Preparation And Directional Evolution Of The Receptor Of Tetracyclines

Veterinary drugs play an irreplaceable role in the rapid development of animal husbandry.Tetracycline drugs,as a class of broad-spectrum antibiotics,are widely used to treat multiple infectious diseases in animals.However,their residues in food of animal origins can cause a range of potential health hazards to consumers.The purpose of this thesis is to prepare the tetracycline receptor TetR protein and evolve it,and use it as a recognition element to establish a new immune method to detect the residue of such drugs in animal foods.First of all,the researchers extracted total DNA,from tetracycline-resistant bacteria and amplified the gene of TetR protein by PCR,then inserted it into expression vector and transferred it into BL21(DE3)competent cells for expression.The results showed that the molecular weight of the TetR protein was 44 KDa and could recognize five tetracyclines at the same time,and IC50 was 2.37-31 ng/mL.The results of molecular docking show that the binding site of the receptor is composed of 12 amino acids,which bind to the D ring of the drug molecule mainly through hydrophobic force and pi-pi bond.The competitive chemiluminescence immunoassay established with the TetR protein as the core reagent can be used to detect the five tetracyclines in milk.The detection limit is 1.24-77 ng/mL and the recovery rate is 77-96%.Then the researchers made virtual mutations of the key contact amino acids in the TetR protein.The results showed that the stability of the receptor was significantly improved when 105 proline was mutated into phenylalanine and tyrosine.So the researchers carried out site-directed mutagenesis of TetR gene and prepared two kinds of TetR mutants.The second molecular docking showed that the number of key contact amino acids of the mutant increased by 8 kinds,and the receptor-drug binding energy increased significantly.At the same time,the two mutants could recognize 7 kinds of tetracyclines,and the IC50 was 3.1-1 7.2 ng/mL,which was 3-10 times higher than that of the prototype TetR.The competitive fluorescence immunoassay established with mutant 2 as the recognition element can be used to detect seven tetracyclines in eggs.The detection limit is 2.0-8.7 ng/mL,and the recovery rate is 68.4-91.2%.Finally,the researchers synthesized a molecularly imprinted-metal-organic framework material complex,established a solid phase dispersion extraction method and combined with ultra-high performance liquid chromatography to detect seven tetracyclines in chicken.The results showed that the complex could specifically recognize tetracycline drugs with high adsorption capacity(3 ?g/g),high recovery rate(95%-98%),high enrichment factor(17-37),and could be reused for 7 times.The detection limit of this method for seven kinds of drugs is 3-16 ng/mL,and the recovery rate is 89%-96%.In this thesis,tetracycline receptor TetR proteins were successfully prepared and evolved,and their molecular recognition mechanism was preliminarily explored.The new immunoassay based on receptor/mutant can quickly detect the residues of these drugs in milk and eggs,which is of great significance to ensure the safety of animal food.