A monoclonal antibody raised against adultSchistosoma mansoni which recognizes a surface antigen on schistosomula

A monoclonal antibody has been raised by immunizing a mouse with an isolated tegumental preparation of adultSchistosoma mansoni. The hybridoma, designated NIMP/M.47, secreted an IgG_2a antibody which was positive by indirect immunofluorescence with live schistosomula ofS. mansoni, but not with live schistosomula ofS. bovis, or with other living life cycle stages ofS. mansoni. In complement-dependent, or cell-mediated in vitro cytotoxicity assays, the monoclonal antibody mediated levels of schistosomular killing as high as those obtained with sera from infected mice. No significant protection, however, was obtained in passive transfer experiments. NIMP/M.47 was specific for a 20,000 dalton polypeptide in the schistosomular surface, which was also recognized by serum from infected mice.     

A model system for the study of antigen secretion by adultSchistosoma mansoni in vivo

Schistosoma mansoni (6 weeks old) were surgically transferred from donor C57BI/6 mice to the hepatic portal veins of naive recipients of the same inbred strain. Between 70% and 100% of the parasites were alive 15 days later, and egg deposition was observed after transfer of worm pairs. The physiological status of the parasites was monitored by measuring the levels of a schistosome gut antigen, circulating anodic antigen (CAA), in the serum of the recipients. When only male worms were transferred, serum CAA levels increased slowly to a peak 9 days later, which was followed by a rapid decline. When worm pairs were transferred, there was an early peak in serum CAA levels followed by a gradual decline, but these levels were always higher than those recorded after male-only transfer; in two mice the pattern was similar to that observed following receipt of male worms. More CAA and eggs were produced after transfer of paired versus separated worms. It was concluded that although worm pairs can be successfully transferred, their physiological status may be sub-optimal. In contrast, male worms survive consistently well, and their transfer to a naive recipient provides a convenient model with which to study the release of antigens by schistosomes in vivo.     

cDNA clone encoding a high molecular weight antigen of Babesia bovis

An expression library was constructed by inserting cDNA copied from mRNA of the blood stages of Babesia bovis isolate KA into bacteriophage lambda gt11-amp3. An antigen-positive cDNA clone detected by screening the library with antibodies from cattle vaccinated with the KA isolate was shown to encode part of a high-molecular weight polypeptide antigen of B. bovis. This molecule was a dominant immunogen and was found by immunofluorescence to be within the parasite in infected erythrocytes.     

A novel cDNA clone encoding a specific excretory/secretory antigen of larval Trichinella pseudospiralis

A cDNA library for Trichinella pseudospiralis was constructed to study the expression of specific antigens. Four positive clones were identified using antibodies against the excretory/secretory (ES) products of the nematode as probe. Sequence analysis showed that they contained identical cDNA inserts of 606 bp, including a 5′ non-translated region of 96 bp, a core translated segment of 408 bp and a poly(A)⁺ 3′ terminus. It encoded a novel 136-amino-acid polypeptide. Southern blot analysis indicated that the cDNA did not cross-hybridize to the genomic digests of T. spiralis, mouse, or rat. A single copy only of its complementary sequence was found in the genome of T. pseudospiralis. Using the lambda ZAP expression system, the cDNA was induced to express a 23-kDa β-galactosidase-fusion protein which did not cross-react with polyclonal and monoclonal antibodies against T. spiralis, heat shock proteins, or four heterologous species of nematodes. The antiserum against the fusion protein recognized a 15-kDa band from the ES products of T. pseudospiralis in immunoblotting. Immunocytolocalization demonstrated that the anti-fusion protein serum only recognized an epitope in the stichosome of T. pseudospiralis and not in T. spiralis. The protein can therefore serve as a specific antigen for the differential diagnosis of trichinellosis. Received: 22 December 1998 / Accepted: 17 February 1999     

Identification by monoclonal antibody of a major (28 kDa) surface membrane antigen of Schistosoma mansoni

Monoclonal antibody M.1 was generated from mice immunized with membrane enriched extracts of mechanically transformed schistosomula. M.1 bound to the surface membranes of cercariae and young (0-24 h post-transformation) schistosomula but did not bind to older schistosomula or cultured worms. M.1 immunoprecipitated an antigen of approximate molecular weight 27-28 kDa from schistosomula. Experiments using metabolic labeling showed that the antigen was actively synthesized by developing schistosomula. Further M.1 immunoprecipitated a similar 27-28 kDa antigen from membrane-enriched extracts of miracidia, lung and adult worms as well as from schistosomula.     

Cloning of the gene encoding a 50 kilodalton potential surface antigen of Schistosoma mansoni

A cDNA library was constructed from the mRNA of adult worms of Schistosoma mansoni, in the expression vector lambda gt11. This library was screened with a pool of sera raised against either soluble egg antigens or purified schistosomulum tegumental membranes. An antiserum raised against the fusion protein of one clone immunoprecipitated a 45 kDa polypeptide from the in vitro translation products of adult worm mRNA and recognised a 50 kDa antigen in homogenates of adult worms. This serum gave positive fluorescence of the surface of schistosomula in indirect immunofluorescence assays and was able to mediate killing of schistosomula by human eosinophils in vitro, suggesting that this clone contained part of a gene encoding a surface antigen.     

Identification and characterization of an Onchocerca volvulus cDNA clone encoding a microfilarial surface-associated antigen.

