|Abstract||蛙虹彩病毒（Rana grylio virus, RGV）是本实验室从患病沼泽绿牛蛙中分离到的病毒病原，为虹彩病毒科蛙病毒属的成员。本文试图在分子水平对RGV的功能基因进行探索性研究，为病毒的防治提供理论基础。从RGV克隆到两个新基因，并对其进行了特征分析和功能探索，主要结果如下：
1、对RGV 3β-羟基类固醇脱氢酶（3β-hydroxysteroid dehydrogenase, 3β-HSD）基因进行了转录、亚细胞定位等特征分析和过量表达的功能研究。
（1）RGV 3β-HSD基因的ORF全长1068bp，编码一个长为355aa，分子量大小为39.3KDa的推定蛋白。氨基酸序列同源搜索及比对表明，RGV 3β-HSD的氨基酸序列与蛙（Xenopus laevis）3β-HSD同源性最高，达到47%；其氨基酸序列中含有与3β-HSD功能密切相关的保守氨基酸和模体（motif）。
（2）通过RT-PCR和5′-RACE分析表明，RGV 3β-HSD基因在感染后4h即从翻译起始密码子ATG上游第19个核苷酸处开始转录，在感染后4h至48h一直保持高水平转录。用放线菌酮（cycloheximide, CHX）和阿糖胞苷（cytosine arabinoside, AraC）进行药物抑制实验，揭示RGV 3β-HSD基因是立即早期（immediate early, IE）基因。
（3）通过真核转染和荧光显微镜观察，显示RGV 3β-HSD在鲤鱼上皮瘤细胞（Epithelioma papulosum cyprini, EPC）、肥头鲤细胞（fathead minnow, FHM）和幼仓鼠肾细胞（baby hamster kidney, BHK-21）中均定位于细胞质。采用两种质粒共转染和激光共聚焦显微镜观察，表明RGV 3β-HSD与细胞线粒体共定位，这是关于病毒编码的3β-HSD具有不同于脊椎动物3β-HSD的亚细胞定位的首例报道。
（4）由真核转染和G418筛选得到过量表达RGV 3β-HSD的EPC细胞（EPC/pcDNA3.1-HSD）和转染空载体的EPC对照细胞（EPC/ pcDNA3.1）。经形态学观察和MTT实验，表明过量表达RGV 3β-HSD能显著抑制RGV和大菱鲆弹状病毒（Scophthalmus Maximus Rhabdovirus, SMRV）感染导致的细胞死亡。通过DNA琼脂糖凝胶电泳和流式细胞术分析，揭示过量表达RGV 3β-HSD能够显著抑制SMRV诱导的细胞凋亡，表明RGV 3β-HSD是一个新型的凋亡抑制因子。
2、对RGV增殖细胞核抗原（proliferating cell nuclear antigen, PCNA）基因进行了基因结构、转录和细胞内定位等特征分析。
（1）RGV PCNA基因的ORF全长738bp，编码一个长为245aa，理论分子量为26KDa的蛋白。氨基酸序列同源搜索显示，RGV PCNA的氨基酸序列和真核生物PCNA的同源性很低，与蛙（Xenopus laevis）PCNA同源性最高仅为18%。通过二级结构分析，表明RGV PCNA具有其它真核生物PCNA所特有的二级结构。
（2）将RGV PCNA基因插入到表达质粒pET32a中，构建了原核表达重组质粒pET32a-PCNA。将其转化表达宿主菌并诱导表达，经SDS-PAGE分析，在预计大小处有明显的融合蛋白表达条带。进一步采用Ni-IDA His•Bind亲和层析柱对重组蛋白进行了纯化，获得了较高纯度的重组蛋白。用纯化的重组蛋白作为抗原免疫小鼠，制备了鼠抗RGV PCNA抗血清。
（3）通过RT-PCR和western blot分析表明，RGV PCNA基因在感染后4h即开始转录并合成蛋白，并且在感染后4h至48h持续进行转录及合成蛋白。CHX和AraC药物抑制实验表明，RGV PCNA基因是晚期（late, L）基因。
（4）通过真核转染和荧光显微镜观察，表明RGV PCNA在EPC、FHM、BHK-21三种细胞内均分布于细胞质中。进一步采用免疫荧光细胞化学显示，在病毒感染后的细胞中，RGV 表达的PCNA特异性的与病毒装配位点的病毒核酸共定位，表明RGV PCNA可能是一个与病毒核酸相互作用的蛋白。|
|Other Abstract||Rana grylio virus (RGV), a member of Ranavirus (family Iridoviridae), was isolated from diseased pig frog Rana grylio. This study is attempting to explore the functional genes from RGV, and then provide some fundamental knowledge for the control of RGV infection. Two novel viral genes were cloned from RGV using conserved oligonucleotide primers, and then several experiments have been carried out to identify their characterization. The main research results are as following.
