原位多聚酶链反应在人巨细胞病毒诊断中的应用

逝年来人巨细胞病毒（ＨＣＭＶ）感染率高,检测方法进展人,有巨细胞包涵体细胞（ＣＣＩＣ）检测、病毒分离、免疫检测、原位杂交（ＩＳＨ）、多聚酶链反应（ＰＣＲ）等法。目前有人把ＩＳＨ与ＰＣＲ结合创立了原位多聚酶链反应法（ＩＳＰＣＲ）,此法更准确、更敏感特异,更有地ＨＣＭＶ感染的早期快速诊断。本文就ＩＳＰＣＲ的原理、程序、特征主临床应用作一综述。     

大鼠诱导型一氧化氮合酶基因转录调控区的克隆与鉴定

采用单特异引物ＰＣＲ克隆法,得到大鼠诱导型一氧化氮合酶（ｉＮＯＳ）基因转录调控区ＤＮＡ片段．核酸序列分析证实,大鼠ｉＮＯＳ基因的５′－侧翼区含有ＩＦＮ－γ和ＴＮＦ－α应答元件及ＮＦ－κＢ结合位点的保守序列．这些保守序列的位置及排列显著区别于人和小鼠的ｉＮＯＳ基因．电泳迁移率改变分析（ＥＭＳＡ）表明,ＶＳＭＣ受ＩＬ－１和ＩＦＮ－γ刺激后,细胞核内产生某种可与ｉＮＯＳ基因５′－侧翼区特异结合的核蛋白因子．     

大鼠诱导型一氧化氮合酶基因转录调控区的克隆与鉴定

采用单特异引物ＰＣＲ克隆法,得到大鼠诱导型一氧化氮合酶（ｉＮＯＳ）基因转录调控区ＤＮＡ片段。核酸序列分析证实,大鼠ｉＮＯＳ基因的５＇－侧翼区含有ＩＦＮ－γ和ＴＮＦ－α应答元件及ＮＦ－ｋＢ结合位点的保守序列。这些保守序列的位置及排列显区别于人和小鼠的ｉＮＯＳ基因。电泳迁移率改变分析（ＥＭＳＡ）表达,ＶＳＭＣ受ＩＬ－１和ＩＦＮ－γ刺激后,细胞核内产生某种可与ｉＮＯＳ基因５＇－侧翼区特异结合的核蛋白因     

犬细小病毒中国内蒙株VP2基因克隆及序列分析

从我国内蒙古地区流行的犬细小病毒病病犬的肠溶物中分离提纯犬细小病毒（ＣＰＶ）。提取病毒基因组ＤＮＡ,并以此ＤＮＡ为模板,采用人工合成的引物进行ＰＣＲ扩增,ＰＣＲ产物经ＢａｍＨＩ、ＳａｃＩ双酶切后,克隆于ｐＵＣ１９质粒的ＢａｍＨＩ／ＳａｃＩ位点。重组质粒ｐＵＣＶＰ２经ＰＣＲ鉴定、限制酶切分析和序列分析,结果表明：获得了犬细小病毒内蒙株（ＣＰＶ－ＩＭ）ＶＰ２基因的全长克隆,ＶＰ２基因全长１７５５ｎｔ,     

EB病毒胸苷激酶基因的定向克隆

采用异硫氰酸胍（ＧｕＳＣＮ）和硅藻从Ｂ９５－８细胞中快速抽摸板ＤＮＡ。根据ＥＢ病毒（ＥＢＶ）Ｂ９５－８株ＤＮＡ全序列及编码ＥＢＶ胸苷激酶（ＴＫ）的开放读框ＢＸＬＦ１的结构,设计合成一对引物,并在引物的５′一端分别引入ＥｃｏＲＩ和ＰｓｔＩ切点,用ＰＣＲ技术扩增出一含完整的ＥＢＶＴＫ基因的１．８４３ＫｂＤＮＡ片段,ＮｃｏＩ酶切分析鉴定,ＥｃｏＲＩ／ＰｓｔＩ双酶切ＰＣＲ产物和载体,使目的基因定向克隆至选     

