200 205
Phe?Ser?Gly?Ser?Lys?Ser?Gly?Thr?Ser?Ala?Ser?Leu?Ala?Ile?Ser?Gly
210 215 220
Phe?Arg?Ser?Glu?Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Ala?Ser?Trp?Asp?Tyr
225 230 235 240
Thr?Leu?Ser?Gly?Trp?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu
245 250 255
Gly?Ala?Ala?Ala?Ser?Gly?Ser?Gly?Ser?Gly?Ser?Gly?Ser?Gly?Glu?Phe
260 265 270
Glu?Ser?Gln?Pro?Asp?Pro?Thr?Pro?Asp?Glu?Leu?His?Lys?Ser?Ser?Glu
275 280 285
Phe?Thr?Gly?Thr?Met?Gly?Asn?Met?Lys?Tyr?Leu?Tyr?Asp?Asp?His?Tyr
290 295 300
Val?Ser?Ala?Thr?Lys?Val?Met?Ser?Val?Asp?Lys?Phe?Leu?Ala?His?Asp
305 310 315 320
Leu?Ile?Tyr?Asn?Ile?Ser?Asp?Lys?Lys?Leu?Lys?Asn?Tyr?Asp?Lys?Val
325 330 335
Lys?Thr?Glu?Leu?Leu?Asn?Glu?Asp?Leu?Ala?Lys?Lys?Tyr?Lys?Asp?Glu
340 345 350
Val?Val?Asp?Val?Tyr?Gly?Ser?Asn?Tyr?Tyr?Val?Asn?Cys?Tyr?Phe?Ser
355 360 365
Ser?Lys?Asp?Asn?Val?Gly?Lys?Val?Thr?Gly?Gly?Lys?Thr?Cys?Met?Tyr
370 375 380
Gly?Gly?Ile?Thr?Lys?His?Glu?Gly?Asn?His?Phe?Asp?Asn?Gly?Asn?Leu
385 390 395 400
Gln?Asn?Val?Leu?Ile?Arg?Val?Tyr?Glu?Asn?Lys?Arg?Asn?Thr?Ile?Ser
405 410 415
Phe?Glu?Val?Gln?Thr?Asp?Lys?Lys?Ser?Val?Thr?Ala?Gln?Glu?Leu?Asp
420 425 430
Ile?Lys?Ala?Arg?Asn?Phe?Leu?Ile?Asn?Lys?Lys?Asn?Leu?Tyr?Glu?Phe
435 440 445
Asn?Ser?Ser?Pro?Tyr?Glu?Thr?Gly?Tyr?Ile?Lys?Phe?Ile?Glu?Asn?Asn
450 455 460
Gly?Asn?Thr?Phe?Trp?Tyr?Asp?Met?Met?Pro?Ala?Pro?Gly?Asp?Lys?Phe
465 470 475 480
Asp?Gln?Ser?Lys?Tyr?Leu?Met?Met?Tyr?Asn?Asp?Asn?Lys?Thr?Val?Asp
485 490 495
Ser?Lys?Ser?Val?Lys?Ile?Glu?Val?His?Leu?Thr?Thr?Lys?Asn?Gly
500 505 510
<210>3
<211>774
<212>DNA
＜213〉the single-chain antibody gene ml3.9 of the anti-HER-2 of humanized
<220>
<221>CDS
<222>(1)..(774)
<223>
<400>3
atg?gcc?cag?gtg?cag?ctg?gtg?cag?tct?ggg?gca?gag?gtg?aaa?aag?ccc 48
Met?Ala?Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro
1 5 10 15
ggg?gag?tct?ctg?aag?atc?tcc?tgt?aag?ggt?tct?gga?tac?agc?ttt?acc 96
Gly?Glu?Ser?Leu?Lys?Ile?Ser?Cys?Lys?Gly?Ser?Gly?Tyr?Ser?Phe?Thr
20 25 30
agc?tac?tgg?atc?gcc?tgg?gtg?cgc?cag?atg?ccc?ggg?aaa?ggc?ctg?gag 144
Ser?Tyr?Trp?Ile?Ala?Trp?Val?Arg?Gln?Met?Pro?Gly?Lys?Gly?Leu?Glu
35 40 45
tac?atg?ggg?ctc?atc?tat?cct?ggt?gac?tct?gac?acc?aaa?tac?agc?ccg 192
Tyr?Met?Gly?Leu?Ile?Tyr?Pro?Gly?Asp?Ser?Asp?Thr?Lys?Tyr?Ser?Pro
50 55 60
tcc?ttc?caa?ggc?cag?gtc?acc?atc?tca?gtc?gac?aag?tcc?gtc?agc?act 240
