CN111534531A - A design method for blue light-induced pyroptosis - Google Patents

Abstract

The invention belongs to the field of nano medicine, and relates to a blue light induced scorching method. A method for inducing apoptosis by blue light in vitro is designed by utilizing a cell apoptosis sensitive plasmid A (Cry 2-GSDMDM-CAAX) and an accessory protein plasmid B (Cib-Caspase-1). The design method of blue light induced cell scorching comprises the following steps: 1) constructing precursor genes Cib-GSDMDM-CAAX and Cry2-Caspase-1 for light control; 2) plasmid A, B was transfected into B16 cells and cultured at 37 ℃ for 24 h. 3) Upon irradiation of transfected B16 cells with blue light, the cells were charred.

Description

A design method for blue light-induced pyroptosis

technical field

The invention belongs to the field of nanomedicine, and relates to a method for inducing pyroptosis by blue light. The system can induce pyroptosis of cells under blue light irradiation, and cells are normal without blue light irradiation.

Background technique

Pyroptosis is a way of cell death in the body, which is naturally uncontrollable and is a programmed cell necrosis mediated by GSDMD. When cells undergo pyroptosis, GSDMD is divided into two segments, GSDMD-C and GSDMD-N. When GSDMD-N is localized on the cell membrane, cells undergo pyroptosis. Optogenetics, also known as optogenetic manipulation technology, is a new technology that combines light control technology with genetics for cell biology research. Usually, the photosensitive protein gene is transferred into the target cell, and the light beam has high spatial and temporal resolution. The characteristic of high rate is to achieve precise targeted aggregation of target proteins at the molecular level. Light-sensitive protein is a protein that polymerizes under specific light, and Cry2 and Cib are a pair of proteins that can bind under blue light. Using pyroptosis-sensitive plasmid A (Cry2-GSDMD-CAAX) and accessory protein particle B (Cib-Caspase-1), an in vitro blue light-induced pyroptosis method was designed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a design method for blue light-induced cell pyroptosis.

The technical scheme of the present invention is a design method for blue light-induced cell pyroptosis. It includes the following steps:

1) Construction of the precursor genes Cib-GSDMD-CAAX and Cry2-Caspase-1 for light control: mainly through the light control of the precursor proteins A (Cib-GSDMD-CAAX) and B (Cry2-Caspase-1) Achieve selective tumor cell pyroptosis. Total RNA was extracted from normal tissues or cells, and it was intended to be transcribed into cDNA. Using this as a template, primers for GSDMD and Caspase-1 were designed for PCR amplification, and then ligated to Cib and Caspase-containing cDNA, respectively. The pigment 2 (Cry2) gene and the eukaryotic expression vector carrying the GFP and mCherry fluorescent protein genes respectively were transformed into E. coli, and then positive clones were picked and sequenced, and finally the target plasmid was extracted. The two plasmids are called the photo-controlled pyroptosis precursor protein A (Cib-GSDMD-CAAX) and the accessory protein B (Cry2-Caspase-1), respectively.

2) B16 cells were transfected with plasmids A and B, and cultured at 37°C for 24h.

3) When the transfected B16 cells are irradiated with blue light, the cells undergo pyroptosis.

Beneficial effects: The prepared nano-light and system have the following major advantages: 1) cells undergo pyroptosis when irradiated with blue light, while cells are normal without blue light and have strong controllability; 2) blue light-induced cell pyroptosis is suitable for any cell , is more universal.

Detailed ways

The present invention is further described below with reference to the embodiments.

Example 1:

Design method for blue light-induced pyroptosis:

1) Construction of the precursor genes Cib-GSDMD-CAAX and Cry2-Caspase-1 for light control: mainly through the light control of the precursor proteins A (Cib-GSDMD-CAAX) and B (Cry2-Caspase-1) Achieve selective tumor cell pyroptosis. Total RNA was extracted from normal tissues or cells, and it was intended to be transcribed into cDNA. Using this as a template, primers for GSDMD and Caspase-1 were designed for PCR amplification, and then ligated to Cib and Caspase-containing cDNA, respectively. The pigment 2 (Cry2) gene and the eukaryotic expression vector carrying the GFP and mCherry fluorescent protein genes respectively were transformed into Escherichia coli, and then the positive clones were picked and sequenced, and finally the target plasmid was extracted. The two plasmids are called the photo-controlled pyroptosis precursor protein A (Cib-GSDMD-CAAX) and the accessory protein B (Cry2-Caspase-1), respectively.

