Cord blood serum affects T cells ability to produce and respond to IL-2

The current literature suggests that cord blood (CB) cells are functionally immature. We previously reported that CB sera inhibit T cell proliferation and suggested that the microenvironment in which CB T cells reside may be, in part, responsible for their reduced function. In this study we have tried to explain some of the actions of the CB sera on peripheral blood mononuclear cells (PBMC). We showed that, as expected CB sera decreased the anti-CD3 and anti-CD28-induced proliferative response of PBMC (p < 0.01) but unexpectedly, increased the interleukin-2 (IL-2) specific proliferation of both a human T cell line (p < 0.005) and T cells within a mononuclear cell population (p < 0.05). These findings prompted us to analyse the effect of CB sera on the T cell ability to make and respond to IL-2. Stimulation of T cells in the presence of CB sera increased the frequency of IL-2 producing cells (p < 0.005) (but not the amount of IL-2 secreted) and resulted in a higher expression of CD25 (p < 0.05). Furthermore CB sera (in the presence and absence of IL-2) made the cells apoptose less (p < 0.005) than adult sera. Our results go some way to explaining the effect of the CB microenvironment on CB cellular function.     

Characterization of an immunoreactive 93-kDa core protein of Borrelia burgdorferi with a human IgG monoclonal antibody

Lyme borreliosis is an infectious disease caused by the tick-borne spirochete Borrelia burgdorferi, which carries the potential for chronic infection. Ag on the etiologic Borrelia are currently being defined structurally and their ability to elicit immune responses delineated. EBV can be used to immortalize human B. burgdorferi-specific B cells from infected donors and generate antibodies against antigenic epitopes encountered in natural infection. A human mAb secreting EBV-transformed B cell line, D7, has been developed that is specific for a 93-kDa B. burgdorferi protein and has been used to characterize this potentially important Ag. D7 produces an IgG3 antibody that detects the 93-kDa Ag as well as smaller fragments at 46 kDa and lower molecular mass. The antibody detects similar epitopes on all B. burgdorferi isolates tested and on a Borrelia hermsii protein with molecular mass greater than 100 kDa but binds poorly to Treponema species. In contrast, polyclonal sera from Lyme disease patients show little binding to the homologous Ag in B. hermsii. Structurally, the 93-kDa protein is associated with the flagellum and may be firmly anchored in the protoplasmic cylinder. It is not solubilized by nonionic detergent treatment of the whole Borrelia. Antibodies against a comparable m.w. protein are present in sera from patients with both early and late infection. Thus, antibodies against this Ag are a sensitive and specific marker of Borrelia infection. This Ag is likely of structural importance and may represent a target of host defenses.     

大鼠大麻素Ⅰ型受体绿色荧光融合蛋白真核表达载体的构建与鉴定

目的:构建大鼠大麻素型Ⅰ受体绿色荧光融合蛋白真核表达载体并观察其在细胞中的表达。方法:大鼠CB1基因序列设计引物,以大鼠脑组织为模板扩增CB1基因编码区片段,克隆至增强型绿色荧光蛋白表达载体pEGFP-N3中,构建重组融合蛋白表达载体pCB1-EGFP。将pCB1-EGFP质粒转染HeLa细胞,通过观察EGFP报告基因的表达以及免疫荧光,Western Blot方法鉴定CB1可在真核细胞中过表达情况。结果:构建重组融合蛋白表达载体pCB1-EGFP,单双酶切和测序验证正确。将pCB1-EGFP质粒转染HeLa细胞,荧光显微镜下观察到融合表达的绿色荧光蛋白,且呈胞膜表达。免疫荧光试验也证明重组载体转染后,CB1基因和GFP共同定位于胞膜部分。Western Blot实验证明表达CB1蛋白。结论:成功构建了高表达的CB1-EGFP融合蛋白真核表达载体。     

Fetal immune responses to Plasmodium falciparum antigens in a malaria-endemic region of Cameroon

