合成肽抗原在戊型肝炎病毒感染诊断中的应用

An ELISA for the detection of anti HEV using synthetic peptide antigens was developed. The synthetic antigens were encoded by OFR2 and OFR3 genes of HEV. The purpose of this study was to determine the applicability of the synthetic antigens in the serodiagnosis of hepatitis E. The anti HEV detection using synthetic antigens was carried out in 47 healthy subjects and 89 patients with acute or chronic viral hepatitis. The results showed that the positive rate of anti HEV IgG in healthy subjects was 4.2%(2/47), and no IgM antibody to HEV was found. The positive rates of IgG and IgM antibodies to HEV in the hepatitis patients were 8.9% and 10% respectively. In addition, we compared the detecting efficacy of the synthetic antigens with that of the market reagent in 57 serum samples, the total coincident rate was 87.7% (50/57). All of the results accorded with the literatures reported. This study suggests that the ELISA based on the synthetic peptide antigens was specific, sensitive and convenient in diagnosis of HEV infection, it can be widely used in both clinical and epidemiological reseaches.     

Serodiagnosis of Mycoses Using Recombinant Antigens

The early diagnosis of mycoses is important for the institution of an effective antifungal therapy. Detection of antibodies against crude antigenic extracts is one of the standard techniques for the diagnosis of most mycoses. However, while these crude antigenic extracts are relatively easy to obtain, they usually show low reproducibility and are not very specific, since antibodies from patients with different mycoses may show cross-reactivity. The application of molecular biology techniques to the study of fungal antigens has allowed the production of recombinant antigens that may help to solve these problems. The purpose of this review is to discuss the use of recombinant fungal antigens in the diagnosis of mycoses.     

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

Background

Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved.

Methodology/Principal Findings

Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus.

Conclusions/Significance

This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.     

Utilization of Proteinase K-Treated Cells as Lipopolysaccharide Antigens for the Serodiagnosis of Helicobacter pylori Infections

We have evaluated the use of proteinase K (PK)-treated cells isolated from Helicobacter pylori as lipopolysaccharide (LPS) antigens in an immunoblot assay and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of H. pylori infection. The sera from patients with chronic gastritis, gastric ulcer, duodenal ulcer or gastric cancer, and from healthy adults with or without H. pylori infection were assayed with three commercial serodiagnostic kits (HM-CAP, Helico-G, and G.A.P. II) and novel methods relying on the use of PK-treated cells. The PK-treated cells used in these assays were selected on the basis of their possibility to possess a common epitope in the O-polysaccharides of H. pylori, which is known to be highly immunogenic in humans. Of the sera from these patients, 71-94% were positive with the commercial kits, 97% with immunoblot assay, and 90% with ELISA. On the other hand, of the healthy adults infected with H. pylori, 72-97% were positive with the commercial kits, 86% with immunoblot assay, and 72% with ELISA. PK-treated cells that did not contain the common epitope were unsuitable as an antigen for immunoblot assay or ELISA. Furthermore, the reactivity of these sera reacted specifically with H. pylori PK-treated cells but not with LPSs from other gram-negative bacteria, such as Campylobacter, Proteus, Bordetella, and Salmonella. These results demonstrate that the serological assays relying on the use of H. pylori PK-treated cells possessing a highly antigenic epitope are potentially useful as a serodiagnostic test for H. pylori infection.     

The Principal Neutralizing Determinant of HIV-1IIIB: Conformation of the Peptide Bound to a Neutralizing Antibody Studied by 2D-NMR

Summary RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5β raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser⁶-Thr¹⁹ are part of the epitope, while Lys⁵ and Ile²⁰ are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel β-strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5β.     

The Principal Neutralizing Determinant of HIV-1IIIB: Conformation of the Peptide Bound to a Neutralizing Antibody Studied by 2D-NMR

RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5 raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser⁶ - Thr¹⁹ are part of the epitope, while Lys⁵ and Ile²⁰ are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel -strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5.     

