Method for producing blood vessel diseases gene medicine-blood vessel endothelium growth gene-2 naked DNA by microorganism cloning vehicle

A carrier and gene technology, applied in the field of recombinant DNA and its construction

Inactive Publication Date: 2005-11-02
甘肃亚盛盐化工业集团有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Prior to this, they had undergone various treatments, but to no avail

Method used

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  • Method for producing blood vessel diseases gene medicine-blood vessel endothelium growth gene-2 naked DNA by microorganism cloning vehicle
  • Method for producing blood vessel diseases gene medicine-blood vessel endothelium growth gene-2 naked DNA by microorganism cloning vehicle
  • Method for producing blood vessel diseases gene medicine-blood vessel endothelium growth gene-2 naked DNA by microorganism cloning vehicle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Mouse-derived VEGF-2 gene and its protein identification in microbial expression system

[0034] A pair of specific primers were designed according to the known mVEGF-2 nucleotide sequence (GenBank: S38100, gi: 249860), and BamH I and EcoR I sites were respectively introduced at the 5' ends of the pair of primers.

[0035] Bam HI

[0036] VP-1: 5'-GCC TCC GGA TCC ATG AAC TTT CTG-3'

[0037] VP-2: 5'- GAA TTC ACC GCC TCG GCT TGT C-3'

[0038] Eco RI

[0039] The embryos were taken out from the mice that had been sacrificed and conceived for 2 weeks, and the total RNA extraction kit (GIBCO company product-TRIZOL) was used to extract the embryos.  Reagent, Cat.No.15596) to extract total RNA. Using total embryonic RNA as a template, Oligo(dT) 15 As a primer, the first strand of cDNA was synthesized under the action of AMV Reverase (AMV-RT). Then, the reverse transcription product was diluted 50 times as a template, and the ...

Embodiment 2

[0046] Example 2. Cloning of human VEGF promoter and construction of transient expression vector for green fluorescent protein (GFP) gene and its functional identification

[0047] The upstream sequence of the human VEGF gene (hVEGF) has been reported (GenBank: AF095785, gi: 4154290), which is 3401bp, and it is difficult to clone such a large fragment. According to the general characteristics of eukaryotic promoters, the main elements of the promoter are usually about 1kb upstream of the transcription initiation site. According to the research of Hideo et al. (Hideo K, et al. Blood, 2000, 95: 189-197), we further analyzed that the fragment -1041~-1 (sorted from the transcription start site) includes the basic elements of the promoter. element. However, in the study of Hideo et al. (Hideo K, et al. Blood, 2000, 95: 189-197), the 5' untranslated region (5'-UTR) (referring to the + 1~+1038 fragments), and now it is increasingly recognized that 5'-UTR plays a very important role...

Embodiment 3

[0063] Example 3. Construction of hVEGF promoter and VEGF-2 mammalian expression vector and ischemic model animal experiment

[0064] 1. Construction of hVEGF promoter and mVEGF-2 mammalian expression vector

[0065] pEGSH (product of STRATAGENE Company of the United States, agent of MERCK Company of Germany, Cat. No. #217461) was selected as the expression vector for inserting hVEGF promoter and mVEGF-2. The reason for choosing this vector is mainly due to three considerations: ① The plasmid has a ColE1 replication origin and an Amp selection marker. It is a relaxed plasmid and can exist in multiple copies in microorganisms, which is conducive to the efficient production of microorganisms hVEGF promoter and mVEGF-2 naked DNA; the presence of selectable markers can prevent the loss of recombinant plasmids in the host bacteria, which is conducive to maintaining the stability of engineering bacteria; ②Multiple cloning sites (MCS) are available to facilitate the insertion of fore...

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Abstract

A process for constructing the vector pEGVPV, which can copy a lot of DNAs in procaryotic cell and is able to express in procaryon and mammal cell, includes such steps as cloning murinal VEGF-2 (mVEGF-2) gene from mouse embryo, cloning human VEGF promoter, linking them and inserting the gene into mammal's expression carrier pEGSH. After said carrier is used to transform E. coli DH5 alpha, the output of DNA separated from the culture medium of cells can reach 1.5 mg / L. A process for preparing the vascular endothelial growth factor 2 as DNA medicine from microbe for treating vascular diseases is also disclosed.

Description

1. Technical field [0001] The invention relates to a recombinant DNA and its construction, and a method for producing the recombinant DNA with microorganisms and using it as DNA medicine. Specifically, the present invention relates to the construction of a eukaryotic expression vector comprising human VEGF promoter (hVEGF promoter) capable of expressing murine VEGF-2 cDNA (mVEGF-2) in mammalian cells, and using microorganism A method for producing the vascular endothelial growth factor-2 (VEGF-2) DNA medicine. 2. Background technology [0002] Vascular disease is a common and frequently-occurring disease that seriously threatens human health. Among them, cerebral thrombosis and coronary heart disease are the most common. They have the characteristics of "high incidence, high recurrence rate, high mortality rate, high disability rate, and many complications". The "first killer" that threatens human health accounts for 40.72% of the total death toll (Wu Zhongxiang. To choose ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P9/10C12N15/12C12P19/34C12Q1/68
Inventor 周长生张金文陈正华
Owner 甘肃亚盛盐化工业集团有限责任公司
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