从噬菌体文库中筛选与表皮生长因子结合的多肽

与许多疾病相关的血管生成作用是由一些血管生成因子介导的 ,其中就包括表皮生长因子 .在肿瘤生长、关节炎等疾病中 ,表皮生长因子参与了其中的血管生成作用 ,拮抗表皮生长因子介导的血管生成就有可能对与其相关的疾病起到治疗作用 ,因此 ,表皮生长因子的拮抗剂可能具有重要的临床价值 .拮抗表皮生长因子的作用可以通过许多途径 ,其中之一就是找到能与表皮生长因子结合并能干预其与受体结合的分子 ,因而表皮生长因子可作为药物靶分子 .从噬菌体文库中筛选药物靶分子的拮抗剂和激动剂已被证明是一种有效的方法 .以表皮生长因子作为药物靶分子 ,从多肽噬菌体文库中筛选与表皮生长因子结合的噬菌体多肽 ,这些潜在的表皮生长因子拮抗剂先导分子经过优化可能具有重要的临床价值 .     

从噬菌体文库中筛选潜在的GMCSF的拮抗剂(英文)

以粒细胞巨噬细胞集落刺激因子（ＧＭＣＳＦ） 为筛选文库的靶分子, 通过高效筛选（Ｈｉｇｈ ｔｈｒｏｕｇｈｐｕｔｓｃｒｅｅｎｉｎｇ, ＨＴＳ） 方法来筛选多种多肽噬菌体文库, 在一个以噬菌体主要蛋白质为载体的多肽噬菌体文库中筛选到了一些与ＧＭＣＳＦ结合的多肽, 并通过了ＥＬＩＳＡ和微淘选（ｍｉｃｒｏｐａｎｎｉｎｇ） 实验的证实。这些多肽先导化合物经过进一步的优化, 可能成为ＧＭＣＳＦ细胞因子的拮抗剂     

从噬菌体文库中筛选潜在的GM—CSF的拮抗剂

以粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）为筛选文库的靶分子,通过高效筛选（Ｈｉｇｈｔｈｒｏｕｇｈｐｕｔｓｃｒｅｅｎｉｎｇ,ＨＴＳ）方法来筛选多种多肽噬菌体文库,在一个以噬菌体主要蛋白质为载体的多肽噬菌体文库中筛选到了一些与ＧＭ－ＣＳＦ结合的多肽,并通过了ＥＬＩＳＡ和微淘选（ｍｉｃｒｏｐａｎｎｉｎｇ）实验的证实,这些多肽先导化合物经过进一步的优化,可能成为ＧＭ－ＣＳＦ细胞因子的拮抗剂。     

从噬菌体多肽文库中筛选α—葡萄糖苷酶的抑制剂

以黑曲霉葡糖淀粉酶为靶分子,筛选噬菌体展示的多肽文库,经过3轮生物淘选法筛选,得到3个特异性结合的噬菌体克隆,分别命名为GB-1、GB2和GB-3。其插入多肽中都含有一对二硫键,破坏二硫键将大大降低噬菌体与葡糖淀粉酶的结合。化学合成的环状多肽KCHFEECLAY,其序列对应于结合力最强的一个克隆GB-1的N端的10个氨基酸残基,该多肽可以竞争性地抑制黑曲霉葡糖淀粉酶（Ki=0.2mmol/L)以及大鼠小肠的α-葡萄糖苷酶（Ki=1.4mmol/L)。     

用氨甲蝶呤偶联琼脂糖亲和吸附法从人肝脏cDNA噬菌体展示文库中筛选氨甲蝶呤相互作用蛋白

目的:利用氨甲蝶呤(MTX)偶联琼脂糖凝胶吸附法从人肝脏细胞cDNA噬菌体展示文库中筛选与MTX相互作用的蛋白.方法:以偶联于琼脂糖凝胶表面的MTX为配基,通过"结合-洗脱-扩增"过程筛选与MTX相互作用的噬菌体.利用PCR对筛选结果进行监测,对筛选得到的噬菌体PCR产物进行序列测定和基因同源性分析.结果:通过五轮亲和筛选富集到特异噬菌体克隆,再通过PCR获得cDNA插入片段.通过BLAST程序搜索GenBank,证明筛选到的片段与人PI-3K相关蛋白激酶SMG-1异构体1蛋白同源性达100%.结论:利用偶联MTX的琼脂糖凝胶作为筛选基质,从T7噬菌体展示cDNA文库中富集特异噬菌体是一种方便、高效的MTX相互作用靶蛋白筛选方法,可为探讨小分子药物的分子作用机制提供借鉴和参考.     

