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

An ML-L-SEC2, immunotoxin technology, applied in the field of genetic engineering pharmacy, can solve the problems of side effects, lack of specificity in tumor treatment, and achieve the effect of eliminating side effects

Inactive Publication Date: 2007-01-10
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, MHC class II molecules are mainly expressed in normal human cells, while the expression in tumor tissues is very small, which makes the tumor treatment of SEC2 lack specificity and cause side effects

Method used

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  • Fusion immunotoxin ML-L-SEC2 and gene and its preparation
  • Fusion immunotoxin ML-L-SEC2 and gene and its preparation
  • Fusion immunotoxin ML-L-SEC2 and gene and its preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A humanized anti-HER-2 single-chain antibody gene ml3.9 has the base sequence of SEQ ID No: 3 in the sequence table.

[0025] ATGGCCCAGG TGCAGCTGGT GCAGTCTGGG GCAGAGGTGA AAAAGCCCGG 50

[0026] GGAGTCTCTG AAGATCTCCT GTAAGGGTTC TGGATACAGC TTTACCAGCT 100

[0027] ACTGGATCGC CTGGGTGCGC CAGATGCCCG GGAAAGGCCT GGAGTACATG 150

[0028] GGGCTCATCT ATCCTGGTGA CTCTGACACC AAATACAGCC CGTCCTTCCA 200

[0029] AGGCCAGGTC ACCATCTCAG TCGACAAGTC CGTCAGCACT GCCTACTTGC 250

[0030] AATGGAGCAG TCTGAAGCCC TCGGACAGCG CCGTGTATTT TTGTGCGAGA 300

[0031] CATGACGTGG GATATTGCAG TAGTTCCAAC TGCGCAAAGT GGCCTGAATA 350

[0032] CTTCCAGCAT TGGGGCCAGG GCACCCTGGT CACCGTCTCC TCAGGTGGAG 400

[0033] GCGGTTCAGG CGGAGGTGGC TCTGGCGGTG GCGGATCGCA GTCTGTGTTG 450

[0034] ACGCAGCCGC CCTCAGTGTC TGCGGCCCCA GGACAGAAGG TCACCATCTC 500

[0035] CTGCTCTGGA AGCAGCTCCA ACATTGGGAA TAATTATGTA TCCTGGTACC 550

[0036] AGCAGCTCCC AGGAACAGCC CCCAAACTCC TCATCTATGA TCACACCAAT 600

[0037] CGGCCCGCAG GGGTCCCTGA CCGATTCTCT G...

Embodiment 2

[0062] Staphylococcus aureus enterotoxin C2 (SEC2) gene sec2 is derived from Staphylococcus aureus and has the base sequence of SEQ ID No: 5 in the sequence table.

[0063] GAGAGTCAACCAGACCCTACGCCAGATGAGTTGCACAAATCAAGTGAGTTTACTGGTACGATGGGTAATATGAAATATTTATATGATGATCATTATGTATCAGCAACTAAAGTTATGTCTGTAGATAAATTTTTGGCACATGATTTAATTTATAACATTAGTGATAAAAAACTAAAAAATTATGACAAAGTGAAAACAGAGTTATTAAATGAAGATTTAGCAAAGAAGTACAAAGATGAAGTAGTTGATGTGTATGGATCAAATTACTATGTAAACTGCTATTTTTCATCCAAAGATAATGTAGGTAAAGTTACAGGTGGTAAAACTTGTATGTATGGAGGAATAACAAAACATGAAGGAAACCACTTTGATAATGGGAACTTACAAAATGTACTTATAAGAGTTTATGAAAATAAAAGAAACACAATTTCTTTTGAAGTGCAAACTGATAAGAAAAGTGTAACAGCTCAAGAACTAGACATAAAAGCTAGGAATTTTTTAATTAATAAAAAAAATTTGTATGAGTTTAACAGTTCACCATATGAAACAGGATATATAAAATTTATTGAAAATAACGGCAATACTTTTTGGTATGATATGATGCCTGCACCAGGCGATAAGTTTGACCAATCTAAATATTTAATGATGTACAACGACAATAAAACGGTTGATTCTAAAAGTGTGAAGATAGAAGTCCACCTTACAACAAAGAATGGA

[0064] (1) Information of SEQ ID No: 5 (see sequence listing)

[0065] (A) Sequential features

[...

Embodiment 3

[0086] The DNA Linker sequence encoding the connecting short peptide has the following base sequence (oligonucleotide chain):

[0087] GGCCGCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGAATTC

[0088] (1) Preparation of DNA Linker sequence gene

[0089] 1) Design and synthesis of DNA Linker sequence gene:

[0090] Design and synthesize two oligonucleotide strands:

[0091] Forward strand L-F: 5'- G GCC GC A AGC GGC TCA GGA TCT GGA TCA GGA TCTGGC G-3';

[0092] Reverse strand L-R: 5'- AAA TTC GCC AGA TCC TGA TCC AGA TCC TGA GCCGCT TGC-3'

[0093] Add NotI and EcoRI sticky ends to the upstream and downstream respectively, and the encoded glycine serine short peptide is (GS) 2 sequence.

[0094] 2) Annealing of oligonucleotide chains:

[0095] 10×Annealing Hybridization Buffer (SSC) Components: 1.5mol / L NaCl, 0.3M Trisodium Citrate·2H 2 O, adjust the pH to 7.0 with HCl;

[0096] Annealing reaction system: 10×SSC buffer 1μl, positive and negative oligonucleotide chains 5pmol ...

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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

technical field [0001] The present invention relates to genetic engineering pharmaceuticals, specifically a novel anti-human epidermal growth factor receptor HER-2-Staphylococcus aureus enterotoxin C2 fusion immunotoxin ML-L-SEC2 gene ml-l-sec2 and its preparation . Background technique [0002] Immunotoxin is a new therapeutic strategy proposed on the basis of the development of modern medical biology, and has been widely used in tumor treatment. Fusion immunotoxin is the product of the combination of targeting molecules and cytotoxic molecules. Antibodies or genetically engineered antibody variable region fragments are often used as targeting molecules. In recent years, single-chain antibody fragments (Single Chain Variable Fragment, scFv) have It is widely used in the research of drug targeting carriers because of its small size, strong tumor penetration, fast blood clearance and short half-life. [0003] Human Epidermal Growth Factor Receptor-2 (HER-2) is a tumor-assoc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62
Inventor 徐明恺张成刚
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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