基于pDS132的基因敲除中创伤弧菌氯霉素的耐药性变化及影响

[背景]基于自杀载体的基因敲除在单基因敲除上的应用较为常见,但在多基因敲除过程中细菌耐药性的变化及对后续敲除的影响尚未明确.[目的]探究基于自杀载体pDS132的创伤弧菌vvhA与rtxA1双基因敲除株构建过程中,创伤弧菌对氯霉素耐药性的变化及对后续基因敲除的影响.[方法]基于自杀载体pDS132的同源重组法构建创伤弧...     

溶瘤腺病毒ZD55对类肝癌干细胞的抑制效应

溶瘤腺病毒能够靶向和杀死癌症干细胞,被认为是一种很有前景的抗癌药物.已有研究表明,溶瘤腺病毒ZD55能够靶向肝癌,并且表现出明显的细胞毒性效应.然而,其对肝癌干细胞是否具有同样地杀伤效力仍需进一步探讨.利用悬浮培养富集类肝癌干细胞,并验证其肝癌干细胞的特征.进一步通过MTT、结晶紫染色、Hoechst染色、Western blot和流式细胞术等检测ZD55对类肝癌干细胞的细胞存活率、凋亡诱导和病理效应等.结果发现,悬浮培养的类肝癌干细胞具有自我更新和分化能力、高表达干细胞相关转录因子(如NANOG和OCT4)、处于静息状态和具有耐药性等特性,溶瘤腺病毒处理后表现出明显的细胞毒性效应和杀伤特性,类肝癌干细胞的最低生存率仅为26.7%.ZD55能够非常明显地诱导类肝癌干细胞凋亡,其凋亡率最高达到60%.因此,ZD55可能会成为靶向肝癌干细胞的一种很有前景的治疗药物,对肝癌的临床治疗具有一定的应用价值.     

番茄ARF2蛋白的生物信息学分析与亚细胞定位

克隆番茄(Solanum lycopersicum) ARF2基因,并分析其分子特性和亚细胞定位,为研究其功能提供基础．通过生物信息学方法分析SlARF2基因编码蛋白的理化性质和分子特性．采用RT-PCR技术从番茄果实cDNA中扩增SlARF2基因全长,并构建与黄色荧光蛋白(YFP)融合的pBA-ARF2-YFP表达载体,进而再通过农杆菌介导的遗传转化方法,将重组质粒转化到野生型番茄中,将得到的T1代转基因种子萌发,然后取根尖通过荧光显微镜观察了融合蛋白在活细胞内分布的特点．生物信息学分析结果表明,SlARF2是富含Ser、Leu、Gly和Pro以及具有ARF家族典型结构域的可溶性蛋白,其氨基酸序列与葡萄、木薯和拟南芥的同源性分别为70.08 %、66.94 %和60.87 %．经酶切和测序分析证实pBA-ARF2-YFP融合表达载体构建成功,此外,PCR分析表明融合蛋白在转基因植株中得到表达．经荧光显微镜观察,ARF2定位在细胞核中．表明转录因子SlARF2定位在细胞核中,对番茄果实发育和成熟起重要作用．     

The fate of sheep-dispersed seeds: Plant species emergence and spatial patterns

Sheep epizoochory has often been proposed as an important vector which can help to overcome the dispersal limitation of plants in fragmented landscapes. In order to evaluate the contribution of herbivores to recruitment especially of target species, the dispersal and post-dispersal fate of such seeds must be known. In a field experiment sheep with seeds of mainly target species (experimentally attached to their coats) were present at three sand plots for 24 h. Natural epizoochorous dispersal was already shown for most of the species in our study area. Seed detachment, trampling intensity and seed shadow were measured; seedling emergence and survival were recorded over an 8-month period. In addition, the effect of sheep trampling on seedling emergence and survival of two threatened species, Jurinea cyanoides and Koeleria glauca, were studied.     

Phylogeny of Prokaryotes and Chloroplasts Revealed by a Simple Composition Approach on All Protein Sequences from Complete Genomes Without Sequence Alignment

The complete genomes of living organisms have provided much information on their phylogenetic relationships. Similarly, the complete genomes of chloroplasts have helped to resolve the evolution of this organelle in photosynthetic eukaryotes. In this paper we propose an alternative method of phylogenetic analysis using compositional statistics for all protein sequences from complete genomes. This new method is conceptually simpler than and computationally as fast as the one proposed by Qi et al. (2004b) and Chu et al. (2004). The same data sets used in Qi et al. (2004b) and Chu et al. (2004) are analyzed using the new method. Our distance-based phylogenic tree of the 109 prokaryotes and eukaryotes agrees with the biologists tree of life based on 16S rRNA comparison in a predominant majority of basic branching and most lower taxa. Our phylogenetic analysis also shows that the chloroplast genomes are separated to two major clades corresponding to chlorophytes s.l. and rhodophytes s.l. The interrelationships among the chloroplasts are largely in agreement with the current understanding on chloroplast evolution.Reviewing Editor: Dr. John Oakeshott     

Glycogen storage in multiple muscles of old GSD-II mice can be rapidly cleared after a single intravenous injection with a modified adenoviral vector expressing hGAA

BACKGROUND: Glycogen storage disease II (GSD-II) is an autosomal recessive lysosomal storage disease, due to acid-alpha-glucosidase (GAA) deficiency. The disease is characterized by massive glycogen accumulation in the cardiac and skeletal muscles. There is early onset (infantile, also known as Pompe disease) as well as late onset (juvenile and adult) forms of GSD-II. Few studies have been published to date that have explored the consequences of delivering a potential therapy to either late onset GSD-II subjects, and/or early onset patients with long-established muscle pathology. One recent report utilizing GAA-KO mice transgenically expressing human GAA (hGAA) suggested that long-established disease in both cardiac and skeletal muscle is likely to prove resistant to therapies. To investigate the potential for disease reversibility in old GSD-II mice, we studied their responsiveness to exogenous hGAA exposure via a gene therapy approach that we have previously shown to be efficacious in young GAA-KO mice. METHODS: An [E1-, polymerase-] adenoviral vector encoding hGAA was intravenously injected into two groups of aged GAA-KO mice; GAA expression and tissue glycogen reduction were evaluated. RESULTS: After vector injection, we found that extremely high amounts of hepatically secreted hGAA could be produced, and subsequently taken up by multiple muscle tissues in the old GAA-KO mice by 17 days post-injection (dpi). As a result, all muscle groups tested in the old GAA-KO mice showed significant glycogen reductions by 17 dpi, relative to that of age-matched, but mock-injected GAA-KO mice. For example, glycogen reduction in heart was 84%, in quadriceps 46%, and in diaphragm 73%. Our data also showed that the uptake and the subsequent intracellular processing of virally expressed hGAA were not impaired in older muscles. CONCLUSIONS: Overall, the previously reported 'resistance' of old GAA-KO muscles to exogenous hGAA replacement approaches can be rapidly overcome after a single intravenous injection with a modified adenoviral vector expressing hGAA.     

