Studies On The Recombinant Enolase Of Schistosome Japonicum

Schistosomiasis caused by infection with Schistosome japonicum, is the most harmful parasitic zoonosis in China. The current control efforts depend mainly on praziquantel chemotherapy. However, praziquantel,the sole wide-spread efficient drug for schistosomasis, can't avoid high rates of re-infection after mass treatment. Moreover, the development of resistance to praziquantel limits strategies based on chemotherapy. Therefore, it is necessary to develop an efficient safe vaccine and new anthelmintic drugs against schistosome. Schistosomiasis likes other parasite, which has the exchange of substances and energy by metabolism in the host. At present, people are convinced that the energy acquisition of Schistosomiasis relys mainly on glycolysis pathway. The enolase plays a role in glycolysis pathway that is a key pathway in energetic metabolism of cell. It's very important in the growth and development of animal. So, we cloned, over -expressed, detected the position of this enolase and did some basic biologic function research in this article.1 According to GenBank ID , a specific primer was designed .Then we got the DNA Sequence of Schistosoma japonicum Enolase (SjEno) .The sequence including a complete open reading frame contains 1305 nucleotides, and encodes 434 amino acids. Its molecular weight in theory is about 47 KD. The SjEno cDNA fragment was subcloned into expression vector to construct prokaryotic expressing plasmids pET28a(+)-SjEno, and transformed to E. coli BL21(DH5α). The recombination protein SjEno/HIS was successfully purified .2 The enolase protein was recognized by the rabbit sera against to adult worm antigen in western analysis .It suggested good antigenicity. In the vaccine trial, the purified rSjEno/HIS was used to immune Balb/c mice .It showed 24.28% liver egg reduction and 21.45% faeces egg reduction, compared with those of the control mice. ELISA result suggested that high levels of specific IgG induced by the recombitant protein may play an important role for the protective effect on the vaccinated mice.3 Detection of SjEno by immunofluorescence microscopy using a mouse antibody raised against recombinant SjEno/HIS showed that SjEno was expressed in the outer tegument28d and 42d adult worm;The significant immunofluorescence signal was also observed at the other inner part of parasite such as muscle layer and gut.4 Kinetic measurement the activity of rSjEno was carried out. The test showed that using 2-PGA as substrate gave a specific activity of nearly 35.81 U(mg protein)^-1; and using PEP as substrate gave a specific activity of nearly 15.87 U(mg protein)^-1.PH-dependent activity measurements gave a maximum at pH 6.5 to 7.0 irrespective of the direction of catalysis. The activity of this enzyme is inhibited by KCL,LiCL,NaCL,MgCL₂,CaCL₂ in the detection range of ion concentration. When 2-PGA was used as substrate ,the ZnCL₂ showed effect of inhibition.whereas, When PEP was used as substrate ,the concentration of ZnCL₂ ranging from 2.5 to 7.5mM showed activiating effect. The variety of temperature from 15 to 45 centigrade in the reaction system have no significant infuence on the activity of enolase.