RACK1蛋白在毕赤酵母GS115中的表达研究

本文克隆得到了拟南芥和水稻的活化态C激酶1受体基因,AtRACK1/OsRACK1,用电转化的方法成功将AtRACK1/OsRACK1基因整合到毕赤酵母GS115的染色体上,经菌体培养和甲醇诱导后获得了有效分泌表达,表达产物存在于培养液上清中,表达蛋白经SDS-PAGE鉴定显示其分子量为36kD左右,在诱导72小时后AtRACK1/OsRACK1蛋白表达量最大,该结果为进一步纯化RACK1蛋白,获得植物RACK1蛋白抗体奠定了基础.     

PPP1R3C基因在3T3-L1前脂肪细胞分化过程中的功能研究

为研究小鼠的蛋白磷酸酶1调节亚基3C(protein phosphatase 1 regulatory subunit 3C,PPP1R3C)基因在脂肪细胞分化中的功能,本研究以小鼠3T3-L1前脂肪细胞为材料,通过基因过表达、干扰及实时定量PCR等实验,分析了PPP1R3C基因对成脂分化的影响,并初步探讨了它对成脂分...     

与阿尔茨海默病发病机制相关的microRNA的研究现状及进展

阿尔茨海默病(Alzheimer’s Disease,AD)是一种复杂的慢性神经系统退行性疾病,病因和发病机制迄今尚未明确,可能与多种因素有关,如基因突变、氧化应激、线粒体功能障碍、胶质细胞活化、细胞凋亡等.MicroRNA(miRNA)是神经细胞分化、生长以及神经系统可塑性调节的关键因子,miRNA的异常表达与AD的发病机制密切相关.综述了近年来miRNA在调节β淀粉样前体蛋白(APP)表达、调节淀粉样前体蛋白β位分解酶1(BACE1)表达、调节微管相关蛋白Tau表达以及调节其他非特异性致病因子等与AD发病机制相关因素等方面的研究现状和进展,将为深入系统研究阿尔茨海默病的发病机制提供参考.     

MiRdetector：一个预测与搜寻miRNA基因的计算工具

MicroRNA（miRNA）是一类内源性的长约21～24 nucleotide(nt)的非编码小分子RNA,能够调节其它基因的表达活性,在生物体的发育过程中发挥重要的作用.每种生物体中miRNA的基因总数还未知,生物信息学分析手段为发现新的miRNA基因提供了有效的方法.作者开发了一个预测与搜寻miRNA基因的完全自动化系统“MiRdetector”.MiRdetector系统是基于计算机比对和miRNA前体茎环结构判断的.用水稻miRNA基因对系统的预测精度进行了检验,系统正确识别了155个样本中的140个,预测精度达到90.32％,并且搜寻到了95个可能的水稻miRNA基因.由于系统的假阳性率较低,因此能够为进一步证实miRNA基因的实验研究提供高质量的数据集.     

阿尔茨海默症miRNA靶基因预测研究

为深入了解阿尔茨海默症的发病机制,利用匹配的基因表达数据和miRNA表达数据,对miRNA的靶基因进行预测分析.首先对选择出的差异表达miRNA进行靶基因预测;然后利用HCTarget算法验证预测的靶基因;最后对miRNA及其靶基因构建调控网络.调控网络中包含11个已确定的与AD有关的miRNA及6个AD致病基因.通过分析网络发现miRNA及其靶基因在正常与患病两种情况下的活性变化趋势,生物学分析也证实了它们在AD中的重要作用,为AD发病机制研究提供了依据.     

蛋白磷酸酶2C(PP2C)的表达、纯化与催化活性

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E.coli 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.     