甲型肝炎病毒衣壳蛋白融合基因植物表达载体的构建 与遗传转化柑桔的研究

基因工程领域的研究进展使得植物体成为具有重要经济价值的药用蛋白的生产体系。以含甲型肝炎病毒结构基因cDNA的克隆载体pCDNAⅡA16为模板,用甲型肝炎病毒衣壳蛋白融合基因特异引物进行PCR扩增,得到全长2.2kb衣壳蛋白融合基因序列。经测序鉴定后正向克隆于植物表达载体pBI121中,衣壳蛋白融合基因位于pBI121质粒T-DNA左右边界区间内,处于CaMV35S启动子控制之下。经限制性内切酶分析和PCR鉴定后利用冻融法将重组质粒pBI121-A导入根癌农杆菌LBA4404。以锦橙 (Citrus. Sinensis Osbeck) 上胚轴为转化材料,通过根癌农杆菌介导法将衣壳蛋白融合基因转 化到植物基因组中。120株转化外植体经卡那霉素50 mg/L筛选,其中13株生长状况良好未出现白化现象的拟转化芽微嫁接到实生砧木继续培养。PCR分析证明,13株拟转化植株中有5株植物基因组中已导入甲型肝炎病毒衣壳蛋白融合基因,转化率为4.1%。此研究是对遗传转化柑桔表达外源蛋白的初步探讨,为进一步研究食用疫苗开辟了新途径。Abstract: The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. The construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins. The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter. The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404. The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments. 13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings. The presence and integration of the transgene had been verified by PCR analysis. The result showed that five transformants were integrated and the transformation efficiency was 4.1%.     

一种多基因串联共表达载体的构建

为了构建一种可用于串联共表达多个基因的原核表达载体,通过PCR引入点突变,对商业载体pET-22b的酶切位点进行定向改造,设计独特的XbaⅠ/SpeⅠ模块。获得改造成功的载体pET-m22b,测序结果显示启动子、核糖体结合位点、多克隆位点及终止子均位于XbaⅠ和SpeⅠ两酶切位点间。选取不同来源的基因进行串联组合及表达鉴定,四个基因成功组合于同一载体中,表达效果良好,各基因表达效率不受明显影响。研究构建的载体pET-m22b,使多基因的共表达更加便捷,为蛋白复合物的表达与纯化、代谢通路的异源重建及其优化等研究奠定了良好的基础。     

基于支持向量机方法的蛋白可溶性预测

按照蛋白质序列中残基的相对可溶性,将其分为两类(表面／内部)和三类(表面／中间／内部)进行预测。选择不同窗宽和参数对数据进行训练和预测,以确保得到最好的分类效果,并同其他已有方法进行比较。对同一数据集不同分类阈值的预测结果显示,支持向量机方法对蛋白质可溶性的整体预测效果优于神经网络和信息论的方法。其中,对两类数据的最优分类结果达到79．0％,对三类数据的最优分类结果达到67．5％,表明支持向量机是蛋白质残基可溶性预测的一种有效方法。     

大鼠Bdnf基因慢病毒载体的构建及其在骨髓间质干细胞中的表达

骨髓间质干细胞（MSCs）是目前基因工程正在探讨应用的靶细胞,为构建带有脑源性神经营养因子（Bdnf）基因慢病毒载体并使其在大鼠骨髓间质干细胞中表达,采用RT-PCR技术获得大鼠Bdnf基因编码区（CDS）片段,限制性内切酶酶切和基因重组构建慢病毒载体质粒PNL-BDNF-IRES₂-EGFP,在脂质体介导下与包装质粒HELPER,包膜质粒VSVG共转染293T细胞包装生产慢病毒。所获慢病毒感染大鼠MSCs（rMSCs）后,PCR和免疫细胞化学法检测在rMSCs中Bdnf基因的插入和表达。结果显示所获的Bdnf基因经测序后与GenBank报道序列完全一致。重组慢病毒载体质粒PNL-BDNF-IRES2-EGFP经鉴定正确。三质粒共转染293T细胞成功,收集、浓缩病毒后测定其滴度为6.7×10⁷TU/mL, PCR证实Bdnf基因插入病毒基因组。感染rMSCs后RT-PCR、免疫细胞化学染色及Western检测各组细胞均有BDNF蛋白表达,其中试验组BDNF-rMSCs更大量表达BDNF,与其余2组(Mock-rMSCs、rMSCs)比较差异具有统计学意义。构建带有Bdnf基因慢病毒载体并在大鼠骨髓间质干细胞中成功表达,为今后基因修饰干细胞的移植后长期观察研究奠定了基础。