Cloning, expression, and cDNA sequence of surface antigen P22 from Toxoplasma gondii

Immunoblot, immunofluorescence, and complement-mediated cytolytic assays revealed that two new monoclonal antibodies raised against a membrane-enriched fraction of Toxoplasma gondii tachyzoites recognize protein P22 on the surface of the parasite. Using these monoclonal antibodies to screen a cDNA expression library in lambda gt11, several clones expressing recombinant fusion proteins were isolated. Subsequent screening of the library with a synthetic oligonucleotide derived from the 5' end of one of these cDNAs permitted the isolation of additional nonexpressing clones containing the entire translated sequence. Blots of parasite RNA and DNA suggested that the corresponding gene occurs as a single copy in the tachyzoite genome. The amino acid sequence deduced from the composite cDNA indicates a primary translation product with a theoretical molecular weight of 18,959. As expected for surface protein P22, the putative polypeptide contains a predicted N-terminal signal sequence and a C-terminal hydrophobic region characteristic of proteins attached to the membrane by a glycophospholipid anchor. Recombinant fusion proteins produced by the expressing clones were recognized on immunoblots by IgG antibodies in the sera of humans with acute and chronic T. gondii infection. Antibodies selected by the fusion protein reacted predominantly with a 22-kDa antigen on immunoblots of parasite lysate.     

弓形虫RH株微线体蛋白MIC3基因的克隆与表达

目的 克隆和表达弓形虫微线体蛋白MIC3基因。方法 从弓形虫RH株分离总的RNA,反转成cDNA.根据MIC3基因序列,设计合成一对引物,用聚合酶链式反应（PCR）方法从弓形虫cDNA中扩增MIC3基因片段,插入pGEM-T载体,并转化大肠杆菌Top10,经PCR、双酶切、测序验证后,将MIC3基因片段定向亚克隆到载体pET-28a中构建原核表达重组质粒pET-28a-MIC3,重组子在E.coli BL21中经IPTG诱导表达,并对表达产物进行SDS-PAGE和Western-blot分析。结果 从弓形虫RH株cDNA中扩增出792bp大小的MIC3基因片段并诱导表达27 300 Mr的重组MIC3蛋白。结论 成功构建和表达了弓形虫pET-28a-MIC3重组质粒,为弓形虫病诊断抗原和疫苗的研究奠定了基础。     

Identification and characterization of antigenic determinants of Mycobacterium leprae that react with antibodies in sera of leprosy patients.

Antigenic determinants of Mycobacterium leprae were identified by screening a lambda gt11::M. leprae genomic library with two separate pools of sera from leprosy patients. A total of 45 recombinant clones were detected with pooled sera from 21 lepromatous (LL) leprosy patients and 5 additional clones specified polypeptides that reacted with antibodies in pooled sera from 30 borderline tuberculoid or tuberculoid leprosy patients. The recombinant clones that specified antigenic determinants that reacted with sera from LL patients were condensed into eight groups on the basis of DNA hybridization experiments among the M. leprae DNA insert fragments. In addition, 11 of the 45 recombinant clones did not hybridize to members of the eight groups nor to one another; these represent unique recombinant clones. None of the recombinant clones identified by screening with sera from tuberculoid leprosy patients hybridized to each other or to any of the 45 LL recombinant clones. The polypeptides specified by the recombinant clones were usually fusion proteins with beta-galactosidase, ranging in size from 117 to 175 kilodaltons (kDa). Members of hybridization group III specified nonfusion proteins of 45 kDa. Only members of hybridization group I reacted with any of 30 monoclonal antibodies prepared against M. leprae proteins; recombinant proteins from these clones reacted with a single monoclonal antibody directed against the M. leprae 65-kDa protein. Thus, at least 22 new antigenic determinants of M. leprae have been identified on the basis of their reactivity to antibodies in sera from LL patients or sera from tuberculoid leprosy patients or both.     

