基于iTRAQ技术研究猪细小病毒感染ST细胞后的蛋白变化

发布时间：2018-04-29 16:50

本文选题：猪细小病毒 + 同位素标记相对和绝对定量技术　；参考：《湖南农业大学》2015年硕士论文

【摘要】：研究目的：本研究主要运用同位素标记相对和绝对定量技术(isobaric Tags for Relative and Absolute Quantitation, iTRAQ)和相关分子生物学方法,以期筛选并获得PPV感染后引起细胞病变和功能变化的关键蛋白,同时也为研究PPV引起母猪繁殖障碍的致病机制奠定基础,并为研究病毒性疾病引起的家畜繁殖障碍研究提供新的思路。材料与方法：本实验采用PCR技术和间接免疫荧光检测PPV感染猪睾丸细胞(ST)后12 h,24 h,36 h,48 h和60 h时间段PPV VP2的表达量,确定病毒在感染细胞后病毒最佳表达时间点。在这个时间点收集感染和未感染细胞的蛋白,采用iTRAQ技术结合LC-ESI-MS/MS分析技术检测感染组与未感染组之间蛋白表达差异。进一步运用生物信息学分析,从相关差异蛋白参与的生物学过程、其细胞结构成分和分子功能分析差异蛋白的功能,并构建差异蛋白间相互作用网,获得参与PPV感染的重要蛋白。另外,分别采用实时荧光定量技术和免疫印迹验证了部分差异蛋白对应的基因水平和蛋白水,其中包括5个上调蛋白和4个下调蛋白。研究结果：PPV在感染ST细胞后在60 h检测到其VP2核酸有较高表达量；通过iTRAQ结合LC-ESI-MS/MS技术共获得27608个肽段,共计5002个蛋白；依据蛋白质丰度水平变化(定义差异倍数达到1.2倍以上为差异蛋白)共获得218个差异蛋白,其中130个为感染后下调的蛋白,88个为感染后上调的蛋白；这些差异蛋白主要与细胞的周期、生长、运动和能量代谢、细胞的骨架、胞内的内分泌以及转录翻译等密切相关；荧光定量和免疫印迹对差异蛋白的验证结果基本与iTRAQ的变化趋势一致。结论：通过本研究得到了PPV感染ST细胞的蛋白图谱及其差异表达蛋白,构建了蛋白作用网络,并获取了PPV感染ST细胞后的一些关键蛋白,但对于这些蛋白在PPV感染中的作用还需进一步的研究。
[Abstract]:Objective: in this study, the relative and absolute quantitative techniques of isobaric Tags for Relative and Absolute Quantitation, iTRAQ) and related molecular biology were used to screen and obtain the key proteins which caused cytopathic and functional changes after PPV infection. At the same time, it lays a foundation for the study of the pathogenic mechanism of reproductive disorders in sows caused by PPV, and provides a new idea for the study of reproductive disorders in domestic animals caused by viral diseases. Materials and methods: in this experiment, PCR technique and indirect immunofluorescence were used to detect the expression of PPV VP2 at 12 h, 24 h, 36 h, 48 h and 60 h after PPV infection in porcine testicular cells, and the optimal time point for virus expression was determined. The proteins of infected and uninfected cells were collected at this time point, and the difference of protein expression between infected and uninfected cells was detected by iTRAQ and LC-ESI-MS/MS analysis. Further more, bioinformatics was used to analyze the function of differential protein from the biological process, cell structure and molecular function, and to construct the interaction network of differential protein. To obtain the important protein involved in PPV infection. In addition, the gene level and protein water of partial differential proteins were verified by real-time fluorescence quantitative technique and Western blotting, including 5 up-regulated proteins and 4 down-regulated proteins. Results the VP2 nucleic acid of VP2 was detected at 60 h after infection of St cells, 27608 peptides were obtained by iTRAQ combined with LC-ESI-MS/MS technique, a total of 5002 proteins were obtained. A total of 218 proteins were obtained according to the changes of protein abundance (defined as differential proteins by more than 1.2 times), of which 130 were down-regulated after infection and 88 were up-regulated after infection. These differential proteins are mainly related to cell cycle, growth, movement and energy metabolism, cytoskeleton, intracellular endocrine and transcriptional translation. The results of fluorescent quantitative analysis and Western blotting were consistent with the trend of iTRAQ. Conclusion: the protein map and differentially expressed proteins of PPV infected St cells were obtained, and the protein action network was constructed, and some key proteins of PPV infected St cells were obtained. However, the role of these proteins in PPV infection needs further study.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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