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1.
人脑内-含有ACP样结构域新基因的发现   总被引:20,自引:2,他引:18  
为寻找脑内新基因,以正常成人全脑cDNA为模板,采用锚定PCR方法进行扩增,将经琼脂糖DNA电泳 鉴定获得的一约1200bp大小的特异性条带回收,并克隆入Teasy载体.用310 Genetic Analyzer进行自动测序. 所得序列进行生物信息学分析BLAST相似性分析结果证明所得序列为新序列,读框分析表明,该序列中存在 一完整编码区,编码含357个氨基酸的蛋白质.ProDom软件分析发现其含有酰基携带蛋白(ACP)样结构域. 随后,经3'RACE法克隆到该基因的全长cDNA,其全长为2024bp,染色体定位在14q11.2,含有16个外显子, 15个内含子,该基因已登录到GenBank.经设计编码区引物,从Teasy载体扩增出编码区后再克隆入pGEX-4T1 表达载体,经异丙基硫代-D-乳糖苷(IPTG)化学诱导表达.其编码区克隆人pGEX-4T1表达载体后,转入 JM109宿主菌,经IPTG诱导已得到表达.点杂交及RNA印迹表明,该基因在正常成人脑内广泛高表达.  相似文献   

2.
近来的研究表明干扰素在哺乳动物早期胚胎发育中有重要的作用。我们首次克隆了兔早期胚胎发育相关新基因IFRG(干扰素应答基因)的全长cDNA序列(AJ584672),根据该cDNA序列以兔卵巢cDNA为模板经PCR扩增后克隆了兔IFRG cDNA的完整开放阅读框(396bp)。将其克隆到原核表达载体pGEX-4T-2上,在大肠杆菌BL21中进行了GST-IFRG融合蛋白的表达。 经IPTG诱导培养后,SDS-PAGE电泳检测结果显示在41kDa处有特异性表达蛋白,回收融合蛋白作为抗原免疫小鼠,制备多克隆抗体,通过Western杂交表明该融合蛋白具有生物免疫活性。  相似文献   

3.
利用RT-PCR技术扩增了编码烟实夜蛾 Helicoverpa assulta 触角化学感受蛋白(chemosensory protein)的全长cDNA。克隆和测序结果表明,烟实夜蛾化学感受蛋白基因核苷酸序列全长384 bp(GenBank序列号: DQ285667),编码127个氨基酸残基,预测N-末端包含16个氨基酸组成的信号肽序列,因此估测其成熟蛋白分子量为12.97 kD,等电点为5.32。将该基因重组到表达载体pGEX-4T2中,并转入原核细胞中进行表达。SDS-PAGE和Western印迹分析表明,经IPTG诱导后,烟实夜蛾化学感受蛋白基因能在大肠杆菌BL21中表达,电泳检测到一条约39 kD的外源蛋白,与预测的融合蛋白分子量大小相符。  相似文献   

4.
运用RT-PCR技术扩增编码烟夜蛾Helicoverpaassulta(Guen啨e)幼虫几丁质酶基因的cDNA片段,将其克隆至pMD18-T载体,获得该基因的成熟蛋白阅读框序列。将该基因重组到表达型质粒pGEX-4T-2中,并转化入原核细胞中表达,序列测定结果表明,烟夜蛾幼虫几丁质酶基因的成熟蛋白阅读框全长1338bp,编码445个氨基酸残基,预测分子量和等电点分别为50.1kDa和9.26;推导的氨基酸序列与其近缘种棉铃虫几丁质酶氨基酸序列的一致性达99%,与其他6种昆虫几丁质酶的氨基酸序列也高度一致(65%~76%),并具有几丁质酶的典型特征。将该基因克隆到原核表达载体pGEX-4T-2上并转化BL21,SDS-PAGE和Western印迹分析表明,经IPTG诱导,76kDa附近没有特异蛋白条带出现,表明烟夜蛾几丁质酶基因不能在原核表达载体pGEX-4T-2中表达。  相似文献   

