棉铃虫核型多角体病毒的血清学研究

用提纯的棉铃虫核型多角体病毒(NPV)的多角体蛋白和病毒粒子免疫家兔制备抗血清,用免疫扩散和对流免疫电泳对四林棉铃虫NPV的多角体蛋白和病毒粒于进行了血清学比较研究。四株棉铃虫NPV分为两个包埋类型：单粒包埋型和多粒包埋型。soI．{3株和H．M株属前者,VHA 273株和XIA 10株属后者。同一株NPV的多角体蛋白或病毒粒子只与它们同源抗血清有反应。它们之间无交叉反应,表明同一株NPV的多角体蛋白和病毒粒子各具有特异的抗原。四株NPV的多角体蛋白不仅与同源的多角体蛋白抗血清有反应,而且也与异 源的多角体蛋白抗血清有交叉反应,说明四株NPV多角体蛋白具有共同的抗原。而四株病毒粒子与同源的病毒粒子抗血清有反应,在它们之间无交叉反应,表明四株NPv病毒粒子各具有自己特异的抗原。     

棉铃虫核型多角体病毒在生态环境中的滞留及作用

棉铃虫核型多角体病毒杀虫剂可湿性粉剂防治第三代棉铃虫,防治效果为８６．２％。使用酶联免疫法（ＥＬＩＳＡ）检测棉铃虫核型多角体病毒在土壤中的滞留,结果表明：施用病毒杀虫剂１、３、５、７天和两个月后都可检测到该病毒的存在。１９９３年９月上旬从河南封丘棉田采集的露水,ＰＨ为７．９８,病毒多角体在露水中保存３天,其生物活性无明显下降。     

中国棉铃虫单粒包埋核型多角体病毒多角体蛋白基因的序列分析

以AcMNPV的多角体蛋白基因为探针,定位了中国棉铃虫单粒包理核型多角体病毒（HaSNPV）的多用体蛋白基因。序列测定表明,HaSNPV的多角体蛋白基因编码区为738个核苷酸,编码246个氨基酸,预计蛋白质分子量为29kDa。同源性分析表明,HaSNPV与美洲棉铃虫单粒包理核型多角体病毒（HzSNPV）具有最高的同源性,在整个阅读框架中只有4个碱基的差异,其中第179位碱基的变化导致唯一的氨基酸变化,这种变化并未导致其二级结构的改变。此外,两种病毒该基因的启动子结构也完全一样。HaSNPV与其它的SNPV和MNPV的同源性明显要低。结果预示HaSNPV与HzSNPV可能为同种病毒的不同变种。     

茶毛虫核型多角体病毒的血清学特性

用免疫双扩散、对流免疫电泳、酶联免疫吸附试验(ELISA),固相免疫电镜(SPIEM)等技术,对茶毛虫核型多角体病毒(EpNPV)的抗原特性及与其它10种核型多角体病毒的血清学关系进行分析。结果表明,EpNPV粒子的抗血清只能与EpNPV粒子起反应,不与EpNPV的多角体蛋白及其它10种昆虫核型多角体病毒(NPV)粒子发生交叉反应;EpNPV多角体蛋白抗血清除了和其同源的多角体蛋白起反应外,还能和其它两种NPV的多角体蛋白起反应。以上结果说明了EpNPV的结构蛋白具有较高的抗原特异性,而多角体蛋白则没有种间特异性。同时将固相免疫电镜技术应用到昆虫病毒的血清学检测中,取得了较为理想的结果。     

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0.36-1.3μm,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。     

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     

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。     

Baculoviral polyhedrin as a novel fusion partner for formation of inclusion body in Escherichia coli

Baculoviral polyhedrin, which originated from Autographa californica nuclear polyhedrosis virus (AcNPV), was employed for the first time as a novel fusion partner for expression of foreign proteins in an Escherichia coli system. We characterized the expression of recombinant polyhedrin protein fused to green fluorescent protein (GFP). The polyhedrin fusion protein ( approximately 58 kDa) was successfully expressed as an insoluble inclusion body comprising approximately 30% of the total cellular protein. The E. coli expressing polyhedrin-GFP fusion protein showed higher cell growth ( approximately 1.8-fold) and higher GFP yield ( approximately 3.5-fold) than the strain expressing soluble single GFP. Interestingly, the polyhedrin fusion portion showed almost the same characteristics as the native baculoviral polyhedrin; it was rapidly solubilized under alkaline conditions, similar to the conditions found in the insect midgut. In addition, the polyhedrin fusion portion was rapidly digested by alkaline proteases in insect Plutella xylostella midgut as well as by alpha-chymotrypsin, a protease that has similar properties to insect midgut polyhedra-associated alkaline proteases. These unique properties suggest that baculoviral polyhedrin might be an advantageous fusion partner for production of foreign proteins, especially harmful proteins, in E. coli expression systems.     

