蚯蚓纤溶酶组分的抗原性分析

  用热变性蚯蚓纤溶酶(earthworm fibrinolitic enzyme,EFE)组分EfP-0-2、EfP-Ⅰ-1、EfP-Ⅱ-1, 以及重组蛋白EfP-Ⅲ-2为抗原,免疫家兔获得了抗血清,利用获得的抗血清对部分组分的免疫学同一性进行了验证.EfP-0、EfP-Ⅰ、EfP-Ⅱ的免疫双扩散反应表明,EfP-Ⅰ和EfP-Ⅱ具有部分免疫学同一性. 利用DNAMAN软件对获得的EfP-0、EfP-Ⅰ、EfP-Ⅱ、EfP -Ⅲ的蛋白质序列进行了初步分析,包括整个序列相似性比对、氨基酸组成、蛋白酶消化、抗原肽、疏水性和亲水性轮廓等,并对N端氨基酸序列进行了分析.结果表明,EfP-Ⅰ和EfP- Ⅱ具有部分免疫学同一性是由它们蛋白质序列的组成和结构决定的.     

双链核糖核酸病毒的研究 Ⅲ.水稻普通矮缩病毒与家蚕细胞质多角体病毒间的免疫交叉反应

水稻普通矮缩病毒(RDV)的兔抗血清能分别与家蚕细胞质多角体病毒(CPV)颗粒及其双链RNA在免疫对流电泳中产生沉淀线。用水稻普通矮缩病毒的抗血清中和后的家蚕CPV的感染力与对照相比降低二个数量级。     

葡萄卷叶病毒的纯化、血清学研究及其在脱毒组培苗检测中的应用

将具有典型葡萄卷叶病(Grapevine leafroll diseas,GLRD)症状的葡萄组织,经差速和硫酸铯—蔗糖密度梯度离心,提纯了GLRV,并制备了兔抗血清。电镜下可观察到长度从600～2000nm的线形病毒颗粒,其中以1400nm左右为主。免疫电镜结果表明线形病毒颗粒能被美国的NY-1分离株抗血清(Ⅲ型)所修饰。在间接ELISA中提纯制品与GLRV的Ⅲ、Ⅳ、Ⅱ型抗血清均能产生免疫反应。与Ⅲ型抗血清产生较强的免疫反应,Ⅳ型次之,Ⅱ型最弱。在SDS-免疫双扩散实验中病组织韧皮部粗提液与GLRV的Ⅲ,Ⅳ、Ⅱ型抗血清均产生免疫沉淀线。从而推测我国葡萄园内的葡萄卷叶病很可能由2种或3种卷叶病毒感染所致.采用A蛋白夹心酶联免疫吸附试验(PAS-ELISA)检测葡萄试管苗,Ⅲ型抗血清和自制抗血清的平行测试结果基本相符,共获得11个生食葡萄和10个山葡萄品种的脱葡萄卷叶病毒和扇叶病毒的组培苗,扩繁后田间试种表现出良好的农艺性状。     

葡萄卷叶病毒的纯化,血清学研究及其在脱毒组培苗检测中的应用

将具有典型葡萄卷叶病(Grapevine leafroll diseas,GLRD)症状的葡萄组织,经差速和硫酸铯—蔗糖密度梯度离心,提纯了GLRV,并制备了兔抗血清。电镜下可观察到长度从600～2000nm的线形病毒颗粒,其中以1400nm左右为主。免疫电镜结果表明线形病毒颗粒能被美国的NY-1分离株抗血清(Ⅲ型)所修饰。在间接ELISA中提纯制品与GLRV的Ⅲ、Ⅳ、Ⅱ型抗血清均能产生免疫反应。与Ⅲ型抗血清产生较强的免疫反应,Ⅳ型次之,Ⅱ型最弱。在SDS-免疫双扩散实验中病组织韧皮部粗提液与GLRV的Ⅲ,Ⅳ、Ⅱ型抗血清均产生免疫沉淀线。从而推测我国葡萄园内的葡萄卷叶病很可能由2种或3种卷叶病毒感染所致.采用A蛋白夹心酶联免疫吸附试验(PAS-ELISA)检测葡萄试管苗,Ⅲ型抗血清和自制抗血清的平行测试结果基本相符,共获得11个生食葡萄和10个山葡萄品种的脱葡萄卷叶病毒和扇叶病毒的组培苗,扩繁后田间试种表现出良好的农艺性状。     

依赖于DNA的RNA聚合酶的研究——Ⅴ.615小鼠肝RNA聚合酶B(Ⅱ)的免疫学特性研究

用615小鼠肝RNA聚合酶B免疫母鸡获得了抗血清。在免疫扩散实验中,这种抗血清和615小鼠肝RNA聚合酶B之间形成清晰的沉淀线;与L615(可移植性小鼠白血病)小鼠及大鼠肝RNA聚合酶B之间形成弱的沉淀线;而与615小鼠肝RNA聚合酶A和C以及大肠杆菌RNA聚合酶之间不形成沉淀线。这种抗血清对615小鼠肝RNA聚合酶B离体转录活性有明显的抑制作用,而对大肠杆菌的RNA聚合酶没有抑制作用。在免疫扩散实验中,这种抗血清可以和不同批号的615小鼠肝RNA聚合酶B产生沉淀线。 这种抗血清和615小鼠肝RNA聚合酶B形成免疫沉淀后的离心上清液电泳图谱中,血清免疫球蛋白的区带消失了。     

艾氏腹水癌患鼠腹水中一种高分子量DNA结合蛋白的分离

 本文利用免疫吸收法和免疫亲和层析法,从艾氏腹水癌患鼠腹水DNA结合蛋白中,分离得到了一种高分子量DNA结合蛋白。在免疫双扩散反应中,它与抗艾氏腹水癌患鼠血清DNA结合蛋白的兎抗血清反应形成一条沉淀线,但与正常小鼠血清DNA结合蛋白的兎抗血清不形成沉淀线。该DNA结合蛋白样品用2-巯基乙醇还原后,经SDC-PAGE分析,测得其分子量约为41000。     

黑曲霉病毒的形态和特性及其在细胞内的表现

从产生糖化酶的黑曲霉(Aspergillus niger)菌株中分离到一种等轴对称、衣壳表面有突起、内含双链RNA的病毒颗粒。病毒颗柱在电镜下直径为28—33nm,呈六面体晶格排列。病毒在蔗糖密度梯度离心中有三个分部,分析超离心所得沉降系数为161S,118S和94S。病毒在凝胶电泳中为一条带,在SDS-聚丙烯酰胺凝胶电泳中外壳蛋白分子量为90,000、82,000、76,000、52,0000、42,000 道尔顿。提取的病毒与其抗血清在免疫双扩散实验中出现一条沉淀线。病毒核酸有5个组分,与聚肌苷：聚胞苷[poly(1)：poly?]抗血清反应中出现一条沉淀线。在早期菌丝细胞超薄切片中,病毒颗粒多近似球状紧密聚集,外被以膜结构,聚生或散生于胞质中;后期菌丝细胞中病毒颗粒则多散生于胞质中。     

抗精氨酸加压素血清的制备及其初步应用

用戊二醛为偶联剂将精氨酸加压素(AVP)与甲状腺球蛋白连接合成AVP免疫原,免疫家兔获得了高滴度和良好特异性与亲和力的抗精氨酸加压素血清。用此抗血清进行了大鼠和豚鼠下丘脑内AVP样免疫反应物质的定位研究,测定了正常人血浆及大鼠部分脑区AVP免疫活性物质含量,结果与国外报道相近,初步应用结果表明此抗血清适用于放射免疫测定(RIA)和免疫细胞化学(ICC)研究。     

中国大陆水稻黄矮病与台湾省水稻暂黄病的血清学鉴定

以前的文献报道我国大陆水稻黄矮病和台湾省的水稻暂黄病的病状、传毒介体和病毒形状均相似或相同,被认为是同一病害,但没有做过血清学反应的鉴定比较。本试验采用琼脂扩散法和双抗体夹心法(PAS-ELISA),对上述两病原的抗血清与黄矮病株提取液和带毒黑尾叶蝉研磨液进行了琼脂双扩散和酶联免疫反应的比较研究。黄矮病毒提取液与暂黄病毒抗血清的琼脂双扩散产生清晰的沉淀带,在ELISA试验中均为典型的阳性反应;黄矮病毒抗血清和暂黄病毒抗血清对生物测定虫的同一头黑尾叶蝉研磨液的测定结果,阳性虫附合率98％,病原田捕捉虫的符合率为100％。根据以上结果可以认为两种抗血清同源,即中国大陆水稻黄矮病与台湾省水稻暂黄病为同一病害。     

蚯蚓纤溶酶新基因EfP-0的电子克隆及其编码区序列RT-PCR验证

利用生物信息学手段,以期获得蚯蚓纤溶酶F-Ⅰ-0组分的基因。根据从粉正蚓(Lumbricusrubellus)中分离的F-Ⅰ-0组分的N端氨基酸序列VVGGSDTTIGQYPHQL,利用DNAMAN软件通过电子克隆方法,从Lumbricidae的dbEST中获得该组分的核酸序列信息,设计特异引物,经过RT-PCR,成功地从赤子爱胜蚓(Eiseniafoetida)中克隆到一条蚯蚓纤溶酶新基因,命名为EfP-0。EfP-0基因全长678bp,编码225个氨基酸的成熟肽,属丝氨酸蛋白酶,胰蛋白酶家族,与F-Ⅰ-0组分的氨基酸组成非常接近。BLAST证明,EfP-0与已报道的蚯蚓纤溶酶基因之间的相似性均低于40%,因此为蚯蚓纤溶酶中的一个新基因,GenBank登录号为DQ836917。构建的pMAL-c2x-EfP-0重组质粒,在大肠杆菌TB1中获得融合蛋白MBP-EfP-0的可溶性表达,表达产物有酪蛋白平板溶解活性。     

The immunological activity of some of the chymotryptic peptides of sperm-whale myoglobin

1. Sperm-whale apomyoglobin was digested with chymotrypsin in a dialysis sac. The ultrafiltrate contained incompletely hydrolysed fragments which partially inhibited the precipitation of metmyoglobin and apomyoglobin by some antisera produced against metmyoglobin. The inhibitory activity was stable to heating at 100 degrees and depended on the peptide structure. 2. The fragments were fractionated according to molecular size and were purified by ion-exchange chromatography. Six pure peptides and two peptides which contained a minor impurity were isolated. Their amino acid compositions and N-terminal amino acid sequences were determined and their entire amino acid sequences deduced from the known amino acid sequence of sperm-whale myoglobin. 3. The peptides formed no detectable precipitates with the antisera. Five of the eight peptides partially inhibited the precipitation of apomyoglobin and/or metmyoglobin by one antiserum. Six of the peptides inhibited the precipitation of apomyoglobin by one or other of two antisera; at least two of these peptides inhibited both antisera. One peptide failed to inhibit the precipitation of either antigen by either antiserum. Two of the peptides possessed the same serological specificity. 4. The molar ratios of inhibitors to antigen for 50% of the maximum inhibition decreased as the molecular size of the inhibitor increased. With one antiserum and with apomyoglobin as the antigen, molar ratios 12 and 80 were obtained for peptides with molecular weights 2051 and 793 respectively. 5. The size and structure of an antigenic site is discussed in relation to the known steric configuration of myoglobin.     

