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Display of aggregation-prone ligand binding domain of human PPAR gamma on surface of bacteriophage lambda

Acta Pharmacologica Sinica 2006年第1期27卷 91-99页

作者： Bo KONG~(2,4) Wei-jun MA~(2,3,5)2 Institute of Health sciences.shanghai institutes for biological sciences,Chinese academy of sciences and Medical School of shanghai Jiao Tong University.shanghai 200025.china 3 shaughai research center of biotechnology,shanghai institutes for biological sciences,chiese academy og sciences,shanghai 200233.china 4 Graduate School of the Chinese academy of sciences Institute of Health sciences Shanghai Institutes for Biological Sciences Chinese Academy of Sciences and Medical School of Shanghai Jiao Tong University Shanghai China Graduate School of the Chinese academy of sciences China shanghai research center of biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

Aim:To display the aggregation-prone ligand binding domain (LBD) of the hu-man peroxisome proliferator-activated receptor gamma (PPARγ) on the surface ofbacteriophages to establish an easy screening assay for the identification of PPARγ***:Plasmids were constructed for the expression of the PPARγLBD as a fusion to the N-terminus of the g3p protein of filamentous phage or theC-terminus of the capsid protein D (pD) of phage *** fusion proteinswere expressed in E coli and solubility characteristics were *** against the LBD as well as the pD protein were prepared for Westernblot analysis and phage capture ***:The pD-LBD fusion protein waspartially soluble,whereas the LBD-g3p fusion protein was detected only in theinsoluble *** pD-LBD fusion protein was efficiently incorporated inphage ***,the LBD was shown to be displayed on the surfaceof bacteriophage *** average,the pD-LBD fusion protein accounted for28% of the total pD protein in the lambda head ***:The hydro-phobic PPARγ LBD was expressed as a soluble form of fusion protein in E coli anddisplayed on the surface of bacteriophage lambda when it was fused to the lambdapD *** lambda pD fusion system could be used for improving the solu-bility of proteins that tend to form inclusion bodies when expressed in E *** phage particles displaying the LBD of PPARy may be of great value for theidentification of novel PPARγ ligands.

关键词： protein expression lambda phage surface display nuclear receptor peroxisome proliferator-activated receptors ligand binding domain

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TNF receptor-associated factor-2 binding site is involved in TNFR75-dependent enhancement of TNFR55-induced cell death

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Cell research 2001年第3期11卷 217-222页

作者： LU FANG, JIA FANG, CHANG QING CHEN shanghai research center of biotechnology, shanghai institutes for biological sciences the Chinese academy of sciences, shanghai 200233, china shanghai research center of biotechnology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai China

TNF recepter-55 is the main mediator of TNF-induced apoptosis. TNF receptor-75-dependent induction or enhancement of cytotoxicity has been explained by intracellular signaling, 'ligand passing' , or induction of endogenous TNF. To study the function of human TNF receptor-75 (hTR75) and the interaction between human TNF receptor-55 (hTR55) and hTR75 in hTNFa-induced cytotoxicity, HEp-2 cells were transfected with bicistronic expression vector of hTR75 and its deletion mutants genes. hTNFa-induced cytotoxicity was determined by crystal violet colorimetric method. The expression of hTR75 and its deletion mutants in HEp-2 cells was demonstrated by RT-PCR and indirect ELISA. We found that the overexpressed hTR75 could significantly increase the susceptibility of HEp-2 cells to hTNFa which especially required TRAF2 binding site. hTR75 could not only mediate partial hTNFa-induced cytotoxicity independently but also fulfill an accessory role in enhancing or synergizing hTR55-mediated cytotoxicity

关键词： hTNFa hTNF receptors TRAF2 cytotoxcity signal transduction.

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Recombinant scFv Antibodies against E Protein and N Protein of Severe Acute Respiratory Syndrome Virus

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 541-547页

作者： Hui LIU , Yan-Li DING, Wei HAN, Mei-Yun LIU, Rui-Yang TIAN, Sheng-Li YANG, and Yi GONGshanghai research center of biotechnology, shanghai institutes of biological sciences, Chinese academy of sciences,Graduate School of the Chinese academy of sciences, shanghai 200233, china shanghai research center of biotechnology Shanghai Institutes of Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences Shanghai 200233 China. huiliu@***

Three single chain antibodies (scFv) against the proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated by phage display from an scFv antibody library. Bio-panning was carried out against immobilized purified envelope (E) and nucleocapsid (N) proteins of SARS-CoV. Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA. Two of them, B10 and C20, could recognize non-overlapping epitopes of the E protein according to the two-site binding test result. Clone A17 could recognize N protein. The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ. The affinity (equilibrium dissociation constant, Kd) of SARS-CoV E protein was 5.7×10–8 M for B10 and 8.9×10–8 M for C20. The affinity of A17 for N protein was 2.1×10–6 M. All three scFv antibodies were purified with affinity chromatography and determined by Western blot.

