本文關(guān)鍵詞：RNA可視化原位雜交技術(shù)對(duì)感染細(xì)胞中豬瘟病毒RNA定位與分布　出處：《中國(guó)農(nóng)業(yè)科學(xué)》2016年12期 　論文類型：期刊論文

  更多相關(guān)文章： 豬瘟病毒 定位 分布 RNA可視化原位雜交 熒光抗體試驗(yàn)(FAT)

【摘要】：【目的】為研究CSFV RNA在體外感染細(xì)胞中的分布及定位,建立了一種準(zhǔn)確、敏感的RNA可視化原位雜交技術(shù)�！痉椒ā勘狙芯客ㄟ^比對(duì)Gen Bank中公布的CSFV、BVDV和BDV全序列,避開BVDV和BDV的同源區(qū),設(shè)計(jì)了CSFV RNA及內(nèi)參基因β-actin的特異探針。以CSFV中等致病力毒株(He BHH1/95)為參考毒株,在PK15細(xì)胞中培養(yǎng)病毒,加入RNA可視化原位雜交的特異探針和相應(yīng)試劑,采用熒光共聚焦顯微鏡進(jìn)行成像觀察。通過綜合分析觀測(cè)結(jié)果、熒光強(qiáng)度、重復(fù)性等因素,采用正交試驗(yàn)優(yōu)化了對(duì)原位雜交過程中具有重要影響的蛋白酶K濃度和甲醛固定時(shí)間,建立了CSFV RNA可視化原位雜交技術(shù),并與FAT方法比較該技術(shù)靈敏度;用我國(guó)目前流行的CSFV 1.1、2.1、2.2、2.3基因亞型及BVDV、PPV、PRV和PCV-2病毒進(jìn)行特異性試驗(yàn)。最終,以CSFV強(qiáng)致病力毒株(SM)接種PK15細(xì)胞,病毒感染后0.5、1、3、6、8、10、14、18、24、36、48、72、96h(hours post inoculation,hpi)取樣,每個(gè)時(shí)間點(diǎn)2個(gè)重復(fù),采用CSFV RNA可視化原位雜交技術(shù)進(jìn)行檢測(cè)。為佐證病毒蛋白在細(xì)胞中的定位及分布,同時(shí)采用FAT方法對(duì)SM株E2蛋白在PK15細(xì)胞中的表達(dá)情況進(jìn)行動(dòng)態(tài)研究。【結(jié)果】采用該技術(shù)在熒光共聚焦顯微鏡下可觀察到CSFV RNA在細(xì)胞中的定位;當(dāng)?shù)鞍酌窴濃度為1:1 000、甲醛固定時(shí)間為30min時(shí)為最優(yōu)反應(yīng)條件;靈敏度試驗(yàn)表明該技術(shù)對(duì)病毒的檢測(cè)極限為10-8/200μL,比FAT高3.5個(gè)數(shù)量級(jí);特異性試驗(yàn)結(jié)果顯示該探針能與CSFV 1.1、2.1、2.2、2.3亞型結(jié)合,與BVDV、PPV、PCV-2、PRV無交叉反應(yīng)。采用該技術(shù)對(duì)CSFV RNA感染后在靶細(xì)胞中的定位與分布研究結(jié)果顯示:0.5hpi在胞核和胞漿均能檢測(cè)到RNA,0.5—6hpi RNA主要分布于胞核內(nèi)并在核內(nèi)富集;10hpi胞漿內(nèi)RNA逐漸增多,胞核內(nèi)RNA逐漸減少,24hpi RNA主要集中在胞漿內(nèi)細(xì)胞核周圍;36hpi核外RNA大量聚集增多,72hpi達(dá)到峰值;96hpi RNA總量有所下降。而FAT結(jié)果顯示:8hpi在少數(shù)細(xì)胞的胞漿內(nèi)檢測(cè)到病毒E2蛋白,10—24hpi蛋白表達(dá)數(shù)量一直較少;36hpi蛋白表達(dá)量不斷增多,72hpi達(dá)到最大值;96hpi熒光信號(hào)由強(qiáng)變?nèi)酢２《镜鞍自诩?xì)胞漿內(nèi)的聚集數(shù)量與細(xì)胞漿中RNA含量成正相關(guān)�！窘Y(jié)論】首次建立了CSFV RNA可視化原位雜交檢測(cè)技術(shù),并對(duì)CSFV強(qiáng)致病力毒株在細(xì)胞中的RNA定位分布進(jìn)行了研究,發(fā)現(xiàn)病毒RNA吸附和進(jìn)入靶細(xì)胞的時(shí)間早于0.5hpi,觀察到CSFV RNA有在細(xì)胞核內(nèi)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.
【作者單位】： 中國(guó)獸醫(yī)藥品監(jiān)察所/國(guó)家豬瘟參考實(shí)驗(yàn)室;
【基金】：國(guó)家自然科學(xué)基金(31372434)
【分類號(hào)】：S852.651
【正文快照】： 0引言【研究意義】豬瘟(classical swine fever,CSF)是由豬瘟病毒(classical swine fever virus,CSFV)引起的豬的高度接觸性、致死性傳染病[1],造成了養(yǎng)豬業(yè)的極大損失[2-3],被世界動(dòng)物衛(wèi)生組織(OIE)列為必須報(bào)告的法定傳染病之一。致病機(jī)理的研究是從根本上控制疫病的途徑之，

本文編號(hào)：1395853