| 摘要: |
| 【目的】探究猪细胞周期素Cyclin A（pCyclinA）及其依赖性激酶2（pCDK2）对猪细小病毒（PPV）增殖的调控作用,为开展PPV复制机制研究提供依据。【方法】采用RT-PCR扩增pCyclinA和 pCDK2基因，克隆至真核表达载体pEGFP-C1中构建重组载体，转染猪睾丸细胞（ST），经新霉素（G418）筛选获得pCyclinA或pCDK2过表达的ST细胞株。设计pCyclinA和pCDK2基因特异性干扰片段（shRNA）CycA894 和CDK1163，构建干扰表达载体，转染ST细胞，经G418筛选得到pCyclinA和pCDK2低表达的ST细胞株，实时荧光定量 PCR检测其干扰效率。流式细胞术分析pCyclinA和pCDK2过表达和低表达对ST细胞周期的影响。将猪细小病毒NY毒株（PPV-NY）接种过表达和低表达的ST细胞，用免疫过氧化物酶单层细胞染色法（IPMA）检测病毒滴度。【结果】pCyclinA和pCDK2基因的开放阅读框（ORF）分别为1 299 和897 bp，构建了重组载体pEGFP-pCyclinA和pEGFP-pCDK2。荧光显微镜观察结果显示，融合蛋白EGFP-pCycA和EGFP-pCDK2分别在过表达细胞株ST-pEGFP-pCyclinA和ST-pEGFP-pCDK2中稳定表达。构建了干扰载体pGPU6-GFP/CycA894、pGPU6-GFP/CDK1163和pGPU6-GFP/-shNC（阴性对照）；低表达细胞株ST-pGPU6-GFP/CycA894和ST-pGPU6-GFP/CDK1163中的pCyclinA、pCDK2基因的mRNA表达量均极显著降低（P＜0.01）。pCyclinA或CDK2过表达分别极显著或显著降低ST细胞S期的比例（P＜0.01或P＜0.05）。pCyclinA低表达能极显著降低ST细胞G0/G1期的比例（P＜0.01）、极显著增加S期和G2/M期的细胞比例（P＜0.01）；pCDK2低表达能显著增加ST细胞G0/G1期的比例（P＜0.05），但却显著降低S期的细胞比例（P＜0.05）。PPV在过表达细胞株ST-pEGFP-pCyclinA和ST-pEGFP-pCDK2中的滴度均极显著低于对照细胞（P＜0.01）。低表达细胞ST-pGPU6-GFP/CycA894中PPV滴度极显著增加（P＜0.01），而ST-pGPU6-GFP/CDK1163中的PPV滴度无显著变化（P＞0.05）。【结论】pCyclinA或pCDK2过表达均能抑制PPV增殖；pCyclinA低表达可促进PPV增殖，pCDK2低表达对PPV增殖无显著影响；pCyclinA低表达极显著降低G0/G1期细胞比例、但极显著增加S期和G2/M期细胞比例。 |
| 关键词: 猪Cyclin A 猪CDK2 细胞周期 猪细小病毒 病毒增殖 |
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| 基金项目:国家自然科学基金青年基金项目（31101837）；湖南省教育厅优秀青年基金项目（17B041）；衡阳师范学院校企合作项目（ZXXQ201701，18H03）；衡阳师范学院引进人才专项（16D20） |
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| Regulation effect of porcine Cyclin A and CDK2 on proliferation of porcine parvovirus |
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TANG Qinghai,YANG Hai,PENG Yonggang,et al
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| Abstract: |
| 【Objective】This study investigated the regulatory effect of porcine Cyclinin A (pCyclinA) and its dependent kinase 2 (pCDK2) on porcine parvovirus (PPV) proliferation,providing the basis for the study off PPV replication mechanism.【Method】The pCyclinA and pCDK2 genes were amplified by reverse transcription polymerase chain reaction (RT PCR),and then cloned into the eukaryotic expression vector pEGFP-C1.The constructed recombinant vectors were then transfected into porcine testicular cells (ST).Neomycin (G418) was used to screen overexpress ST cell lines of pCyclinA and pCDK2.The shRNA sequences of CycA894 and CDK1163 targeting of pCyclinA or pCDK2 genes were designed,and interference expression vectors were constructed and transfected into ST cells.ST cell lines with low expression of pCyclinA and pCDK2 were screened by G418,and interference efficiency was detected by real-time fluorescence quantitative PCR.The effect of overexpression and low expression of pCyclinA and pCDK2 on ST cell cycle was analyzed by flow cytometry.Porcine parvovirus NY strain (PPV-NY) was inoculated to overexpression and low expression ST cells,and the virus titer was detected by immunoperoxidase monolayer staining (IPMA).【Result】The open reading frame (ORF) values of pCyclinA and pCDK2 genes were 1 299 and 897 bp,respectively.The recombinants pEGFP-pCyclinA and pEGFP-pCDK2 were constructed successfully.Observation of fluorescence microscopy showed that the fusion proteins EGFP-pCycA and EGFP-pCDK2 were stably expressed in the over-expression cell lines ST-pEGFP-pCyclinA and ST-pEGFP-pCDK2,respectively.Interference vectors pGPU6 GFP/CycA894,pGPU6-GFP/CDK1163 and pGPU6-GFP/-shNC (control) were constructed.The mRNA expression levels of pCyclinA and pCDK2 genes in pGPU6-GFP/CycA894 and pGPU6-GFP/CDK1163 cells were significantly decreased (P<0.01).The overexpression of pCyclinA or CDK2 proteins significantly decreased the S phase of ST cells (P<0.01).Low expression of pCyclinA could significantly reduce the proportion of ST cells in G0/G1 phase (P<0.01),and significantly increase the proportion of ST cells in S phase and G2/M phase (P<0.01).Low expression of pCDK2 significantly increased the proportion of ST cells in G0/G1 phase (P<0.05),but significantly reduced the proportion in S phase (P<0.05).The titers of PPV in the over-expression cell lines ST-pEGFP-pCyclinA and ST-pEGFP-pCDK2 were significantly lower than that in the control cells (P<0.01).The PPV titers in ST-pGPU6-GFP/CycA894 cells with low expression of pCyclinA were significantly increased (P<0.01),while the PPV titers in ST-pGPU6-GFP/CDK1163 cells showed no significant change (P>0.05).【Conclusion】The overexpression of pCyclinA or pCDK2 could inhibit the proliferation of PPV.Low expression of pCyclinA could promote PPV proliferation,while low expression of pCDK2 had no effect on PPV proliferation.When the proportion of cells in the G0/G1 phase of cell cycle was significantly decreased,and cells in the S phase and G2/M phase were significantly increased simultaneously with low expression of pCyclinA,the proliferation efficiency of PPV was significantly promoted. |
| Key words: porcine Cyclin A porcine Cyclin A-dependent kinase 2 cell cycle porcine parvovirus proliferation of virus |
