两株蓖麻蚕核型多角体病毒DNA同源性的研究

两株不同来源的蓖麻蚕核型多角体病毒(ArscsNPV和ArNPV)经提纯后,使用SDS—苯酚抽提病毒核酸,并使用限制性内切酶EcoRI,BamHI酶解后,用分子杂交方法与缺口平移标记的ArscsNPV-DNA探针杂交,分析了两株蓖麻蚕NPV病毒核酸的同源性。EcoRI酶解的ArNPV-DNA产生8个片段,其中5个片段能与ArscsNPV-DNA探针杂交。BamHI酶解ArNPV-DNA产生7个片段,其中6个片段能与ArscsNPV-DNA探针杂交。结果表明:两株蓖麻蚕NPV之间病毒核酸具有很高的同源性。使用斑点杂交方法分析了ArscsNPV与ArNPV,柞蚕NPV及家蚕NPV之间的核酸同源性,结果表明:ArscsNPV与ArNPV,柞蚕NPV具有同源性。而与家蚕NPV无核酸同源性。     

两株粘虫核型多角体病毒理化性质的比较研究

电镜观察粘虫(Leucania separala)核型多角体病毒的不同株系内蒙株(简称N株)和阜阳株(简称F株)的多角体、病毒粒子及核衣壳在形态大小方面都没有很大差别,DNA分子大小也相近。但在SDS-PAGE多肽图谱及DNA酶切图谱上则表现出一定的差异。经SDS-PAGE所得病毒粒子多肽图谱N株有17条多肽,F株有19条多肽。两株病毒DNA经BamHI酶切N株获得7条带,F株则有8条带,说明DNA酶切位点不尽相同。据此,生化分析能更快速、准确地进行株系分类。     

蓖麻蚕核型多角体病毒多角体蛋白基因的克隆和核苷酸序列分析

蓖麻蚕(Attacus ricini)是我国特有蚕种,以其核多角体病毒(ArNPV)为载体有可能发展成为新的基因工程表达系统,我们建立了ArNPV基因库,并亚克隆了含多角体蛋白(Ph)基因DNA片段。对该1.1kb全长DNA片段进行序列分析,确定ArNPV Ph结构基因全长735bp,与苜蓿尺蠖NPV(AcNPV)、家蚕NPV(BmNPV)同源性分别为76%和81%,ArNPV 5'端调控结构Rohrmann box与各类NPV的Ph基因相似,但3'下游序列几无同源,显示了ArNPV Ph基因结构的特征性。同时,我们还对Ph基因启动子的其它结构特点作了剖析。     

棉铃虫（Heliothisspp）核型多角体病毒四个分离株的比较研究

本文从形态结构、生物活性、结构多肽、核酸限制性内切酶图谱和核酸同源性等方面对棉铃虫核型多角体病毒四个分离株（两个ＳＮＰＶ分离株：ＨａＳ、ＨｚＳ,和两个ＭＮＰＶ分离株：ＨａＭ１、ＨａＭ２）进行了比较研究。它们对中国棉铃虫二龄末三龄初幼虫的ＬＤ５０佰依次为３６１、３８７、２６３３、３５６０ＰＩＢｓ／克饲料,当感染剂过为５×ｌ０３ＰＩＢｓ／克饲料时,其ＬＴ５０值依次为４．６、４．９、６．４和６．６天。两个ＳＮＰＶ分离株的生物活性显著高于两个ＭＮＰＶ分离株。经ＳＤＳ－ＰＡＧＥ分析,四个分离株多角体蛋白均为一条带,两个ＭＮＰＶ分离株结构多肽均具有相同的迁移率,两个ＳＮＰＶ分离株的结构多肽图谱也颇相似,但ＳＮＰＶ与ＭＮＰＶ分离株之间带型相差较大。各分离株基出组经ＢａｍＨＩ、ＥｃｏＲＩ、ＨｉｎｄⅢ和ＸｂａＩ消化后,得到的内切酶图谱表现为两个ＮＭＰＶ分离株一致,两个ＳＮＰＶ分离株也很相似,而ＳＮＰＶ与ＭＮＰＶ分离株之间相差很大。严格条件杂交结果表明：两个ＳＮＰＶ分离株的基因组有较高的同源性,而ＳＮＰＶ与ＭＮＰＶ基因组之间的同源性极低。     

