用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物,用ＰＣＲ技术,从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中,分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体,获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明,该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点,而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明,质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒,插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。     

单纯疱疹病毒通用及Ⅱ型特异性DNA探针的制备及应用研究

单纯疱疹病毒（ＨＳＶ）Ⅰ型及Ⅱ型之间有很多共同抗原,能引起血清学交叉反应,鉴别诊断比较困难。本实验利用重组ＤＮＡ技术,将部分ＨＳＶ－２ＤＮＡ的ＰｓｔＩ片段克隆到载体质粒ＰＳＫ中,并筛选出两个重组质粒（Ｐ和Ｐ）只与ＨＳＶ－２反应,与ＨＳＶ－１不反应,这两个重组质粒中所含的ＨＳＶ－２ＤＮＡ片段大小分别是３．１和４．３ｋｂ,另外,还筛选了一个重组质粒（ＰＨＳＶ２－１,含５．８ｋｂＨＳＶ－２ＤＮＡ片段）与ＨＳＶ－１和ＨＳＶ－２均反应。将４．３ｋｂ的片段用光生物素标记后作为探针检测了１５９份人阴道拭子,其中２３份样品呈阳性反应,其余均为阴性,从２３份阳性样品中挑选１２价涂片用间接荧光抗体法检测也都呈阳性反应,随机挑选的几份杂交反应阴性样品在间接荧光试验中也是阴性。本实验制备的ＨＳＶ通用及ＨＳＶ－２型特异性探针将比常规的血清学方法诊断和鉴别ＨＳＶ－１和ＨＳＶ－２感染更为可靠。     

广西HBsAg阳性母亲的胎儿肝,肾细胞中乙肝病毒DNA存在状态的研究

要用核酸分子杂交Ｓｏｕｔｈｅｒｎ印迹法,以＾３２Ｐ标记的ＨＢＶＤＮＡ为探针,检测ＨＢｓＡｇ阳性母亲引产的４０例胎儿的肝,肾组织。结果有２例胎肝和１例胎肾细胞ＤＮＡ出现大于３．２ｋｂ的杂交带,表明的ＨＢＶＤＮＡ已处于整合状态,胎肾细胞基因组中查出ＨＢＶＤＮＡ整合为首次报道。     

广西HBsAg阳性母亲的胎儿肝、肾细胞中乙肝病毒DNA存在状态的研究

采用核酸分子杂交Ｓｏｕｔｈｅｒｎ印迹法,以３２Ｐ标记的ＨＢＶＤＮＡ为探针,检测ＨＢｓＡｇ阳性母亲引产的４０例胎儿的肝、肾组织。结果有２例胎肝和１例胎肾细胞ＤＮＡ出现大于３．２ｋｂ的杂交带,表明ＨＢＶＤＮＡ已处于整合状态。胎肾细胞基因组中查出ＨＢＶＤＮＡ整合为首次报道。     

中国棉铃虫核型多角体病毒不同基因型的虫体克隆

首次利用虫体克隆技术,对野生型中国棉铃虫核型多角体病毒（ＨａＳＮＰＶＷ）的不同基因型进行了分离纯化。以较低浓度的ＨａＳＮＰＶＷ感染三龄初中国棉铃虫幼虫,病毒致死幼虫单虫收集,分别提取相应的病毒核酸,进行限制性内切酶分析,得到了ＨａＳＮＰＶ的七个不同基因型（ＨａＳＮＰＶＧ１至Ｇ７）,其ＥｃｏＲＩ酶切图谱均不相同。用ＢａｍＨＩ酶切分析,七个基因型的酶切图谱只在一个片段上有显著区别：Ｇ１、Ｇ２、Ｇ３、Ｇ４ＤＮＡ的ＢａｍＨＩｈ片段大小是３．３０ｋｂ,Ｇ５、Ｇ６的这一片段的大小为３．５ｋｂ,而Ｇ７的这一片段为２．７ｋｂ。结果表明,野生型ＨａＳＮＰＶ至少包含七个不同的基因型。     

紫云英根瘤菌Exo^—变种的生理遗传及胞外多糖组分分析

从质粒ｐＪＢ１１－Ｂ６的８．５ｋｂ野生型ＤＮＡ片段中克隆到５．８ｋｂ和２．６ｋｂ的ＤＮＡ片段,其中５．８ｋｂ的片段互补紫云英根瘤菌１０７菌株的胞外多糖（ＥＰＳ）缺陷型变种ＮＡ０６和ＮＡ１２,２,２．６ｋｂ的片段只能互补变种ＮＡ１２。回接实验和电镜切片显示,这种个变种的根瘤与野生型显著不同,无固氮活性,根瘤内基本不含类菌体;它们的Ｅｘｏ＾＋回复子的根瘤与野生型根瘤相似,多数细胞胞含有类菌体,但仍无固     