人RACK1基因的克隆及原核表达

克隆人RACK1基因,构建原核表达载体pET-30a(+)-RACK1,诱导表达重组蛋白,纯化后Western blot鉴定目的蛋白.根据GeneBank提供的RACK1基因序列及pET-30a(+)载体上的多克隆位点设计引物,以人胎肝cDNA文库为模板,钓取RACK1cDNA全长序列.将目的片段与原核表达载体pET-30a(+)连接,转入E.coli DH5α感受态细胞,筛选阳性克隆.将测序正确的重组质粒转入E.coli BL21(DE3)细胞,IPTG诱导表达,利用镍柱对表达的蛋白进行纯化,Western blot法检测纯化的蛋白.结果显示,克隆得到的目的基因序列与GeneBank中已报道的序列完全一致,成功构建了pET-30a(+)-RACK1原核表达载体.重组蛋白主要以包涵体形式存在,经镍离子亲和层析柱纯化后,Western blot检测证实纯化的蛋白能与特异性的抗体发生反应.本研究获得了人RACK1蛋白,为深入研究RACK1的功能奠定基础.     

采用反向PCR技术优化适合毕赤酵母表达的截短型HPV58型L1基因

目的:探讨利用反向聚合酶链反应(反向PCR)技术根据毕赤酵母偏爱密码子优化截短型人乳头瘤病毒58型(HPV58)L1基因的研究.方法:设计PCR引物扩增截短型HPV58L1目的基因,将其克隆入毕赤酵母分泌表达载体pPICZαC;测序并对目的基因进行序列分析;根据毕赤酵母偏爱密码子利用反向PCR技术设计引物对目的基因进行扩增.结果:扩增了截短型HPV58L1基因并将其克隆入毕赤酵母分泌表达载体pPICZαC中;根据毕赤酵母偏爱密码子优化了截短型HPV58L1基因.结论:成功构建经密码子优化截短型HPV58L1基因的毕赤酵母分泌表达载体pPICZαC-HPV58L1.     

RACK1基因在急性冠脉综合征行PCI术患者外周血中的表达及意义

选取在我院行PCI术的急性冠脉综合征患者66例,用实时荧光定量PCR法分别测定PCI术前及术后3个月时患者外周血单个核细胞中RACK1基因的表达。40名健康者作为对照组。测定结果是:(1)急性冠脉综合征组患者外周血单个核细胞中RACK1 mRNA表达水平明显高于对照组(17.56±4.98 vs 3.11±1.24,P0.05);(2)急性冠脉综合征患者行PCI术3月后,外周血单个核细胞中RACK1 mRNA表达水平较术前明显降低(10.84±3.71 vs 17.56±4.98,P0.05),但仍高于健康者(10.84±3.71 vs 3.11±1.24,P0.05)。RACK1在急性冠脉综合征患者外周血中表达上调,其表达水平可能和病情进展相关。     

基于转录组测序初步筛选维生素C延缓秀丽隐杆线虫衰老的相关基因

为探究维生素C调控秀丽隐杆线虫寿命通路相关基因，设置空白对照组(0 mg/mL维生素C组)、0.4 mg/mL维生素C组、0.8 mg/mL维生素C组以及1.6 mg/mL维生素C组，构建维生素C处理秀丽隐杆线虫模型，检测维生素C与秀丽隐杆线虫寿命关系。空白对照组和处理组利用Illumina HiSeq基因转录组测序。通过本位数据库(Gene Ontology, GO)和通路显著性富集分析数据库(Kyoto Encyclopedia of Genes and Genomes, KEGG)挖掘影响秀丽隐杆线虫寿命的差异表达基因。研究发现，0.8 mg/mL维生素C处理下，秀丽隐杆线虫寿命相较于其他浓度明显延长，lips-10基因表达明显上调且参与秀丽隐杆线虫寿命相关通路调节过程，表明维生素C能够影响秀丽隐杆线虫寿命，调控其相关衰老过程。     

