Method for building neuronal nicotinic acetylcholine subtype receptor cell model

Abstract

The invention relates to a method to build a recipient cell model of neuronal nicotinic type acetylcholine subtype of the technical field. The method comprises the following steps: (1) gene APP695 is cloned into plasmid pcDNA3.1 (-); (2) pcDNA3.1(-)-APP695 is stably transferred into SH-EP1-Alpha4 Beta2 cell through a liposome method and is screened by adding pressure and by screening antibiotics; (3) expression of APP695 mRNA cloned by sable transfection cell is identified; (4) expression of APP695 and ABetaprotein cloned by stable transfection cell is identified; (5) receptor function of upper nicotineAlpha4 Beta2 cloned by the stable transferction cell is identified; patch-clamp is used in the process of receptor function identification of upper nicotine cloned by stable transfection cell. The stable expression nicotineAlphaBeta2 recipient and A Beta cell line are obtained in the invention, and central neuronal nicotinic acetylcholine Alpha4 Beta2 subtype recipient and cell models of A Beta co-expression are built, and the invention provides a powerful tool for solving the problems of AD incidence and focusing problem about nicotinic recipients subtype, providing an effective channel for AD prevention and treatment medicine.

Description

technical field

[0001] The invention relates to a method in the field of biotechnology, in particular to a method for establishing a neuron nicotinic acetylcholine subtype receptor cell model. Background technique

[0002] Amyloid protein precursor (APP) APP gene exists mainly in three forms, namely APP695, APP751 and APP770, and APP695 is mainly present in brain tissue. Amyloid (Aβ) is formed by hydrolytic cleavage by β and γ secretases. The Aβ theory of Alzheimer's disease (AD) believes that under pathological conditions, the production of Aβ40 and 42 increases to form oligomers, and then forms plaques, which have multiple toxic effects on neurons, manifested as a series of symptoms of AD . Neuronal nicotinic acetylcholine receptors widely express multiple subtypes, and the α4β2 subtype (a pentamer assembled from 3 α subunits and 2 β subunits) is the most widely expressed in the central nervous system. The Aβ theory of AD believes that under pathological conditions, the...

Examples

Embodiment 1

[0020] The APP695 gene and pcDNA3.1(-) were digested with Xbal 1 and Hindlll enzymes, ligated with T4 DNA ligase after gel recovery, transformed into DH5α bacteria, and ampicillin resistance was screened for the recombinant pcDNA3.1(-)-APP695 , amplified and extracted plasmids, sequenced and identified. pcDNA3.1(-)-APP695 was transfected into SH-EP1-α4β2 cells with liposome method LipofectamineTM PLUS reagent, neomycin 500g / L pressurized selection 3 days after transfection, cell monoclonal formation after 14 days, limiting dilution Cells were monoclonalized.

Embodiment 2

[0022] Monoclonal cells were collected 34 days after stable transfection, and total cellular RNA was extracted by the Trizol method according to the instructions. Using total RAN as a template, APP695 primer 1 and primer 2 were used for PCR.

[0023] The specific PCR program is: denaturation at 94°C for 3 minutes to enter the cycle process, denaturation at 94°C for 1.5 minutes, annealing at 55°C for 1.5 minutes, extension at 72°C for 3 minutes, and renaturation at 72°C for 10 minutes after 31 cycles. PCR products were identified by gel electrophoresis; PCR was then performed with α4 and β2 gene primers, α4 primers were 5'-CCATCGCTCAGCTCATTGACG-3' and 5'-CTGGTCGGAGGGTGACTTGC-3', and β2 primers were 5'-TATTCCAATGCCGTGGTCTCC-3' and 5'- TGGTCATCGTCCTCGCTC-3', PCR product identified by gel electrophoresis.

