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1.
含有Epstein-Barr病毒膜抗原的重组表达质粒及其基因免疫   总被引:1,自引:0,他引:1  
将Epstein-Bar(EB)病毒主要的膜抗原(MA)BLLF1基因片段插入pHD101-3质粒的CMV启动子下游,构建了真核表达质粒pHD-gp350,并转染293细胞进行瞬间表达。用免疫荧光法从细胞膜检测到表达的抗原能与其单克隆抗体发生特异性结合,Western-blot法证实,表达的抗原分子量为350kD.用能在真核细胞表达的重组质粒pHD-gp350的DNA,经Sepharose2B柱纯化后,注射经普鲁卡因预处理的Balb/C小鼠的四头肌,观察到EBV-IgA/MA抗体水平比EBV-IgG/MA低,而EBV-IgA/MA的持续时间比EBV-IgG/MA长。采用表达EBVMA的质粒DNA与CHO细胞表达的MA蛋白免疫小鼠,均获得抗EBVMA的抗体。  相似文献   

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利用EB病毒转化可产生较高水平人IgG和特异性抗2型登革病毒人抗体的Hu-TLC-SCID小鼠脾细胞,通过免疫组化、免疫荧光和PCR法检测转化细胞的人B细胞表面标志、EB病毒抗原和EB病毒基因。结果表明,被团体的Hu-TLC-SCID小鼠脾细胞能继续产生抗2型登革病毒的特异性人抗体,并具有人B细胞的CD20^+、SmIgG标志及EB病毒潜伏膜蛋白-1(LMP-1)基因,可表达LMP-1和EB病毒核  相似文献   

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利用EB病毒转化可产生较高水平人IgG和特异性抗2型登革病毒人抗体的Hu-TLC-SCID小鼠脾细胞,通过免疫组化、免疫荧光和PCR法检测转化细胞的人B细胞表面标志、EB病毒抗原和EB病毒基因。结果表明,被转化的Hu-TLC-SCID小鼠脾细胞能继续产生抗2型登革病毒的特异性人抗体,并具有人B细胞的、SmIgG标志及EB病毒潜伏膜蛋白-1(LMP-1)基因,可表达LMP-1和EB病毒核抗原(EBNA)。  相似文献   

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刘敬  龚炜 《生物学杂志》1999,16(1):9-10,35
用PCR方法扩增人丙型肝炎病毒(HCV)C抗原部分基因片段,将其克隆到一种新的表达载体PQE中构成重组质粒PQE-Core短,转化大肠杆菌M15株。含PQE-Core重组质粒的工程菌株以2YT中37℃下培养加入IPTG诱导7小时,经15%SDS-PAGE凝胶电泳分离蛋白质,证明工程菌合成了大量改造的HCV Corenhj r  相似文献   

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人GM—CSF cDNA的克隆和在大肠杆菌中的表达   总被引:3,自引:0,他引:3  
从诱导的人胚肺细胞HFL株中提取总RNA.经RT-PCR反应获取了人GM-CSFcDNA,DNA序列测定表明其顺序与文献报道完全一致。为了获得高效表达,应用PCR改造了人GM-CSF的cDNA5’端核苷酸序列,并将改造的人GM-CSF基因插入含T7启动子的质粒pET-11d构建成表达质粒pETC-5,将此质粒转化大肠杆菌株BL21(DE3)得到表达菌株BLEC4。表达菌株用0.5mol/LIPTG诱导2小时后,产生大量重组蛋白并形成包涵体。SDS—PAGE电泳图谱扫描结果表明,rhGM-CSF产量占菌体总蛋白量的16%。ELISA和TF-1细胞培养测定表明,初步纯化和复性的rhGM-CSF具有天然的hGM-CSF生物活性。  相似文献   

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应用基因重组技术,把编码EB病毒早期蛋白的BCRF1基因重组于真核表达载体pSG5中,并使该基因在乳地鼠肾(BHK)传代细胞中获得良好表达,表达率为0.5%。血清学实验证实,鼻咽癌、类风湿性关节炎病人和正常人血清中均不同程度地含有IgG/BCRF1抗体,抗体阳性率分别为92%、86%和77%,几何平均滴度(GMT)分别是1:16.35、1:14.72和1:10.15。两组病人和正常人血清中IgA/BCRF1抗体阳性率和滴度之间有较大差别,它们的阳性率分别是74%、71%和12%,GMT分别是1:12.32,1:10.56和1:2.35。还证实,鼻咽癌和类风湿性关节炎病人血清中IgA/BCRF1和IgA/EA(早期抗原)抗体阳性率和滴度间有很好的相关性,此为首次报导。重组表达质粒pSG5-BCRF1的构建和表达为进一步研究BCRF1基因在病毒感染和肿瘤免疫中的作用创造了条件。本文就质粒的构建和3组血清中IgA/BCRF1、IgA/BCRF1、IgA/EA和IgG/BCRF1抗体间的关系和有关问题进行了讨论。  相似文献   

7.
t-PA cDNA的克隆及其在毕赤酵母中的表达   总被引:5,自引:0,他引:5  
应用PCR方法,在t-PA cDNA5'端引入合适的限制酶位点和酵母分泌信号肽,与酵母表达载体pPIC9重组,构建表达质粒pSTE-Y,利用LiCl转化法转化酵母菌YS108,在MM和MD平板上筛选表型,PCR筛选t-PA cDNA与酵母染色体整合而形成的阳性克隆,阳性克隆经甲醇诱导表达后,用SDS-PAGE证明表达产物的分子量为6kDa左右,用酪蛋白板溶圈法测定t-PA的活性。Mut^s表型菌表  相似文献   

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GDNF重组腺病毒的构建及促进多巴胺能神经元存活的研究   总被引:4,自引:0,他引:4  
利用体内同源重组原理,构建了能介导GDNF基因转移和表达的复制缺陷型重组腺病毒AdCMVgdnf,其中GDNFcDNA插入腺病毒基因组的E1区并由CMV启动子控制在人293细胞内通过同源重组包装生成重组腺病毒后,用形态学方法、病毒DNA酶切分析、PCR和RT-PCR等方法进行鉴定正确.经测定病毒滴度达到1010pfu/ml.用免疫沉淀方法从重组腺病毒感染的293细胞及其培养基上清中均检测到大量GDNF蛋白.用重组腺病毒直接感染或者用其条件培养基处理,分别使胚胎大鼠中脑原代多巴胺能神经元的数目增加88.2%和96.4%,明显增加多巴胺能神经元存活,对帕金森氏病基因治疗具有重要意义  相似文献   

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为研究重组腺病毒接种实验动物后的免疫反应性,利用RT-P C R方法,从HAV的RNA中克隆了结构蛋白基因插入穿梭质粒pXCX2Not I,通过磷酸钙-DNA共 沉淀技术,将复制缺陷型腺病毒载体与线形化的pXCX2-CMV-HAV共转染293细胞。一系列检测方法证明产生了重组腺病毒rAdHAV。纯化后的rAdHAV滴度为1×109TCID50/mL ,腹腔注射免疫昆明种小白鼠后,可诱导产生抗HAV IgG和HAV中和抗体。复制缺陷型腺病毒 可作为发展基因工程病毒疫苗载体的有效系统。  相似文献   

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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   

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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.  相似文献   

16.
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.  相似文献   

17.
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.  相似文献   

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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  相似文献   

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