中华补血草金属硫蛋白基因的克隆及高盐处理下的表达模式分析

以中华补血草叶片为材料,提取其总RNA并反转录cDNA,并以cDNA为模板,克隆得到包含该基因完整开放阅读框的cDNA序列,序列分析表明,该基因开放阅读框长249 bp,编码82个氨基酸;该蛋白含有14个半胱氨酸,主要分布在蛋白质的N端和C端,呈CC,CX,CXXC形式排列.预测该蛋白分子质量为8 133.1 D;等电...     

小鼠胚胎脑特异表达的新基因bsg3的克隆及初步研究

通过消减差异筛选的方法克隆到一个在小鼠胚胎脑特异表达的基因bsg3(brain specific gene 3).它编码的蛋白与人的KIAA0961具有80.3%的同源性.bsg3基因包含一个1644bp的完整阅读框,编码一个含548个氨基酸,具有1个KRAB结构域(kruppel-associated box)和13个C2H2型锌指结构域的蛋白.该基因定位在小鼠第7号染色体上,包含5个外显子,4个内含子.以bsg3基因全长编码区为探针的原位杂交结果显示,bsg3在小鼠胚胎脑及鸡胚脑特异表达.半定量反转录聚合酶链式反应(RT-PCR)的结果表明,在成体小鼠的组织中,bsg3基因脑中表达也较强.这提示bsg3基因可能在小鼠脑发育中起着重要的作用.对bsg3基因时间和空间的表达模式的分析将有助于进一步揭示它在脑发育及功能维持中的作用.     

梅毒螺旋体47ku抗原的分段表达及抗原表位分析

通过大肠杆菌表达系统表达了梅毒螺旋体47 ku膜蛋白及其N端和/或C端缺失的重组蛋白共15个,经亲和层析纯化,并通过免疫印迹和酶联免疫实验检测这组蛋白与梅毒病人血清的反应性.结果发现所有包含D结构域的重组蛋白的免疫反应性显著高于不含D结构域的蛋白,提示D结构域中可能包含47 ku膜蛋白上的一个免疫优势表位.     

水稻白叶枯病菌luxR同源基因的克隆和表达

luxR基因是革兰氏阴性菌感应反应（Quorum Sensing,QS）LuxR／Luxl环路中起重要作用的基因．从水稻白叶枯病菌中分离到一个luxR同源基因,命名为xooR．xooR全长765bp,编码一个由254个氨基酸残基组成的转录因子,分子质量和等电点分别为28．3ku和8．72．XooR蛋白的N一端15AA-171AA是一个Autoindbind结构域,C-端191AA-245AA是一个HTH（helix-turn-helix）结合DNA结构域利用原核表达载体pET-24a（＋）和gfp基因构建融合蛋白表达系统,Western blot分析表明XooR-GFP融合蛋白大小为54ku,xooR基因成功地在E．coliB1．21中实现了表达．     

铜转运蛋白hCtr1结合Cu(I)并转运至铜伴侣蛋白Atox1

铜转运蛋白hCtr1细胞膜内的C端区域对于细胞的铜转运过程具有重要作用,然而这一结构域对铜离子的捕获和胞内蛋白的铜分布机理尚不清楚.为了研究hCtr1蛋白的C端结构域与铜的结合方式,通过紫外-可见吸收光谱、核磁共振谱和质谱等方法,对C端8肽(C8)与铜离子的结合进行了分析.结果表明,一价铜离子对C8有很高的亲和力(log K=16.6).C8能够结合多个铜离子,其HCH基序中的组氨酸和半胱氨酸残基是铜结合位点,而另一个天冬氨酸残基也可能参与铜结合.进一步研究发现,铜离子能够从C8转移到Atox1.实验数据揭示了铜与hCtr1结合的详情,也有助于理解铜在细胞中的分布.     

