棉铃虫核型多角体病毒orf86基因的原核表达、抗体制备与免疫印迹检测

棉铃虫核型多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HearNPV)orf86是一个功能未知的基因。从HearNPV基因组中通过PCR得到orf86基因编码序列,将其构建于原核表达载体pGEX-4T-2,转化大肠杆菌BL21获得融合表达产物。融合表达蛋白经分离纯化后免疫新西兰大白兔,制备其多克隆抗体。用ELISA测定结果表明,ORF86多克隆抗体效价为1:5.12×105。利用该抗体Western印迹检测HearNPV感染的HZAM1细胞,检测到一个36kD的目的蛋白条带,与ORF86预期大小一致。结果表明该多抗可用于对orf86编码蛋白的检测和相关功能研究。     

Production and characterization of poly(3-hydroxypropionate-co-4-hydroxybutyrate) with fully controllable structures by recombinant Escherichia coli containing an engineered pathway

Copolyesters of 3-hydroxypropionate (3HP) and 4-hydroxybutyrate (4HB), abbreviated as P(3HP-co-4HB), was synthesized by Escherichia coli harboring a synthetic pathway consisting of five heterologous genes including orfZ encoding 4-hydroxybutyrate-coenzyme A transferase from Clostridium kluyveri, pcs' encoding the ACS domain of tri-functional propionyl-CoA ligase (PCS) from Chloroflexus aurantiacus, dhaT and aldD encoding dehydratase and aldehyde dehydrogenase from Pseudomonas putida KT2442, and phaC1 encoding PHA synthase from Ralstonia eutropha. When grown on mixtures of 1,3-propanediol (PDO) and 1,4-butanediol (BDO), compositions of 4HB in microbial P(3HP-co-4HB) were controllable ranging from 12 mol% to 82 mol% depending on PDO/BDO ratios. Nuclear magnetic resonance (NMR) spectra clearly indicated the polymers were random copolymers of 3HP and 4HB. Their mechanical and thermal properties showed obvious changes depending on the monomer ratios. Morphologically, P(3HP-co-4HB) films only became fully transparent when monomer 4HB content was around 67 mol%. For the first time, P(3HP-co-4HB) with adjustable monomer ratios were produced and characterized.     

Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli

ABSTRACT: BACKGROUND: Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as gamma-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). RESULTS: Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies. CONCLUSIONS: Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.     

Correction to: Environmental biodegradation of haloarchaea-produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in activated sludge

Applied Microbiology and Biotechnology - The published online version contains mistake in the funding information. Instead of 30370096, it should have been 31370096.     

湖南沅水下游?繁殖期内繁殖力和卵径的变化研究

为分析鱼类繁殖期内繁殖力和卵径变化趋势, 研究于4—10月在沅水下游逐月采集180尾?(Hemiculter leucisculus), 分析81尾雌性成熟个体的繁殖力。研究结果表明: ?的性体指数在5、6月最高, 其次在7、8和9月, 在4和10月最低。绝对繁殖力平均值为(30116±19390)粒, 以10000—30000粒为主, 相对繁殖力的平均值为(650±324)粒/g, 以400—800粒/g为主。绝对繁殖力与体长、空壳重呈幂函数关系(P<0.05)。绝对繁殖力和相对繁殖力在5和6月最大, 7、8和9月次之, 在4和10月最小。不同月份成熟卵子的卵径无显著差异, 补充卵子的卵径在5和6月最大, 暗示5和6月分批繁殖次数较多。不同繁殖时期的分批繁殖力、繁殖次数不一样, 对评估种群的繁殖潜能具有重要的指导意义。比较不同水系繁殖力发现, 湖泊种群的个体繁殖力普遍大于河流种群, 沅水种群的个体繁殖力跟湖泊种群的相似, 可能与其具有类似湖泊的缓流水或静水生境有关。研究揭示了?的基本繁殖特征, 为渔业资源管理提供了基础数据资料。     

Biosynthesis and characterization of polyhydroxyalkanoate block copolymer P3HB-b-P4HB