5.
尹姣  李克斌  曹雅忠 《昆虫学报》2009,52(2):216-222
羧肽酶是昆虫体内重要的消化酶系之一。利用甜菜夜蛾Spodoptera exigua(Hübner)围食膜多克隆抗体免疫筛选草地螟Loxostege sticticalis L.中肠cDNA表达文库, 得到编码羧肽酶A的全长cDNA克隆。该cDNA克隆全长1 380 bp (GenBank登录号EU924506), 开放阅读框长1 302 bp, 编码434个氨基酸, 预测分子量和等电点分别为49.1 kDa和9.56。序列含有胰蛋白酶切割位点和催化特征, 具有典型的羧肽酶A特性。将该基因与pET30载体重组后, 经IPTG诱导, 蛋白在大肠杆菌中获得了表达。  相似文献   

6.
目的:克隆小鼠IL-33基因全长编码区cDNA,并对其进行序列分析。方法:从BALB/c小鼠的脊髓组织中提取总RNA,逆转录为cDNA,用热启动PCR技术,扩增小鼠IL-33基因全长编码区cDNA,经双酶切后,克隆入pcDNA3.1( )载体中,构建真核表达载体pcDNA3.1-mIL-33,然后进行酶切鉴定与序列分析。结果:小鼠IL-33基因的PCR产物和重组载体经凝胶电泳和酶切鉴定、测序分析证实,其序列与GenBank中数据一致。小鼠IL-33基因的全长编码序列为801 bp,编码266个氨基酸。结论:小鼠IL-33基因成功的克隆并构建了其真核表达载体,为进一步进行IL-33的表达与功能研究奠定了基础。  相似文献   

7.
小鼠Klf4基因的克隆及原核表达分析   总被引:2,自引:1,他引:1  
目的克隆Klf4基因并对其重组蛋白进行原核表达及分析。方法提取胎鼠皮肤mRNA后反转录为cDNA序列,用一对两端引入特定酶切位点引物,从该cDNA中扩增出Klf4基因编码区序列,将其克隆到pEasy-T3载体上。对质粒双酶切并回收其中Klf4基因片段后,克隆入pET-52b(+)载体后转化Origmai B(DE3)型大肠杆菌,用IPTG诱导表达,最后采用SDS-PAGE对重组蛋白进行鉴定及分析。结果对所克隆的Klf4mRNA蛋白编码区的DNA序列分析表明,klf4CDS区包括终止密码子在内为1452 bp,与参照序列对比仅有四处存在差异,不仅其同源性达到99.72%,且其氨基酸序列同源性为100%;在IPTG诱导下pET-52b(+)-Klf4重组质粒可表达与预期相符的约为57×103的蛋白质;经IPTG刺激后重组蛋白表达明显上调,其中IPTG为0.4 mol/L时效果最佳。结论从胎鼠皮肤中克隆的Klf4基因可在原核中表达。  相似文献   

8.
CPP32 cDNA的克隆及其表达和活性检测   总被引:1,自引:0,他引:1  
CPP32在细胞编程死亡调控中起重要的作用.利用CPP32专一性引物,用RT-PCR方法从人鼻咽癌CNE细胞中扩增出约830 bp的片段,经序列分析证明与已发表的CPP32序列完全一致.将扩增出的编码人全长CPP32的cDNA片段克隆入pGEX-2T中,转化大肠杆菌DH5α.转化菌经诱导表达出较高含量的GST-CPP32融合蛋白.进一步研究显示,细菌中表达的CPP32蛋白能自我切割,而且能裂解体外翻译的PARP,从而证明其具有生物活性.  相似文献   

9.
白鹅催乳素基因的克隆及诱导表达条件的优化   总被引:2,自引:0,他引:2       下载免费PDF全文
郭丽  杨焕民  李鹏  康波 《遗传》2008,30(11):1433-1438
摘要: 运用RT-PCR方法, 从白鹅脑垂体总RNA中扩增得到了催乳素(Prolactin, PRL)基因编码区序列cDNA, 并将其克隆到pMD18-T载体上。DNA序列分析表明, PRL cDNA包括终止密码子在内的长度为690 bp,编码230个氨基酸残基的蛋白质, 与皖西白鹅的有所差异, 二者碱基同源性在99.57%, 氨基酸同源性达99.56%。将PRL基因编码区序列cDNA定向克隆到表达载体pET-32a (+)中, 构建表达质粒pET-32a(+)-PRL。该质粒的BL21 (DE3)转化菌在IPTG的诱导下可表达PRL基因融合蛋白, IPTG终浓度1 mmol/L, 37℃, 诱导4 h表达量最高, 表达量约占菌体总蛋白的28.96%。  相似文献   

