豇豆病毒病病原的分子鉴定

为澄清浙江省豇豆病毒病病原及其分类,采用马铃薯Y病毒属通用引物Sprimer扩增了浙江豇豆线状病毒基因组3′－末端序列。同时又根据已知CMV RNA3序列设计引物pCMV-44-63/1200-1181,扩增了混合的CMV－ZJ运动蛋白基因全序列。外壳蛋白氨基酸序列分析表明,产中仅存在一种线状病毒（CV－ZJ）,该病毒与一泰国豇豆蚜传花叶病毒（CABMV）具有最高的同源性（98.6%）,与花生条纹病毒（PStV）、石斛花叶病毒（DeMV0．）、赤豆花叶病毒（AZMV）、黑眼豆豆花叶病毒（BICMV）和菜豆普通花叶病毒（BCMV）分离物间的同源性为88.5%－94.4%,而与其它CABMV分离物的同源性均低于70％。进化树分析表明,PStV、AZMV、DeMV、BlCMV和BCMV为同种异名,应统称为BCMV,而CABMV为另一种病毒;CV－ZJ和泰国CABMV分离物应分类为BCMV豇豆株系。CMV－ZJ的运动蛋白序列分析表明,该分离物属于CMV亚组Ⅰ,与其它分离物氨基酸序列同源性为93.1%－97.8%。     

用A蛋白夹层酶联免疫吸附法对黄瓜花叶病毒的血清学鉴定

用A蛋白夹层酶联免疫吸附法(PAS-ELISA)对两个来自日本蓉茄和一个来自中国青椒植物上的黄瓜花叶病毒分离物进行了血清学鉴定和比较。该方法利用A蛋白和酶联A蛋白分别作预包被和检测结合于抗体一抗原一抗体夹层中的抗体,并通过底物反应,间接反应出病毒抗原的量。对经过两次差速离心纯化的病毒进行检测的结果表明,这三种黄瓜花叶病毒(CMV)分离物与黄瓜花叶病毒P、Q两株系同属一个血清型,即可能均属于黄瓜花叶病毒中的ToRS组。讨论了这种间接酶联免疫吸附法检测植物病毒的优越性和几个CMV分离物的提纯、保存方法。     

引起番茄坏死病的黄瓜花叶病毒TN分离物的研究

从南京郊区田间番茄坏死病株中分离出黄瓜花叶病毒ＴＮ分离物,经人工接种１０科３９种植物,能侵染其中的８科２６种,接种番茄,辣椒和普通烟都引起坏死症状。ＴＮ分离物的失毒温度为５０℃,稀释限点５×１０￣（－３）－５×１０￣（－４）,体外存活期１－２天。蚜虫以非持久性方式传毒,经免疫双扩散测定,ＴＮ分离物与黄瓜花叶病毒抗血清呈阳性反应。用差速离心提纯的病毒粒子经电镜观察为廿面体,直径为２８ｎｍ。ＳＤＳ－ＰＡＧＥ分析病毒外壳蛋白为单一组份,分子量２７５００Ｄ。病毒核酸组份分析发现,该病毒分离物含有ＣＭＶ基因组核酸和一个低分子量的卫星ＲＮＡ。以上结果同已报道的引起番茄坏死的带卫星ＲＮＡ的黄瓜花叶病毒株系一致（Ｋｅａｒｎｅｙ,Ｃ．Ｍ．１９９０;Ｊｏｒｄａ,Ｃ．１９９２）。本文还讨论了该病害的发生和流行。     