Human antibody response to the nucleoside triphosphate hydrolase of Toxoplasma gondii

An enzyme-linked immunosorbent assay (ELISA) that uses a recombinant protein of Toxoplasma gondii as antigen was used for the detection of specific antibodies in human sera. An antigenic portion of the T. gondii nucleoside triphosphate hydrolase (NTPase) was expressed in Escherichia coli as a glutathione S-transferase fusion protein with an apparent molecular mass of about 5000. A total of 118 T. gondii positive and 63 negative sera were examined. Seven % of the T. gondii positive sera, but none of the negative sera, reacted with the recombinant NTPase. Advantages and disadvantages that are associated with the use of fusion proteins as antigens in ELISA are discussed.     

Vaccination against Taenia taeniaeformis infection in rats using a recombinant protein and preliminary analysis of the induced antibody response

Primary screening of a cDNA expression library of Taenia taeniaeformis oncospheres in lambda gt11 bacteriophage was carried out using rabbit anti-T, taeniaeformis oncosphere serum affinity-purified from oncosphere pellets. From approximately 1.6 x 10(5) plaques, 21 single clones that were positive with the affinity-purified antibodies were isolated. Sibling analysis revealed that 17 clones out of the 21 could be assigned to five different antigen families. Only family 1 was strongly recognized by a serum prepared in a rabbit against a partially purified host-protective oncosphere antigen fraction. The fragments of lambda DNA were inserted into a pGEX plasmid vector that encodes glutathione S-transferase (GST) of Schistosoma japonicum. Clones designated TtO-18, -49.53 (family 1), 46 (family 2), 15 (family 3), 40 (family 4) and 66 (family 5) were established as subclones in pGEX-1 plasmid vectors which produced GST fusion proteins. All GST fusion proteins were soluble and recognized by anti-GST and anti-TtO sera. Three vaccination experiments with these fusion proteins using specific-pathogen-free Wistar rats revealed that all three fusion proteins of family 1 were exclusively effective against T. taeniaeformis oncosphere challenge with approximately 95% and 91% reductions in cystic metacestode and total metacestode recoveries, respectively. Rats vaccinated with fusion proteins of family 1 produced antibodies which reacted with a 21-kDa oncosphere antigen component which appeared to be a major oncosphere stage-specific antigen.     

Cloning and expression in Escherichia coli of cDNAs encoding a 31-kilodalton surface antigen of Sarcocystis muris.

Using polyadenylated RNA isolated from Sarcocystis muris cyst merozoites, we have constructed a cDNA library in the expression vector lambda ZAP. Immunoscreening with monoclonal and polyclonal antibodies directed against a 31-kDa surface antigen of S. muris [1] yielded a number of clones with insert sizes ranging between 1.1 kb and 1.3 kb. An additional clone with an insert length of 1.55 kb was isolated by screening with a labeled DNA probe derived from one of the cDNA clones. The cDNA sequence was found to contain an open reading frame specifying a polypeptide of 280 amino acids with a predicted size of 29.7 kDa. The deduced amino acid sequence is rich in serine and threonine (22%) and harbors a hypothetical N-terminal signal peptide sequence as well as a C-terminal glycosyl phosphatidylinositol anchor attachment site. The predicted amino acid sequence has been confirmed by peptide sequencing and an analysis of the overall amino acid composition of the 31-kDa protein. A recombinant protein was obtained which was recognized by the polyclonal antibodies directed against the 31-kDa antigen. Antiserum raised against the purified fusion protein specifically reacted with a 31-kDa protein from S. muris cystozoites. Southern blot analysis indicated that the corresponding gene exists as a single copy within the S. muris genome.     

Expression, characterization, and serologic reactivity of recombinant surface antigen P22 of Toxoplasma gondii.

The utility of recombinant Toxoplasma gondii surface antigen P22 for the detection of specific T. gondii antibodies in human sera was evaluated. Polymerase chain reaction was used to produce a 438-bp fragment of the P22 gene; the fragment corresponded to the amino acids predicted to be in the processed, native antigen. The fragment was subcloned into pGEX-2T and was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The fusion protein was purified in a soluble form and was found to be recognized by sera from infected individuals in immunoblots and an enzyme-linked immunosorbent assay. Immunoglobulin G antibodies in sera from 31 acutely infected patients in general reacted more strongly to the fusion protein than did those in sera from 31 patients with the chronic infection. None of the sera from a panel of 26 seronegative controls reacted with the fusion protein was separated from the GST partner by cleavage with thrombin, it retained its immunoreactivity and its electrophoretic mobility in polyacrylamide gels was found to be similar to that of native P22. By a modification of the published method for purification of the foreign polypeptide from the GST carrier, the recombinant P22 was readily purified to homogeneity by thrombin cleavage of the fusion protein while it was adsorbed to glutathione agarose.     