Embodiment 3

[0025] For the positive clones selected by RT-PCR, lyse the cells, extract the cell protein, measure the protein concentration with a microplate reader, take 10 μg of the sample for SDS-polyacrylamide gel electrophoresis, use 12% separating gel, 5% stacking gel , Semi-dry electrotransfer on nitrocellulose membrane, transfer time 2h, mouse anti-human APP695 monoclonal antibody incubation for 3h, goat anti-mouse secondary antibody incubation for 1h, ECL fluorescence detection, developing and fixing, and taking pictures to detect cell clone APP695

[0026] Cells were lysed with a lysate containing protease inhibitors PMSF and AEBSF, and the protein concentration was determined by BCA method. 30.5 μg of the sample was loaded for Tris-tricine electrophoresis, using 16.5% separating gel, 10% interlayer gel, 4% stacking gel, PVDF membrane wet Transfer, membrane transfer time 1h, 6E10 monoclonal antibody incubated at room temperature for 3h, goat anti-mouse secondary antibody incubated...

technical field

[0001] The invention relates to a method in the field of biotechnology, in particular to a method for establishing a neuron nicotinic acetylcholine subtype receptor cell model. Background technique

[0002] Amyloid protein precursor (APP) APP gene exists mainly in three forms, namely APP695, APP751 and APP770, and APP695 is mainly present in brain tissue. Amyloid (Aβ) is formed by hydrolytic cleavage by β and γ secretases. The Aβ theory of Alzheimer's disease (AD) believes that under pathological conditions, the production of Aβ40 and 42 increases to form oligomers, and then forms plaques, which have multiple toxic effects on neurons, manifested as a series of symptoms of AD . Neuronal nicotinic acetylcholine receptors widely express multiple subtypes, and the α4β2 subtype (a pentamer assembled from 3 α subunits and 2 β subunits) is the most widely expressed in the central nervous system. The Aβ theory of AD believes that under pathological conditions, the...

Examples

Embodiment 1

[0020] The APP695 gene and pcDNA3.1(-) were digested with Xbal 1 and Hindlll enzymes, ligated with T4 DNA ligase after gel recovery, transformed into DH5α bacteria, and ampicillin resistance was screened for the recombinant pcDNA3.1(-)-APP695 , amplified and extracted plasmids, sequenced and identified. pcDNA3.1(-)-APP695 was transfected into SH-EP1-α4β2 cells with liposome method LipofectamineTM PLUS reagent, neomycin 500g / L pressurized selection 3 days after transfection, cell monoclonal formation after 14 days, limiting dilution Cells were monoclonalized.

Embodiment 2

[0022] Monoclonal cells were collected 34 days after stable transfection, and total cellular RNA was extracted by the Trizol method according to the instructions. Using total RAN as a template, APP695 primer 1 and primer 2 were used for PCR.

[0023] The specific PCR program is: denaturation at 94°C for 3 minutes to enter the cycle process, denaturation at 94°C for 1.5 minutes, annealing at 55°C for 1.5 minutes, extension at 72°C for 3 minutes, and renaturation at 72°C for 10 minutes after 31 cycles. PCR products were identified by gel electrophoresis; PCR was then performed with α4 and β2 gene primers, α4 primers were 5'-CCATCGCTCAGCTCATTGACG-3' and 5'-CTGGTCGGAGGGTGACTTGC-3', and β2 primers were 5'-TATTCCAATGCCGTGGTCTCC-3' and 5'- TGGTCATCGTCCTCGCTC-3', PCR product identified by gel electrophoresis.

Embodiment 3

[0025] For the positive clones selected by RT-PCR, lyse the cells, extract the cell protein, measure the protein concentration with a microplate reader, take 10 μg of the sample for SDS-polyacrylamide gel electrophoresis, use 12% separating gel, 5% stacking gel , Semi-dry electrotransfer on nitrocellulose membrane, transfer time 2h, mouse anti-human APP695 monoclonal antibody incubation for 3h, goat anti-mouse secondary antibody incubation for 1h, ECL fluorescence detection, developing and fixing, and taking pictures to detect cell clone APP695

[0026] Cells were lysed with a lysate containing protease inhibitors PMSF and AEBSF, and the protein concentration was determined by BCA method. 30.5 μg of the sample was loaded for Tris-tricine electrophoresis, using 16.5% separating gel, 10% interlayer gel, 4% stacking gel, PVDF membrane wet Transfer, membrane transfer time 1h, 6E10 monoclonal antibody incubated at room temperature for 3h, goat anti-mouse secondary antibody incubated...