增强型绿色荧光蛋白的真核表达和纯化

根据编码增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的开放读码框(open reading frame,ORF)设计引物,PCR方法扩增出5'端带His标签的EGF PORF,利用杆状病毒表达系统构建表达EGFP基因的重组杆状病毒DNA分子,转染sf9细胞.取细胞...     

利用基于GFP的FRET探针检测β-分泌酶的活性

β淀粉样肽(amyloidβ-peptide,Aβ)在阿尔茨海默病(Alzheimeir’s disease,AD)患者脑内的异常产生和积累在AD的发病机理中扮演着重要的角色。β-分泌酶对淀粉样前体蛋白的裂解是Aβ产生的关键步骤,因此抑制β-分泌酶的活性成为AD药物治疗的一个重要策略。为了检测β-分泌酶的活性,应用基因工程技术将β-分泌酶作用底物序列(NFEV)的两端分别与绿色荧光蛋白(green fluorescent protein,GFP)的颜色突变体--青色荧光蛋白(cyan fluorescent protein,CFP)和黄色荧光蛋白(yellow fluorescent protein,YFP)相连,构建了一个基于GFP的荧光能量共振转移(fluorescence resonance energy transfer,FRET)探针。荧光光谱分析结果显示,该荧光融合蛋白中CFP和YFP之间存在着较强的FRET。而当与β-分泌酶进行孵育后,FRET逐步降低,证明该探针能被β-分泌酶酶切。SDS-PAGE分析显示探针与β-分泌酶孵育后,荧光融合蛋白由60 kD大小逐渐变成30kD大小的蛋白,表明探针被β-分泌酶酶切成CFP和YFP分子,证实了荧光光谱的结果。这些结果表明,可通过检测探针的FRET效率方便地分析β-分泌酶的活性,为进一步筛选β-分泌酶的抑制剂提供了一个平台。     

变异剪接体的结构、功能及其在疾病治疗中的应用

变异剪接(alternative splicing)是高等真核生物在发育和应激反应中调控基因表达的一种主要机制,能够调控蛋白表达,或产生编码具有不同功能蛋白的pre-mRNAs。剪接体(spliceosome)由五种snRNP(U1、U2、U4、U5和U6)和许多非snRNP蛋白质构成,参与整个剪接过程。变异剪接与诸多疾病有着密切联系,异常的变异剪接会导致疾病的发生,增加疾病的易感性与病变程度,甚至引起癌变。现就剪接体的生物学特征、剪接体与疾病的关系及其在疾病治疗方面的内容进行综述。     

绿色荧光蛋白及其应用

随着对绿色荧光蛋白(green fluorescent protein,GFP)研究的不断深入,人们对其结构、荧光产生机理等已有较为全面的认识。近年来利用GFP及其它荧光蛋白(FPs)发展了诸如荧光互补技术(FC)、荧光共振能量转移技术(FRET)和超分辨成像(super-resolution imaging)等一系列新技术,极大地促进了生物学、医药科学的研究。主要介绍了荧光蛋白的结构,荧光产生的机理,不同类型的荧光蛋白和基于荧光蛋白产生的新技术等方面的最新研究进展。     

根癌农杆菌介导的地衣型真菌Cladonia metacorallifera的转化

以潮霉素抗性和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)作为筛选标记,利用地衣型真菌Cladonia metacorallifera的菌丝,成功实现了根癌农杆菌介导的遗传转化,PCR检测证明转化子中存在潮霉素抗性基因,共聚焦显微镜检测到转化子菌丝能够产生绿色荧光,证明EGFP能够在trp C启动子控制下在地衣型真菌中表达。     

猪瘟病毒Core蛋白核仁定位序列鉴定

黄病毒科病毒核衣壳蛋白的核仁定位在病毒颗粒包装与病毒复制中发挥重要作用。为鉴定黄病毒科的猪瘟病毒Core蛋白核仁定位序列,本研究构建了将Core蛋白、截短突变体和氨基酸位点突变体分别与增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP )融合的真核表达质粒,转染至PK15细胞后进行表达和定位分析,结果显示 Core蛋白核仁定位序列为PESRKKL,其关键氨基酸为R76K77,对理解猪瘟病毒Core蛋白结构与功能和为后续研究Core蛋白在病毒复制及颗粒包装中的作用有重要意义。     

荧光能量转移技术在生物学研究中的应用

荧光能量转移(FRET)是指两个携带不同荧光基团的大分子在相互间距离足够近时(10～100A)所发生的能量非放射性地由一个荧光基团向另一个荧光基团转移的现象。结合绿色荧光蛋白的发现,FRET技术可用于检测生物大分子中不同亚基的位置和生物大分子间的相互作用。近年来,FRET技术在生物学研究中的突破性进展是在活体细胞中实时监测生物大分子之间的相互作用。本文就绿色荧光蛋白的发现,FRET技术的原理、研究进展和应用前景作简要综述。     

单个活细胞中生物分子动态行为的FRET实时检测

分别采用两种不同绿色荧光蛋白(green fluorescent prote in,GFP)突变体作为荧光共振能量转移(fluo-rescence resonance energy transfer,FRET)对的供体和受体,并利用分子生物学技术将供体和受体分子分别与特定的生物分子融合,这种技术已经成为在单个活细胞中实时长时间检测蛋白质间的动态相互作用的主要技术。主要介绍了基于GFPs的FRET技术在单个活细胞中实时长时间研究生物分子动态行为的应用。     

登革2型病毒非结构蛋白NS4B及其突变体的真核表达与鉴定

目的：构建登革2型病毒非结构蛋白NS4B及其突变体Δ2K-NS4B基因的真核载体,并观察二者在哺乳动物细胞内的定位情况。方法：从登革2型病毒43株的全长cDNA克隆载体上扩增获得编码NS4B及缺失2K片段的NS4B突变体Δ2K-NS4B的基因;通过基因重组的方法分别将2段基因克隆入真核表达载体pcDNA6/V5-HisA,获得重组真核表达载体pc/D2-NS4B和pc/D2-Δ2K-NS4B;经脂质体法转染BHK-21细胞后,用RT-PCR、间接免疫荧光和Western印迹鉴定表达的蛋白。结果：重组蛋白D2-NS4B和D2-Δ2K-NS4B可在BHK-21细胞中表达,二者均定位于细胞质中,并具有较好的抗原性,能够被抗登革2型病毒NS4B的多克隆抗体特异识别。结论：重组蛋白D2-NS4B和D2-Δ2K-NS4B在哺乳动物细胞胞质中的正确表达,为深入了解NS4B在登革病毒致病过程中的生物学功能奠定了基础。     

鼻咽癌相关基因NAG7编码产物的表达和定位分析

为了研究NAG7基因编码产物的特性和在活细胞内定位与表达,首先利用生物信息学分析NAG7编码蛋白的一般性质并预测其定位,再通过构建增强型绿色荧光蛋白（Enhance green fluorescent protein,EGFP)与NAG7融合基因的真核表达载体pEGFP-C2-NAG7,通过脂质体介导分别转染非洲绿猴肾细胞系COS7和人鼻咽癌细胞系HNE1,瞬时表达后荧光显微镜观察NAG7基因编码蛋白的活细胞内定位及表达,研究结果表明NAG7的编码产物可以COS7细胞中高表达并定位于细胞浆,而在HNE1细胞中虽也定位于胸浆,但仅有极少数细胞存在表达,因此NAG7编码产物在HNE1中的表达差异是否是NPC发生的原因之一有待深入研究。     

The Survivin Isoform Survivin-3B is Cytoprotective and can Function as a Chromosomal Passenger Complex Protein

Survivin is described as a bifunctional protein inhibiting apoptosis and regulating mitosis. However, the biological functions and contributions to cancer progression of survivin splicevariants are controversially discussed. We here show that the intracellular localization of 5 these splice variants depends on a Crm1-dependent nuclear export signal (NES) present in survivin, survivin-2B and survivin-3B, but absent in survivin-ΔEx3 and survivin-2α. Survivin isoforms lack an active nuclear import signal and are able to enter the nucleus by passive diffusion. Only survivin-3B but none of the other splice variants is cytoprotective and able to efficiently interact with chromosomal passenger complex (CPC) proteins. The NES together 10 with efficient CPC formation is required for the cytoprotective activity of survivin isoforms, aswell as for their correct localization and function during cell division. In the tumours from breast, colorectal, head and neck cancer, lymphoma and leukemia patients, survivin and survivin-2B were found overexpressed. However, survivin was the predominant form detected, and the other survivin isoforms were only expressed at low levels in tumours. Our data 15 provide a molecular rationale for the localization and activity of survivin variants, and conclude that survivin isoforms are unlikely to modulate survivin in trans in cancer patients.     

Survivin and its spliced isoform gene expression is associated with proliferation of renal cancer cells and clinical stage of renal cancer

Background: Survivin has been implicated in inhibition of apoptosis. To date, alternatively spliced isoforms, Survivin-2α, -2B, -ΔEx3, -3B, have been described. We assessed the effect of survivin gene expression on the proliferation of renal cancer (RCC) cells, and studied the association of survivin and its spliced isoform gene expression levels with the clinical stage of RCC. Methods: Gene expression of survivin and its spliced isoform in RCC cells, Caki-1, were performed by RT-PCR. We knocked down the gene expression of Survivin using small interfering RNA (siRNA), and assessed the cell proliferation by MTS assay. Next, we quantified the gene expression levels of survivin and its isoform in nephrectomy samples using quantitative real-time PCR. Results: In Caki-1 cells, survivin and survivin-2α, -2B were expressed higher than survivin-ΔEx3. Decrease of Survivin gene expression by transfection of siRNA was accompanied by inhibition of the proliferation of Caki-1 cell with 36% decrease in comparison with negative control transfected cells (p < 0.01). In clinical RCC tissues, survivin expression levels in metastatic stage were significantly higher compared with those in distant metastasis stage (M0:M1 = 1:4.81, p = 0.014); survivin 2B gene expression levels in pT3 tumors were associated significantly higher than those in pT1 (pT1:pT3 = 1:4.50, p = 0.043). No significant differences were found in survivin-2α expression levels and the ratio of survivin-2B/survivin gene expression levels among any clinical stages. Conclusion: We first demonstrated the gene expression of survivin-2α in renal cancer cells, and also showed that survivin and its spliced isoforms had associations with renal cancer cell proliferation and distant metastases.     

外源Ca2+对水杨酸诱导番茄抗灰霉病的增效机制

探讨了外源Ca²⁺对水杨酸(SA)诱导番茄抗灰霉病的增效机制.以番茄灰霉病敏感型品种‘L402’幼苗为材料,分别进行H₂O(对照)、SA、SA+Ca和SA+EGTA(Ca²⁺螯合剂)处理,期间(1～5 d)分析各处理植株叶片活性氧(ROS)含量,苯丙氨酸解氨酶、几丁质酶和β-1,3-葡聚糖酶活性,以及病程相关蛋白编码基因PR1、PR2和PR3表达水平的变化,并调查处理3 d后灰霉病情指数.结果表明： 与对照(病情指数为74.8)相比,SA、SA+Ca和SA+EGTA处理的植株叶片灰霉病的病情指数分别为46.9、38.5和70.3;SA处理明显提高叶片ROS含量以及苯丙氨酸解氨酶、几丁质酶和β-1,3-葡聚糖酶活性,这些参数在SA+Ca处理的植株中被进一步提高,但在SA+EGTA处理的植株中则被降低;SA处理明显提高了PR1、PR2a和PR3b的表达水平,Ca²⁺进一步加强了这一效果,而EGTA则起抑制作用.SA或SA+Ca处理期间的PR2b和PR3a表达较未处理的对照上调了1～2倍,而PR1、PR2a和PR3b上调了2～5倍.表明Ca²⁺对SA诱导番茄抗灰霉病具有增效作用,其机理至少与Ca²⁺和SA协同作用促进ROS形成有关,而ROS作为信号分子增加植株抗病相关酶活性以及PR1、PR2a和PR3b等防卫基因的表达.     

外源Ca2+对水杨酸诱导番茄抗灰霉病的增效机制

探讨了外源Ca²⁺对水杨酸(SA)诱导番茄抗灰霉病的增效机制.以番茄灰霉病敏感型品种‘L402’幼苗为材料,分别进行H₂O(对照)、SA、SA+Ca和SA+EGTA(Ca²⁺螯合剂)处理,期间(1～5 d)分析各处理植株叶片活性氧(ROS)含量,苯丙氨酸解氨酶、几丁质酶和β-1,3-葡聚糖酶活性,以及病程相关蛋白编码基因PR1、PR2和PR3表达水平的变化,并调查处理3 d后灰霉病情指数.结果表明： 与对照(病情指数为74.8)相比,SA、SA+Ca和SA+EGTA处理的植株叶片灰霉病的病情指数分别为46.9、38.5和70.3;SA处理明显提高叶片ROS含量以及苯丙氨酸解氨酶、几丁质酶和β-1,3-葡聚糖酶活性,这些参数在SA+Ca处理的植株中被进一步提高,但在SA+EGTA处理的植株中则被降低;SA处理明显提高了PR1、PR2a和PR3b的表达水平,Ca²⁺进一步加强了这一效果,而EGTA则起抑制作用.SA或SA+Ca处理期间的PR2b和PR3a表达较未处理的对照上调了1～2倍,而PR1、PR2a和PR3b上调了2～5倍.表明Ca²⁺对SA诱导番茄抗灰霉病具有增效作用,其机理至少与Ca²⁺和SA协同作用促进ROS形成有关,而ROS作为信号分子增加植株抗病相关酶活性以及PR1、PR2a和PR3b等防卫基因的表达.     

