共查询到20条相似文献，搜索用时 359 毫秒
1.
利用甲酸脱氢酶(FDH)催化甲酸脱氢反应是酶促有机合成中重要的还原型烟酰胺辅酶再生系统。实验对E.coli BL21(DE3)/pET32/Bsp-FDH诱导表达NADP+依赖甲酸脱氢酶的表达条件进行了优化,在培养第4h加入0.4 mmol/l的IPTG、诱导时间8h、诱导温度30℃的诱导表达条件下,每升发酵液产甲酸脱氢酶170U。利用亲和层析法一步分离纯化重酶,纯化倍数为9.65、酶活回收达60.43%。实验为重组甲酸脱氢酶应用于有机合成中的NADPH再生奠定基础。 相似文献
2.
《科学中国人》2015,(26)
目的:构建Visfatin原核表达质粒,诱导表达并纯化重组人内脂素(Visfatin)。方法:从人LO2细胞中提取总RNA后,经RT-PCR法得到人Visfatin的c DNA序列,用以构建带HIS标签的Visfatin重组质粒。然后将重组质粒转化到大肠杆菌BL21中,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,用Ni柱亲和层析纯化融合蛋白。结果:PCR扩增出的目的基因片段为1600 bp左右,与理论大小一致。SDS-PAGE、Western blot、质谱分析数据证明了蛋白是Visfatin融合蛋白。结论:成功的表达并纯化得到带HIS标签的Visfatin融合蛋白。 相似文献
3.
4.
一株嗜热梭菌β-葡聚糖酶基因的克隆和表达 总被引：1，自引：0，他引：1
提取嗜热梭菌(Clostridium.sp)基因组DNA,首次通过PCR克隆了该菌的β-葡聚糖酶基因全长,结果表明:该基因全长1040bp,ORF为1003bp,编码334个氨基酸,计算分子量为37.8kD,等电点为7.67。经Blast分析,该序列与热纤梭菌同源性最高(99%),而与基因库中嗜热梭菌的同源性为94%,该基因已被GenBank接受(AY225318)。用BamHⅠ和XhoⅠ双酶切目的片段和表达载体pET鄄30a( )后相连接,构建重组表达载体pET鄄clo,并导入BL21细菌中表达.酶学特性表明:SDS鄄PAGE电泳在37kD左右有表达蛋白带,该工程菌最适酶活29.4U/mL,是出发菌的15倍,最适温度在80℃左右,最适pH在9左右。该工程菌可构建耐热性好、酶活高的杂合基因工程菌。 相似文献
5.
《科学中国人》2015,(26)
目的构建SLO原核表达质粒,重组蛋白的诱导表达并纯化。方法提取链霉菌溶血素O模板DNA,PCR法扩增slo基因。构建融合表达重组质粒p GEX-6p-1-slo和pet32a-tev-slo,将正确的重组质粒转化至大肠杆菌BL21和大肠杆菌BL21-DE3,使用异丙基硫代βD半乳糖苷(IPTG)诱导表达重组融合蛋白。采用亲和层析纯化重组蛋白,切除标签后,再通过亲和层析纯化,获取SLO重组蛋白。结果 PCR扩增出slo基因,基因片段(1700 bp)与理论一致。经SDSPAGE,蛋白质印迹显示重组蛋白相对分子质量为55 k Da,与数据库中的相对分子质量结果相符。结论成功构建了slo原核表达质粒,表达并纯化了SLO重组蛋白。 相似文献
6.
以硫磺矿硫化叶茼基因组DNA为模板进行PCR扩增，得到其淀粉酶基因。将该片段与载体pSBPYF连接后转化枯草茅孢杆菌DB1342，得到重组菌株DB1342（pSBA），对该菌株进行培养，经蔗糖诱导表达，在其上清中可检测到α-淀粉酶活力，比空载体的淀粉酶活力高了5倍多。该酶作用的最适pH值为4．5，最适作用温度为80℃，在酶液中添加不同的金属离子均降低酶活。 相似文献
7.
