弓形虫ROP2酵母双杂交诱饵表达载体构建及毒性和自激活活性检测

目的为筛选弓形虫分泌毒性因子-棒状体蛋白2(ROP2)在终末宿主细胞内的互作因子,构建ROP2的酵母双杂交诱饵表达载体,并检测其表达蛋白对酵母菌生长的毒性作用及自激活活性。方法根据ROP2蛋白的特性选取1~561氨基酸、25~241氨基酸、242~561氨基酸作为目的片段,PCR扩增弓形虫基因组获得3片段,定向克隆到酵母表达载体pGBKT 7上,经测序正确后,将其转化到酵母菌Y187感受态细胞,在缺陷性培养基上观察3个诱饵表达载体在Y187中的表达情况,检测诱饵载体有无毒性作用和自激活功能。结果成功扩增出ROP2基因的3个目的片段,并将其克隆到诱饵表达载体pGBKT7中,经测序及酶切鉴定结果正确,转化到酵母Y187细胞中的诱饵表达载体无毒性和自激活作用。结论成功获得了对宿主酵母细胞无毒性且未自主激活活性的诱饵表达载体pG-BKT7-ROP2_(1-561)、pGBKT7-ROP2_(242-561)、pGBKT7-ROP2_(25-241),可作为酵母双杂合系统中的"诱饵",为下一步利用酵母双杂交系统筛选ROP2在终末宿主细胞内的相互作用蛋白创造了条件。     

细粒棘球蚴14-3-3与MKK2酵母双杂交载体的构建及自激活鉴定

目的构建细粒棘球幼(Eg)14-3-3与MKK2酵母双杂交系统,并对诱饵质粒进行自激活活性及毒性检测。方法从Eg cDNA中扩增Eg14-3-3基因和EgMKK2编码序列,将其克隆入酵母双杂交载体pGADT7和pGBKT7中,经PCR、限制性酶切鉴定及序列测定正确后,PEG/LiAc法转化酵母菌株,检测融合蛋白对酵母菌生长的影响和自激活活性。结果扩增Eg14-3-3和EgMKK2基因编码区全长分别为749bp和1 572bp。构建的pGBKT7-Eg14-3-3及pGADT7-EgMKK2重组质粒转化到酵母细胞Y2HGold中,检测表达蛋白对Y2HGold无毒性及自激活作用。结论 Eg14-3-3基因和EgMKK2编码序列表达的蛋白对酵母菌Y2HGold无毒性和自激活作用,为进一步运用酵母双杂交技术筛选与之相互作用的蛋白奠定了基础。     

禽流感病毒NP基因的酵母双杂交饵载体表达及自激活检测

目的构建H5N1亚型禽流感病毒NP基因的酵母双杂交诱饵载体,验证其在酵母中的表达并检测其自激活作用。方法以PCR法从pGEMT/H5NP扩增NP基因编码区序列,将其定向克隆到PGBKT7载体,经测序鉴定后,PEG/Li-Ac法转化酵母菌株AH109,用表型筛选法及颜色筛选法检测其自激活作用同时Westernblot验证诱饵蛋白的表达。结果获得NP编码区基因,并成功构建酵母双杂交系统中的诱饵载体pGBKT7-NP,对宿主细胞酵母菌株AH109无毒性和自激活作用,并能在酵母细胞中稳定表达。结论诱饵载体pGBKT7-NP可用于GAL4酵母双杂交系统钓取与禽流感病毒核蛋白相互作用的蛋白。     