棉铃虫核型多角体病毒朝鲜分离株的初步研究

为了充分利用棉铃虫核型多角体病毒(Helicoverpa armigera,HaSNPV)资源,为开展害虫生物防治提供依据,对首次在朝鲜分离到的棉铃虫核型多角体病毒进行了研究.本文从形态结构,结构多肽.核酸限制性内切酶图谱等方面进行了研究.多角体直径0.36-1.3 μm,平均1.02μm,病毒粒子大小为326 nm×69nm.经SDS-PAGE分析,棉铃虫核型多角体朝鲜株多角体蛋白为一条带.多角体蛋白分子量为28.7kDa.棉铃虫核型多角体朝鲜株基因组经BamH I,EcoR I,HindⅢ和Pst I消化后,得到的内切酶图谱表现,与已报道的几个分离株比较类似.分子大小均为130.18kb.     

茶尺蠖核型多角体病毒多角体蛋白单克隆抗体的制备

为了建立一种基于免疫反应检测茶尺蠖核型多角体病毒的方法,以纯化后的茶尺蠖核型多角体病毒作为抗原,免疫BALB/c小鼠,将小鼠脾脏细胞与小鼠骨髓瘤细胞Sp2/0融合,经间接ELISA筛选及克隆得到了一株稳定分泌单克隆抗体的杂交瘤细胞株,命名为7D3。同时克隆并在大肠杆菌中表达了EoNPV多角体蛋白基因,获得重组多角体蛋白。经Western blotting鉴定,该抗体可与EoNPV的多角体蛋白特异性结合。利用制备EoNPV多角体蛋白的单克隆抗体,建立了间接ELISA测定EoNPV的方法。     

大尺蠖核型多角体病毒多角体蛋白基因的结构分析

大尺蠖核型多角体病毒(BsNPV)的多角体蛋白基因(ocu)定位在2．3kb的BamHl—H片段上,用xho1、sma1、HindIII和PstI构建了H片段的物理图谱,并测定了两端636bp序列。在5’端发现了杆状病毒ocu基因共有的典型特征,即：ATG起始区;启动子区(14bp保守序列);TATA box和CATA box区等。单一xhoI位点在ATG上游-15bp处,适于作为构建ocu基因转移载体的插入位点。在这一基因5’端序列中发现了五个反转重复单元CGAGC GCTCG,讨论了这一单元在杆状病毒ocu基因高效表达中的调控功能。     

异源多角体蛋白对家蚕核型多角体病毒粒子的包装

利用PCR方法从AcMNPV基因组DNA中分离出多角体蛋白基因 ,将该扩增片段克隆到转移载体pBacPAK8中 ,得到重组转移载体pOAc。将该质粒DNA与线性化的Bm BacPAK6病毒基因组DNA共传染BmN细胞 ,得到了能形成多角体且不产生蓝色空斑的重组病毒hp BmNPV。纯化该重组病毒的多角体颗粒 ,并对多角体蛋白、病毒核酸及多角体病毒颗粒进行分析 ,发现AcMNPV的多角体蛋白能在家蚕细胞中大量表达且能在细胞内识别家蚕核型多角体病毒并组装成多角体颗粒 ;病毒基因组DNA因部分交换 ,其酶切行为发生了相应的变化 ;电镜观察发现经AcMNPV多角体蛋白包装的家蚕核型多角体病毒的多角体颗粒大小为1 2 μm～ 2 9μm ,明显小于野生型家蚕核型多角体病毒的多角体颗粒     