Investigation of serological determinants from single storage plant proteins

The main storage proteins of the angiosperms provide valuable characters for taxonomic research. By comparing the reactivity of one specific protein in different taxa (mainly Ranunculaceae). determinants or determinant groups can be discriminated. Their number is a function of the number of the reference or anti-systems and of the number of taxa compared. These serological determinants prove a posteriori to be good characters and most useful in taxonomy. Their systematic range extends from one genus to the whole of the angiosperms. The resolving power is best between genera within a family. Using such a single protein system, it is possible to co-ordinate the determinants of the taxa investigated and to map their distribution; this is an advantage over the multi-protein systems commonly used. By serological comparison, it should be possible to establish the homology of the main storage globulins throughout the angiosperms.     

日本三株斜纹夜蛾核型多角体病毒的增殖特性及其多角体蛋白基因的序列分析

利用斜纹夜蛾Spodoptera litura培养细胞,对近年来在日本本州、九州和四国等地发现并筛选出的对斜纹夜蛾幼虫具有强烈杀虫活性的3株斜纹夜蛾核型多角体病毒(SpltMNPV)（K-3、G1-2和G10-3）进行了生物学活性和分子生物学的初步研究,克隆了多角体蛋白基因,并进行了序列分析和比较。结果表明:（1）SpltMNPV日本分离株K-3、G1-2和G10-3分别具有不同的特征性酶切图谱,分别属于3种基因型（A型、B型和C型）; （2）3个分离株的芽生型病毒（budded virus）产生能力和多角体产生能力有差异,免疫印迹分析表明,多角体蛋白的分子量也不同;（3）日本株SpltMNPV核型多角体蛋白结构基因由747个核苷酸编码序列（编码249个氨基酸）组成,其序列与中国株SpltMNPV的同源性为98.9%,与其他6种核型多角体病毒有较高的同源性（61.7%～74.2%）,但其5′端侧翼序列(nt-1～-100)与AcMNPV和BmNPV相比差异显著,在对该基因表达调控起决定性作用的8个高度保守核苷酸序列中（nt-44～-51）有2处发生自然突变。     

Characterization of theHelicoverpa assulta nucleopolyhedrovirus genome and sequence analysis of the polyhedrin gene region

A local strain ofHelicoverpa assulta nucleopolyhedrovirus (HasNPV) was isolated from infectedH. assulta larvae in Korea. Restriction endonuclease fragment analysis, using 4 restriction enzymes, estimated that the total genome size of HasNPV is about 138 kb. A degenerate polymerase chain reaction (PCR) primer set for the polyhedrin gene successfully amplified the partial polyhedrin gene of HasNPV. The sequencing results showed that the about 430 bp PCR product was a fragment of the corresponding polyhedrin gene. Using HasNPV partial predicted polyhedrin to probe the Southern blots, we identified the location of the polyhedrin gene within the 6 kbEcoRI, 15 kbNcoI, 20 kbXhoI, 17 kbBgl II and 3 kbClaI fragments, respectively. The 3 kbClaI fragment was cloned and the nucleotide sequences of the polyhedrin coding region and its flaking regions were determined. Nucleotide sequence analysis indicated the presence of an open reading frame of 735 nucleotides which could encode 245 amino acids with a predicted molecular mass of 29 kDa. The nucleotide sequences within the coding region of HasNPV polyhedrin shared 73.7% identity with the polyhedrin gene fromAutographa californica NPV but were most closely related toHelicoverpa andHeliothis species NPVs with over 99% sequence identity.     

Comparative epitope profiles of the particle proteins of whitefly-transmitted geminiviruses from nine crop legumes in India

Geminiviruses associated with yellow or golden mosaic diseases of legume crops in two regions of India were compared by testing their reactivity with 27 monoclonal antibodies (MAbs) prepared to the particles of African cassava mosaic (ACMV) or Indian cassava mosaic (ICMV) viruses. The viruses fell into two main groups. Group 1 comprised isolates of dolichos yellow mosaic virus; these reacted with three or four ACMV MAbs and four ICMV MAbs. Group 2 comprised isolates of horsegram yellow mosaic virus, together with isolates from blackgram, cowpea, French bean, pigeonpea, soybean, Indigofera hirsuta and probably also isolates from mungbean. These reacted with three or four ACMV MAbs but with few or no ICMV MAbs. Isolates within each group differed slightly in epitope profile, depending on the source species (Group 2) or geographical origin (Groups 1 and 2). Isolates from lima bean resembled those in Group 2 but had some antigenic differences, and their status is uncertain. The poor detectability of geminivirus isolates in mungbean may reflect a low virus concentration in this species.     

