关键词: 恶性疟原虫, 信号肽肽酶, 绿色荧光蛋白, 转染

Abstract:

Objective  To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum （PfSPP） and GFP， and transfect into P. falciparum（3D7 strain） to obtain mutant parasites which can express PfSPP-GFP. 　Methods  Plasmodium falciparum（3D7 strain） genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PfSPP， an 883 bp gene fragment was amplified by PCR， and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR， double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol， stained with DAPI， and observed under immunofluorescence microscope. The PfSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting.  Results   The C-terminal region of PfSPP was amplified from P. falciparum（3D7 strain） genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening， double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy， and mainly located in the cytoplasm. The PfSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64 000.  Conclusion  Recombinant vector PfSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.

Key words: Plasmodium falciparum, Signal peptide peptidase, Green fluorescent protein, Transfection