|其他题名: ||Establishment of two new fish cell lines, production of monoclonal antibodies and nucleoprotein analysis of fish rhabdoviruses|
1、两株鱼类新细胞系的建立及其初步应用：分别取来源于淡水的中华鲟（Chinese sturgeon）和来源于海水的赤点石斑鱼（Red spotted grouper）的鳍条和吻端组织为材料，建立用于鱼类病毒分离和鉴定的细胞系，分别称为GS（Grouper snout）和CSTF（Chinese sturgeon tail fin）细胞系，并从细胞形态、生长曲线、核型等角度进行了鉴定，在病毒感染性和外源基因转染方面进行了初步应用。结果显示GS是成纤维细胞，而CSTF是上皮样多边形，25℃下两者都能在含10%小牛血清FBS的DMEM培养液中贴壁生长。在不加FBS的培养液条件下CSTF细胞不能正常贴壁，而GS细胞虽能贴壁但生长几乎停滞。CSTF细胞在同样培养条件下能维持时间较长，耗营养液较少。目前两株细胞系都传代超过了60代，均保持二倍体核型，染色体众数分别达到48条（GS）和264条（CSTF），只是后者包含有更多的点状染色体。细胞对病毒的敏感性测定表明大菱鲆弹状病毒（Scophthalmus maximus rhabdovirus, SMRV）和蛙虹彩病毒（Rana grylio virus, RGV）均能感染GS细胞系，而只有RGV能感染CSTF细胞。荧光显微镜和电镜观察均可见在受感染细胞的胞质内有子代病毒病原出现。eGFP真核表达载体转染到两种细胞系后，24 h内即可明显看到细胞内有绿色信号荧光蛋白的表达，显示其在转基因研究中的潜在应用价值。
2、SMRV和PORV（Paralichthys olivaceus rhabdovirus, PORV）单抗制备及鉴定：在鱼类细胞系上分别扩增两株病毒，并用蔗糖梯度超速离心纯化病毒病原。将病毒抗原免疫Balb/c小鼠后，经细胞融合、筛选和有限稀释等程序分离能分泌SMRV和PORV特异性抗体的细胞株。对所制备抗体从亚类和识别抗原能力等方面进行了鉴定，并在细胞和组织水平上分别进行了应用。结果表明，在所分离的5株SMRV单抗（抗体分别定名为1G8、1H9、2D2、2D3和2F5）和2株PORV单抗（抗体定名为3C7和3C9）中，除了1G8和1H9为IgG2b之外，其它几株均为IgM。Western blotting分析表明，抗SMRV的五株抗体能同SMRV的两条结构蛋白发生反应（约26kDa和30kDa），而
3C7和3C9能识别PORV的三个结构蛋白，分子量分别为42kDa、30kDa和22kDa。利用所制备的抗体做免疫荧光实验，均能在受病毒感染的细胞胞质内看到病毒病原；而用其中的两株抗体2D3和3C7分别去捕获SMRV感染的牙鲆和PORV感染的草鱼鱼体组织冰冻切片上的病原也显示了较强的阳性信号。流式细胞术用于对弹状病毒感染的鱼类细胞进行计数，显示SMRV（0.01PFU/cell）和PORV（0.25PFU/cell）分别感染EPC（Epithelioma papulosum cyprini）和GCO（Grass carp ovary）细胞36 h后，分别用2D3和3C7作为一抗同细胞内的病原反应，细胞感染率分别达到总数的10.8%和23%。
3、鳜鱼弹状病毒（Siniperca chuatsi rhabdovirus, SCRV）核蛋白N的功能初步分析：将四个针对N基因mRNA序列不同位点的特异siRNA分别转染EPC细胞，5 h后用SCRV病毒感染细胞。通过细胞病变观察、Western blotting和病毒滴度测定等角度检验RNA干扰效应，结果最终筛选出si983（距AUG有983bp）具有最好的抑制效果。Western blotting分析显示si983能显著降低N蛋白的表达，而且这种抑制具有剂量依赖的效应。此外，si983和N基因的真核表达载体共转染后N蛋白表达量也显著下降。而特异性沉默N蛋白能显著降低病毒在细胞内的滴度，表明N蛋白对病毒的复制是必需的。
病毒感染和N基因的真核表达载体转染到EPC细胞后，除了全长的约47kDa的N蛋白之外，Western blotting还检测到两个分子量大小约为41kDa和35kDa的小蛋白带。上述两种情况下细胞的caspase-3酶活性上升，当用caspases抑制剂z-VAD-FMK（终浓度100μM）处理后，N蛋白的剪切现象消失或变弱，但不会影响子代病毒在细胞内的产量。据此，确定N蛋白的特异性剪切是由caspases酶介导的。根据caspases酶的切割蛋白特点，我们构建了起始或终止位点不同的N基因缺失片段GFP表达载体，分别为N1-GFP（aa1-376）、C1-GFP（aa377-429）、N2-GFP（aa1-281）、C2-GFP（aa282-429）和C3-GFP（aa325-429）。分别转染EPC细胞48 h后收样做Western blotting分析，比较各片段表达蛋白分子量差异和在细胞内的剪切片段大小，初步鉴定了N蛋白的两个caspases酶切位点，即D324和D374。这也是有关弹状病毒核蛋白剪切现象的首次报道。|
|英文摘要: ||Cell culture and monoclonal antibodies (mAbs) techniques are still now basic approaches on the virological investigation. Though it has a solid research foundation on the establishment systems of cell lines and mAbs for medical virology, much more information on the above two aspects need to be accumulated in lower vertebrate animals (i.g. the fish) virology research. In addition, exploring the functional genes of aquatic viruses could provide some fundamental knowledge for the control of virus infection. In this thesis, combining the fish rhabdoviruses, one of the most important viral pathogens in aquaculture, three major work involved in the establishment of fish new cell lines, virus mAbs production and identification of virus gene were done. The main research results are given as follows:
1. Establishment of two new fish cell lines and their preliminary application. Grouper snout (GS) and Chinese sturgeon tail fin (CSTF) cell lines were successfully established and characterized from healthy red spotted grouper and Chinese sturgeon, respectively. Morphologically, the GS cells were fibroblastic, while the latter consists of epithelial-like polygonal cells. Both of them could be maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium with 10% fetal bovine serum (FBS) for more than 60 subcultures and the highest growth rate was obtained at 25℃. With free-FBS medium, GS cells could not grow well while trypsinized CSTF cells could not adhere to the flask substratum. Karyotypic analysis revealed that both of the two cell lines keep a normal diploid karyotype, with the chromosome modal number 48 (GS) and 264 (CSTF), respectively. The CSTF cells contain large numbers of punctate chromosomes. Two strains of aquatic animal viruses (Scophthalmus maximus rhabdovirus, SMRV; Rana grylio virus, RGV) were used to test the