10.
以球毛壳菌cDNA文库中获得过氧化物膜蛋白(pero)基因片段(GenBank Accn:BP099709)为基础,用RACE 技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3′RACE产物和508bp的5′RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17.5kD,理论等电点为5.75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明:该基因与粗糙脉孢菌(Neurospora crassa)的过氧化物膜蛋白过敏原同源性最高(83%)。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在21kD处有一特异性融合蛋白带,大小与预期相符,说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。  相似文献   

11.
Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.  相似文献   

12.
2018年中国植物科学若干领域重要研究进展   总被引:1,自引:0,他引:1  
2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。  相似文献   

13.
Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.  相似文献   

14.
2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。  相似文献   

15.
2017年中国植物科学若干领域重要研究进展   总被引:1,自引:0,他引:1  
2017年中国植物科学继续保持高速发展态势, 重大成果频出, 具体表现在中国植物学家在国际顶级学术期刊发表的文章数量平稳上升。中国植物科学领域的研究工作者成果精彩纷呈, 如新型广谱抗病机制的发现、水稻广谱抗病遗传基础及机制和疫霉菌诱发病害成灾机制研究等。2017年中国生命科学领域十大进展评选中, 有两项植物科学领域的研究成果入选。水稻生物学、进化与基因组学和激素生物学等领域学科发展突出。另外, 值得一提的是, 长期从事高等植物与代谢途径调控分子网络研究和水稻品种设计育种的李家洋院士的研究成果“水稻高产优质性状形成的分子机理及品种设计”荣获2017年国家自然科学一等奖。这一具有重大国际影响的开创性贡献标志着中国植物科学在该领域的国际科学前沿居于引领和卓越地位。该文对2017年中国本土科学家在植物科学若干领域取得的重要研究成果进行了系统梳理, 旨在全面追踪和报道当前中国植物科学领域发展的最新前沿动态, 与广大读者共同分享我国科学家所取得的辉煌成就。  相似文献   

16.
葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义。综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望。  相似文献   

17.
我国葫芦科植物离体培养研究进展   总被引:5,自引:0,他引:5  
葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义.综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望.  相似文献   

18.
We report the appearance of apoptotic cells in experimental myocardial infarction (rabbit heart) in in situ and in vitro preparations. Apoptosis was recognized by intravital staining with Hoechst 33342 (Ho342), by nick-end labeling (TUNEL) and by DNA laddering. A steady rise in the relative number of apoptotic cardiomyocytes (apoptotic index) was noted in in situ preparations. Apoptosis was first noted 6 h after the onset of ischemia with its highest value occurring after 72 h. Apoptotic nuclei were absent in remote areas of the left and right ventricles. Apoptotic nuclei within the infarcted area showed diminished intensity of Ho342 fluorescence. Three days after ischemia, a border zone adjacent to the infarcted area consisting of apoptotic macrophages was recognized. A novel finding was the appearance of apoptotic cardiomyocytes in the isolated perfused ischemic heart. Occurring as early as 50 min after the onset of ischemia, a high apoptotic index was present adjacent to the ligature placed around the coronary artery. This observation provides the opportunity to selectively examine factors leading to apoptosis in the ischemic heart under controlled experimental conditions.  相似文献   

19.
Abstract. Changes are described in aquatic vegetation in oligotrophic, groundwater-fed Rhine floodplain streams in Alsace (eastern France), resulting from disturbance. Disturbance factors include changes in nutrients, either permanent ones - effluent from a waste water treatment plant or trout hatcheries - or periodic ones: flooding. Regular inputs of high levels of phosphate and ammonia modified the macrophyte vegetation in these streams. The floristic composition, which was characteristic of oligotrophic waters upstream of the eutrophicated sector, changed to that of a eutrophic situation as originally found downstream. Periodic disturbance by floods which normally occur once a year, irregularly eutrophicates the small streams, causing the development of a mixture of eutrophic and oligotrophic species. Six macrophyte communities are distinguished, indicating different trophic levels. The aquatic vegetation is adapted to the variations of phosphate and ammonia levels. Hence, aquatic macrophytes can be used as bio-indicators of fluctuations in water nutrient levels in relation to the type of disturbance.  相似文献   

20.
The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.  相似文献   

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