基于人HMGB1-B box的抑制性小肽筛选及其抗炎功能分析

目的从噬菌体构象型7肽库中筛选人HMGB1-Bbox的抑制性小肽。方法以重组人HMGB1-Bbox为靶分子对噬菌体构象型7肽库进行6轮亲和筛选,获得Bbox结合的克隆,并经ELISA验证。选取亲和力高的克隆进行DNA测序,并推导出呈现的多肽序列,通过IL-6 ELISA检测噬菌体呈现的小肽对人HMGB1-B box致炎功能的抑制作用。结果经过6轮亲和筛选,噬菌体的回收率增加,阳性克隆得到富集。挑选15个结合力强的克隆进行测序,推导出2个多肽序列。所获两个阳性噬菌体克隆能特异性地抑制人HMGB1-B box刺激THP-1细胞产生炎症因子的能力。结论获得了噬菌体呈现的能够抑制人HMGB1-B box的两个小肽。     

噬菌体肽库筛选抗人B7-H4中和抗体的模拟抗原表位及其免疫原性鉴定

目的:用噬菌体呈现随机12肽库筛选能与抗人B7-H4(h B7-H4)中和抗体特异性结合的模拟抗原表位肽,并用其免疫小鼠检测其免疫原性。方法:以抗h B7-H4中和抗体为靶分子,用体外生物淘洗法从噬菌体呈现随机12肽库中筛选与之结合的噬菌体克隆,用竞争性细胞ELISA鉴定阳性噬菌体克隆;化学合成候选多肽,并与钥孔血蓝蛋白或破伤风毒素偶联鉴定多肽的特异性;进一步用融合蛋白免疫小鼠检测其免疫原性和抗血清的补体依赖的细胞杀伤活性(CDC)。结果:经过3轮体外筛选后随机挑取50个阳性噬菌体克隆,其中20个克隆与抗h B7-H4抗体有较强的结合能力,DNA测序得到6组结构相似的肽序列;竞争性ELISA结果显示1号肽噬菌体能与细胞表面的h B7-H4竞争性地结合抗h B7-H4单抗;点杂交结果显示1号肽能特异性结合抗h B7-H4单抗;小鼠免疫实验结果显示1号肽融合蛋白能诱导高滴度的抗h B7-H4抗血清,并且抗血清具有补体依赖的细胞杀伤活性。结论:筛选得到能与抗h B7-H4中和抗体特异性结合的12肽模拟抗原表位序列并且具有免疫原性,为进一步开发h B7-H4相关的多肽疫苗提供了实验依据。     

神经生长因子低亲和力受体(p75NTR)的模拟配基的筛选

人神经生长因子低亲和力受体 (p75NTR)转染R2细胞而建立的R2L1细胞 ,在去血清培养时发生凋亡 ,该作用可被神经生长因子 (NGF)所抑制 .用R2L1和R2两种细胞差式筛选噬菌体随机 7肽库和 1 2肽库 ,获得和p75NTR特异结合的噬菌体 .测定DNA序列后得到有关多肽的氨基酸序列 .7肽库共有序列为C (H D)LP(K M)HPM C ;1 2肽库优势序列为TLPSPLALLTVH .化学合成相应的 2个短肽 .用细胞结合法和ELISA方法证实阳性噬菌体和合成短肽能与p75NTR结合 ,并证实了它们对R2L1细胞去血清培养后的凋亡有抑制作用     

从噬菌体随机肽库中筛选流感病毒神经氨酸酶的抑制多肽

目的:从噬菌体呈现12肽库中筛选与流感病毒神经氨酸酶特异性结合的肽。方法:以甲三型流感病毒裂解疫苗原液为靶分子,经过3轮生物淘选,从噬菌体随机肽库中筛选与之结合的噬菌体。用ELISA方法鉴定噬菌体克隆与靶分子的结合力,用荧光方法测定噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶的抑制活性。对筛选到的阳性克隆进行DNA序列测定并推导出相应的氨基酸序列。结果:经过3轮筛选后,42个噬菌体克隆与靶分子有高度亲和力,23个噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶有抑制活性。对27个噬菌体克隆的测序结果表明,分别有10个和2个克隆的序列是一致的,其氨基酸序列分别为KSLSRHDHIHHH和WPRHHHSASVQT。结论:通过噬菌体肽库筛选到抑制流感病毒神经氨酸酶的12肽,为进一步研究对流感病毒神经氨酸酶有抑制活性的分子药物奠定了基础。     

用随机6肽库筛选HGVE2抗原表位

利用抗 HGV E2区的 3株单克隆抗体 M6、M1 3、M30作为筛选配基 ,对随机 6肽库进行亲和筛选 . 3轮筛选的投入产出比逐轮升高至 3.5× 1 0 -3、假阳性率逐轮降低至 0 .4% ,提示具有良好的富集效果 .从第 3轮随机挑出 1 2个克隆进行功能鉴定 ,结果表明 8个克隆与 M6抗体有较强的特异性结合力并有较好的竞争抑制作用 ,测序发现它们的外源肽具有核心序列 :WA( W/Y) WXH,该序列与 HGV同源性低 ;用外源肽与核心序列相似的噬菌体克隆 P6GC9做竞争抑制试验 ,约 3×1 0 10个噬菌体即可较好地抑制 M6单抗与 HGV抗原结合 .该多肽可能是 HGV E2区识别 M6单抗并具有一定功能的模拟表位 .     