Characterization of cDNA clones encoding ribonucleoprotein antigens expressed in Trypanosoma cruzi amastigotes

Sera from patients with Chagas' disease were used to screen a Trypanosoma cruzi amastigote cDNA library. Characterization of 50 positive clones showed that 21 (42%) encode previously identified T. cruzi ribosomal and flagellar proteins, heat-shock proteins or proteins with repetitive motifs. Twenty-nine clones (58%) correspond to nine genes not previously described in T. cruzi. Three cDNAs, encoding novel repetitive antigens with homology to ribosomal proteins and to other RNA binding proteins, were further characterized. Patient humoral responses against the recombinant proteins encoded by these cDNAs were evaluated in anticipation that they may constitute potential new targets for serodiagnostic assays.     

Stage-specific surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi identified by monoclonal antibodies

Stage-specific Trypanosoma cruzi surface antigens were characterized by using monoclonal antibodies (MAb) which bind specifically to the metacyclic trypomastigotes derived from either the insect vector or acellular cultures. A protein with an apparent molecular weight of 90,000 was detected by the MAb 5E7 on the surface of cultured metacyclics of four strains of T. cruzi: G, CL, Y and Tulahuen. The MAb 1G7, which binds to an epitope of the 90 kDa antigen distinct from that recognized by the MAb 5E7, reacted with metacyclics of the G and Tulahuen strains but not of the Y or CL strain. A polypeptide of approximately 82 kDa, identified by the MAb 3F6, was found in the metacyclics of all four T. cruzi strains. The MAb 3F6 also detected a 75 kDa antigen in the G strain metacyclics. The stage-specific MAb and the polyclonal antibodies from mice protected against acute T. cruzi infection by immunization with killed G metacyclics identified the same set of major surface proteins of G metacyclic trypomastigotes.     

Molecular cloning of cDNAs expressing SS-B/La protein

Using serum from a patient with Sj?gren's syndrome containing a high titer of anti-SS-B/La antibody, cDNA clones (a representative clone was called pA158) were isolated from a human fibroblast cDNA library in lambda gt11 expression vector. After subcloning of pA158 cDNA into an expression plasmid vector pEX-2, a large amount of the recombinant fusion protein with cro-beta-galactosidase (called pA158EX) was obtained in E. coli culture containing the recombinant pEX-2. Antibodies against pA158EX were purified from the patient serum by Sepharose 4B conjugated with the purified pA158EX protein. Immunofluorescent staining of HEp-2 cells with the anti-pA158EX antibodies showed a speckled nuclear staining. In immunoblot analysis, the anti-pA158EX antibodies reacted with 50 kDa protein that was compatible with SS-B/La protein. Immunoprecipitation of leukocyte lysate with the anti-pA158EX antibodies and the following RNA analysis showed that the antibody precipitated Y5 RNA. These findings indicate pA158 is a cDNA for SS-B/La protein. The purified fusion protein was used for enzyme-linked immunosorbent assay (ELISA). Optical density values of anti-SS-B positive sera were high, but those of anti-SS-B negative sera and healthy donor sera were low. In the Northern blot using human RNA and pA158 cDNA, a single band about 1.8 kb was recognized. A full-length cDNA was further obtained by screening of pcD library using pA158 cDNA as a probe.     

