凝血靶向通用效应因子tTF/SA融合蛋白的表达及活性鉴定

制备凝血靶向通用效应因子tTF/SA融合蛋白,并鉴定其生物学活性。利用PCR技术构建tTF与链霉亲和素SA的融合基因,克隆至表达载体pET22b( ),在E.coliBL21(DE_3)中表达,镍亲和层析柱纯化tTF/SA融合蛋白。凝血实验和FⅩ活化实验鉴定融合蛋白中tTF的活性,ELISA鉴定融合蛋白中SA与生物素Biotin结合的活性。获得序列正确的tTF/SA/pET22b( )重组子,融合基因在E.coliBL21(DE_3)中高效表达。纯化后的融合蛋白具有活化FⅩ、引起血液凝固的能力,且能与生物素结合。融合基因已成功在E.coliBL21(DE_3)中表达,tTF/SA融合蛋白具有TF和SA活性。融合蛋白tTF/SA可作为通用效应因子,与生物素化的肿瘤组织血管特异性载体联用,实现选择性诱发肿瘤组织血管栓塞的多点治疗。     

一种新型融合蛋白(RGD)3/tTF的基因表达与活性分析

为了发展一种新型的融合蛋白(RGD)3/tTF用于肿瘤血管的选择性栓塞治疗,利用PCR技术重组(RGD)3/tTF融合基因,克隆于pET22b( )载体,表达于E.coliBL21(DE3)。用镍柱纯化融合蛋白。凝血实验与FⅩ活化实验检测融合蛋白tTF组分的活性。间接ELISA分析(RGD)3/tTF与αvβ3的特异结合能力。pET22b( )/(RGD)3/tTF重组质粒成功获得并表达于E.coliBL21(DE3)。纯化蛋白(RGD)3/tTF能有效诱发血液凝固,活化FⅩ。(RGD)3/tTF与αvβ3的特异结合能力比RGD/tTF提高了32%。新型融合蛋白(RGD)3/tTF已在E.coli系统成功表达,表达蛋白保持tTF的活性并显示比RGD/tTF更高的与αvβ3的结合能力。     

重组葡激酶和水蛭素融合蛋白的血栓靶向性机制

为解释以凝血因子Xa(FXa)的识别序列为连接肽的葡激酶和水蛭素的融合蛋白(命名为SFH)在体内的强溶栓和低出血的特征,研究分析了SFH的两个血栓靶向性作用机理.首先采用ELISA和免疫组化的方法在体外分析了由水蛭素游离的C末端赋予的SFH对血栓的靶向性,结果显示SFH对凝血酶和富含凝血酶的血栓具有更高的亲和力.为阐明SFH抗凝活性在血栓部位的靶向性释放,构建表达了仅在水蛭素N末端连接FXa识别序列的水蛭素衍生物(命名为FH).体外试验结果表明完整的FH无抗凝活性,在体内FH可以发挥抗栓作用,且出血副作用较低,这些结果说明FXa的识别序列可以封闭水蛭素的抗凝活性,在体内FH可以由于FXa的成功裂解而释放其抗凝活性,且其抗凝活性可能仅局限于血栓局部.这就间接说明了SFH的抗凝活性可以在血栓局部进行靶向性释放.以上两个血栓靶向性作用机理是SFH在体内发挥更高溶栓效率和降低出血副作用的重要机制.     

携带白介素-18基因的人脐带间充质干细胞的构建及其意义(英文)

目的:构建携带白介素-18(IL-18)基因人脐带间充质干细胞(hUMSCs),为肿瘤靶向性基因治疗研究提供一种工具。方法:体外分离培养hUMSCs,流式细胞仪(FACS)检测hUMSCs的细胞免疫表型。应用基因重组技术将表达IL-18基因的慢病毒转染至hUMSCs,利用RT-PCR及Western blot法检测IL-18的蛋白及mRNA表达水平。结果:成功在体外分离和培养了hUMSCs,流式细胞仪检测结果显示hUMSCs表达CD29、CD44和CD105,而不表达CD34和CD45,符合hUMSCs的表型。成功构建携带IL-18基因的hUMSCs,RT-PCR及Western blot法检测结果提示IL-18基因转染至hUMSCs并能稳定表达。结论:构建携带IL-18基因的hUMSCs并稳定表达IL-18,为肿瘤靶向性基因治疗实验性研究提供了一种新实验工具。     