HIV—1融膜肽的编码DNA序列比较

通过分析GenBank中的全部95个HIV－1完整基因组序列,设计融膜肽“探针序列”,对所获得的95段融膜肽的编码DNA序列进行了翻译、对准和分析。得到融膜肽及其编码序称的“优势序列”及突变分布。     

Peptide nucleic acids as epigenetic inhibitors of HIV-1

The advent of highly active antiretroviral therapy (HAART) was once perceived to havetransformed deadly HIV/AIDS into a treatable, chronic infectious disease. However, mountingevidence now suggests that the prevalence of multi-drug resistant HIV (MDR-HIV) infection issteadily rising among newly infected individuals in the HAART-experienced countries, raising aconcern for a future outbreak of MDR-HIV/AIDS. Our global fight against AIDS must include sustainedeffort to search and discover a new therapeutic modality for HIV infection. Of plausible viraltargets explored to date, HIV gene-targeting approach has not yet seen a considerable success invivo. The pursuit of anti-HIV gene intervention should include the identification of critical genetargets as well as the optimization of biomolecules that can effectively interact with theintended targets. Using unmodified peptide nucleic acids (PNA) as a biomolecular tool, we discovereda potentially critical HIV gene segment within gag-polencoding gene. Antisense PNA targetingthis specific region effectively disrupted a translation of HIV gag-polmRNA, abolishing thevirion production from chronically HIV-infected cells. This exemplifies the possibility that epigenic HIV inhibitors may be developed in the coming years, if emerging novel technologies permitsufficient and stable in vivo delivery of PNA or other similarly effective biomolecules.     

Peptide nucleic acids as epigenetic inhibitors of HIV-1

Summary The advent of highly active antiretroviral therapy (HAART) was once perceived to have transformed deadly HIV/AIDS into a treatable, chronic infectious disease. However, mounting evidence now suggests that the prevalence of multi-drug resistant HIV (MDR-HIV) infection is steadily rising among newly infected individuals in the HAART-experienced countries, raising a concern for a future outbreak of MDR-HIV/AIDS. Our global fight against AIDS must include sustained effort to search and discover a new therapeutic modality for HIV infection. Of plausible viral targets explored to date, HIV gene-targeting approach has not yet seen a considerable success in vivo. The pursuit of anti-HIV gene intervention should include the identification of critical gene targets as well as the optimization of biomolecules that can effectively interact with the intended targets. Using unmodified peptide nucleic acids (PNA) as a biomolecular tool, we discovered a potentially critical HIV gene segment within gag-pol encoding gene. Antisense PNA targeting this specific region effectively disrupted a translation of HIV gag-pol mRNA, abolishing the virion production from chronically HIV-infected cells. This exemplifies the possibility that epigenic HIV inhibitors may be developed in the coming years, if emerging novel technologies permit sufficient and stable in vivo delivery of PNA or other similarly effective biomolecules.     

Peptide nucleic acids as epigenetic inhibitors of HIV-1

Summary The advent of highly active antiretroviral therapy (HAART) was once perceived to have transformed deadly HIV/AIDS into a treatable, chronic infectious disease. However, mounting evidence now suggests that the prevalence of multi-drug resistant HIV (MDR-HIV) infection is steadily rising among newly infected individuals in the HAART-experienced countries, raising a concern for a future outbreak of MDR-HIV/AIDS. Our global fight against AIDS must include sustained effort to search and discover a new therapeutic modality for HIV infection. Of plausible viral targets explored to date, HIV gene-targeting approach has not yet seen a considerable success in vivo. The pursuit of anti-HIV gene intervention should include the identification of critical gene targets as well as the optimization of biomolecules that can effectively interact with the intended targets. Using unmodified peptide nucleic acids (PNA) as a biomolecular tool, we discovered a potentially critical HIV gene segment within gag-pol encoding gene. Antisense PNA targeting this specific region effectively disrupted a translation of HIV gag-pol mRNA, abolishing the virion production from chronically HIV-infected cells. This exemplifies the possibility that epigenic HIV inhibitors may be developed in the coming years, if emerging novel technologies permit sufficient and stable in vivo delivery of PNA or other similarly effective biomolecules.     

