本文選題：PRRSV粒子 + 胞外體(Exosome)�。� 參考：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：胞外體(Exosome)是真核細(xì)胞分泌的脂質(zhì)雙層膜性小囊泡,主要功能是介導(dǎo)細(xì)胞間的通訊,其形成和特點(diǎn)具有細(xì)胞特異性。Exosome在病毒感染中的作用被稱為病毒感染的Exosome途徑。PRRSV粒子和Exosome的浮力密度相同、尺寸大小相近,傳統(tǒng)的分離方法不能達(dá)到分離目的。本研究旨在：建立Exosome研究的Marc-145細(xì)胞培養(yǎng)模式,觀察細(xì)胞形態(tài);收集細(xì)胞上清液,提取總Exosome,電鏡負(fù)染色法和Western blot鑒定;設(shè)計(jì)碘克沙醇速率區(qū)帶離心分離Exosome,采用Western b lot鑒定其分布區(qū)間；建立Exosome研究的PRRSV感染模型,IFA觀察結(jié)果；收集上清液,粗提取總Exosome,免疫磁珠捕獲技術(shù)分離純化Exosome與PRRSV粒子,Western blot分析純化結(jié)果。實(shí)現(xiàn)在體外培養(yǎng)的Marc-145細(xì)胞中PRRSV粒子與Exosome的分離。結(jié)果顯示：細(xì)胞形態(tài)觀察顯示消耗血清胞外體培養(yǎng)的Marc-145細(xì)胞形態(tài)飽滿,折光率強(qiáng),與無消耗胎牛血清胞外體培養(yǎng)的細(xì)胞相比無明顯差別,提示該模式適合后續(xù)研究；電鏡負(fù)染色法結(jié)果發(fā)現(xiàn)Marc-145產(chǎn)生的胞外體符合其一般特性,尺寸為40-1 OOnm,形態(tài)呈圓形或橢圓形；Western blot結(jié)果顯示存在Exosome標(biāo)志蛋白CD63、Alix、Tsg-101及P-actin,不存在非Exosome標(biāo)記蛋白EEA1、GRP94、Cytochrome c,表明得到較純凈Exosome;梯度樣品通過VVestern blot分析標(biāo)志蛋白CD63,得到Exosome分布于3-12區(qū)帶；IF結(jié)果提示Exosone研究的PRRSV感染模型建立成功；免疫磁珠捕獲技術(shù)結(jié)合Western blot實(shí)驗(yàn)結(jié)果分析顯示,同型對(duì)照磁珠系統(tǒng)不存在Exosome表面標(biāo)記蛋白Tsg-101,提示成功純化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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28

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本文編號(hào)：1747137