用DREAM技术纠正人工合成基因中的突变

目的：介绍一种简便、有效的定点突变技术。方法：根据突变位点附近的DNA序列推导出氨基酸序列,再以此氨基酸序列进行逆翻译,这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体（silent mutants）,这些突变体中包含大量的限制性内切酶位点,选择合适的酶切位点设计引物用PCR技术扩增两侧DNA片段,然后以相应酶切融合这两个片段即可完成定点突变。结果：用该方法成功地在人工合成的含有缺失的可溶性组织因子基因的472位插入C,T两个碱基,校正了阅读框架,获得了预期的目的基因。结论：该方法简便、有效, 避免了多轮PCR和合成长引物导致突变的可能性,这种改进的PCR 定点诱变技术我们称之为“设计限制酶辅助突变”（Designed Restriction Enzyme Assisted Mutagenesis, DREAM）。此技术简单方便, 诱变的成功率高, 适于实验室常规应用。     

Molecular characterization of porcine MMP19 and MMP23B genes and its association with immune traits

MMP19 and MMP23B belong to the Matrix metalloproteases (MMPs) family, which are zinc-binding endopeptidases that are capable of degrading various components of the extracellular matrix. They are thought to play important roles in embryonic development, reproduction and tissue remodeling, as well as in cell proliferation, differentiation, migration, angiogenesis, apoptosis and host defense. However, they are poorly understood in pigs. Here, we obtained the full length coding region sequence and genomic sequence of the porcine MMP19 and MMP23B genes and analyzed their genomic structures. The deduced amino acid sequence shares similar precursor protein domains with human and mouse MMP19 and MMP23B protein, respectively. Using IMpRH panel, MMP19 was mapped to SSC5p12-q11 (closely linked to microsatellite DK) and MMP23B was mapped to SSC8q11-q12 (linked to microsatellite Sw2521). Quantitative real-time PCR showed that MMP19 was abundantly expressed in the liver, while MMP23B was strongly expressed in the ovarian and heart. Furthermore, both genes were all expressed increasingly in prenatal skeletal muscle during development. Three SNPs were detected by sequencing and PCR-RFLP methods, and association analysis indicated that C203T at exon 5 of MMP19 has a significant association with the blood parameters WBC (G/L) and IgG2 (mg/mL) (P<0.05), SNP C131T at exon 3 of MMP23B is significantly associated with the blood parameters HGB (g/L) and MCH (P<0.05), and A150G in exon 4 has no significant association with the economic traits in pigs.     

猪繁殖与呼吸综合征病毒ORF2～4特异siRNA的筛选及其抑制病毒复制效果的研究

RNA干扰(RNAi)技术是基因功能研究的有效工具,为了了解猪繁殖与呼吸综合征病毒(PRRSV)次要结构蛋白GP2、GP3、GP4在病毒复制中的作用,针对各自的编码基因ORF2、ORF3、ORF4分别选取4个小干扰RNA(siRNA)位点(共12个),构建相应的短发夹RNA(shRNA)表达载体,转染MARC-145细胞后,通过荧光定量PCR和病毒滴度检测干扰效果。筛选了可以减少GP2、GP3、GP4相应基因mRNA含量的ORF2、ORF3、ORF4特异shRNA表达载体,病毒效价滴定表明shRNA表达载体处理细胞可以减少GP2、GP3、GP4相应基因mRNA含量,细胞培养上清中的病毒滴度比对照低184~4.65倍。     

Hydroxyethylamine-based inhibitors of BACE1: P1–P3 macrocyclization can improve potency,selectivity, and cell activity

We describe a systematic study of how macrocyclization in the P₁–P₃ region of hydroxyethylamine-based inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity. This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid β-peptide (Aβ)-lowering activity in HEK293T cells stably expressing APP_SW. However, for several closely related analogs the benefits of macrocyclization are attenuated by the effects of other structural features in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhibitors bound to BACE1 revealed their binding conformations and interactions with the enzyme.     

豆大蓟马对寄主植物及挥发性化合物的趋性

为明确不同寄主和不同信息化合物对豆大蓟马的行为影响,本文应用"Y"型嗅觉仪测定了豆大蓟马雌成虫对两种寄主植物花和不同浓度(v/v)下11种化合物的趋向性反应,结果表明:豆大蓟马对豇豆花、10-4烟酸乙酯、10-6烟酸乙酯、10-2橙花醇、10-2芳樟醇、10-6邻茴香醛和10-23-苯丙醛、10-43-苯丙醛具有极显著的趋向反应;对四季豆花和10-4橙花醇具有显著的趋向反应;10-4β-香茅醇、10-6β-香茅醇和10-6苯甲醛对豆大蓟马具有显著的驱避反应。不同浓度同一化合物对豆大蓟马的吸引作用也存在显著差异。实验结果可为进一步开发利用信息化合物田间防治豆大蓟马提供参考。     

Clinical,Virological and Immunological Features from Patients Infected with Re-Emergent Avian-Origin Human H7N9 Influenza Disease of Varying Severity in Guangdong Province

BackgroundThe second wave of avian influenza H7N9 virus outbreak in humans spread to the Guangdong province of China by August of 2013 and this virus is now endemic in poultry in this region.MethodsFive patients with H7N9 virus infection admitted to our hospital during August 2013 to February 2014 were intensively investigated. Viral load in the respiratory tract was determined by quantitative polymerase chain reaction (Q-PCR) and cytokine levels were measured by bead-based flow cytometery.ResultsFour patients survived and one died. Viral load in different clinical specimens was correlated with cytokine levels in plasma and broncho-alveolar fluid (BALF), therapeutic modalities used and clinical outcome. Intravenous zanamivir appeared to be better than peramivir as salvage therapy in patients who failed to respond to oseltamivir. Higher and more prolonged viral load was found in the sputum or endotracheal aspirates compared to throat swabs. Upregulation of proinflammatory cytokines IP-10, MCP-1, MIG, MIP-1α/β, IL-1β and IL-8 was found in the plasma and BALF samples. The levels of cytokines in the plasma and viral load were correlated with disease severity. Reactivation of herpes simplex virus type 1(HSV-1) was found in three out of five patients (60%).ConclusionExpectorated sputum or endotracheal aspirate specimens are preferable to throat swabs for detecting and monitoring H7N9 virus. Severity of the disease was correlated to the viral load in the respiratory tract as well as the extents of cytokinemia. Reactivation of HSV-1 may contribute to clinical outcome.     

Design and synthesis of a new fluorescent probe for cascade detection of Zn2+ and H2PO4− in water and targeted imaging of living cells

Design and synthesis of new fluorescence probes with good water‐solubility is of great importance to better understanding the significant role of ions which are related to biology and the environment. As important ions, zinc ion (Zn²⁺) and dihydrogen phosphate ion (H₂PO₄^?) display essential roles in living systems, and quantitative detection of these ions in water is still a challenge. In order to consider the significant role of the galactose moiety in the design of a water‐soluble fluorescence sensor, herein, we have developed a novel probe, Gal‐AQTF, for the cascade detection of Zn²⁺ and H₂PO₄^? with excellent selectivity in water. Through the introduction of the galactose moiety onto the sensor AQTF, which has been reported earlier by us, the water‐solubility, cell compatibility and targeting ability were enhanced. Gal‐AQTF has been successfully applied in the imaging of the living cells of HepG2 and A549, and illustrated good selectivity for the HepG2 cells which overly express the asialoglycoprotein (ASGP) receptor.     

Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromosome 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed. Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.