人NANOGP8蛋白的原核表达及多克隆抗体的制备

为获得抗人 NANOGP8基因的多克隆抗体,采用PCR法扩增了NANOGP8基因,经序列测定正确后,连接到pET-28a质粒上,将获得的重组质粒pET-28a-NANOGP8转化到大肠杆菌BL21（DE3）中,在16℃和37℃下分别进行诱导表达,经SDS-PAGE检测发现融合蛋白以包涵体形式存在于37℃诱导表达的沉淀中,大小约为45kDa。对包涵体进行洗涤、溶解处理,经Chelating Sepharose Fast Flow亲和层析柱进行纯化。对纯化得到的蛋白进行SDS-PAGE及Western blot免疫印记杂交检测,得到了单一条带。用获得的高纯度融合蛋白免疫新西兰兔,得到兔来源的抗人NANOGP8血清。对抗血清进行蛋白免疫印记杂交反应和酶联免疫吸附反应检测,结果显示成功制备了高滴度和特异性的兔抗人NANOGP8多克隆抗体,与市售抗人Nanog多克隆抗体相比,杂交更迅速,效果更明显。为进一步研究NANOGP8基因在肿瘤发生、发展中的作用奠定了基础。     

果蝇心脏发育标记基因lbe的多克隆抗体制备

lbe是已经证明的心脏标记基因。为了深入研究lbe在心脏发育中的功能,需要获得lbe蛋白并制备其抗体。首先提取野生型成体果蝇的总RNA,反转录获得其cDNA文库,通过PCR克隆出lbe编码区序列,将其连接到pET-28a原核表达载体上。经酶切及测序鉴定后,质粒构建成功。将重组质粒（pET-28a-lbe）转化大肠杆菌菌株Rosseta,用IPTG诱导表达出融合蛋白,经Ni-IDA凝胶柱纯化后,最后将纯化的融合蛋白免疫新西兰大白兔制备lbe多克隆抗体,并用Western blotting检测抗体的效价和特异性。结果显示获得了lbe原核表达重组融合蛋白及高效价的特异性兔抗lbe多克隆抗体,为lbe功能的进一步研究奠定了基础。     

猪FcγRIII的原核表达及多克隆抗体的制备

目的：构建猪FcγRIII 基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪 FcγRIII 抗血清。方法：从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII 原核表达载体pET-FcγRIII ,转化大肠杆菌BL21 (DE3) ,IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His 为抗原免疫小鼠,获得抗血清。Western blotting、ELISA 法鉴定获得的抗血清,ELISA 结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果：成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA 法证实多克隆抗体制备成功。结论：成功获得了猪 FcγRIII 多克隆抗体,为进一步研究猪FcγRIII 蛋白的功能奠定了基础。     

猪FcγRⅢ的原核表达及多克隆抗体的制备

目的:构建猪FcγRIII基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪FcγRIII抗血清。方法:从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII原核表达载体pET-FcγRIII,转化大肠杆菌BL21(DE3),IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His为抗原免疫小鼠,获得抗血清。Western blotting、ELISA法鉴定获得的抗血清,ELISA结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果:成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论:成功获得了猪FcγRIII多克隆抗体,为进一步研究猪FcγRIII蛋白的功能奠定了基础。     

Nono蛋白的原核表达、纯化和多克隆抗体的制备

目的表达和纯化带多聚组氨酸（6×His）标签的Nono （ non-POU-domain-containing, octamer-binding protein ）融合蛋白并制备抗Nono多克隆抗体。方法构建pET-28a（＋）-Nono重组表达质粒,转入Rosetta（DE3）大肠埃希菌,以IPTG诱导6×His-Nono融合蛋白表达,经镍离子金属螯合树脂纯化后,用纯化出的蛋白免疫BALB／C小鼠制备多克隆抗体,并用ELISA检测多克隆抗体的效价,Western印迹检测多克隆抗体的特异性。结果在大肠埃希菌中诱导出高水平表达的His-Nono融合蛋白,经亲和树脂纯化后免疫小鼠,获得了高特异性的抗Nono抗血清。结论成功构建pET-28a（＋）-Nono原核表达质粒,表达并纯化出高纯度的目标蛋白,制备出高滴度、高特异性的多克隆抗体。     