人源锌指蛋白ZNF24及其锌指结构域的表达、纯化及DNA结合性质的研究

转录因子是调控基因表达的关键蛋白，能够以序列特异性方式结合DNA。ZNF24是Krüppel-like锌指转录因子家族成员之一，在细胞增殖和分化中起重要作用，能促进肝癌细胞的增殖，抑制神经元细胞的分化。ZNF24蛋白N端包含一个SCAN结构域，C端为锌指区，由4个串联的C2H2型锌指结构组成。ZNF24能特异性识别和结合基因的启动子，参与基因的表达调控，但其发挥功能的具体分子机制并不清楚。本文我们在大肠杆菌中成功表达了人源锌指蛋白ZNF24全长和其锌指结构域ZNF24(243～368),ZNF24全长蛋白在溶液中以三聚体形式存在，而锌指结构域在溶液中为单体，表明ZNF24蛋白N端的SCAN结构域能够促进全长蛋白的寡聚化。凝胶迁移实验验证ZNF24全长蛋白和锌指结构域与双链DNA底物结合能力，为进一步研究锌指蛋白ZNF24调控基因表达提供理论基础，相关结果表明SCAN结构域介导的蛋白寡聚可能代表了一种新的转录活性调节机制。     

SBD在重组蛋白质中位移对其酶活性及与淀粉粒结合性能的影响

将环状糊精糖基转移酶的SBD基因编码序列连接到萤光素酶基因的5′-端(SBD-LUC)后,重组基因通过农杆菌介导导人马铃薯植株中．对重组蛋白在转基因淀粉粒中的定位与积累、重组蛋白中SBD与淀粉的亲和性以及萤光素酶的活性进行了分析比较．结果表明：重组蛋白在马铃薯淀粉粒中获得了特异性表达,当C-端SBD移位到重组萤光素酶蛋白的N-端后,SBD和淀粉的亲和性与LUC-SBD重组蛋白质比较有所下降,但萤光素酶活性则与马铃薯遗传背景有关。     

一个推测的马氏珠母贝肿瘤抑制子QM的cDNA分析

从马氏殊母贝（P．martensif）足的SMARTcDNA文库中得到了一个与肿瘤抑制子QM基因同源的克隆,测序获得了757bp的全长cDNA序列，推测的开放阅读框位于23-673bp，编码217个氨基酸，由20种氨基酸组成，最少的为色氨酸，仅占1．4％，最多的为精氨酸，有24个，占11．1％；同GenBank数据库中查找到的马氏珠母贝（P．fucata）QM基因仅在末尾有4个氨基酸存在差异．P.fucata缺少P．mariensii在开放阅读框的第637处对应的碱基C，导致P．fucala的QM蛋白在3′端多了13个氨基酸和缺乏多聚腺苷酸加尾信号AATAAA．因而，已报道的P.fitcataQM基因很可能由于测序等原因导致在开放阅读框第637处缺失了碱基C．     

锌指结构基因HKR1在脑胶质瘤中的表达分析

从人胎脑cDNA文库中筛选到2612nt的锌指蛋白HKR1克隆,C端含有10个C2H2锌指结构,N端含有2个KRAB区域,基因芯片的原位杂交检测显示,HKR1基因表达在脑胶质瘤中明显升高,提示该基因可能是一条潜在的肿瘤相关基因。     

人FOXA1蛋白的原核表达、纯化及蛋白互作分析

构建FOXA1的原核表达载体,诱导表达并纯化后获得人FOXA1的C端和N端蛋白片段,为寻找FOXA1的互作蛋白提供材料基础.提取人乳腺癌细胞MCF7的总RNA,逆转录成cDNA,设计引物PCR克隆FOXA1的cDNA,再以此为模板扩增FOXA1的C端、N端cDNA片段,分别并克隆进带有GST标签的原核表达载体pGEX-4T-1,构建重组表达质粒.双酶切及测序鉴定正确后,转化至大肠杆菌BL21Rosetta DE3,经IPTG诱导,Glutathione Sepharose 4B亲和纯化,通过SDS-PAGE电泳及Western blot确定FOXA1蛋白的表达.与MCF7细胞裂解液孵育,进行GST-Pull down试验,检测纯化蛋白与FOXA1已知互作蛋白TLE3的相互作用.成功构建pGEX-4T-1-FOXA1-C和pGEX-4T-1-FOXA1-N的原核表达载体;在大肠杆菌中诱导目的蛋白表达;经Glutathione Sepharose 4B纯化后,获得了人FOXA1的C端蛋白片段GST-FOXA1-C与N端蛋白片段GSTFOXA1-N;证实GST-FOXA1-C能与FOXA1已知互作蛋白TLE3的相互作用.     

