猪囊虫体表抗原用于 ELISA 诊断囊虫病的研究

应用猪囊尾蚴体表抗原对66例临床确诊的囊虫病人用 ELISA 进行检测,同时以囊液粗抗原作比较观察。阳性率分别为95.5%和68.2%,两者差异非常显著(P<0.005)。两种抗原对同批血清的反应强度也呈现明显差异(P<0.05)。用囊虫体表抗原和囊液粗抗原同时检测50名健康人血清,假阳性率分别为2%与12%;两种抗原与10例包虫病人血清分别有8例和4例出现交叉反应。

Preparation and Identification of Specific Monoclonal Antibody against Porcine Circovirus Type 2

BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.

Analysis of BAC_5 mcAb-Related Epitope Using Random Peptide library

Objective To identify epitope relating to BAC 5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopharyngeal carcinoma (NPC) cells. Methods Using BAC 5 mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presented by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibody capture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce and compare the order of the amino acids of exogenous peptides among the phage clones. Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC 5 mcAb. The 3 kinds of the peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of '-P-V-'structure existed near N-terminus of the 3 different peptides, i.e. -H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8) -Q-N-Q-A-W-F-S-Q-P-V-R-M- (3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide ' -N-H-Q-S-T-F-W-Q-K-W-T-A-' displayed by M13 phages from the negative clones (6/6). Conclusion BAC 5 mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic the structure of the epitope on the surface of NPC cells recognized by BAC 5 mcAb.

Diversity of Helicobacter pylori isolates in expression of antigens and induction of antibodies

AIM: To obtain evidence for selection of antigens used in genetically engineered vaccine against Helicobacter pylori (H pylori). METHODS: Enzyme linked immunoabsorbent assay (ELISA) was established on the basis of recombinant protein antigens rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB of H pylori to detect expression rates of the antigens in bacterial isolates as well as positive rates of the antibodies in sera from H pylori-infected patients. PCR was applied to the detection of carrying rates of the genes encoding antigens in the isolates. RESULTS: The outputs of rUreB, rHpaA, rVacA, rCagA1, rNapA, rFlaA and rFlaB were approximately 35%, 32%, 15%, 23%, 56%, 25% and 20% of the total bacterial proteins, respectively. One hundred and fifty-one strains of H pylori were isolated from 347 biopsy specimens of chronic gastritis, peptic ulcer or gastric adenocarcinoma, with a positive rate of 43.5%. All of the isolates expressed UreB, HpaA, FlaA and FlaB while 52.3%, 92.1% and 93.4% of the isolates expressed VacA, CagA and NapA, respectively. In the sera of 151 H pylori-infected patients, the positive ratesof IgG antibodies against UreB, HpaA, VacA, CagA, NapA, FlaA and FlaB were 100%, 87.4%, 43%, 71.5%, 89.4%, 84.8% and 79.5%, respectively. Furthermore, the expression frequencies of VacA and NapA were found to be relative to the severity of gastric diseases (P = 0.016 and P < 0.0001, respectively). CONCLUSION: UreB antigen is the top option of developing genetically engineered vaccine against H pylori followed by NapA or HpaA.

Disulfide-stabilized single-chain antibody-targeted superantigen: Construction of a prokaryotic expression system and its functional analysis

AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

Baculovirus Host-Range

Baculoviruses are used as microbial insecticides, protein expression vectors, epitope display platforms, and most recently as vectors for gene therapy. Understanding the mechanisms that control baculovirus host-range and tissue tropisms are important for assessing their safety and for improving their properties for these biotechnology applications. In the past two decades some progress has been made and several baculovirus genes that influence host-range have been identified. Despite this progress, our understanding of the underlying mechanisms that restrict baculovirus host-range is still limited. Here we review what is currently known about baculovirus genes that influence virus host-range.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185^(c-erbB-2)

The c-erbB-2 proto-oncogene encodes a 185kDa protein p!85, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p!85. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.

