利用TALEN打靶技术建立果蝇心脏发育候选基因Yippee突变品系

研究工作者们一直致力于寻找一种在模式生物中进行精确基因修饰的方法.近期基于TALENs的基因打靶方法受到广泛的注意,并在包括果蝇、斑马鱼、小鼠及人类多潜能细胞的多物种中取得成功.在这里,我主要介绍基于TALENs的基因修饰在果蝇模型中的应用以及果蝇心脏发育候选基因Yippee的敲除.首先,我们设计并拼接好了Yippee基因的TALENs靶位点序列,连接在核酸酶XmnⅠ的催化区域.在核酸酶的作用下产生双链的断裂,随后在非同源末端连接物修复系统（NHEJ）的修复下导致Yippee基因的消除或部分缺失.经过检测,得到Yippee基因的TALENs打靶效率约为9％.     

果蝇心脏发育标记基因lbe的多克隆抗体制备

lbe是已经证明的心脏标记基因。为了深入研究lbe在心脏发育中的功能,需要获得lbe蛋白并制备其抗体。首先提取野生型成体果蝇的总RNA,反转录获得其cDNA文库,通过PCR克隆出lbe编码区序列,将其连接到pET-28a原核表达载体上。经酶切及测序鉴定后,质粒构建成功。将重组质粒（pET-28a-lbe）转化大肠杆菌菌株Rosseta,用IPTG诱导表达出融合蛋白,经Ni-IDA凝胶柱纯化后,最后将纯化的融合蛋白免疫新西兰大白兔制备lbe多克隆抗体,并用Western blotting检测抗体的效价和特异性。结果显示获得了lbe原核表达重组融合蛋白及高效价的特异性兔抗lbe多克隆抗体,为lbe功能的进一步研究奠定了基础。     

城市景观格局视角下空气微生物研究进展

城市化进程将原有自然生态系统改造为以不透水面为主的人工景观,这种变化影响了空气微生物群落的生存环境及其时空异质性。空气微生物受城市化的影响程度与其生态功能的发挥关系密切,微生物群落特征的改变会在一定程度上影响当地生态环境质量并给人群健康带来潜在威胁。城市化进程和空气微生物群落动态分属两个时空尺度差异巨大的生态过程,二者的联合分析已成为目前生态安全与环境健康领域的研究热点。从空气微生物的来源及其组成特征、空气微生物群落的时空异质性及其影响因素以及空气微生物的生态环境效应三个方面系统梳理和总结了近年来探索城市化和空气微生物群落动态之间关系的研究,从宏观视角探讨了当前空气微生物研究的不足,并引入社会-经济-自然复合生态系统、景观格局与过程等相关理论和方法来分析城市化对空气微生物群落特征的影响,旨在明确城市景观格局作用下的空气微生物群落对人群健康的潜在威胁程度,为快速城市化过程中的人居环境的改善提供参考。     

果蝇odd蛋白的原核表达及多克隆抗体的制备研究

odd是一个新发现的心脏特异表达基因。为了进一步研究odd在心脏发育中的功能,根据已报道的odd基因序列,以果蝇cDNA文库为模板进行PCR扩增,将所得的odd部分编码区序列连接到pET-28a载体上构建原核表达载体;重组子经酶切测序鉴定后,转化到大肠杆菌BL21菌株,IPTG诱导融合蛋白表达,Ni-IDA凝胶柱亲和纯化,纯化后的His-odd融合蛋白免疫新西兰大白兔以制备多克隆抗体,Western Blot检测抗体活性。结果表明,获得了odd原核表达重组融合蛋白以及高效价的、特异性兔抗Odd多克隆抗体,为后续的odd功能研究奠定了基础。     

Anti-nociceptive effect of Portulaca oleracea L. ethanol extracts attenuated zymosan-induced mouse joint inflammation via inhibition of Nrf2 expression

The aim of this study was to explore the effects of ethanol extracts from Portulaca oleracea L. (ePO) on joint inflammation and to explain the underlying mechanisms. A joint inflammation mouse model was constructed by injecting zymosan, and the Von Frey method was employed and the joint thickness measured. The numbers of leukocytes, neutrophils, and monocytes were counted in the joint cavity and the infiltration of inflammatory cells was assessed by joint histopathological analysis. The mRNA levels of inflammatory cytokines were determined by quantitative RT-PCR and their secretion levels were determined by specific ELISAs. Pre-treatment with ePO inhibited articular mechanical hyperalgesia and edema and ameliorated the recruitment of mononuclear neutrophils and leukocytes. In addition, pre-treatment with ePO improved pathological alternations in the joint tissues by reducing the number of inflammatory cells. Pre-treatment with ePO regulated the nuclear factor erythroid 2-related factor 2 (Nrf2)-related proteins and thereby inhibited oxidative stress. In addition, ePO inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome-related genes (NLRP3, ASC, pro-caspase-1 and pro-IL-1ß), modulated inflammatory cytokines and the activation of NF-κB. ePO attenuated zymosan-induced joint inflammation by regulating oxidative stress, NLRP3 inflammasome, and NF-κB.