靶向抗肿瘤蛋白iRGD-CDD的原核表达及生物活性鉴定

目的: 原核表达并纯化具有特异性成纤维细胞激活蛋白(FAPα)酶切位点的靶向抗肿瘤GP-CDD-iRGD融合蛋白,利用FAPα的酶切功能切除融合标签,检测其对FAPα阳性肿瘤细胞株的毒性。方法: 设计并合成GP-CDD-iRGD基因,插入pGEX-4T3 载体,构建重组表达质粒,转化至BL21大肠杆菌感受态细胞中,IPTG诱导表达,SDS-PAGE分析重组融合蛋白的表达,Western blot检测融合蛋白的表达,经GST亲和柱纯化融合蛋白后,通过体外细胞毒性试验(MTT法)和细胞凋亡实验评价该融合蛋白的靶向抗肿瘤活性。结果: 成功构建重组原核表达质粒pGEX-GP-CDD-iRGD,可溶性表达相对分子量约为36 kDa的融合蛋白GST-GP-CDD-iRGD,纯化后蛋白纯度约为90%,经MTT实验测定其对FAPα阳性4T1细胞株的ED50约为18.5μmol/L,流式细胞术检测到其对FAPα阳性4T1细胞株具有选择性毒性作用,早期凋亡比例达到约28%。结论: 原核表达的重组融合蛋白GP-CDD-iRGD对FAPα阴性4T1细胞株未显示毒性,而对FAPα阳性4T1细胞株具有显著的促凋亡作用,为进一步研究其在体内的靶向抗肿瘤活性提供了依据。     

重组毒素rCCK_(96-104)PE38靶向结肠癌生物特性研究

旨在原核表达并纯化新型重组毒素rCCK_(96-104)PE38,验证其对结肠癌细胞的靶向杀伤作用。使用基因扩增技术将反向编译的胆囊收缩素CCK_(96-104)与绿脓杆菌外毒素PE38融合,构建原核表达载体。利用Ni-NTA亲和层析进行纯化。通过结肠癌细胞体外杀伤实验以及结肠癌裸鼠模型的治疗验证rCCK_(96-104)PE38的抗肿瘤能力。结果显示,扩增出的rCCK_(96-104)PE38序列正确,成功利用pET-28a原核表达系统实现了活性表达。纯化后的重组蛋白能够在体外诱导结肠癌细胞凋亡,并能抑制裸鼠模型体内的肿瘤生长。成功制备高纯度、无标签的rCCK_(96-104)PE38重组免疫毒素,体内、体外实验证实其具有抑制结肠癌的能力。     

人tBID蛋白的表达纯化及其偶联的新型分子探针对前列腺癌细胞的促凋亡作用研究

目的:以PQE-30为原核表达载体,构建PQE30-tBID重组表达载体,表达和纯化目的蛋白tBID,并利用金磁纳米微粒将tBID蛋白与人HER2抗体偶联成分子探针Anti HER2-Gold Mag-tBID,以探究其对前列腺癌细胞的促凋亡作用。方法:根据tBID的基因序列设计特异性的上下游引物,利用普通PCR扩增目的基因tBID,构建重组表达载体PQE30-tBID,将其转化到BL21(DE3)中,IPTG诱导表达,经SDS-PAGE凝胶电泳和Western Blot鉴定分析,验证目的蛋白tBID的表达,并对其进行纯化。利用金磁纳米微粒与蛋白质之间的静电相互作用以及疏水相互作用,将人HER2抗体与tBID蛋白偶联在其表面,构建分子探针Anti HER2-Gold Mag-tBID。流式细胞术检测该分子探针与前列腺癌PC-3细胞的特异性亲附结合能力,通过Annexin V-FITC细胞凋亡检测试剂盒分析分子探针对PC-3细胞的促凋亡作用。结果:普通PCR扩增后得到了411 bp的DNA片段,经双酶切鉴定以及菌液测序,表明重组表达载体PQE30-tBID构建成功。促凋亡蛋白tBID成功地在大肠杆菌中表达,蛋白相对分子量约15 KD,经过纯化,得到了纯度较高的tBID蛋白。经过与金磁纳米微粒的偶联,成功构建出一种新型的分子探针Anti HER2-Gold Mag-tBID。该分子探针可与PC-3细胞特异性结合,且经Annexin V-FITC染色分析可见PC-3细胞发生明显凋亡,凋亡率达62.9%,与未处理组(3.79%)和对照组(4.33%)相比,具有显著的统计学差异。结论:PQE30-tBID重组表达载体能在大肠杆菌中高效表达,且成功得到了纯度较高的人促凋亡蛋白tBID。经金磁纳米微粒偶联,该蛋白能够与人HER2抗体重组成新型的分子探针,且能特异性地靶向前列腺癌PC-3细胞并显著促进其凋亡。     

