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NMT在大肠杆菌中的His6融合表达及其纯化研究 总被引:2,自引:0,他引:2
将啤酒酵母NMT基因以N端融合6个组氨酸的形式在E.coliBL21(DE3)中进行了IPTG诱导的表达研究。SDS-PAGE分析确定有与His。-rNMT理论分子量一致的诱导表达条带,其表达量占全菌蛋白的10%左右;表达产物性质分析表明His-rNMT主要以可溶形式存在。在此基础上不受利用固定化金属离子配体亲和层析一步纯化目的蛋白,纯度可达95%以上。体外标脾性是His6-rNMT具有与成熟NM 相似文献
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人Mn—SOD cDNA的克隆及高效表达 总被引:13,自引:0,他引:13
用逆转录-聚合酶链反应(RT-PCR),以人肝细胞株(L02)总RNA为模板,扩增了人锰超氧化物歧化酶(hMn-SOD)的cDNA。重组到T7启动子控制下的表达载体pET-24a(+)中,构建表爱质粒pET-MnSOD,并转化大肠杆菌BL21(DE3)。SDS-PAGE及蛋白质印迹分析表明,经1mmol/L异丙基硫代-β-D-半乳糖苷(IPTG)诱导后,可高效表达一分子量为22kD的蛋白质,与抗人 相似文献
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利用基因工程技术,将质粒PYX382用XbaI和EcoRI切下插入的TGFa-PE40融合基因片段,连接到可表达载体PCB604的XbaI/EcoRI位点中,构建成新的重组质粒P2X-TP1。P2X-TP1转化E.coliBL21感受态菌后,在IPTG诱导下Ipp启动子转寻表达TGFa-PE40融合蛋白。表达产生物主要以包涵体形式沉积在细胞内,隔合蛋白表达量的高低与诱导时的细胞密度,诱导的温度以及 相似文献
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缩短的人巨噬细胞集落刺激因子(M—CSF)在大肠杆菌中… 总被引:1,自引:1,他引:0
本文用聚合酶链反应(PCR)获得了一个缩短的人巨噬细胞集落刺激因子cDNA基因,并克隆在质粒pAET3d中,在T7启动子指导下,在大肠杆菌BL2LysE中获得了和一个6组氨酸短肽标签的融合表达。重组的融合M-CSF表达量占菌体总蛋白的12%,表达产物一部分以不溶性包涵体形式存在,另一部分则以可溶性蛋白存在。经过金属螯合新和层析一步纯化,所得的融合(His)6-M-CSF在还原型SDS-PAGE上基 相似文献
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抗结肠癌相关抗原单链抗基因的构建和表达 总被引:3,自引:2,他引:1
通过PCR扩增和酶切分别得到抗结肠癌相关抗原抗体的重链可变区序列,轻链可变区序列及连接肽序列,将它们构建成为VH-linker-VL形式的单链抗体基因片段,并在大肠杆菌中进行了表达,SDS-PAGE分析结果表明,以pComb3为载体,在大肠杆菌JM83中,scFv未获得有效表达,而以pET-22b(+)为载体的scFv在大肠杆菌BL21(DE3)中,30℃诱导培养获得了高效表达,表达水平占全菌蛋白 相似文献
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人乳头瘤病毒16碧蓝晚期基因L1在大肠杆菌中的表达 总被引:1,自引:0,他引:1
人乳头瘤病毒(HPV)感染与宫颈癌的发生关系密切。其晚期蛋白L1是主要结构蛋白,可以刺激机体产生中和抗体,对病毒攻击可以起到保护作用。本研究将HPV16型中国分离株的L1基因定向克隆到原核表达载体pET-21c质粒中,建立HPV16 L1在大肠杆菌中的高效表达系统pET21c-HPV16 L1,该系统在IPTG的诱导下可表达60ku的L1蛋白,表达效率为23%,此蛋白通过Western-blot得 相似文献
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利用RT-PCR技术成功地从人胎盘组织中克隆了膜联蛋白V的cDNA,测序分析表明,与文献报道的核苷酸序列完全一致,交膜联蛋白V的cDNA克隆进T7启动子控制下的表达质粒pET-24a(+)中,经大肠杆菌BL1(DE3)后,经IPTG诱导可高效表达分子量为36kD的膜联蛋白V蛋白,表达产物以可溶形式存在。表达产物占菌体总蛋白的38%左右。表达产物经肝素亲和层析纯化后具有明显的延长部分凝血活酶时间的作 相似文献
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人GM—CSF cDNA的克隆和在大肠杆菌中的表达 总被引:3,自引:0,他引:3
从诱导的人胚肺细胞HFL株中提取总RNA.经RT-PCR反应获取了人GM-CSFcDNA,DNA序列测定表明其顺序与文献报道完全一致。为了获得高效表达,应用PCR改造了人GM-CSF的cDNA5’端核苷酸序列,并将改造的人GM-CSF基因插入含T7启动子的质粒pET-11d构建成表达质粒pETC-5,将此质粒转化大肠杆菌株BL21(DE3)得到表达菌株BLEC4。表达菌株用0.5mol/LIPTG诱导2小时后,产生大量重组蛋白并形成包涵体。SDS—PAGE电泳图谱扫描结果表明,rhGM-CSF产量占菌体总蛋白量的16%。ELISA和TF-1细胞培养测定表明,初步纯化和复性的rhGM-CSF具有天然的hGM-CSF生物活性。 相似文献
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In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980. 相似文献
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N. P. Vesselkin Yu. V. Natochin 《Journal of Evolutionary Biochemistry and Physiology》2010,46(6):592-603
Evolution of living organisms is closely connected with evolution of structure of the system of regulations and its mechanisms.
The functional ground of regulations is chemical signalization. As early as in unicellular organisms there is a set of signal
mechanisms providing their life activity and orientation in space and time. Subsequent evolution of ways of chemical signalization
followed the way of development of delivery pathways of chemical signal and development of mechanisms of its regulation. The
mechanism of chemical regulation of the signal interaction is discussed by the example of the specialized system of transduction
of signal from neuron to neuron, of effect of hormone on the epithelial cell and modulation of this effect. These mechanisms
are considered as the most important ways of the fine and precise adaptation of chemical signalization underlying functioning
of physiological systems and organs of the living organism 相似文献
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