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点状产气单胞菌脯氨酰内肽酶在大肠杆菌中的高效表达

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生物化学与生物物理学报 2000年第2期32卷 183-186页

作者：李民徐皓陈常庆中国科学院上海生物工程研究中心!上海200233

采用PCR分段克隆法将点状产气单胞菌点状亚种 (Aeromonaspuctatasubsp .puctata)脯氨酰内肽酶 ( pro lylendopeptidase ,apPEP)的编码区基因分成 3段扩增并拼接成编码 690个氨基酸的完整基因apPEP ,将其克隆在表达载体 pBL和 pKKH上 ,... 详细信息

采用PCR分段克隆法将点状产气单胞菌点状亚种 (Aeromonaspuctatasubsp .puctata)脯氨酰内肽酶 ( pro lylendopeptidase ,apPEP)的编码区基因分成 3段扩增并拼接成编码 690个氨基酸的完整基因apPEP ,将其克隆在表达载体 pBL和 pKKH上 ,构建成温度和IPTG诱导型高效表达apPEP的重组大肠杆菌BL2 1/ pBL PEP和BL2 1/pKKH PEP。诱导后 ,重组apPEP的表达量占菌体总蛋白质的 3 0 %左右 ,活力是野生菌的 10 0倍左右 ,表达产物为可溶性的胞内蛋白 ,非还原SDS PAGE显示为单体 ,分子量为 76kD ,与基因序列预测的分子量一致。采用阴离子HPLC纯化得到了纯度大于 90 %的重组apPEP ,酶比活力为 67u/mg。N端氨基酸序列测定的结果证实了表达的正确性。

关键词：脯氨酰内肽酶大肠杆菌高效表达纯化

过量表达的hTR75可独立介导hTNFα的细胞毒性

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生物化学与生物物理学报 1999年第6期31卷 637-642页

作者：方佳朱蓓王德宝陈常庆中国科学院上海生物工程研究中心!上海200233 中国科学院上海生物化学研究所分子生物学国家重点实验室!上海200031

利用细小ＲＮＡ病毒属脑炎和心肌炎病毒（ＥＭＣＶ）的内核糖体进入位点（ＩＲＥＳ）元件构建了人肿瘤坏死因子受体７５基因（ｈＴＲ７５）和新霉素抗性基因（ｎｅｏＲ）的双顺反子表达载体，通过电脉冲转染ＢＨＫ２１细胞， ... 详细信息

利用细小ＲＮＡ病毒属脑炎和心肌炎病毒（ＥＭＣＶ）的内核糖体进入位点（ＩＲＥＳ）元件构建了人肿瘤坏死因子受体７５基因（ｈＴＲ７５）和新霉素抗性基因（ｎｅｏＲ）的双顺反子表达载体，通过电脉冲转染ＢＨＫ２１细胞，筛选出分别和同时稳定过量表达两种ｈＴＮＦ受体的细胞株。这些细胞在ｍＲＮＡ和蛋白质水平均可检测到ｈＴＲ７５的表达。利用野生型和具有两种受体选择性结合活性的ｈＴＮＦα突变体对这些细胞株进行测定，发现过量表达的ｈＴＲ７５可以独立地介导ｈＴＮＦα对ＢＨＫ２１细胞的细胞毒性，而ｈＴＲ５５的存在并不能显著增强这种作用。上述结果为进一步阐明ＴＲ７５的功能以及两种受体间的相互作用提供了一条新的途径。

关键词：人肿瘤坏死因子受体内核糖体进入位点表达信号转导

细菌对氨基糖苷类抗生素产生抗性的机理及其控制策略

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中国生物工程杂志 2004年第11期24卷 9-12页

作者：周勤华张毅中国科学院上海生命科学研究院生物工程研究中心上海200233

氨基糖苷类抗生素在治疗感染性疾病尤其是革兰氏阴性菌引起的严重感染方面起着重要作用 ,但是耐药菌株的出现较大地限制了此类抗生素的发展 ,因此 ,如何控制耐药性已经成为一项迫切需要解决的任务。细菌对氨基糖苷类抗生素产生抗性的机... 详细信息