一个引起玉米矮花叶病的甘蔗花叶病毒基因组全序列测定及其结构分析

测定了从浙江省呈现矮花叶病症状的玉米上分离得到的一个马铃薯Y病毒属病毒RNA的核苷酸全序列. 该病毒分离物的RNA基因组由9596个核苷酸组成(不包括polyA尾). 单一的ORF由9192个核苷酸组成, 编码一个分子量为346.1 ku的聚合蛋白. 该蛋白结构特征与高粱花叶病毒(SrMV)中国甘蔗分离物和一个玉米矮花叶病毒(MDMV)保加利亚分离物基因组编码的蛋白非常相似. 序列分析表明, 该病毒分离物与甘蔗花叶病毒(SCMV)各分离物(已报道的仅为基因组3′末端序列)同源性最高, 与SrMV和MDMV同源性次之, 而与约翰逊草花叶病毒(JGMV)同源性最低. 根据马铃薯Y病毒属区分不同病毒和株系的分类标准, 报道的玉米病毒分离物应当鉴定为SCMV的一个株系. 然而, 该分离物在HC-Pro, P3和CI蛋白区域和SrMV中国甘蔗分离物具有极高的氨基酸同源性.     

一个侵染山东大葱的胡葱黄条病毒分离物的鉴定

在山东章丘大葱上发现了一种引起畸形黄条症状的线状病毒。该病毒容易摩擦接种侵染大葱和胡葱,但不侵染其它22种测试植物,能经由豌豆蚜传播。病毒粒子形态和细胞病理特征具有马铃薯Y病毒科成员的典型特征。Western blot分析表明,提纯病毒制备的抗血清能与病毒自身外壳蛋白起特异性的强反应,与薤花叶病毒、晚香玉轻斑驳病毒、小西葫芦黄花叶病毒和芋花叶病毒外壳蛋白有较弱的反应。采用马铃薯Y病毒科简并引物PCR技术扩增了该病毒的基因组3′-末端部分序列,序列比较表明它为胡葱黄条病毒(SYSV),系统进化树分析表明世界范围内的SYSV分离物可以区分为4个主要群体,在一定程度上与寄主植物种类及地理分布相关。     

山东省玉米矮花叶病毒的生物学特性及基因组全序列测定

对山东省玉米矮花叶病毒原分离物(SD)进行了寄主范围、血清学等普通生物学鉴定,测定了该病毒的基因组核苷酸全序列。该病毒基因组RNA由9596个核苷酸组成(不包括3’—polyA的长度),整个基因组按一个ORF编码一个3063个氨基酸的多聚蛋白。序列比较表明,该病毒分离物(SD)与玉米矮花叶病河南分离物(HN,EMBL登录号：AF94510)核苷酸全序列同源性最高,为98．2％,与已报道的甘蔗花叶病毒(SCMV)7个分离物同源性也高达79．5％-98．2％,但与玉米矮花叶病毒保加利亚分离物(MDMV—B8,AJ001691)和高梁花叶病毒萧山甘蔗分离物(SrMV—XoS,AJ310197)的同源性仅为67．8％和69．3％,与约翰逊草花叶病毒(226920,JGMV)差异最大。系统进化树分析也表明,该病毒与SCMV分离物位于同一进化簇,而与MDMV进化关系很远。     

蚕豆萎蔫病毒纯化和病毒蛋白质及RNA组份分析

属蚕豆萎蔫病毒（ＢＢＷＶ）血清ＩＩ型的分离物Ｂ－９３４接种昆诺藜,采收的病叶先用含蔗糖的高浓度磷酸钾盐缓冲液,后用ＴｒｉｔｏｎＸ－１００处理,成功地提取了大量病毒粒子,提纯病毒得率为１３４ｍｇ／ｋｇ病叶,提纯病毒在电镜下可观察到大量球状病毒粒子,直径约２５ｎｍ,提纯的ＢＢＷＶ经甘油梯度超离心后在可观察到３个组份,分别称Ｔ,Ｂ和Ｍ组份,其中Ｔ为空壳蛋白,Ｂ和Ｍ为核蛋白,各组份经ＳＤＳ－ＰＡＧＥ测定,     

