高滴度单克隆抗体杂交瘤细胞系获得途径的探讨

为探索获得能分泌较高价单克隆抗体杂交瘤细胞系的途径,比较了用不同免疫方法得到的小鼠脾细胞制备杂交瘤,对其产生有效瘤和高价抗体分泌瘤的影响。用不使小鼠致病的病毒(如NDV)或巳灭活的病毒作为抗原时,以较高浓度加佐剂、长间隔(30天)、多次免疫效果较好。用能使小鼠感染的病毒作为免疫原时,以活病毒的亚致死剂量感染小鼠,取其脾细胞制备杂交瘤,效果最理想。有效杂交瘤产率高,分泌高效价抗体的杂交瘤细胞系也多。探讨了从免疫方法着手,定向研制高价单克隆抗体杂交瘤细胞系的可行性。     

Effect of drought on ear and flag leaf photosynthesis of two wheat cultivars differing in drought resistance

We investigated net photosynthetic rate (P_N) of ear and two uppermost (flag and penultimate) leaves of wheat cultivars Hongmangmai (drought resistant) and Haruhikari (drought sensitive) during post-anthesis under irrigated and non-irrigated field conditions. The P_Nof ear and flag leaf were significantly higher and less affected by drought in Hongmangmai than in Haruhikari. The rate of reduction in stomatal conductance (g_s) was similar for the two cultivars, but intercellular CO₂concentration (C_i) in the flag leaf of Hongmangmai was lower than that of Haruhikari in non-irrigated treatment. No differences were observed in leaf water potential (ψ₁) and osmotic adjustment of the flag leaf of the cultivars. These results imply that differences in photosynthetic inhibition on the flag leaf at low leaf ψ₁between the cultivars were primarily due to non-stomatal effects. Hence the main physiological factor associated with yield stability of Hongmangmai under drought stress may be attributed to the capacity for chloroplast activity in the flag leaf, which apparently allows sustained P_Nof flag leaf during grain filling under drought stress. The higher P_Nof ear in Hongmangmai under drought could also be related to its drought resistance.     

Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon

We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.     

An SEIS epidemic model with transport-related infection

In this paper, an SEIS epidemic model is proposed to study the effect of transport-related infection on the spread and control of infectious disease. New result implies that traveling of the exposed (means exposed but not yet infectious) individuals can bring disease from one region to other regions even if the infectious individuals are inhibited from traveling among regions. It is shown that transportation among regions will change the disease dynamics and break infection out even if infectious diseases will go to extinction in each isolated region without transport-related infection. In addition, our analysis shows that transport-related infection intensifies the disease spread if infectious diseases break out to cause an endemic situation in each region, in the sense of that both the absolute and relative size of patients increase. This suggests that it is very essential to strengthen restrictions of passengers once we know infectious diseases appeared.     

Spartina alterniflora invasions and effects on crab communities in a western Pacific estuary

We investigated Spartina alterniflora invasions, their relationship with shoreline dynamics and effects on crab communities in the Yellow River estuary, China, where shoreline dynamics have been accelerated due to human-mediated estuarine sediment deposition and sea-level rise. We determined the distribution of Spartina with extensive ground surveys, and quantified shoreline dynamics between 2001 and 2009 by interpreting satellite images. We used pitfall traps to sample crab populations in Spartina-invaded habitats and non-invaded mudflats in 2009 and 2010. Large areas (>0.5 km²) of Spartina plants were found at three locations in the estuary. The seaward limit of Spartina at each location generally coincided with the present shoreline, regardless of historical shoreline advance or retreat. Crab communities in Spartina-invaded habitats significantly differed from those in non-invaded habitats. The total number and biomass of crabs caught per trap were much higher in Spartina-invaded habitats than in non-invaded habitats, however, species richness and Shannon diversity were much lower. These results suggest that Spartina invasions are likely to keep pace with shoreline dynamics accelerated by global change and have significant ecological consequences for crab communities. These issues should be taken into account to improve the use and management of Spartina, especially in rapidly accreting estuaries with large-area mudflats that are important habitats but prone to Spartina invasions.     

大肠杆菌中高效表达rhBMP-4的探讨及初步活性测定

目的 在大肠杆菌中表达具有生物活性的rhBMP-4。方法 在不改变氨基酸序列的前提下,以全基因合成的方式对人BMP-4成熟肽基因全长进行定点突变,将之重组入pET-3c表达载体并转化至大肠杆菌BL21(DE)plysS。IPTG诱导和包涵体复性后,利用C2C12细胞横向成骨细胞分化实验以及小鼠肌袋异位骨形成实验检测其活性。 结果 获得0.348 kb的BMP-4 DNA序列,表达的目的蛋白主要以包涵体的形式存在。经纯化及复性后,体内与体外的活性检测表明rhBMP-4有良好的诱骨生成活性。结论 该方案能够实现rhBMP-4在大肠杆菌中的高效表达。     

Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells

Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-αscFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.     

Esophageal cancer‐derived microvesicles induce regulatory B cells

The role of B cells in the generation of cancer‐immune tolerance is unclear. This study aims to investigate the role of cancer‐derived microvesicles (Mvcs) in the generation of transforming growth factor (TGF)‐β⁺ B cells. In this study, esophageal cancer (Eca) cells were isolated from surgically removed cancer tissue. Mvcs were purified from the culture supernatant and characterized by Western blotting. The immune suppression assay was carried out with a cell culture model and flow cytometry. The results showed that Eca‐derived Mvcs were LAMP1 positive and carried MMP9. Exposure to the Mvcs induces naive B cells to differentiate into TGF‐β‐producing regulatory B cells; the latter show immune suppressor functions on CD8⁺ T‐cell proliferation. In conclusion, Eca‐derived Mvc can induce TGF‐β⁺ B cells; the latter suppress CD8⁺ T‐cell activities. The MMP9‐laden Mvcs may be a new therapeutic target in the treatment of Eca. Copyright © 2015 John Wiley & Sons, Ltd.     

Small G Rac1 is involved in replication cycle of dengue serotype 2 virus in EAhy926 cells via the regulation of actin cytoskeleton

Bleeding is a clinical characteristic of severe dengue and may be due to increased vascular permeability. However, the pathogenesis of severe dengue remains unclear. In this study, we showed that the Rac1-microfilament signal pathway was involved in the process of DENV serotype 2 (DENV2) infection in EAhy926 cells. DENV2 infection induced dynamic changes in actin organization, and treatment with Cytochalasin D or Jasplakinolide disrupted microfilament dynamics, reduced DENV2 entry, and inhibited DENV2 assembly and maturation. Rac1 activities decreased during the early phase and gradually increased by the late phase of infection. Expression of the dominant-negative form of Rac1 promoted DENV2 entry but inhibited viral assembly, maturation and release. Our findings demonstrated that Rac1 plays an important role in the DENV2 life cycle by regulating actin reorganization in EAhy926 cells. This finding provides further insight into the pathogenesis of severe dengue.