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Connection of horseradish peroxidase gene and vascellum esoderma growth factor gene and clone thereof

A technology of horseradish peroxidase and vascular endothelium, applied in the field of gene composition of fusion proteins, can solve problems such as embryonic vascular malformation and large angiogenesis barriers

Inactive Publication Date: 2008-06-11
邵金辉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Injecting soluble VEGFR1 into bird embryos by Drake et al. resulted in embryonic vascular malformations and impairment of macroangiogenesis

Method used

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  • Connection of horseradish peroxidase gene and vascellum esoderma growth factor gene and clone thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The amplification of embodiment 1 horseradish peroxidase gene fragment

[0020] Using the extracted horseradish genomic DNA as a template, the PCR method was used to amplify the remaining part of the HRPC3 gene except the signal peptide sequence and the stop codon sequence, a total of 864bp. Restriction sites EcoRI and Bgl II were added at both ends of the upstream and downstream primers, respectively, and the downstream primers had no stop codon sequence. The primer at the 5' end is SEQ ID NO.1 in the sequence listing, and the primer at the 3' end is SEQ ID NO.2 in the sequence listing.

Embodiment 2

[0021] Embodiment 2 Amplification of VEGF165 gene fragment

[0022] The VEGF165 gene fragment was amplified by PCR, a restriction site BamHI was added to the 5' end of the upstream primer, and a DNA sequence encoding six consecutive histidine residues and a restriction site NotI were added to the 5' end of the downstream primer. The 5' end primer is SEQ ID NO.3 in the sequence listing, and the 3' end primer is SEQ ID NO.4 in the sequence listing.

Embodiment 3

[0023] Embodiment 3 horseradish peroxidase gene is connected with connecting peptide gene

[0024] Two primers Link1 and Link2 were synthesized, and 16 bases at the 3' end of Link1 were complementary to the 3' end of the horseradish peroxidase gene. The 5' end of Link1 and the 3' end of Link2 have the same sequence of 15 bp. There is a Bgl II restriction site in Link1, and a BamHI restriction site at the 5' end of Link2. Using the horseradish peroxidase gene amplified in Example 1 as a template, using SEQ ID NO.1 as an upstream primer, and using Link1 as a downstream primer to carry out PCR, then using the PCR product as a template, using SEQ ID NO. 1 and Link2 were used as primers, PCR was performed with Pyrobest DNA polymerase, and A was added to the DNA end with Taq DNA polymerase and dATP. The primer Link1 is shown as SEQ ID NO.5 in the sequence listing, and the primer Link2 is shown as SEQ ID NO.6 in the sequence listing.

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Abstract

The invention relates to the gene preparation of fusion protein with the function of killing tomour cells. The recombination genes of target tropism fusion protein is constructed through the invention, which have a growth factor gene of the human blood vessel endothelium, a horse radish peroxidase gene, and a middle connecting peptide DNA sequence. The preparation method of the fusion protein gene comprises the following steps: firstly, the blood vessel endothelial cell growth factor gene and the horse radish peroxidase gene are separately obtained; secondly, the blood vessel endothelial cell growth factor gene is connected with the horse radish peroxidase gene through a micromolecule connecting peptide gene; thirdly, the blood vessel endothelial cell growth factor gene and the horse radish peroxidase gene are connected on the expression carrier pPIC9K of pichia pastoris; fourthly, the connected recombination carrier is cloned in escherichia coil. The recombination gene of the invention can be used for expressing the fusion protein with the function of killing the tumour cells.

Description

Technical field: [0001] The invention relates to a gene composition of a fusion protein capable of killing tumor cells, in particular to the connection and cloning of human vascular endothelial growth factor gene and horseradish peroxidase gene. Background technique: [0002] Many enzymes and prodrugs have been used to kill tumor cells with much success. Horseradish peroxidase is being used to kill tumor cells. Indole-3-acetic acid (IAA), as a ubiquitous plant growth hormone, can be catalyzed by horseradish peroxidase to form reactive oxygen species carrying molecules, including reactive oxygen species radicals (ROS), which can lead to lipid hypertrophy. Oxidation, causing changes in the structure of the cell membrane, resulting in intracellular damage and apoptosis, resulting in animal cytotoxicity. And because IAA cannot be oxidized by endogenous peroxidase of animal cells, it has no effect on the growth of normal cells, therefore, it can become a candidate drug for enzy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/12C12N15/53C12N1/21
Inventor 邵金辉
Owner 邵金辉
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