Plasmodium falciparum infection during pregnancy can lead to the transplacental passage of malarial Ags that are capable of inducing acquired immune responses in the fetus. Studies have identified cytokines produced by malaria-specific cord blood (CB) T cells, but information on fetal B cells is limited. Thus, CB mononuclear cells from 120 Cameroonian newborns were cultured for 7 days in vitro and supernatants were assessed by ELISA for Abs to an extract of malarial schizonts (MA), recombinant apical merozoite Ag 1 (AMA-1), the 42-kDa C-terminal region of merozoite surface protein 1 (MSP-1(42)), a B epitope of ring-infected erythrocyte surface Ag (RESA), and the dominant B epitope of the circumsporozoite protein (CSP). Only 12% of supernatants contained IgM to MA but 78% had IgG to one or more malarial Ags, with 53% having IgG to AMA-1, 38% to MSP-1(42), 3% to RESA, and 0% to CSP. The Abs to AMA-1 and MSP-1(42) were predominantly IgG1 and IgG3. CB mononuclear cells were also tested for the ability to secrete cytokines in response to MA and a pool of conserved MSP-1 T cell epitopes. Among the Ag-reactive samples, 39.3% produced only Th2-type cytokines, whereas 60.6% produced a combination of Th1- and Th2-type cytokines. Although a Th2 bias was observed, the in utero cytokine environment was adequate to support isotype switching to cytophilic IgGs, the isotypes that are protective in adults. Because many infants living in a low transmission area are born with malaria-specific B and T cells, the influence of in utero priming on neonatal immunity merits further investigation.     

The Labeling of Proliferating Cells by Ki67 and MIB-1 Antibodies Depends on the Binding of a Nuclear Protein to the DNA

The antigen Ki-67 Ag, regarded as a marker for proliferating cells, was identified as a protein(s) (pKi-67) which can exist free or associated with DNA as evidenced by DNA digestion of cells before or after immunolabeling with Ki-67. The dual nature of this antigen was also supported by reconstitution of Ki-67 Ag from purified DNA and nuclear proteins extracted from the K562 cell line. The immunoreactivity of the resulting complexes was examined in solution using Ki-67 and MIB-1 antibodies. The interaction between Ki-67 or MIB-1 antibodies and pKi-67 was enhanced in the presence of undegraded ds DNA, indicating that ds DNA modulates the conformation of pKi-67 and that the altered conformation of pKi-67 is more reactive than the pure protein to both Ki-67 and MIB-1 antibodies.     

Alpha-actinin is a cross-reactive renal target for pathogenic anti-DNA antibodies

Anti-DNA Abs commonly found in patients with systemic lupus erythematosus are thought to play an important pathogenic role in lupus nephritis. Anti-DNA Abs may contribute to renal disease by cross-reactivity with renal Ags, the identity of which remain elusive. To identify a target Ag for pathogenic anti-DNA Abs, we performed Western blotting and immunoprecipitations of mesangial cell lysates from the lupus-prone MRL-lpr/lpr mouse and a nonautoimmune BALB/c mouse with the pathogenic anti-DNA Ab R4A. We found that R4A (but not a nonpathogenic Ab mutant of R4A) binds to and immunoprecipitates a 100-kDa protein expressed on the cell surface and in lysates of MRL-lpr/lpr mesangial cells. DNase treatment of the lysate and of the R4A Ab did not effect binding, indicating that the binding of R4A to the 100-kDa protein was direct and not mediated by an antigenic bridge containing DNA. Binding was greatly diminished in BALB/c lysates, suggesting that Ag expression or availability at the level of the target organ may be a factor in determining susceptibility to lupus nephritis. Following identification of this 100-kDa protein as nonmuscle alpha-actinin, binding of R4A to alpha-actinin was confirmed by Western blot, ELISA, inhibition studies, and immunofluorescence. High titers of anti-alpha-actinin Abs were present in sera and kidney eluates of lupus mice with active nephritis. These results indicate that the nephritogenicity of some anti-DNA Abs may be mediated via cross-reactivity with alpha-actinin. Furthermore, variations in target Ag display between individuals may underlie differential susceptibility to anti-DNA Ab-induced renal disease.     

Cell transformation by a virus containing a molecularly constructed gag-erbB fused gene.

A provirus DNA that contains a gag-erbB fused gene as the sole and transforming gene was molecularly constructed from plasmid pSRA2 containing the entire genome of Rous sarcoma virus and pAE7.7 containing the entire genome of an avian erythroblastosis virus (AEV), AEV-H. A virus containing the gag-erbB fused gene (GEV) was recovered from chicken embryo fibroblasts transfected with the proviral DNA and a helper virus DNA. GEV could transform chicken embryo fibroblasts as efficiently as could AEV-H. Anti-erbB and anti-gag sera immunoprecipitated a protein with a molecular weight of about 110,000 from GEV-transformed cells. The erbB and gag-erbB fused-gene products in AEV-H- and GEV-transformed cells were analyzed.     

