Constructing The CDNA Library Of Liver-stage Schistosomulum And Identifying And Expressing The Triosephosphate Transferase Gene Of Schistosoma Japonicum

Schistosomasis is one of the most harmful parasitic diseases. After many years exterminating the snail and chemical treating, A substancial progress has been made in the prevention and the treatment of schistosomasis. But, because of the obvious defection of the common ways , such as kidney and liver damage , it is necessary for schistosomiasis control to develop schistosoma vaccine. With the development of molecular biology and technique related, it is feasible to prepare the molecular vaccine . Molecular vaccine has many virtues such as easy in mass production, preservation and usage . But, all the present molecular vaccine is not good enough . The reason is that most of present molecular vaccine come from adult worm mRNA or cDNA library . So , it is necessary to look for the molecular vaccine against schistosomulum . Schistosomulum is on the stage of development and differentiation and is easier to be killed than adult worm . In order to look for new vaccine molecular and earlier period diagnosis reagents . Four rabbits were infected experimentally by cercariae releasedfrom positive snails and were sacrificed fifteen days later. Schistosomulum were collected by douching the portal vein. Total RNA was extracted from schistosomulum and cDNA was synthesised by RT-PCR. The cDNA was recombinated oriently in to phage vector with sfi A.B adaptor . A schistosomulum cDNA library was constructed after packaging. The cDNA molecule weight range from 400-3 500bp was identified with agar electrophoresis. The recombination rate were 90% measured by plate with IPTG and x-Gal. Six recombinated clones were pick out randomly and were self-cycled into plasmid. The plasmid were digested by two enzymes, the inserted fragments range from 400 to 1 200bp. The triosephosphate transferase gene of schistosoma was cloned and expressed in Prokaryotic in order to look for the new vaccine molecule to prevent schistosoma japonicum infection . A schistosomulum cDNA library was screened by using the serum mixture of schistosomiasis patient , the positive clone was sequenced and the deduced protein was taken as homology comparison by using Genebank BLAST. The positive clone has high homology with the TPM gene of many other species. After cloned into T-vector the positive clone was subcloned into pBK-CMV expression vector . The recombinant was induced by IPTG and the expressed protein was assayed by SDS-PAGE and Western blot . The recombinant of TPM gene was testified by PCR and EcoR I, Xho I digesting . The expressed protein was recognized by the serum mixture of schistosomiasis patients . The recombined expression vector of TPM gene of schistosoma japonicum has been successfully constructed for the first time . On the base of this research, taking the gene as a potential vaccine molecule is possible.