鸡传染性法氏囊病病毒内蒙古毒株VP2基因的克隆和序列分析

以鸡传染性法氏囊病病毒内蒙古毒株（ＩＢＤＶ-ＮＭ）ｄｓＲＮＡ为模板,用ＲＴ-ＰＣＲ法扩增其主要寄主保护抗原ＶＰ２基因的全长ｃＤＮＡ,克隆于ｐＵＣ１９的ＸｂａＩ／ＫｐｎＩ位点,进行了全序列分析。序列比较发现ＩＢＤＶ-ＮＭ毒株ＶＰ２基因与已报道的其它７个ＩＢＤＶＣＪ８０１ｂｋｆ、Ｃｕ１、ＰＢＧ９８、５２／７０、００２—７３、ＳＴＣ及ＶａｒｉａｎｔＥ毒株之间高度同源,其核苷酸序列的同源率为９１.６％～９６．２％,推测的氨基酸序列的同源率为９６.２％～９８．６％。ＩＢＤＶ-ＮＭ毒株ＶＰ２高变异区的第一个亲水区氨基酸序列与ＣＪ８０１ｂｋｆ、Ｃｕ１、ＰＢＧ９８、５２／７０、ＳＴＣ、００２—７３比较,有一个氨基酸差异,第二个亲水区氨基酸序列与上述６个毒株完全相同。而与ＶａｒｉａｎｔＥ比较,两个亲水区内各有两个氨基酸差异。此外,ＩＢＤＶ-ＮＭ毒株ＶＰ２具有强毒株所特有的７肽保守区：ＳＷＳＡＳＧＳ。这些结果表明,ＩＢＤＶ-ＮＭ毒株为标准血清Ⅰ型ＩＢＤＶ强毒株。     

中国人白细胞介素-12 cDNA基因的克隆及序列分析与比较

为研究中国人ＩＬ－１２的基因特征,采用逆转录巢式聚合酶链反应（ＲＴ－ｎＰＣＲ）从中国人脐带血单核细胞中分别克隆了Ｐ３５、Ｐ４０两亚基ｃＤＮＡ基因,包括完整的前体蛋白编码序列,其中Ｐ３５ ｃＤＮＡ编码２１９个氨基酸的多肽,Ｐ４０ ｃＤＮＡ编码３２８个氨基酸的多肽,与国外序列（ＮＫＳＦ、ＣＬＭＦ）比较结果发现：所克隆序列Ｐ３５同ＮＫＳＦ相比,第４４ａａ密友子由ＧＴＣ（Ｖａｌ）→ＧＴＧ（Ｖａｌ）,但未改     

黑麦1R染色体特异性PCR引物的分子证据

根据黑麦（ＳｅｃａｌｅｃｅｒａｌｅＬ．）与小麦（ＴｒｉｔｉｃｕｍａｅｓｔｗｕｍＬ．）ｒＲＮＡ基因间隔区序列差异,按Ｋｏｅｂｎｅｒ设计的引物序列,合成了黑麦特异引物ＮＯＲ－Ｒ１。运用该引物对不同植物材料进行ＰＣＲ扩增,观察表明,含有黑麦１Ｒ染色体的植物均扩增出黑麦的特异带,但含有其他黑麦染色体的小麦种质,普通小麦品种及其近缘物种长穗偃麦草（Ａｇｒｏｐｙｒｏｎｅｌｏｎｇａｔｕｍ（Ｈｏｓｔ）Ｂｅａｕｖ）     

猪繁殖和呼吸综合征病毒膜蛋白和核衣壳蛋白基因在大肠杆菌中的表达

以 Ｐ Ｒ Ｒ Ｓ Ｖ 弱毒株膜蛋白（ Ｍ） 和核衣壳（ Ｎ） 蛋白基因为模板,设计的一对含有 Ｅｃｏ Ｒ Ｉ 和 Ｂａｍ Ｈ Ｉ酶切位点的引物,通过 Ｒ Ｔ Ｐ Ｃ Ｒ 扩增出一约９００ ｂｐ 的 Ｍ Ｎ 基因片段,将此基因片段成功克隆于高效表达载体ｐ Ｂ Ｖ２２０ ,构建成重组质粒ｐ Ｂ Ｖ Ｍ Ｎ,导入大肠杆菌 Ｄ Ｈ５α,经温敏诱导,成功地表达了 Ｍ Ｎ 基因。表达产物经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 电泳和 Ｗｅｓｔｅｒｎ ｂｌｏｔ 印迹分析,其分子量约３４ ｋ Ｄ,与兔抗 Ｐ Ｒ Ｒ Ｓ Ｖ 高免血清发生反应,经光密度扫描分析,表达产物量占菌体总蛋白的１２ ％ 。该研究为 Ｐ Ｒ Ｒ Ｓ 基因诊断抗原的研制奠定了基础     

PCR法快速检测临床标本中结核杆菌DNA

应用聚合酶链反应（ＰＣＲ）快速检测临床标本（脑脊液、胸水、腹水、血、痰液）中的结核杆菌ＤＮＡ,特异性扩增片段１２３ｂｐ,为结核杆菌的特异性重复序列ＩＳ６１１０部分基因。ＰＣＲ检测人型结核杆菌的敏感性达１０ｆｇＤＮＡ。临床标本的ＰＣＲ检测阳性率（２３．３％）明显高于抗酸染色涂片（２．９％）和细菌培养（５．７％）的阳性率（Ｐ〈０．０５）。通过设立对照系统及对扩增产物酶切分析,表明该法无假阴性结果（特异     