建兰花哇病毒运动蛋白基因克隆及序列分析

从建兰花叶病毒（CyMV）石斛兰分离物中提取病毒RNA,用反转录－－聚合酶链式反就（RT－PCR）方法获得约500bp的运动蛋白基因片段,插入pGEM-T载体克隆并测序,序列分析表明,该基因片数由474个核苷酸组成,和CyMV美国夏威夷分离物、新加坡分离物相应基因核甘酸序列分别具有97.8%同源性;根据核酸序列推导该片断含有3个部分重叠的开放阅读框架（ORF）,分别编码14kD、12kD和10kD的多肽。     

黄瓜花叶病毒亚组I和Ⅱ分离物外壳蛋白基因的序列分析与比较

对我国分离的经生物学和血清学鉴定为黄瓜花叶病毒（ＣＭＶ）亚组Ｉ和Ⅱ的各一分离物（ＧＢ、ＸＢ）的外壳蛋白（ＣＰ）基因进行了序列分析和比较。以提纯病毒ＲＮＡ为模板,进行逆转录及ＰＣＲ扩增,并通过常规基因克隆方法得到插入ＣＰ基因片段的重组克隆。对插入ＧＢ和ＸＢ两个分离物ＣＰ基因片段的重组克隆进行全序列测定,结果表明重组克隆序列长分别为７７７ｂｐ和７９２ｂｐ均只含一个开放读框（ＯＲＦ）,长度为６５７ｎｔ,     

菜豆黄色花叶病毒(BYMV)—新疆苜蓿分离株的鉴定

从新疆苜蓿黄斑花叶病株上分离到病毒分离物M-4,该分离物能引起多种豆科值物系统花叶,并在藜科植物上产生局部褪绿斑,易经汁液摩擦接种和蚜虫传毒,不经菜豆种子传毒。病毒致死温度60—65℃,体外保毒期4—5天,稀释限点10~(-3)—10~(-4)。病毒粒体线状,长约660—740nm,宽15nm;在感病的寄主叶片细胞中,电镜观察到风轮状、带状和环状内含体。免疫电镜法测定,该分离物与菜豆黄色花叶病毒(BYMV)抗血清有血清反应。经SDS-聚丙烯酰胺凝胶电泳和氨基酸自动分析仪分析分别测得该病毒的衣壳蛋白亚基分子量为16,200道尔顿,氨基酸残基数128个。鉴定结果认为,分离物M-4是BYMV的一个株系。     

易发生聚集的重组HBcAg病毒样颗粒的纯化*

目的:探索针对易发生聚集的重组HBcAg病毒样颗粒(VLP)的有效纯化方案。方法:培养的大肠杆菌经IPTG诱导重组HBcAg蛋白的表达,菌体超声破碎后的离心沉淀用含有不同浓度尿素的PBS缓冲液重悬溶解,经密度梯度离心并结合电镜观察对VLPs行为进行分析鉴定。以Sepharose 4 FF凝胶过滤层析在选定的尿素条件下纯化沉淀溶解液,纯化获得的目的蛋白进一步在含30%山梨醇的PBS中脱盐去除尿素。整个过程以SDS-PAGE及电镜进行各步骤样品中目的蛋白的分析。结果:含有1mol/L尿素的PBS缓冲溶液重悬超声沉淀,可有效溶解聚集的VLPs,在蔗糖密度梯度离心中显示典型HBcAg VLPs的行为,且电镜观察颗粒形态结构完整。经1mol/L尿素下凝胶过滤,VLPs进一步获得纯化。在脱尿素过程中流动相采用含30%山梨醇的PBS,有效避免了VLPs在尿素去除后重新聚集。结论:尿素与山梨醇的联合应用,为具有聚集现象的VLPs纯化制备提供了一种有效解决方案。     