Characteristics of the binding features of Nelin with F-actin and screening Nelin interactive proteins

The interaction of nelin, a cardiac-specific expressed protein of human novel gene nelin, with F-actin was studied by both F-actin cosedimentation in vitro and colocalization assays. The results showed that nelin is a new F-actin binding protein and is colocolized with F-actin in cytoplasm of cells. Three new nelin binding proteins, filamin C subtype, titin N2B subtype and inter-alpha trypsin inhibitor heavy chain precursor (ITIH), were identified from human heart cDNA library using yeast two-hybrid screening. The binding activity of filamin C with nelin was confirmed by coimmunoprecipitation. Filamin C binds to nelin through its C-terminal region. It is indicated that nelin is a cytoskeleton associated protein and acts as a membrane-cytoskeleton associated protein involved in the formation of focal adhesions.     

人穿孔素羧基端肽段的表达纯化与活性鉴定

穿孔素,即成孔蛋白（pore forming protein,PFP),其溶细胞作用与免疫调节和自身免疫病以及其它多种疾病过程中的免疫性病理损伤相关。为得到足够量的PFP建立与之相关的免疫学研究手段用于基础和临床研究,在已克隆人PFP cDNA的基础上,用基因工程方法表达了人PFP C端124个氨基酸肽段（hPFP-C),并通过谷胱甘肽琼脂糖新和层析获得纯化的GST/hPFP-C融合蛋白,经凝血酶酶切和再次北极和层析去除GST部分,得到了纯化的hPFP-C蛋白。纯化的hPFP-C蛋白与兔红细胞共育,呈现钙依赖的溶血活性。     

Activated CD8 binding to class I protein mediated by the T-cell receptor results in signalling

The CD8 glycoprotein of T cells bind nonpolymorphic regions of class I major histocompatibility complex proteins on target cells and these interactions promote antigen recognition and signalling by the T-cell receptor. Studies using artificial membranes indicated that effective CD8/class I interaction is critical for response by alloantigen-specific cytotoxic T lymphocytes when class I protein is the only ligand on the antigen-bearing surface. But significant CD8-mediated binding of cytotoxic T lymphocytes to non-antigenic class I protein could not be detected in the absence of the alloantigen. These apparently contradictory findings indicate that CD8 binding to class I protein might be activated through the T-cell receptor and the results reported here demonstrate that this is the case. Treatment of cytotoxic T lymphocytes with soluble anti-T-cell receptor antibody activates adhesion of the cytotoxic T lymphocytes to class I, but not class II proteins. The specificity of this binding implies that it is mediated by CD8 and blocking by anti-CD8 antibodies confirmed this. Furthermore, binding of CD8 to class I protein resulted in generation of an additional signal(s) necessary to initiate response at low T-cell receptor occupancy levels.     

Chaetoglobosins E诱导乳腺癌MCF-7细胞凋亡作用机制研究

为探究Chaetoglobosins E(ChE)对肿瘤细胞增殖和凋亡的影响,体外培养人乳腺癌MCF-7细胞、人膀胱癌T-24细胞、人黑色素瘤C8161细胞、人白血病U937细胞,用不同浓度的ChE分别作用于4种细胞24 h或48 h,MTT法检测4种肿瘤细胞的增殖情况;为进一步研究其作用机制,Hoechst 3334...     

Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis

Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohn's disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohn's disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8(ΔIEC)) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8(ΔIEC) mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8(ΔIEC) mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-α, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohn's disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IECs from TNF-α-induced necroptotic cell death.     

