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Construction of 21ShRNA fluorescent expression vector of long-term silencing esophageal cancer cell HIF-1Alpha gene

A technology of HIF-1 and expression vector, applied in the field of biomedicine, can solve problems such as decreased PDT effect

Inactive Publication Date: 2010-10-20
河南省医药科学研究院
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Problems solved by technology

Since the photodynamic effect depends on the presence of molecular oxygen, but in most solid tumors there is often a hypoxic state, so the lack of oxygen molecules may lead to a decline in the effect of PDT, and the hypoxic state also leads to tumor cells being resistant to many therapeutic methods. (eg, radiation and chemotherapy) against

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  • Construction of 21ShRNA fluorescent expression vector of long-term silencing esophageal cancer cell HIF-1Alpha gene
  • Construction of 21ShRNA fluorescent expression vector of long-term silencing esophageal cancer cell HIF-1Alpha gene
  • Construction of 21ShRNA fluorescent expression vector of long-term silencing esophageal cancer cell HIF-1Alpha gene

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Embodiment Construction

[0022] The construction of a 21ShRNA fluorescent expression vector for long-term silencing of esophageal cancer cell HIF-1α gene according to the present invention comprises the following steps:

[0023] (1) Design of HIF-1α interference sequence

[0024] The HIF-1α gene ID and mRNA full-length sequence number were obtained from NCBI (NM_001530.3), and the WHITEHOUSE software was used to predict and analyze the interference sequence consisting of 21 nucleotides, and the GC content was controlled at 30%-50%; at the same time, the s iDirect online design software, input the full length of the obtained mRNA sequence into the sequence box to be predicted, select the design policy as the Reynolds equation, control the GC content at 30%-50%, run the remote prediction program, and obtain the prediction result;

[0025] (2) BLAST analysis

[0026] Using NCBI's Nucleotide Blast to compare the predicted nucleotide sequences, design a 21-nucleotide HIF-1α interference sequence, the sens...

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Abstract

The invention discloses construction of a 21ShRNA fluorescent expression vector of a long-term silencing esophageal cancer cell HIF-1Alpha gene. The construction method comprises the following steps of: acquiring HIF-1Alpha gene ID NM_001530.3 from NCBI (National Center of Biotechnology Information), designing 21 oligonucleotide siRNA interference sequences by software forecasting, designing a shRNA-encoded DNA sequence according to plasmid insertion sites and joint structures, and connecting pEZsiRNA6.1 vectors to obtain pEZsiRNA6.1hif-1Alpha recombinant plasmid of the long-term silencing HIF-1Alpha gene. The invention designs the HIF-1Alpha interference sequence and constructs the shRNA long-term interference fluorescence expression vector by utilizing a shRNA principle, can obtain a stable cell line expressed by long-term silencing HIF-1Alpha protein after cell transfection, screening and identification and provide a new material for new drug development and gene therapy which take HIF-1Alpha as a target.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and in particular provides the construction of a 21ShRNA fluorescent expression vector for long-term silencing of the HIF-1α gene of esophageal cancer cells. Background technique [0002] Esophageal cancer is one of the common malignant tumors of the digestive tract. The key to improving the therapeutic effect of esophageal cancer is early detection, early diagnosis and early treatment. [0003] Hypoxia-inducible factor-1 is a DNA-binding protein. It was first discovered that it can activate the transcription of human EPO gene in response to hypoxic environment, and then it was found that HIF-1 can activate the transcription of human VEGF gene. HIF-1 activity is regulated by the HIF-1α subunit at the cellular level, which dimerizes with hypoxia-inducible factor-1β (HIF-1β) to form an active transcription factor. HIF-1 is difficult to detect in normoxic cells, and is quickly activated under h...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/65C12N15/113
Inventor 汲振余康巧珍郭欢孙蕾赵立群杨观瑞阎红霞张亚冰张聚真杨小静裘一兵
Owner 河南省医药科学研究院
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