人肠三叶因子酵母双杂交载体的构建及其自激活作用鉴定

目的:构建人肠三叶因子(hITF/hTFF3)酵母双杂交诱饵载体并鉴定其自激活作用.方法:从人结肠黏膜提取总RNA.RT-PCR制备总cDNA,PCR扩增hTFF3基因.TA克隆至pGEMT载体并测序鉴定.经NcoⅠ/XhoⅠ双酶切,连接到pENTR11质粒构建入门克隆.LR反应获得酵母双杂交诱饵载体pDEST32一hTFF3,测序正确后与酵母双杂交空猎物载体pDEST22共同转化Mav203酵母细胞,SD/-Leu/- Trp固体培养基上生长.挑取单克隆划线接种到分别含有不同浓度氨基三唑(3AT)的SD/- Leu/-Trp/-His培养基上,观察重组诱饵载体的自激活情况.结果:从人正常结肠黏膜成功克隆了hTFF3基因,构建了pDEST32-hTFF3酵母表达质粒.转化pDEST32-hTFF3和pDEST22的Mav203酵母细胞可在3AT浓度为30 mmol/L以下的SD/- Leu/-Trp/-His培养基上生长,在3AT浓度为30 mmol/L以上的培养基上则未见酵母细胞生长结论:所构建的pDEST32-hTFF3可作为酵母双杂交的诱饵质粒.     

细粒棘球蚴TGF—βⅠ型受体全长和胞内域酵母双杂交载体的构建及白激活鉴定

目的构建细粒棘球绦虫（Eg）转化生长因子I型受体（EgTβRI）全长及胞内域酵母双杂交真核表达载体,愉测重组诱饵质粒对酵母菌株的毒性作用及自激活活性。方法采用TRIzol法提取细粒棘球蚴总RNA;采用RT—PCR扩增EgTβRI全长基因及EgTβRI胞内域（EgTβRIintracellulardomain,EgTβRII）基因片段,分别构建pGBKT7-EgTβRI和pGBKT7-EgTβRI—I真核表达载体,经PCR、限制性酶切鉴定及序列测定正确后,PEG／LiAc法转化人酵母菌株,检测诱饵蛋白对酵母菌的毒性作用及其自激活活性。结果成功构建了pGBKT7-EgTβRI及pGBKT7EgTβRI—I真核表达载体,经双酶切,分别得到1662bp和1314bp目的基因片段,大小与预测值相符。两重组质粒转化Y2HGold酵母菌形成的菌落与pGBKT7质粒转化酵母菌菌落大小一致,直径为1．5～2．0mm;两重组质粒转化酵母菌在SD／Trp／X／A平板上无蓝色菌落生长,在SD／-Trp平板上形成直径为2mill的菌落。结论成功构建了pGBKT7-EgTβRI及pGBKT7-EgTβRI—I真核表达载体,其表达蛋白对Y2HGold酵母菌无毒性作刷和无自激活活性;该表达载体可以用于酵母双杂交系统,为进一步研究EgTβRI与其配体之间的交互作用奠定了基础。     

酵母双杂交诱饵蛋白LASS1融合表达质粒的构建及鉴定

目的 构建酵母双杂交系统诱饵蛋白大鼠LASS1融合表达质粒,为进一步筛选大鼠脑神经元内与LASS1p相互作用的蛋白奠定基础.方法 应用PCR方法获得大鼠LASS1基因2个片段,分别克隆入酵母双杂交系统诱饵蛋白质粒载体pGBKT7,转染酵母菌AH109并检测重组质粒的自激活现象及毒性.利用Western印迹检测重组质粒在AH109的表达情况.结果 LASS1基因的PCR产物片段大小分别为Flag(111 bp)、Slag(109 bp);Western印迹结果表明2个重组质粒表达的蛋白均可以与抗c-Myc抗体在22 kU处特异性反应;重组质粒转化酵母后无自激活作用.结论 重组质粒pGBKT7-Flag、pGBKT7-Slag均能够在AH109内正确表达,可作为酵母双杂交系统诱饵蛋白使用.     

弓形虫ROP21诱饵质粒的构建与自激活鉴定

目的筛选与弓形虫ROP21相互作用的宿主蛋白。方法构建弓形虫ROP21基因的三个诱饵载体,通过酵母双杂交技术分别对其在Y2H Gold酵母菌中的毒性和自激活进行鉴定。提取弓形虫RNA并进行反转录,采用RT-PCR方法扩增得到弓形虫ROP21全长序列,根据其不同功能区构建全长或部分序列诱饵质粒,测序后分别命名为pGBKT7-ROP21、pGBKT7-LC和pGBKT7-ST。将重组的诱饵载体转化到Y2H酿酒酵母菌,在营养缺陷型培养基上观察该重组诱饵载体的毒性和自激活现象。结果发现三个质粒转化酵母菌后,pGBKT7-ROP21和pGBKT7-ST存在自激活现象,而pGBKT7-LC转化酵母菌不存在,因此其可以作为酵母双杂交筛选的诱饵质粒。结论为进一步运用酵母双杂交技术筛选与弓形虫ROP21互作的宿主蛋白奠定了基础。     

