Construction method of drug screening system for treating senile valvular heart disease by single traditional Chinese medicine

Abstract

The invention discloses a construction method of a drug screening system for treating senile valvular heart disease by using a single traditional Chinese medicine. The construction method specifically comprises the following steps of constructing pmirGLO-RAGE5'-UTR and pmirGLO-mut-RAGE vectors; annealing the target fragment of the gene to synthesize double-stranded DNA, performing thermal denaturation at 95 DEG C for 2 minutes, lowering the temperature by 1 DEG C every 90 seconds to 25 DEG C, standing for 30 minutes, storing at 4 DEG C, and performing cryopreservation at -20 DEG C for later use; carrying out double enzyme digestion on the pmir GLO vector by using Sac I and Xho I enzymes so as to unlink the pmir GLO vector to be linearized; carrying out DNA recovery on a double-enzyme digestion product through agarose gel electrophoresis, and connecting the linear vector with the RAGE5 '-UTR vector; connecting the mutant target fragments by the same method to construct mutant recombinant vector plasmids; transforming the two recombinant vector plasmids and the no-load plasmid into escherichia coli DH5 alpha, picking bacterial colonies, and extracting and identifying the plasmids; and culturing 293T cells, transferring the 293T cells into plasmids containing recombinant pmirGLO-RAGE5'-UTR or pmirGLO-mut-RAGE through LipofectamineTM2000, transfecting for 24 hours, detecting the transfection efficiency, and selecting stably transfected cells, thereby successfully constructing the screening system. The screening system is simple and effective, and target sites can be replaced for popularization and application.

Description

technical field [0001] The invention relates to a construction method of a screening system, in particular to a construction method of a single traditional Chinese medicine drug screening system for treating senile valvular disease. Background technique [0002] Senile valvular disease is a group of degenerative diseases involving multiple valves, especially valve stenosis or insufficiency caused by aortic valve calcification. As my country gradually enters an aging society, the incidence of senile aortic valve calcification (aortic valve calcification) is increasing year by year. The detection rate of valve calcification in people over 50 years old reaches 12.5%, of which aortic valve involvement accounts for 94.4%. At present, calcified aortic valve disease (CAVD) in the elderly can only be treated with surgery or interventional valve replacement. The current replacement valves are divided into two types: biological valves or mechanical valves. Open the chest to replace t...

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Problems solved by technology

[0005] However, as mentioned above, the current screening of anti-CAVD traditional Chinese medicine is mainly based on the inference of traditional Chinese medicine theory, lacking objective scientific experimental evidence

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