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CN-121975870-A - Grin2b stable transgenic cell line, construction method and application thereof, and pollutant high-throughput neurotoxicity or learning memory disorder screening method

CN121975870ACN 121975870 ACN121975870 ACN 121975870ACN-121975870-A

Abstract

The invention discloses a Grin2b stable transgenic cell strain, a construction method and application thereof, and a method for screening pollutant high-throughput neurotoxicity or learning and memory disorder, belonging to the technical field of biology, wherein the construction method comprises the following steps of (1) cell culture; the method comprises the steps of (1) constructing a plasmid vector, wherein the plasmid vector comprises a Grin2b coding sequence, (3) virus coating and virus collection, (4) testing slow virus titer, (5) screening of stable transgenic plants, and (6) functional verification of the stable transgenic plants. The Grin2b stable transgenic cell strain based on the Grin2b related ion channel constructed by the invention provides a brand-new technical platform for high-throughput neurotoxicity identification, study memory screening and mechanism research of organic pollutants and degradation products thereof.

Inventors

  • Chang Junzhuang
  • CHENG SHUJUN
  • Bo Ruihong
  • DENG XINYI
  • Liu Aolu

Assignees

  • 上海华代生物科技有限公司
  • 广州市华代生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260205

Claims (10)

  1. 1. The construction method of the Grin2b stable transgenic cell strain is characterized by comprising the following steps: (1) Culturing cells; (2) Constructing a plasmid vector, wherein the plasmid vector comprises a Grin2b coding sequence; (3) Virus coating and virus collection; (4) Testing lentivirus titer; (5) Screening the stable transgenic plants; (6) Performing function verification of stable transformation plants; The Grin2b coding sequence in the step (2) is a human sequence, and NCBI is numbered as NM_001363750.1.
  2. 2. The method of claim 1, wherein the plasmid in step (2) is a lentivirus expression plasmid.
  3. 3. The method of claim 1, wherein the plasmid vector in step (2) is Flag-tagged.
  4. 4. The method of claim 1, wherein the method of testing lentivirus titer in step (4) is qPCR.
  5. 5. The method of claim 1, wherein the tranquilizer selection in step (5) is a drug selection, and the drug is puromycin.
  6. 6. The method according to claim 1, wherein the functional test of the tranquilizer in the step (6) comprises extracting membrane proteins and detecting the expression level of the membrane proteins.
  7. 7. A Grin2b stably transformed cell line obtained by the construction method of any one of claims 1-6.
  8. 8. A method for screening high-throughput neurotoxicity or learning and memory disorders of pollutants is characterized in that the Grin2b stable transgenic cell line in claim 7 is subjected to cell culture, a calcium ion fluorescent probe is added, and fluorescence is detected after the pollutants are exposed, so that the high-throughput screening is realized.
  9. 9. Use of the Grin2b stably transformed cell line of claim 7 in contaminant high-throughput neurotoxicity or learning memory disorder screening.
  10. 10. The use according to claim 9, wherein the contaminants are organic contaminants and degradation products thereof.

Description

Grin2b stable transgenic cell line, construction method and application thereof, and pollutant high-throughput neurotoxicity or learning memory disorder screening method Technical Field The invention relates to the technical field of biology, in particular to a Grin2b stable transgenic cell strain, a construction method and application thereof, and a method for screening pollutant high-throughput neurotoxicity or learning and memory disorders. Background With the rapid development of industrial manufacturing, agricultural production and chemical material industry, a large amount of organic pollutants having a complex structure and strong stability are continuously released into the natural environment. The compound contains pesticide, herbicide, dye intermediate, plasticizer, fire retardant and partial new industrial additive, and features that it is difficult to degrade completely under natural condition, easy to stay in ecological system for long period and accumulated stage by stage via food chain. More critical, the contaminants themselves and their partial degradation products often carry potential nervous system toxic, cytotoxic or metabolic interference effects, which not only cause ecological imbalances (e.g., reduced species numbers, reduced biodiversity), but also may pose a direct or indirect threat to human health through environmental exposure pathways, e.g., interfering with nervous system development, inducing chronic diseases, etc. Therefore, establishing an evaluation system capable of accurately reflecting the actual biological effects of pollutants and degradation products thereof has become a core requirement for improving the environmental treatment efficiency and the risk assessment accuracy. Currently, the mainstream environmental toxicity detection method in industry mainly depends on two technologies, namely a physicochemical analysis technology (such as high performance liquid chromatography, gas chromatography-mass spectrometry and the like), the technology can realize quantitative detection of pollutants, determine the concentration level of the pollutants in the environment, but cannot directly reflect the toxic effect of the pollutants on a biological organism, and a biological evaluation system based on a general cell model (such as HEK293 cells, vero cells and the like), wherein the model can provide basic toxicity information (such as cell survival rate and proliferation inhibition rate) of the pollutants, but has obvious limitations in analyzing disturbance of cell signal paths, identifying toxic target effects (especially neurotoxicity) and distinguishing differential effects caused by a prototype of the pollutants and degradation products thereof. From the molecular mechanism point of view, the NMDA receptor NR2B subunit encoded by the Grin2B gene (Glutamate Ionotropic Receptor NMDA Type Subunit B, glutamate ionotropic receptor NMDA subtype 2B subunit gene) plays a core role in the central nervous system, and the regulated ion channel is directly involved in synaptic plasticity regulation, neural development process and cell excitability control and has high sensitivity to the stimulation of various exogenous chemical factors. It has been proved by studies that Grin2b related signal pathway shows specific response to toxic effects of various environmental chemicals (such as plasticizer phthalate esters and organotin compounds), and especially in mechanism studies of glutamate neurotoxicity mediation, calcium ion channel interference, oxidative stress activation and the like, the change of the pathway can be used as early warning index of neurotoxicity. Therefore, a cell model carrying the functional characteristics of Grin2b is constructed, and the method is hopeful to accurately capture the fine influence of pollutants on key molecular nodes of a nervous system, and fundamentally improves the sensitivity, selectivity and mechanism directivity of neurotoxicity detection. However, existing research systems still generally face the following challenges: 1. the existing monitoring means have obvious defects in the aspect of mechanism-oriented neurotoxicity judgment by focusing on the change of the physical and chemical layers; 2. The different pollutants and degradation paths thereof are complex and various, and a sensitive cell tool which can be widely applied to screening of various compounds is needed. Disclosure of Invention In order to solve the technical problems, the invention provides a Grin2b stable transgenic cell strain, a construction method and application thereof, and a pollutant high-flux neurotoxicity or learning memory disorder screening method, and the Grin2b stable transgenic cell strain based on the Grin2b related ion channel constructed by the invention provides a brand-new technical platform for high-flux neurotoxicity identification, learning memory screening and mechanism research of organic pollutants and degradation products thereof. In order to ach