Secretion of firefly luciferase in E.coli

Summary A DNA segment carrying the full-length, intronless firefly luciferase gene was inserted into the high expression secretion vector, pIN-III -ompA. Upon induction of gene expression, luciferase activity was detected in extracts prepared from periplasmic fractions. The results indicated that the OmpA signal peptide was able to direct secretion of firefly luciferase across the cytoplasmic membrane. This has important implications for using this luciferase as a reporter in studying protein export and targeting.     

应用GeneXpert MTB/RIF对肺外结核的快速检测及评估

目的 评估GeneXpert MTB/RIF检测肺外结核分枝杆菌的准确性,并与传统方法进行比较。 方法 选取2016年6月至2017年6月在本院就诊的144例疑似肺外结核病患者,对所有标本分别进行金胺“O”荧光染色镜检、液体培养及药敏试验、固体培养及比例法体外药敏试验和Xpert法检测。 结果 收集的144例疑似肺外结核标本中,确诊108例,以胸水、淋巴结活检和脓液感染较多,另36例阴性患者中,10例为非结核分枝杆菌感染。Xpert试验的敏感性为28.73%,特异性为96.00%,其阳性预测值和阴性预测值均高于其他3种检测方法。在阳性检出率方面,Xpert试验高于涂片镜检(χ2=17.39,P2=8.64,P2=2.56,P>0.05)。固体培养、液体培养和Xpert试验3种方法对结核分枝杆菌利福平耐药率检测差异无统计学意义(P>0.05),耐药率分别为8.33%、9.68%和11.11%,且Xpert试验方法检测出2株耐多药结核分枝杆菌,平均耗时2.5 h。 结论 GeneXpert MTB/RIF可以作为一种筛选及快速检测工具应用于肺外结核的诊断,同时可作为检测MDR TB的一种指标。     

利用肠激酶加工融合蛋白的方法制备重组人甲状旁腺素（1—34）肽

采用全基因分段合成和补丁连接相结合的方法构建了人甲状旁腺素 (hPTH) (1～ 34)肽基因 ,并利用GST融合表达系统 ,在大肠杆菌中克隆和可溶性表达了hPTH(1～ 34)肽基因。融合蛋白表达量占菌体总蛋白质的 2 5 %以上 ,经高密度发酵、亲和纯化后 ,在每升发酵液中得到了 10 g融合蛋白。融合蛋白经肠激酶一步加工并亲合纯化和反相脱盐后 ,最终可以得到约 0 .6g /L人甲状旁腺素 (1～ 34)肽纯品 ,样品的理论回收值达到了4 8.75 %。产物的纯度和性质经HPLC、毛细管电泳、质谱分析、N端测序等得到证明 ,并与化学合成产物的结果相吻合。兔肾皮质细胞测活表明 ,重组产物与化学合成人甲状旁腺素 (1～ 34)肽具有相近的腺苷酸环化酶激活活性。     

Sex-specific dynamics of global chromatin changes in fetal mouse germ cells

Mammalian germ cells undergo global reprogramming of DNA methylation during their development. Global DNA demethylation occurs around the time when the primordial germ cells colonize the embryonic gonads and this coincides with dynamic changes in chromatin composition. Global de novo DNA methylation takes place with remarkably different dynamics between the two sexes, prospermatogonia attaining methylation during fetal stages and oocytes attaining methylation postnatally. Our hypothesis was that dynamic changes in chromatin composition may precede or accompany the wave of global DNA de novo methylation as well. We used immunocytochemistry to measure global DNA methylation and chromatin components in male and female mouse fetal germ cells compared to control somatic cells of the gonad. We found that global DNA methylation levels sharply increased in male germ cells at 17.5 days post coitum, but remained low in female germ cells at all fetal stages. Global changes in chromatin composition: i, preceded global DNA methylation in fetal germ cells; ii, sex specifically occurred in male but not in female germ cells; iii, affected active and repressive histone marks and iv, included histone tail and histone globular domain modifications. Our data suggest that dynamic changes of chromatin composition may provide a framework for the pattern of male-specific de novo DNA methylation in prospermatogonia.     

Characterization of fibroblasts recruited from bone marrow-derived precursor in neonatal bronchopulmonary dysplasia mice

We sought to determine whether the extrapulmonary origin of fibroblasts derived from bone marrow (BM) progenitor cells is essential to lung fibrosis in bronchopulmonary dysplasia (BPD). Neonate mice were durably engrafted with BM isolated from transgenic reporter mice that expressed green fluorescent protein (GFP). Such chimera mice were subjected to 60% O(2) exposure for 14 days. A large number of fibroblast-specific protein-1 (FSP1) and GFP-positive fibroblasts were identified in active fibrotic lesions. More surprisingly, however, FSP1(+) fibroblasts also arose in considerable numbers from BM-derived alveolar type II cells (AT2) through epithelial-mesenchymal transition (EMT) during lung fibrogenesis. Cultured lung fibroblasts could express the CXC chemokine receptor (CXCR4) and responded chemotactically to their cognate ligand, chemokine (C-X-C motif) ligand 12 (CXCL12), which were elevated in the serum of BPD mice. These data suggest that lung fibroblasts in BPD fibrosis could variably arise from BM progenitor cells. This finding, which suggests the pathophysiological process of fibrosis, could contribute to a therapy for BPD that is characterized by extensive interstitial fibrosis.     

Enhanced immunodetection of cell-free synthesized erythropoietin under denaturing conditions

A monoclonal antibody produced by hydridoma cell line, ATCC HB8209, was used to detect and purify erythropoietin synthesized in a cell-free system. The antibody was raised against the N-terminal 20 residues of erythropoietin. It retained anti-erythropoietin activity in 6 M urea in which most of the cell-free synthesized erythropoietin became soluble and gave an enhanced activity of the antibody.