Objective: To subclone a novel gene of Schistosoma japonicum (Sj), adenylate kinase (AK) cDNA, which was identified through expressed sequence tag (EST) strategy and homology search, so as to prepare for further functional study of this gene.
Method: The inserted cDNA fragment was sequenced and searched with BLASTn program. Two PCR primers were designed according to the sequence of this Sj AK cDNA and the cloning sites in pET32a (+) plasmid, with the product purified before linkage with pMD 18-T vector. The recombinant T-vector was digested with EcoRI /XhoI to obtain Sj AK cDNA, which was then introduced into the expression plasmid pET32a (+).
Results: The novel gene possessed 86% homology with Sm AK cDNA, and the PCR product is of expected length. Double digestion with EcoR I and Xho I proved that the recombinant T-vector and the expression plasmid had the insert with length identical to that of the target fragment.
Conclusion: The novel cDNA codes for adenylate kinase of Schistosoma japonicum, and the recombinant expression plasmid pET32a (+)-Sj AK have been successfully constructed.