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毕赤酵母优化表达外源蛋白策略   总被引:2,自引:1,他引:1       下载免费PDF全文
毕赤酵母(Pichia pastoris)表达系统是一种异源蛋白表达的理想系统,但目前并非所有的外源蛋白都能在毕赤酵母中成功高效表达,不同的蛋白表现为不同表达水平、生物活性及稳定性。从遗传因素和表达条件综述了外源蛋白在毕赤酵母中的优化表达策略。  相似文献   

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毕赤酵母高效表达策略概述   总被引:1,自引:0,他引:1  
毕赤酵母表达系统是外源蛋白表达的较为理想的系统,但是并不是所有蛋白都能利用此系统获得高效表达,不同来源的蛋白,其表达水平、生物活性和稳定性均存有明显差别。概述了影响毕赤酵母高效表达的主要因素以及外源蛋白在毕赤酵母中的高效表达策略。  相似文献   

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毕赤酵母(Pichia pastor)表达系统是近年发展起来的一种高效表达外源蛋白的系统,利用该系统表达外源基因具有良好的应用前景。尽管毕赤酵母表达系统具有比较完备的基因表达调控机制和对真核基因表达产物的加工修饰能力,但由于基因本身及表达系统等诸多因素,仍然存在外源蛋白表达产量很低甚至不表达的情况。针对毕赤酵母表达系统这一因素,对表达载体的优化,毕赤酵母菌株优化及发酵条件优化进行了综述,以期为外源基因在毕赤酵母中的高效表达提供理论基础。  相似文献   

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真菌免疫调节蛋白家族(Fungi immunoregulatory proteins,FIPs)各成员所具有的免疫调节和抗肿瘤活性已被广泛研究。本研究利用毕赤酵母表达系统对其成员Lz-8进行了重组表达。以毕赤酵母突变株GS115为表达宿主细胞,PCR和DNA测序结果均显示Lz-8的cDNA已被成功地整合入酵母基因组。聚丙烯酰胺凝胶电泳(SDS-PAGE)、激光解析飞行时间质谱(MALDI-TOF-MS)和免疫学实验均被用于重组表达蛋白的检测。实验结果表明Lz-8在毕赤酵母表达系统中得到成功表达,在SDS-PAGE中可观察到分子量为14000D的单一条带,MALDI-TOF-MS的实验结果显示rLz-8的分子量为12722D。在相关的免疫学实验中,rLz-8可引起绵羊血红细胞凝集,但对人血4种血型的红细胞并没有凝集作用,rlz-8还可诱导巨噬细胞吞噬作用,均与其他报道中的实验结果吻合。以上结果表明,本实验已成功地利用毕赤酵母表达系统对Lz-8进行重组表达。  相似文献   

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毕赤酵母表达系统是近年来发展起来的一种高效表达外源基因的表达系统。综述了毕赤酵母表达系统的起源、生物学特性、融合蛋白的表达以及影响蛋白表达量的因素。  相似文献   

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毕赤酵母作为一种重要的表达外源蛋白的宿主,提高其外源蛋白的分泌量非常有必要。近年来很多学者报道了与毕赤酵母外源蛋白分泌相关的基因、蛋白质,同时毕赤酵母基因组的公布加快了这方面的研究进展。文章根据外源蛋白分泌的途径,分步骤地总结了涉及的基因和蛋白,有利于分析控制蛋白分泌效率的具体步骤,为构建更加高效的毕赤酵母表达系统提供参考。  相似文献   

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巴斯德毕赤酵母表达系统在外源基因表达中的研究进展   总被引:7,自引:0,他引:7  
巴斯德毕赤酵母是目前应用最广泛的外源蛋白表达系统。分别从的菌株、载体、外源基因整合、表达产物糖基化和外源基因高效表达等方面综述了毕赤酵母表达系统的研究进展。  相似文献   

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毕赤酵母表达系统是近年来发展最为迅速的一种新型外源蛋白真核表达系统,被广泛应用于多种不同领域且成功表达了许多基因工程产品。高密度发酵技术已被广泛运用到毕赤酵母工程菌发酵工程当中。主要从毕赤酵母的表达常用菌株、载体及表型等方面介绍了其表达系统,从外源基因自身的特性、培养基的组成、温度、p H、溶氧量及补料流加策略方面阐述了对毕赤酵母高密度发酵的过程及蛋白质表达结果的影响,并对毕赤酵母工程菌高密度发酵进行了展望,为其今后的研究及应用提供借鉴。  相似文献   

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幽门螺杆菌感染Beagle犬动物模型的建立   总被引:1,自引:0,他引:1  
人白细胞介素 10 (IL 10 )是一种免疫调节细胞因子 ,生物学活性广泛 ,包括免疫抑制、抗炎及免疫调节特性。动物试验及临床试验表明 :重组人IL 10 (Tenovil)在治疗自身免疫疾病如炎性肠炎、类风湿性关节炎、克隆病、银屑病、器官移植以及治疗丙型肝炎患者的肝纤维化方面有一定的疗效。我们将hIL 10基因重组到酵母表达载体pPIC9K中 ,酶切鉴定和测序正确后通过电穿孔转化毕赤酵母 ,涂布MD选择性平板 ,挑取生长快速的大菌落进一步用G4 18筛选多拷贝表达盒的酵母转化子。抗高浓度G4 18的酵母转化子用甲醇诱导表达 ,酵母培养上清液经SDS P…  相似文献   

10.
依据已报道的地鳖虫成熟肽cDNA序列设计引物,通过RT-PCR法从地鳖虫(Eupolyphage sinensis Walker)中克隆得到675 bp地鳖虫纤溶活性蛋白 (fibrinolytic protein,EFP)成熟肽编码序列.将此片段克隆到表达载体pPICZα-A中,转化毕赤酵母GS115,甲醇诱导表达得到重组表达蛋白,经SDS-PAGE电泳和活性鉴定,表明重组EFP在毕赤酵母中均获得表达,重组表达蛋白相对分子质量为28.2 kD,表达产物分子质量与理论分子质量相符.重组蛋白在毕赤酵母中以分泌形式表达,具有纤溶活性.  相似文献   

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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   

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The lactate dehydrogenase activity in reactions of lactate oxidation and synthesis was studied in subfractions of the chicken brain, heart and liver at the embryonal, early postembryonal and adult stages of development after thyroxine administration. It has been shown that during embryogenesis thyroxine predominantly enhanced the rate of lactate oxidation in the mitochondrial tissues. A marked increase in the lactate synthesis was found in cytoplasm of the adult chicken tissues. Specificity of enzyme activity alterations was detected in the chicken brain during ontogenesis after thyroxine administration.  相似文献   

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In order to determine if the absence of vitamin C in the diet of capybaras (Hydrochoerus hydrochaeris) causes scurvy, a group of seven young individuals were fed food pellets without ascorbic acid, while another group of eight individuals received the same food with 1 g of ascorbic acid per animal per day. Animals in the first group developed signs of scurvy-like gingivitis, breaking of the incisors and death of one animal. Clinical signs appeared between 25 and 104 days from the beginning of the trial in all individuals. Growth rates of individuals deprived of vitamin C was considerably less than those observed in the control group. Deficiency of ascorbic acid had a severe effect on reproduction of another population of captive capybaras. We found that the decrease in ascorbic acid content in the diet affected pregnancy, especially during the first stages. The results obtained suggest that it is necessary to supply a suitable quantity of vitamin C in the diet of this species in captivity.  相似文献   

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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   

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