恶性疟原虫信号肽肽酶-绿色荧光蛋白突变株的建立及其在疟原虫体内的表达分析

Construction of Plasmodium falciparum Signal Peptide Peptidase-GFP Mutant and its Expression Analysis in the Malaria Parasite

目的构建恶性疟原虫(Plasmodium falciparum)信号肽肽酶(PfSPP)基因转染载体,筛选可在体内表达疟原虫信号肽肽酶-绿色荧光蛋白(PfSPP-GFP)的疟原虫。方法提取Trager-Jensen法培养的恶性疟原虫3D7株基因组DNA,PCR扩增PfSPP C端不含终止密码子的883 bp基因片段,克隆构建重组转染载体pSPPcGT。重组载体经PCR和双酶切鉴定后送测序。电转化法将重组载体转染入恶性疟原虫体内,采用5 nmol/L恶性疟原虫二氢叶酸还原酶抑制剂WR99210筛选转染后,恶性疟原虫经无水乙醇固定、4′,6-二脒基-2-苯基吲哚(DAPI)染色后,荧光显微镜下观察PfSPP-GFP在其体内的表达分布情况。提取筛选后恶性疟原虫全蛋白,Western blotting分析虫体内PfSPP-GFP蛋白的表达情况。结果 PCR扩增获得PfSPP基因C端不含终止密码子的DNA片段,大小为883 bp。构建的重组转染载体pSPPcGT经PCR鉴定、双酶切鉴定和测序鉴定均正确。荧光显微镜观察结果显示,筛选后恶性疟原虫细胞内有绿色荧光蛋白表达,主要位于细胞质中,表明PfSPP-GFP已成功转染至恶性疟原虫并表达。Western blotting分析结果显示,转染的恶性疟原虫可表达含PfSPP-GFP融合蛋白,与预期相对分子质量(Mr)64 000大小一致。结论构建了恶性疟原虫PfSPP-GFP重组转染载体,筛选获得了能在疟原虫体内表达PfSPP-GFP的突变株。

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 pPM2 GT 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 of883 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 Mr64 000. Conclusion Recombinant vector PfSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.