鳜鱼传染性脾肾坏死病毒的胞嘧啶5-甲基转移酶基因结构及其序列分析

对鳜鱼传染性脾肾坏死病毒（infectious spleen and kidney necrosis virus,ISKNV）的胞嘧啶5－甲基转移酶（MTase）基因的结构及序列进行了分析。序列比较分析表明,ISKNV MTase编码区全长684bp,编码长227个氨基酸的蛋白质,推测分子量为25855D。与一些细菌的MTase比较,ISKNV MTase也含有负责转移甲基的4个保守区,但缺乏识别靶序列的保守区。比较ISKNV与其它6种脊椎动物虹彩病毒的MTase序列并建立系统树,ISKNV显著不同于蛙病毒属和淋巴囊肿病毒属。7种脊椎动物虹彩病毒MTase具有高度保守区,可以此设计引物用PCR方法鉴定脊椎动物虹彩病毒。     

Outbreaks and risks of infectious spleen and kidney necrosis virus disease in freshwater ornamental fishes

We examined the distribution of iridoviruses in 10 freshwater ornamental fish species hatched in Korea and imported from other Asian countries using both 1-step and 2-step polymerase chain reation (PCR). None of the 10 fish species analyzed were free of iridovirus as shown by 2-step PCR positive results, and 3 species yielded 1-step PCR positive results with associated mortality. Cloned PCR amplicons of the adenosine triphosphatase (ATPase) and major capsid protein (MCP) genes in genomic DNA of iridovirus showed the same nucleotide sequences as that of infectious spleen and kidney necrosis virus (ISKNV) isolated from the mandarinfish Siniperca chuatsi. These results indicate the presence of ISKNV disease in various ornamental fish as new host species and that the disease is widespread throughout different Asian countries including Korea, Singapore and China. Such infections were either clinical with associated mortality (and 1-step PCR positive) or asymptomatic in fish that were externally healthy (and only positive in 2-step PCR). Molecular analyses of the K2 region performed on iridovirus samples isolated from freshwater ornamental fishes revealed deletion/insertion of repetitive sequences of various lengths (42 to 339 bp), depending on the ISKNV isolates, without substitutions. Experimental infection of pearl gourami Trichogaster leeri and silver gourami T. microlepis with a tissue homogenate of pearl gourami infected by ISKNV induced 70 and 20% cumulative mortalities in the pearl and silver gourami, respectively.     

Infectious spleen and kidney necrosis virus (a fish iridovirus) enters Mandarin fish fry cells via caveola-dependent endocytosis

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. Megalocytiviruses have been implicated in more than 50 fish species infections and currently threaten the aquaculture industry, causing great economic losses in China, Japan, and Southeast Asia. However, the cellular entry mechanisms of megalocytiviruses remain largely uncharacterized. In this study, the main internalization mechanism of ISKNV was investigated by using mandarin fish fry (MFF-1) cells. The progression of ISKNV infection is slow, and infection is not inhibited when the cells are treated with ammonium chloride (NH(4)Cl), chloroquine, sucrose, and chlorpromazine, which are inhibitors of clathrin-dependent endocytosis. The depletion of cellular cholesterol by methyl-β-cyclodextrin results in the significant inhibition of ISKNV infection; however, the infection is resumed with cholesterol replenishment. Inhibitors of caveolin-1-involved signaling events, including phorbol 12-myristate 13-acetate (PMA), genistein, and wortmannin, impair ISKNV entry into MFF-1 cells. Moreover, ISKNV entry is dependent on dynamin and the microtubule cytoskeleton. Cofraction analysis of ISKNV and caveolin-1 showed that ISKNV colocates with caveolin-1 during virus infection. These results indicate that ISKNV entry into MFF-1 cells proceeds via classical caveola-mediated endocytosis and is dependent on the microtubules that serve as tracks along which motile cavicles may move via a caveola-caveosome-endoplasmic reticulum (ER) pathway. As a fish iridovirus, ISKNV entry into MFF-1 cells is different from the clathrin-mediated endocytosis of frog virus 3 entry into mammalian cells (BHK-21) at 28°C, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection.     

Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders Paralichthys olivaceus in Korea

In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea. The full open reading frames (ORFs) encoding the major capsid protein (MCP) (1362 bp) from the 13 flounder iridoviruses (FLIVs) were sequenced and the deduced amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the MCP revealed that all 13 FLIVs were the same species as rock bream iridovirus (RBIV), red sea bream iridovirus (RSIV), and infectious spleen and kidney necrosis virus (ISKNV), and were grouped into an unknown genus which was different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus. This is the first report on the isolation and phylogenetic analysis of the iridovirus of unknown genus from flounders during epizootics.     