Ser?Phe?Gln?Gly?Gln?Val?Thr?Ile?Ser?Val?Asp?Lys?Ser?Val?Ser?Thr
65 70 75 80
gcc?tac?ttg?caa?tgg?agc?agt?ctg?aag?ccc?tcg?gac?agc?gcc?gtg?tat 288
Ala?Tyr?Leu?Gln?Trp?Ser?Ser?Leu?Lys?Pro?Ser?Asp?Ser?Ala?Val?Tyr
85 90 95
ttt?tgt?gcg?aga?cat?gac?gtg?gga?tat?tgc?agt?agt?tcc?aac?tgc?gca 336
Phe?Cys?Ala?Arg?His?Asp?Val?Gly?Tyr?Cys?Ser?Ser?Ser?Asn?Cys?Ala
100 105 110
aag?tgg?cct?gaa?tac?ttc?cag?cat?tgg?ggc?cag?ggc?acc?ctg?gtc?acc 384
Lys?Trp?Pro?Glu?Tyr?Phe?Gln?His?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
115 120 125
gtc?tcc?tca?ggt?gga?ggc?ggt?tca?ggc?gga?ggt?ggc?tct?ggc?ggt?ggc 432
Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly
130 135 140
gga?tcg?cag?tct?gtg?ttg?acg?cag?ccg?ccc?tca?gtg?tct?gcg?gcc?cca 480
Gly?Ser?Gln?Ser?Val?Leu?Thr?Gln?Pro?Pro?Ser?Val?Ser?Ala?Ala?Pro
145 150 155 160
gga?cag?aag?gtc?acc?atc?tcc?tgc?tct?gga?agc?agc?tcc?aac?att?ggg 528
Gly?Gln?Lys?Val?Thr?Ile?Ser?Cys?Ser?Gly?Ser?Ser?Ser?Asn?Ile?Gly
165 170 175
aat?aat?tat?gta?tcc?tgg?tac?cag?cag?ctc?cca?gga?aca?gcc?ccc?aaa 576
Asn?Asn?Tyr?Val?Ser?Trp?Tyr?Gln?Gln?Leu?Pro?Gly?Thr?Ala?Pro?Lys
180 185 190
ctc?ctc?atc?tat?gat?cac?acc?aat?cgg?ccc?gca?ggg?gtc?cct?gac?cga 624
Leu?Leu?Ile?Tyr?Asp?His?Thr?Asn?Arg?Pro?Ala?Gly?Val?Pro?Asp?Arg
195 200 205
ttc?tct?ggc?tcc?aag?tct?ggc?acc?tca?gcc?tcc?ctg?gcc?atc?agt?ggg 672
Phe?Ser?Gly?Ser?Lys?Ser?Gly?Thr?Ser?Ala?Ser?Leu?Ala?Ile?Ser?Gly
210 215 220
ttc?cgg?tcc?gag?gat?gag?gct?gat?tat?tac?tgt?gcc?tcc?tgg?gac?tac 720
Phe?Arg?Ser?Glu?Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Ala?Ser?Trp?Asp?Tyr
225 230 235 240
acc?ctc?tcg?ggc?tgg?gtg?ttc?ggc?gga?gga?acc?aag?ctg?acc?gtc?cta 768
Thr?Leu?Ser?Gly?Trp?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu
245 250 255
ggt?gcg 774
Gly?Ala
<210>4
<211>258
<212>PRT
＜213〉the single-chain antibody gene ml3.9 of the anti-HER-2 of humanized
<400>4
Met?Ala?Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro
1 5 10 15
Gly?Glu?Ser?Leu?Lys?Ile?Ser?Cys?Lys?Gly?Ser?Gly?Tyr?Ser?Phe?Thr
20 25 30
Ser?Tyr?Trp?Ile?Ala?Trp?Val?Arg?Gln?Met?Pro?Gly?Lys?Gly?Leu?Glu
35 40 45
Tyr?Met?Gly?Leu?Ile?Tyr?Pro?Gly?Asp?Ser?Asp?Thr?Lys?Tyr?Ser?Pro
50 55 60
Ser?Phe?Gln?Gly?Gln?Val?Thr?Ile?Ser?Val?Asp?Lys?Ser?Val?Ser?Thr
65 70 75 80
Ala?Tyr?Leu?Gln?Trp?Ser?Ser?Leu?Lys?Pro?Ser?Asp?Ser?Ala?Val?Tyr
85 90 95
Phe?Cys?Ala?Arg?His?Asp?Val?Gly?Tyr?Cys?Ser?Ser?Ser?Asn?Cys?Ala
100 105 110
Lys?Trp?Pro?Glu?Tyr?Phe?Gln?His?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
115 120 125
Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly
130 135 140
Gly?Ser?Gln?Ser?Val?Leu?Thr?Gln?Pro?Pro?Ser?Val?Ser?Ala?Ala?Pro
145 150 155 160
Gly?Gln?Lys?Val?Thr?Ile?Ser?Cys?Ser?Gly?Ser?Ser?Ser?Asn?Ile?Gly
165 170 175
Ash?Asn?Tyr?Val?Ser?Trp?Tyr?Gln?Gln?Leu?Pro?Gly?Thr?Ala?Pro?Lys
180 185 190
Leu?Leu?Ile?Tyr?Asp?His?Thr?Asn?Arg?Pro?Ala?Gly?Val?Pro?Asp?Arg
195 200 205
Phe?Ser?Gly?Ser?Lys?Ser?Gly?Thr?Ser?Ala?Ser?Leu?Ala?Ile?Ser?Gly
210 215 220
Phe?Arg?Ser?Glu?Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Ala?Ser?Trp?Asp?Tyr
225 230 235 240
Thr?Leu?Ser?Gly?Trp?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu
245 250 255
Gly?Ala
<210>5
<211>717
<212>DNA
<213>Staphylococcus?aureus
<220>
<22l>CDS
<222>(1)..(717)
<223>
<400>5
gag?agt?caa?cca?gac?cct?acg?cca?gat?gag?ttg?cac?aaa?tca?agt?gag 48
Glu?Ser?Gln?Pro?Asp?Pro?Thr?Pro?Asp?Glu?Leu?His?Lys?Ser?Ser?Glu
1 5 10 15
ttt?act?ggt?acg?atg?ggt?aat?atg?aaa?tat?tta?tat?gat?gat?cat?tat 96
Phe?Thr?Gly?Thr?Met?Gly?Asn?Met?Lys?Tyr?Leu?Tyr?Asp?Asp?His?Tyr
20 25 30
gta?tca?gca?act?aaa?gtt?atg?tct?gta?gat?aaa?ttt?ttg?gca?cat?gat 144
Val?Ser?Ala?Thr?Lys?Val?Met?Ser?Val?Asp?Lys?Phe?Leu?Ala?His?Asp
35 40 45
tta?att?tat?aac?att?agt?gat?aaa?aaa?cta?aaa?aat?tat?gac?aaa?gtg 192
Leu?Ile?Tyr?Asn?Ile?Ser?Asp?Lys?Lys?Leu?Lys?Asn?Tyr?Asp?Lys?Val
50 55 60
aaa?aca?gag?tta?tta?aat?gaa?gat?tta?gca?aag?aag?tac?aaa?gat?gaa 240
Lys?Thr?Glu?Leu?Leu?Asn?Glu?Asp?Leu?Ala?Lys?Lys?Tyr?Lys?Asp?Glu
65 70 75 80
gta?gtt?gat?gtg?tat?gga?tca?aat?tac?tat?gta?aac?tgc?tat?ttt?tca 288
Val?Val?Asp?Val?Tyr?Gly?Ser?Asn?Tyr?Tyr?Val?Asn?Cys?Tyr?Phe?Ser
85 90 95
tcc?aaa?gat?aat?gta?ggt?aaa?gtt?aca?ggt?ggt?aaa?act?tgt?atg?tat 336
Ser?Lys?Asp?Asn?Val?Gly?Lys?Val?Thr?Gly?Gly?Lys?Thr?Cys?Met?Tyr
100 105 110
gga?gga?ata?aca?aaa?cat?gaa?gga?aac?cac?ttt?gat?aat?ggg?aac?tta 384
Gly?Gly?Ile?Thr?Lys?His?Glu?Gly?Asn?His?Phe?Asp?Asn?Gly?Asn?Leu
115 120 125
caa?aat?gta?ctt?ata?aga?gtt?tat?gaa?aat?aaa?aga?aac?aca?att?tct 432
Gln?Asn?Val?Leu?Ile?Arg?Val?Tyr?Glu?Asn?Lys?Arg?Asn?Thr?Ile?Ser
130 135 140