The expression and antigenicity identification of recombinant rat TGF-β1 in bacteria

In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

A Case of Hepatitis B Reactivation due to the Hepatitis B Virus Escape Mutant in a Patient undergoing Chemotherapy

A 62-year-old man had chronic hepatitis B virus （HBV） infection and was diagnosed with liver cirrhosis. At the time of diagnosis the patient＇s virologic markers were positive for hepatitis B surface antigen （HBsAg）, antibody to hepatitis B e antigen （anti-HBe） and antibody to hepatitis B core antigen （anti-HBc）, while antibody to hepatitis B surface antigen （anti-HBs） and HBV DNA were negative. Later the patient received chemotherapy for malignancy. However, this was interrupted due to elevated liver enzymes. At the same time HBV DNA became positive. Lamivudine （LMV） therapy was administered immediately. However, the levels of serum aminotransferase and total bilirubin （TB） were still rising. Finally the patient died of fulminant hepatic failure. A sequence revealed HBV genotype C （HBsAg subtype adw） with immune escape mutations, F8L, $34L, F41S, G44V, F93C, V96G, Lll0I, C149Y and F161Y. The high morbidity and mortality of this complication is one of the major obstacles to completing the standard treatment for malignancy in HBV carriers. Therefore, the relative risk of antiviral prophylactic failure should be further assessed and the optimal strategy for antiviral prophylaxis in HBsAg-positive patients with oncologic and hematologic malignancies undergoing chemotherapy should be revised.

A prophylactic approach for bone marrow transplantation from a hepatitis B surface antigen-positive donor

It has been accepted that bone marrow transplantation （BMT） is the only curative therapeutic option for certain hematologic malignancies. The southeast Asia region is an endemic area of hepatitis B virus （HBV） infection; thus, BMT using a hepatitis B surface antigen （HBsAg）- positive donor is occasionally unavoidable. Organ transplantation using a HBsAg-positive donor can lead to post-transplantation de novo HBV infection and severe HBV-related hepatitis if no effective prophylactic measures are taken prior to and after transplantation. In this report, a four-level approach was designed for a patient with chronic myeloid leukemia, beginning with a booster HBV vaccination before performing BMT with a HBsAg-positive donor. Prior to BMT, the HBV viral load of the donor was reduced to an undetectable level by anUviral therapy. After BMT, hepatitis B immunoglobulin was administered intramuscularly for 1 wk together with a long-term antiviral drug, lamivudine. One year after discontinuation of lamivudine, the patient is still free of HBV infection.

E2 GLYCOPROTEIN OF GENOTYPE Ⅲ CHINESE ISOLATES OF HEPATITIS C VIRUS EXPRESSED IN MAMMALIAN CELL AS ANTIGEN FOR ANTI-E2 ANTIBODY DETECTION

Expression vector inserted with E2/NS1 gene derived from genotype Ⅲ Chinese isolates of HCV was transfected into mammalian cells to express E2 glycoprotein. Expressed protein was used as antigen for an- ti-E2 antibody detection in 19 cases of hepatitis C patients by Western blot. It was first to express E2 gly- coprotein of genotype Ⅲ Chinese hepatitis C virus isolates. For anti-E2 detection, 14 cases of patients were positive of antibodies against E2(73. 7 % ). These results indicated that E2 glycoprotein expressed in mam-malian cells had good immunogenicity and cross reactivity to serum infected with genotype Ⅱ Chinese hep-atitis C virus isolates.