The identification and characterization of a recombinant cDNA clone (OV103) expressing a microfilarial surface-associated antigen of Onchocerca volvulus is described. OV103 was identified and isolated from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA using a chimpanzee antiserum, taken 2 years after infection with third-stage larvae of O. volvulus. The cDNA clone encodes a 12.5-kDa protein that corresponds to a 15-kDa parasite protein present in microfilariae and adult female worms. The antigen encoded by this clone is located in the basal layer of the cuticle and the hypodermis of the female adult worm, and on the surface of microfilariae. OV103 fusion polypeptide is recognized only by some sera from onchocerciasis infected subjects (57%), but more significantly (89%) by sera from individuals that have low levels of patent infection. In addition, the antibody response to this protein developed before appearance of microfilariae in the skin of chimpanzees that had developed non-patent or low level patent infections, while the antibody response in chimpanzees with high levels of microfilariae appeared later at the time of appearance of microfilariae. Preliminary experiments indicated that affinity purified antibodies directed against OV103 fusion polypeptide mediated killing of nodular microfilariae in vitro in the presence of normal peripheral blood granulocytes.     

cDNA encoding the major mite allergen Der f II

cDNA encoding the major house dust mite allergen Der f II from Dermatophagoides farinae was amplified using the polymerase chain reaction and cloned into E. coli. It encoded a 129-residue protein with a calculated molecular weight of 14,021 D and had the expected high homology (88%) with Der p II including the absence of N-glycosylation sites and conserved cysteine residues. These results are consistent with the high degree of antibody crossreactivity and may help identify the differences in T-cell epitopes revealed for these molecules so far.     

Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms

The isolation and characterization of a recombinant cDNA clone (OV7) expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Using chimpanzee antiserum generated against irradiated infective larvae, we isolated a cDNA clone from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA. The open reading frame encodes 131 amino acids corresponding to a 15.2-kDa protein. Affinity purified antibodies which bound specifically to OV7 fusion polypeptide recognized a single antigen with an apparent molecular weight of 17,000 in extracts of L3, L4 and adult worms. Immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis and the basal layer of the cuticle of L3 and female adult worm, and in the egg shell around developing microfilariae. Since the OV7 fusion polypeptide is onchocerca-specific and is recognized specifically by sera from onchocerciasis patients, and sera from non-patent but infected chimpanzees, and not by sera from patients with other filarial parasites, it may have potential as an antigenic component in a test for detection of non-patent and patent infections of O. volvulus. The OV7 amino acid sequence contains residues that have a probable homology with the cysteine proteinase inhibitor superfamily.     

Identification by message selection of cDNA clones encoding antigens of Schistosoma mansoni

cDNA clone banks have been constructed in a plasmid vector (pJSC73) using mRNA isolated from adult worms and eggs of Schistosoma mansoni. These clone banks have been screened by message selection (hybrid release translation) and clones containing fragments of genes encoding schistosome antigens have been identified by immunoprecipitation of the corresponding mRNA in vitro translation product. Over 50 clones encoding schistosome antigens have been identified including those of the 100 000, 86 000, 28 000, and 27 000 molecular weight surface antigen precursors.     

Caveolae-like structures in the surface membrane of Schistosoma mansoni

Specialized regions of cellular membranes termed detergent-insoluble glycosphingolipid-enriched membrane domains (DIG) have been identified in mammalian cells and shown to contain signalling molecules, cholesterol, sphingolipids and caveolae. Here we report that the unusual double surface membrane of the tegument of the trematode parasite Schistosoma mansoni possesses biochemically distinct domains analogous to DIG. When subjected to sucrose density gradient centrifugation, a detergent-extracted tegument from adult parasites yielded a low-density fraction consisting of detergent-insoluble complexes (DIC). Several tegument proteins were concentrated in DIC and a subset of these were labelled when adult schistosomes were biotinylated using a membrane-impermeant reactive biotin prior to extraction. The GPI-linked proteins alkaline phosphatase (SmAP), Sm200, the membrane-bound protein Sm23, and a protein recognized by an antibody against human caveolin, co-purified with DIC whereas soluble proteins, such as paramyosin and aldolase, were found at the bottom of the gradient. Antibodies against DIC immunoprecipitated a subset of worm surface proteins and immunolabeled the dorsal tegument of adult worms. Transmission electron microscopy of DIC revealed caveolae-like structures in the double bilayer surface structure. These results suggest that the tegument of adult S. mansoni possesses specialized membrane domains that are resistant to detergent-extraction, contain a subset of total tegument membrane proteins, and bear caveola-like invaginations, and thus are analogous to DIG.     

用负向选择增富法筛选人GM-CSF cDNA

用负向选择增富法成功地获得人GM-CSF cDNA克隆,筛选阳性率提高至1/170。用该cDNA转染的CDS-7细胞上清可测出集落刺激活性。经亚克隆化后在大肠杆菌表达出重组人GMCSF,可维持细胞株BMU-2的生长并促进急性非淋巴细胞白血病(ANLL)患者骨髓细胞的集落形成。     

编码人分化抗原5C5全长cDNA的克隆及功能初探

目的 编码５Ｃ５分化抗原全长ｃＤＮＡ的克隆及功能探索。方法 构建人活化Ｂ细胞株３Ｄ５细胞的λｇｔ１１ ｃＤＮＡ文库，以核苷酸杂交法从ｃＤＮＡ文库中筛选阳性克隆、作核苷酸序列分析，编码蛋白质氨基酸序列的亲疏水分析，Ｎｏｒｔｈｅｒｎ ｂｌｏｔ测５Ｃ５ ｍＲＮＡ转录本长度，用ＲＴ－ＰＣＲ检测５Ｃ５ ｍＲＮＡ在不同细胞株的表达，观察单抗５Ｃ５－Ｇ１对３Ｄ５细胞增殖的影响。结果 从人活化Ｂ细胞株３Ｄ５的λｇ