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Alternative TitlePreliminary research on fish antiviral immunity in response to virus induction
Thesis Advisor张奇亚
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword鱼类 水生病毒 细胞凋亡 皮肤培养 抗病毒免疫
Abstract本文分别以牙鲆、草鱼等为主要研究对象,进行鱼类的抗病毒免疫应答研究,得到主要结果如下: 1. 克隆鉴定的牙鲆PoVDAC基因cDNA长1380 bp,最大ORF长852 bp,编码283氨基酸,推测PoVDAC蛋白包括一个α 螺旋,13个跨膜 折叠结构和一个真核生物线粒体孔蛋白标签序列。PoVDAC基因在牙鲆心脏、肌肉和鳃中丰富表达,经SMRV病毒诱导FEC细胞PoVDAC的表达水平升高。PoVDAC在牙鲆组织中主要形成32 kDa单体蛋白。 PoVDAC在鱼类细胞中过量表达能诱导细胞凋亡,而且在病毒诱导FEC细胞凋亡中PoVDAC分布发生明显变化,提示PoVDAC可能通过诱导细胞凋亡介导牙鲆抗病毒免疫反应。 2. SMRV病毒诱导的草鱼血清及皮肤培养上清液中抗体能识别SMRV的磷酸化蛋白(P)和基质蛋白(M)抗原。经腹腔注射免疫诱导的草鱼皮肤培养上清液中抗体水平较低。用皮肤培养液进行IFA结果显示,在 SMRV 病毒感染 EPC 细胞有病毒包涵体荧光信号。进一步用FCM分析 SMRV 病毒感染36h的 EPC 细胞,可以检测4.39%的病毒感染细胞。 3. RGV病毒诱导的草鱼皮肤培养上清液中存在抗RGV抗体成分。Western blot分析显示,兔抗草鱼IgM 多克隆抗体主要识别草鱼抗RGV血清中IgM 的重链(88 kDa)和轻链(21 kDa)分子。用皮肤培养上清液进行IFA结果显示,在RGV病毒感染EPC细胞核附近的胞质区有荧光信号。 4. 未诱导的赤点石斑鱼皮肤培养上清液对 SMRV 和 RGV 病毒有非特异性抑制作用;经56℃处理30 min 仍有抗病毒活性,提示皮肤上清液中存在非热依赖性抗病毒物质。
Other AbstractIn the present study, we aims to prevention and control Scophthalmus maximus rhabdovirus (SMRV) and Rana grylio virus (RGV), using flounder Paralichthys olivaceus, grouper Epinephelus akaara and grass carp Ctenopharyngodon idellus as the experimental models to study fish antiviral immune response, respectively. The main results were described as follows: Firstly, in this study, the full length of PoVDAC cDNA is 1380 bp with an open reading frame of 852 bp encoding a 283 amino acid protein. The deduced PoVDAC contains one α-helix, 13 transmembrane -strands and one eukaryotic mitochondrial porin signature motif. Constitutive expression of PoVDAC was confirmed in all tested tissues by real-time PCR. Further expression analysis revealed PoVDAC mRNA was upregulated by viral infection. We prepared fish antiserum against recombinant VDAC proteins and detected the PoVDAC in heart lysates from flounder as a 32 kDa band on western blot. Overexpression of PoVDAC in fish cells induced apoptosis. Immunofluoresence localization indicated that the significant distribution changes of PoVDAC have occurred in virus-induced apoptotic cells, suggesting that PoVDAC might be mediated flounder antiviral immune response through induction of apoptosis. Secondly, this study determined whether specific antibodies were present in the excised skin explants of grass carp, immune to SMRV and RGV viruses, respectively. The antiserum and culture fluid from immune skin explants are detected by iELISA, western blot, IFA and flow cytometry, respectively. The results of iELISA showed that cutaneous antibody titer levels were much lower (1:12) than antiserum titer (1:1458) from intraperitoneal (IP) immunized grass carp. The phosphoprotein (P) and matrix protein (M) antigens of purified SMRV proteins were recognized by cutaneous antibodies from skin culture fluid using western blot. The skin culture fluid produced staining signals in viral assembly sites and cytoplasm of SMRV-infected EPC cells by IFA technique. Results of flow cytometry showed that 4.39% SMRV-infected EPC cells were detected, while control cells only come to 2.0% in view of the unspecific reaction. In addition, following injection with RGV virus, specific antibody levels of immunised fish skin culture supernatant were determined by iELISA. Specific antibody can be detected in immunised fish skin culture supernatant. The immunised grass carp serum IgM heavy chain (88 kDa)and light chain(21 kDa) proteins were recognized by rabbit anti-fish antibody using western blot. The skin culture fluid produced staining signals in the perinuclear region of cytoplasm of RGV-infected EPC cells by IFA technique, indicating the viromatrix of RGV distributed in the perinuclear region of cytoplasm. The above findings indicated that skin mucosal immunity play important roles in fish against virus infection. Thirdly, to further extend the above findings, skin culture supernatant from non-immunized grouper Epinephelus akaara collected and tested for antiviral activity against RGV and SMRV viruses using cell-based assays, respectively. The results demonstrated that fish skin supernatant fluids have non-specific antiviral activities against RGV and SMRV, respectively. Heat-treated supernatant fluids (56℃, 30 min) displayed both the anti-RGV and anti-SMRV activities, indicating that were not heat labile and thus probably not complement mediated.
Document Type学位论文
Recommended Citation
GB/T 7714
吕爱军. 三种养殖鱼类抗病毒免疫的初步研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2007.
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