梅毒螺旋体重组抗原酶联免疫吸附试验的建立及应用

建立梅毒螺旋体重组抗原酶联免疫吸附试验（ELISA）,用于梅素血清学诊断和调查。其法,用表达的重组抗原IPN17和TmpA,建立检测血清特异抗体的间接ELISA,并与其它检测方法比较,分别检测梅毒参比血清、病人及献血员血清。其结果,敏感性、特异性均为100％。新建ELISA与TPHA的总符率为95．7％,明显高于RPR与TPHA的总符合率（89．1％）。献血员人群抗体阳性率为0．3％－0．69％,健康人群中抗体阳性率较低。     

HIV-1 TAT蛋白转导肽的研究进展

TAT蛋白转导肽是人类免疫缺陷病毒1型(human immunodeficiency virus type 1, HIV-1)编码的一段富含碱性氨基酸、带正电荷的多肽,属于蛋白转导域家族的一员。长期研究发现其全长及11个碱性氨基酸富集区的核心肽段(YGRKKRRQRRR)不仅能够在包括蛋白质、多肽及核酸等多种外源生物大分子的跨膜转导过程中具有重要作用,而且能够携带这些外源生物大分子通过活体细胞的各种生物膜性结构(如细胞膜和血脑屏障等)并发挥生理功能,但其跨膜转导机制仍不明确。新近研究还发现TAT核心肽段在促进外源蛋白高效表达过程中也具有重要作用,能够显著增加外源蛋白高效、可溶性表达的水平,显示了TAT蛋白转导肽的新功能。以TAT蛋白转导肽跨膜转导作用的长期研究背景为基础,分别从TAT蛋白转导肽的结构特点、其跨膜转导作用的影响因素及其作用机制等方面进行了系统综述,进一步结合TAT蛋白转导肽的最新研究进展分别从药物研发、机制探索及新功能的开发等方面展望了后续研究方向与应用价值,不仅为深入阐述TAT蛋白转导肽的跨膜转导作用的功能意义提供了参考依据,而且为TAT蛋白转导肽在微生物工程及蛋白质工程等领域的潜在应用价值提供了重要参考信息。     

Evidence for Interference by Immune Complexes in the Serodiagnosis of Cryptococcosis

The latex agglutination test was used to compare cryptococcal antigen titers before and after protease treatment in 19 patients with pulmonary cryptococcosis. Antigen was detected by the LA test in 13 of 33 serum samples before protease treatment, and in an additional 13 samples following treatment. Of 26 antigen-positive serum samples, 22 (84.6%) showed an increased antigen titer after protease treatment. Using the cell slide agglutination test, antibody was detected in 3 of 19 cases. In one of these 3, antigen could only be detected after protease treatment. Soluble immune complex was prepared in vitro using anti-C. neoformans rabbit antiserum and polysaccharide antigen of C. neoformans, and the effects of immune complexes on the LA test were examined. In this experimental model, soluble immune complexes seemed to be observed in antibody excess region, because the antigen titers were increased after the protease treatment. We concluded that C. neoformans capsular polysaccharide antigen and anti-C. neoformans antibody formed soluble immune complexes in patients' sera, which interfered with antigen detection by the latex agglutination test without protease treatment.     

Antibody Responses Raised against a Conformational V3 Loop Peptide of HIV-1

The amino acid sequence of the principal neutralizing determinant (PND) of 224 cases of human immunodeficiency virus type 1 (HIV-1) was determined and the most frequently occurring sequence was used as a peptide antigen for studying virus-specific antibody responses. In our present study, a linear peptide of the most frequent PND was first synthesized and then oxidized to create a disulfide-bridged loop conformation. Then, in order to construct a macromolecular structure for the purpose of increasing anti-genicity, the synthetic peptide was conjugated to a core peptide. We compared the immunogenicity of the disulfide-bridged loop PND peptide antigen (AG4) and the linear PND peptide antigen (AG5). After immunizing rabbits 5 and 6 times with both peptides, the results obtained using ELISA revealed that AG4 (conformational-loop type) was more capable of inducing a high titer of antigen-specific antibodies than was AG5 (linear type). Despite an amino acid sequence homology of 72%, a 1:8 dilution of serum raised against AG4 inhibited 81.9% of HIV-1_IIIB-mediated cell fusion, suggesting that conformational V3 loop peptide is able to elicit an antibody response which is strongly HIV-1-specific.     