Molecular cloning and bioinformatics analysis of a novel spliced variant of survivin from human breast cancer cells.

Survivin gene and its two alternatively spliced variants, survivin-2 B and survivin- Delta Ex3 gene were cloned from human breast cancer cell lines B-cap37 firstly. A new gene designated as survivin-image (SI) was cloned from above cell lines, which has not been reported yet to clone from any cell lines. It was found that the novel gene 507 bp comprises partial survivin gene (345 bp), partial image gene (155 bp) of eye cancer and other insertion of 7 bp by analyzing with a series of recent bioinformatics software at the level of nucleotide and protein deduced. Predicted 3-D structures of the new molecule showed greatly similar to that of survivin in N-terminal containing BIR by homology modeling. These results suggested SI gene (GenBank accession No.AY830084) might be a novel alternatively spliced isoforms of the survivin gene involved in other functional significances related to tumorigenesis.     

川西亚高山3个优势树种不同径级根系分解特征

采用埋袋法对川西亚高山3个优势树种(岷江冷杉、粗枝云杉和红桦)细根(≤2 mm)、中根(2～5 mm)和粗根(≥5 mm)质量损失率及碳(C)、氮(N)、磷(P)在经历生长季和非生长季之后释放特征进行研究.结果表明： 红桦质量残留率低于岷江冷杉和粗枝云杉.同一树种,质量残留率总体上随根系径级增加而增加.非生长季的质量损失率占全年质量损失率的52.1%～64.4%.红桦C释放率最高,岷江冷杉最低,且随根系直径的增粗,C释放率降低.岷江冷杉和粗枝云杉N释放模式为非生长季富集、生长季释放,而红桦反之.富集期间,径级越大,富集量越多.3个树种各径级根系P动态在非生长季和生长季皆表现为富集-释放模式,且岷江冷杉P富集程度显著高于粗枝云杉和红桦,但各径级之间差异总体不显著.根系径级对川西亚高山森林根系分解具有显著影响,而径级影响与树种和分解时期有一定关系.     

Roles of survivin isoforms in the chemopreventive actions of NSAIDS on colon cancer cells

NSAIDs downregulate survivin (an apoptosis inhibitor), increase apoptosis and reduce growth of colon polyps and cancers. Recently, anti- and pro-apoptosis isoforms of survivin were identified. The roles of these isoforms in NSAID-induced colon cancer cell death have not been examined, and is the focus of this study. The anti-apoptosis isoforms, wild-type (WT) survivin and survivin-ΔEx3, and the pro-apoptosis isoform, survivin-2b, were present in HT-29 and RKO cells. Indomethacin treatment significantly decreased WT survivin and survivin-ΔEx3 (30.5±10.4% and 20.3±6.7%, respectively) but not survivin-2b mRNA in RKO cells. In HT-29 cells, all three isoform mRNAs were slightly decreased by indomethacin treatment. Consistently, indomethacin treatment dramatically reduced WT survivin protein in RKO but not HT-29 cells. Indomethacin treatment increased apoptosis and general cell death more significantly in RKO cells (75.7±1.1% cell death at 48 h) than in HT-29 cells (25.4±3.7% cell death at 48 h). Anti-sense suppression of survivin-2b mRNA increased resistance of both RKO and HT-29 cells to indomethacin. These data support a role for survivin isoforms in colon cancer cell apoptosis, and thus in prevention of colon cancer growth by NSAIDs.     

陕北沙地3种典型灌木根木质部解剖结构及水力特性

比较了陕北沙地沙柳、柠条和沙棘3种典型灌木不同土壤深度(0～20和30～50 cm)根木质部解剖结构和水力特性.结果表明： 沙柳具有较高的叶水势,根木质部导管平均最大直径(d_max)、平均最小直径(d_min)、平均导管面积(A_lum)以及导管面积占木质部面积比例(A_ves/A_xyl)显著高于柠条和沙棘,根导管密度(VD)与沙棘相当但显著高于柠条;沙柳根的比导水率分别为柠条和沙棘的5.0和2.8倍;沙柳根栓塞脆弱性指数与柠条根相当,但显著高于沙棘根.表明沙柳属耗水型水分利用策略,而柠条和沙棘属节水型水分利用策略,且柠条更耐旱.3种灌木在2个土层深度的d_max、d_min和A_lum无显著差异,但30～50 cm土层根VD和A_ves/A_xyl显著高于表层;30～50 cm土层根比导水率显著高于表层根,但脆弱性指数小于表层根,深层根具有高的水分传输效率和低的水力脆弱性.