地衣芽孢杆菌α—淀粉酶基因在枯草芽孢杆菌中的诱导表达

采用PCR技术扩增了sacB基因的启动子-信号序列,并将扩增的序列重组进含地衣芽孢杆菌α-淀粉酶基因的质粒载体上构建了含α-淀粉酶基因的分泌型表达载体pSA60。将pSA60转化枯草芽孢杆菌QB1098后,α-淀粉酶基因在sacB基因启动子-信号序列的调控和蔗糖的诱导下获得表达,表达产物分泌至胞外。     

枯草芽孢杆菌碱性蛋白酶基因的克隆和表达

目的:获得碱性蛋白酶基因。方法:用PCR的方法从枯草芽孢杆菌A-109中扩增碱性蛋白酶基因(apr),并进行测序分析,构建表达载体,最后转化大肠杆菌BL21,SDS-聚丙烯酰胺凝胶电泳检测该基因的表达情况。结果:apr基因片段含1092个碱基对。该基因片段核苷酸序列与Bacillus amyloliquefaciens subtilisin DFE precursor有99%的同源性,对应的氨基酸序列与Bacillussp.DJ-4有99%的同源性。apr基因在大肠杆菌BL21中获得表达,并表现出蛋白酶活性。结论:获得了具有活性的新的碱性蛋白酶基因。     

碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。     

嗜碱性芽孢杆菌PB92碱性蛋白酶分别在大肠杆菌和枯草芽孢杆菌中表达

从Bacillus alcalophillus PB92中扩增出碱性蛋白酶基因Mapr,Mapr分别插入到大肠杆菌载体pET-22b( )和枯草芽孢杆菌载体pWB980中构建成重组分泌型表达载体pET22b( )-Mapr、pWB980-Mapr。碱性蛋白酶基因分别在大肠杆菌宿主BL21和枯草芽孢杆菌DB104中得到表达。SDS-PAGE分析,重组蛋白酶的分子量为28kD。在大肠杆菌,所得酶活为231U/ml,而在枯草芽孢杆菌,其酶活为1563U/ml。大概是由于碱性蛋白酶在枯草芽孢杆菌折叠成熟机制与大肠杆菌的不同造成的。     

青霉素酶基因在枯草芽孢杆菌中的高效表达

以获得大量胞外青霉素酶为目的,将青霉素酶基因克隆至表达载体pWB980中,并转化到双蛋白酶缺陷的Bacillus subtilis DB104。重组菌在LB培养基中培养24小时后, SDS-PAGE分析发现目的蛋白分子量为28kDa,酶活力为339U/mL;通过筛选7种不同的发酵培养基发现4#培养基更利于青霉素酶的表达,最大酶活力为1580U/mL,较优化前提高了3.66倍,并对该重组菌进行了7L罐放大实验,结果显示在培养24小时产酶达到高峰,酶活力为1255.8 U/mL。     

利用degQ基因提高枯草芽孢杆菌纤溶酶表达

从枯草芽孢杆菌(Bacillus subtilis)中通过PCR扩增得到degQ基因,将其克隆到含有枯草杆菌纤溶酶基因的蔗糖诱导表达载体pUBS中,并转化至B．subtillis DB403受体菌,得到基因工程菌DB403(pUBSD)。通过发酵表达证实degQ基因能增强枯草杆菌纤溶酶的表达,酶活提高了2．2倍。同时还对不同种类的糖、不同浓度蔗糖、不同诱导时间等发酵条件进行优化和比较研究。     

枯草芽孢杆菌(Bacillus subtilis)碱性脂肪酶基因在毕赤酵母中的表达

将来自枯草芽孢杆菌的碱性脂肪酶基因经密码子优化,全基因合成后克隆到pPICZαA载体,构建了pPICZαA-bsl分泌型重组质粒,该重组质粒经限制性内切酶PmeI线性化后使用LiCl法转化到毕赤酵母X-33,经过筛选获得分泌表达碱性脂肪酶的重组毕赤酵母X-33/pPICZαA-bsl。摇瓶发酵液上清酶活最高可达4.78 U/mL,初步研究了该脂肪酶的酶学性质,其最适作用温度为40-60℃,最适pH9.0,且具有高度耐碱的特性。该重组脂肪酶对旧新闻纸具备较明显的脱墨能力。     