酵母双杂交诱饵蛋白LASS1合表达质粒的构建及鉴定

目的构建酵母双杂交系统诱饵蛋白大鼠LASS1融合表达质粒，为进一步筛选大鼠脑神经元内与LASS1p相互作用的蛋白奠定基础。方法应用PCR方法获得大鼠LASS1基因2个片段，分别克隆人酵母双杂交系统诱饵蛋白质粒载体pGBKT7，转染酵母菌AH109并检测重组质粒的自激活现象及毒性。利用Western印迹检测重组质粒在AH109的表达情况。结果LASS1基因的PCR产物片段大小分别为Flag（111bp）、Slag（109bp）；Western印迹结果表明2个重组质粒表达的蛋白均可以与抗c-Myc抗体在22ku处特异性反应；重组质粒转化酵母后无自激活作用。结论重组质粒pGBKT7-Flag、pGBKT7-Slag均能够存AH109内正确表达，可作为酵母双杂交系统诱饵蛋白使用。     

细粒棘球蚴TGF-βⅡ型受体不同结构域酵母双杂交载体的构建和自激活鉴定

目的构建细粒棘球蚴转化生长因子βⅡ型受体全长(EgTβRⅡ)、受体结合域(EgTβRⅡ-A)和激酶域(EgTβRⅡ-K)的酵母双杂交真核表达载体,检测重组融合蛋白对酵母Y2HGold菌株的毒性作用和自激活活性。方法采用Trizol法提取细粒棘球绦虫总RNA,反转录合成cDNA;PCR扩增EgTβRⅡ-A、EgTβRⅡ-K和EgTβRⅡ基因,分别克隆入pGADT7、pGBKT7载体中,经PCR、限制性内切酶鉴定及序列测定正确后,采用PEG/LiAc法转入酵母菌,检测重组融合蛋白对酵母菌毒性和自激活活性。结果构建EgTβRⅡ-A、EgTβRⅡ-K和EgTβRⅡ的pGADT7、pGBKT7重组质粒,经双酶切和1%琼脂糖凝胶电泳鉴定,得到长度为406bp、1 023bp和1 824bp的目的基因片段,与预期结果一致;重组质粒转化酵母菌后形成的菌落与对照质粒pGBKT7转化菌的菌落生长状况一致,直径1.5~2.0mm,而在SD/-Leu/X/AbA、SD/-Trp/X/AbA平板上无菌落生长。结论成功构建EgTβRⅡ-A、EgTβRⅡ-K和EgTβRⅡ基因的pGADT7、pGBKT7真核表达载体,其表达蛋白对酵母菌Y2HGold无毒性作用和自激活活性,重组质粒载体可用于酵母双杂交系统,为鉴定EgTβRⅡ在TGF-β/Smad通路中的生物学功能奠定了理论基础。     

酵母双杂交筛选Clathrin相互作用蛋白

目的 应用酵母双杂交技术从小鼠肺cDNA文库中筛选与Clathrin相互作用蛋白,进一步阐明Clathrin在急性肺损伤和急性呼吸窘迫症（ALI/ARDS）发生时肺泡上皮极性损伤中的具体作用机制。方法 首先构建酵母双杂交pSos-Clathrin诱饵载体,酶切鉴定,然后确定Clathrin诱饵蛋白无自激活特性并检测了Clathrin诱饵蛋白的表达;最后筛选小鼠肺cDNA文库并对筛选得到的阳性克隆进行回转验证,对回转验证结果为阳性的文库克隆质粒送检测序,分析克隆序列。结果 酵母双杂交筛选小鼠肺cDNA文库得到4个与Clathrin相互作用蛋白,分别是：腺苷酸环化酶关联蛋白1,细丝蛋白α,DAP凋亡诱导蛋白激酶2和G蛋白耦联受体激酶6。结论 Clathrin参与细胞极性调节、炎症损伤和细胞凋亡等过程。     