Serodiagnosis of recently acquired Toxoplasma gondii infection with a recombinant antigen

A portion of a cDNA encoding a 35-kDa antigen from Toxoplasma gondii was cloned into the CKS expression vector and expressed in Escherichia coli. By using the enzyme-linked immunosorbent assay (ELISA), the recombinant protein (rP35 antigen) was examined for reactivity with immunoglobulin G (IgG) antibodies in the sera of pregnant women. Of these women, 41 had a toxoplasma serologic profile suggestive of recently acquired T. gondii infection (Sabin-Feldman dye test [DT] titers from 1:256 to 1:32,000, positive IgM ELISA titers from 2.3 to 9.7, positive IgA ELISA from 1 to >28, and acute patterns in the differential agglutination [AC/HS] test) (group I), and 50 women had a toxoplasma serologic profile suggestive of infection acquired in the distant past (low DT titers from 1:16 to 1:512, negative IgM ELISA titers from 0 to 0.8, and chronic patterns in the AC/HS test) (group II). The classification of acute or chronic profile was based on the individual's clinical history as well as the combination of the results of the toxoplasma serological profile. An additional group (group III) was composed of sera from 50 women who were seronegative for T. gondii antibodies in the DT. The results revealed that whereas 85.3% of women in group I had IgG antibodies that reacted with the rP35 antigen, only 8% of women in group II had IgG antibodies that reacted with the same antigen. In immunoblots, the rP35 antigen was recognized by IgG antibodies in a pool of sera from individuals with a toxoplasma serologic profile compatible with acute infection but not in a pool of sera from individuals with a serologic profile characteristic of a chronic infection. These results reveal that IgG antibodies against the P35 antigen are produced during the acute stage of the infection but are uncommon in the latent or chronic phase of the infection. Thus, the rP35 antigen may be a useful serologic marker to differentiate between recently acquired infection and that acquired in the more distant past.     

重组弓形虫棒状体蛋白5诱导小鼠的免疫应答及其抗弓形虫感染的保护作用

为探讨重组弓形虫棒状体蛋白5诱导的免疫应答及其免疫保护效应,本研究克隆表达了弓形虫棒状体蛋白5,并分析其诱导的细胞免疫、体液免疫应答和抗弓形虫感染的保护作用。采用PCR方法从刚地弓形虫cDNA中扩增出ROP5基因片段,将该基因片段克隆至pET-28a原核表达载体表达,用重组ROP5蛋白免疫BALB／c小鼠,ELISA检测免疫后小鼠抗体和细胞因子的变化。以强毒型RH株弓形虫攻击感染免疫小鼠,统计小鼠不同时间存活率,评价重组蛋白产生的免疫保护性。结果表明ROP5重组蛋白疫苗免疫BALB／c小鼠诱导机体产生高水平的IgG、IgG1、IgG2a抗体和IFN—y、IL-2及IL-10细胞因子。与PBS及佐剂对照组相比,重组蛋白免疫组小鼠的存活时间明显延长。本实验证实了ROP5重组蛋白免疫BALB／c小鼠能够诱导其产生高水平的体液免疫和细胞免疫应答,以及一定的抗弓形虫感染保护作用。     

Isolation of T-Cell Antigens by Using a Recombinant Protein Library and Its Application to the Identification of Novel Vaccine Candidates against Schistosomiasis

We present here a novel approach to identify T-cell antigens from any infectious agent by use of a library of purified recombinant proteins. Essential features of this strategy include (i) a highly efficient cDNA cloning system which negatively selects against nonrecombinant transformants by making use of the bacterial EcoK restriction system, (ii) affinity staining of cDNA clones expressing recombinant proteins, and (iii) a procedure of simultaneous purification of recombinant proteins from large numbers of isolated clones (representing the protein library) in a single step from pools consisting of up to 24 individual clones. The feasibility of the screening system was confirmed by constructing a protein library of the human parasite Schistosoma mansoni. The recombinant antigens of this library were used to stimulate CD4(+) T cells derived from the axillary lymph nodes of mice vaccinated with irradiated cercariae. In initial screening experiments, we detected parasite-specific proliferation and gamma interferon (IFN-gamma) secretion in response to several pools of cDNA clones. Further analysis of one particular pool revealed that only one of its constituents stimulated considerable IFN-gamma secretion by CD4(+) T cells and that the expressed antigen is identical to a small fragment of myosin heavy chain.     

Monoclonal antibody selection and analysis of a recombinant DNA-derived surface immunogen of Treponema pallidum expressed in Escherichia coli.