Human immunodeficiency virus induces phosphorylation of its cell surface receptor

AIDS is an immunoregulatory disorder characterized by depletion of the CD4+, helper/inducer lymphocyte population. The causative agent of this disease is the human immunodeficiency virus, HIV, which infects CD4+ cells and leads to cytopathic effects characterized by syncytia formation and cell death. Recent studies have demonstrated that binding of HIV to its cellular receptor CD4 is necessary for viral entry. We find that binding of HIV to CD4 induces rapid and sustained phosphorylation of CD4 which could involve protein kinase C. HIV-induced CD4 phosphorylation can be blocked by antibody against CD4 and monoclonal antibody against the HIV envelope glycoprotein gp120, indicating that a specific interaction between CD4 and gp120 is required for phosphorylation. Electron microscopy shows that a protein kinase C inhibitor does not impair binding of HIV to CD4+ cells, but causes an apparent accumulation of virus particles at the cell surface, at the same time inhibiting viral infectivity. These results indicate a possible role for HIV-induced CD4 phosphorylation in viral entry and identify a potential target for antiviral therapy.     

Functional interaction between human T-cell protein CD4 and the major histocompatibility complex HLA-DR antigen

Mature T cells segregate phenotypically into one of two classes: those that express the surface glycoprotein CD4, and those that express the glycoprotein CD8. The CD4 molecule is expressed primarily on helper T cells whereas CD8 is found on cytotoxic and suppressor cells. A more stringent association exists, however, between these T-cell subsets and the major histocompatibility complex (MHC) gene products recognized by their T-cell receptors (TCRs). CD8+ lymphocytes interact with targets expressing class I MHC gene products, whereas CD4+ cells interact with class II MHC-bearing targets. To explain this association, it has been proposed that these 'accessory' molecules bind to monomorphic regions of the MHC proteins on the target cell, CD4 to class II and CD8 to class I products. This binding could hold the T cell and its target together, thus improving the probability of the formation of the trimolecular antigen: MHC: TCR complex. Because the TCR on CD4+ cells binds antigen in association with class II MHC, it has been difficult to design experiments to detect the association of CD4 with a class II molecule. To address this issue, we devised a xenogeneic system in which human CD4 complementary DNA was transfected into the murine CD4-, CD8- T-cell hybridoma 3DT-52.5.8, the TCR of which recognizes the murine class I molecule H-2Dd. The murine H-2Dd-bearing target cell line, P815, was cotransfected with human class II HLA-DR alpha, beta and invariant chain cDNAs. Co-culture of the parental T-cell and P815 lines, or of one parental and one transfected line resulted in a low baseline response. In contrast, a substantial increase in response was observed when CD4+ 3DT-52.5.8 cells were co-cultured with HLA-DR+ P815 cells. This result strongly indicates that CD4:HLA-DR binding occurs in this system and that this interaction augments T-cell activation.     

复性的乙型肝炎病毒X蛋白突变体HBxΔNΔC具有体外生物学活性

乙型肝炎病毒X蛋白(HBx)在肝炎引起的肝癌病变中作为一个多功能调节因子起着关键的作用.对HBx的功能有非常多的体外研究报导,而由于HBx蛋白难以溶解和稳定使得体外的研究报导非常少.我们根据肝癌患者中最常见的截短突变体,在大肠杆菌包涵体中重组表达了一个保留了第12～127位氨基酸的、N端和C端截断的突变体HBxΔNΔC,并使用变复性的方法将其复性溶解.分子筛显示HBxΔNΔC为单体;圆二色谱显示HBxΔNΔC包含有5％的α-螺旋,30％的β-折叠和65％的其他结构.复性的HBxΔNΔC能从人HepG2细胞中拉下(pull-down)肿瘤抑制因子p53和DDB1蛋白,它还能刺激肝细胞的迁移.腹腔注射1μg/g的HBxΔNΔC能明显刺激小鼠肝的生长.这些结果显示HBxΔNΔC具有一定的生物学活性,它可以用来研究体外的HBx结构和分子作用机制.