关键词: 日本血吸虫, 谷胱甘肽-S-转移酶, 霍乱毒素B亚基, 杆状病毒, 真核表达

Abstract:

To induce mucosal protective immunity for schistosomiasis, 4 major epitopes from a leading vaccine antigen of Schistosoma japonicum 28 000 glutathione-S-transferase (Sj28GST) was fused with cholera toxin B subunit (CTB) as a mucosal adjuvant and the fusion protein was expressed as recombinant protein in insect cell Sf9 using Bac-to-Bac baculovirus expression system. The coding DNA for fusion CTB-Sj28GST was PCR amplified from previous constructed plasmid and then subcloned into transposed plasmid pFastBac using SalⅠand SphⅠsites. The recombinant pFastBac-CTB-Sj28GST plasmid DNA was transformed into DH10Bac for homologous recombination. The successfully obtained recombinant viruses were identified by PCR using M13 universal primers and CTB-Sj28GST specific primer and then transfected into Sf9 insect cells. The recombinant viruses were amplified by repetitively infecting Sf9 cells. The expressed recombinant fusion CTB-Sj28GST in infected insect cells was identified by indirect immunofluorescent assay (IFA), and the expressed fusion protein in the cell lysates identified by Western blotting with polyclonal antibody against Sj28GST. Results demonstrated that a 519 bp DNA fragment containing 4 epitopes of Sj28GST (189 bp) fused with CTB coding DNA was successfully amplified and cloned into pFastBac plasmid to construct pFastBac-CTB-Sj28GST. The obtained recombinant viruses from infected Sf9 cells contained the same size of fusion DNA identified by PCR. The transfected Sf9 insect cells enabled to express fusion CTB-Sj28GST identified by IFA with anti-Sj28GST specific antibody. Western blotting showed a specific band at the M_r 22 000, the same size as the expected molecular weight of fusion CTB-Sj28GST, which could be recognized by the polyclonal antibody against-Sj28GST.

Key words: Schistosoma japonicum, Glutathione-S-transferase, Cholera toxin B, Baculovirus, Eukaryotic expression