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131.
132.
利用PCR方法对单纯疱疹病毒Ⅱ型糖蛋白D(HSV-2gD)基因进行了修饰,在其5'端删去约500bp的非编码区,仅保留ATG上游7个bp。将修饰后的HSV-2gD基因插入到带有痘苗病毒天坛株TK基因区段的痘苗表达质粒pJSA1175,置于痘苗病毒P7.5k早/晚期启动子控制下。将此重组质粒用脂质体Lipofectin方法转染已受野型TK ̄+痘苗病毒天坛株感染的TK ̄-143细胞,通过同源重组机制和标志基因LacZ产物的蓝斑显色作用,以及BudR试剂对TK表型的选择压力,筛选出整合有HSV-2gD基因的重组痘苗病毒。Southem杂交表明,HSV-2gD基因已正确地插入痘苗病毒TK基因区内;间接免疫荧光检测显示,HSV-2gD蛋白已得到有效表达,且主要分布于细胞膜。重组病毒免疫家兔可产生明显的抗HSV-2gD中和抗体。用重组病毒免疫小鼠,3周后可使94%(17/18)的小鼠对抗HSV-2的致死量攻击,表明重组病毒具有明显的免疫保护作用。 相似文献
133.
酪氨酸羟化酶基因载体-pCMVTH 质粒的构建及在大鼠骨骼肌内的表达 总被引:4,自引:0,他引:4
为用转基因方法治疗巴金森氏病大鼠模型,本研究采用分子克隆技术,将合成多巴胺的关键酶-酪氨酸羟化酶(TH)的基因,克隆进入以巨细胞病毒CMV为启动子的载体质粒内,经限制性内切酶定位分析证实该重组的DNA质粒的可靠性。携带TH基因的PCMVTH质粒以LIPO-FECTIN介导,在培养的原代骨骼肌细胞中高效表达。本研究为进一步用转基因的细胞植入脑内以治疗巴金森氏病打下一定基础。 相似文献
134.
PCR法快速检测临床标本中结核杆菌DNA 总被引:2,自引:0,他引:2
应用聚合酶链反应(PCR)快速检测临床标本(脑脊液、胸水、腹水、血、痰液)中的结核杆菌DNA,特异性扩增片段123bp,为结核杆菌的特异性重复序列IS6110部分基因。PCR检测人型结核杆菌的敏感性达10fgDNA。临床标本的PCR检测阳性率(23.3%)明显高于抗酸染色涂片(2.9%)和细菌培养(5.7%)的阳性率(P〈0.05)。通过设立对照系统及对扩增产物酶切分析,表明该法无假阴性结果(特异 相似文献
135.
介绍用交流示波极谱滴定法测混合氨基酸的含量。在KCl底液中,利用HCHO将-NH2封闭,用KOH标准液滴定,HCHO同时做指示剂,以极谱图形的敏锐变化指示终点的到过,测定结果准确。 相似文献
136.
Bi(Ⅲ)与金属硫蛋白作用性质研究张保林,黄辉,朱凌燕,岳晟,唐雯霞(南京大学配位化学研究所,配位化学国家重点实验室,南京210093)如何降低顺铂或其它抗癌铂的毒性,一直是癌症化疗中的重要课题之一,最近研究发现预先给大鼠或肺癌病人服用铋盐,可以极大... 相似文献
137.
