分泌型Survivin真核表达载体的构建及其在HeLa细胞中的表达

目的:构建含6个组氨酸标签(His6)的小鼠存活素(Survivin)融合蛋白的真核表达载体,及其在真核细胞中的表达。方法:根据小鼠Survivin序列设计引物,经过RT-PCR克隆Survivin的cDNA并进一步构建分泌型Sur-vivin的真核表达载体,经测序鉴定后在大肠杆菌中扩增在HeLa细胞中表达;以金属离子螯合层析富集,再用免疫印迹法鉴定。结果:克隆到Survivin编码区全长序列,经DNA测序后证明与已报道序列相同。构建氨基端融合小鼠IL-2信号肽,羧基端带有His6标签的Survivin融合蛋白的真核表达载体,在HeLa细胞中转染成功并且表达;细胞上清经HisTrap HP柱层析富集后,进行免疫印迹分析,表明该融合蛋白以分泌型方式在HeLa细胞中表达。结论:成功克隆Survivin的cDNA,并构建其分泌型真核表达载体。     

Structure of Sindbis virus core protein reveals a chymotrypsin-like serine proteinase and the organization of the virion

Sindbis virus consists of a nucleocapsid core surrounded by a lipid membrane through which penetrate 80 glycoprotein trimers. The structure of the core protein comprising the coat surrounding the genomic RNA has been determined. The polypeptide fold from residue 114 to residue 264 is homologous to that of chymotrypsin-like serine proteinases with catalytic residues His 141, Asp 163 and Ser 215 of the core protein positioned as in other serine proteinases. The C-terminal tryptophan remains in the P1 substrate site subsequent to the autocatalytic cis cleavage of the capsid protein, thus rendering the proteinase inactive. Model building of the Sindbis core protein dimer shows that the nucleocapsid is likely to have T = 4 quasisymmetry.     

Cloning and sequence analysis of cDNAs encoding two distinct somatostatin precursors found in the endocrine pancreas of anglerfish

Complementary DNAs for two distinct anglerfish somatostatin peptides (termed I and II) have been cloned in bacterial plasmids and sequenced. The nucleotide sequence for somatostatin I encodes a large percursor peptide (molecular weight 13,300) in which the somatostatin hormones is at the carboxyl terminus. The predicted 14-amino acid sequence for anglerfish somatostatin I is the same as mammalian somatostatin. Somatostatin II is also synthesized as part of a larger precursor (molecular weight 14,100) with the presumptive somatostatin hormone also at the carboxyl terminus. The 14-amino acid sequence of somatostatin II differs from somatostatin I at two internal residues (Tyr in place of Phe 7 and Gly in place of Thr 10). The two different somatostatins may have distinct biological activities. Homologies in the amino acid sequences of the two peptides outside the somatostatin moiety suggest other regions of the molecules have biological functions.     

A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission.

The mammalian central nervous system (CNS) contains an abundance of the transition metal zinc, which is highly localized in the neuronal parenchyma. Zinc is actively taken up and stored in synaptic vesicles in nerve terminals, and stimulation of nerve fibre tracts that contain large amounts of zinc, such as the hippocampal mossy fibre system, can induce its release, suggesting that it may act as a neuromodulator. The known interaction of zinc with the major excitatory and inhibitory amino-acid neurotransmitter receptors in the CNS supports this notion. That zinc has a role in CNS synaptic transmission, however, has so far not been shown. Here we report a physiological role for zinc in the young rat hippocampus (postnatal, P3-P14 days). Our results indicate that naturally occurring spontaneous giant depolarizing synaptic potentials (GDPs) in young CA3 pyramidal neurones, mediated by the release of GABA (gamma-aminobutyric acid), are induced by endogenously released zinc. These synaptic potentials are inhibited by specific zinc-chelating agents. GDPs are apparently generated by an inhibitory action of zinc on both pre- and postsynaptic GABAB receptors in the hippocampus. Our study implies that zinc modulates synaptic transmission in the immature hippocampus, a finding that may have implications for understanding benign postnatal seizures in young children suffering with acute zinc deficiency.     

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.     

