In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjc14FABP and Sjc26GST genes and identify their expression in vitro, Sj 14 and Sj26 genes were ob- tained by RT-PCR with total RNA ...In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjc14FABP and Sjc26GST genes and identify their expression in vitro, Sj 14 and Sj26 genes were ob- tained 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-Sjl4 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 （IL-2） upstream Sj 14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyi-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 plRES, resulting in another new recombinant plasmid plRES-Sj26-Sj14. The expression of Sj 14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays （IFA） when the plasmid plRES-Sj26-Sj 14 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-Sj 14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj 14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.展开更多
基金This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30471603).
文摘In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjc14FABP and Sjc26GST genes and identify their expression in vitro, Sj 14 and Sj26 genes were ob- tained 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-Sjl4 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 （IL-2） upstream Sj 14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyi-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 plRES, resulting in another new recombinant plasmid plRES-Sj26-Sj14. The expression of Sj 14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays （IFA） when the plasmid plRES-Sj26-Sj 14 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-Sj 14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj 14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.
