弹性蛋白样生物材料的制备及性质鉴定

组织工程器官重建因不同的用途而对材料的弹性、刚度、生物活性等有一系列的需求,但当前所应用的材料多难以同时满足,例如弹性材料聚乙二醇双丙烯酸酯等高分子材料并不具备较高的生物活性,而生物材料胶原等生物活性好而弹性较差。弹性蛋白(elastin)作为广泛存在于动物体内的一种弹性极佳的功能蛋白,因其可承受较大形变而不破坏本身结构,故应用于弹性器官的组织工程重建中。为获得高生物活性的弹性蛋白样多肽(elastin-like polypeptides,ELP),根据其基本重复单元Val-Pro-Gly-Xaa-Gly设计了氨基酸序列,并进行优化,构建成原核表达质粒,通过大肠杆菌BL21(DE3)菌株进行诱导表达并收集,SDS-PAGE鉴定。通过流变检测、扫描电镜检测、细胞活性检测等方法鉴定材料的弹性性能、物理结构及生物活性,为下一步通过调整交联方法提高其弹性提供了材料,也为ELP作为生物材料应用于组织工程器官重建奠定了基础。     

自组装ELP多肽的从头设计及其在生物医药领域中的应用进展

重组类弹性蛋白多肽(elastin-like polypeptides,ELPs)是一种通过基因工程方法合成的多肽聚合物,其结构由类弹性蛋白的肽段单元重复串连组成,具有刺激响应性、自组装特性、显著的弹性和良好的生物学特性,如低血小板黏附性和低免疫原性等,因此ELPs材料已被广泛应用于组织工程、药物输送和纳米生物器件制备...     

ELP[Ⅰ]50标签在重组硫氧还蛋白分离纯化中的应用及PEG对其相变温度的影响

[目的]使用自行设计的类弹性蛋白(Elastin-like protein,ELP) ELP[Ⅰ]50作为非色谱纯化标签,分离纯化重组硫氧还蛋白(Thioredoxin,Trx),并研究聚乙二醇(Polyethyleneglycol,PEG)对ELP[Ⅰ]50-Trx相变温度(Inverse temperature transition,Tt)的影响.[方法]人工合成Trx基因,将其亚克隆到自行构建的表达载体pET28编码ELP[Ⅰ]50标签下游,转入大肠杆菌BLR(DE3)进行表达.融合蛋白表达后,采用可逆相变循环(Inverse transition cycling,ITC)分离纯化,并检测不同浓度PEG时的Tt值.[结果]成功表达、分离纯化出融合蛋白ELP[Ⅰ]50-Trx,检测出该蛋白浓度为25 μmol/L时,Tt为28.6℃;而当PEG的浓度为5％、10％、15％、20％时,Tt分别降至22.3℃、15.9℃、6℃、0℃.[结论]ELP[Ⅰ]50标签高效纯化重组蛋白具有操作简便、成本较低、易于扩大的优势,而PEG能降低蛋白的Tt值,进一步增强分离纯化效果,扩大使用范围,可望应用于分离纯化多种重组蛋白.     

ELP[I]50标签在重组硫氧还蛋白分离纯化中的应用及PEG对其相变温度的影响

【目的】使用自行设计的类弹性蛋白(Elastin-like protein, ELP) ELP[I]50作为非色谱纯化标签, 分离纯化重组硫氧还蛋白(Thioredoxin, Trx), 并研究聚乙二醇(Polyethylene glycol, PEG)对ELP[I]50-Trx相变温度(Inverse temperature transition, Tt)的影响。【方法】人工合成Trx基因, 将其亚克隆到自行构建的表达载体pET28编码ELP[I]50标签下游, 转入大肠杆菌BLR(DE3)进行表达。融合蛋白表达后, 采用可逆相变循环(Inverse transition cycling, ITC)分离纯化, 并检测不同浓度PEG时的Tt值。【结果】成功表达、分离纯化出融合蛋白ELP[I]50-Trx, 检测出该蛋白浓度为25 μmol/L时, Tt为28.6 °C; 而当PEG的浓度为5%、10%、15%、20%时, Tt分别降至22.3 °C、15.9 °C、6 °C、0 °C。【结论】ELP[I]50标签高效纯化重组蛋白具有操作简便、成本较低、易于扩大的优势, 而PEG能降低蛋白的Tt值, 进一步增强分离纯化效果, 扩大使用范围, 可望应用于分离纯化多种重组蛋白。     

疏水性ELP基因设计与基因库构建

[目的]设计合成疏水性类弹性蛋白(Elastin-like polypeptide,ELP)基因,建立ELP基因库.[方法]选择疏水性最强的异亮氨酸(I1e,I)(疏水参数:4.5)取代ELP五肽重复序列单元(缬氨酸-脯氨酸-甘氨酸-客座氨基酸-甘氨酸,VPGXG)中客座残基X.全基因合成一段含编码(VPGIG)10序列,上游含Dra Ⅲ酶切位点、下游含BglⅠ酶切位点的ELP单元.将Dra Ⅲ和BglⅠ设计为一对同尾酶(Isocaudarner),利用这对同尾酶定向克隆(Recursive directional ligation,RDL)一系列不同拷贝数的ELP基因.为鉴定基因库有效性,随机选取库中ELP[Ⅰ]50基因进行蛋白表达、纯化并测定其相变温度(Inverse temperature transition,Tt).[结果]建立了ELP[Ⅰ]n(n=10、20、30、40、50、60、70、80、90、100、110、120)基因库.测定ELP[Ⅰ]50的Tt为24.3℃.[结论]首次单独选用I1e(I)作为ELP蛋白质标签中客座残基氨基酸,增加了ELP中疏水性氨基酸的含量,为进一步筛选出表达量高、Tt低、分子量小的ELP标签奠定基础.     