草鱼出血病病毒多肽的基因定位

用聚丙烯酰胺凝胶电泳分离纯化的草鱼出血病病毒ＧＣＨＶ８７３的基因组ｄｓ－ＲＮＡ的１１个片段,分别在麦胚无细胞翻译体系中进行翻译。其翻译产物经ＳＤＳ－ＰＡＧＥ系统分析。结果表明,基因组片段１、２、３、４、５、和１０分别编码病毒核心衣壳的结构多肽ＶＰ１、ＶＰ２、ＶＰ３、ＶＰ４、ＶＰ５和ＶＰ１０,片段６和７分别编码病毒外层衣壳的结构多肽ＶＰ６和ＶＰ７。片段８和９分别编码５２ｋＤ和４１ｋＤ的多肽,片段１１编码两种多肽,分子量分别为２９ｋＤ和１９．５ｋＤ,它们与病毒结构多肽无明显对应关系。病毒基因组与多肽大体是一一对应的关系。     

曼氏血吸虫发育过程中的基因组DNA变异

曼氏血吸虫（Ｓｃｈｉｓｔｏｓｏｍａｍａｎｓｏｎｉ）和日本血吸虫（Ｓ．ｊａｐｏｎｉｃｕｍ）成虫和曼氏血吸虫尾蚴基因组ＤＡＮ经限制性内切酶ＢａｍＨＩ消化后,分别与＾３２ｐ－ｄＣＴＰ标记的来源于核糖体ＤＮＡ的ＰＳＭＨＣＲ５、ＰＳＭＨＣＲ４ＰＳＭ８８９探针杂交,曼我血吸虫尾蚴和成虫在ＰＳＭＨＣＲ４杂交带型的１．６－２．８ｋｂ之间,存在有明显不同的次杂带;而ＰＳＭＨＣＲ５和ＰＳＭ８８９的杂交带型,无明显差异     

鸡减蛋综合征病毒（EDSV—76）基因组E1区结构特点分析

ＥＤＳＶ－７６病毒中国株ＡＡ－２经常规方法提取其病毒ＤＮＡ后,建立了限制性内切酶ＰｓｔＩ水解片段的全基因文库。对其中ＰｓｔＩ－Ｇ片段和ＰｓｔＩ－Ａ片段的正反链进行序列测定,获得ＥＤＳＶ Ｅ１区（０－８．８ｍ．ｕ）的核苷酸序列。经分析,ＥＤＳＶ Ｅ１区具有与其他腺病毒Ｅ１区类似的结构。以大于６０个氨基酸残基为标准,ＥＤＳＶ Ｅ１区共有７个开放读码框架（ＯＲＦ）,其中Ｒ１、Ｒ２、ＥｌｂｓＴ和Ｅ１ｂ１Ｔ     

兔出血症病毒结构多肽分析

粗提病毒经Ｓｅｐｈａｒｏｓｅ４Ｂ层析后,获得纯化的兔出血症病毒（ＲＨＤＶ）。提纯病毒经过ＳＤＳ－ＰＡＧＥ经考马斯亮蓝染色显示Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆ和Ｇ７条多肽,凝胶扫描显示Ａ为ＲＨＤＶ主要结构多肽。用多抗和单抗作免疫转印分析,证实Ａ、Ｂ、Ｃ、Ｄ、Ｅ、和Ｇ为结构多肽,此６条结构多肽间的抗原关系十分密切。     

European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses.

Monoclonal antibodies directed against the capsid protein of rabbit hemorrhagic disease virus (RHDV) were used to identify field cases of European brown hare syndrome (EBHS) and to distinguish between RHDV and the virus responsible for EBHS. Western blot (immunoblot) analysis of liver extract of an EBHS virus (EBHSV)-infected hare revealed a single major capsid protein species of approximately 60 kDa that shared epitopes with the capsid protein of RHDV. RNA isolated from the liver of an EBHSV-infected hare contained two viral RNA species of 7.5 and 2.2 kb that comigrated with the genomic and subgenomic RNAs of RHDV and were recognized by labeled RHDV cDNA in Northern (RNA) hybridizations. The nucleotide sequence of the 3' 2.8 kb of the EBHSV genome was determined from four overlapping cDNA clones. Sequence analysis revealed an open reading frame that contains part of the putative RNA polymerase gene and the complete capsid protein gene. This particular genome organization is shared by RHDV but not by other known caliciviruses. The deduced amino acid sequence of the capsid protein of EBHSV was compared with the capsid protein sequences of RDDV and other caliciviruses. The amino acid sequence comparisons revealed that EBHSV is closely related to RHDV and distantly related to other caliciviruses. On the basis of their genome organization, it is suggested that caliciviruses be divided into three groups.     