Ⅰ型单纯疱疹病毒包膜糖蛋白L基因片段的克隆及高效表达

 采用PCR方法扩增HSV-1病毒型特异性包膜糖蛋白L(gL)基因片段并克隆至原核表达载体pGEX-5X-1获得重组质粒pGEX-5X-1-gL,将重组质粒转化E.coli BL21表达菌后经IPTG诱导表达目的蛋白.SDS-PAGE蛋白检测表明,在分子质量56 ku处有HSV-1 GST-gL融合蛋白的高效表达,通过IPTG浓度筛选和诱导前表达菌扩增培养时间的比较分析对诱导条件进行了优化,GST-gL融合蛋白表达量可达到菌体蛋白总量的48.65%.Western blot中利用HSV-1灭活病毒获得的多克隆抗体确证所表达蛋白为HSV-1病毒组分.这一表达系统的建立和优化对进一步探讨HSV-1 gL蛋白功能及其免疫原性提供了有利条件.     

Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly

The assembly of 80S ribosomes requires joining of the 40S and 60S subunits, which is triggered by the formation of an initiation complex on the 40S subunit. This event is rate-limiting for translation, and depends on external stimuli and the status of the cell. Here we show that 60S subunits are activated by release of eIF6 (also termed p27BBP). In the cytoplasm, eIF6 is bound to free 60S but not to 80S. Furthermore, eIF6 interacts in the cytoplasm with RACK1, a receptor for activated protein kinase C (PKC). RACK1 is a major component of translating ribosomes, which harbour significant amounts of PKC. Loading 60S subunits with eIF6 caused a dose-dependent translational block and impairment of 80S formation, which were reversed by expression of RACK1 and stimulation of PKC in vivo and in vitro. PKC stimulation led to eIF6 phosphorylation, and mutation of a serine residue in the carboxy terminus of eIF6 impaired RACK1/PKC-mediated translational rescue. We propose that eIF6 release regulates subunit joining, and that RACK1 provides a physical and functional link between PKC signalling and ribosome activation.     

全基因组关联分析显示基因ANXA8和C10orf11为影响肌少症的候选基因

为了寻找与瘦体重(lean body mass,LBM)相关的单核苷酸多态性(single nucleotide polymorphism, SNP)位点及易感基因,在1 000个不相关的白人中采用Affymetix 500K芯片扫描了500 000个SNPs,并进行全基因组关联分析(genome-wide association study,GWAS),显著结果在1 625个中国人样本和2 283个欧洲白人样本中进行验证,并将验证结果与研究结果进行荟萃分析。研究发现SNPsrs7905603,rs9416083,rs4409772,rs2894310与LBM关联,其中rs7905603位于基因ANXA8,其他3个SNPs位于基因C10orf11。荟萃分析得到的合并p值分别为2.08×10-5,7.44×10~(-6),6.73×10~(-6),6.76×10~(-6)。ANXA8和C10orf11基因是影响LBM变异的候选基因,这对肌少症的认识提供了新的理论依据。     

Non-viral gene delivery carrier of probe type host molecule ——Interactions between DNA and β-cyclodextrin derivative complexes (I)

GENE THERAPY IS A COMMON PROCESS TO DELIVER EXTRIN- SIC GENES INTO TARGET CELLS FOR FURTHER GENE EXPRESSION, FOR THE PURPOSE OF TREATING DISEASES. A WELL DESIGNED GENE DELIVERY CARRIER CAN EFFICIENTLY PACKAGE AND PRO- TECT NUCLEIC ACID FROM BEING DIGESTED BY A VARIETY OF ENZYMES IN VIVO AND SHOULD BE ABLE TO SPECIFICALLY LO- CATE EXTRINSIC GENES INTO THE TARGET ORGANISMS. S…     