表位九肽库的构建及人Ⅳ型胶原酶特异结合肽的筛选

将人工合成的编码九肽的随机序列DNA片段克隆进丝状噬菌体表达载体FUSE5,经多次电击转化和表达,获得肽段与噬菌体pⅢ蛋白融合并展示在噬菌体表面的随机序列九肽表位肽库。库容量达10 10个克隆。以Ⅳ型胶原酶为靶蛋白,采用亲和纯化筛选模式,从中筛选出Ⅳ型胶原酶结合肽。进一步ELISA检测筛选出与Ⅳ型胶原酶特异结合的20个阳性克隆。序列分析发现一组肽含有WDXXD的共同序列,一组含有WVGXXR的共同序列。其中WDXXD的序列与Ⅳ型胶原酶单链抗体可变区序列同源。结果表明,多肽库是筛选蛋白特异结合肽的有力工具,表位九肽库的构建和筛选方法的建立为进一步应用筛选具有高亲和力的特异结合肽奠定了基础。     

趋化因子受体 CCR5 亲合短肽的筛选

趋化因子受体 5 (CCR5) 是 HIV-1 与宿主细胞结合的辅助因子之一,其功能缺失或被 CCR5 拮抗剂封闭则会阻止 HIV-1 感染细胞 . 为得到与 CCR5 特异结合的肽类拮抗剂,采用噬菌体展示技术,以稳定表达 CCR5 的 CHO 细胞 (CHO/CCR5) 作为靶标,通过噬菌体随机 12 肽库筛选与 CCR5 特异结合的多肽;经过四轮筛选后,挑选 20 个阳性噬菌体克隆进行测序,从中得到 11 个含有 AFDWTFVPSLIL 序列的小分子肽 . 含该序列的噬菌体能与抗人 CCR5 单抗 (2D7) 竞争性结合 CCR5 ,且合成肽 AFDWTFVPSLIL 对趋化因子 RANTES 与 CHO/CCR5 的结合具有明显的抑制作用,初步证明该小肽与 CCR5 具有特异性结合作用 .     

抗人白细胞介素15中和抗体识别抗原表位的噬菌体肽库筛选与鉴定

目的:用噬菌体呈现随机七肽库筛选能与抗人白细胞介素15(h IL-15)中和抗体特异性结合的模拟抗原表位肽,并初步鉴定其免疫原性。方法:以抗h IL-15中和抗体为靶分子,用生物淘洗法从噬菌体呈现线性七肽库中筛选与之结合的噬菌体克隆,用噬菌体ELISA和竞争抑制ELISA鉴定阳性噬菌体克隆;化学合成筛选得到的多肽,并与匙孔血蓝蛋白(KLH)偶联免疫BALB/c小鼠,检测其免疫原性。结果:经过3轮体外筛选后随机挑取50个阳性噬菌体克隆,ELISA检测结果显示其中15个克隆与抗h IL-15抗体有较强的结合能力,DNA测序结果得到的结构相似群为MTPFWQK、MSPFNQK、MIPYWQK和MIPFHQK;竞争性ELISA结果显示4个序列均能与IL-15竞争性地结合抗IL-15单抗;小鼠免疫实验结果显示4组多肽均能诱导IL-15特异性免疫反应。结论:筛选得到能与抗h IL-15中和抗体特异性结合,且具有免疫原性的模拟抗原7肽序列,为进一步开发h IL-15相关的多肽疫苗提供了依据。     

从七肽噬菌体展示库中筛选内毒素结合蛋白质配基

目的:研究从噬茵体展示库中筛选内毒素结合蛋白质配基,为其在内毒素致病作用机理及在内毒素血症防治研究中的应用奠定基础.方法:以内毒素为靶分子从随机七肽噬菌体展示库中筛选内毒素的高亲和力噬菌体配体,通过ELISA鉴定,DNA测序及相关软件分析.结果:所筛选的亲和力最高的噬菌体的ELISA检测值A405nm可达1.965通过比较亲和性噬菌体外源插入肽的DNA序列,认为FHENWPS肽段中包含有与内毒素分子发生亲和结合的一个共有序列.该序列展示肽的等电点为5.36,具有双嗜性,这有利于肽与LPS分子表面的位点相互作用从而产生亲和吸附.结论:运用亲和筛选方法从肽库中筛选内毒素结合蛋白质配基是可行的.     

利用噬菌体随机肽库筛选可结合志贺毒素B亚单位的短肽序列

以制备的重组志贺毒素B亚单位(StxB)为靶标,利用噬菌体展示亲和淘选技术,经4轮筛选,从随机十二肽库中筛选到与StxB结合的一批噬菌体克隆,对特异结合活性较高的27个噬菌体克隆的表面展示肽进行序列测定,其中A6序列出现16次,A9和A3序列分别出现2次和3次。为评价筛选克隆中和毒素毒性的能力,将展示肽出现频率最高的A6噬菌体克隆,体外与志贺毒素孵育进行动物试验,动物存活率达33.3%,表明毒素的毒性得到部分抑制,A6短肽可能发展成为志贺毒素的拮抗剂。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

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.     

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     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.