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  

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

作　　者：张玉杰[1] 赵燕[1] 徐璐[1] 张乾义[1] 陈锴[1] 孙永芳[1] 邹兴启[1] 朱元源[1] 赵启祖[1] 宁宜宝[1] 王琴[1] 

机构地区：[1]中国兽医药品监察所/国家猪瘟参考实验室,北京100081

出　　处：《中国农业科学》2016年第12期2397-2407,共11页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31372434)

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FA【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（He BHH1/95） 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 96 hpi.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 10 hpi,there were more CSFV RNA gathered in cytolymph than before and less CSFV RNA were detected than before in nucleus;at 24 hpi,CSFV RNA mainly gathered in cytoly

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(FAT) 

分 类 号：S852.651[农业科学—基础兽医学]