RNA可视化原位杂交技术对感染细胞中猪瘟病毒RNA定位与分布

发布时间：2018-01-08 05:31

  本文关键词：RNA可视化原位杂交技术对感染细胞中猪瘟病毒RNA定位与分布　出处：《中国农业科学》2016年12期 　论文类型：期刊论文

  更多相关文章： 猪瘟病毒 定位 分布 RNA可视化原位杂交 荧光抗体试验(FAT)

【摘要】：【目的】为研究CSFV RNA在体外感染细胞中的分布及定位,建立了一种准确、敏感的RNA可视化原位杂交技术。【方法】本研究通过比对Gen Bank中公布的CSFV、BVDV和BDV全序列,避开BVDV和BDV的同源区,设计了CSFV RNA及内参基因β-actin的特异探针。以CSFV中等致病力毒株(He BHH1/95)为参考毒株,在PK15细胞中培养病毒,加入RNA可视化原位杂交的特异探针和相应试剂,采用荧光共聚焦显微镜进行成像观察。通过综合分析观测结果、荧光强度、重复性等因素,采用正交试验优化了对原位杂交过程中具有重要影响的蛋白酶K浓度和甲醛固定时间,建立了CSFV RNA可视化原位杂交技术,并与FAT方法比较该技术灵敏度;用我国目前流行的CSFV 1.1、2.1、2.2、2.3基因亚型及BVDV、PPV、PRV和PCV-2病毒进行特异性试验。最终,以CSFV强致病力毒株(SM)接种PK15细胞,病毒感染后0.5、1、3、6、8、10、14、18、24、36、48、72、96h(hours post inoculation,hpi)取样,每个时间点2个重复,采用CSFV RNA可视化原位杂交技术进行检测。为佐证病毒蛋白在细胞中的定位及分布,同时采用FAT方法对SM株E2蛋白在PK15细胞中的表达情况进行动态研究。【结果】采用该技术在荧光共聚焦显微镜下可观察到CSFV RNA在细胞中的定位;当蛋白酶K浓度为1:1 000、甲醛固定时间为30min时为最优反应条件;灵敏度试验表明该技术对病毒的检测极限为10-8/200μL,比FAT高3.5个数量级;特异性试验结果显示该探针能与CSFV 1.1、2.1、2.2、2.3亚型结合,与BVDV、PPV、PCV-2、PRV无交叉反应。采用该技术对CSFV RNA感染后在靶细胞中的定位与分布研究结果显示:0.5hpi在胞核和胞浆均能检测到RNA,0.5—6hpi RNA主要分布于胞核内并在核内富集;10hpi胞浆内RNA逐渐增多,胞核内RNA逐渐减少,24hpi RNA主要集中在胞浆内细胞核周围;36hpi核外RNA大量聚集增多,72hpi达到峰值;96hpi RNA总量有所下降。而FAT结果显示:8hpi在少数细胞的胞浆内检测到病毒E2蛋白,10—24hpi蛋白表达数量一直较少;36hpi蛋白表达量不断增多,72hpi达到最大值;96hpi荧光信号由强变弱。病毒蛋白在细胞浆内的聚集数量与细胞浆中RNA含量成正相关。【结论】首次建立了CSFV RNA可视化原位杂交检测技术,并对CSFV强致病力毒株在细胞中的RNA定位分布进行了研究,发现病毒RNA吸附和进入靶细胞的时间早于0.5hpi,观察到CSFV RNA有在细胞核内的生活史。
[Abstract]:[objective] to establish an accurate method to study the distribution and localization of CSFV RNA in infected cells in vitro. The sensitive RNA visual in situ hybridization technique. [methods] the whole sequence of CSFV Bank was compared with that of BDV published in this study. Avoid the homology of BVDV and BDV. The specific probes of CSFV RNA and 尾 -actin were designed. He BHH 1 / 95, a medium virulence virus strain of CSFV, was used as reference strain. The virus was cultured in PK15 cells and the specific probes and corresponding reagents of RNA visual in situ hybridization were added. The fluorescence confocal microscope was used for imaging observation. CSFV RNA visual in situ hybridization was established by optimizing the concentration of protease K and the fixation time of formaldehyde in situ hybridization by orthogonal test. The sensitivity of the method is compared with that of FAT method. The specific tests were carried out by using the CSFV 1.1 1. 1 2. 2 and 2. 3 gene subtype and the PCV-2 virus and PCV-2 virus. PK15 cells were inoculated with CSFV virulent virulence strain. After infection, the virus was infected with PK15 cells. Hours post inoculation hpi. sampling, 2 repeats at each time point. In order to confirm the localization and distribution of virus protein in cells, CSFV RNA in situ hybridization technique was used to detect it. At the same time, the expression of E2 protein of SM strain in PK15 cells was studied by FAT method. [results] CSFV could be observed under fluorescent confocal microscope. The localization of RNA in cells; When the concentration of protease K was 1: 1 000 and the fixed time of formaldehyde was 30 min, the optimal reaction condition was obtained. The sensitivity test shows that the detection limit of the virus is 10-8 / 200 渭 L, which is 3.5 orders of magnitude higher than that of FAT. The results of specificity test showed that the probe could bind to the subtype of CSFV 1.1 ~ 2.1 ~ 2.22 ~ (2. 3), and it could be combined with CSFV _ (VV) ~ (2) PPV-PCV-2. There was no cross reaction in PRV. The results of localization and distribution of CSFV RNA in target cells showed that RNA could be detected in nucleus and cytoplasm of 0. 5 hpi. 0.5-6 hpi RNA were mainly distributed in the nucleus and enriched in the nucleus. The RNA in the cytoplasm increased gradually at 10 h pi, and the RNA in the nucleus decreased gradually. The 24 h pi RNA was mainly concentrated around the nucleus in the cytoplasm. The accumulation of RNA outside the nucleus of 36hpi was increased to 72hpi and the peak value was 72hpi. The total amount of 96hpi RNA decreased, while the FAT results showed that the expression of E2 protein 10-24hpi protein was very small in the cytoplasm of a few cells. The expression of 36hpi protein increased continuously, and 72hpi reached the maximum value. The fluorescence signal of 96hpi became weak from strong to weak. There was a positive correlation between the amount of viral protein aggregation in cytoplasm and the content of RNA in cytoplasm. [conclusion] CSFV was established for the first time. RNA visual in situ hybridization technique. The localization and distribution of RNA of CSFV virulent strains in the cells were studied. It was found that the time of RNA adsorption and entry into target cells was earlier than 0.5 hpi. It was observed that CSFV RNA had a life history in the nucleus.
【作者单位】： 中国兽医药品监察所/国家猪瘟参考实验室;
【基金】：国家自然科学基金(31372434)
【分类号】：S852.651
【正文快照】： 0引言【研究意义】猪瘟(classical swine fever,CSF)是由猪瘟病毒(classical swine fever virus,CSFV)引起的猪的高度接触性、致死性传染病[1],造成了养猪业的极大损失[2-3],被世界动物卫生组织(OIE)列为必须报告的法定传染病之一。致病机理的研究是从根本上控制疫病的途径之，

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