Monoclonal antibodies directed against a 34-kilodalton (kDa) surface immunogen of Treponema pallidum were used to select 12 unique T. pallidum DNA-containing Escherichia coli recombinant clones expressing the recombinant form of the 34-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmids in the host cell. Restriction enzyme analyses and Southern hybridization of plasmid DNA demonstrated that all recombinant clones contained common DNA sequences of T. pallidum origin. Further hybridization analyses revealed that the cloned T. pallidum DNA sequences were an accurate representation of the T. pallidum genomic DNA arrangement. Purified immunoglobulin G (IgG) from pooled immune rabbit serum reacted with the clones, while IgG from pooled normal rabbit serum did not. Results of immunological experiments and Southern hybridization indicated that a similar 34-kDa immunogen was present in T. pallidum subsp. pertenue, but it was absent from four species of nonpathogenic treponemes tested, as well as from homogenates of normal rabbit testicular tissue. Metabolic labeling of the E. coli clones with [35S]methionine followed by radioimmunoprecipitation with monoclonal antibodies revealed that the 35S-labeled recombinant and 125I-labeled native (T. pallidum) forms of the antigen had identical electrophoretic mobilities. The production of a complete antigen by E. coli was independent of the orientation of the foreign gene sequence with respect to vector DNA. T. pallidum also produced an apparently identical immunoprecipitable 34-kDa antigen after metabolic labeling with [35S]methionine in the presence of cycloheximide. The apparent specificity of the 34-kDa immunogen for pathogenic treponemes and its native cell surface association on T. pallidum justifies a more intense study of this antigen and its corresponding gene.     

Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli.

Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials.     

Conservation of calnexin in the early branching protozoan Tritrichomonas suis

The cloning and characterization of Ts-p66, a calcium-binding protein representing calnexin of the protozoan parasite Tritrichomonas suis is described. A T. suis cDNA expression library was screened with monospecific antibodies affinity-purified on an immuno-reactive 66 kDa antigen in a Triton X-114 membrane-protein fraction. The deduced amino acid sequence of the resulting cDNA clones revealed that Ts-p66 belongs to the calreticulin protein family and represents calnexin of T. suis. The key structural features and sequence motifs of the calnexins were all conserved. By lectin-blotting we demonstrated that the native protein is glycosylated. Northern and Southern hybridizations showed that T. suis calnexin was highly expressed and encoded by a single or low copy number gene. A cDNA encoding Ts-p66 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant proteins bind Ca(2+). Using immunofluorescence with affinity-purified antibodies, a staining pattern was observed which points towards a putative localization of Ts-p66 in the nuclear membrane and endoplasmic reticulum. Demonstration of a structurally conserved calnexin in the amitochondriate protist T. suis indicates the very early evolutionary origin of the machinery for quality control of protein folding in the endoplasmic reticulum and the molecules involved hereby.     

Molecular cloning of GRA4, a Toxoplasma gondii dense granule protein, recognized by mucosal IgA antibodies.

Clones which were selected from a Toxoplasma gondii expression library with the immune serum from a T. gondii-infected rabbit, were further screened using milk and intestinal secretions from mice which had been orally infected with T. gondii cysts. The gene products of several clones reacted strongly with milk IgA and weakly with intestinal IgA. Three of these clones (5.1, 36.1, 37.4) were shown to encode a dense granule protein of 40 kDa (GRA4). The GRA4 protein co-migrates with one of the T. gondii antigens recognized by mucosal IgA. The complete nucleotide sequence of GRA4 has been obtained by cloning genomic T. gondii BamHI fragments containing the 37.4 DNA insert. The coding sequence contains no intron. The deduced amino acid sequence indicates a proline rich (12%) product with an internal hydrophobic region of 19 amino acids and a potential site of N-glycosylation. The primary translation product with a theoretical size of 36,260 Da contains a putative N-terminal signal sequence of 20 amino acids but no apparent glycolipid anchor sequence. Quantitation of the GRA4 gene and Southern blot analysis suggested that the GRA4 gene is single copy. GRA4 gene is translated in tachyzoites to yield a single mRNA species of about 1900 bases.     

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.