关键词： SARS-CoV antibody scFv phage display affinity chromatography

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Two Observed Regions in B Lymphocyte Stimulator Important for Its biological Activity

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Acta Biochimica et Biophysica Sinica 2006年第4期38卷 227-232页

作者： Qiong SHEN~(1,2) Su-Xia LI~1 Fei-Hao FU~1 Qin-Sheng YUAN~1* Yi GONG~2* 1 State Key Laboratory of Bioreactor Engineering,East china University of Science and Technology,shanghai 200237,china 2 research center of biotechnology,shanghai institutes for biological sciences,Chinese academy of sciences,shanghai 200233.china State Key Laboratory of Bioreactor Engineering East China University of Science and TechnologyShanghai 200237 China research center of biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200233 China

B lymphocyte stimulator （BLyS）,a member of the tumor necrosis factor superfamily ofligands,is a crucial survival factor for B *** successfully constructed seven mutants of the functionalsoluble fragment of human BLyS （named cBLyS,amino acid 134-285）,including three deletion mutants andfour site-directed *** the mutant proteins were expressed in Escherichia coli and purified by Ni-NTA affinity *** biological activities of these mutants were assessed by the ligand-recep-tor binding assay,B cell proliferation assay and immune effect response in *** results indicated thatfour residues,H218,F220,T228and L229,are indispensable for the biological activity of cBLyS,whereas tworegions,amino acid 134-148 and amino acid 271-285,are related to the biological activity of *** of deletion of amino acid 134-148 leads to a complete defection in raising the antigen-specific *** deletion of amino acid 271-285 reduces the effectiveness compared with the native *** that the region of amino acid 134-148 is indispensable for cBLyS to function normally.

关键词： B lymphocyte stimulator (BLyS) deletion mutant biological activity

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Fusion Protein of Interleukin 4 and Diphtherial Toxin with High Cytotoxicity to Cancer Cells

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Acta Biochimica et Biophysica Sinica 2004年第6期36卷 437-442页

作者： Yu-Jian ZHANG, Xiao-BoHU, Su-Xia LI1, Li-Ping TIAN, Sheng-Li YANG, and Yi GONG* research center of biotechnology, shanghai institutes for biological sciences,Chinese academy of sciences, Graduate School of the Chinese academy of sciences, shanghai 200233, china State Key Laboratory of Bioreactor Engineering, East china University of Science and Technology, shanghai 200237, china research center of biotechnology Shanghai Institutes for Biological SciencesChinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200233 China State Key Laboratory of Bioreactor Engineering East China University of Science and TechnologyShanghai 200237 China

Receptor of human interleukin 4 (hIL4R) has been found to be present on many types of cancer, so it may be a good target for cancer therapy. Here, fusion toxin gene DT4H has been constructed by fusing DNA sequence encoding the first 389 amino acids of diphtherial toxin (DT), which can not bind its own receptor, to human interleukin 4 (hIL4) gene. In order to improve the affinity of fusion toxin for hIL4R, a circularly permuted form of hIL4 (cpIL4) was used. The fusion gene was expressed in Escherichia coli where the fusion toxin DT4H was highly expressed. Purified DT4H was very cytotoxic to cancer cell line U251 cells, and moderate cytotoxic to HepG2 and MCF-7 cells. SGC-7901 cells were insensitive to it. The cytotoxic action of DT4H was specific because it was blocked by excess hIL4. These results suggest that DT4H may be a useful agent in the treatment of certain malignancies.

关键词： fusion toxin diphtherial toxin cancer therapy interleukin 4 IL-4 receptor

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Expression and Comparative Analysis of Genes Encoding Outer Membrane Proteins LipL21,LipL32 and OmpL1 in Epidemic Leptospires

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 7+650-656页

作者： Xiang-Yan ZHANG~(1#) Yang YU~(2,3#) Ping HE~1 Yi-Xuan ZHANG~(2,3) Bao-Yu HU~1 Yang YANG~1 Yi-Xin NIE~4 Xiu-Gao JIANG~4 Guo-Ping ZHAO~(2,3) Xiao-Kui GUO~(1*) 1 Department of Medical Microbiology and Parasitology,shanghai Jiaotong University,shanghai 200025,china 2 research center of biotechnology,shanghai institutes for biological sciences,Chinese academy of sciences,shanghai 200031,china 3 Chinese National Human Genome center at shanghai,Zhangjiang High Tech Park,shanghai 201203,china 4 Institute for Infectious Disease Control and Prevention,Chinese center for Disease Control and Prevention,Beijing 102206,china Department of Medical Microbiology and Parasitology Shanghai Jiaotong UniversityShanghai 200025 China research center of biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China Chinese National Human Genome center at shanghai Zhangjiang High Tech ParkShanghai 201203 China Institute for Infectious Disease Control and Prevention Chinese Center for Disease Control and PreventionBeijing 102206 China

Leptospiral outer membrane proteins (OMPs) are highly conserved in different species,andplay an essential role in the development of new immunoprotection and serodiagnosis *** genesencoding LipL21,LipL32 and OmpL1 were cloned from the complete genome sequence of Leptospirainterrogans serovar lai strain Lai and expressed in *** comparison analysis revealed that the threegenes were highly conserved among distinct epidemic leptospires,including three major epidemic speciesLeptospira interrogans,Leptospira borgpetersenii and Leptospira weilii,in *** analysis wasfurther performed to scrutinize 15 epidemic Leptospira reference strains using the antisera of the *** immunoblot assay and reverse transcription-polymerase chain reaction demonstrated that thesethree OMPs were conservatively expressed in pathogenic *** strains and other pathogenic ***,the use of these recombinant OMPs as antigens in enzyme-linked immunosorbent assay (ELISA)for serodiagnosis of leptospirosis was *** recombinant LipL32 and OmpL1 proteins showed ahigh degree of ELISA reactivity with sera from patients infected with *** strain Lai and otherpathogenic *** results may contribute to the identification of candidates for broad-rangevaccines and immunodiagnostic antigens in further research.

关键词： outer membrane protein expression comparative analysis epidemic leptospire

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Identification and Analysis of Genes Present in Leptospira interrogans serovar lai but Absent in L. biflexa serovar monvalerio

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Acta Biochimica et Biophysica Sinica 2004年第12期36卷 832-839页

作者： Ping HE, Xiang-Yan ZHANG, Xiao-Kui GUO*, Bao-Yu HU, Xiao-Tian HUANG, Yang YANG, and Guo-Ping ZHAO1,2* Department of Microbiology and Parasitology, shanghai Second Medical University, shanghai 200025, china 1 research center of biotechnology, institutes for biological sciences, Chinese academy of sciences, shanghai 200031, china 2Chinese National Human Genome center at shanghai, Zhangjiang High Tech Park, shanghai 201203, china Department of Microbiology and Parasitology Shanghai Second Medical UniversityShanghai 200025 China research center of biotechnology Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China Chinese National Human Genome center at shanghaiZhangjiang High Tech Park Shanghai 201203 China

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogans serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridiza- tion is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.

关键词： Leptospira interrogans serovar lai suppression subtractive hybridization (SSH) specific genes

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Generation and Characterization of C305,a Murine Neutralizing scFv Antibody That Can Inhibit BLyS Binding to Its Receptor BCMA

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Acta Biochimica et Biophysica Sinica 2005年第6期37卷 415-420页

作者： Mei-Yun LIU~(1.2) Wei HAN~(2.3) Yan-Li DING~(2.3) Tian-Hong ZHOU~1 Rui-Yang TIAN~2 Sheng-Li YANG~2 Hui LIU~2 Yi GONG~2 1 College of Life Science and Technology,Jinan University. Guangzhou 510632, china 2 shanghai research center of biotechnology.shanghai institutes for biological sciences. Chinese academy of sciences. shanghai 200233.china 3 School of Pharmaceatical Engineering, Shenyang Pharmaceutical University. Shenyang 110016. china College of Life Science and Technology Jinan UniversityGuangzhou 510632 China shanghai research center of biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200233 China School of Pharmaceutical Engineering Shenyang Pharmaceutical UniversityShenyang 110016 China

B-lymphocyte stimulator(BLyS)is a member of the tumor necrosis factor(TNF)family anda key regulator of B cell *** single-chain fragment variable(scFv)antibody against BLySbinding to its receptor BCMA has the potential to play a prominent role in autoimmune disease *** display scFv library constructed on pⅢ protein of M13 filamentous phage was screened using *** five rounds of panning,their binding activity was characterized by *** sequencingrevealed that at least two different scFv gene fragments(C305 and D416)were *** two differentscFv gene fragments were expressed to obtain the soluble scFv antibodies,then the soluble scFv antibodieswere characterized by means of competitive ELISA and in vitro neutralization *** results indicatedthat C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.

关键词： phage display neutralizing scFv antibody BLyS

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Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome

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Cell research 2004年第3期14卷 208-216页

作者： Yi Xuan ZHANG, Xiao Kui GUO, Chuan WU, Bo BI, Shuang Xi REN, Chun Fu WU, Guo Ping ZHAO Pharmaceutical Department, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, china. research center of biotechnology, shanghai institutes for biological sciences, Chinese academy of sciences, 500 Caobao Road, shanghai 200233, china. Department of Microbiology and Parasitology, shanghai Second Medical University, 280 Chongqingnan Road, shanghai 200025, china. Chinese National Human Genome center at shanghai (CHGCS), 250 Bibo Road, ZhangJiang High Tech Park, shanghai, 201203, china. Pharmaceutical Department Shenyang Pharmaceutical University 103 Wenhua Road Shenhe District Shenyang China research center of biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China Department of Microbiology and Parasitology Shanghai Second Medical University Shanghai China Chinese National Human Genome center at shanghai (CHGCS) 250 Bibo Road ZhangJiang High Tech Park Shanghai China

Comparative genomic analysis of the coding sequences （CDSs） of Leptospira interrogans revealed a pair of closely linked genes homologous to the vapBC loci of many other bacteria with respect to both deduced amino acid sequences and operon organizations. Expression of single vapC gene in Escherichia coli resulted in inhibition of bacterial growth, whereas co-expression of vapBC restored the growth effectively. This phenotype is typical for three other characterized toxin-antitoxin systems of bacteria, i.e., mazEF[1], relBE[2] and chpIK[3]. The VapC proteins of bacteria and a thermophilic archeae, Solfolobus tokodaii, form a structurally distinguished group of toxin different from the other known toxins of bacteria. Phylogenetic analysis of both toxins and antitoxins of all categories indicated that although toxins were evolved from divergent sources and may or may not follow their speciation paths （as indicated by their 16s RNA sequences）, co-evolution with their antitoxins was obvious.

关键词： Leptospria toxin-antitoxin system VapBC co-evolution speciation.

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Effect of GNTI,a kappa opioid receptor antagonist,on MK-801-induced hyperlocomotion and stereotypy in mice

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Acta Pharmacologica Sinica 2006年第11期27卷 1401-1408页

作者： Chun-ting QI~2 Hong ZOU~2 Chen-hao ZHANG~2 Qing-lian XIE~2 Mei-lei JIN~(2,4)Lei YU~(3,4)2 research center for biotechnology,shanghai institutes for biological sciences,Chinese academy of sciences,shanghai 200233.china 3 Department of Genetics and center of Alcohol Studies,Rutgers,the State University of New Jersey 08854-8001,USA research center for biotechnology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China Department of Genetics and center of Alcohol Studies Rutgers the State University of New Jersey USA

Aim:To examine the effect of GNTI [5′-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5α-epoxy-3,14-dihydroxy-6,7-2′,3′-indolomorphinan],a selective antago-nist for the kappa opioid receptor,in the MK-801(dizocilpine maleate)-inducedbehavioral model of psychosis in schizophrenia as a way to explore the involve-ment of the kappa opioid receptor in modulating psychotic symptoms ***:Two doses of MK-801(0.3 mg/kg and 0.6 mg/kg)wereadministered by systemic injection in mice to induce psychosis-like behavior as arodent schizophrenia model,preceded by an injection of different doses of *** locomotion and stereotypy were measured as the behavioral endpoints forquantitative ***:GNTI inhibited MK-801-induced hyperlocomotionand *** particular,GNTI showed differential modulation of stereotypyinduced by 0.3 mg/kg vs 0.6 mg/kg ***:Antagonism of kappaopioid receptors attenuates MK-801-induced behavior,suggesting a potentialinvolvement of the kappa opioid receptor in psychosis-like symptoms *** appears to be a useful pharmacological tool to explore thekappa opioid receptor function in vivo.

关键词： GNTI kappa opioid receptor MK-801 schizophrenia model

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