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Targeted anti-tumour fused protein containing adenovirus E40rf4 protein

A fusion protein and adenovirus technology, which is applied in the field of targeted anti-tumor fusion proteins containing adenovirus E4orf4 protein, which can solve the problems of immaturity and safety risks.

Inactive Publication Date: 2008-11-26
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adenovirus is a promising gene therapy vector, but in view of the fact that adenovirus gene therapy provides many benefits, the relevant technology is not yet fully mature, and there are certain risks in the safety of treatment

Method used

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  • Targeted anti-tumour fused protein containing adenovirus E40rf4 protein
  • Targeted anti-tumour fused protein containing adenovirus E40rf4 protein
  • Targeted anti-tumour fused protein containing adenovirus E40rf4 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of α-factor leader peptide-EGF-E4orf4 gene fragment by overlapping PCR

[0037] In this example, the following PCR primers were used:

[0038] Primer 1 (31-mer):

[0039] 5'- GGAATTC ACC ATG AGA TTT CCT TCA ATT TTT-3'

[0040] EcoRI Kozak

[0041] Primer 2 (45-mer):

[0042] 5’-ACC ACC ACC GGA ACC ACC ACC ACC TTC CCA CCACTT CAA GTC TCT-3’

[0043] Primer 3 (45-mer):

[0044] 5’-GGT TCC GGT GGT GGT GGT TCC TCC ATG GTT CTTCCA GCT CTT CCC-3’

[0045] Primer 4 (31-mer):

[0046] 5'- GGAATTC TCA TTA CTG TAC GGA GTG CGC CGA-3’

[0047] EcoRI kill cipher

[0048] Primer 1 contains an EcoRI restriction site and Kozak sequence, and is complementary to the 5' end of the α-factor leader peptide; primer 2 is complementary to the 3' end of EGF, and partly coded for the amino acid arm consisting of glycine and serine. sequence; primer 3 is complementary to the 5' end of E4orf4, and a part of the sequence encoding the amino acid arm composed ...

Embodiment 2

[0050] Example 2: Enzyme digestion identification of recombinant plasmid pUC-EGF-E4orf4

[0051] Identification method: 2 μg of the recombinant plasmid pUC-EGF-E4orf4 was digested with EcoRI enzyme at 37°C for 2 hours, electrophoresed on a 1.0% agarose gel, stained with ethidium bromide, and a total of 261 fragments of the pUC18 vector and the leader peptide encoding α-factor-EGF-E4orf4 were seen. An about 800bp DNA clone fragment of amino acids, 2 terminators, Kozak sequence and EcoRI site.

[0052] For enzyme digestion identification of this plasmid, see figure 2 , in the figure: Lane 1: λDNA / HindIII, the fragments from large to small are: 23130bp, 9414bp, 6557bp, 4361bp, 2322bp, 2021bp, 564bp. Lane 2: recombinant plasmid pUC-EGF-E4orf4. Lane 3: pUC-EGF-E4orf4 / EcoRI 2.69 kb (vector), 800 bp (insert fragment). Lane 4: DNA Marker DL2000, the fragments from large to small are: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.

Embodiment 3

[0053] Example 3: Site-directed mutagenesis of recombinant plasmid pUC-EGF-E4orf4

[0054] Since the construction of a multi-copy expression vector requires the use of the homologous enzymes Bg1II and BamHI for the tandem connection of the expression units, the exogenous gene fragments cannot contain the cutting points of BglII and BamHI. The E4orf4 gene fragment contains a BglII recognition site, which needs to be removed by site-directed mutagenesis without changing the amino acid sequence.

[0055] Site-directed mutagenesis was performed using Stratagene kit (QuickChange TM Site-Directed Mutagenesis Kit), strategy see image 3 . The primer sequences for site-directed mutagenesis are as follows:

[0056] Primer 1:

[0057] 5’-CGG AGA CGC AGA TCG GTT TGT CAC GCC CGC-3’

[0058] Primer 2:

[0059] 5’-GCG GGC GTG ACA AAC CGA TCT GCG TCT CCG-3’

[0060] Using the plasmid pUC-EGF-E4orf4 obtained in Example 1 as a template, PCR amplification of site-directed mutagenesis wa...

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Abstract

The invention relates to recombination human epidermis growth factor - gland virus E4orf4 fusion protein, fusion gene which codes the fusion protein, the fusion gene contained express carrier, using methyl nutritional yeast to make secretary express method of this recombination fusion protein, and the protein contained medicine combination and its treating application.

Description

technical field [0001] The invention relates to recombinant human epidermal growth factor-adenovirus early transcription region 4 open reading frame protein product (E4orf4) fusion protein, a fusion gene encoding the fusion protein, an expression vector containing the fusion gene, and methyl nutrition A method for secretory expression of the recombinant fusion protein by type yeast, a pharmaceutical composition containing the protein and its therapeutic application. Background technique [0002] The human adenovirus gene early transcription region 4 (E4) transcription unit contains at least 7 open reading frames, and their respective products have no obvious similarity. The product encoded by the fourth open reading frame (E4open reading frame 4, E4orf4) is a 14kDa small molecule protein consisting of 114 amino acids, and its amino acid sequence has no homology with any known protein. E4orf4 protein has a highly conserved sequence, which is RXKRRXRRRR, and it also has a pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N15/81A61K38/18A61P35/00
Inventor 陈虹黄秉仁王欣马晓骊
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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