Construction and expression of bivalent membrane-anchored DNA vaccine encoding Sj14FABP and Sj26GST genes

Abstract

In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjcl4FABP and Sjc26GST genes and identify their expression in vitro, Sj14 and Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sj14 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 (IL-2) upstream Sj14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyl-terminal of human placental alkaline phosphatase (PLAP) downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid pIRES, resulting in another new recombinant plasmid pIRES-Sj26-Sj14. The expression of Sj14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays (IFA) when the plasmid pIRES-Sj26-Sj14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and plRES-Sj26-Sj14 were successfully constructed and the expression of modified Sj14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.

Protective Effects of Membrane-Anchored and Secreted DNA Vaccines Encoding Fatty Acid-Binding Protein and Glutathione S-Transferase against Schistosoma japonicum

“…The membrane-anchored pIRES-sjFABP-sj26GST DNA vaccine encoding a fatty acid-binding protein and the 26-kDa S. japonicum glutathione S-transferase gene was previously constructed in our laboratory [12]. The secreted pIRES-sjFABP-sj26GST plasmid was constructed using the same protocol as the one used for the membrane-anchored vaccine, except that the placental alkaline phosphatase (PLAP) membrane-anchored sequence at the 3′-end of the designed primer was removed (Fig.…”