Cloning And Expression Of Recombinant Snails Hemocyanin Functional Domains

Schistosomiasis is one kind of parasitic zoonosis. In China, people are infected bySchistosomiasis japonicum. In the life cycle of Schistosoma japonicum, Oncomelania hupensis isthe only intermediate host. The geographical distribution of Schistosomiasis japonicum iscoincided with Oncomelania. An effective way to reduce Schistosomiasis transmission is controlof the reproduction and dissemination of Oncomelania. At the present time, the chemicalmolluscacide is the most common method to kill Oncomelania hupensis. Because it can save timeand labor, bear fruit soon and has a great scope of application. Niclosamide, sodiumpentachlorophenol and other drugs can effectively kill Oncomelania. However, a large of thosedrugs is requred, has low specificity and emerge some toxic effects on humans and animals.Therefore, it is a great significance for us to expoit a specific drug.Oncomelania belongs to Mollusca Genus Oncomelania, which is amphibious and ofteninhabits in fresh waters. Previous reports have shown that hemocyanin will responsible for oxygentransport in Oncomelania. At present, there are few reports about the Oncomelania hemocyanin. Inthis paper, the hemocyanin has been extracted from Oncomelania hupensis. A full-lengthfunctional domain gene was cloned from Oncomelania hemocyanin, and then the gene wasexpressed in E.coli. In order to successfully obtain the protein crystal, a large amount of proteincrystallization conditions have been screened. Analysis of data collection and protein structureswould lay the foundation to clarify the mechanism of oxygen transportation and basic functions ofOncomelania hemocyanin. Besides, it can provide a basis for discovering drugs with specifictarget.Specifically, the method of CODEHOP primer design method was used to design primers, andthe hemocyanin gene from Oncomelania hupensis was successfully cloned. According to theanalysis by BLAST soft on the web of NCBI, a complete functional domain of Oncomelaniahupensis hemocyanin was found and was named P, which is about1.3kb. Other primers were alsodesigned to extract Oncomelania hupensis hemocyanin domains P （OhHP）. Secondly, the OhHPgene was inserted into prokaryotic expression vector pET28a by double digesting with BamHI andNdeI. The recombinant vector OhHP-pET28a has been obtained. Eventually the correctrecombinant vector was identified through double digesting and sequencing. Then therecombinant vector was transformed into BL21and induced by IPTG. An about47KD of theprotein was identified by SDS-PAGE, which is consistent with the expected sizes. Using Ni²⁺affinity chromatography and DEAE affinity chromatography to purify proteins, a large number ofrelatively pure proteins could be obtained. The purified protein was concentrated to about25mg/mL, and then screened the condition to crystallize protein by the Hanging drop vapordiffusion method. Finally, the protein crystal has been successfully obtained. The collection of X-ray diffraction data and the structural determination are going on.