Zhao Hong-Xi, Li Xia, Liu Jun-Long, Guan Gui-Quan, Luo Jian-Xun
School of Agriculture, Ningxia University, Yinchuan, China.
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
Front Vet Sci. 2022 Oct 20;9:997294. doi: 10.3389/fvets.2022.997294. eCollection 2022.
schizont-infected host cells in culture show unlimited proliferation similar to tumor cells; thus far, are the only eukaryotes that have been found to transform mammalian cells (immortalized). The transformation of these cells is reversible; when the parasite is eliminated in transformed cells by buparvaquone (BW720c), the host cells show normal growth and apoptosis. TFG is a tropomyosin-receptor kinase fused gene that is conserved among many species and is an important proto-oncogene. In this study, the bovine TFG gene was amplified by PCR from the cDNA of schizont-transformed cells, cloned into the pGEX-4T-1 vector and expressed in BL21 (DE3). After purification, the fusion protein was injected into rabbits to produce polyclonal antibodies. Using -transformed cells together with BW720c treatment to kill the parasite, we aimed to identify changes in TFG gene expression by real-time PCR and Western blotting. The results showed that the bovine TFG gene was ~582 bp in size; SDS-PAGE analysis showed that the fusion protein was expressed in BL21 (DE3) cells with a molecular mass of 48 kD, and Western blotting indicated that the polyclonal antibodies could react with bovine TFG proteins from -transformed cells and showed high specificity. Compared with that in the control group, the transcription level of the host TFG gene decreased significantly in the BW720c test group, and the expression of host tumor-related TFG protein decreased sharply after 72 h of drug treatment, suggesting that the TFG protein expression in transformed cells was directly related to . This finding laid a foundation for further study on the interaction between and host cells.
培养物中被裂殖体感染的宿主细胞表现出与肿瘤细胞相似的无限增殖能力；迄今为止，它们是唯一被发现能转化哺乳动物细胞（永生化）的真核生物。这些细胞的转化是可逆的；当通过丁萘醌（BW720c）在转化细胞中消除寄生虫时，宿主细胞表现出正常生长和凋亡。TFG是一种原肌球蛋白受体激酶融合基因，在许多物种中保守，是一种重要的原癌基因。在本研究中，通过PCR从裂殖体转化细胞的cDNA中扩增牛TFG基因，克隆到pGEX-4T-1载体中并在BL21(DE3)中表达。纯化后，将融合蛋白注射到兔子体内以产生多克隆抗体。使用裂殖体转化细胞并结合BW720c处理来杀死寄生虫，我们旨在通过实时PCR和蛋白质免疫印迹法鉴定TFG基因表达的变化。结果表明，牛TFG基因大小约为582 bp；SDS-PAGE分析表明融合蛋白在BL21(DE3)细胞中表达，分子量为48 kD，蛋白质免疫印迹表明多克隆抗体可与裂殖体转化细胞中的牛TFG蛋白反应并显示出高特异性。与对照组相比，BW720c试验组宿主TFG基因的转录水平显著降低，药物处理72 h后宿主肿瘤相关TFG蛋白的表达急剧下降，表明转化细胞中TFG蛋白的表达与……直接相关。这一发现为进一步研究……与宿主细胞之间的相互作用奠定了基础。
