Expression And Characterization Of ?-carbonic Anhydrase,a Potential Drug Target From Schistosima Japanicum

Schistosomiasis iscaused by parasitic flatworms of the Schistosoma kind,and it is widely distributed alone the Yangtze River Valley and the south of our country.It seriously impeded China's social and economic development and threated to people's health,as one of important public health problem for China,the state is working to develop a new 10-year plan for control of schistosomiasis.Only Praziquantel is safe and effective for the schistosomiasis treatment,and it has been used for 30 years since the late 70 s of last century.However,in recent years several geographical strains displaying less sensitive to praziquantel and even invalid treatment were reported in some S.mansoia endemic areas in Africa and South America.So find a new drug target and developanew therapeutic agentsfor schistosomiasis is the public health needs?-carbonic anhydrases??-CA?is a zinc metalloenzyme which catalyze CO₂ hydration and widely distribute in nature.It's physiological function is diverse,and has an important role in many organisms.?-CAscan be inhibited by drugs,such as amino sulfonic acid and anion,and some present studys show that the activity of ?-CA could be inhibitedon several organisms in vivo.Because of the critical role of ?-CA and it is distribution in invertebrates,it promise an attractive drug target for developing a new broad-spectrum antibacterial and anti-parasitic drugsin new mechanism.This study is to express thepotential drug target?-CAof S.japonicum,analyze its catalytic activity and expect to establisha platform for screening new compoundsthat against S.japonicum.Firstly,the cDNA sequence of ?-CA from Schistosoma japonicum was obtained by the method of bioinformatics.Then the sequence Sjp₀056790.1 was expressed in the prokaryotic expression system.SDS-PAGE and Western blotting was taken for identifyingrecombined protein,and it waspurified by Ni affinity chromatography,then refolded the recombinant protein and performed the carbonic anhydrase activity assay.An in vitro inhibition study was taken to varify that taking ?-CAof Schistosama japonicun.as a drugs target is feasible.Finally we confirmed that the amino acid sequence from S.japonicumwas similar tosequences of ?-CAsfrom the other organisms.The recombinant expression vector pET-32a?+?-SjaCA was successfully constructed and confirmed by PCR and double enzyme restricted digestion analysis.SDS-PAGE and Western blotting results showed that the recombinantprotein has a molecular weight of about 38 kDa which consistent with expectation,and it can be recognized by anti-his-tag antibody.Then the carbonic anhydrase activity assay showed that the expressed protein possess a similar catalyzing activity as the standard carbonic anhydrase did,and the activity could be inhibited by acetazolamidewhich is a commercial inhibitor for ?-carbonic anhydrases.Then the phamacological inhibition study showed that the Schistosama japonicuncould be inhibited by AAZ.Our study successfully identified the cDNA sequence of Schistosama japonicunandexpressed it by prokaryotic expression system with catalytic activity.The activity of this expressed protein could be inhibited by carbonic inhibitor AAZ,and the AAZ also inhibit Schistosama japonicun in vitro.?-CA promise an attractive drug target for developing a new anti-schistosomal drug.Our study laid a foundation for the subsequent drug inhibition research and contributed to the searching and developing for a new mechanism anti-schistosomal drugs.