克隆、表达及纯化核糖体蛋白S6用于体外激酶活性检测

目的:构建40S核糖体蛋白S6的原核表达载体,表达并纯化S6蛋白,将其作为底物用于S6激酶(S6K)的体外活性测定。方法:采用RT-PCR方法从人胚肾细胞HEK293中获取S6 cDNA,将扩增产物克隆至大肠杆菌表达载体中,进行酶切及测序鉴定;IPTG诱导GST-S6融合蛋白在大肠杆菌中表达,用谷胱甘肽亲和层析纯化GST-S6,免疫沉淀法检测该蛋白是否可作为底物用于S6K的体外激酶活性测定。结果:酶切及测序鉴定表明构建了S6原核表达载体,并表达及纯化出GST-S6融合蛋白,相对分子质量为55×103。该蛋白可用于S6K的体外激酶活性测定,特异性强。结论:S6蛋白的克隆、表达与纯化成功,可用于S6K的体外激酶活性测定,为研究S6K的功能奠定了基础。     

RGD序列修饰的MS2噬菌体样颗粒的构建及表达

目的:构建并表达衣壳蛋白表面展示有精氨酸-甘氨酸-天冬氨酸(RGD)序列的MS2噬菌体样颗粒,以期获得肿瘤细胞靶向性RNA递送载体。方法:用分子克隆技术将RGD4C编码序列插入MS2衣壳蛋白编码基因,然后构建到p ETDuet-1质粒多克隆位点MCS1上;将含有MS2噬菌体包装位点的pre-let-7编码基因构建到p ETDuet-1质粒多克隆位点MCS2上;重组质粒转化大肠杆菌,表达产物纯化后经琼脂糖凝胶电泳、电子显微镜鉴定。结果和结论:采用分子克隆技术和原核细胞单质粒双表达系统获得表面展示有RGD序列,内部包装有pre-let-7 RNA的MS2噬菌体样颗粒,为体内和体外研究其肿瘤细胞靶向递送和功能奠定了基础。     

基于4-1BBL和CD3双信号的融合蛋白协同抗肿瘤作用研究

旨在研究4-1BBL/CD20融合蛋白增强抗CD3/抗CD20 diabody介导的特异性靶向杀伤活性。采用亲和层析法纯化本室构建的抗-CD3/抗-CD20 diabody和4-1BBL/CD20融合蛋白可溶性表达产物;采用calcein释放试验测定其介导的体外靶向杀伤活性;采用人B淋巴瘤细胞系Raji裸鼠移植瘤模型测定其介导的体内靶向杀伤活性。纯化4-1BBL/CD20融合蛋白在体外能增强抗-CD3/抗-CD20 diabody介导激活的T细胞杀伤Raji细胞;在人B淋巴瘤细胞系Raji裸鼠移植瘤模型联合人T淋巴细胞4-1BBL/CD20融合蛋白增强抗-CD3/抗-CD20 diabody高效抑制Raji细胞裸鼠移植瘤的生长,明显延长荷瘤裸鼠的生存时间。在体外和体内4-1BBL/CD20融合蛋白均能增强抗-CD3/抗-CD20 diabody介导激活的T细胞杀伤表达CD20抗原的肿增细胞,是一个有望用于B细胞恶性肿瘤临床治疗的特异性融合蛋白。     

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.