Human monoclonal antibodies reactive with antigens of the group A Streptococcus and human heart

Human mAb were produced from tonsillar or PBL of normal individuals or patients infected with group A streptococci. Lymphocytes were purified on Ficoll-Hypaque gradients and stimulated in vitro with purified group A streptococcal membranes or M protein extracts. The mAb were selected for study based on their reaction with group A streptococci, pep M5 protein, and/or M6 Escherichia coli protein. Further analysis by Western immunoblot or competitive inhibition ELISA revealed that there were two types of antibodies: one type that reacted with myosin and DNA and the other type that reacted with myosin, keratin, and/or actin. The specificities of these human mAb are similar to specificities observed in our previous studies of murine mAb reactive with group A streptococci and heart Ag. For comparison, anti-myosin antibodies were affinity purified from the sera of infected or acute rheumatic fever patients and were shown to react with myosin and DNA as well as with group A streptococci and M protein. To affinity purify these antibodies from normal sera, five times the amount of sera was required to obtain detectable quantities. These data suggest that the human mAb reactive with group A streptococci and myosin reflect the antibodies seen in sera from infected patients or acute rheumatics and that the B lymphocyte clones capable of producing these cross-reactive antibodies are also present in normal individuals.     

Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.     

A panel of candidate tumor antigens in colorectal cancer revealed by the serological selection of a phage displayed cDNA expression library

In the last few years it has been shown that the humoral immune response in cancer patients is a rich source of putative cancer vaccine candidates. To fully explore the complex information present within the Ab repertoire of cancer patients, we have applied a method, serological Ag selection, to molecularly define tumor Ags recognized by the humoral immune response in colorectal cancer (CRC). First, we built a cDNA display library by cloning a cDNA library from CRC cell line HT-29 for expression as a fusion protein with a filamentous phage minor coat protein, pVI. This cDNA display library was then enriched on pooled sera from CRC patients who had undergone active specific immunization with autologous tumor. We identified a panel of 19 clones reactive with the serum pool. Seventeen of 19 (89%) clones showed reactivity with one or more of the eight Ag-reactive sera, conversely six of eight (75%) sera were reactive with at least one of the 19 clones. Sequencing revealed that these 19 clones represented 13 different Ags. A detailed serological analysis of the 13 different Ags showed preferential reactivity to sera of cancer patients for six different Ags. Four of these Ags displayed increased serum reactivity after the active specific immunization procedure. Furthermore, one of the six Ags, a novel Ag homologous to HSPC218, showed restricted expression in normal testis, suggesting that it belongs to the cancer-testis Ag family. Some of the Ags we have identified may be candidates for tumor vaccination, for sero-diagnosis of cancer, as prognostic markers, or as probes for monitoring tumor cell-based vaccination trials.     

Apoptotic cell death by the novel natural compound, cinobufotalin

Cinobufotalin (CB), one of the bufadienolides prepared from toad venom, was investigated for its cytotoxicity, and the underneath mechanism involved. We primarily utilized DNA fragmentation assay and microscopic observation to assess the effect of various doses of CB in human lymphoma U937 cells. Following that, we investigated other parameters involved in cell death mechanism such as reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and apoptotic proteins activation. HeLa cells were concomitantly used to generalize the data observed. Our results show that CB caused significant DNA fragmentation, decrease of MMP, and an increase in the intracellular Ca(2+) ion and ROS production. In addition, CB induced upregulation of Fas protein, proteolytic activation of cytochrome c, caspase-2, -3, -8 and -9 together with the activation of Bid and Bax. Our findings were further validated using either Fas/FasL antagonist or pan-caspase inhibitor to significantly inhibit CB-induced DNA fragmentation. In our study, we suggest that CB induces caspase dependent cell death in U937 cells, and that Fas plays a role in CB-induced apoptosis. Altogether, our data provides novel insights of the mechanism of action of CB and its potential as a future chemotherapeutic agent.     