杆状病毒AcMNPV vp39基因的克隆、表达与抗体制备

目的:构建苜蓿丫纹夜蛾核多角体病毒(Autographa californica nucleopolyhedro virus,AcMNPV)VP39的原核表达载体,表达、纯化蛋白并制备多克隆抗体。方法:用PCR方法扩增vp39基因,并将其克隆至pET-21a( )上,转化到大肠杆菌BL21(DE3)中进行诱导表达,采用割胶回收的方法纯化融合蛋白,纯化的融合蛋白作为抗原,免疫新西兰大白兔,Western blot检测抗体活性。结果:构建了pET-VP39原核表达质粒,含有该质粒的大肠杆菌经IPTG诱导超量表达了一个与预期理论值相符的约为40kDa的融合蛋白。对制备的抗体进行免疫印迹分析表明该抗血清能与感染苜蓿丫纹夜蛾核多角体病毒的细胞蛋白样品发生特异性反应。结论:获得了兔抗AcMNPV-VP39多克隆抗体,为进一步深入研究VP39在病毒侵染过程中与宿主因子的相互作用提供了检测工具。     

棉铃虫病毒gp41基因的克隆表达及抗体制备

根据棉铃虫单核衣壳核多角体病毒(Helicoverpa armigerasingle nucleocapsid nucleopolyhedrovirus,HaSNPV)gp41基因的序列,设计引物,引入适当的酶切位点,通过PCR的方法扩增目的片段。将扩增出的基因片段克隆至原核表达载体pET-28a,构建重组质粒并转化至大肠杆菌中,经IPTG诱导表达。纯化蛋白产物并免疫家兔产生抗血清。该抗血清可与原核表达的His-GP41融合蛋白及在感染的昆虫细胞中表达的GP41蛋白发生特异性免疫反应。该抗体的获得为深入研究GP41的功能提供了基础。     

重组丙型肝炎病毒核心蛋白的原核表达、纯化和抗体制备

本文旨在获得纯化丙型肝炎病毒(HCV)核心蛋白(HCV-C)及抗HCV-C多克隆抗体,为深入研究HCV-C与肝细胞相互作用的分子机制奠定基础。首先以HCV1b亚型HC-J4-91全基因组质粒为模板,聚合酶链反应(PCR)扩增HCV-C基因,构建重组质粒pQE31-HCV-C。融合蛋白经原核表达、纯化后,免疫BALB/c小鼠,制备抗HCV-C多克隆抗体。利用酶联免疫吸附试验(ELISA)检测抗体效价,蛋白免疫印迹(Westernblot)和间接免疫荧光染色鉴定抗体特异性。结果显示,表达HCV-C的原核表达质粒pQE31-HCV-C构建正确,获得相对分子质量约22000的纯化融合蛋白。ELISA检测重组蛋白免疫小鼠的抗血清效价达1:12800。结果显示,自制的抗HCV-C多克隆抗体能特异性识别HCV-C。本研究获得了纯度较好、原核表达的HCV-C,并成功制备了抗HCV-C多克隆抗体,为深入研究HCV-C的致病机制提供了有实用价值的研究工具。     

Tamdy病毒糖蛋白基因的重组表达及抗体制备

原核表达Tamdy病毒（Tamdy virus,TAMV）糖蛋白Gn和Gc,分别制备兔抗融合蛋白Gn和Gc多克隆抗体。利用RT-PCR扩增Gn和Gc基因片段,分别构建原核表达质粒pET-28a-Gn和pET-32a-Gc,并在E.coli BL21(DE3)中诱导表达,镍柱亲和层析纯化融合蛋白Gn和Gc之后,以皮下注射方式分别免疫新西兰兔,制备兔抗融合蛋白Gn和Gc多克隆抗体。经Western Blotting和间接免疫荧光（IFA）鉴定多克隆抗体抗原识别能力,ELISA法测定抗体效价。结果显示,pET-28a-Gn、pET-32a-Gc原核表达质粒构建正确,融合蛋白Gn和Gc大小分别约为45 kD、74 kD,制备的兔抗融合蛋白Gn和Gc多克隆抗体能够分别识别原核表达的融合蛋白Gn和Gc和真核表达产物,效价分别为1∶409 600、1∶204 800。制备的多克隆抗体效价高,能够特异性识别原核系统和真核系统表达的TAMV糖蛋白,为TAMV的流行学分析提供基础。     

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