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Objective: This study aimed to construct the prokaryotic expression and purification system of non-selective cation channel TRPV4 protein and to carry out bioinformatics analysis to provide technical support for the study of diseases related to the target protein and the screening of drug active ingredients. Methods: The human TRPV4 gene was cloned into pET-28a(+), pET-32a, pET-15b, pGEX-5X-1, pEX-4T and pGEX-6p-1, respectively, and the prokaryotic expression system of TRPV4 was constructed using two E.coli expression hosts, BL21 and Rossetta. The target protein was isolated and purified by glutathione affinity chromatography and nickel column affinity chromatography. The target protein was analyzed by bioinformatics technology to obtain its corresponding physical, chemical properties and structural parameters. Results: The prokaryotic expression system of GST-TRPV4 and GST-TRPV4-6his fusion proteins was constructed using pGEX-6p-1 vector and Rossetta. The GST-TRPV4 fusion protein was significantly expressed when the IPTG concentrationwas 0.6 mmol/L, and the GST-TRPV4-6his fusion protein was significantly expressed when the IPTG concentration was 0.4 mmol/L, and the expression temperature was 18 ℃. When GST-TRPV4-6his fusion protein was purified, the complete GST-TRPV4-6his fusion protein could be obtained by imidazole solution at 100 mmol/L or 200 mmol/L. Conclusion: In this paper, human TRPV4 ion channel protein was successfully constructed, expressed and purified in prokaryotic expression system for the first time, and the physicochemical properties of the protein were analyzed by bioinformatics, which laid a good foundation for subsequent high-purity mass expression and further study.

Objective To establish a method for determination of 6 kinds of carbamate pesticides and 3 kinds of chloronicotinyl pesticides in Chinese Kushui rose by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled with QuEChERS. Methods After extracted by acetonitrile, the Chinese Kushui rose was cleaned by QuEChERS. The target compounds were separated by C18 column (2.1 mm×100 mm, 1.7 μm) using 10 mmol/L ammonium acetate solution (0.1% formic acid) with acetonrtrile as mobile phase for gradient elution, and analyzed by MS/MS system with electrospray ionization (ESI+) under muti-reaction monitoring mode and quantified by external standard method. Results All the 9 kinds of pesticides showed good linear relationships in range of 0.01-0.50 μg/mL, and the correlation coefficients were above 0.990, the recoveries at different spiked levels for all target compounds in blank matrices were 76.3%-102%, and the relative standard deviation (RSD) were 1.3%-9.0% (n=6). The limits of detection and quantification of the method were 0.001 6-0.003 2 and 0.005 4-0.010 mg/kg. Conclusion The method was suitable for rapid screening and analysis of 9 pesticide residues in Chinese Kushui rose with the advantage of accuracy, rapidity, simplicity and high sensitivity.