新型乳腺癌标志基因C35的研究进展

C35基因是近年来发现的在乳腺癌细胞中特异性高表达,而在正常乳腺上皮细胞中不表达的基因,且C35基因的高度表达较Her2/-neu更为直接,表达谱更为广泛和稳定。C35基因可通过其保守的ITAM结构域促使细胞癌变,已成为一种新的特异性乳腺癌标志基因。本文综述了C35基因的序列分析、表达调控、致癌机制、互作因子和结构研究等方面的研究进展。     

变性高压液相色谱法分析肺癌患者痰标本Rb2/p130、p53基因突变

目的 探讨变性高压液相色谱(DHPLC)法分析肺癌患者痰标本Rb2／p130和p53突变及其作为分子诊断标记的可行性。方法 DHPLC法分析47例患者痰标本[35例肺癌(男22例,女13例,年龄16～81岁;其中腺癌17例,鳞癌12例,小细胞癌4例,肺泡癌1例,腺鳞癌1例),12例对照(男7例,女5例,年龄38～68岁;其中结核1例,肺炎1例,支气管扩张症2例,肺泡蛋白沉积症1例,结节病1例,多发性软骨炎1例,慢性阻塞性肺疾病5例)]Rb2／p130基因Exon19～22和p53基因Exon5～9突变。结果 肺癌患者Rb2／p130突变检出率为22．86％(8／35),p53突变检出率为28．57％(10／35),对照者均未检出1例(P=0．049,P=0．046)。作为肺癌诊断分子标记,Rb2／p130特异度100％,灵敏度22．86％;p53特异度100％,灵敏度28．57％;联合检测Rb2／p130和p53特异度100％,灵敏度51．43％。结论 联合分析痰标本Rb2／p130和p53基因突变可提高诊断的灵敏度。     

Prospective identification of tumorigenic breast cancer cells

Breast cancer is the most common malignancy in United States women, accounting for >40,000 deaths each year. These breast tumors are comprised of phenotypically diverse populations of breast cancer cells. Using a model in which human breast cancer cells were grown in immunocompromised mice, we found that only a minority of breast cancer cells had the ability to form new tumors. We were able to distinguish the tumorigenic (tumor initiating) from the nontumorigenic cancer cells based on cell surface marker expression. We prospectively identified and isolated the tumorigenic cells as CD44(+)CD24(-/low)Lineage(-) in eight of nine patients. As few as 100 cells with this phenotype were able to form tumors in mice, whereas tens of thousands of cells with alternate phenotypes failed to form tumors. The tumorigenic subpopulation could be serially passaged: each time cells within this population generated new tumors containing additional CD44(+)CD24(-/low)Lineage(-) tumorigenic cells as well as the phenotypically diverse mixed populations of nontumorigenic cells present in the initial tumor. The ability to prospectively identify tumorigenic cancer cells will facilitate the elucidation of pathways that regulate their growth and survival. Furthermore, because these cells drive tumor development, strategies designed to target this population may lead to more effective therapies.     

Co-localization of stanniocalcin-1 ligand and receptor in human breast carcinomas

Stanniocalcin-1 (STC1) is a new polypeptide hormone that has metabolic effects on target cell mitochondria. Recent studies have shown that the STC1 gene is upregulated in primary breast tumors and co-expressed with the estrogen receptor. In this report we have demonstrated the histological co-localization of STC1 and its receptor in invasive and non-invasive human mammary gland ductal carcinomas. Analysis of 58 malignant breast biopsies revealed that STC1 and its receptor co-localized to cancer cells in 91% of cases. The study therefore reveals that in breast carcinomas STC1 signals in an autocrine feedback loop and opens up the possibility that it may be sequestered by neoplastic cells in much the same manner as it is by non-malignant cells. The data further supports the notion that STC1 plays a role in breast cancer and that it may prove to be a novel diagnostic and prognostic marker, and potential therapeutic target.     

Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady- state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.