枯芽孢杆菌α—淀粉酶基在在大肠杆菌中的表达及其产物的分泌

The E. coli which carrying the alpha-amylase gene fragment cloned from B. subtilis secreted the gene products into the medium. The reason is the exogenous gene fragment act on the cell wall of E. coli by some way, gives rise to the change of its structure. It leads up to the alpha-amylase and some periplasm proteins passing through the cell wall into the medium. It also causes the change of host colonial morphology. The secrete process are non-specific.     

α-淀粉酶在短短芽孢杆菌中的分泌表达

应用PCR技术从枯草杆菌168中分离出α－淀粉酶基因。将其引入分泌表达载体pBKE50后,用Tris-PEG法转入短短芽孢杆菌50中,发现α－淀粉酶以活性形式被分泌表达。酶测活分析表明α－淀粉酶活性约为出发菌枯草杆菌168的1.7倍。     

Authentic human basic fibroblast growth factor produced by secretion in Bacillus subtilis

Bacillus subtilis is generally accepted as an inborn host candidate employed for secretory production of heterologous proteins. However, this ideal host system has never been employed for commercial production of medically useful proteins. In this communication, we report for the first time the employment of an engineered B. subtilis system, in conjunction with a facile cell-wall destabilization protocol, to successfully obtain an alluring yield of 40 mg?l^?1 of secreted human basic fibroblast growth factor (hbFGF) in the culture supernatant. The product was not only shown to exhibit potent bioactivity but also revealed to possess a protein sequence identical to that of mature native hbFGF (Mat-hbFGF). Our findings may pave way for the development of a cost-effective process for producing Mat-hbFGF, which is currently sold at an unusually expensive price of over US $1 million?g^?1, for medical and skin care applications.     

枯草芽孢杆菌BS224的16S rDNA序列测定及系统进化分析

利用细菌通用引物,通过PCR的方法,对菌株BS224的16S rDNA基因进行扩增,PCR产物经胶回收试剂盒纯化后测序,测序结果与GenBank上已登录的高同源16S rDNA序列进行比较,并构建系统进化树,结果BS224与Bacillus subtilisstrain ZHA9相似性最高,达到99.38%,与模式菌株Bacillus subtilisstrain 168的相似性也达到98.55%,结合其形态特征、培养特征、生理生化特性的分析结果,可以确定BS224属于枯草芽孢杆菌属。     

碱性成纤维细胞生长因子与肿瘤

碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF) 作为多种细胞的致裂源,与肿瘤的关系近几年来受到关注,本文概述了有关bFGF与肿瘤关系的研究进展。     

Basic fibroblast growth factor in Alzheimer's disease

We have examined the presence of basic fibroblast growth factor (FGF) in normal and in Alzheimer brains, studied the distribution of the mitogen by immunohistochemical techniques, measured the quantities of growth factor in selected areas of the brain (Brodmann areas 10/11 and 20/21), characterized the molecular forms by Western blotting and determined its sites of synthesis by in situ hybridization. Although the same molecular forms of basic FGF are found in control and Alzheimer brains, basic FGF is increased in the brains of Alzheimer's patients. Furthermore, basic FGF is not distributed in an identical fashion to normal and Alzheimer brains, but is found in association with the lesions that characterize this disease. In normal controls (n = 5), basic FGF was found to be widely distributed throughout the three brain regions examined (prefrontal cortex, hippocampus, and hypothalamus). Immunoreactivity was observed within astrocytes in both the grey and white matter, as well as within neuronal perikarya. Brain tissues that were obtained from Alzheimer patients (N = 4) showed a substantial increase in the overall specific staining of astrocytes and neurons, particularly in areas of reactive gliosis. Focal concentration of immunoreactive basic FGF was evident within the neuritic plaques, and could be clearly seen in association with the neurofibrillary tangles present within neuronal perikarya. The possibility that basic FGF expression in the CNS is linked to the pathogenesis of the disease is discussed.     