蚯蚓纤溶酶的成分分析

蚯蚓纤溶酶（ｅａｒｔｈｗｏｒｍｆｉｂｒｉｎｏｌｙｔｉｃｅｎｚｙｍｅｓ,ＥＦＥ）是从蚯蚓体内提取的一类纤维蛋白水解酶,是一种新的溶栓药．利用亲和层析技术,自蚯蚓匀浆液一步提取蚯蚓纤溶酶,并对该酶的成分进行了分析．实验表明,蚯蚓纤溶酶是一组非均一的糖蛋白,含糖量为５％左右,以中性已糖为主;等电点（ｐＩ）在４．０以下;富含Ａｓｐ,而Ｍｅｔ、Ｔｒｐ和Ｌｙｓ含量很少;ｌｍｇ蚯蚓纤溶酶相当于２５０～３００尿激酶单位．     

Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit.

Biosynthesis of the phytohormone ethylene in higher plants proceeds via the following pathway: S-adenosylmethionine----1-aminocyclopropane-1-carboxylic acid (ACC)----ethylene. Ethylene-forming enzyme (EFE), the enzyme responsible for the oxidation of ACC to ethylene, has been only partially characterized in vitro. We have obtained authentic EFE activity in vitro from extracts of avocado fruit (Persea americana Mill. cv Hass). Ammonium sulfate fractionation revealed the presence of two EFE activities, which we designate as EFE1 and EFE2. EFE1 activity utilizes ACC and O2 as substrates and requires Fe(II) and ascorbate as cofactors. The enzyme has a relatively low Km (32 microM) for ACC, discriminates diastereomers of 1-amino-2-ethyl-cyclopropane-1-carboxylic acid, and is inhibited competitively by 2-aminoisobutyric acid, thus confirming its identity with authentic EFE. Activity is retained in a 100,000 x g supernatant and has a pH optimum of 7.5-8.0, suggesting a cytosolic localization.     

Isolation and characterization of a tumor-associated NADH oxidase (tNOX) from the HeLa cell surface.

Cell-surface-located, drug-responsive and tumor-associated NADH oxidase (tNOX) proteins were purified and characterized from HeLa cells. The proteins isolated exhibited NADH oxidase activity inhibited by capsaicin and were resistant to heating and to protease digestion. The activity was purified 200- to 500-fold to provide apparently homogeneous gel bands for N-terminal sequencing using three different protocols. All three protocols involved heat (50 degrees C) and proteinase K treatment. Recovery of the total NADH oxidase activity was 86% and inhibition by capsaicin was 60 to 80%. After 450-fold purification, a 52-kDa component was obtained as a single gel band that retained the capsaicin-inhibited NADH oxidase activity. Amino acid composition and partial amino acid sequences were obtained. The partial amino acid sequences were used to generate peptide antisera. Both the peptide antisera and polyclonal antisera to the 52-kDa component immunoprecipitated capsaicin-inhibited NADH oxidase activity and reacted with 52-, 34-, and 17-kDa components on Western blots from different steps of the purification. The tNOX protein exhibited immunological cross-reactivity and amino acid sequence identity with tNOX cloned from a HeLa cDNA library using a monoclonal antibody to tNOX from sera of cancer patients. The results provide a direct sequence link between tNOX of the HeLa cell surface and the cloned tNOX representative of patient sera. The tNOX form from the surface of HeLa cells yielded N-terminal sequence consistent with a coidentity of the cell surface and serum forms of the two activities.     

Distribution of Cereal Luteoviruses and Molecular Diversity of BYDV‐PAV Isolates in Central and Southern Iran: Proposal of a New Species in the Genus Luteovirus

