CS3抗原基因的表达及纤毛装配元件的研究

在肠毒素大肠杆菌(ETEC)的已知定居因子中,CS3是临床分离株中最常见的抗原之一。为了研究CS3纤毛装配的基本元件,绘制了CS3亚基结构基因和辅助蛋白编码区的限制酶谱。通过亚克隆的亚基基因和不同辅助蛋白基因之间的互补性表达结果,确定了CS3纤毛装配所需要的辅助蛋白的DNA功能片段。微细胞分析结果显示,CS3基因的有效表达和纤毛装配至少需要6条蛋白多肽,分子量分别为(×10~3)15、17、24、27、48和90。除了15×10~3/17×10~3的蛋白多肽为CS3亚基外,其余的蛋白多肽参与CS3亚基的转运及纤毛的装配。根据以上结果初步确定了上述相关基因的相对位置。     

Antibodies to meningococcal iron regulated protein FbpA and minor proteins in the sera of patients with meningococcal meningitis

Altogether 7 blood serum specimens taken from 3 patients with meningococcal meningitis were studied by the method of immunoblotting. The study revealed that on day 7 and especially on day 10 from the onset of the disease antibodies to periplasmatic iron-regulated protein FbpA with a molecular weight of 34 kD appeared in the serum of one patient. In the sera of two other patients the appearance of antibodies to minor iron-regulated proteins with molecular weights of 43 kD and 46 kD, absent at the acute stage of the disease, were detected on day 7. As the process of convalescence was progressing, in all patients under observation an increase in the specific immune response to proteins of class 5 with molecular weights of 20-14 kD was observed.     

Biochemical and immunochemical analyses of the protein components of the cell wall in group-A streptococci isolated by a sparing chemical method]

To study the protein components of the cell wall of group A streptococci, type M 29, a special preparative method was developed (extraction with 1 M hydroxylamine solution, pH 6.0, and subsequent purification). Altogether six protein fractions were obtained. The isolated proteins were found to be a heterogeneous group of molecules, consisting of 25-40 individual proteins with molecular weights ranging between 13 and 94 kD. The study of the protein fractions thus obtained in the immunodiffusion test with rabbit antiserum to the initial protein preparation revealed that these proteins contained type-specific components, 3-6 type-nonspecific protein antigens common with protein antigens of M 1 and M 12, as well as one protein antigen common with type M 1. Fc receptor was shown to be absent. The detected type-nonspecific protein antigens were partially separated by ion-exchange chromatography and some of them could be purified from the admixtures of nucleic acids and group-specific polysaccharide.     

HCV核心蛋白在大肠杆菌中的表达及免疫学特性分析

目的:表达HCV核心蛋白,为检测丙肝病毒提供合适抗原。方法:以含HCV核心全长cDNA克隆的pMD18T/core质粒为模板,PCR扩增全长的HCV核心抗原基因,插入表达载体pQEN1构建重组质粒pQEN1/Core,转化BL-21(DE3)大肠杆菌,IPTG诱导表达6×His融合蛋白,表达产物经SDS-PAGE及Western blot检测和鉴定。结果:经SDS-PAGE及Western blot显示HCV核心蛋白在大肠杆菌中正确表达,融合蛋白分子量约为22 kD,表达量约占菌体蛋白总量的30%。纯化后的C蛋白能与慢性丙型肝炎患者有血清反应。结论:HCV核心蛋白在大肠杆菌中成功表达并具有较强的抗原性。     

Cytokinin Binding Proteins from Phaseolus vulgaris Hypocotyls

t-Zeatin (t-Z) and isopentenyladenosine (iPA) occur naturally as highly active plant cell division regulators, t-Z-Sepherose-4B and iPA-Sepherose-4B affinity column were constructed to isolate and purify the cytokinin-binding proteins from etiolated hypocotyl of Phaseolus vulgaris. Two kinds of cytokinin-binding proteins were obtained. One was 15.5 kD in molecular weight (named ZBP) with only one peptide. The other (named IBP), 165 kD in molecular weight, contained two different subunits (40 kD and 43 kD respectively). The binding activity of ZBP was tested and the dissociation constant (Kd) was determined to be 3.2 × 10-7 mol/L. There was one binding site for t-Z in each molecule of ZBP.     