Novel strategy for expression of authentic and bioactive human basic fibroblast growth factor in Bacillus subtilis

Applied Microbiology and Biotechnology - Inteins, also known as “protein introns,” have been found to be present in many microbial species and widely employed for the expression and...     

Basic fibroblast growth factor in rat salivary glands

We studied the occurrence and localization of basic fibroblast growth factor (bFGF) in rat salivary glands using a specific monoclonal antibody. It was shown that the extract of rat salivary glands has a pronounced stimulatory activity on the growth of bovine capillary endothelial cells, which is blocked by the addition of an antibody against bFGF. The concentration of bFGF in the submandibular/sublingual gland, as determined by radioimmunoassay, was 80% that in the brain. Immunocytochemistry revealed bFGF-immunoreactivity localized primarily in the epithelial cells lining the striated ducts and excretory ducts of the parotid, sublingual and submandibular glands. In addition, intense bFGF-immunoreactivity was observed in the granular convoluted tubule of the submandibular gland, localized predominantly in the agranular pillar cells, which lay in small numbers among the majority of weakly immunostained cells containing many apical secretory granules. At the electron-microscopic level, the immunoreactive material was distributed diffusely in the cytoplasmic matrix and nuclei of all immunoreactive cells, whereas it was absent from all cytoplasmic organelles including the secretory granules. These results indicate that bFGF is localized in different cellular and subcellular compartments from those of other growth factors in the duct system of rat salivary glands.     

Basic fibroblast growth factor inhibits osteoclast-like cell formation

Basic fibroblast growth factor (bFGF) inhibited osteoclast-like cell formation in co-cultures of mouse bone marrow cells either with the mouse stromal cell line, ST2, or with primary osteoblastic cells. Basic FGF significantly inhibited the osteoclast-like cell formation, induced by 1α,25-dihydroxyvitamin D₃[1α, 25(OH)₂D₃] when the cytokine was added to the culture, at an intermediate stage, suggesting that bFGF inhibits the differentiation of the osteoclast progenitors. With regard to target cells, bFGF directly affected ST2; it increased [³H]thymidine uptake and decreased the number of alkaline phosphatase-positive cells. In contrast, bFGF had no inhibitory effect on the colony formation of bone marrow cells induced by macrophage colony stimulating factor in methylcellulose culture. In addition, ST2 cells treated with bFGF produced similar amounts of colony forming activity to those without the cytokine. These findings indicated that the bFGF is not involved in the proliferation of progenitor cells even in the presence of ST2 cells. Furthermore, bFGF inhibited osteoclast-like cell formation induced not only by 1α,25(OH)₂D₃, but also by prostaglandin E₂ and by interleukin-11. These results suggest that bFGF inhibits the common site of osteoclast-like cell formation, as induced by different mechanisms. Our data also indicated that the target cells for bFGF in inhibiting osteoclast formation are not osteoclast progenitors but stromal cells such as ST2 and osteoblastic cells, which support osteoclast development. © 1996 Wiley-Liss, Inc.     

Basic fibroblast growth factor induces retinal regeneration in vivo

In the present study, we have investigated the effect of basic fibroblast growth factor (bFGF) on retinal regeneration in the stage 22-24 chick embryo. The neural retina was surgically removed in ovo leaving the retinal pigment epithelium (RPE) intact and then slow-release, plastic implants containing bFGF were inserted into the eye. Light microscopic examination of eyes 7 days later revealed that bFGF induced retinal regeneration in a dose-dependent manner. The absence of the RPE in these eyes and the reversed polarity of the regenerated neural retina is consistent with the hypothesis that this process occurs by transdifferentiation of the RPE. This represents the first time that a known molecule has been shown to induce retinal regeneration in vivo.     

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury

To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8±1.5% and 96.5±3.5% of preischemic levels in control and bFGF-treated hearts (10 g/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 g bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease.