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Aβ28-Induced Specific and Effective Serum Antibodies Against Aβ42

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Chemical Research in Chinese Universities 2010年第6期26卷 974-980页

作者： WANG Jia-peng CUI Li-li HUANG Xue-mei zhang ying-jiu Key Laboratory for Molecular Enzymology ＆ Engineering Ministry of Education Jilin University Changchun 130021 P. R. China

Though phase I clinical trial of immunotherapy for Alzheimer＇s disease（AD） with inoculated Aβ42 in humans had been proven to be effective,phase II immunotherapy trial was discontinued in 2002 because a few patients developed significant inflammatory reactions caused by C-terminal domain of Aβ*** aim of the study was to investigate the levels and the abilities of antibodies induced by C-terminal truncated Aβ species to inhibit Aβ42 aggregation and cytotoxity in vitro.46-week-old male BALB/c mice,Aβ42 and Aβ28/Aβ35/Aβ42 with a C-terminal eight-histidine tag（Aβ28H8,Aβ35H8,and Aβ42H8） were applied in the present *** mice were randomly divided into 5 groups（n=8）,control mice immunized with the normal saline,Aβ42-immunized mice and Aβ42H-/Aβ35H-/Aβ28H-immunized *** the serum antibodies were evaluated by measuring their abilities to inhibit Aβ42 aggregation and cytotoxity in *** mouse was vaccinated with purified Aβ peptide emulsified with Freund＇s adjuvant（volume ratio 1：1）.Titers,concentrations and isotypes of serum antibodies against Aβ42 were measured by indirect *** of serum antibodies on Aβ42 aggregation and disassembly of Aβ42 fibrils in vitro were observed by electron *** of serum antibodies on Aβ42 cytotoxicity were determined by MTT assay.Aβ42 or Aβ28 could induce higher anti-Aβ42 antibody titer（1：6400） than Aβ35（1：3200）.Significantly,Aβ28 induced more IgG1 and IgG2b isotype antibodies and less IgG2b isotype antibody than other Aβ species though all the induced serum antibodies could inhibit Aβ42 aggregation or fibrillogenesis,could induce the disassembly of Aβ42 aggregates,and neutralized or inhibited the cytotoxicity of Aβ42 in vitro.C-Terminal truncated Aβ28 could induce the same effective but safer serum antibodies against Aβ42 than full length Aβ42 by eliciting more Th2-type immune responses.

关键词： Alzheimer's disease Amyloid peptide Antibody Epitope Immunization

Expression Characterization and Preparation of Human Amyloid Precursor Protein in Escherichia coli

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Chemical Research in Chinese Universities 2009年第1期25卷 64-68页

作者： XU Guang-wei WANG Jia-peng HUANG Xue-mei zhang ying-jiu Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education Jilin University Changehun 130021 P R. China

To analyze whether expressed amyloid precursor protein（APP） existed in hydrophilic（cytoplasmid） or hydrophobic（lipid bilayer） environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coll. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hydrophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni^2＋ agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coli cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.

关键词： Amyloid precursor protein Expression characterization Preparation Membrane Inclusion body

Synthesis of Codon-optimized Human Interleukin-18 Gene by Combination of Chemical and Enzymatic Method

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Chemical Research in Chinese Universities 2008年第4期24卷 487-490页

作者： GAO Chao-hui SHI Xiao-yue HOU Xin-tong MENG Qing-fan zhang ying-jiu TENG Li-rong College of Life Science Jilin University Changehun 130012 P. R. China Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education Jilin University Changehun 130021 P. R. China Changehun Diseases Control and Prevention Center Changehun 130033 P. R. China College of Animal Science and Veterinary Medicine Jilin University Changehun 130062 P. R. China

According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.

关键词： Human IL-18 Gene synthesis One-step method Oligodeoxynucleotide synthesis

决定细胞形状和运动性的蛋白质palladin

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生物化学与生物物理进展 2005年第7期32卷 607-611页

作者：张应玖 YANMIN YANG 吉林大学生命科学学院分子酶学工程教育部重点实验室长春130023Department of Neurology Stanford University Stanford CA 94305 USA Department of Neurology Stanford University Stanford CA 94305 USA

Palladin是肌动蛋白结合蛋白家族的新成员,广泛分布于平滑肌、中枢神经系统和胚胎的各种组织中,其主要的生物功能是参与构建肌动蛋白骨架系统,并在细胞骨架的动态变化中起作用.在肌动蛋白细胞骨架中palladin与alpha-辅肌动蛋白共存在.... 详细信息

Palladin是肌动蛋白结合蛋白家族的新成员,广泛分布于平滑肌、中枢神经系统和胚胎的各种组织中,其主要的生物功能是参与构建肌动蛋白骨架系统,并在细胞骨架的动态变化中起作用.在肌动蛋白细胞骨架中palladin与alpha-辅肌动蛋白共存在.目前发现,palladin在决定细胞的形态和迁移或运动等过程中起关键的作用.在转移性癌细胞和中枢神经受损伤后的星形胶质细胞中,都有palladin的特殊表达.Palladin的表达使星形胶质细胞形成了神经胶质疤痕.

关键词： palladin 形状运动神经胶质疤痕

Molecular Design and Immunogenicity of a Multiple-epitope FMDV Antigen and DNA Vaccination

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Chemical Research in Chinese Universities 2008年第1期24卷 69-74页

作者： MA Ming-xiao JIN Ning-yi LIU Hui-juan ZHENG Mini YIN Ge-fen TIAN Ming-yao JIN Ming-lan LU Hui-jun SHEN Guo-shun ZHU Guang-ze JIN Hong-tao JIN Kuo-shi zhang ying-jiu Genetic Engineering Laboratory Academy of Military Medical Seienees Changehun 130062 P. R. China College of Animal Seienee and Veterinary Medieine Liaoning Medieal University Jinzhou 121001 P. R. China College of Animal Seienee and Veterinary Medieine Yunnan Agrieultural University Kunming 650201 P R. China Key Laboratory for Moleeular Enzymology and Engineering of Ministry of Edueation Jilin University Changehun 130021 P. R. China

This article reports the design and construction of a multiple-epitope foot and mouth disease virus （FMDV） antigen, designated as OAAT. This recombinant antigen consists of the structural protein VP1 genes from serotypes A and O FMDV, five major VP1 immunodominant epitopes from two genotypes of Asial serotype, and three Th2 epitopes originating from the nonstructural protein, three ABC gene and structural protein VP4 gene. Expressions of target gene from these plasmids in HeLa cells were verified by Western-blot. BALB/c mice were immunized intramuscularly with the DNA vaccines thrice every two weeks. We found that pA could induce simultaneously specific antibodies against serotypes A, Asial, and O FMDV. Compared to those of the controls, the spots of FMDV-specific IFN-7 and cytotoxic activity from mice immunized with pA were significantly increased, pA provided full protection in 2/4 guinea pigs from challenge with FMDV O/NY00 and Asial/YNBS/58, respectively. The results show that although pA did not give full protection in 100% immunized guinea pigs from challenge with type O and Asial FMDV, respectively, OAAT may be potential immunogen against FMDV and pA may be potential DNA vaccines against FMDV.

关键词： Multiple-epitope Foot and mouth disease virus DNA vaccine

膜蛋白Presenilin 1的跨膜结构及催化机制

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中国生物化学与分子生物学报 2007年第11期23卷 894-898页

作者：徐广伟张应玖吉林大学分子酶学工程教育部重点实验室长春130021

膜蛋白presenilin1(PS1)是γ分泌酶的催化组分,是催化产生β淀粉样蛋白(β-amyloid,Aβ)的关键蛋白酶,因此也是治疗阿尔茨海默病(Alzheimer’s disease,AD)的主要靶点.PS1属于膜内裂解蛋白酶家族,这是一类在膜脂双层内部催化肽键水解断... 详细信息

膜蛋白presenilin1(PS1)是γ分泌酶的催化组分,是催化产生β淀粉样蛋白(β-amyloid,Aβ)的关键蛋白酶,因此也是治疗阿尔茨海默病(Alzheimer’s disease,AD)的主要靶点.PS1属于膜内裂解蛋白酶家族,这是一类在膜脂双层内部催化肽键水解断裂的蛋白酶.PS1其独特的跨膜结构和催化机制虽然还未完全揭示,但近期相关的研究取得了重要成果:PS1有10个疏水区,跨膜9次,其N端位于胞内,C端位于胞膜外或者内质网腔内,亦或不同程度地插入膜内,2个起催化作用的天冬氨酸残基都位于疏水性的膜内,膜蛋白底物被催化水解时必须先结合到酶的疏水表面上来,然后再进入位于活性部位.虽然PS1的晶体从未获得,但2006年首次解析的膜内裂解蛋白酶GlpG的晶体结构和所提出的催化机理为PS1催化机理的揭示奠定了基础,也为设计和筛选PS1/γ分泌酶的特异性抑制剂提供了理论依据.

关键词： presenilin 1(PS1) γ分泌酶跨膜结构催化机制

蛋白磷酸酶2C(PP2C)的表达、纯化与催化活性

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吉林大学学报（理学版） 2009年第1期47卷 144-149页

作者：周旺亓晓艳张应玖吉林大学分子酶学工程教育部重点实验室长春130021

从小鼠肌肉组织中提取了总RNA,经RT-PCR,扩增出蛋白磷酸酶2C(PP2C)基因,并构建了pUCm-T载体.经核酸序列分析证明PP2C基因序列正确后,将pUCm-T中的PP2C基因插入pET28a载体中,构建了表达载体pET28a-PP2C,并转化到*** BL21(DE3)进行表达.... 详细信息

从小鼠肌肉组织中提取了总RNA,经RT-PCR,扩增出蛋白磷酸酶2C(PP2C)基因,并构建了pUCm-T载体.经核酸序列分析证明PP2C基因序列正确后,将pUCm-T中的PP2C基因插入pET28a载体中,构建了表达载体pET28a-PP2C,并转化到*** BL21(DE3)进行表达.经测定,该菌株表达目的蛋白PP2C的最适条件为:诱导物IPTG的终浓度为0.8 mmol/L,37℃,诱导时间为20 h.在此条件下,PP2C实现了高效表达,表达的PP2C蛋白约占菌体总蛋白的18.2%,其中在裂解上清液、沉淀中分别占总蛋白的7.1%和11.1%.经SDS-PAGE分析,表达产物分子量约为42 000.应用Ni-NTA柱实现了可溶性PP2C的纯化,收率达80.5%,纯化的PP2C比活力达34.5 U/mg.

关键词：蛋白磷酸酶2C(PP2C) 表达纯化活性

生物膜电极反应器降解对氨基二甲基苯胺的研究

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高等学校化学学报 2007年第3期28卷 556-558页

作者：康博黄卫民张应玖张雪娜林海波吉林大学化学学院吉林大学分子酶学工程教育部重点实验室长春130021

4-Amino-dimethyl-aniline hydrochloride is one of the main components of the wastewater from vanillin production plant. So it is used for the target contamination to be degraded by microorganism, electrochemical oxidation and bio-electro reactor in this paper. We found out a economical, efficient and non-polluted methods for the treatment of wastewater from vanillin production plant. The results indicate that there are a lot of special microbes and bacteria eating 4-amino-dimethyl-aniline hydrochloride in the activated sludge after 20 days′ incubation, at the same time, a stable bio-filmed electrode was formed. Under the condition of 25 ℃, pH=6—7. The use of bio-electro reactor not only increased the biodegradability and decreased the electrical energy consumption, but also solved the problem which the second pollution of organic compounds containing chlorine in the process of indirect electrochemical degradation on the organic pollutants.

关键词：香兰素生物膜电极反应器降解对氨基二甲基苯胺

Rho/ROCK2通路在阿尔茨海默病病理进程中的重要作用

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国际药学研究杂志 2016年第1期43卷 33-38页

作者：张天弼张云龙胡警文张应玖吉林大学分子酶学工程教育部重点实验室、吉林大学生命科学学院长春130012

Rho/Rho相关卷曲螺旋形成蛋白激酶(ROCK)通路是生物体中广泛存在的经典信号通路,参与多种生理调节过程。ROCK有2种亚型,即ROCK1和ROCK2。在阿尔茨海默病(AD)患者脑中,Rho/ROCK2表现为活性上调,并伴有Aβ42水平升高,以及神经细胞突起的... 详细信息

Rho/Rho相关卷曲螺旋形成蛋白激酶(ROCK)通路是生物体中广泛存在的经典信号通路,参与多种生理调节过程。ROCK有2种亚型,即ROCK1和ROCK2。在阿尔茨海默病(AD)患者脑中,Rho/ROCK2表现为活性上调,并伴有Aβ42水平升高,以及神经细胞突起的形态与功能异常,推测AD的发生、发展与Rho或ROCK2的高表达或过度激活有关。目前Rho/ROCK2通路被认为是预防和治疗AD的一个有效的靶通路,而Rho或ROCK2,也成为药物研发的重要靶点。研究发现,降低Rho或ROCK2的表达,或者抑制Rho或ROCK2的活性均可减少Aβ42诱导的神经毒性,保护神经元,减缓AD的发展。因此,Rho/ROCK2的特异性抑制对中枢神经损伤修复及AD的治疗有重要的意义。为此,本文针对Rho/ROCK2通路在阿尔茨海默病发展中的作用做一综述。

关键词： Rho Rho相关卷曲螺旋形成蛋白激酶阿尔茨海默病抑制剂