P2X7受体短肽单克隆抗体的制备及鉴定

旨在制备与鉴定鼠抗P2X7受体的蛋白单克隆抗体.以人P2X7受体的胞外段制备短肽作为抗原,皮下注射免疫Balb/c小鼠.分离小鼠脾脏B淋巴细胞与骨髓瘤细胞融合并培养,挑选阳性杂交瘤细胞,扩大培养,制备和鉴定P2X7其生物学效应.结果显示,获得1株稳定分泌抗人P2X7受体的单克隆抗体的杂交瘤细胞株,所分泌的单抗类型重链为IgG1,轻链为κ;该株杂交瘤细胞腹水效价为1∶6.4×104;传30代及液氮中保存6个月,抗体效价稳定;Western blotting检测证明该单抗与人细胞表面的P2X7受体蛋白特异地结合.所制备的抗人P2X7受体的单克隆抗体具有高度的特异性及稳定性,为针对P2X7受体为靶点的抗体药物的开发应用、疾病的辅助诊断奠定了基础.     

Quorum Sensing System Used as a Tool in Metabolic Engineering

Quorum sensing (QS) is a ubiquitous cell–cell communication mechanism in microbes that coordinates population‐level cell behaviors, such as biofilm production, virulence, swarming motility, and bacterial persistence. Efforts to engineer QS systems to take part in metabolic network regulation represent a promising strategy for synthetic biology and pathway engineering. Recently, design, construction, and implementation of QS circuits for programmed control of bacterial phenotypes and metabolic pathways have gained much attention, but have not been reviewed recently. In this article, the architectural organizations and genetic contributions of the naturally occurring QS components to understand the mechanisms are summarized. Then, the most recent progress in application of QS toolkits to develop synthetic networks for novel cell behaviors creation and metabolic pathway engineering is highlighted. The current challenges in large‐scale application of these QS circuits in synthetic biology and metabolic engineering fields are discussed and future perspectives for further engineering efforts are provided.     

人Leptin基因在大肠杆菌中的高效表达、纯化及其生物学活性研究

将人Leptin表达质粒pBV220-OB转化E.coliJM109,经热诱导获得了目的蛋白的表达。经SDS-PAGE鉴定分析,表达产物以包涵体形式存在,目的蛋白表达量占菌体总蛋白的40%以上。通过包涵体分离,Sephacryl S200HR凝胶和DEAE52离子交换层析及Hypersil C18柱反相色谱纯化,获得纯度在95%以上,内毒素含量小于10EU/mg的高纯度的重组人Leptin。Western-blot鉴定表明,纯化表达产物能和抗Leptin抗体特异性结合;蛋白质N端15个氨基酸序列分析结果和预期的序列一致。纯化产物经复性处理,其分子中Cys96和Cys146形成二硫键。体内活性检测显示,纯化和复性的rh-Leptin明显抑制BALB/c小鼠的进食和体重增长,提示其具有明显的生物学活性。     

Molecular genetic characterization of rice seed lipoxygenase 3 and assessment of its effects on seed longevity

Lipoxygenases (LOXs) are enzymes involved in lipid peroxidation. Here we reported the identification, molecular and functional characterization of the gene encoding rice (Oryza sativa L.) seed LOX3 (sLOX3). Via a map-based cloning strategy we identified Os03g0700400 as the candidate gene encoding sLOX3. Further functional complementary test and biochemical characterization of the recombinant Os03g0700400 protein verified the identification. The sLOX3 gene was highly expressed in roots, moderately in embryos and very weakly in leaves, leaf sheaths and stems. Transient expression experiment (in rice protoplasts) and subsequent laser confocal microscopic analysis demonstrated that the sLOX3 protein was localized into the cytosol. We next showed that overexpression of sLOX3 in a japonica sLOX3-normal rice cultivar, Wuyunjing 7 accelerated the decrease of seed germination ability when the seeds were routinely stored, which demonstrated that sLOX3 had a negative effect on seed longevity (storability). Meanwhile, an increased occurrence of embryo decay was observed in the same transgenic seeds, suggesting that sLOX3 might negatively affect seed longevity by facilitating colonization of particular seed pathogens. Our result forwarded the understanding of the effects of 9-LOX on rice seed longevity.     

Pyrosequencing Reveals Contrasting Soil Bacterial Diversity and Community Structure of Two Main Winter Wheat Cropping Systems in China

Microbes are key components of the soil environment, playing an important role in maintaining soil health, sustainability, and productivity. The composition and structure of soil bacterial communities were examined in winter wheat–rice (WR) and winter wheat–maize (WM) cropping systems derived from five locations in the Low-Middle Yangtze River plain and the Huang-Huai-Hai plain by pyrosequencing of the 16S ribosomal RNA gene amplicons. A total of 102,367 high quality sequences were used for multivariate statistical analysis and to test for correlation between community structure and environmental variables such as crop rotations, soil properties, and locations. The most abundant phyla across all soil samples were Proteobacteria, Acidobacteria, and Bacteroidetes. Similar patterns of bacterial diversity and community structure were observed within the same cropping systems, and a higher relative abundance of anaerobic bacteria was found in WR compared to WM cropping systems. Variance partitioning analysis revealed complex relationships between bacterial community and environmental variables. The effect of crop rotations was low but significant, and interactions among soil properties, locations, and crop rotations accounted for most of the explained variation in the structure of bacterial communities. Soil properties such as pH, available P, and available K showed higher correlations (positive or negative) with the majority of the abundant taxa. Bacterial diversity (the Shannon index) and richness (Chao1 and ACE) were higher under WR than WM cropping systems.     

A型流感病毒持续感染细胞系来源的IVpi-189病毒生物学性状的初步研究

为明确E61-24-P15 A型重组流感病毒的第189代传代子病毒(IVpi-189)是否具备流感病毒温度敏感减毒活疫苗候选株的特点,将IVpi-189病毒感染MDCK细胞,并于不同培养温度条件下培养,观察其致细胞病变效应,病毒合成、释放情况,以及不同温度条件下病毒存活时间。结果显示32℃培养温度下,IVpi-189病毒具有等同于亲代野生病毒株的诱导细胞病变能力,而当培养温度上调至38℃,IVpi-189病毒致细胞病变效果出现缓慢且程度明显减轻。空斑形成单位实验发现IVpi-189病毒在38℃培养条件下增殖能力明显下降,其原因与病毒灭活速度及子病毒释放无关,但与感染细胞病毒合成能力下降有关。上述实验结果初步证实流感病毒持续感染细胞系来源的IVpi-189病毒具有温度敏感减毒活疫苗的生物学特性,在许可培养温度条件下具有良好的增殖能力,而在非许可培养温度下,病毒增殖活性受到明显抑制。本研究为流感病毒减毒活疫苗的开发研制提供实验佐证。     

Inhibition of Filamin-A Reduces Cancer Metastatic Potential

Filamin-A cross-links actin filaments into dynamic orthogonal networks, and interacts with an array of proteins of diverse cellular functions. Because several filamin-A interaction partners are implicated in signaling of cell mobility regulation, we tested the hypothesis that filamin-A plays a role in cancer metastasis. Using four pairs of filamin-A proficient and deficient isogenic cell lines, we found that filamin-A deficiency in cancer cells significantly reduces their migration and invasion. Using a xenograft tumor model with subcutaneous and intracardiac injections of tumor cells, we found that the filamin-A deficiency causes significant reduction of lung, splenic and systemic metastasis in nude mice. We evaluated the expression of filamin-A in breast cancer tissues by immunohistochemical staining, and found that low levels of filamin-A expression in cancer cells of the tumor tissues are associated with a better distant metastasis-free survival than those with normal levels of filamin-A. These data not only validate filamin-A as a prognostic marker for cancer metastasis, but also suggest that inhibition of filamin-A in cancer cells may reduce metastasis and that filamin-A can be used as a therapeutic target for filamin-A positive cancer.     

芽孢杆菌β—淀粉酶基因在大肠杆菌中的克隆与表达

本工作采用新设计的营养缺陷型淘汰筛选法,从本实验室筛选的芽孢杆菌Bacillus R2中分离到能够水解生淀粉的β-淀粉酶基因。该基因所在的DNA片段为5.25kb,在大肠杆菌(E.coli)中的表达产物具有与供体菌相同的淀粉酶特性,实验室条件下酶产量为500IU/ml以上,RDA值为57%,并且可以全部分泌到培养基中。