Infectious spleen and kidney necrosis virus ORF48R functions as a new viral vascular endothelial growth factor

Infectious spleen and kidney necrosis virus (ISKNV) causes a pandemic and serious disease in fish. Infection by ISKNV causes epidermal lesions, in which petechial hemorrhages and abdominal edema are prominent features. ISKNV ORF48R contains a domain similar to that of the platelet-derived growth factor and vascular endothelial growth factor (VEGF) families of proteins. ISKNV ORF48R showed higher similarity to the VEGFs encoded by Megalocytivirus and Parapoxvirus than to those encoded in fish and mammals. We used zebrafish as a model and constructed a recombinant plasmid containing the DNA sequence of ISKNV ORF48R to study ISKNV infection. The plasmid was microinjected into zebrafish embryos at the one-cell stage. Overexpression of the ISKNV ORF48R gene results in pericardial edema and dilation at the tail region of zebrafish embryos, suggesting that ISKNV ORF48R induces vascular permeability. ISKNV ORF48R is also able to stimulate a striking expression of flk1 in the zebrafish dorsal aorta and the axial vein. Furthermore, ISKNV ORF48R, while cooperating with zebrafish VEGF(121), can stimulate more striking expression of flk1 than can either ISKNV ORF48R or zebrafish VEGF(121) alone. However, decreased expression of FLK-1 by gene knockdown results in the disappearance of pericardial edema and dilation at the tail region of zebrafish embryos induced by overexpression of ISKNV ORF48R in the early stages of embryonic development.     

云斑尖塘鳢肿大细胞病毒属虹彩病毒的分离与鉴定

2009年10月, 广东顺德地区一云斑尖塘鳢养殖场暴发不明病因疾病, 发病尖塘鳢体长15-18 cm不等,死亡率约85%, 濒死尖塘鳢从池塘底层游至水面, 呈现游动失衡状态直至死亡。死亡尖塘鳢腹部膨大,剖检可见肝脏、脾脏、肾脏肿大, 有出血斑点, 从内脏器官肝脏、脾脏和肾脏未分离到致病菌。病鱼内脏组织研磨过滤除菌后,腹腔注射20尾尖塘鳢, 7d后开始出现死亡, 10d后全部死亡, 对照组无死亡。自然发病鱼和人工感染鱼的病理切片显示肝脏、脾脏和肾脏出现大量肿大细胞,超薄切片经电子显微镜观察, 肝脏、脾脏和肾脏观察到大量病毒颗粒。电镜下病毒呈六边形,直径约135 nm,形态与虹彩病毒相似。针对虹彩病毒主衣壳蛋白(Major capsid protein,MCP)序列设计引物,提取自然发病鱼和人工感染鱼的DNA作为模板, 均能扩增出预期大小的特异性产物。利用NCBI的Blast搜索, 结果显示扩增序列与肿大细胞病毒属的传染性脾肾坏死病毒(ISKNV)、闪电丽鱼虹彩病毒(DGIV)和条石鲷虹彩病毒(RBIV)MCP核苷酸序列同源性分别为98.8%、98.1%和94.7%。利用MCP序列构建的系统发育树显示, 导致云斑尖塘鳢发病死亡的病毒为肿大细胞病毒属虹彩病毒, 暂命名云斑尖塘鳢虹彩病毒(MSGIV)。       

Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection

An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue–green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140 nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0 kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e−124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.     

Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection

An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue-green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e-124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.     

Iridovirus disease in two ornamental tropical freshwater fishes: African lampeye and dwarf gourami

Many species of ornamental freshwater fishes are imported into Japan from all over the world. We found African lampeye Aplocheilichthys normani and dwarf gourami Colisa lalia suffering from an iridovirus infection just after being imported by tropical fish wholesalers from Singapore. African lampeye were cultured on the Indonesian Island of Sumatra and dwarf gourami were cultured in Malaysia before export. Diseased fishes displayed distinct histopathological signs of iridovirus infection: systemic appearance of inclusion body-bearing cells, and necrosis of splenocytes and hematopoietic cells. Electron microscopy revealed viral particles (African lampeye:180 to 200 nm in edge to edge diameter; dwarf gourami: 140 to 150 nm in diameter) in an inclusion body within the cytoplasm of inclusion body-bearing cells as well as in the cytoplasm of necrotized cells. Experimental infection with an iridovirus isolate from African lampeye (ALIV) revealed pathogenicity of ALIV to African lampeye and pearl gourami Trichogaster leeri. Polymerase chain reaction (PCR) products from ALIV and an iridovirus isolate from dwarf gourami (DGIV) using iridovirus-specific primers were indistinguishable. The nucleotide sequence of PCR products derived from ALIV (696 base pairs) and DGIV (701 base pairs) had 95.3% identity. These results indicate that ALIV and DGIV have a single origin.