Marc-145细胞中猪繁殖与呼吸综合症病毒粒子与胞外体的分离与鉴定

发布时间：2018-04-14 01:32

本文选题：PRRSV粒子 + 胞外体(Exosome)　；参考：《山西农业大学》2015年硕士论文

【摘要】：胞外体(Exosome)是真核细胞分泌的脂质双层膜性小囊泡,主要功能是介导细胞间的通讯,其形成和特点具有细胞特异性。Exosome在病毒感染中的作用被称为病毒感染的Exosome途径。PRRSV粒子和Exosome的浮力密度相同、尺寸大小相近,传统的分离方法不能达到分离目的。本研究旨在：建立Exosome研究的Marc-145细胞培养模式,观察细胞形态;收集细胞上清液,提取总Exosome,电镜负染色法和Western blot鉴定;设计碘克沙醇速率区带离心分离Exosome,采用Western b lot鉴定其分布区间；建立Exosome研究的PRRSV感染模型,IFA观察结果；收集上清液,粗提取总Exosome,免疫磁珠捕获技术分离纯化Exosome与PRRSV粒子,Western blot分析纯化结果。实现在体外培养的Marc-145细胞中PRRSV粒子与Exosome的分离。结果显示：细胞形态观察显示消耗血清胞外体培养的Marc-145细胞形态饱满,折光率强,与无消耗胎牛血清胞外体培养的细胞相比无明显差别,提示该模式适合后续研究；电镜负染色法结果发现Marc-145产生的胞外体符合其一般特性,尺寸为40-1 OOnm,形态呈圆形或椭圆形；Western blot结果显示存在Exosome标志蛋白CD63、Alix、Tsg-101及P-actin,不存在非Exosome标记蛋白EEA1、GRP94、Cytochrome c,表明得到较纯净Exosome;梯度样品通过VVestern blot分析标志蛋白CD63,得到Exosome分布于3-12区带；IF结果提示Exosone研究的PRRSV感染模型建立成功；免疫磁珠捕获技术结合Western blot实验结果分析显示,同型对照磁珠系统不存在Exosome表面标记蛋白Tsg-101,提示成功纯化Exosome。
[Abstract]:Exosome is a lipid bilayer vesicle secreted by eukaryotic cells, whose main function is to mediate intercellular communication.Its formation and characteristics are cell-specific. The role of exosome in virus infection is called the Exosome pathway of virus infection. PRRSv particles and Exosome have the same buoyancy density and similar size. The traditional separation method can not achieve the purpose of isolation.The purpose of this study was to establish Marc-145 cell culture model studied by Exosome, observe cell morphology, collect cell supernatant, extract total exosome, electron microscope negative staining and Western blot identification.The isolated exosomees were isolated by centrifugation in the rate zone of iodoxanol, and their distribution was identified by Western b lot. The PRRSV infection model studied by Exosome was established, and the supernatant was collected.Crude extraction of total exosome, immunomagnetic bead capture technique were used to isolate and purify Exosome and PRRSV particles by Western blot.The separation of PRRSV particles from Exosome in cultured Marc-145 cells was realized.The results showed that the morphology of Marc-145 cells cultured with consuming serum was full, and the refractive index was strong. The results showed that there was no significant difference between the cells cultured with the extracellular body of fetal bovine serum and that without consuming fetal bovine serum. The results suggested that the model was suitable for further study.The results of electron microscopy negative staining showed that the extracellular bodies produced by Marc-145 accord with its general characteristics.The size is 40-1 OOnm, and the morphology is round or elliptical. The results of Western blot show that there are Exosome marker proteins CD63Tsg-101 and P-actin.There is no non- marker protein EEA1GP94Cytochrome, which indicates that the purified exosome is obtained, and the gradient sample is characterized by VVestern blot analysis of the marker protein CD63.The results of 3-12 banding showed that the PRRSV infection model studied by Exosone was successful.The results of immunomagnetic bead capture and Western blot test showed that there was no Exosome surface marker protein Tsg-101 in the same type control system, which suggested that exosome was purified successfully.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

【参考文献】