《科技风》2017,(17)
对一株产纤维素酶的黄曲霉进行发酵条件优化和酶学性质初步探究,利用DNS法测定CMC酶活、滤纸酶活,结果显示此菌株纤维素酶最佳反应温度为50℃,最佳反应pH为7.0。在金属离子中,发现Zn~(2+)具有抑制作用,最大抑制浓度为0.4 mg/m L,Mg~(2+)、Mn~(2+)、Ca~(2+)、Fe~(3+)、Fe~(2+)具有促进作用,最适浓度分别为0.4、1、1、0.8、1.4 mg/mL。蛋白胨和酵母粉具有促进作用,最适浓度分别为1.0%、0.6%,最适pH为6.0。综合优化后,测得滤纸酶活和CMC酶活分别为7.73 U/mL、11.28 U/mL,相比优化前提高了38.8%和43.7%。 相似文献
8.
9.
本研究根据马、驴线粒体DNA中的保守区段设计了一对引物，通过聚合酶链式反应（polymerase chain reaction，PCR）采取马、驴、牛、羊、猪、鸡、兔、鹿、火鸡等实验材料进行扩增，可以专一地扩增出马、驴源性成分的DNA片段，再经限制性内切酶Sau3A和Alu I的酶切鉴定可以区分马源性成分和驴源性成分，PCR扩增产物的测序结果验证了酶切鉴定结果的正确性。引物灵敏度测试结果表明该方法的检测低限均达0．3％，该对引物可以成为检测马、驴源性成分高效准确的检测工具。 相似文献
10.
本研究根据马、驴线粒体DNA中的保守区段设计了一对引物,通过聚合酶链式反应(polymerase chain reaction,PCR)采取马、驴、牛、羊、猪、鸡、兔、鹿、火鸡等实验材料进行扩增,可以专一地扩增出马、驴源性成分的DNA片段,再经限制性内切酶Sau 3A和Alu Ⅰ的酶切鉴定可以区分马源性成分和驴源性成分,PCR扩增产物的测序结果验证了酶切鉴定结果的正确性.引物灵敏度测试结果表明该方法的检测低限均达0.3%,该对引物可以成为检测马、驴源性成分高效准确的检测工具. 相似文献
11.
12.
《Electronic Journal of Biotechnology》2014,17(6):311-316
BackgroundLysozyme plays a crucial role in innate immunity with its well-recognized bacteriolytic activity. In this study, the influence of expression parameters (inoculation volume, culture volume, growth time, induction temperature and time, initial pH and methanol concentration) on human lysozyme (HLZ) production in recombinant P. pastoris SMD1168 was investigated through Plackett–Burman (PB) design and response surface methodology (RSM).ResultsIt was revealed that induction temperature, induction time and culture volume had significant influence (P < 0.01) on HLZ expression level, which were elected for further optimization with three-dimensional response surface designs for enhanced HLZ production. The highest lysozyme activity reached 3301 U/mL under optimized conditions (at 23.5°C for 90 h with culture volume of 48 mL) in shake flask, which increased 2.2 fold compared with that achieved with the standard protocol (Invitrogen). When high-cell-density fermentation of the recombinant Pichia pastoris was performed in a 15 L fermenter under optimized conditions, the extracellular lysozyme activity reached 47,680 U/mL. SDS-PAGE analysis of the product demonstrated that HLZ was produced as a single major protein with a molecular weight of approximately 14.7 kDa, consistent with its expected size.ConclusionsThe results indicated that the optimized culture conditions using PB design and RSM significantly enhanced the expression level of HLZ, and the Pichia expression system for HLZ production was successful and industrially promising. 相似文献
13.
BackgroundPhospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD.ResultsStreptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20°C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105.81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression.ConclusionsAfter combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.How to cite: Wu R, Cao J, Liu F, et al. High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization. Electron J Biotechnol 2021;50.https://doi.org/10.1016/j.ejbt.2020.12.002 相似文献
14.