氨基糖苷类抗生素在治疗感染性疾病尤其是革兰氏阴性菌引起的严重感染方面起着重要作用 ,但是耐药菌株的出现较大地限制了此类抗生素的发展 ,因此 ,如何控制耐药性已经成为一项迫切需要解决的任务。细菌对氨基糖苷类抗生素产生抗性的机制很多 ,目前普遍接受的主要有三种 :1 通过减少对氨基糖苷类抗生素的摄取或减少药物在体内的累积而产生抗性。 2 通过改变核糖体结合位点而产生抗性。 3 通过表达氨基糖苷类抗生素修饰酶而产生抗性。目前细菌耐药性的控制主要集中在对原有氨基糖苷类抗生素进行改造或合成新的抗生素 ,开发氨基糖苷类抗生素修饰酶抑制剂。

关键词：氨基糖苷类抗生素治疗修饰酶细菌过表达严重感染体内抗性累积耐药菌株

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五步蛇毒血小板聚集抑制因子cDNA的克隆及表达

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生物化学与生物物理学报 1999年第5期31卷 531-536页

作者：范春阳钱友存沈雁杨胜利龚毅中国科学院上海生物工程研究中心!上海200233

采用一步法抽提五步蛇毒腺总ＲＮＡ，通过ＲＴ ＰＣＲ扩增出低分子量金属蛋白酶酶原的ｃＤＮＡ，克隆并测定了全序列。根据推导的氨基酸序列，发现其中一个ｃＤＮＡ除编码一个低分子量金属蛋白酶外，羧基端还包括... 详细信息

采用一步法抽提五步蛇毒腺总ＲＮＡ，通过ＲＴ ＰＣＲ扩增出低分子量金属蛋白酶酶原的ｃＤＮＡ，克隆并测定了全序列。根据推导的氨基酸序列，发现其中一个ｃＤＮＡ除编码一个低分子量金属蛋白酶外，羧基端还包括一个血小板聚集抑制因子（称为ａｃｕｇｒｉｎ），这一结果证实了蛇毒金属蛋白酶和血小板聚集抑制因子起源于蛇毒金属蛋白酶酶原的前体。Ａｃｕｇｒｉｎ与其他蛇毒来源的血小板聚集抑制因子具有一定的同源性，有４对二硫键，具有一个蛇毒血小板聚集抑制因子保守的结构域：ＲＧＤ序列。将ａｃｕｇｒｉｎ的ｃＤＮＡ克隆到ｐＴ７ＺＺａ融合表达载体，在大肠杆菌系统中得到融合表达，融合蛋白具有较强的抑制ＡＤＰ诱导的血小板聚集的活力，半抑制剂量ＩＣ５０为４ｎｍｏｌ／Ｌ。

关键词：蛇毒血小板聚集抑制因子五步蛇融合表达抗血栓

中国人胃癌染色体11_p15.5处杂合性缺失分析

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复旦学报（医学版） 2003年第1期30卷 24-26,29页

作者：刘韬韬史耀舟沈锡中孔祥银王吉耀复旦大学附属中山医院消化科上海200032 中国科技大学生命科学院合肥230027 中国科学院上海生物工程研究中心上海200233

目的　通过对中国人胃癌染色体 11p15 .5处杂合性缺失的研究 ,了解中国人群中胃癌病人在此区域杂合性缺失的情况。方法　从 66例胃癌病人的石蜡包埋的手术病理标本中 ,提取肿瘤及相应正常组织的DNA ,用荧光标记的方法选取 11p15 .5处的... 详细信息

目的　通过对中国人胃癌染色体 11p15 .5处杂合性缺失的研究 ,了解中国人群中胃癌病人在此区域杂合性缺失的情况。方法　从 66例胃癌病人的石蜡包埋的手术病理标本中 ,提取肿瘤及相应正常组织的DNA ,用荧光标记的方法选取 11p15 .5处的微卫星DNA标记进行PCR扩增 ,再将PCR产物进行变性聚丙烯酰胺凝胶电泳 ,电泳后行杂合性缺失分析。结果　 11/ 3 6( 3 0 .6% )的病人在D11S13 18处存在杂合性缺失 ;而D11S40 46位点处 ,13 / 60 ( 2 1.7% )的病例存在杂合性缺失 ;2 4/ 61( 3 9.3 % )的病例在 11p15 .5处至少有 1个位点存在杂合性缺失。杂合性缺失的频率与肿瘤病人的年龄、性别、肿瘤大小、肿瘤部位及淋巴结是否转移均无关。结论　高频杂合性缺失的区域可能有抑癌基因的存在。这将有助于阐明胃癌发生发展的分子机制 ,进而为对胃癌进行风险预测。

关键词：中国人染色体杂合性缺失抑癌基因胃癌

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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.

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

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

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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

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