Antibacterial hemolymph proteins of Manduca sexta

Exclusion column fractionated immune hemolymph of the M. sexta larva contains five peaks of anti-E. coli activity with molecular weights of greater than 140 kD and approximately 91, 54, 14 and 4 kD, plus one peak of lysozyme activity with a molecular weight of 17 kD. Purification of the 54 kD peak showed that this peak consists of the previously described M18 proteins which have monomeric weights of approximately 20 kD and had antibacterial activity against certain gram negative bacteria. Approximately 80% of the total hemolymph antibacterial activity was detected in the 14 and 4 kD peaks. These proteins, which kill both gram negative and gram positive bacteria, appeared to be directly analogous to the cecropins of H. cecropia. The greater than 140 and 91 kD peaks constituted only a minor part of the total antibacterial activity.     

Investigations on the aminoacid content of tumor associated antigens of rat sarcoma cells induced by virus

In our previous work (Alexandrov et al., 1996) was reported that the rat sarcoma cells induced by SR-RSV express two tumor associated antigens (TAA). The one TAA has a molecular weight of 52 kD and is detected by the help of a monoclonal antibody 2C2 only on the outer side of the plasma membrane of the sarcoma cells. The other antigen, with molecular weight of 28 kD, is expressed on the outher and inner side of the membrane. The antigens were isolated as a pure fraction by polyacrylamide electrophoresis and prepared for aminoacid analysis after that. The consisting 16 bound aminoacids were in different amounts. Both antigens are rich in glycine and poor in aromatic and sulphur-containing aminoacids. The presence of glucosamine and galactosamine in the antigens proves their glycoprotein nature. The received data show that the both TAA-s differ not only in molecular weights, place of expression and functional activity, but also in the amount of the bound aminoacids which constitute their proteins.     

Molecular Weight and Subunit Composition of the Sensitive to GABA-Ergic Ligands Cl<Superscript>−</Superscript>/HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-Stimulated Mg<Superscript>2+</Superscript>-ATPase from Plasma Membrane of Rat Brain

The molecular weight and subunit composition of Cl-,HCO3(-)- and picrotoxin-stimulated Mg2+-ATPase from rat brain plasma membrane solubilized in sodium deoxycholate were studied by gel filtration chromatography. The enzyme activity eluted from a Sephacryl S-300 column in a single peak associated with a protein of molecular weight approximately 300 kD and a Stokes radius of 5.4 nm. The enzyme-enriched fraction, concentrated and denatured by SDS, migrated through a Sephacryl S-200 column as three peaks with molecular weights of approximately 57, 53, and 45 kD. SDS-PAGE also showed three major protein bands with molecular weights of about 57, 53, and 48 kD. The molecular weight and subunit composition of the Cl- and HCO3(-)-stimulated Mg2+-ATPase from neuronal membrane of rat brain are similar with the molecular properties of GABA(A)-benzodiazepine receptor complex from mammalian brain but are different from those of P-type transport ATPases.     

Comparative analysis of the immune response of a rabbit to antigens to live and killed Francisella species bacteria]

Serum antibodies were analyzed in rabbits immunized with live and formalin-killed Francisella (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia). Passive hemagglutination test with erythrocytes sensitized by these bacteria' LPS showed much higher titers of species-specific antibodies in all sera to live microorganisms than sera to killed bacteria. The results of immunoblotting with purified LPS and bacterial lysates indicate that sera to live bacteria contained mainly immunoglobulins to species-specific antigenic epitopes of LPS O-polysaccharide chain and few antibodies to the protein component of the cell. By contrast, killed bacterial cells induced weak production of antibodies to S-LPS and a pronounced antibody response to protein antigens. Besides the quantitative differences, live and killed bacteria differed by the qualitative spectrum of immunodominant proteins. Serum to live F. tularensis 15/10 contained antibodies to at least 3 immunodominant antigens of the cell, while serum to killed bacteria contained antibodies to only two of these. Immunoglobulins to protein antigens, absent in homologous sera to live bacteria, were detected in the sera to killed F. novicida and F. novicida-like bacteria. Both sera to F. philomiragia had antibodies reacting with LPS epitopes and immunodominant complex containing protein. In contrast to other Francisella, F. philomiragia was found to synthesize an uncommon LPS representing two major lipooligosaccharides with different molecular weights and antigenic specificity. Therefore, immune response of the host to live and killed Francisella is different: live cells more effectively induce the production of antibodies to S-LPS epitopes, while killed ones to protein antigens.     