Construction of a novel bifunctional biogenic amine receptor by two point mutations of the H2-histamine receptor. 下载免费PDF全文
J. DelValle I. Gantz L. Wang Y. J. Guo G. Munzert T. Tashiro Y. Konda T. Yamada 《Molecular medicine (Cambridge, Mass.)》1995,1(3):280-286
BACKGROUND: H2-histamine receptors mediate a wide range of physiological functions extending from stimulation of gastric acid secretion to induction of human promyelocyte differentiation. We have previously cloned the H2-histamine receptor gene and noted that only three amino acids on the receptor were sufficient to define its specificity and selectivity. Despite only modest overall amino acid homology (34% amino acid identity and 57.5% similarity) between the H2-histamine receptor and the receptor for another monoamine, the beta 2-adrenergic receptor, there is remarkable similarity at their critical ligand binding sites. We hypothesized that, if the specificity and selectivity of both receptors are invested in just three amino acids, it should be possible to convert one of the receptors into one that recognizes the ligand of the other by simple mutations at only one or two sites. MATERIAL AND METHODS: We explored the effect of two single mutations in the fifth transmembrane domain of the H2-histamine receptor, which encompasses the sites that determine H2 selectivity. The canine H2 receptor gene was mutated at Asp186 and Gly187 (Asp186 to Ala186 and Gly187 to Ser187) by oligonuceotide directed mutagenesis. The coding region of both the wild-type and mutated H2 receptors was subcloned into the eukaryotic expression vector, CMVneo, and stably transfected into Hepa cells and L cells. The biological activity of histamine and epinephrine on the expressed receptor was examined by measurement of cellular cAMP production and inositol trisphosphate formation. RESULTS: Hepa cells transfected with the Ala186-Ser187 mutant H2 receptor demonstrated a biphasic rise in cAMP in response to epinephrine with an early phase (ED50 approximately 10(-11) M) that could be inhibited by both propranolol and cimetidine. Epinephrine also induced IP3 generation in the same cells, a biological response that is characteristic of activation of the wild-type H2 but not of the beta-adrenergic receptor. L cells transfected with the Ala186-Ser187 mutant H2 receptor also responded to epinephrine in a cimetidine and propranolol inhibitable manner. CONCLUSIONS: We converted the H2-histamine receptor into a bifunctional one that has characteristics of both histamine and adrenergic receptors by two simple mutations. These results support the hypothesis that ligand specificity is determined by only a few key points on a receptor regardless of the structure of the remainder of the molecule. Our studies have important implications on the design of pharmacological agents targeted for action at physiological receptors. 相似文献
138.
小麦条锈菌毒性小种及其无毒性突变型侵染初期,是不亲和反应的小麦叶片内可翻译mRNA水平迅速增加,而呈亲和反应叶片的增加幅度小且滞后。同时前者的Poly(A+)-RNA水平高于未接种对照,后者低于对照。32P标记实验证实不亲和反应叶片Poly(A+)-RNA的合成增加早于亲和反应叶片。Poly(A+)-RNA体外翻译产物经SDS-PAGE分离后,放射自显影图谱显示一些多肽条带的35S-Met相对掺入量有定量差异。 相似文献
139.
S. W. Guo 《American journal of human genetics》1994,54(6):1104-1109
I provide a novel approach to computing the mean and variance of the proportion of genetic material shared identical by descent (IBD) by sibling pairs in a specified chromosomal region, conditional on observed marker data. I first show that each chromosome in an offspring can be represented by a two-state Markov chain, with the time parameter being the map distance along the chromosome. On this basis, I show that IBD proportion can be written as a stochastic integral and that the computation of its mean and variance can be reduced to evaluation of an integral of some elementary functions. In addition, I show how Goldgar's model can be extended to include dominance effects. Several examples are provided to illustrate the calculation. 相似文献
140.
Studies of cell pellets: I. Electrical properties and porosity. 总被引:2,自引:1,他引:1
Cell pellets formed by centrifugation provided a good system to study the osmotic behavior, electroporation, and interaction between cells. Rabbit erythrocyte pellets were used in this study because they were simpler than nucleated cells to model analytically. Structurally, cell pellets possessed properties of porous solid bodies and gels. Electrically, cell pellets were shown to behave as a parallel set of resistance, Rp, and capacitance, Cp. Information on pellet structures was obtained from electric measurements. The pellet resistance reflected the intercellular conductivity (porosity and gap conductivity), whereas the pellet capacitance depended mostly on membrane capacitance. The pellet resistance was more sensitive to experimental conditions. The intercellular gap distance can be derived from pellet porosity measurements, providing the cell volume and surface area were known. Rp increased and relaxed exponentially with time when centrifugation started and stopped; the cycles were reversible. When supernatants were exchanged with solutions containing hypotonic electrolytes or macromolecules (such as PEG) after the pellets were formed, complicated responses to different colloidal osmotic effects were observed. A transient decrease followed by a large increase of Rp was observed after the application of a porating electric pulse, as expected from a momentary membrane breakdown, followed by a limited colloidal-osmotic swelling of pelleted cells. The equilibrium values of Rp, Cp, pellet porosity, and intercellular distances were measured and calculated as functions of cell number, centrifugation force, and ionic strength of the exchanged supernatant. Thus, the structure and properties of cell pellets can be completely characterized by electrical measurements. 相似文献