The Ag85B protein of Mycobacterium tuberculosis may turn a protective immune response induced by Ag85B-DNA vaccine into a potent but non-protective Th1 immune response in mice

Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy is essential to understand tuberculosis pathology and to develop novel vaccines. In mice, a primary vaccination with Ag85B-encoding plasmid DNA (DNA-85B) was protective against Mycobacterium tuberculosis (MTB) infection and associated with Ag85B-specific CD4+ T cells producing IFN-gamma and controlling intramacrophagic MTB growth. Surprisingly, this protection was eliminated by Ag85B protein boosting. Loss of protection was associated with a overwhelming CD4+ T cell proliferation and IFN-gamma production in response to Ag85B protein, despite restraint of Th1 response by CD8+ T cell-dependent mechanisms and activation of CD4+ T cell-dependent IL-10 secretion. Importantly, these Ag85B-responding CD4+ T cells lost the ability to produce IFN-gamma and control MTB intramacrophagic growth in coculture with MTB-infected macrophages, suggesting that the protein-dependent expansion of non-protective CD4+ T cells determined dilution or loss of the protective Ag85B-specific CD4+ induced by DNA-85B vaccination. These data emphasize the need of exerting some caution in adopting aggressive DNA-priming, protein-booster schedules for MTB vaccines. They also suggest that Ag85B protein secreted during MTB infection could be involved in the instability of protective anti-tuberculosis immune response, and actually concur to disease progression.     

Enhancing DNA vaccine potency by combining a strategy to prolong dendritic cell life with intracellular targeting strategies

We have recently shown that intradermal coadministration of DNA encoding Ag with DNA encoding inhibitors of apoptosis, including Bcl-x(L), prolongs dendritic cell (DC) life and thereby enhances the potency of DNA vaccines in vivo. We have also demonstrated that DNA vaccines targeting Ag to subcellular compartments, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, or the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhanced DNA vaccine potency. In this study, we reasoned that the combination of a strategy to prolong DC life with intracellular targeting strategies might produce a more effective DNA vaccine against human papillomavirus E7. We showed that coadministration of DNA encoding Bcl-x(L) with DNA encoding E7/heat shock protein 70, calreticulin/E7, or Sig/E7/LAMP-1 resulted in further enhancement of the E7-specific CD8(+) T cell response for all three constructs. Of these strategies, mice vaccinated with Sig/E7/LAMP-1 DNA mixed with Bcl-x(L) DNA showed the greatest increase in E7-specific CD8(+) T cells ( approximately 13-fold increase). This combination of strategies resulted in increased CD8(+) T cell functional avidity, an increased E7-specific CD4(+) Th1 cell response, enhanced tumor treatment ability, and stronger long-term tumor protection when compared with mice vaccinated without Bcl-x(L) DNA. Therefore, DNA vaccines that combine strategies to enhance intracellular Ag processing and prolong DC life have potential clinical implications for control of viral infection and neoplasia.     

Inhibition of mycoplasma cell division by cytochalasin B

Mycoplasma gallisepticum has subcellular organelles which may function as a primitive "mitotic-like" apparatus. To investigate these further, we have studied the effects of cytochalasin B (CB) on M. gallisepticum. We found that CB inhibits cell division; this is the only procaryote thus far reported to be inhibited by CB. CB does not inhibit glucose or macromolecule precursor uptake. It stops cellular DNA synthesis, however, although RNA and protein synthesis continue (at a reduced rate). CB removal results in a resumption of DNA synthesis, followed by cell division. There appears to be some degree of cell synchrony in this first division after CB removal. These results, together with morphological data, indicate that CB blocks at two points in the cell cycle: at the time "mitotic-like" structures are formed and at the time of cell division. It is suggested that the CB blocks may result from a disruption of actin-like protein structures required at these points in the cell cycle.     

Functional analysis of a T cell line specific for antiidiotypic antibodies to a Schistosoma mansoni protective epitope. II. Induction of protective immunity in experimental rat schistosomiasis.