1. Characterization of RGV 3β-hydroxysteroid dehydrogenase (3β-HSD) gene and functional analyses of RGV 3β-HSD overexpression.
(1) The full length of RGV 3β-HSD ORF has 1068bp, encoding a 355aa deduced protein with a theoretical molecular weight of 39.3 KDa. Upon the homology search and amino acid alignment, the deduced amino acid sequence of RGV 3β-HSD shows the highest identity of 47% with frog (Xenopus laevis) 3β-HSD. Also, it was revealed that the deduced amino acid sequence of RGV 3β-HSD contains some conserved amino acids or motifs that are critical for the function of 3β-HSD.
(2) By RT-PCR analysis, it was revealed that the transcript of RGV 3β-HSD could be detected at 4h post-infection (h p.i.) and continued to be present at high level during 4-48 h post-infection (h p.i.). By rapid amplification of 5’-terminal cDNA end (5’-RACE), the transcription initiation site of RGV 3β-HSD was identified to located at 19 nucleotides (nt) upstream of the translation start site. Furthermore, cycloheximide (CHX), a de novo protein synthesis inhibitor, and cytosine arbinoside (AraC), a DNA synthesis inhibitor, were utilized to classify the transcript of RGV 3β-HSD gene. The result showed that RGV 3β-HSD should be an immediate-early (IE) viral gene.
(3) Upon eukaryotic transfection and fluorescence microscopy observation, the RGV 3β-HSD with a C-terminal enhanced green fluorescent protein (EGFP) tag was revealed to locate in the cytoplasm of the transfected cells, including epithelioma papulosum cyprini (EPC), fathead minnow (FHM), and baby hamster kidney (BHK-21). To further evaluate the subcellular localization of RGV 3β-HSD, two organelle-specific markers, pDsRed2-ER (endoplasmic reticulum) and pDsRed2-Mito (mitochondia), were used. Observation of the cotransfected EPC cells by confocal microscopy showed that RGV 3β-HSD was exclusively associated with cellular mitochondria, suggesting that the subcellular localization of a virus-encoded 3β-HSD is quite different from that of vertebrate 3β-HSD.
(4) EPC cells were trasfected with pcDNA3.1-HSD and empty vector pcDNA3.1, selected by G418, and confirmed by RT-PCR to obtain two stable transfectants, EPC/pcDNA3.1-HSD and EPC/pcDNA3.1. Upon the morphological observation and MTT quantitative analyses, it was found that overexpression of RGV 3β-HSD could significantly inhibit the cell death induced by either RGV or Scophthalmus Maximus Rhabdovirus (SMRV). Furthermore, the results of DNA agarose gel electrophoresis and flow cytometry revealed that overexpression of RGV 3β-HSD could significantly inhibit SMRV-induced apoptosis. All these suggest that RGV 3β-HSD might be a novel anti-apoptosis factor.
2. Characterization of RGV proliferating cell nuclear antigen (PCNA) gene.
(1) The full length of RGV PCNA ORF was identified to be 1068bp, encoding a 245aa deduced protein with a theoretical molecular weight of 26KDa. The deduced amino acid sequence of RGV PCNA shows poor identity with eukaryotic PCNA, and has the highest identity of only 18% with frog (Xenopus laevis) PCNA. However, the secondary structure of RGV PCNA was revealed to have striking similarity with that of eukaryotic PCNA, and contain the conserved domains that are critical for the function of PCNA.
(2) A prokaryotic expression recombinant plasmid, pET32a-PCNA, was constructed and introduced into the E. coli. strain BL21(DE3) for expression. The fusion protein band was detected by SDS-PAGE and purified by Ni-IDA His•Bind affinity column. The purified fusion protein was utilized to immunize mouse, and then mouse anti-PCNA serum was prepared.
(3) By RT-PCR and western blot analyses, the expression of RGV PCNA could be detected as early as 4h p.i. and continued to be present during the infection at both RNA level and protein level. CHX and AraC inhibition assay showed that RGV PCNA should be a late (L) viral gene.
(4) By eukaryotic transfection and fluorescence microscopy, it was found that RGV PCNA with a C-terminal EGFP tag is aggregated in the cytoplasm of the transfected cells of EPC, FHM, and BHK-21. Moreover, using immunofluorescence assay on RGV-infected FHM cells, it was showed that the PCNA expressed by RGV is exclusively distributed with the viral nuclei at viral assembly sites. Therefore, the result suggests that RGV PCNA might be a viral protein interacting with viral nuclei.|