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Expression and purification of the complete PreS region of hepatitis B Virus

World Journal of Gastroenterology 2005年第20期11卷 3060-3064页

作者： QiangDeng Yu-YingKong you-huaxie YuanWang StateKeyLaboratoryofMolecularBiology InstituteofBiochemistryandCellBiologyShanghaiInstitutesofLifeScienceChineseAcademyofSciences.Shanghai200031.China

AIM: To express the complete PreS region of HBV in *** with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6× histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization. RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in ***. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain. CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in *** can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.

关键词： Hepatitis B virus PreS Expression Purification

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Screening for PreS specific binding ligands with a phage displayed peptides library

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World Journal of Gastroenterology 2005年第26期11卷 4018-4023页

作者： QiangDeng MingZhuang Yu-YingKong you-huaxie YuanWang StateKeyLaboratoryofMolecularBiology InstituteofBiochemistryandCellBiologyShanghaiInstitutesofLifeScienceChineseAcademyofSciencesShanghai200031China Sino-FranceCenterforLifeScienceandGenomeResearch

AIM:To construct a random peptide phage display library and search for peptides that specifically bind to the PreS region of hepatitis B virus (HBV). METHODS: A phage display vector, pFuse8, based on the gene 8 product (pⅧ) of M13 phage was made and used to construct a random peptide library. Ecoli derived thioredoxin-PreS was purified with Thio-bond beads, and exploited as the bait protein for library screening. Five rounds of bio-panning were performed. The PreS-binding specificities of enriched phages were characterized with phage ELISA assay. RESULTS: A phage display vector was successfully constructed as demonstrated to present a pⅧ fused HBV PreSl epitope on the phage surface with a high efficiency. A cysteine confined random peptide library was constructed containing independent clones exceeding 5±108 clone forming unit (CFU). A pool of phages showing a PreS-binding specificity was obtained after the screening against thio-PreS with an enrichment of approximately 400 times. Five phages with high PreS-binding specificities were selected and characterized. Sequences of the peptides displayed on these phages were determined. CONCLUSION: A phage library has been constructed, with random peptides displaying as pVIII-fusion proteins. Specific PreS-binding peptides have been obtained, which may be useful for developing antivirals against HBV infection.

关键词： Hepatitis B virus Phage Peptide

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A candidate DNA vaccine elicits HCV specific humoral and cellular immune responses

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World Journal of Gastroenterology 2004年第17期10卷 2488-2492页

作者： Li-XinZhu JingLiu YeYe you-huaxie Yu-YingKong Guang-DiLi YuanWang StateKeyLaboratoryofMolecularBiology InstituteofBiochemistryandCellBiologyShanghaiInstitutesforBiologicalSciencesChineseAcademyofSciencesShanghai200031China

AIM: TO investigate the immunogenicity of candidate DNA vaccine against hepatitis C virus (HCV) delivered by two plasmids expressing HCV envelope protein 1 (El) and envelope protein 2 (E2) antigens respectively and to study the effect of CpG adjuvant on this candidate ***: Recombinant plasmJds expressing HCV EI and E2 antigens respectively were used to simultaneously inoculate mice with or without CpG adjuvant. Antisera were then collected and tJters of antJ-HCV antibodies were analyzed by ELISA. One month after the last injection, animals were sacrificed to prepare single-cell suspension of *** cells were subjected to HCV antigen specific proliferaion assays and cytokine secretion assays to evaluate the cellular immune responses of the vaccinated ***: Antibody responses to HCV EI and E2 antigens were detected in vaccinated animals. Animals receiving CpG adjuvant had slightly lower titers of anti-HCV antibodies in the sera, while the splenocytes from these animals showed higher HCV-antigen specific proliferation. Analysis of cytokine secretion from the splenocytes was consistent with the above results. While no antigen-specific IL-4 secretion was detected for all vaccinated animals, HCV antigen-specific INF-γ, secretion was detected for the splenocytes of vaccinated animals. CpG adjuvant enhanced the secretion of INF-γ, but did not change the profile of IL-4 ***: Vaccination of mice with plasmids encoding HCV E1 and E2 antigens induces humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response.

关键词： DNA疫苗 HCV 特效药体液细胞免疫反应抗体

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Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

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World Journal of Gastroenterology 2004年第20期10卷 2972-2978页

作者： Li-XinZhu JingLiu you-huaxie Yu-YingKong YeYe Chun-LinWang Guang-DiLi YuanWang StateKeyLaboratoryofMolecularBiology InstituteofBiochemistryandCellBiologyShanghaiInstitutesforBiologicalSciencesChineseAcademyofSciencesShanghai200031China

AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis. METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CliO) cell line. Cell proliferation was assessed by ^3H thymidine uptake. Apoptosis was examined by Hoechst 33258 staining, flow cytometry and DNA fragmentation analysis. RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line. CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

关键词：基因表达丙型肝炎病毒蛋白质包膜2 细胞凋亡人工培养哺乳动物细胞 E2 分芽增殖

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Gene expression profile in liver of hB1F transgenic mice

