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首页> 外文期刊>Infection and immunity >Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro
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Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro

机译：从致病性鸡瘟菌克隆的nfa1基因在非致病性猪笼草中的表达增强了对CHO靶细胞的体外细胞毒性

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The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5′ upstream regions, the nfa1 open reading frame, and 3′ downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with na?ve N. gruberi.
机译：致病性变形虫 Naegleria fowleri 具有360 bp的 nfa1 基因，该基因编码位于变形虫假足中的Nfa1蛋白（13.1 kDa）和抗Nfa1抗体可降低 N。 Fowleri诱导的体外哺乳动物细胞毒性。相比之下，抗Nfa1抗体无法检测非致病变形虫变形虫（Naegleria gruberi）中的Nfa1蛋白表达，该变形虫也具有 nfa1 基因。在本研究中， nfa1 基因从致病性 N克隆。将fowleri 转染到非致病性 N中。 gruberi 来确定它是否与致病性有关。首先将 nfa1 基因插入真核转染载体pEGFP-C2，该载体包含巨细胞病毒启动子和绿色荧光蛋白（GFP）基因，并设计为pEGFP-C2 / nfa1 < / em> UTR（ nfa1 UTR包含5'上游区域， nfa1 开放阅读框和3'下游区域）。转染后，在 N的细胞质中观察到绿色荧光。 gruberi 滋养体。这些转染子在选择后被保存超过9个月。使用 nfa1 -和载体特异性引物通过PCR在 N的基因组DNA中观察到转染的 nfa1 基因。 pEGFP-C2 / nfa1 UTR载体转染了gruberi 。另外，通过反转录PCR在转基因 N中鉴定了 nfa1 和GFP基因。 gruberi 。 Nfa1蛋白在转基因 N中表达。通过使用抗Nfa1抗体的蛋白质印迹将gruberi 鉴定为13.1 kDa条带。最后， N。发现用pEGFP-C2 / nfa1 UTR载体转染的gruberi 与单纯的 N相比，对CHO细胞具有增强的细胞毒性。 gruberi 。

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