Rapid Serodiagnosis of Mycobacterium avium-intracellulare Complex Infection by ELISA with Cord Factor (Trehalose 6, 6′-Dimycolate), and Serotyping Using the Glycopeptidolipid Antigen

Mycobacterium avium-intracellulare complex (MAC) is one of the most important opportunistic pathogens, particularly in patients with acquired immunodeficiency syndrome (AIDS). The aim of this study was to determine whether an enzyme-linked immunosorbent assay (ELISA) using trehalose 6,6′-dimycolate (TDM) as an antigen can be used for the rapid serodiagnosis of MAC infection. We also identified MAC serotypes by ELISA using serotype-specific glycopeptidolipid (GPL) antigen. To confirm our findings, the thin-layer chromatographic (TLC) behavior of serotype-specific GPL of the strains isolated from MAC-infected patients was also tested. Forty patients infected with MAC and 30 healthy controls were tested. Thirty-two of the 40 MAC-infected patients had higher titers of serum antibodies against MAC TDM than against MTB TDM, while all 30 healthy control sera were unreactive to MAC TDM and MTB TDM. Results of the GPL ELISA indicated that 20 of the 40 MAC-infected patients' sera were reactive against serotype 4 GPL, 3 against serotype 8 GPL, and 1 against serotype 16 GPL. A TLC analysis of the GPL of the 40 MAC isolates showed that 16 strains were of serotype 4, 5 of serotype 8, and 2 of serotype 16. Results of the GPL ELISA were in good accord with those of the TLC analysis for most patients. Our findings suggest that ELISA using TDM is useful for rapid serodiagnosis of MAC infection, and that complementary ELISA testing using serotype-specific GPL gives additional detailed information concerning MAC serotypes.     

Serodiagnosis of cancers by ELISA of anti-histone H2B antibody

An enzyme-linked immunosorbent assay method for serodiagnosis of cancers was developed by employing histone H2B. This method measures anti-histone H2B antibody levels in sera and includes a device for coating the plastic immunoplate with a mixture of histone H2B and diluted fetal calf serum. The coating of immunoplates with this mixture decreased apparent sensitivity of the assay compared with that in the absence of fetal calf serum, but effective reduction of nonspecific background enabled a specific assay of anti-histone H2B antibody with excellent reproducibility. By this method cancer patients were discriminated from normal healthy subjects at detection rates of 37% for lung cancer, 33% for liver cancer, 50% for pancreatic cancer, 42% for colon cancer, and 78% for cervical cancer. However, stomach and esophagus cancers showed detection rates of less than 17%, which are comparable to the values for benign diseases. It is likely that this assay method detects squamous cell carcinomas at relatively high rates.     

Conformational Plasticity in the HIV-1 Fusion Peptide Facilitates Recognition by Broadly Neutralizing Antibodies

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O1群和O139群霍乱弧菌主要抗原研究进展

霍乱弧菌是引起人和动物烈性肠道传染病霍乱的病原体。在霍乱弧菌的200多个血清群中,只有O1群和O139群霍乱弧菌能引起霍乱。快速准确检测O1群和O139群霍乱弧菌是霍乱防治的关键。表面抗原在O1群和O139群霍乱弧菌检测中发挥着重要作用。简要综述了O1群和O139群霍乱弧菌的脂多糖、霍乱肠毒素、外膜蛋白W、毒素共调菌毛和甘露糖敏感血凝素等5种主要抗原的研究进展。     

结核分枝杆菌Rv3425蛋白的原核表达纯化及其血清学诊断价值

目的：用原核系统表达结核分枝杆菌Rv3425蛋白并纯化,评价该重组蛋白在结核病血清学诊断方面的价值。方法：以结核分枝杆菌H37Rv株基因组为模板,PCR扩增得到Rv3425基因序列,克隆至表达载体pET-28a中,转入大肠杆菌BL21（DE3）进行诱导、表达后纯化,用Western印迹和ELISA法进行抗原性初步评价。结果：在原核系统内经IPTG诱导表达后,Rv3425蛋白主要以包涵体形式存在,经复性和镍柱层析纯化后,纯度达95%以上;Western印迹和ELISA结果证明重组Rv3425具有较强的抗原活性;用纯化的Rv3425蛋白做抗原,临床诊断结核病人血清,阳性率达50%。结论：高纯度的Rv3425蛋白在结核病诊断方面具有很高的应用价值,可作为结核病诊断的备选抗原。     

结核分枝杆菌重要诊断用抗原研究进展

血清学试验是结核诊断的重要依据。随着科学技术的进步,新的结核诊断用抗原不断被发现。我们简要综述了结核菌素蛋白衍生物、抗原85复合体、38kDa磷酸盐转运蛋白、6kDa早期分泌性蛋白、10kDa培养滤液蛋白、免疫性蛋白MPT64、主要分泌性免疫蛋白MPB70、表面脂蛋白MPB83等8种结核分枝杆菌重要抗原作为结核诊断用抗原的研究进展。