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

Objectiveβadrenergic agonists which were illegally added in food effect Chinese health,especially combination uesed of multipleβadrenergic 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 ofβadrenergic agonists was demanded urgently with the development of the society.Artificially expressedβ₂ adrenergic receptor and heat shock protein 27 combined withβadrenergic 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 ofβadrenergic receptor agonists.Methods1.Heat shock protein 27（HSP27）andβ₂ adrenergic receptor（β₂AR）were combined withβadrenergic receptor agonists respectively by molecular docking technology.According to the results of molecular docking,differences of their combining ability were compared.2.Theβ₂AR gene was expressed the functional activity ofβ₂AR protein in Escherichia Coli BL21.The expression condition was optimized andβ₂AR 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 andβ₂ adrenergic receptor were combined withβadrenergic receptor agonists respectively.5.HSP27 was used to detectβadrenergic receptor agonists.Established standard curve and calculated the limit of detection,it’s to explore the possibility that HSP27applied in multi-residues determination ofβadrenergic receptor agonists.Results1.Results of molecular dockingThe diagram of clenbuterol,ractopamine and fenoterol respectively combined with HSP27 andβ₂ adrenergic receptor by molecular docking show that ligands combined with HSP27 in the small bag of transmembrane region.Interaction of HSP27 andβ₂ adrenergic receptor withβadrenergic receptor agonists rely on hydrophobic bond and hydrogen bond.Nine of fifteen total scores of molecular docking of HSP27 were higher thanβ₂ adrenergic receptor.Paired-samples t test analysis the difference of their combining ability is statistically significant（P=0.03）.2.The results ofβ2 adrenergic receptor in expression and purificationThe Restructuredβ₂AR gene were expressed the functional activity ofβ₂AR protein in Escherichia Coli BL21 successfully.The size ofβ₂AR 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 theβ2AR 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 andβ₂ 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 ofβ₂ adrenergic receptor were 0.448,0.320 and 0.362.5.HSP27 applied to detectβadrenergic receptor agonistsHSP27 was used to detect three kinds ofβadrenergic receptor agonists,results show that HSP27 presented better linear relation in the concentration range of 1μg/L¹000μg/L.Cross-reactivity rate of ractopamine and fenoterol with clenbuterol were 52.6%and 29.7%respectively,and the LOD of detection was 1.53μg/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 ofβadrenergic receptor agonists,HSP27 has a good repeatability.ConclusionsThis reseach found that HSP27 has a strong combining ability withβadrenergic receptor agonists and HSP27 could be used to detectβadrenergic receptor agonists.This provides new idea and scientific basis for multi-residues determination ofβadrenergic receptor agonists...