旨在研究大米糖浆取代乳糖后对早期断奶仔猪血液生化及激素指标的影响。方法是采用7d断奶的二元杂交仔猪（长白×大白）32头,随机分为2个处理组,每组16头,每组设16个重复,每个重复1只。日粮成分除乳糖水平外（对照组和处理组分别为43.5%和0%）,其他成分均一致。试验期14d,分别于试验第7天和第14天每组随机抽取5头,颈静脉采血制备血清。结果表明,14d处理组的胆固醇显著低于对照组（p<0.05）,而在21d对照组与处理组间差异不显著（p>0.44）。14d和21d对照组血清总蛋白、总甘油三酯、尿素氮及胰岛素与处理组间差异均不显著（p>0.05）。14d对照组与处理组间血清胃泌素水平差异不显著（p>0.05）,21d对照组血清胃泌素水平显著高于处理组（p<0.05）。14d对照组T3水平显著高于处理组（p<0.05）,21d T3水平对照组与处理组间差异均不显著（p>0.05）。14d对照组血清T4水平比处理组高,但差异不显著（p>0.05）,21d对照组血清T4水平极显著高于处理组（p<0.01）。综合分析认为,乳糖具有促进早期断奶仔猪肠道吸收胆固醇的作用,但对早期断奶仔猪的脂肪和蛋白质代谢没有影响,此外胃可能不是早期断奶仔猪消化乳糖的主要部位,低乳糖水平及大米糖浆具有降低胃PH的作用。 相似文献
15.
16.
肿瘤多药耐药 (multidrugresistance ,MDR)是临床化疗成功最为严重的障碍 .首先阐明了新拓扑异构酶II抑制剂沙尔威辛对MDR肿瘤细胞直接的细胞毒性作用及下调mdr 1基因和P 糖蛋白的作用 .沙尔威辛能有效杀伤MDR细胞株 ,如K5 62 A0 2 ,KB VCR和MCF 7 ADR细胞 ,其杀伤能力与对相应亲本细胞相当 ,而明显强于几种临床常用的抗癌药物 .沙尔威辛下调mdr 1基因和P 糖蛋白的表达 ,但并不影响MRP和LRP基因 .其次 ,揭示了转录因子c jun的激活 ,在沙尔威辛下调K5 62 A0 2细胞内mdr 1基因表达及诱导凋亡过程中起着关键作用 .沙尔威辛增加K5 62 A0 2细胞的c jun表达明显早于其减少mdr 1基因的表达 ;c jun反义寡核苷酸消除沙尔威辛升高c jun蛋白、下调mdr 1基因表达的作用 .沙尔威辛还促进JNK和c jun磷酸化并增强转录因子AP1的DNA结合活性 .此外 ,c jun反义寡核苷酸还抑制沙尔威辛的凋亡诱导和细胞毒性作用 .最后 ,进一步研究发现沙尔威辛本身不引起MDR表型 .成功建立了对沙尔威辛具有 8 91倍耐药的A5 4 9 SAL细胞株 .该细胞株对抗代谢药产生 6.70倍的耐药 ,但对多种其他天然来源的抗肿瘤药物、烷化剂以及铂类化合物则缺乏交叉耐药性 . 相似文献
17.
18.
Little is known about host-parasite inter-relationship in the lymphatic filarial parasites. There is no information available
about the ability of these parasites to acquire cholesterol, though it is known that in general, nematodes lack the ability
to synthesise cholesterolde novo. In this study, we have shown that the filarial parasites also lack the ability to incorporate labelled acetate into cholesterol,
indicating the absence of the machinery for cholesterol biosynthesis. We have further shown that they elaborate a 43 kDa surface
receptor for acquiring LDL-bound cholesterol. We have shown by polymerase chain reaction the presence of a 860 bp fragment
indicating the presence of the gene for LDL-related protein (LRP) in the human filarial parasiteWuchereria bancrofti in the genomic DNA. We have also shown that it is expressed as seen in the cDNA clones identified from an expression library. 相似文献
19.
20.
根据JK触发器的功能,通过观察和分析同步十进制加法驻地数器的输出波形,找出各JK触发器的连接方式,从而完成同步十进制加法计数器的设计。 相似文献