susceptibility of the GS and CSTF cells to infection. SMRV could infect the GS cell line but not produce cytopathic effect (CPE) in CSTF cells. The two cell lines both show sensitivity to RGV. Infection was also confirmed by immunofluorescence and electron-microscope observations, which detected the viral antigens or particles in the cytoplasm of virus-infected cells. Furthermore, after transfection with pEGFP vector DNA, the transfected GS and CSTF cells all produced significant fluorescent signals indicating their potential utility in exogenous studies.
2. Production and characterization of monoclonal antibodies (mAbs) against SMRV or PORV (Paralichthys olivaceus rhabdovirus). The SMRV and PORV antigens were purified with differential centrifugation followed by sucrose density gradient centrifugation from virus-infected fish cells, respectively. Purified virus antigens were used to immunize intraperitoneally Balb/c mice. After fusion, screening and limiting dilution, five mAbs (1G8, 1H9, 2D2, 2D3 and 2F5) specific to SMRV and two mAbs (3C7 and 3C9) against PORV were finally isolated and cloned. Isotype determination showed that the 1G8 and 1H9 belong to IgG2b, while the others are IgM subclass. Western blotting analysis showed that all the 5 SMRV mAbs could react with two viral protein bands of 30 and 26 kDa, while 3C7 and 3C9 reacted with three PORV virus proteins (42 kDa, 30 kDa and 22 kDa). All the mAbs produced staining signals in the cytoplasm of virus-infected EPC or GCO cells. 2D3 and 3C7 were also used to detect the viral antigens in SMRV-infected flounders and PORV-infected carp tissues respectively, which showed positive immunostaining in tissue sections. Furthermore, flow cytometry was applied for detection the virus-infected fish cells with the above two mAbs. The results suggested that 36 h postinfection, the SMRV-infected epithelioma papulosum cyprinid (EPC) (0.01PFU/cell) and PORV-infected grass carp ovary (GCO) (0.25PFU/cell) cell populations could be distinguished from uninfected cells, respectively. Approximately 10.8% of EPC cells were infected with SMRV, while the percentage of PORV-infected GCO cells came to 23%.
3. Inhibition of Siniperca chuatsi rhabdovirus (SCRV) by RNA interference (RNAi) and its specific cleavage of the nucleoprotein in a fish cell line. Several small interfering RNAs (siRNAs) corresponding to nucleoprotein (N) gene of the SCRV were chemically synthesized to explore the possibility of silencing the gene and inhibition the virus replication
in EPC cells. Cytopathic effect observation and Western blotting analysis both suggested apparent inhibition effect of N gene expression and virus replication by si983, which presented a dose dependent activity.
Furthermore, immunoblotting also showed that except to the full-length N protein band, two additional bands (41 kDa and 35 kDa) were detected in SCRV-infected or N-GFP transfected EPC cell line, which had not been recorded before in rhabdoviridae. The caspase-3 activity in infected or transfected EPC cells was higher five times or twice than controls’. When treated with z-VAD-FMK (100 μM, a caspases pan-inhibitor) in the above cells, the cleavage of N protein became fainter, even disappeared. But, treatment of z-VAD-FMK could not reduce the progeny virus production in SCRV-infected cells. Combined with the results of caspase-3 activity assay and z-VAD-FMK inhibition experiments, we indicated that the lower bands of N were the results of caspase-mediated cleavage. To further verify the proteolytically cleavage sites, several kinds of nucleoprotein gene truncated mutant fragments (N1-GFP [aa1-376], C1-GFP [aa377-429], N2-GFP [aa1-281], C2-GFP [aa282-429], C3-GFP [aa325-429]) were constructed and transfected transiently into EPC cells for immunoblotting analysis. According to the differences of every truncated protein fragments molecular weights and cleavage products in transfected cells, processing of N at residues D324 and D374 by caspases was finally identified, which is the first report on the cleavage of rhabdoviruses nucleoproteins.|
|Appears in Collections:||中科院水生所知识产出（2009年前）_学位论文|
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