一种移码差错率大幅度下降的聚合酶N端缺失体的研究

采用双引物定变法,构建了以２３６位氨基酸为起始密码子的Ｔａｑ酶的高表达质粒,并以－１移码突变质粒ｐＦＤＰＭ１１８作为模板,建立了测定ＤＮＡ聚合酶的体外合成精确性的Ｇａｐｐｅｄ－ＤＮＡ系统。通过转化大肠杆菌ＴＧ１菌株皇计数Ｘ－ｇａｌ平板上蓝白菌落的比例,测定了Ｔａｑ和ＴａｑＮＤ２３６两种ＤＮＡ聚合酶在ＤＮＡ体外合成中的称码突变率,发现缺失后的ＴａｑＮＤ２３６复制精确性提高了１０倍以上。     

人GM—CSF cDNA的克隆和在大肠杆菌中的表达

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。     

粒细胞—巨噬细胞集落刺激因子／白细胞介素—3融合蛋白的表达研究

利用ＰＣＲ扩增得到粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）、白细胞介素－３（ＩＬ－３）完整基因片段,将其分别克隆ｐＧＥＭ－Ｔ构建成ＧＭ－ＣＳＦ／ＩＬ－３融合蛋白基因,ＤＮＡ序列与设计预期一致。将得到的融合蛋白基因克隆对７２ＲＮＡ聚合酶表达载体ｐＴ７ｚｚ,得到表达质粒ｐＦｕ,经转化至表达宿主Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）,在ＩＰＴＧ诱导下获得融合蛋白目的产物的直接表达。经ＳＤＳ－ＰＡＧＥ电泳鉴     

p16抑癌基因定点突变及其在大肠杆菌中的表达与纯化

为了研究错义突变对ｐ１６功能的影响,应用ＰＣＲ体外定点突变方法对ｐ１６ｃＤＮＡ进行体外定点突变,并将野生型和突变型ｐ１６ｃＤＮＡ克隆于ｐＧＥＸ－５Ｔ载体,在大肠杆菌中经ＩＰＴＧ诱导表达,Ｗｅｓｔｅｒｎ印迹鉴定确证表达．而后用谷胱甘肽－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化野生型和突变型ｐ１６融合蛋白．得到了第４８位密码子ＣＣＧ（Ｐｒｏ）→ＣＴＧ（Ｌｅｕ）突变的ｐ１６－Ｐ４８突变体,并在大肠杆菌中表达了４２ｋＤ的ＧＳＴ－ｐ１６和ＧＳＴ－ｐ１６Ｐ４８Ｌ融合蛋白．最后经纯化得到了野生和Ｐ４８Ｌ突变的ｐ１６融合蛋白     

人NMT的表达,纯化和活性研究

利用ＰＣＲ技术并进行ＤＮＡ序列测定,从人脑ｃＤＮＡ库中扩增得到人豆蔻酰ＣｏＡ蛋白Ｎ端豆蔻酰转移酶的编码基因,构建其在Ｔ７启动子控制下的成熟型和Ｈｉｓ６融合型的表达质粒ｐＭＦ－ｈＮＭＴ３和ｐＭＦＨＴ－ｈＮＭＴ２。转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。ＳＤＳ－ＰＡＧＥ分析结果显示,在３７℃条件下表达的各种重组ｈＮＭＲ几乎全是不溶性产物,但在较低温度条件下表达的Ｈｉｓ６－ｈＮＭＲ绝大     

人重组GDNF及其生物活性研究

从人胎脑组织中提取总ＲＮＡ,以ＲＴ－ＰＣＲ方法获取编码胶质细胞源神经营养因子（ＧＤＮＦ）成熟蛋白的ｃＤＮＡ。将人ＧＤＮＦｃＤＮＡ插入含Ｔ７启动子的质粒ｐＥＴ－２８ａ（＋）,构建表达质粒ｐＥＴ－ＧＤＮＦ,转化大场杆菌获得表达菌株ＢＬＧＤＮＦ,经诱导表达的ＧＤＮＦ形成包含体。凝胶自动扫描分析表明,表达量约占菌体总蛋白的３０％以上。用纯化的ＧＤＮＦ蛋白免疫新西兰兔制备了ＧＤＮＦ抗血清。纯化和复性的ＧＤＮ     

人Mn—SOD cDNA的克隆及高效表达

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     

重组FAS分子的表达、纯化及抗体制备

用ＰＣＲ法扩增出编码人ＦＡＳ分子胞外区的ｃＤＮＡ片段,直接克隆到ｐＧＥＭ－Ｔ载体上,经ＤＮＡ序列测定后,再插入到谷胱甘肽转硫酶（ＧＳＴ）融合蛋白表达载体ｐＧＥＸ－ＫＧ的ＥｃｏＲⅠ和ＳａｌⅠ位点之间,构成重组质粒ｐＫＧ－ｈＦＡＳ,将此质粒导入大肠杆菌,经ＩＰＴＧ诱导后获得ＧＳＴ－ｈＦＡＳ重组融合蛋白的表达,用谷胱甘肽偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ经亲合层析获得纯化的ＧＳＴ－ｈＦＡＳ蛋白,经凝血酶酶切和二次亲合层析去除ＧＳＴ部分,得到纯化的ＦＡＳ蛋白．用纯化的ＦＡＳ抗原免疫家兔制备了抗ＦＡＳ抗体,经检测发现抗ＦＡＳ抗体能诱导Ｕ９３７细胞发生细胞凋亡     

丙型肝炎病毒非结构区NS4b基因片段的克隆和表达

采用聚合酶链反应（ＰＣＲ）技术和ＤＮＡ体外重组方法,克隆出５７９ｂｐ的丙型肝炎病毒（ＨＣＶ）ＮＳ４ｂ基因片段,插入到原核高效表达载体ｐＥＴ－２８ａ中,构建重组质粒ｐＥＴ／ＮＳ４ｂ,转化大肠杆菌ＢＬ２１（ＤＥ３）菌株,经ＩＰＴＧ诱导培养后,获得了目的蛋白的高效表达。ＳＤＳ－ＰＡＧＥ分析显示在３０ｋＤ处有一条表达的目的蛋白区带。通过固定化金属配体亲和层析（ＩＭＡＣ）纯化目的的蛋白,ＥＬＥＳＡ检测结果表     

胰岛素受体的配体结合结构域的克隆与表达

用ＰＣＲ方法从人胎盘ｃＤＮＡ 中获得编码胰岛素受体α亚基中结合胰岛素的相对独立的结构域Ｌ１、Ｌ２以及人工设计的Ｌ１－（Ａｌａ）１０－Ｌ２的基因,克隆入含Ｔ７噬菌体ＲＮＡ聚合酶启动子的表达质粒ｐＥＴ－３ａ中,转化大肠杆菌ＢＬ２１（ＤＥ３）,用ＩＰＴＧ诱导表达成功。ＤＮＡ测序、氨基酸组成分析以及蛋白质Ｎ端测序证明所表达的蛋白质正确。经过包涵体的分离、洗涤、溶解和纯化,得到了纯的变性状态受体的胰岛素高亲     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

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.     

Principles of organization and evolution of systems of regulation of functions

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