磷酸乙醇胺转移酶MCR1结构与功能的研究

本实验克隆了来源于E.coli的MCR1基因酶催化结构域,以E.coli表达系统表达蛋白.采用亲和层析、阴离子交换层析、分子筛层析等纯化方法获得纯度高、均一性好的蛋白;采用座滴和悬滴法,获得酶催化区域的蛋白质晶体;收集X射线数据后,分子置换法解析出酶催化区域的结构,其分辨率达到1.63埃.反常散射信号检测到4个锌离子信号,结构分析锌离子与周围Thr285、His465、His466、His395位氨基酸联系紧密,且Thr285被磷酸化.Thr285、His465、His466和His395点突变为丙氨酸后,宿主菌粘菌素抗性显著降低,表明该区域与酶活密切相关.本实验解析出MCR1酶活性区域的结构,鉴定出酶活性中心,为寻找抗MCR1靶向药物提供可用信息.     

高产杆菌肽菌株的筛选及培养条件研究

采用细胞融合技术得到的菌株与不同来源地衣芽孢杆菌菌株,进行深层液体发酵生产杆菌肽,研究了不同菌株,不同碳源,不同量锌和磷量诸因素对杆菌肽高产的影响.结果表明,1号菌比B-1号、B-2号、3号、4号株菌的效价高,为5.80×102Iu.mL-1.以柠檬酸为碳源时,磷的用量为0.10~0.20 mmol.L-1的最佳培养基中培养时,最有利于杆菌肽的合成,锌的用量10%,其发酵液中杆菌肽效价最高,可达8.86×102~9.85×102Iu.mL-1.     

Raman spectroscopy study of zinc finger ZNF191（243-368）

The structure of ZNF191(243-368), the zinc finger region protein of zinc finger protein ZNF191, and its structural change upon thermal and EDTA-induced denaturation were investigated by the Raman spectroscopy. It was demonstrated that the coordination between Zn^2 and His/Cys in ZNF191(243-368) is the essential factor to the stability of zinc finger, which plays an important role in maintaining the hydrophobic core and the secondary structure in zinc finger, and the Raman spectroscopy is a powerful tool for investigating the structure of ZNF191(243-368).     

Crystallographic analysis of mutant human haemoglobins made in Escherichia coli

The expression of beta-globin in Escherichia coli has enabled us to study the functional role of individual amino-acid residues in haemoglobin (Hb) by site-directed mutagenesis. In contrast to mammalian Hbs, some teleost fish haemoglobins show a drastic lowering of oxygen affinity and cooperativity at low pH, a phenomenon known as the Root effect. We have produced the two mutant haemoglobins Hb Nymphéas [Cys(F9)93 beta----Ser] and Hb Daphne [His(H21)143 beta----Arg, Cys(F9)93 beta----Ser] to investigate this allosteric property. Although these substitutions were thought to be responsible for the Root effect, Hb Nymphéas and Hb Daphne show an increased oxygen affinity and a reduced effect of pH on oxygen affinity. Our X-ray crystallographic studies show that the hydroxyl group of Ser 93 beta forms a hydrogen bond with Asp 94 beta which is in equilibrium with the salt bridge between Asp 94 beta and His 146 beta. The oxygen-binding properties of Hbs Nymphéas and Daphne are accounted for by the partial disruption of the salt bridge.     

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。     

X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes

Knowledge of the tertiary structure of the proteinase from human immunodeficiency virus HIV-1 is important to the design of inhibitors that might possess antiviral activity and thus be useful in the treatment of AIDS. The conserved Asp-Thr/Ser-Gly sequence in retroviral proteinases suggests that they exist as dimers similar to the ancestor proposed for the pepsins. Although this has been confirmed by X-ray analyses of Rous sarcoma virus and HIV-1 proteinases, these structures have overall folds that are similar to each other only where they are also similar to the pepsins. We now report a further X-ray analysis of a recombinant HIV-1 proteinase at 2.7 A resolution. The polypeptide chain adopts a fold in which the N- and C-terminal strands are organized together in a four-stranded beta-sheet. A helix precedes the single C-terminal strand, as in the Rous sarcoma virus proteinase and also in a synthetic HIV-1 proteinase, in which the cysteines have been replaced by alpha-aminobuytric acid. The structure reported here provides an explanation for the amino acid invariance amongst retroviral proteinases, but differs from that reported earlier in some residues that are candidates for substrate interactions at P3, and in the mode of intramolecular cleavage during processing of the polyprotein.     