ttt?gaa?gtg?caa?act?gat?aag?aaa?agt?gta?aca?gct?caa?gaa?cta?gac 480
Phe?Glu?Val?Gln?Thr?Asp?Lys?Lys?Ser?Val?Thr?Ala?Gln?Glu?Leu?Asp
145 150 155 160
ata?aaa?gct?agg?aat?ttt?tta?att?aat?aaa?aaa?aat?ttg?tat?gag?ttt 528
Ile?Lys?Ala?Arg?Asn?Phe?Leu?Ile?Asn?Lys?Lys?Asn?Leu?Tyr?Glu?Phe
165 170 175
aac?agt?tca?cca?tat?gaa?aca?gga?tat?ata?aaa?ttt?att?gaa?aat?aac 576
Asn?Ser?Ser?Pro?Tyr?Glu?Thr?Gly?Tyr?Ile?Lys?Phe?Ile?Glu?Asn?Asn
180 185 190
ggc?aat?act?ttt?tgg?tat?gat?atg?atg?cct?gca?cca?ggc?gat?aag?ttt 624
Gly?Asn?Thr?Phe?Trp?Tyr?Asp?Met?Met?Pro?Ala?Pro?Gly?Asp?Lys?Phe
195 200 205
gac?caa?tct?aaa?tat?tta?atg?atg?tac?aac?gac?aat?aaa?acg?gtt?gat 672
Asp?Gln?Ser?Lys?Tyr?Leu?Met?Met?Tyr?Asn?Asp?Asn?Lys?Thr?Val?Asp
210 215 220
tct?aaa?agt?gtg?aag?ata?gaa?gtc?cac?ctt?aca?aca?aag?aat?gga 717
Ser?Lys?Ser?Val?Lys?Ile?Glu?Val?His?Leu?Thr?Thr?Lys?Asn?Gly
225 230 235
<210>6
<211>239
<212>PRT
<213>Staphylococcus?aureus
<400>6
Glu?Ser?Gln?Pro?Asp?Pro?Thr?Pro?Asp?Glu?Leu?His?Lys?Ser?Ser?Glu
1 5 10 15
Phe?Thr?Gly?Thr?Met?Gly?Asn?Met?Lys?Tyr?Leu?Tyr?Asp?Asp?His?Tyr
20 25 30
Val?Ser?Ala?Thr?Lys?Val?Met?Ser?Val?Asp?Lys?Phe?Leu?Ala?His?Asp
35 40 45
Leu?Ile?Tyr?Asn?Ile?Ser?Asp?Lys?Lys?Leu?Lys?Asn?Tyr?Asp?Lys?Val
50 55 60
Lys?Thr?Glu?Leu?Leu?Asn?Glu?Asp?Leu?Ala?Lys?Lys?Tyr?Lys?Asp?Glu
65 70 75 80
Val?Val?Asp?Val?Tyr?Gly?Ser?Asn?Tyr?Tyr?Val?Asn?Cys?Tyr?Phe?Ser
85 90 95
Ser?Lys?Asp?Asn?Val?Gly?Lys?Val?Thr?Gly?Gly?Lys?Thr?Cys?Met?Tyr
100 105 110
Gly?Gly?Ile?Thr?Lys?His?Glu?Gly?Asn?His?Phe?Asp?Asn?Gly?Asn?Leu
115 120 125
Gln?Asn?Val?Leu?Ile?Arg?Val?Tyr?Glu?Asn?Lys?Arg?Asn?Thr?Ile?Ser
130 135 140
Phe?Glu?Val?Gln?Thr?Asp?Lys?Lys?Ser?Val?Thr?Ala?Gln?Glu?Leu?Asp
145 150 155 160
Ile?Lys?Ala?Arg?Asn?Phe?Leu?Ile?Asn?Lys?Lys?Asn?Leu?Tyr?Glu?Phe
165 l70 175
Asn?Ser?Ser?Pro?Tyr?Glu?Thr?Gly?Tyr?Ile?Lys?Phe?Ile?Glu?Asn?Asn
180 185 190
Gly?Asn?Thr?Phe?Trp?Tyr?Asp?Met?Met?Pro?Ala?Pro?Gly?Asp?Lys?Phe
195 200 205
Asp?Gln?Ser?Lys?Tyr?Leu?Met?Met?Tyr?Asn?Asp?Asn?Lys?Thr?Val?Asp
210 215 220
Ser?Lys?Ser?Val?Lys?Ile?Glu?Val?His?Leu?Thr?Thr?Lys?Asn?Gly
225 230 235

Claims (3)

1. fusion immunotoxin ML-L-SEC 2 is characterized in that: have protein sequence among the sequence table SEQ ID No:2.
2. the fusion immunotoxin ML-L-SEC 2 gene is characterized in that: have base sequence among the sequence table SEQ IDNo:1.
