中国农业科学 ›› 2016, Vol. 49 ›› Issue (12): 2397-2407.doi: 10.3864/j.issn.0578-1752.2016.12.015

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

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

张玉杰,赵燕,徐璐,张乾义,陈锴,孙永芳,邹兴启,朱元源,赵启祖,宁宜宝,王琴   

  1. 中国兽医药品监察所/国家猪瘟参考实验室,北京 100081
  • 收稿日期:2015-12-31 出版日期:2016-06-16 发布日期:2016-06-16
  • 通讯作者: 王琴,Tel:010-61255400;E-mail:wq551@vip.sina.com
  • 作者简介:张玉杰,E-mail:zyj-0811@sohu.com。赵 燕,E-mail:zhaoyan82882@126.com。张玉杰、赵燕为同等贡献作者。
  • 基金资助:
    国家自然科学基金(31372434)

Study of Location and Distribution of Classical Swine Fever Virus RNA in PK15 Cells by Visualization in Situ Hybridization Technology

ZHANG Yu-jie, ZHAO Yan, XU Lu, ZHANG Qian-yi, CHEN Kai, SUN Yong-fang, ZOU Xing-qi, ZHU Yuan-yuan, ZHAO Qi-zu, NING Yi-bao, WANG Qin   

  1. China Institute of Veterinary Drug Control/National Classical Swine Fever Reference Lab, Beijing 100081
  • Received:2015-12-31 Online:2016-06-16 Published:2016-06-16

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GenBank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(HeBHH1/95)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。

关键词: 猪瘟病毒, 定位, 分布, RNA可视化原位杂交, 荧光抗体试验

Abstract: 【Objective】In order to make researches on the RNA location and distribution of Classical Swine Fever Virus (CSFV)in infected PK15 cells, a rapid, accurate and sensitive in situ hybridization (ISH) technology was established. 【Method】After comparison with the complete sequences of CSFV, BVDV and BDV to avoid the homology regions, a set of specific probes of CSFV RNA andβ-actin were designed and synthesized. A reference strain CSFV (HeBHH1/95) was used to optimize the ISH technology through comparison several parameters such as fluorescence intensity, repeatability, protease K concentration and formalin fixation time. After these parameters were determined, an ISH technology was established. Fluorescent antibody test (FAT) was used to compare the sensitivity with the ISH technology. All the sub genotypes of CSFV (sub genotype 1.1, 2.1,2.2 and 2.3) which are present in China and other normal pig infectious virus (BVDV, PPV, PRV,PCV-2) were used to detect specificity of this ISH technology. A high virulent strain of CSFV (SM) was inoculated in PK15 cells. Infected cells were sampled at 0.5 hours post inoculation (hpi)、1, 3, 6, 8, 10, 14, 18, 24, 36, 48, 72, and 96hpi. Then FAT was performed in parallel to detect the expression and location of CSFV E2 protein. 【Result】CSFV RNA were detected in infected PK15 cells by using the ISH technology. The optimal concentration of protease K was 1:1 000 and the optimal time of formalin fixation was 30 minutes. The minimum of detection is 10-8/200μL which is 3.5 orders of magnitude higher than FAT. The specific tests showed that the ISH technology could react with sub genotypes 1.1, 2.1,2.2 and 2.3 of CSFV in China and has no cross reaction with BVDV, PPV, PRV,PCV-2 viruses. CSFV RNA ISH test results showed that CSFV RNA were firstly detected in nucleus at 0.5 hpi and gathered in nucleus from 0.5hpi to 6hpi; at 10hpi, there were more CSFV RNA gathered in cytolymph than before and less CSFV RNA were detected than before in nucleus;at 24hpi, CSFV RNA mainly gathered in cytolymph around nucleus; at 36hpi, more and more CSFV RNA gathered in cytolymph and got maximum at 72hpi; at 96hpi, the amount of CSFV RNA declined. The results of FAT showed that little E2 protein was detected in cytolymph at 8hpi. From 10hpi to 24 hpi, only few cells were detected positive. After 36hpi, the expression of E2 protein increased gradually and got to the maximum at 72hpi. But after 96hpi, the amount of E2 protein declined. The trend of E2 protein expression was in accord with CSFV RNA. 【Conclusion】 A visualization in situ hybridization technology was firstly established to detect CSFV RNA. The location and distribution of CSFV RNA was studied by using this technology. The results proved that CSFV RNA enters cell early than 0.5hpi and CSFV RNA has ever existed in nucleus.

Key words: Classical Swine Fever Virus (CSFV), location, distribution, in situ hybridization (ISH), Fluorescent antibody test (FAT)