高效价甘薯羽状斑驳病毒抗血清的制备

用嫁接方法将甘薯羽状斑驳病毒（ＳＰＦＭＶ）接种到Ｉ．ｓｅｔｏｓａ上扩繁,以０．２ｍｏｌ／ＬｐＨ７．２ＰＢＫ缓冲液、垫层差速离心、蔗糖密度梯度离心提取纯化ＳＰＦＭＶ。纯化的ＳＰＦＭＶＯＤ２６０／２８０的比值为１．２５。将纯化的ＳＰＦＭＶ免疫家兔制备抗血清,在环状沉淀和微量沉淀试验中,用提纯病毒测定抗血清的效价均为１：４０９６;以ＳＰＦＭＶ－ＩｇＧ为第一抗体,应用Ｄｏｔ－ＥＬＩＳＡ对甘薯和Ｉ．ｓｅｌｏｓａ叶片中的ＳＰＦＭＶ分别作了测定。     

两步串联层析法纯化鼠抗人CD80单克隆抗体4E5

采用阴离子交换与凝胶过滤两步串联层析法,纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后,在Tris-HCl缓冲溶液(pH8.0, 50mmol/L)条件下上阴离子交换柱对目的单抗进行捕集,采用0-0.5 mol/L NaCl浓度分步洗脱;含目的单抗的洗脱馏分再上凝胶过滤柱纯化,用PB缓冲溶液（pH7.2, 20mmol/L）洗脱,获得目的单抗4E5,其生物学活性高、纯度大于95%,抗体总回收率达61%。     

Surface-displayed porcine reproductive and respiratory syndrome virus from cell culture onto gram-positive enhancer matrix particles

Vaccine immunization is now one of the most effective ways to control porcine reproductive and respiratory syndrome virus (PRRSV) infection. Impurity is one of the main factors affecting vaccine safety and efficacy. Here we present a novel innovative PRRSV purification approach based on surface display technology. First, a bifunctional protein PA-GRFT (protein anchor-griffithsin), the crucial factor in the purification process, was successfully produced in Escherichia coli yielding 80 mg/L of broth culture. Then PRRSV purification was performed by incubation of PA-GRFT with PRRSV and gram-positive enhancer matrix (GEM) particles, followed by centrifugation to collect virions loaded onto GEM particles. Our results showed that most of the bulk impurities had been removed, and PA-GRFT could capture PRRSV onto GEM particles. Our lactic acid bacteria-based purification method, which is promising as ease of operation, low cost and easy to scale-up, may represent a candidate method for the large-scale purification of this virus for vaccine production.     

丙型肝炎病毒核心重组抗原C_（27）的纯化

高效表达了ＨＣＶ核心区基因抗原之后,对表达蛋白Ｃ_（２７）进行了纯化。经研究,重组蛋白是以包涵体形式存在于宿主菌内的。Ｃ_（２７）重组蛋白分别经过包涵体洗涤、ＤＥＡＥ阴离子交换层析和Ｓ－２００分子筛两步柱层析纯化之后,纯度大于９５％,纯化得率为５３．２％,总回收率为１７．９％。纯化工艺流程简单、得率高,适合向规模化生产发展。     

Evaluation of viral clearance in the production of HPV-16 L1 virus-like particles purified from insect cell cultures

Biopharmaceutical products produced from cell cultures have a potential for viral contamination from cell sources or from adventitious introduction during production. The objective of this study was to assess viral clearance in the production of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs). We selected Japanese encephalitis virus (JEV), bovine viral diarrhea virus (BVDV), and minute virus of mice (MVM) as relevant viruses to achieve the aim of this study. A downstream process for the production of purified HPV-16 L1 VLPs consisted of detergent lysis of harvested cells, sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. The capacity of each purification/treatment step to clear viruses was expressed as reduction factor by measuring the difference in log virus infectivity of sample pools before and after each process. As a result, detergent treatment (0.5% v/v, Nonidet P-40/phosphate-buffered saline) was effective for inactivating enveloped viruses such as JEV and BVDV, but no significant reduction (< 1.0 log(10)) was observed in the non-enveloped MVM. The CsCl equilibrium density centrifugation was fairly effective for separating all three relevant adventitious viruses with different CsCl buoyant density from that of HPV-16 L1 VLPs (JEV, BVDV, and MVM = 4.30, 3.10, > or = 4.40 log(10) reductions). Given the study conditions we used, overall cumulative reduction factors for clearance of JEV, BVDV, and MVM were > or = 10.50, > or = 9.20, and > or = 6.40 log(10) in 150 ml of starting cell cultures, respectively.     

