Clonorchis sinensis: expression, characterization, immunolocalization and serological reactivity of one excretory/secretory antigen-LPAP homologue

From a Clonorchis sinensis adult cDNA plasmid library, a cDNA clone encoding a novel lysophosphatidic acid phosphatase (LPAP) homologue was isolated. The predicted molecular weight of putative protein was 48.8 kDa and the deduced amino acid sequence had 45%, 32%, and 29% identity with LPAP of Schistosoma japonicum, Danio rerio, and Homo sapiens, respectively. Prediction of signal peptide and Western blot analysis indicated that the CsLPAP homologue was an excretory-secretory antigen (ES antigen) of C. sinensis. Immunostaining revealed that the CsLPAP was markedly localized in the intestinal cecum, seminal receptacle and eggs of the adult worm. The recombinant CsLPAP showed slightly higher sensitivity (82.14%) and specificity (85.86%) than the crude worm antigen by enzyme-linked immunosorbent assay (ELISA), a result which suggested that the recombinant antigen might be valuable in the serodiagnosis of human clonorchiasis.     

Fasciola hepatica: heterologous expression and functional characterization of a thioredoxin peroxidase

A Fasciola hepatica cDNA clone of 779 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 582 bp which encoded a 194-amino-acid-residue polypeptide (M(r) 21,723 Da) showing a high degree of homology to thioredoxin peroxidases. This putative antioxidant protein gene was expressed in Escherichia coli as a GST fusion protein. The recombinant fusion protein showed in vitro antioxidant properties and protected rabbit muscle enolase and E. coli glutamine synthetase from inactivation by nonenzymatic Fe(3+)/O(2)/DTT or Fe(3+)/O(2)/ascorbate metal-catalyzed oxidation systems.     

Immunisation of cattle with recombinant acetylcholinesterase from Dictyocaulus viviparus and with adult worm ES products

Dictyocaulus viviparus causes a serious lung disease of cattle. For over 30 years, a radiation-attenuated larval vaccine has been used with success; however, this vaccine has several disadvantages. A more stable vaccine against D. viviparus, capable of stimulating prolonged protective immunity, would be beneficial. Recent research has been directed at adult worm ES components that may be involved in parasite survival in the host. One component is the secreted enzyme, acetylcholinesterase (AChE), a target for circulating antibody in infected calves. Here, we describe a study where protection was investigated in calves immunised with either native adult ES products or a recombinant parasite AChE. These antigens were administered twice with Freund's incomplete adjuvant. Subsequently, all calves were challenged with 700 L3 and their worm burdens and immune responses compared with those in calves that received an anthelmintic-abbreviated infection and challenge control calves. Significant levels of protection were not obtained in the immunised groups but significant immunity was achieved in the calves that received the anthelmintic abbreviated infection. Antibody responses amongst the groups were different, with significantly higher IgG1 responses in the immune, infected group and in adult ES recipients. Significantly higher IgG2 responses were found in the latter group. Following challenge, the groups that received the abbreviated infection and the fusion protein produced specific antibody that bound the native enzyme. No differences were observed between groups in peripheral blood mononuclear cell responsiveness to either antigen. However, adult ES products appeared to have a mitogenic effect on these cells, whilst the fusion protein exhibited an inhibitory effect. These results suggest that in this form, AChE is not a potential vaccine candidate and that adult ES products, in contrast to previous experiments in guinea pigs, do not contain protective components.     

Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.     

Identification of bacterial clones that encode cow's caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant DNA plasmids carrying cDNA fragments of bovine caseins in the cDNA library from rRNA of bovine mammary glands. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter paper. Antigens covalently bound to the CNBr-activated paper or bound to the nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. Four clones were positive among 5400 bacterial clones of the cDNA library--al, b2, b5, h7. Molecular weights of chimeric proteins pre-beta-lactamase:casein synthesized in Escherichia coli were determined by immunoblotting. Colony hybridization and DNA sequence analysis showed that clone b5 contained cDNA fragment of bovine kappa-caseins and clone h7 cDNA fragment of beta-casein. The last clone was designated pKcas beta-7.     

Leishmania donovani: identification of novel vaccine candidates using human reactive sera and cell lines

Leishmaniasis is a complex of diseases caused by protozoan parasites belonging to the genus Leishmania. The development of specific resistance against re-infection after cure suggests that a vaccine approach is feasible. Various studies in humans and experimental animals strongly suggest that Th1 type of cell-mediated immune response is important for protection against the disease. A defined antigen that could elicit a specific T-cell-mediated immune response in the host would be an ideal candidate for the vaccine against this parasite. In order to select a candidate antigen, we established a screening system to identify the recombinant clone, expressing antigen having T-cell epitopes from a cDNA library. We screened the library using an established Leishmania specific cell line (LSCL) from a naive healthy human subject. The cell line with predominantly CD4+ cells behaved in a Leishmania specific manner. Fifty-two immuno-reactive clones were screened against the LSCL in vitro and we identified three cDNA clones expressing recombinant antigens that could induce proliferation of these cells to produce INFgamma. The protective efficacy of one of these recombinant proteins was investigated in a hamster model of experimental visceral leishmaniasis and showed protection against a virulent challenge. The identified antigens might be potential candidates for vaccine against Leishmania.     