2) B16 cells were transfected with plasmids A and B, and cultured at 37°C for 24h.

3) When the transfected B16 cells are irradiated with blue light, the cells undergo pyroptosis.

Example 2:

1) Construction of the precursor genes Cib-GSDMD-CAAX and Cry2-Caspase-1 for light control: mainly through the light control of the precursor proteins A (Cib-GSDMD-CAAX) and B (Cry2-Caspase-1) Achieve selective tumor cell pyroptosis. Total RNA was extracted from normal tissues or cells, and it was intended to be transcribed into cDNA. Using this as a template, primers for GSDMD and Caspase-1 were designed for PCR amplification, and then ligated to Cib and Caspase-containing cDNA, respectively. The pigment 2 (Cry2) gene and the eukaryotic expression vector carrying the GFP and mCherry fluorescent protein genes respectively were transformed into Escherichia coli, and then the positive clones were picked and sequenced, and finally the target plasmid was extracted. The two plasmids are called the photo-controlled pyroptosis precursor protein A (Cib-GSDMD-CAAX) and the accessory protein B (Cry2-Caspase-1), respectively.

2) B16 cells were transfected with plasmids A and B, and cultured at 37°C for 24h.

3) When the transfected B16 cells are irradiated with near-infrared light, the cells undergo pyroptosis.

Example 3:

1) Construction of the precursor genes Cib-GSDMD-CAAX and Cry2-Caspase-1 for light control: mainly through the light control of the precursor proteins A (Cib-GSDMD-CAAX) and B (Cry2-Caspase-1) Achieve selective tumor cell pyroptosis. Total RNA was extracted from normal tissues or cells, and it was intended to be transcribed into cDNA. Using this as a template, primers for GSDMD and Caspase-1 were designed for PCR amplification, and then ligated to Cib and Caspase-containing cDNA, respectively. The pigment 2 (Cry2) gene and the eukaryotic expression vector carrying the GFP and mCherry fluorescent protein genes respectively were transformed into E. coli, and then positive clones were picked and sequenced, and finally the target plasmid was extracted. The two plasmids are called the photo-controlled pyroptosis precursor protein A (Cib-GSDMD-CAAX) and the accessory protein B (Cry2-Caspase-1), respectively.

2) Hela cells were transfected with plasmids A and B, and cultured at 37°C for 24h.

3) When the transfected Hela cells are irradiated with blue light, the cells undergo pyroptosis.

Claims (2)

1. a design method of blue light-induced pyroptosis, is characterized in that, comprises the steps: 1) Construction of precursor genes Cib-GSDMD-CAAX and Cry2-Caspase-1 for light control 2) B16 cells were transfected with plasmids A and B, and cultured at 37°C for 24h; 3) When the transfected B16 cells are irradiated with blue light, the cells undergo pyroptosis. 2. the design method of a kind of blue light-induced pyroptosis according to claim 1, is characterized in that, described step 3) is concrete: (1) Selective tumor cell pyroptosis is finally achieved mainly through light-controlled photosensitive pyroptosis precursor proteins A (Cib-GSDMD-CAAX) and B (Cry2-Caspase-1); (2) Extracting total RNA from normal tissues or cells, and transcribe it into cDNA; (3) take this as template again, design primers for GSDMD and Caspase-1 to carry out PCR amplification to it; (4) are subsequently connected to eukaryotic expression vectors containing Cib and cryptochrome 2 (Cry2) genes and respectively carrying GFP and mCherry fluorescent protein genes, and transformed into Escherichia coli; (5) Then pick positive clones and perform gene sequencing; (6) Finally, extract the target plasmid. The two plasmids are called the photo-controlled pyroptosis precursor protein A (Cib-GSDMD-CAAX) and the accessory protein B (Cry2-Caspase-1), respectively.