一株兔出血症病毒2型毒株的分离鉴定

【背景】我国于2020年4月突发兔出血症病毒2型(Rabbit Hemorrhagic Disease Virus 2,RHDV2)疫情,严重威胁兔养殖业和生态平衡,而且目前国内对RHDV2的病原学以及遗传特征等基础研究匮乏。【目的】分离鉴定RHDV2毒株,对分离株进行全基因测序与遗传进化分析。【方法】对成都某兔场疑似RHDV2感染致死的家兔进行病理剖检,通过RT-qPCR检测和动物回归试验,分离鉴定得到RHDV2毒株,进一步进行全基因测序及遗传进化分析。【结果】病死兔剖检表现为各实质器官出血肿大,以心、肺、肝脏尤为明显,经RT-qPCR确诊为RHDV2,而且不存在其他病原混合感染,试验感染家兔可致相似病变。将分离株命名为SCCN03,其基因序列全长为7464bp,与参考毒株(GenBank登录号为MN901451.1)一致性为99.21%。对比参考株氨基酸序列,分离株的非结构蛋白和结构蛋白氨基酸序列发生了多处错义突变,其中非结构蛋白p16和结构蛋白的几处错义突变可能与毒株变异有关。进化树显示毒株SCCN03属于GI.2基因型。【结论】分离鉴定出一株RHDV2毒株,获得其基因序列,丰富了RHDV2的全基因数据资料,为后续RHDV毒力相关研究和相关疫苗研发奠定了基础。     

兔出血症病毒2型理化特性研究

[目的]本研究旨在研究该病毒的理化特性,评价不同处理条件对RHDV2的杀灭效果.[方法]本研究拟对临床疑似RHDV2感染致死的家兔进行RT-PCR鉴定病原,并利用不同pH值、不同温度、常用兽用消毒剂、不同浓度甲醛处理RHDV2,通过PMA-RT-qPCR对病毒理化特性进行研究.[结果]经RT-PCR检测与测序分析,确诊...     

A Comparison of Digoxigenin and Biotin Labelled DNA and RNA Probes for in Situ Hybridization

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes acheived maximal sensitivity without use of biotin-streptavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.     

A direct effect of growth hormone on the incorporation of precursors into proteins and nucleic acids of perfused rat liver

1. The livers of rats were perfused in situ. When the amino acid concentration in the perfusing medium was that present in rat plasma, the addition of growth hormone to the medium stimulated the incorporation of labelled amino acids into liver protein only marginally and not to a statistically significant extent. When, however, the amino acid concentration was raised to three times that present in rat plasma, growth hormone significantly and substantially stimulated amino acid incorporation into protein within 30min. of perfusion of normal rat liver. 2. A significant effect of growth hormone on labelling of normal rat-liver protein was seen with concentrations not much greater than those reported to be present in rat plasma. 3. The labelling of nucleic acids of normal and hypophysectomized rat liver by [(3)H]orotic acid was enhanced by addition of growth hormone to the perfusing medium when normal concentrations of amino acids were used. 4. At elevated concentrations of amino acids, growth hormone stimulated labelling of nucleic acids of hypophysectomized rat liver at 30 and 60min. of perfusion. Under these conditions, nucleic acids of normal rats were labelled to about the same extent in control and hormone-treated livers at 30min. and, because of a fall in the radioactivity of the control livers, there was more labelled nucleic acids in growth-hormone-treated livers at 60min. than in the control livers. 5. Growth hormone, unlike insulin, had no inhibitory effect on the release of glucose by the perfused liver. 6. It is concluded that growth hormone can stimulate the incorporation of precursor into proteins and nucleic acids of liver directly and without the mediation of other organs or of insulin.     

兔的一种新病毒：Ⅱ.一株兔出血症...

In this paper a strain of Rabbit Hemorrhagic Disease Virus (RHDV) was isolated and purified from the diseased rabbit livers with a method of using chloroform, two-phase of polyethylene-glycol-dextran sulfate sodium and sucrose density gradient centrifugation. Purified virus was nonenveloped, icosahedeal symmetry with a triangulation number of 3, and 33-37 nm in diameter. The capsid was composed of 32 capsomeres with central holes in an outer diameter of about 9nm. Two types of viral particles having different sedimentation coefficient, 130s and 166s could be identified after sucrose density gradient centrifugation. Probably no less than four virion proteins with molecular weight of 66.4, 65.0, 63.5, 41.0 x 10(3) dalton were detected by SDS-polyacrylamide gel electrophoresis. Viral nucleic acid was extracted from purified virus by using SDS-proteinase K-phenol. Tests with diphenylamine, formaldehyde, and staining with acridine orange as well as the curves of thermal denaturation showed that this kind of virus had a single-stranded DNA. The molecular weight of the ssDNA was approx 2.1 x 10(6) dalton as determined by electron microscopy. Data indicate that the RHDV may like the parvovirus of the family Parvoviridae.