以噬菌体作为配基的一种新型亲和材料的制备方法研究

目的:针对亲和层析材料制备困难的问题,本文拟研究以聚丙烯酰胺冷凝胶为基质,以噬菌体展示多肽为配基制备亲和材料方法的可行性.方法:以低温聚合制备的聚丙烯酰胺冷凝胶作为基质,将筛选噬菌体展示人肝脏cDNA文库获得的噬菌体作为配基,通过两种不同的化学键合方法键合于凝胶表面,制备亲和材料.通过高效液相色谱法(HPLC)分析此亲和材料对药物分子是否具有亲和能力.结果:聚丙烯酰胺冷凝胶具有较大的孔径及良好的亲水性,适用于生物大分子如噬菌体的键合,键合特异噬菌体展示多肽的凝胶材料与键合未筛选噬菌体文库的亲和材料比较,对药物分子具有明显的高亲和力.结论:以展示特异多肽的噬菌体为亲和配基,以聚丙烯酰胺冷凝胶为基质,通过化学键和方法制备亲和材料是具有可行性的,此种亲和材料在生物分子纯化特别是药物分析纯化、蛋白质分离纯化以及细胞分离分析等方面将具有一定的应用前景.     

利用氨甲蝶呤－琼脂糖凝胶进行噬菌体展示人肝脏cDNA文库筛选的方法研究

目的：利用氨甲蝶呤（MTX）偶联琼脂糖凝胶吸附法从人肝脏细胞cDNA噬菌体展示文库中筛选与MTX相互作用的蛋白。方法：以偶联于琼脂糖凝胶表面的MTX为配基,通过"结合－洗脱－扩增"过程筛选与MTX相互作用的噬菌体。利用PCR对筛选结果进行监测,对筛选得到的噬菌体PCR产物进行序列测定和基因同源性分析。结果：通过五轮亲和筛选富集到特异噬菌体克隆,再通过PCR获得cDNA插入片段。通过BLAST程序搜索GenBank,证明筛选到的片段与人PI-3K相关蛋白激酶 SMG-1异构体1 蛋白同源性达100％。结论：利用偶联MTX的琼脂糖凝胶作为筛选基质,从T7噬菌体展示cDNA文库中富集特异噬菌体是一种方便、高效的MTX相互作用靶蛋白筛选方法。本方法可为探讨小分子药物的分子作用机制提供借鉴和参考.     

从噬菌体展示的构象限制性随机肽库中筛选有 trkB 结合活性的小肽

脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)能够促进多种神经元的存活和分化, 在神经退行性疾病的治疗中具有广阔的应用前景, 但由于其分子量大而无法穿过血脑屏障, 限制了其临床应用. 以表达于NIH 3T3细胞上的BDNF受体trkB为靶分子, 从噬菌体展示的随机肽库中筛选与trkB有亲和力的小肽, 采用不表达trkB 的NIH 3T3细胞预吸附和最后一轮筛选以1 μg/mL的BDNF竞争性洗脱策略, 使能与trkB天然构象结合、又能有效拮抗BDNF与trkB结合的特异性噬菌体得到了有效富集, 获得了5种trkB结合小肽, 它们有核心序列CRA/TXφXXφXXC(X代表随机残基, φ为T, L或I). 对展示有5种外源小肽的噬菌体进行了初步生物活性测定, 未发现有BDNF样活性, 反而能剂量依赖性地拮抗BDNF的生物功能. 依据其中一个克隆序列(C1)合成的肽也证实有拮抗BDNF的作用, 表明获得的5种小肽可能为trkB受体拮抗剂. 这些trkB拮抗性小肽在治疗神经母细胞瘤和慢性痛以及神经生物学研究中具有应用前景, 同时也为构建二级噬菌体展示肽库、继续筛选BDNF模拟小肽奠定了基础.     

噬菌体展示肽文库及其应用

肽库是研究与特定靶分子高亲和结合配体的有力工具,肽文库可被分为合成文库和噬菌体肽文库两类,作者综述了噬菌体肽文库及其蛋白质结构域分析定位,疫苗研制及抗原表位分析和药物设计等方面的应用。     

结核病阳性血清特异结合蛋白的筛选与鉴定

[目的]从T7噬菌体展示的结核分枝杆菌基因组DNA文库中筛选结核病阳性血清特异结合蛋白。[方法]以饱和硫酸铵法初步纯化的结核病阳性血清为靶分子,对结核分枝杆菌基因组DNA文库进行3轮生物淘选; PCR扩增阳性噬菌体的外源DNA片段,测序后进行BLAST分析;间接ELISA和斑点免疫印迹试验检测阳性噬菌体与结核病阳性血清能否特异结合。[结果]经过3轮生物淘选,与结核病阳性血清特异结合的噬菌体得到明显富集; BLAST结果表明随机挑取的19个阳性噬菌体包括4种不同的序列,其中编码核糖激酶的序列出现次数最多;这些代表性噬菌体均能与结核病阳性血清特异结合。[结论]成功筛选到能与结核病阳性血清特异结合的4种蛋白,其中核糖激酶可能为与结核病阳性血清特异结合的优势抗原。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细