Induction of hepatitis C virus-specific cytotoxic T and B cell responses by dendritic cells expressing a modified antigen targeting receptor

AIM: To find a novel antigen (Ag) presentation strategy to improve the immune responses induced by dendritic cell (DC)vaccine expressing hepatitis C virus (HCV) core antigen (pcDNA3HCV C-Fc) in Balb/c mice (H-2d).METHODS: pcDNA3HCV C-Fc plasmid and eukaryotic expression vector pcDNA3 were injected into mice sc. Immune responses to pcDNA3HCV C-Fc were studied. Meanwhile the effect of pcDNA3HCV C-Fc on anti-translated subcutaneous tumor of SP2/0 cells stably expressing HCV C Ag (SP2/0-HCV C-FC) was also studied. Anti-HCV C in serum was detected by enzyme-linked immunoadsordent assay (ELISA) and HCV specific cytotoxic T lymphocyte (CTL) activity was measured by LDH release assay. After 3 wk of DNA immunization,the cells of SP2/0-HCV C-FC were inoculated into mice subcutaneously and tumor growth was measured every 5 d.The survival rate and living time of mice were also calculated.RESULTS: After 4 wk of DC immunization, the A450 nm values of sera in mice immunized with pcDNA3HCV C-Fc-DC and pcDNA3-DC were 0.56±0.17 and 0.12±0.03 respectively. The antibody titres in mice codeliveried with pcDNA3HCV C-Fc with DC were significantly higher than those of mice injected with pcDNA3-DC. The HCV specific CTL activities in mice coinjected with DC and pcDNA3HCV C-Fc or empty expression vectors were(73.2±3.1) % and (24.4±8.8) %, which were significantly higher than those of mice injected with water.The DC vaccine could evidently inhibit tumor growth, prolong the survival time of mice and improve the survival rate of mice and these effects could be improved by HCV C-Fc (pcDNA3HCV C-Fc) gene codelivered.CONCLUSION: DC vaccine has a strong antigenicity in humoral and cellular immunities, which can be promoted by transduced pcDNA3HCV C-Fc expressing HCV C or Fc.Thus, pcDNA3HCV C-Fc-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.

Identification of the Epitopes of Monoclonal Antibodies against P74 of Helicoverpa armigera Nucleopolyhedrovirus

P74 is a per os infectivity factor of baculovirus.Here,we report the production of three monoclonal antibodies (mAbs),denoted as 20D9,20F9 and 21E1,raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV),and the identification of their recognition epitopes.The full-length P74,without the transmembrane domains at the C-terminus,was first divided into three segments (N,M and C,respectively),based on the proposed cleavage model for the protein,which were then expressed individually.Western blot analyses revealed specific cross-reactions with the N fragment,for both 20D9 and 21E1.Extensive truncation,followed by prokaryotic expression,of the P74 N fragment was then performed in order to screen for linear epitopes of P74.The recognition regions of 20D9 and 21E1 were revealed to be localized at R144-T153 and T199-C219,respectively.In addition,immunofluorescence microscopy indicated that 20D9 and 20F9 could recognize native P74 in HearNPV-infected cells.These findings will facilitate further investigations of the proteolytic processing of HearNPV P74,and of its involvement in virus-host interactions.

Isolation and Characterization of Exosomes Derived from Tumor Cells Genetically Expressing Model Antigen

Tumor cell-derived exosomes have been proposed as non-cellular nanomeric vaccine which could induce potent anti- tumor immune response in mice. In order to develop the protocols to prepare tumor cell-derived exosomes for basic research and clinical trail, we isolated exosomes from ovalbumin (OVA)-expressing thymoma cells EG.7-OVA by various preparation methods. We demonstrate the non-sedimentation method is simple, rapid, efficient with higher yield and purity of exosomes. EG.7-OVA-derived exosomes are 40-100 nm in diameter sequestered by lipid bi-layer, and contain rich heat shock protein (HSP) and OVA. The result of the size distribution determination is consistent with the calculation by the visual microscopic inspection, with 90.4% particles at the range of 50-90 nm. Moreover, as a model antigen of the EG.7 cells, OVA concentra- tion in EG.7-derived exosomes can be regarded as a good quality control parameter. Therefore, we have established a platform to efficiently prepare exosomes for tumor immunotherapy.