In our previous work on the idiotypic network in the rat model of schistosomiasis we showed that immunization with an IgE mAb specific for 26/56-kDa parasitic Ag resulted in the production of anti-anti-Id antibodies of both the IgG and IgE classes. Further studies demonstrated that anti-Ab2 T cell lines, obtained by immunization with Ab2 antibodies, functioned as conventional Th cells; they were MHC-restricted and required APC to proliferate in the presence of the native schistosomula Ag and the Ab2 antibodies. We report the involvement of these anti-Ab2 cells in the regulation of protective immunity. The transfer of long term culture anti-Ab2 T cell lines into LOU/M rats, followed by a challenge infection by Schistosoma mansoni 1 day after the cell transfer led to a slight increase in the worm burden. On the contrary, the transfer of anti-Ab2 T cells 90 days before S. mansoni infection induced a significant reduction of the worm burden (up to 57%). T cells recovered from the protected rats were stimulated by the native schistosomula Ag as well as by tryptic fragments of IgG isolated from the Ab2 sera, in the presence of irradiated thymic cells as APC. We also analyzed the humoral response developed by the rats after transfer with the anti-Ab2 T cell lines. The sera induced various inflammatory cells into cytotoxic effectors against the larvae of S. mansoni, arguing for the presence of functional IgE in the sera. Moreover, when these sera were passively transferred into rats infected 1 day later, a significant reduction of the worm burden was observed. However, antibody-dependent cytotoxic mechanisms efficient 10 days after the anti-Ab2 T cell transfer did not correlate with the protective immunity which required a 90-day delay to be established. These data suggest that the protective immunity induced by the anti-Ab2 cells is supported both by the cellular and humoral components and that in a future vaccinating strategy the idiotypic network may play a crucial role.     

Antigenic and immunogenic properties of cyanogen bromide peptides from a serotype 5 gonococcal outer membrane protein I

Treatment of a gonococcal major outer membrane protein IB (serotype 5) with cyanogen bromide (CNBr) resulted in cleavage of PIB into three major fragments of apparent molecular weight of 15, 13, and 8 kD. The location of these peptides in the intact protein was determined by analysis of partial cleavage products. The 8 kD peptide (CB2) was found to be located in the central region of the protein. Chymotrypsin cleavage of PIB revealed a cleavage site near one of the CNBr cleavage sites. Trypsin was found to cleave the protein, either in outer membranes (OMC) or in detergent micelles, in the central CB2 fragment. These result suggest that CB2 is a part of the surface exposed region of PIB.Immunization of mice with purified PIB (serotype 5) induced antibodies against all three CB-peptides. Absorption of the sera with homologous intact OMC resulted in a complete removal of antibodies against CB2, supplying further evidence for its surface exposed nature. Antibodies against the 13 kD peptide (CB1) could not be absorbed with intact OMC, suggesting that this peptide is buried within the outer membrane.Antisera raised against CB2 of serotype 5 demonstrated a considerable cross-reactivity with heterologous outer membranes. On the contrary, intact OMC induced mainly type-specific antibodies. These data demonstrate the presence of conserved epitopes on the surface exposed CB2 peptide. These conserved epitopes are generally not very immunogenic when present in intact OMC.     

Induction of protective immunity in mice using a 62-kDa recombinant fragment of a Schistosoma mansoni surface antigen.

Mice exposed to radiation-attenuated cercariae of Schistosoma mansoni are highly resistant to challenge infection, and sera from these mice can confer partial resistance when transferred to naive recipients. These sera recognize Ag present in schistosomular and adult worms, among them an Ag of 200 kDa. A cDNA encoding a 62-kDa portion of this Ag was cloned; the deduced amino acid sequence of this cDNA clone shares homology with myosins of other species. To assess the immunoprophylactic potential, we carried out vaccination trials in mice using the recombinant polypeptide expressed as a fusion protein with beta-galactosidase presented in the form of proteosome complexes with the outer membrane protein of meningococcus. The level of protection achieved was 32%, and this level could be increased to 75% by removal of those amino acids included in the fusion protein that were derived from the vector to yield a polypeptide, designated rIrV-5. A similar level of protection was achieved when mice were immunized with the same dose of rIrV-5 in the form of protein complexes but without outer membrane protein, suggesting that protection did not require the use of adjuvant. However, at least three immunizations were necessary to achieve protection. Using mAb and sera from mice vaccinated with rIrV-5, we demonstrated that the native protein recognized by antibodies against rIrV-5 is a 200-kDa protein that is expressed on the surface of newly transformed schistosomula. The protection achieved with rIrV-5 in mice encourages additional studies of its potential as a vaccine candidate for the prevention of schistosomiasis.     