聚乙二醇对ELP[I]40相变温度的影响

目的:研究聚乙二醇(polyethylene glycol,PEG)对类弹性蛋白(elastin-like protein,ELP)ELP[I]40相变温度(inverse temperature transition,Tt)的影响.方法:设计并合成ELP[I]40基因(由40个(VPGIG)五肽单元串联组成),表达纯化后,检测不同浓度PEG条件下ELP[I]40的Tt.结果:在ELP[I]40终浓度为25 μmol/L时,PEG浓度为5％,10％,15％,20％,25％时分别使Tt由29℃降至26.5℃,22℃,15.2℃,8.8℃,2.5℃.结论:PEG可降低ELP的Tt,可通过PEG促进ELP重组蛋白分离纯化.     

组织工程的一般考虑

组织工程的最终目标是通过体外增减活细胞与其胞外环境相互作用而发育成具胡生物活性的组织或器官替代物,替换、修复组织或器官,或强化其生物学功能。本文主要讨论细胞移植和组织重建的影响因素和可能的方法。     

高中综合实验设计——以"表达和纯化红色荧光蛋白标记的类弹性蛋白"为例

以类弹性蛋白(elastin-like polypeptide,ELP)作为非色谱纯化标签,分离纯化红色荧光蛋白 mCherry.ELP 与△ I-CM(intein cleavage mutant)的 N 端连接,mCherry 片段与△I-CM的C端连接,在ELP的N端插入GFP片段,用于检测蛋白纯度.采用低温诱导...     

类弹性蛋白标签在大肠杆菌周质空间中表达研究

旨在研究类弹性蛋白[I]_(40)(elastin-like polypeptide[I]_(40),ELP[I]_(40))在大肠杆菌周质空间的表达。构建表达载体pIG6LH/ELP[I]_(40)及pIG6LH/ELP[I]_(40)+Trx,分别将构建的表达载体转化入表达宿主菌E.coli BLR(DE3),IPTG诱导表达,采用可逆相变循环(Inverse transition cycling,ITC)技术纯化蛋白。测定ELP[I]_(40)及ELP[I]_(40)+Trx蛋白相变温度(T_t),检测ELP[I]_(40)、ELP[I]_(40)+Trx蛋白浓度及NaCl对相变温度的影响。结果显示,经3轮ITC纯化得到了ELP[I]_(40)、ELP[I]_(40)+Trx蛋白。分别测定ELP[I]_(40)和ELP[I]_(40)+Trx在10、25、50、75和100μmol/L浓度下的T_t,其T_t依次为31.5℃、29℃、27℃、26℃、25℃和31.8℃、29.5℃、27.5℃、26℃、25.5℃;测定了不同浓度的NaCl对T_t影响,在ELP[I]_(40)和ELP[I]_(40)+Trx终浓度为25μmol/L,NaCl浓度为0.25、0.5、0.75、1.00和1.25 mol/L时,分别使ELP[I]_(40)的T_t由29℃降至24.5℃、22℃、19℃、15℃和11.5℃,使ELP[I]_(40)+Trx的T_t由29.5℃降至25℃、23℃、20.2℃、15.5℃和11.8℃。在大肠杆菌周质空间表达的ELP[I]_(40)与胞内表达的具有相同的理化性质,ELP可作为在大肠杆菌周质空间表达的蛋白质分离纯化标签。     

一种含ELP60基因的载体pRELPN促进大肠杆菌的外源基因的高表达

类弹性蛋白多肽（ELP）为含有人工合成的ELP60基因的表达载体pRELPN,能促使外源基因在大肠杆菌中的高表达。当ELP60在大肠杆菌表达载体pET28a的多克隆位点被克隆后,其自身的表达低,也不与目的基因构成ELP融合蛋白质,而是促进克隆在ELP60基因后的含起始密码ATG的外源目的基因独立高表达。外源目的基因表达量占宿主蛋白的20% ～ 60%,比用pET28a载体表达的外源基因表达量高2～10倍。此类表达载体pRELPN适合于表达包括抗体、抗原、酶、重组蛋白质、多肽及ELP融合蛋白质等的外源基因的独立高表达。这些结果表明,pRELPN代表了一种有效的表达载体,有助于解决在原核表达中,所受限的普通载体对外源基因低表达或不表达所导致的不能产业化的问题。     

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.