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World Journal of Gastroenterology 2004年第20期10卷 3006-3010页

作者： Shui-LiangWang HuaYang you-huaxie YuanWang Jian-ZhongLi LongWang Zhu-GangWang Ji-LiangFu DepartmentofMedicalGenetics SecondMilitaryMedicalUniversityShanghai200433China StateKeyLaboratoryforMolecularBiology InstituteofBiochemistryandCellBiologyShanghaiInstitutesforBiologicalSciencesChineseAcademyofSciencesShanghai200031China ShanghaiNanfangResearchCenterforModelOrganisms Shanghai201203China

AIM: To analyze the tissue morphologic phenotype and liver gene expression profile of hBIF transgenic mice. METHODS: Transgene expression was analyzed with RT-PCR and Western blotting. For one of the transgenic mouse lines, tissue expression pattern of the transgene was also examined with immunochemical methods. Pathological analysis was used to examine the tissue morphologic phenotype of established transgenic mice. The liver gene expression profile of transgenic mice was analyzed with microchip, and some of bhe differentially expressed genes were verified with RT-PCR. RESULTS: The expressions of hBIF were shown in livers from 6 of 7 transgenic mouse lines. The overexpression of hB1F transgene did not cause pathological changes. Expressions of three genes were up-regulated, while down-regulation was observed for 25 genes. CONCLUSION: The overexpression of hBIF transgene may cause changes of gene expression profiles in the liver of transgenic mice.

关键词：基因表达肝脏 hB1F 遗传转移老鼠病理学

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Establishment of transgenic mice carrying the gene of human nuclear receptor NR5A2 (hB1F)

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World Journal of Gastroenterology 2003年第6期9卷 1333-1336页

作者： Shui-LiangWang HuaYang you-huaxie YuanWang Jian-ZhongLi LongWang Zhu-GangWang Ji-LiangFu DepartmentofMedicalGenetics SecondMilitaryMedicalUniversityShanghai200433China InstituteofBiochemistryandCellBiology ShanghaiInstituteforBiologicalSciencesChineseAcademyofSciencesShanghai200031China ShanghaiNanfangResearchCenterforModelOrganisms Shanghai201203China

AIM: Human hepatitis B virus enhancer II B1 binding factor (hBIF) was cloned and characterized as a novel member of the Ftz-F1 (NR5A) nuclear receptor subfamily. Although progresses have recently been made, its biological function remains largely unidentified. The aim of this study was to establish an hBIF transgenic mouse model to promote the functional study of ***: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female *** offsprings were identified by PCR and Southern blot analysis. Transgene expression was analyzed with RT-PCR and Western blot analysis. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 and F2 mice were identified by PCR ***: Seven mice were identified as carrying copies of transgene. RT-PCR and Western blotting results showed that the transgene was expressed in heart, liver, lung, kidney and stomach in one of the transgenic mouse *** analysis of the transgenic mice demonstrated that the transgene was integrated into the chromosome at a single site, and was transmitted ***: In this study we established an hB1F transgenic mouse model, which will facilitate the investigation of the biological function of hB1F in vivo.

关键词：乙型肝炎病毒 hBIF NR5A 生物学功能动物模型

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Gamma-ray Emission from the γ-ray-loud BL Lac Objects

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Chinese Journal of Astronomy and Astrophysics 2001年第6期1卷 494-500页

作者： Guang-ZhongXie Ben-ZhongDai En-WeiLiang Zhao-huaxie YunnanAstronomicalObservatory ChineseAcademyofScienceKunming650011 DepartmentofPhysics UniversityofHongKong

Using the HST observation data of BL Lac objects by Urry et al. and γ-ray observation data, we find that there is a correlation between Fγ and Fonuclei forγ-ray-loud BL Lac objects (correlation coefficients: 7γ=0.63,p = 4.0 × 10-2), but no correlation between Fγ and Fohost, where Fonuclei and Fohost are the fluxes of nuclei and host galaxy in V-band. For 19γ-ray-loud BL Lac objects with observed spectral index in multi-wavebands, the spectral index correlations between any two bands are as follow: (1) there is a strong correlation between aγ and aK for 15 BL Lac objects(γ = 0.84,p = 3.11 × 10-4); (2) the correlation between aγ and ao for 12 BL Lac objects isγ = 0.82, p = 1.5 × 10-3; (3) there is no correlation between a7 and ax for 16 BL Lac objects. The results, together with characteristic double-humped shape of their SEDs, show that the synchrotron self-Compton mechanism might be a main mechanism for theγ-ray emission of the BL Lac objects. The electrons emitting JR and optical radiation via synchrotron are also responsible for upscattering these photons toγ-rays, and a variability in IR-optical regime should be accompanied by a change in theγ-rays.

关键词： gamma rays - BL Lacertae objects: general

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Roles of histamine and its receptors in allergic and inflammatory bowel diseases

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World Journal of Gastroenterology 2005年第19期11卷 2851-2857页

作者： huaxie Shao-HengHe AllergyandInflammationResearchInstitute ShantouUniversityMedicalCollegeShantou515031GuangdongProvinceChina

Mast cell has a long history of being recognized as an important mediator-secreting cell in allergic diseases, and has been discovered to be involved in IBD in last two decades. Histamine is a major mediator in allergic diseases, and has multiple effects that are mediated by specific surface receptors on target cells. Four types of histamine receptors have now been recognized pharmacologically and the first three are located in the gut. The ability of histamine receptor antagonists to inhibit mast cell degranulation suggests that they might be developed as a group of mast cell stabilizers. Recently, a series of experiments with dispersed colon mast cells suggested that there should be at least two pathways in man for mast cells to amplify their own activation-degranulation signals in an autocrine or paracrine manner. In a word, histamine is an important mediator in allergic diseases and IBD, its antagonists may be developed as a group of mast cell stabilizers to treat these diseases.

关键词： Allergic diseases Immunoglobulin

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Inhibition of tryptase release from human colon mast cells by protease inhibitors

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World Journal of Gastroenterology 2004年第3期10卷 332-336页

作者： Shao-HengHe huaxie ImmunopharmacologyGroup UniversityofSouthamptonSouthamptonUK AllergyandInflammationResearchInstitute ShantouUniversityMedicalCollegeShantou51503lGuangdongProvinceChina

AIM: To investigate the ability of protease inhibitors to modulate tryptase release from human colon mast ***: Enzymatically dispersed cells from human colon were challenged with anti-IgE or calcium ionophore A23187 in the absence or presence of tryptase and chymase inhibitors,and tryptase release was ***:IgE dependent tryptase release from colon mast cells was inhibited by up to approximately 37%, 40% and 36.6% by chymase inhibitors Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPFM), N-tosyI-L-phenylalanyl-chloromethyl ketone (TPCK), and α1-antitrypsin, respectively. Similarly, the inhibitors of tryptase leupeptin, N-tosyI-L-lysine chloromethyl ketone (TLCK) and lactoferrin were also able to inhibit anti-IgE induced tryptase release by a maximum of 39.4%,47.6% and 36.6%, respectively. The inhibitory actions of chymase inhibitors, but not tryptase inhibitors on colon mast cells were enhanced by preincubation of them with cells for 20min before challenged with anti-IgE. At a concentration of 10μg/mL, protamine was able to inhibit anti-IgE and calcium ionophore induced tryptase release. However, at 100μg/mL, protamine elevated tryptase levels in supematants.A specific inhibitor of aminopeptidase amastatin had no effect on anti-IgE induced tryptase release. The significant inhibition of calcium ionophore induced tryptase release was also observed with the inhibitors of tryptase and chymase examined. The inhibitors tested by themselves did not stimulate tryptase release from colon mast ***:It was demonstrated for the first time that both tryptase and chymase inhibitors could inhibit IgE dependent and calcium ionophore induced tryptase release from dispersed colon mast cells in a concentration dependent of manner, which suggest that they are likely to be developed as a novel class of anti-inflammatory drugs to treat chronic of colitis in man.

关键词：类胰蛋白酶肥大细胞蛋白酶抑制剂免疫球蛋白E 炎症病理机制

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Modulation of tryptase secretion from human colon mast cells by histamine

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World Journal of Gastroenterology 2004年第3期10卷 323-326页

作者： Shao-HengHe huaxie AllergyandInflammationResearchInstitute MedicalCollegeShantouUniversityShantou515031GuangdongProvinceChina ImmunopharmacologyGroup UniversityofSouthamptonSouthamptonUK

AIM: TOtryptasepotentialinvestigate the ability of histamine to modulate release from human colon mast cells and the ***: Enzymatically dispersed cells from human colons were challenged with histamine, anti-IgE or calcium ionophore A23187 (CI), and the cell supernatants after challenge were collected. Tryptase release was determined with a sandwich ELISA ***:Histamine at concentrations from 1ng/mL was able to induce a “bell” shape dose related release of tryptase from colon mast cells. The maximum release of tryptase was approximately 3.5 fold more than spontaneous release. As little as 10ng/ml histamine showed a similar potency to 10μg/mL anti-IgE in induction of tryptase release. Histamine induced release of tryptase initiated at 10s when histamine (100ng/mL) was added to cells, gradually increased thereafter, and completed at 5 *** pertussis toxin or metabolic inhibitors were able to inhibit histamine induced tryptase release. When histamine and anti-IgE were added to colon mast cells at the same time, the quantity of tryptase released was similar to that induced by anti-IgE *** similar results were observed with CI. However, when various concentrations of histamine were incubated with cells for 20min before adding anti-IgE or CI, the quantity of tryptase released was similar to that was induced by histamine ***:Histamine is a potent activator of human colon mast cells, which represents a novel and pivotal selfamplification mechanism of mast cell degranulation.

关键词：类胰蛋白酶组胺肥大细胞免疫球蛋白E 酶联免疫法细胞调节炎症

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