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目的了解华支睾吸虫ATP合酶b亚基(CsATP-synt_B)核定位序列(NLS)介导该蛋白进入细胞核与细胞周期的关系,及该蛋白对自身和宿主同源基因表达的影响。方法将已构建的CsATP-synt_B与绿色荧光蛋白(GFP)融合表达质粒(pEGFP-N1-CsATP-synt_B)转染Hela细胞,转染细胞经同步化处理后,在激光共聚焦显微镜下观察在不同细胞周期相,细胞不同部位的荧光分布。流式细胞技术检测该融合蛋白表达量的变化,并通过半定量RT-PCR分析不同细胞周期相CsATP-synt_B和Hela细胞同源基因的表达。结果转染细胞经同步化处理后,流式细胞检测结果显示,空载体pEGFP-N1在G0/G1期Hela细胞中的表达量明显下降,而pEGFP-N1-CsATP-synt_B在该时期表达量呈上升趋势;在其他细胞周期时相,两者的荧光表达量的变化趋势相似。显微镜观察发现,在G0/G1期、S期和G2/M期CsATP-synt_B集中在线粒体表达,G1/S期细胞核内见绿色荧光,且多数细胞的绿色荧光集中见于核仁。在不同的细胞周期,半定量RT-PCR分析结果显示,该重组蛋白进入细胞核后,目的基因CsATP-synt_B和人源性ATP-synt_B(Ho-moATP-synt_B)mRNA的表达均呈上升趋势,CsATP-synt_B上升幅度较大,但两者的表达变化趋势一致。结论 CsATP-synt_B在G1/S期进入细胞核,受细胞能量需求和细胞周期的调控。
Objective To investigate the relationship between the nuclear localization of CsATP-synt_B nuclear localization sequence (NLS) and the cell cycle and the effect of this protein on the expression of self-host homologous genes. Methods Hela cells were transfected with constructed CsATP-synt_B and green fluorescent protein (GFP) fusion plasmid (pEGFP-N1-CsATP-synt_B). The transfected cells were treated with the same method and observed under confocal laser scanning microscope Cell cycle phase, the fluorescence distribution of different parts of the cell. The expression of fusion protein was detected by flow cytometry. The homologous gene expression of CsATP-synt_B and Hela cells in different cell cycle phases was analyzed by semi-quantitative RT-PCR. Results After the transfected cells were synchronized, the expression of pEGFP-N1 in Hela cells at G0 / G1 phase was significantly decreased by flow cytometry, while the expression of pEGFP-N1-CsATP-synt_B in Hela cells at G0 / The trend of fluorescence expression in the two cell lines was similar at other cell cycle phases. Microscopically, CsATP-synt_B was mainly expressed in mitochondria in G0 / G1 phase, S phase and G2 / M phase. Green fluorescence was found in nuclei in G1 / S phase, and green fluorescence in most cells was found in nucleoli. Semi-quantitative RT-PCR analysis showed that the expression levels of CsATP-synt_B and Ho-moATP-synt_B mRNA increased after the recombinant protein entered the nucleus. CsATP -synt_B larger increase, but the expression of both changes in the same trend. Conclusions CsATP-synt_B enters the nucleus at G1 / S phase and is regulated by cell energy requirement and cell cycle.