Method for screening protease inhibitor

Abstract

The invention provides a method for screening a protease inhibitor. The method comprises the following steps of: (1) adding a substance to be tested in protease solution to be uniformly mixed and meanwhile setting the protease solution in which the substance to be tested is not added as a blank control; (2) adding substrate protein solution into the protease solution to react so as to obtain reaction solution; (3) adding reaction end solution in the reaction solution; (4) adding ninhydrin solution to react; and (5) adding double distilled water, judging a result according to an absorbance value OD570 nm, and ensuring that a sample to be tested is positive if the absorbance of the solution of the matter to be tested is less than that of the blank control. By using the method, the protease inhibitor can be conveniently and quickly screened, a substrate is not required to be manually synthesized, the cost is low, the flexibility is high, and a new approach is provided for finding a medicament for inhibiting tumor invasiveness and metastasis.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for screening protease inhibitors. Background technique [0002] Cancer is one of the major diseases that seriously affects the quality of life and survival of human beings. There is no feasible way for human beings to prevent cancer cells from infiltrating and metastasizing to other tissues and organs. Matrix metalloproteinases (MMP) are one of the main enzymes involved in cancer metastasis, which can degrade the tissues around cancer cells, allowing cancer cells to spread to a larger area or enter the blood circulation to metastasize to other organs. Inhibiting the activity of matrix metalloproteinases can effectively control the metastasis of cancer cells, so screening inhibitors that can inhibit the activity of matrix metalloproteinases from known or newly synthesized compounds is a hot spot in the current anticancer drug research. For this reason, it is necessary to estab...

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Problems solved by technology

[0004] Although the fluorescence method (or spectrophotometry) has been used in the screening of matrix metalloproteinase inhibitors, which has high sensitivity and large-throughput screening, there are still some shortcomings: 1) Due to oversensitivity, artificial false phases are common in experiments In the process, a reasonable control group should be designed; 2) If the inhibitor has fluorescence or can quench the fluorescence, it cannot be used; 3) It needs artificially synthesized substrates, which is expensive; 4) It needs special instruments; 5) Fluorescence is easier Quenched or enhanced by other impurities or reaction buffer components, causing artifacts

At the same time, ninhydrin is also used in the determination of protein content, but the protein is completely hydrolyzed into amino acids (digested and hydrolyzed for 24 hours), and then reacted with ninhydrin. The early reaction is time-consuming and laborious, which increases the cost of the reaction and is not conducive to large Flux of proteins reacted with ninhydrin

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