棉花类耐盐锌指蛋白基因的克隆与结构分析

从棉花花瓣cDNA文库中随机挑选部分克隆，经测序发现一个与拟南芥耐盐锌指蛋白基因同源的cDNA(CSTZ),CSTZ序列全长1012bp，开放阅读框共编码272个氨基酸，含典型的植物双锌指（Cys2/His2)结构区，Northern杂交证实，CSTZ的表达随棉花幼苗钠盐处理浓度的升高而增强，在棉花花龄期，CSTZ基因在叶片，根，花瓣和花药组织中大量表达，在柱头组织中表达相对较弱。     

乙酰氨基己酸锌的急性毒性试验和Ames实验

目的与方法:本文研究乙酰氨基己酸锌对小鼠的急性毒性作用以及用鼠伤寒沙门氏菌突变株的回复突变性检测乙酰氨基己酸锌是否具有致突变性.结果与讨论:结果表明,乙酰氨基己酸锌的小鼠腹腔注射的半数致死量LD50=364.69mg/kg;在加与不加S9的条件下,对TA97,TA98,TA100和TA102的致突变作用为阴性.     

钠离子依赖的中性氨基酸转运蛋白SNAT2-HA融合蛋白的构建及鉴定

钠离子依赖的中性氨基酸转运蛋白SNAT2在哺乳动物组织中广泛表达,具有转运中性氨基酸的功能,在谷氨酸-谷氨酰胺循环、肝脏糖质新生等生物通路中发挥重要作用．为了方便测定SNAT2在细胞膜上的表达和定位,本研究采用PCR扩增和酶切连接将HA标签蛋白与SNAT2的C末端连接,构建了真核生物表达载体pBK-CMVA（1098—1300）-SNAT2-HA表达载体．用脂质体转染法将该表达载体瞬时转染到HEK293T细胞中,通过Westernblot法检测到SNAT2-HA融合蛋白在细胞膜上的正确表达和定位．pBK-CMV△（1098-1300）-SNAT2-HA表达载体的成功构建,为今后对SNAT2的结构和功能的研究提供了有效方法．     

八肋游仆虫eRF1a的C端是肽链释放因子eRF3的结合区(英文)

真核生物蛋白质合成终止需要两类肽链释放因子,eRF1和eRF3.研究表明八肋游仆虫的两类肽链释放因子在体内和体外都能形成复合物,且第一类肽链释放因子eRF1a与第二类肽链释放因子eRF3的C端结合.为了确定eRF3在eRF1a上的结合区,本研究以八肋游仆虫第一类肽链释放因子eRF1a基因为模板,用PCR的方法获得了N端分别截短140和206个氨基酸的eRF1a片段,同时在这两个片段的3'端融合了编码6个组氨酸残基的核苷酸序列,将这两个序列分别插入原核表达载体pTWIN1,并构建重组表达质粒pTWIN1-eRF1aC1 his6和pTWIN1-eRF1 aC2his6,转入大肠杆菌BL21( DE3)中获得了可溶性表达,通过一步His60 Ni Superflow柱亲和层析,重组蛋白CBD-intein-eRF1 aC1 his6和CBD-intein-eRF1aC2 his6获得纯化.体外pull down分析显示eRF1aC1和eRF1aC2均能与八肋游仆虫第二类释放因子eRF3相互作用,这表明八肋游仆虫eRF1a的C端是肽链释放因子eRF3的结合区.     

Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.     

Cloning and characterization of a gene that regulates cell adhesion.

Molecules of the cadherin and integrin families involved in cell-cell and cell-matrix adhesion have been implicated in epithelial differentiation, carcinogenesis and metastasis. Having observed that a colon cancer cell line bound avidly to collagen type I, inducing integrin-triggered glandular differentiation, we investigated the regulation of integrin function in these cells. We modified a mammalian expression cloning system that used monoclonal antibody selection to clone cell surface molecules. Using attachment to collagen type I to select for adhesive phenotype, we isolated a complementary DNA clone that increases cell adhesion to components of the extracellular matrix. The corresponding gene (cell adhesion regulator, CAR) is located on the long arm of chromosome 16 (16q) and encodes a protein of 142 amino acids, which has an N-terminal myristoylation motif and a consensus tyrosine-kinase phosphorylation site at the C terminus. Removal of this tyrosine residue abolishes enhancement of cell-matrix adhesion. This gene may encode an adhesion signal transduction molecule that functions in the suppression of tumour invasion.