3. the preparation of the described fusion immunotoxin ML-L-SEC 2 gene of claim 2, it is characterized in that: the single-chain antibody gene ml3.9 of the anti-HER-2 of humanized connects with the restriction endonuclease interconnection technique with staphylococcus aureus enterotoxin C 2 gene sec2 is connected small peptide by coding DNA Linker, and resulting fusion immunotoxin gene has base sequence among the sequence table SEQ ID No:1;
The base sequence of described connection small peptide DNA Linker is,
GGCCGCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGAATTC；
Described staphylococcus aureus enterotoxin C 2 has base sequence among the sequence table SEQ ID No:5.
CN 200510046819 2005-07-06 2005-07-06 Fusion immunotoxin ML-L-SEC2 and gene and its preparation Pending CN1891718A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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Publication Number Publication Date
CN1891718A true CN1891718A (en) 2007-01-10

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101249262B (en) * 2008-04-08 2011-02-16 中国人民解放军第二军医大学 Targeted nano granule of humanization monoclonal antibody trastuzumab modified packaged toxin protein, and preparation and applications thereof
CN101597612B (en) * 2008-06-06 2011-05-11 中国科学院沈阳应用生态研究所 SEC2 mutant gene with increased activity of super-antigen and preparation method thereof
CN102718846A (en) * 2011-03-29 2012-10-10 中国科学院沈阳应用生态研究所 Activity-increased SEC 2 mutant protein, coding gene, preparation and application

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101249262B (en) * 2008-04-08 2011-02-16 中国人民解放军第二军医大学 Targeted nano granule of humanization monoclonal antibody trastuzumab modified packaged toxin protein, and preparation and applications thereof
CN101597612B (en) * 2008-06-06 2011-05-11 中国科学院沈阳应用生态研究所 SEC2 mutant gene with increased activity of super-antigen and preparation method thereof
CN102718846A (en) * 2011-03-29 2012-10-10 中国科学院沈阳应用生态研究所 Activity-increased SEC 2 mutant protein, coding gene, preparation and application
CN102718846B (en) * 2011-03-29 2014-10-22 中国科学院沈阳应用生态研究所 Activity-increased SEC 2 mutant protein, coding gene, preparation and application

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