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Fusion immunotoxin B-L-Vpr genes, and cloning and heterologous expression methods

An immunotoxin and gene technology, applied in the field of genetic engineering, can solve the problems of limiting the development and application of Vpr protein and the lack of specificity of the target of Vpr protein, so as to avoid toxic effects, eliminate side effects, and have obvious anti-tumor effects

Inactive Publication Date: 2009-06-24
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the target of Vpr protein lacks specificity. Most fast-growing cells and most lymphocytes in the body can be recognized by Vpr protein and produce toxic effects. This lack of specificity severely limits the clinical application of Vpr protein as an anti-tumor agent. development and application

Method used

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  • Fusion immunotoxin B-L-Vpr genes, and cloning and heterologous expression methods
  • Fusion immunotoxin B-L-Vpr genes, and cloning and heterologous expression methods
  • Fusion immunotoxin B-L-Vpr genes, and cloning and heterologous expression methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The fusion immunotoxin B-L-Vpr gene has the base sequence of SEQ ID No: 1 in the sequence table.

[0035] ATGGCCCAGG TGCAGCTGGT GCAGTCTGGG GCAGAGGTGA AAAAGCCCGG 50

[0036]GGAGTCTCTG AAGATCTCCT GTAAGGGTTC TGGATACAGC TTTACCAGCT 100

[0037] ACTGGATCGC CTGGGTGCGC CAGATGCCCG GGAAAGGCCT GGAGTACATG 150

[0038] GGGCTCATCT ATCCTGGTGA CTCTGACACC AAATACAGCC CGTCCTTCCA 200

[0039] AGGCCAGGTC ACCATCTCAG TCGACAAGTC CGTCAGCACT GCCTACTTGC 250

[0040] AATGGAGCAG TCTGAAGCCC TCGGACAGCG CCGTGTATTT TTGTGCGAGA 300

[0041] CATGACGTGG GATATTGCAC CGACCGGACT TGCGCAAAGT GGCCTGAATA 350

[0042] CTTCCAGCAT TGGGGCCAGG GCACCCTGGT CACCGTCTCC TCAGGTGGAG 400

[0043] GCGGTTCAGG CGGAGGTGGC TCTGGCGGTG GCGGATCGCA GTCTGTGTTG 450

[0044] ACGCAGCCGC CCTCAGTGTC TGCGGCCCCA GGACAGAAGG TCACCATCTC 500

[0045] CTGCTCTGGA AGCAGCTCCA ACATTGGGAA TAATTATGTA TCCTGGTACC 550

[0046] AGCAGCTCCC AGGAACAGCC CCCAAACTCC TCATCTATGA TCACACCAAT 600

[0047] CGGCCCGCAG GGGTCCCTGA CCGATTCTCT GGCTCCAAGT CTGGCACCTC ...

Embodiment 2

[0088] 1) The humanized anti-HER-2 single-chain antibody gene b has the base sequence of SEQ ID No: 3 in the sequence table.

[0089] ATGGCCCAGG TGCAGCTGGT GCAGTCTGGG GCAGAGGTGA AAAAGCCCGG 50

[0090] GGAGTCTCTG AAGATCTCCT GTAAGGGTTC TGGATACAGC TTTACCAGCT 100

[0091] ACTGGATCGC CTGGGTGCGC CAGATGCCCG GGAAAGGCCT GGAGTACATG 150

[0092] GGGCTCATCT ATCCTGGTGA CTCTGACACC AAATACAGCC CGTCCTTCCA 200

[0093] AGGCCAGGTC ACCATCTCAG TCGACAAGTC CGTCAGCACT GCCTACTTGC 250

[0094] AATGGAGCAG TCTGAAGCCC TCGGACAGCG CCGTGTATTT TTGTGCGAGA 300

[0095] CATGACGTGG GATATTGCAC CGACCGGACT TGCGCAAAGT GGCCTGAATA 350

[0096] CTTCCAGCAT TGGGGCCAGG GCACCCTGGT CACCGTCTCC TCAGGTGGAG 400

[0097] GCGGTTCAGG CGGAGGTGGC TCTGGCGGTG GCGGATCGCA GTCTGTGTTG 450

[0098] ACGCAGCCGC CCTCAGTGTC TGCGGCCCCA GGACAGAAGG TCACCATCTC 500

[0099] CTGCTCTGGA AGCAGCTCCA ACATTGGGAA TAATTATGTA TCCTGGTACC 550

[0100] AGCAGCTCCC AGGAACAGCC CCCAAACTCC TCATCTATGA TCACACCAAT 600

[0101] CGGCCCGCAG GGGTCCCTGA CCGATTCTCT GG...

Embodiment 3

[0127] 1) HIV-1 Vpr protein coding gene vpr has the base sequence of SEQ ID No: 5 in the sequence table.

[0128] ATGGAACAAG CCCCAGAAGA CCAGGGGCCG CAGAGGGAAC CATACAATGA 50

[0129] ATGGACATTA GAGCTTTTAG AGGAACTTAA GCATGAAGCT GTTAGACACT 100

[0130] TTCCTAGGCC ATGGCTCCAT GGCTTAGGAC AATATATCTA TGAAACCTAT 150

[0131] GGGGATACTT GGGCAGGAGT TGAAGCTATA ATAAGAATTT TGCAACAACT 200

[0132] ACTGTTTGTT CATTTCAGAA TTGGGTGCCA ACATAGCAGA ATAGGCATTA 250

[0133] TGCAACAGAG AAGAGCAAGA AATGGAGCCA GTAGATCCTA A 291

[0134] (1) Information of SEQ ID No: 5

[0135] (a) Sequential features

[0136] * Length: 291 bp

[0137] *Type: nucleic acid

[0138] * Chain type: double chain

[0139] *Topology: Linear

[0140] (b) Molecular type: cDNA

[0141] (c) Assumption: No

[0142] (d) Antisense: No

[0143] (e) Original source: HIV-1 virus

[0144] 2) Preparation of HIV-1 Vpr gene vpr:

[0145] Upstream primer V-F: 5'-ATA GAA TTC ATG GAA CAA-3'

[0146] Downstream primer V-R: 5'-GCG ...

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Abstract

The invention relates to the field of genetic engineering, in particular to a fusion immunotoxin B-L-Vpr gene as well as cloning and heterologous expression. The gene has a base sequence in Sequence Table SEQ ID No:1, meanwhile, protein encoded by the fusion immunotoxin B-L-Vpr gene has an amino acid sequence in SEQ ID No:2. In the invention, a single-chain antibody for resisting HER-2 is regarded as a targeted molecule and Vpr protein is regarded as an effector molecule. The targeted molecule and the effector molecule form the fusion immunotoxin B-L-Vpr through the connection of small peptides and effectively express the fusion immunotoxin protein in colon bacillus. The fusion immunotoxin can have a specific inhibitive effect on a tumor cell over-expressed by a HER-2 receptor.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a fusion immunotoxin B-L-Vpr gene and a method for cloning and heterologous expression. 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-associated antigen, which is overexpressed in 25-30% of breast and ovarian cancer pati...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/10C07K19/00C12N15/70
Inventor 徐明恺张惠文张成刚王小刚
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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