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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 Plasmid Plasmodium falciparum signal peptide peptidase (PfSPP) gene transfection vector and screen for plasmodium plasmodium expressing Plasmodium falciparum signal peptide peptidase - green fluorescent protein (PfSPP-GFP) in vivo. Methods Genomic DNA of Plasmodium falciparum 3D7 strain cultured by Trager-Jensen method was extracted. The 883 bp fragment without Cterminal codon of PfSPP was amplified by PCR, and the recombinant plasmid pSPPcGT was cloned. Recombinant vector was identified by PCR and double digestion after sequencing. The recombinant vector was transfected into P. falciparum by electroporation and was transfected with 5 nmol / L Plasmodium falciparum dihydrofolate reductase inhibitor WR99210. Plasmodium falciparum was fixed with anhydrous ethanol, then 4 ’, 6-bis After amidino-2-phenylindole (DAPI) staining, the expression and distribution of PfSPP-GFP in the body were observed under a fluorescence microscope. The whole protein of Plasmodium falciparum was extracted and the expression of PfSPP-GFP protein was analyzed by Western blotting. Results The DNA fragment containing the stop codon at the C terminus of PfSPP gene was amplified by PCR and the size was 883 bp. The constructed recombinant plasmid pSPPcGT was identified by PCR and identified by double enzyme digestion and sequencing. Fluorescence microscopy showed that the expression of green fluorescent protein was mainly located in the cytoplasm of Plasmodium falciparum after screening, indicating that PfSPP-GFP was successfully transfected into Plasmodium falciparum and expressed. Western blotting analysis showed that the transfected P. falciparum could express PfSPP-GFP fusion protein, which was consistent with the expected relative molecular mass (Mr) 64,000. Conclusion PfSPP-GFP recombinant plasmids were constructed and the mutants that can express PfSPP-GFP in Plasmodium were screened out.