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

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 takeHIF-1Alpha as a target.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and in particular provides a method for constructing 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 u...

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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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