Cloning,expression and preliminary identification of the coding genetic sequences of the Tsunagi protein from Schistosoma japonicum

To obtain the coding genetic sequence of the Tsunagi protein from Schistosoma japonicum and to identify its antigenicity,Degenerate PCR,5'end and 3'end anchored PCR were used to isolate the whole open reading frame(ORF) for this protein from S.japonicum cDNA library and adult cDNA.After confirmed by DNA sequencing,the encoding ORF was amplified and inserted into expression vector pET28a(+) and then expressed as a fusion protein.The recombinant protein was purified by an affinity column and specific antiserum was obtained from the mice immunized with this protein.The titer and specificity of the antiserum was determined by ELISA and Western blotting assay.It was found that the S.japonicum Tsunagi gene has an ORF of 531bp encoding 177 amino acids.The titer of the antiserum was about 1∶51 200.Western blotting analysis showed that the antibody could bind to the Tsunagi protein specifically.These results showed that the coding gene was cloned and expressed successfully,which pave the way for the further study in the developmental biology of S.japonicum.