Exploration For Combining Ability Of HSP27 And ?-agonists By Molecular Docking Technology

Objective?adrenergic agonists which were illegally added in food effect Chinese health,especially combination uesed of multiple?adrenergic agonists and the emergence of new structural analogues increased people's fears of food safety problem and the difficulty of market regulation.Therefore,a rapid screening approach for multi-residues determination of?adrenergic agonists was demanded urgently with the development of the society.Artificially expressed?₂ adrenergic receptor and heat shock protein 27 combined with?adrenergic agonists by molecular docking technology.The differences of their combining ability were respectively compared.It was to explore the possibility that HSP27 was applied in multi-residues determination of?adrenergic receptor agonists.Methods1.Heat shock protein 27?HSP27?and?₂ adrenergic receptor??₂AR?were combined with?adrenergic receptor agonists respectively by molecular docking technology.According to the results of molecular docking,differences of their combining ability were compared.2.The?₂AR gene was expressed the functional activity of?₂AR protein in Escherichia Coli BL21.The expression condition was optimized and?₂AR protein was purified.3.The HSP27 gene was expressed the functional activity of HSP27 protein in Escherichia Coli BL21.The expression condition was optimized and HSP27 protein was purified.4.The ELISA was to verify the ability of heat shock protein 27 and?₂ adrenergic receptor were combined with?adrenergic receptor agonists respectively.5.HSP27 was used to detect?adrenergic receptor agonists.Established standard curve and calculated the limit of detection,it's to explore the possibility that HSP27applied in multi-residues determination of?adrenergic receptor agonists.Results1.Results of molecular dockingThe diagram of clenbuterol,ractopamine and fenoterol respectively combined with HSP27 and?₂ adrenergic receptor by molecular docking show that ligands combined with HSP27 in the small bag of transmembrane region.Interaction of HSP27 and?₂ adrenergic receptor with?adrenergic receptor agonists rely on hydrophobic bond and hydrogen bond.Nine of fifteen total scores of molecular docking of HSP27 were higher than?₂ adrenergic receptor.Paired-samples t test analysis the difference of their combining ability is statistically significant?P=0.03?.2.The results of?2 adrenergic receptor in expression and purificationThe Restructured?₂AR gene were expressed the functional activity of?₂AR protein in Escherichia Coli BL21 successfully.The size of?₂AR protein is about 47KD.The induced condition:bacterial grow in logarithmic phase?OD=0.6⁰.9?,and IPTG concentration is 1.0 mmol/L,and the condition of shaking culture is 37?,220r/min,16 h.After ultrasonic broken and centrifugation,the supernatant fluid purified the?2AR protein in the condition of 300 mmol/L imidazole eluent.3.The results of HSP27 in expression and purificationThe Restructured HSP27 gene were expressed the functional activity of HSP27protein in Escherichia Coli BL21 successfully.The size of HSP27 protein is about 27KD.The induced condition:bacterial grow in logarithmic phase?OD=0.6⁰.9?,and IPTG concentration is 0.8 mmol/L,and the condition of shaking culture is 37?,220r/min,16 h.After ultrasonic broken and centrifugation,the supernatant fluid purified the HSP27 protein in the condition of 250 mmol/L imidazole eluent.4.The ELISA results of active identificationThe results of ELISA show that HSP27 protein and?₂ adrenergic receptor are able to react with clenbuterol,ractopamine and fenoterol characteristically.The OD values of HSP27 were 0.685,0.662 and 0.525.The OD values of?₂ adrenergic receptor were 0.448,0.320 and 0.362.5.HSP27 applied to detect?adrenergic receptor agonistsHSP27 was used to detect three kinds of?adrenergic receptor agonists,results show that HSP27 presented better linear relation in the concentration range of 1?g/L¹000?g/L.Cross-reactivity rate of ractopamine and fenoterol with clenbuterol were 52.6%and 29.7%respectively,and the LOD of detection was 1.53?g/L.Fortified recovery rates of clenbuterol,ractopamine and fenoterol were 52.7%⁷7.3%,51.7%⁷1.3%and 52%⁷6.8%,and all coefficient of variation were within 15%,suggest that detecting three kinds of?adrenergic receptor agonists,HSP27 has a good repeatability.ConclusionsThis reseach found that HSP27 has a strong combining ability with?adrenergic receptor agonists and HSP27 could be used to detect?adrenergic receptor agonists.This provides new idea and scientific basis for multi-residues determination of?adrenergic receptor agonists...