Construction And Study Of TRPV4 Ion Channel Protein Expression Model

Objective: Fever is one of the common manifestations of many clinical diseases,and its impact on the body does more harm than good.As aspirin and other antipyretic drugs are widely used,adverse reactions occur in varying degrees in human body,so it is necessary to explore new antipyretic drug targets.Transient receptor potential vanilloid 4（TRPV4）is a member of transient receptor potential ion channel family and belongs to a non selective cation channel.TRPV4 is widely expressed in the body.It can be activated by heating and participate in the pathophysiological process of many diseases.In this study,TRPV4 channel protein was expressed in E.coli and 293 T cells,and aspirin was used as a representative drug to explore the expression of TRPV4 at different temperatures and the effect of aspirin on the expression of TRPV4 in 293 T cells,so as to provide a theoretical basis for clinical research on new antipyretic pathways.Methods: 1.p ET-28a（+）-TRPV4,p GEX-6P-1-TRPV4 and p GEX-6P-1-TRPV4-6His recombinant plasmids were constructed and transferred into E.coli DH10 B competent cells.The correct recombinant plasmid,which was screened by sequencing,was transferred into E.coli Rosetta（DE3）competent cells.The transformants were selected for expanded culture.Under the induction of different temperatures and IPTG concentrations,it was cultured overnight to screen the optimal expression conditions.After the culture under the optimum expression conditions,according to the GST-TRPV4 fusion protein with GST label,the target protein was isolated and purified by glutathione affinity chromatography;and according to GST-TRPV4-6His fusion protein with His tag,the target protein was isolated and purified by Ni column affinity chromatography.The interaction between purified protein and saikosaponin A or saikosaponin D was detected by microscale thermophoresis（MST）.2.To determine whether 293 T cells can be used as a cell model for subsequent experiments,the effects of temperature on the survival rate of 293 T cells and the expression of TRPV4 protein,as well as the effects of aspirin on TRPV4 m RNA transcription and protein expression were tested under simulated human fever conditions.Results: 1.The recombinant plasmid p ET-28a（+）-TRPV4 could not express the target protein after being introduced into E.coli.The recombinant plasmids p GEX-6P-1-TRPV4 and p GEX-6P-1-TRPV4-6His could express the target protein after being introduced into E.coli.When GST-TRPV4 fusion protein was purified,10 m L of reduced glutathione with a concentration of 20 m M was used to elute the target protein,which was obviously broken and could not be obtained;and GST-TRPV4-6His fusion protein can be obtained by eluting the protein with 100 m M or 200 m M imidazole,but a small amount of protein was still broken.2.At 37℃,the dissociation equilibrium constant（Kd）of the purified protein bound to saikosaponin A was（56.026 ± 225.33）m M,and the signal-to-noise was 10.2;the signal-to-noise of protein to saikosaponin D was 0.6,which was less than 5,there was no Kd value,and the fitting curve was close to a straight line.At 42℃,there was no Kd value and signal-to-noise between protein and saikosaponin A;The Kd of protein binding to saikosaponin D was（4.8313 ± 4.6048）m M,and the signal-to-noise was 5.5.3.With the increasing of culture temperature of 293 T cells,the expression of TRPV4 protein was significantly increased compared with 37.0℃.A small amount of aspirin promoted TRPV4 m RNA transcription and protein expression.10 m M aspirin increased TRPV4 expression from 37.0℃ to 39.0℃;at 40.0℃,5 m M aspirin increased the expression of TRPV4.The effect of culture conditions at 39.0℃ on the expression of TRPV4 was stronger than the other three temperatures conditions.Conclusion: 1.By constructing the prokaryotic expression system of TRPV4 protein,under the experimental conditions of this study,it was found that Rosetta expression system constructed with p ET-28a（+）vector could not express the target protein,and Rosetta expression system constructed with p GEX-6P-1-TRPV4 vector successfully expressed the target protein.2.GST-TRPV4 fusion protein was purified by glutathione affinity chromatography,and the target protein could not be obtained;GST-TRPV4-6His fusion protein was purified by Ni column affinity chromatography,and a complete target protein could be obtained,but the target protein was still degraded.3.Under the experimental conditions of this study,at 37℃,the purified protein interacts with saikosaponin A,and may not interact with saikosaponin D;at 42℃,there is interaction between purified protein and saikosaponin D,and there may be no interaction between purified protein and saikosaponin A.4.From 37.0℃ to 40.0℃,high temperature will lead to the increase of TRPV4 expression.Aspirin will promote TRPV4 m RNA transcription and protein expression.293 T cells can be used as cell models for subsequent drug experiments.