Molecular Cloning And Inhibition Of Onchocerca Volvulus Chitinase OvCht1

Onchocerca volvulus is a kind of human parasite which can cause visual impairment or blindness.Thus Onchocerciasis is also called River Blindness.Blackflies play an important role in its transmission.The third-stage infective larvae would migrate to human through blackflies’ biting.Ivermectin,originally used as veterinary anthelmintic,is the only drug available to kill the adult worms,but having been used for decades,resistance is emerging.O.volvulus chitnase Ov Cht1 is a stage-specific product which can only express in L3 infective larvae.According to previous researches,Ov Cht1 participates in L3 moulting and might involve in many other vital development processes,such as host infection.Thus it could be regarded as a novel potential target.In this work,expression vectors were constructed to express Ov Cht1,and several inhibitors of Ov Cht1 were obtained through screening the compound library of our lab.Furthermore,the inhibition mechanisms were studied through computational analysis.The main work of this thesis includes follows:(1)Construction of Escherichia coli expression vectors to obtain full-length Ov Cht1.The gene of Ov Cht1,Ov CHT,encodes 497 amino acids and does not have transmenbrane domain.E.coli expression vector p ET22b-Ov Cht1L-C and p ET28a-Ov Cht1(L-N,S-N,L-C,S-C)were constructed after sequence analysis.The full-length Ov Cht1 with high purity were obtained using chelating chromatography.The purified proteins were resolved as a single band by SDS-PAGE,detected concentration by Coomassie brilliant blue method.Chtinase activity was detected using MU-β-(Glc NAc)3 as substrate.(2)Screening of inhibitors against Ov Cht1.Based on Ov Cht1,compounds were screened under 20 μM compared with the activity of Closantel.Compounds with inhibitory rate higher than 50% were screened under 10 μM.The Ki values of compounds with high inhibitory activity were measured in order to compare their activity.The selective inhibition of the compounds was also studied by testing theirs inhibitory activity against human chitotriosidase h Cht and chitinase from Caenorhabditis elegans Ce T1.(3)Mechanism analyse of compounds inhibition using Auto Dock.The structure of Ov Cht1 was modeled by SWISS-MODEL.The inhibitors were docked into the crystal structure of the Ov Cht1 using Auto Dock.The best docking conformation of compound was determined in accordance with both its binding energy and clustering result.Thus the binding mode of these inhibitors could be compared.Compound 2-8 was docked into Ov Cht1,h Cht and Ce T1 using Auto Dock in order to discover the characteristics at theirs active sites.