日本血吸虫中国株次黄嘌呤鸟嘌呤磷酸核糖转移酶的克隆、表达及其免疫保护性研究

根据基因库中日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HGPRT) EST (BU803192) 以及日本血吸虫成虫cDNA文库载体λgt11多克隆位点邻近核苷酸序列设计引物,以日本血吸虫成虫cDNA文库为模板,采用锚式PCR对SjHGPRT基因不完整的3′端和5′端进行扩增、测序,用电子软件拼接,获得SjHGPRT全长cDNA (1 270 bp),经序列分析,推断该片段含有编码SjHGPRT基因的完整阅读框,其编码基因与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SmHGPRT) 全长编码基因碱基一致性为82%,其理论推导的氨基酸组成与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶的一致性约为83%. 将其编码基因克隆到表达载体pQE30上,在大肠杆菌M15中获得准确、高效表达,表达产物分子质量约为28 ku. 用日本血吸虫成虫抗原免疫血清对表达产物进行蛋白质印迹检测,在预测位置上出现明显的识别条带. 重组蛋白动物免疫保护性结果显示：在虫荷、每克肝卵、每克粪卵和雌子宫内卵数方面,疫苗组与对照组比较差异均具有显著性 (P < 0.05,P < 0.01). 结果表明,日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SjHGPRT) 全长cDNA成功克隆并在大肠菌中得到表达,表达产物具有良好的抗原性和动物免疫保护效果,是一种潜在的具有部分免疫保护性的抗血吸虫病疫苗候选分子.     

Schistosoma mansoni heat shock protein 70 elicits an early humoral immune response in S. mansoni infected baboons

A study was undertaken to search for DNA recombinant Schistosoma mansoni proteins responsible for eliciting an antibody response from the host at a very early phase after infection. A S. mansoni adult worm cDNA expression library was screened using pooled sera from baboons with four weeks of infection. Based on their specific reactivity with the S. mansoni infected sera and no reactivity when tested against the pre-infection sera from the same baboons, four clones were selected for further studies. Sequence analysis revealed that they were homologous to the S. mansoni heat shock protein 70 (hsp70). The insert sizes of the four selected clones varied from 1150 to 2006 bp. The preliminary characterization for antibody reactivity against a panel of baboon sera showed that the longest clone was the most reactive, eight out of eight acute and three out of four chronic sera reacting positively to this clone. The shortest clone was the least reactive. Our results suggest that the S. mansoni hsp70 elicits an early and strong antibody response in baboons and that antibodies to this protein can be detected in chronically infected animals. Therefore S. mansoni hsp70 may be a valid target for immunodiagnosis. However further studies are needed to identify the portion of the hsp70 that best fits the requirements for a valuable diagnostic antigen.     

Immunodiagnosis of clonorchiasis using a recombinant antigen

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a β-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.     

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

We have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase [Van den Bosch, H., Aarsman, A. J., DeJong, G. N., & Van Deenen, L. M. (1973) Biochim. Biophys. Acta 296, 94-104].     

Serological differentiation of acute and chronic schistosomiasis using Schistosoma mansoni recombinant protein RP26

We obtained a recombinant protein encoded by Schistosoma mansoni gene which was able to differentiate acute from chronic schistosomiasis when applied as antigen in enzyme-linked immunosorbent assay (ELISA). A cDNA clone encoding a 26 kDa recombinant protein (RP26) was selected by screening of an adult worm S. mansoni λZAP expression library with rabbit sera produced against PIII, an adult worm protein fraction already known to possess protective and immunomodulating effects. The clone cDNA presented 99% identity with S. mansoni Sm22.3 gene. We assayed IgG reactivity of sera from 18 patients with acute, 25 patients with chronic S. mansoni infection and 20 uninfected donors with RP26 in ELISA. Our results showed that 89% of sera were positive in acute schistosomiasis group, and only 26% in chronic group, without false-positive reactions in uninfected group. In mice the immune response to RP26 increased up to week 9 after infection and then diminished. We proposed that production of antibodies binding to RP26 stopped at the chronic stage of disease. The testing of sera from eight other parasitic infections with RP26 revealed no positive reactions in majority of sera. However, we observed low positive reaction in sera from 20% of leishmaniasis patients. Our results indicate that a recombinant protein RP26 can be used as immunodiagnostic reagent for detection of acute phase of schistosomiasis mansoni.     

Identification of a human immunodominant B-cell epitope within the GRA1 antigen of Toxoplasma gondii by phage display of cDNA libraries.

Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambdaD-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide).     