Measuring Telomere Length in Proliferating Cells by Flow-FISH Method

The purpose of present work is a measurement of telomere length dynamic in proliferating cells in vitro by modified flow-FISH method. This method is a combination of two modifications： telomere length measurement in differentiated cells by surface antigen and analysis of cells divisions＇ number by vital dye dilution. Lymphocytes were activated by anti-CD3 Abs with IL-2 presents and grown in vitro for 7 days. Cells division＇s number was measured by dilution of CFSE vital dye which cells were stained previously activation. For telomere length measurement we used flow-FISH method with Cy3 labeled telomere PNH probe. Using this method we evaluated the dynamic of telomere length in CD4＋ and CD8＋ T-cells after 7 days culturing in vitro and revealed the difference in telomere lengthening and shortening versus division rounds in cell subsets. In CD8＋ cells telomeres start lengthen on a second division with the maximum on 4th division round becoming more that 20% longer compared with undividing cells. In CD4＋ cells telomeres did not have any length peculiarities through all division rounds demonstrating different telomere regulation in subsets. This probably occurs due to the higher level ofhTERT protein expression in CD8＋ than CD4＋ cells do.

Synthetic immunity to break down the bottleneck of cancer immunotherapy

As a result of recent breakthroughs in cancer immunotherapies, unprecedented and durable remission, and even cure, has been reported in some patients. Importantly, this progress has been achieved, not by the induction of immunity, but by the delivery of immunity in the form of engineered antibodies （cAbs） or effector T cells. However, these single-target technologies have failed to result in a therapeutic effect in some patients, and evidence suggests that further advances depend on an effective strategy for coping with cancer heterogeneity and dynamics. A synthetic immunity （SI） strategy is proposed to achieve this goal. The fundamental basis of SI involves the generation of a panel of cAbs and antibody-retargeted CTLs designed to destroy all cell lineages of a cancer with high specificity. This goal can be achieved only when the composition of the cAbs is determined using a systematic approach, i.e., selecting the antigens targeted by the cAbs based on an epitope-tree illustrating the clonal antigen architecture of the cancer. Integration of technologies that increase the epitope breadth, cAb affinity and T cell activity will further enhance the efficacy of SI. Using DNA vectors to express the eAbs will be a safe, effective and affordable solution.

Analysis on molecular interaction mechanism of four hapten flavonoids in Shuang-huang-lian powder injection with bovine serum albumin

Baicalein, baicalin, scutellarin and Chrysin-7-O-β-D-glucuronide are the major flavonoids of the Shuang-huang-lian powder injection. These flavonoids are thought to be haptens that can induce allergic reactions. The interaction mechanism of these haptens with bovine serum albumin （BSA） was investigated by surface plasmon resonance （SPR） and molecular modeling method. The SPR study indicated that these compounds could specifically bind to the BSA with one binding site and equilibrium dissociation constant （KD） values were determined. Molecular modeling explored the mechanism of interaction under simulated physiological condition. The result of molecular modeling indicated that flavonoids could bind with BSA in the hydrophobic pocket of sub-domain II with hydrogen bonding as the main acting force.

Epitope variation in the Newcastle disease virus HN gene under antibody immune selective pressure in cell culture

The influence of antibody immune selective pressure on Newcastle disease virus (NDV) HN and F gene mutations was studied in cell cultures.NDV field strain TZ060107 was inoculated into chicken embryo fibroblast cells and continuously passaged with (group A) or without (group B) anti-NDV monospecific serum.Each group contained three independent passage series.HN and F genes were amplified and sequenced for the 10th,20th,30th,40th and 50th generations of each serial passage,and compared with the original strain.The results demonstrated that increased HN gene mutations were observed in group A with the antibody than in group B without the antibody.The nonsynonymous (NS) to synonymous (S) mutations ratio was 6 for group A,significantly higher than 3.4 in group B.In group A with the antibody,there were five stable NS mutations in HN gene,three of which (related to aa#353,521 and 568) were related to known epitopes.There were two stable NS mutations in F gene in group A,but no stable NS mutations in group B.The NS/S ratios of F gene were less than 2.5 for both groups A and B.Our results suggested that the antibody strongly influenced HN gene mutations,while the F gene was less influenced by the same antibody.