Protective and other biological properties of Bacillus anthracis soluble antigens

The soluble antigens were explored of the culture filtrate (CF) derived during static growth of B. anthracis vaccine strain 34F2 on a medium containing casein hydrolysate. Electrofocusing of CF preparations revealed that the protective activity was distributed over a wide range of pH 3-7. The most pronounced and stable protective activity was observed at pH 4.6-4.8. Following toxin factors were isolated and identified: protective antigen (87 kD), oedema factor (87 kD) and lethal factor (78-81 kD). The greatest protective activity was associated with antigens characterized by a molecular weight of 78-87 kD and toxic activity. Preparations of the oedema and lethal factors had the same protective activity as protective antigen (PA) preparations. Other CF soluble antigens protected about 30% of immunized guinea pigs. A protein was isolated with a molecular weight of 80 kD and isoelectric point at pH 5.3-5.7 which was not toxic and did not form toxic mixtures in association with other toxin factors; this protein featured a high immunogenic activity, however, it protected only 31% of immunized animals. Factors are analyzed which determine differences in the protective effects of live and chemical vaccines.     

Klebsiella pneumoniae secreted protein-containing antigens in the system of adaptive immunity

The study was aimed at the evaluation of the antigenic properties of K. pneumoniae secreted protein-containing antigens with a molecular weightt of 21 and 34-35 kD, obtained from supernatant culture fluid. As confirmed by the method of flow cytofluorimetry, the protein-containing fractions belonged to the secreted components of the microbial cell. The fraction with a molecular weight of 34-35 kD possessed high antigenic activity and contributed to the formation of specific antibodies after the immunization of mice. At the same time none of the protein fractions lead to an increase in the level of autoantibodies in mouse blood sera to organ-unspecific and organ-specific antigens. As revealed by the method of solid-phase, in 6 (27.3%) from 22 patients of patients with rhizomelic spondylitis had an increased level of IgG to K. pneumoniae cell-wall antigens with a molecular weight of 34-35 kD. An increase in the level of IgG to the secreted protein-containing fraction with a molecular weight of 34-35 kD was detected only in one patient (4.5%) (p<0.05).     

Tyrosine phosphorylated proteins in different tissues during chick embryo development

A high affinity polyclonal antibody specific for phosphotyrosyl residues has been used in immunoblotting experiments to survey developing embryonic chicken tissues for the presence and characteristics of tyrosine phosphorylated proteins. Proteins phosphorylated on tyrosine were found to be present in all the embryonic tissues examined, including heart, thigh, gizzard, intestine, lung, liver, kidney, brain, and lens, from 7 to 21 d of development in ovo, but were greatly reduced or absent in the same tissues taken from adult chickens. A limited number of major tyrosine phosphorylated proteins were seen in all the tissues examined and they ranged in molecular mass from 35 to 220 kD. Most of the tissues contained proteins phosphorylated on tyrosine with apparent molecular masses of 120, 70, 60, and 35 kD, suggesting that the substrates of tyrosine protein kinases in different tissues may be related proteins. One-dimensional peptide mapping of the 120- and 70-kD protein bands indicated a close structural relationship among the phosphotyrosine-containing proteins of 120 kD, and similarly among those of 70 kD, from the different tissues.     

Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis.

Outer membrane fractions were prepared from 11 bacteria in the family Enterobacteriaceae: Escherichia coli serotypes O1K-, O4K2, O26K60, O75K-, and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumonia, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providencia stuartii. All strains studied were found to contain one non-peptidoglycan-bound, heat-modifiable outer membrane protein, and one or two peptidoglycan-associated major outer membrane proteins in the 27,000- to 40,000-dalton range. Crossed immunoelectrophoresis using sodium dodecyl sulfate-polyacarylamide gel electrophoresis for separation of the antigens in the first dimension of the procedure was shown to provide a useful model system for studying the antigenic relationships of the major outer membrane proteins in Enterobacteriaceae species. Peptidoglycan-bound major outer membrane proteins of all bacteria studied reacted with antiserum against the purified peptidogylcan-bound matrix protein I of E. coli O26K60 in this system. Non-peptidoglycan-associated proteins of all strains cross-reacted with protein II of E. coli O26K60 in both their unmodified and their heat-modified forms. These results indicate that the genes coding for the major outer membrane proteins in the family Enterobacteriaceae have been well enough conserved during the course of evolution to allow significant antigenic cross-reactivity between the corresponding proteins in different enterobacterial species.     

矮牵牛(Petunia hybrida)开花过程中的可溶性蛋白质分析

在进行矮牵牛(Petunia hybrida)光周期诱导开花的过程中,对叶子中的可溶性蛋白质进行了分析,其中有4条与开花有关的特异蛋白,分子量分别为49.45 kD(a)、35.45 kD(b)、17.98 kD(c)和11.74 kD(d).在不开放的花苞中不含有蛋白质a和d,只有这4种蛋白质全出现时,才能形成花苞并且开放.花开了以后,蛋白质c和d就消失.即使在开花的植株中,各组织中的蛋白质也是不同的.茎中完全不含有蛋白质c和d,叶子和花中的蛋白质组成也是不同的.     