Microbiological and serological diagnosis of Lyme borreliosis

In Europe, Lyme borreliosis is caused by Borrelia burgdorferi sensu stricto, B. afzelii, B. garinii and the recently described species B. spielmanii. For the development of diagnostic tools, the heterogeneity of the causative agents must be considered. The serological diagnosis should follow the principle of a two-step procedure: a sensitive enzyme-linked immunosorbent analysis as the first step, followed by immunoblot (IgM and IgG) if reactive. The sensitivity and standardization of immunoblots have been enhanced by the use of recombinant antigens instead of whole cell lysates. Improved sensitivity has resulted from the use of recombinant proteins primarily expressed in vivo (e.g. VlsE) and the combination of homologous proteins from different strains (e.g. DbpA). At present, detection rates for serum antibodies are 20-50% in localized, 70-90% in disseminated early and nearly 100% in late disease. Detection of the borreliae by culture or PCR should be confined to specific indications. The best results are obtained from skin biopsies (50-70% with culture or PCR) and synovial tissue or fluid (50-70% with PCR). Cerebrospinal fluid is positive in only 10-30%. Methods that are not recommended for diagnostic purposes include antigen tests in body fluids, PCR of urine and lymphocyte transformation tests.     

A comparison of serological techniques for detecting and identifying honeybee viruses

The sensitivity and specificity of conventional Ouchterlony gel-diffusion, immuno-osmoelectrophoresis (IO), immune serum electron microscopy (ISEM), “decoration,” radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) tests for detecting black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), and sacbrood virus (SBV) particles in extracts of diseased honeybees were compared. A “slow” ISEM method detected virus particles in extracts of individuals or groups of individuals diluted to 10^?3 and 10^?4, respectively, whereas the IO method and a “fast” ISEM method using protein A were one-tenth as sensitive, and Ouchterlony gel-diffusion tests were only one-thousandth as sensitive. Using the antibody “decoration” technique, mixtures of serologically unrelated virus particles could be resolved. RIA and ELISA were found to be one thousand times more sensitive than ISEM in detecting the particles of BQCV, CBPV, KBV, and SBV; however, nonspecific reactions occurred when using RIA with very dilute particle suspensions, and this made dilution endpoints difficult to assess, but this did not occur when using the ELISA method. There was little difference in the effectiveness of rabbit or hen antisera in the tests, except when protein A was used as it does not combine with hen antibodies.     

Cancer-associated antigens and antigen arrays in serological diagnostics of malignant tumors

The appearance of antibodies to cancer-associated antigens in biological fluids (particularly, in blood sera) of cancer patients is now a well-established fact, and their detection by immunochemical methods is a promising approach to diagnostics of malignant neoplasms. In this review, we consider some immunobiological aspects of the most extensively studied cancer-associated B-cell antigens, various applications of autoantibodies as cancer biomarkers, and prospects for the use of antigen arrays for improving diagnostic sensitivity.     

HaNPV在宿主内的复制及其对宿主蛋白质代谢的影响

对棉铃虫Helicoverpaarmigera的中肠、血淋巴及脂肪体进行SDS-PAGE蛋白质分析表明：感染早期,HaNPV对棉铃虫的蛋白质合成有刺激作用,晚期则表现出抑制作用。感染96 h和120 h脂肪体有较高水平的多角体蛋白合成。电镜观察表明：①HaNPV感染棉铃虫除病毒在中肠复制后经出芽进入血腔感染脂肪体等组织外,还可能存在病毒粒子直接经中肠进入血腔而感染脂肪体这样的途径;②中肠与血腔之间存在屏障结构;③中肠不仅是一次感染时病毒粒子复制的场所,它完全可能被病毒粒子二次感染。在中肠细胞中没有发现多角体的形成。     

Serological Relationships Among the Over-expressed Coat Proteins of Allexiviruses

Coat protein genes of six members of the genus Allexivirus, Garlic viruses ‐A, ‐B, ‐C, ‐D, ‐E and ‐X, were over‐expressed in bacteria and used to prepare polyclonal antibodies. In western blot analysis, Garlic virus D (GarV‐D) and GarV‐E were closely related serologically and there were weaker relationships between some other pairs, particularly GarV‐E with GarV‐A, GarV‐X with GarV‐A, GarV‐X with GarV‐B and GarV‐X with GarV‐C. Sequence analysis suggested that there was no simple relationship between amino acid identity and serological properties.