Construction and expression of bivalent membrane-anchored DNA vaccine encoding Sjl4FABP and Sj26GST genes.
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.
Guo
Ping
P
Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Tongii Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Dai
Wuxing
W
Liu
Shuojie
S
Yang
Ping
P
Cheng
Jizhong
J
Liang
Liang
L
Chen
Zhihao
Z
Gao
Hong
H
D000818:Animals; D000947:Antigens, Helminth / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D006367:HeLa Cells; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012554:Schistosomiasis japonica / Q000235:genetics* / Q000276:immunology / Q000517:prevention & control; D019444:Vaccines, DNA / Q000096:biosynthesis* / Q000276:immunology
D000947:Antigens, Helminth; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D005982:Glutathione Transferase