Polyhydroxyalkanoates (PHA) synthesis genes phbC and orfZ cloned from Ralstonia eutropha H16 were transformed into beta-oxidation weakened Pseudomonas putida KTOY08ΔGC, a mutant of P. putida KT2442. The recombinant P. putida strain termed KTHH06 was able to produce a short-chain-length PHA block copolymer consisting of poly(3-hydroxybutyrate) (P3HB) as one block and poly(4-hydroxybutyrate) (P4HB) as another block. One-dimensional and two-dimensional nuclear magnetic resonance (NMR) clearly indicated the polymer was a diblock copolymer consisting of 20 mol % P3HB as one block and 80 mol % P4HB as another one. Differential scanning calorimetric (DSC) showed that P3HB block melting temperatures (T(m)) in the block copolymer P3HB-b-P4HB was shift to low temperature compared with homopolymer P3HB and a blend of P3HB and P4HB. The block copolymer with a number average molecular weight of 50000 Da and a polydispersity of 3.1 demonstrated a better yield and tensile strength compared with that of its related random copolymer and blend of homopolymers of P3HB and P4HB.     

富含miR-30a的牛初乳组分通过抑制癌细胞自噬促进顺铂化疗的肿瘤消亡

本实验首次发现牛初乳中富含miR-30a.取牛初乳中富含miR-30a的组分处理用顺铂化疗的癌细胞,能显著地抑制癌细胞自噬,促进癌细胞凋亡:单独使用顺铂化疗18 h的癌细胞凋亡率为12.5%,而化疗的同时施加富含miR-30a的牛初乳组分18 h后癌细胞凋亡率为56.5%.用这种富含miR-30a的牛初乳组分饲喂顺铂化疗的荷瘤小鼠,能更有效地抑制肿瘤生长:化疗小鼠饲喂牛初乳组分后,肿瘤直径自6 mm缩小为4.5 mm,而单独用顺铂化疗的肿瘤直径约8 mm.因此富含miR-30a的牛初乳组分能增进化疗药物的疗效.施用牛初乳组分,还可减少化疗药物的用量:单独用顺铂处理细胞18 h需20 mg/L剂量,加牛初乳组分后10 mg/L即可取得明显凋亡效果;单独用顺铂化疗荷瘤小鼠10天每次需500μg,而添加富含miR-30a的牛初乳组分后,顺铂剂量减半即可明显缩小肿瘤.     

5,7-双羟色胺损毁大鼠中缝背核后底丘脑核的神经活动增强

采用玻璃微电极在体细胞外记录法,观察了5,7-双羟色胺(5,7-dihydroxytryptamine,5,7-DHT)损毁大鼠中缝背核(dorsalraphenucleus,DRN)后,底丘脑核(subthalamicnucleus,STN)神经元电活动的变化。结果发现,对照组和DRN损毁组大鼠STN神经元的放电频率分别是(6.93±6.55)Hz和(11.27±9.31)Hz,DRN损毁组大鼠的放电频率显著高于对照组(P<0.01)。在对照组大鼠,13%的神经元呈现规则放电,46%为不规则放电,41%为爆发式放电;而在DRN损毁组大鼠,具有规则、不规则和爆发式放电的神经元比例分别为9%、14%和77%,爆发式放电的STN神经元比例明显高于对照组(P<0.01)。结果显示,DRN损毁后大鼠STN神经元的放电频率增高,爆发式放电增多,提示在正常大鼠DRN抑制STN神经元的活动。     

13,28-Epoxy triterpenoid saponins from Ardisia japonica selectively inhibit proliferation of liver cancer cells without affecting normal liver cells

Twenty 13,28-epoxy and related triterpenoid saponins from Ardisia japonica were evaluated for their anti-proliferative activity on human liver cancer cells and normal liver cells. Eight saponins selectively inhibited the growth of liver cancer Bel-7402 and HepG-2 cells without affecting the survival of normal liver HL-7702 cells. The structure-activity relationship analyses indicated that the 13,28-epoxy, 16α-hydroxy, and C-30 methyl moieties in the sapogenin parts and the glycosyl moiety consisting from tetra- to hepta-saccharide units are important for this activity. Among the active saponins, ardisianoside B (2) and 3β-O-β-d-glucopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)]-α-l-arabinopyranosyl-13β,28-epoxy-16α-hydroxyoleanane (3) showed the most potent anti-proliferative activity against Bel-7402 cells in a dose- and time-dependent manner. The selective anti-proliferative activity is attributed to the different cellular responses (CDKs and cyclins levels, cell cycle arrest and apoptosis) between tumor and normal liver cells. Exposure to 2 and 3 selectively led to cell cycle arrest and apoptosis in Bel-7402 cells together with the increased pro-apoptotic caspase-8 and the decreased anti-apoptotic Cdc25A levels.