日本血吸虫期别差异表达基因文库的构建及分析

为从期别差异表达基因分析入手研究血吸虫的生长发育机制,应用抑制性消减杂交 (suppressed subtractive hybridization , SSH) 技术首次构建了日本血吸虫尾蚴、虫卵和成虫的期别差异表达基因文库 . 经消减效率分析和三种文库克隆的 EST 的期别差异性鉴定,表明所建文库质量较高,为在整个基因组水平分离血吸虫的差异表达基因提供了重要材料 . 由三个文库选择 257 个插入片段大于 500 bp 的克隆测定了 EST 序列 . 同源性分析结果表明 257 个 EST 代表 182 种血吸虫基因,其中有 22 种为血吸虫已知基因,有 128 种为血吸虫已知 EST ,有 32 种为新发现的血吸虫基因 . 对 EST 编码蛋白的功能预测结果显示：尾蚴消减文库的基因多与运动、能量代谢、转录调节及致病性相关;虫卵消减文库的基因可能参与信号转导、细胞粘附、蛋白质和碳水化合物的代谢以及抗氧化反应;成虫消减文库的基因多参与蛋白质的合成、转运及分解代谢,参与虫体的运动等 . 大规模分离、分析血吸虫期别差异表达基因将对从分子水平去解读血吸虫的生长发育机制,筛选高效疫苗候选抗原、药物靶标及诊断制剂有重要意义 .     

Molecular cloning of the cDNA encoding an immunodominant antigen of Dirofilaria immitis.

An immunodominant antigen of Dirofilaria immitis was studied using recombinant DNA techniques. The mRNA from D. immitis adult female worms was translated in vitro and a major 34 kDa antigenic polypeptide product was demonstrated by immunoprecipitation. cDNA was synthesized from mRNA and a lambda gt11 expression library was constructed and immunoscreened with dirofilariasis positive serum. A positive clone containing a nearly full length cDNA was isolated. The cDNA was 2415 bp in length and consisted of a single open reading frame followed by a long 3' non-coding region of 1446 bp. The open reading frame of 969 bp encoded a polypeptide of 322 amino acids with a molecular weight of 34,400. A cDNA fusion protein synthesized by bacteria (Escherichia coli JM109) using the expression vector pGEMEX-1 was identified as an immunodominant antigen by absorption experiments and had no cross-reactivity with sera from patients with other filarial species.     

Identification of bacterial clones encoding bovine caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by ¹²⁵I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-β-lactamase: casein proteins synthesized in Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine χ-casein, clone 3-H7 contained a cDNA fragment of β-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of α_si-casein.     

Cloning of a Partial Length cDNA Encoding the C-Terminal Portion of the 75-77-kDa Antigen of Trypanosoma cruzi

ABSTRACT It has been suggested that several Trypanosoma cruzi antigens have possible protective epitopes which may be suitable vaccine candidates. We found previously that animals resistant to T. cruzi infection produced antibodies against the 75-77-kDa parasite antigen. To test the ability of the recombinant form of this antigen to protect animals from T. cruzi infection, the cDNA which encodes a portion of the 75-77-kDa antigen was cloned using a cDNA library constructed in an orientation-specific bacteriophage expression vector (λgt11) from poly (A)⁺ RNA of Brazil strain epimastigotes. One clone, named SFS-40, was selected by screening the library using affinity purified antibodies specific for the 75-77-kDa parasite antigen as probe. The cDNA corresponding to the 1.7-kilobase insert of SFS-40 was subcloned into plasmid vectors and characterized. The cDNA sequence encodes a polypeptide of about 40 kDa. The putative product of the cDNA was homologous to members of the 70-kDa stress protein family. When epimastigotes were shifted from 29° C to 37° C, there was no change in the level of SFS-40 mRNA. In contrast, the 70-kDa heat shock protein mRNA of the parasite was increased about four fold by this treatment.     

Human T cell clones recognize two abundant Mycobacterium tuberculosis protein antigens expressed in Escherichia coli

Human T cells reactive to Mycobacterium tuberculosis were cloned from peripheral blood mononuclear cells (PBMC) of four tuberculosis patients by using whole irradiated bacilli as the in vitro stimulatory agent. Twenty-two T cell clones (CD4+) were tested for their reactivity to 12 different mycobacterial species and showed a distribution from limited to broad cross-reactivity. These T cell clones were also tested for their reactivity to three abundant M. tuberculosis proteins of 65, 19, and 14 kD, each expressed from recombinant DNA in Escherichia coli. The three proteins were expressed from DNA clones that were previously isolated from a lambda gt11 genomic DNA library of M. tuberculosis by using monoclonal antibodies directed against this pathogen. A T cell clone from one patient was stimulated by an E. coli lysate containing the 65 kD antigen, and a T cell clone from a second patient was stimulated by an E. coli lysate containing the 19 kD antigen in an in vitro proliferation assay. Both T cell clones showed very limited cross-reactivity when tested against other mycobacteria. We conclude that some patients with tuberculosis exhibit a T cell response to the 65 and 19 kD M. tuberculosis proteins defined by these isolated genes.     

Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae.

The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from T. spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed. A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing nearly full-length cDNA for a 46 kDa protein was isolated. The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide. The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.     

Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.