雌雄蓖麻蚕减数分裂染色体的比较研究

以蓖麻蚕Philosamia cynthia riciniBoisduval为材料,全面比较了雌雄蓖麻蚕减数分裂前期I染色体的行为变化,从细胞学角度证明了雌蚕减数分裂染色体完全连锁,而雄蚕发生连锁交换;详细观察了雌蚕减数分裂I双线期过程中性染色体的动态变化,发现其存在着自身配对的过程,进而对其起源进化假说进行了探讨,认为其可能来源于一对同源常染色体片段的易位融合。     

蓖麻蚕核型多角体病毒DNA转染四种昆虫细胞系的研究

用蓖麻蚕核型多角体病毒(PcrNPV)DNA转染草地贪夜蛾(SF)、家蚕(Bm)、斜纹夜蛾(SL)和菜粉蝶(Pr)四种昆虫细胞系,结果表明,PcrNPV DNA能在SF细胞内复制增殖并形成多角体,Pr细胞系对PcrNPV DNA转染不敏感;Bm和SL细胞出现病变症状,但在电镜下未观察到病毒粒子或多角体。     

Performance of Ooencyrtus kuvanae (Hymenoptera: Encyrtidae) on two host species,Halyomorpha halys and Philosamia ricini

The brown marmorated stink bug, Halyomorpha halys, is an invasive agricultural pest of fruit trees and vegetables. Egg parasitoids play a key role in the reducing of H. halys populations. Ooencyrtus kuvanae (Howard) (Hymenoptera: Encyrtidae) can parasitize H. halys and complete its life cycle in this host species. Many factors can influence this parasitoid–host relationship. Of these factors, we evaluated the effect of female age, exposure time, and host species on the biological characteristics and fecundity of O. kuvanae reared on eggs of H. halys as well as another previously known host Philosamia ricini (Lepidoptera: Saturniidae). In this study, we used a 3-year-old laboratory colony of O. kuvanae. Parasitism rates positively affected by exposure time in P. ricini. The highest parasitism rates were obtained in 5- and 7-day-old females of both hosts. The highest emergence rates were recorded on P. ricini for 5- and 7-day-old female P. ricini (81.8% and 84.8%, respectively). The development time of O. kuvanae ranged from 18.4 to 19.1 days on H. halys and 17.7 to 18.3 days on P. ricini. The longevity of O. kuvanae that were provided honey was 38.5 and 47.8 days on H. halys and P. ricini, respectively. The longevity of O. kuvanae that were not provided honey was 2.3 and 2.8 days on H. halys and P. ricini, respectively. The sex ratio was male-biased (36.5% female) on H. halys and female-biased (55.2% female) on P. ricini. Fecundity of O. kuvanae was 37.7 and 59.6 progeny per female for H. halys and P. ricini, respectively. The performance of O. kuvanae was lower when compared with its performance on the host P. ricini. Our results suggest that O. kuvanae has potential as new biological control agent for H. halys.     

兔出血症病毒四个分离株的病毒多肽和血清学关系研究

使用PEG-DS两相系统和超离心提纯的兔出血症病毒(RHDV)四个分离株病毒,再经Sepharose 4B柱层析进一步提纯后,得到较纯的病毒粒子,回收率可达70％以上。应用常规双向免疫扩散试验,交叉血凝抑制试验和酶联免疫吸附试验(ELISA)对四个不同地区分离株间的血清学关系进行了比较研究。结果表明实验中的四个分离株病毒均属同一血清型。SDS-PAGE结果表明,这四个分离株病毒均含有四条多肽,分子量为28—64KD。各株病毒多肽的分子量和各多肽在病毒粒子总蛋白中所占比例略有差异。因此四个分离株的RHDV在蛋白结构上可能存在地区差异。     

Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non-Catalytic Subunit (P Protein)

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [³H]leucine and [³²P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).