大豆降解组文库microRNAs的启动子分析

研究目的：创新要点：通过分析miRNA的核心启动子和顺式作用元件为进一步解析大豆（Glycinemax）miRNAs表达调控及其功能研究提供重要信息。利用生物信息学方法全面解析了大豆降解组文库miRNA的启动子特征,并依据顺式作用元件及靶基因构建了miRNA的表达与生长素响应因子、赤霉素响应因子之问存在潜在的负反馈调控网络。研究方法：本研究利用TSSP程序和PlantCARE数据库预测了来自大豆降解组文库的440个miRNA的核心启动子以及369个miRNAs的顺式作用元件,并依据顺式作用元件及靶基因构建miRNA调控网络。重要结论：83．86％的miRNA在其上游序列中含有启动子,8．64％的miRNA在其下游序列中含有启动子,21．59％的miRNA包含增强子。核心启动子的TATA盒与转录起始位点（TSSs）的分布相似（见图2）。此外,对转录起始位点5’端的顺式作用元件预测为miRNAs的可能功能和表达的时空性提供了线索。miRNAs的顺式作用元件和靶基因的分析显示,部分miRNA的表达与生长素响应因子、赤霉素响应因子之间存在潜在的负反馈调控（见图3）。     

Cloning the interferon regulatory factor I gene in lungfish （Protopterus annectens） and its molecular evolution among sarcopterygians

Sarcopterygians is an important vertebrate clade that includes crossopterygians and tetrapods. Crossopterygians are lobe-finned fish that include lungfish and coelacanths. Tetrapods include amphibians, reptiles, avians and mammals. To compare the interferon regulatory factor 1 (irf-1) gene structure and to explore phylogenetic relationships among sarcopterygians, we cloned the cDNA sequence of irf-1 from lungfish and compared it with irf-1 orthologs in other sarcopterygian species. The lungfish is a primitive sarcopterygian that occupies a very important position in vertebrate phylogeny. Interferon regulatory factors (IRFs) are a family of proteins involved in innate immunity. To date, 11 IRF family members have been reported. All IRFs share homology in the first 115 amino acids, which encompasses a DNA binding domain containing a characteristic repeat of 5 tryptophan residues separated by 10–18 amino acids. IRF-1 and IRF-2 were the first members of this family to be reported and they have a very important role in innate immunity. However, studies of the irf-1 and irf-2 genes are mostly confined to mammals; very few non-mammalian irf-1 genes have been reported. Consistent with the irf-1 gene sequences already published, the first 345 nucleotides of lungfish irf-1 are highly conserved. At the carboxyl terminal a C-terminal transactivating region motif and an interferon associated domain (IAD2) were identified. 417 million years separate the present from the closest common ancestor of lungfish and tetrapods; however, the irf-1 genes among sarcopterygians are highly conserved and have very obvious phylogenetic relationships. Also the interrelationship tree of sarcopterygians, based on IRF-1 amino acid sequences, is identical with trees produced using other data, such as morphological characteristics or mitochondrial gene sequences.     

Correlation of microRNAs responding to high dose γ-irradiation with predicted target mRNAs in HeLa cells using microarray analyses

An increasing data indicates that altered microRNAs （miRNAs） participate in the radiation-induced DNA damage response. However, a correlation of mRNA and miRNA profiles across the entire genome and in response to irradiation has not been thor- oughly assessed. We analyzed miRNA microarray data collected from HeLa cells after ionizing radiation （IR）, quantified the ex- pression profiles of mRNAs and performed comparative analysis of the data sets using target prediction algorithms, Gene Ontol- ogy （GO） analysis, pathway analysis, and gene network construction. The results showed that the altered miRNAs were involved in regulation of various cellular functions, miRNA-gene network analyses revealed that miR- 186, miR- 106b, miR- 15 a/b, CCND 1 and CDK6 played vital role in the cellular radiation response. Using qRT-PCR, we confirmed that twenty-two miRNAs showed differential expression in HeLa cells treated with IR and some of these miRNAs affected cell cycle progression. This study demonstrated that miRNAs influence gene expression in the entire genome during the cellular radiation response and suggested vital pathways for further research.