黄粉甲幼虫抗菌物质的诱导及其抗菌活性

采用饥饿法、紫外线照射法和针刺法处理黄粉甲Tenebriomolitor 6龄幼虫后均能诱导其 产生抗菌物质,收集的血淋巴上清液对真菌有抑制作用,对细菌无抑制作用;经热处理后的血 淋巴上清液则对细菌有抑制作用,而对真菌无抑制作用。SDS-PAGE检测结果发现,与未诱导的 对照相比经诱导的黄粉甲幼虫血淋巴中,原有的一类大分子蛋白质如分子量分别为97kD、44 kD和37 kD左右的蛋白质缺失;而ESI-MS分析结果显示诱导后比诱导前黄粉甲幼虫血淋巴中有 小分子物质产生,推测可能是此类缺失蛋白质分解为小分子量的抗菌肽,从而表现出抗菌活性 。     

Antigenic cross-reactivity of major outer membrane proteins in enterobacteriaceae species.

The protein constituents in the outer membrane (OM) of several serotypes of Escherichia coli and some other Enterobacteriaceae cross-reacted antigenically. Solubilized OM preparations of these bacteria were applied in interfacial precipitin tests to antisera elicited in rabbits against whole bacterial cells, absorbed with their appropriate lipopolysaccharide before testing. The resulting immunecomplexes were analysed on polyacrylamide gels. Protein profiles of the immunoprecipitates showed a considerable antigenic cross-reactivity of outer membrane proteins between most E. coli serotypes. Cross-reactivity, though substantially lower, was also found with OM from three other Enterobacteriaceae species, but was not detectable with Pseudomonas aeruginosa OM. When OM preparations were solubilized at room temperature, the peptidoglycan-bound proteins in the molecular weight range 37,000 to 41,000 predominated in the protein profiles of the immunecomplexes. In profiles of immunecomplexes obtained with boiled OM preparations, a heat-modifiable protein (mol. wt 33,000) predominated. The major OM proteins of the Gram-negative bacterium may therefore play a role as common surface antigens of the family of Enterobacteriaceae.     

九香虫血淋巴及其纯化蛋白抑菌活性的研究

对九香虫AsporgopuschinensisDallas血淋巴及其血淋巴蛋白质分离物的抗菌活性进行了研究,抗菌活性检测指示菌为大肠杆菌Escherichiacoli和金黄色葡萄球菌Staphilocalliesacereus。测定结果表明,九香虫血淋巴及其离心上清液都具有明显的抗菌活性。用凝胶过滤法从血淋巴蛋白分离提纯获得一种小分子肽,SDS PAGE电泳为单一带,分子量约为1～1 4 4kD。该小分子蛋白对大肠杆菌和金黄色葡萄球菌都有抑菌作用,与血淋巴对2种细菌的抗菌性一致,表明其是九香虫血淋巴中具抗菌作用的主要物质之一。     

Calmodulin-Binding Proteins Within the Slow Phase of Axonal Transport in the Rabbit Vagus Nerve Per Ekstrom and Martin Kanje

Calmodulin-binding proteins (CBPs) in the rabbit vagus nerve were studied by means of calmodulin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis. The soluble fraction (10(5) g supernatant) of a nerve homogenate contained four CBPs with molecular weights of 44, 55, 91, and 93 kD, respectively. Slowly transported proteins were recovered in the vagus 3 days after injection of [35S]methionine into the nodose ganglion. Four labelled CBPs with molecular weights of 44, 55, 69, and 83 kD, respectively were found. The nodose ganglion contained two labelled CBPs, 44 and 55 kD. The 55-kD CBP was identified as tubulin after immunoblotting. In separate experiments it was also shown that bovine brain tubulin bound to the calmodulin column.     

The LPS-protein complex from the outer membrane of Francisella tularensis]

LPS-protein complex containing proteins of 15 kD, 17 kD and 19 kD was isolated from F. tularensis outer membrane by solving with sodium deoxycholate with the subsequent gel filtration on Sephacryl S-200. Protein of 17 kD constituted the main protein component of the complex. The LPS-protein ratio of this complex was 1:1. Proteins contained in LPS-protein complex have mainly the alpha-spiral structure. In the absence of detergent these proteins and LPS formed micelles with molecular weight exceeding 10(7) D. LPS-protein complex was shown to have a protective effect in mice infected with F. tularensis virulent strain 503.     

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。