During a survey , 148 wheat, 70 barley and 24 wild grass samples of plants showing symptoms of yellowing or reddening of leaves and general stunting were collected in central and southern provinces of Iran and tested for Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV) infection by enzyme‐linked immunosorbent assay (ELISA) and tissue print immunoassay (TPIA). The results showed the presence of the viruses in most regions. Positive reactions to BYDV‐PAV, BYDV‐MAV, CYDV‐RPV and BYDV‐SGV antisera were recorded. BYDV‐PAV was the most prevalent virus. The genetic diversity of BYDV‐PAV isolates in central and southern provinces was studied by analysing ORF1 (903 nt) and read through domain (RTD) (575 nt) of 13 and nine isolates respectively. Sequence analysis of RTD at nucleotide and amino acid levels revealed a high identity (91.8–97.2% and 91.4–100% respectively) between Iranian and other available isolates in the GenBank. However, in regards to ORF1, a high genetic diversity among Iranian and other known PAV isolates at both amino acid (2–16.9%) and nucleotide (4.1–16.5%) levels were detected. Based on phylogenetic analysis of ORF1, two major groups of BYDV‐PAV isolates were distinguished. The Iranian isolates were divided between the two clusters. Our results suggest that the occurrence of two genetically distinct groups of PAV isolates in central and southern Iran, from which according to the ICTV criteria for species demarcation in the family Luteoviridae, four isolates from central parts of the country, qualify for designation as new species.     

Host-Specific Hemagglutination of Influenza A (H1N1) Virus

H1N1 strains of influenza A virus isolated during the influenza season of 1991–92 were divided into two groups according to the property of host-specific hemagglutination. Group 1 viruses agglutinated human and chicken red blood cells. Group 2 viruses agglutinated human but not chicken red blood cells. The viruses of both groups, however, showed the same antigenic structure determined with ferret antisera. The virus clones which were plaque-purified twice from a group 2 virus retained the characteristic of host-specific hemagglutination after five successive passages in MDCK cells, indicating that this phenomenon is genetically determined. However, the amino acid, sequences of the hemagglutinin (HA) polypeptides deduced from the nucleotide sequences of the HA gene of the two groups did not show any differences between them. This suggests a difference in amino acids in some other polypeptide(s), which affects the host-specific hemagglutination.     

Immunological and structural relatedness of catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria.

Purified catabolic ornithine carbamoyltransferase of Pseudomonas putida and anabolic ornithine carbamoyltransferase (argF product) of Escherichia coli K-12 were used to prepare antisera. The two specific antisera gave heterologous cross-reactions of various intensities with bacterial catabolic ornithine carbamoyltransferases formed by Pseudomonas and representative organisms of other bacterial genera. The immunological cross-reactivity observed only between the catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria suggests that these proteins share some structural similarities. Indeed, the amino acid composition of the anabolic ornithine carbamoyltransferase of E. coli K-12 (argF and argI products) closely resembles the amino acid compositions of the catabolic enzymes of Pseudomonas putida, Aeromonas formicans, Streptococcus faecalis, and Bacillus licheniformis. Comparison of the N-terminal amino acid sequence of the E. coli anabolic ornithine carbamoyltransferase with that of the A. formicans and Pseudomonas putida catabolic enzymes shows, respectively, 45 and 28% identity between the compared positions; the A. formicans sequence reveals 53% identity with the Pseudomonas putida sequence. These results favor the conclusion that anabolic ornithine carbamoyltransferases of enterobacteria and catabolic ornithine carbamoyltransferases derive from a common ancestral gene.     

Cloning and expression of earthworm fibrinolytic enzyme PM(246) in Pichia pastoris

We have cloned, expressed, and purified a novel earthworm fibrinolytic enzyme (EFE) of Lumbricus rubellus in Pichia pastoris. Its cDNA sequence revealed a 747bp region containing an intact ORF that encodes a protein of 246 amino acid residues, designated as EFE PM(246). While EFE PM(246) is distinct, its cDNA shows a high degree of sequence homologies with four other EFE cDNAs registered in GenBank. The recombinant EFE PM(246) was active, showing a fibrinolytic activity of 7.5 x 10(6)U/L in basal salts medium, a higher fibrinolytic activity than those produced in other expression systems. The recombinant EFE PM(246) expressed in basal salts medium was purified by a three-step purification procedure with a recovery rate of about 20%. This is the first report detailing the successful purification of a genetically engineered earthworm fibrinolytic enzyme. The main physiochemical features of the EFE PM(246), including temperature stability, pH resistance, and sensitivity to some protein inhibitors, were also characterized.