Protective immunity against Brugia malayi infective larvae in mice. II. Induction by a T cell-dependent antigen isolated by monoclonal antibody affinity chromatography and SDS-PAGE

A mAb directed against filarial worm secretory/excretory product and reactive with Brugia malayi larval worm surface was used in conjunction with preparative SDS-PAGE to isolate protective Ag from extracts of adult B. malayi. The IgM mAb OVH bound to a repeating carbohydrate epitope present in adult, infective, and fourth stage larvae and microfilariae of B. malayi, and on the surface of fourth stage larvae. Ag bearing this epitope were also present in the sera of hosts infected with a variety of helminths, including Brugia, Onchocerca, Dirofilaria, and Paragonimus. Affinity chromatography of SDS extract of adult Brugia, using mAb OVH immobilized on agarose beads, isolated several Ag that separated into multiple protein staining bands on SDS-PAGE. In comparing SDS-PAGE-fractionated Ag from the crude SDS extract with fractionated mAb OVH-isolated Ag for the ability to protect BALB/c mice from challenge with B. malayi-infective larvae, it was found that of the mAb OVH-isolated Ag only those at a molecular mass of 26 to 32 kDa were protective while the original SDS extract yielded protective Ag at the following molecular mass: greater than 200, 170 to 200, 40 to 44, 33 to 36, 23 to 28, 20 to 22, and 17 to 19 kDa. Although Ag isolated by mAb OVH were highly protective, they failed to induce high antibody levels against the immunogen or SDS extracts compared to crude SDS extract immunized mouse sera, as determined by immunoblot and ELISA. Transfer of nylon wool non-adherent T cells from BALB/c mice immunized with the 26- to 28-kDa fraction of mAb OVH-isolated Ag to naive mice just before challenge with infective larvae of B. malayi resulted in a 70% reduction in larvae recovered 14 days after challenge.     

新型抗结核蛋白亚单位疫苗Ag85A-γ干扰素的免疫效果评价

本研究旨在评价新型抗结核融合蛋白亚单位疫苗Ag85A-γ干扰素(Ag85A-IFN-γ)的免疫效果.在成功表达并纯化了去除Ag85A信号肽的融合蛋白Ag85A-IFN-γ后,将其与免疫佐剂二甲基三十六烷基铵(DDA)混合,皮下免疫C57BL/6小鼠3次,每次间隔2周,末次免疫2周后进行效果评价.采用酶联免疫吸附试验(ELISA)检测血清中IgG、IgG1和IgG2c水平.结果显示,融合蛋白Ag85A-IFN-γ组的IgG水平均高于单独抗原组,且融合蛋白组IgG2c/IgG1 比值显著高于对照组,表明融合蛋白能刺激宿主产生更强烈的体液免疫反应,更倾向于激发T辅助细胞1型(Th1型)免疫反应.流式细胞术检测特异性CD4+和CD8+ T细胞比例,发现Ag85A-IFN-γ组CD8+/CD4+最高,显示该融合蛋白能显著增强CD8+ T细胞增殖.上述结果表明,融合蛋白Ag85A-IFN-γ有望成为有效的新型抗结核融合蛋白亚单位疫苗.     

The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein.

cDNA libraries have been constructed in the plasmid vector pUC18 with mRNA isolated from both epimastigotes and trypomastigotes of the Peru strain of Trypanosoma cruzi. Pools of randomly selected clones were analyzed by hybridization-selection-translation. Translation products were immunoprecipitated either with normal human sera or with sera from patients with Chagas' disease (chagasic sera), and the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this approach, a cDNA clone (pEC5) was identified which encodes a portion of an 85,000-Mr polypeptide. A genomic clone was subsequently isolated (FG1) by using oligonucleotide probes derived from the DNA sequence of this cDNA clone. A portion of this clone was isolated and sequenced, and the coding region for the protein was identified. Computer analysis of the predicted protein sequence indicates that this protein is closely related to the 83,000-Mr heat shock protein (hsp83) of Drosophila melanogaster, the hsp90 of Saccharomyces cerevisiae, and the hsp90 of chicken. This gene is tandemly organized in the T. cruzi genome as a cluster of 6 to 10 copies.