Isolation of the GDP binding protein from brown adipose tissue mitochondria of several animals and amino acid composition study in rat

The nucleotide binding protein (uncoupling protein, GDP binding protein) of brown adipose tissue mitochondria has been isolated from cold adapted rat, newborn guinea pig and newborn rabbit. The purification, using hydroxyapatite in sucrose gradient centrifugation, follows the procedures established previously for the isolation of this protein from cold adapted hamster. A similar degree of purification was obtained, reaching 60 μmol GDP bound/g protein. In SDS gel electrophoresis the purified protein gave a single band of M_r 32 000 from all species.     

产热凝胶土壤杆菌ATCC31749蛋白质组双向电泳图谱的构建

建立了热凝胶生产茵土壤杆茵茵体总蛋白的蛋白质提取方法和双向电泳方案,确定了使用蛋白质裂解液(7 mol/L尿素,2 mol/L硫脲,1%ASB-14去垢剂,40 mmol/L Tris,0.001%溴酚蓝,1 mmol/L EDTA,1%TBP和1%两性电解质)结合超声破碎法来提取茵体总蛋白的方案为最佳,选择17 cm...     

Hydrangea mosaic virus, a new ilarvirus from Hydrangea macrophylla (Saxifragaceae)

A previously unrecognised virus isolated from Hydrangea macrophylla with chlorotic mosaic leaf symptoms in West Sussex was named hydrangea mosaic virus (HydMV). HydMV was mechanically transmitted without difficulty to four of 16 species from three of five families, and was seed transmitted in Chenopodium quinoa, but was not aphid transmitted. Although relatively unstable in vitro, HydMV was purified by clarifying leaf extracts by emulsification with chloroform and acidification with citric acid, followed by differential centrifugation and sucrose density gradient centrifugation. Purified virus incompletely separated on sucrose density gradients into three components (T. M and B) with sedimentation coefficients (s^o_20w) of 86, 97, and 105 S respectively, but all particles had buoyant densities in caesium chloride of 1.37 g/cm³. Virus contained a single polypeptide species (mol. wt 26.4 times 10³), appeared quasiisometric to ovoid or elliptical, and measured c. 28 times 30 (T), 30 times 30 (M) or 30 times 32–38 nm (B). Single-stranded RNA species or mol. wt 1–25, 1–08, 0–83, 0–36 and 0–27 (RNA-1 to 5 respectively) were obtained from virus preparations but mixtures containing RNA-1 to 3 plus either RNA-4 + 5 or the coat protein, were infective. These properties suggest that HydMV has affinities with ilarviruses, but it showed no serological relationship to any of six ilarviruses or 42 other viruses.     

HIV-1 Gag蛋白在大肠杆菌表达系统中诱导和纯化条件的优化

为探讨诱导温度对于HIV-1 Gag在大肠杆菌中表达产物状态以及尿素浓度对蛋白纯化效果的影响, 将30oC和37oC诱导表达的包涵体分别溶于不同浓度的尿素, 比较溶解性的差异, 并比较复性的不同。将30oC诱导的目的蛋白分别用2 mol/L和8 mol/L尿素溶解后做层析分离, 比较两者的分离效果。结果发现, 与37oC相比, 30oC诱导表达的蛋白能有效溶于低浓度尿素, 并且更容易复性。与8 mol/L尿素溶解相比, 30oC诱导的包涵体用2 mol/L尿素溶解后通过凝胶过滤和离子交换层析纯化能得到更好的分离效果。这提示低温诱导的Gag包涵体中可能含有更多类似天然态构象的蛋白, 而低浓度尿素有利于保持包涵体中蛋白的天然态构象。从而为包涵体蛋白的诱导表达和分离纯化提供了参考。