Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.     

Crisis-Like Behavior in China's Stock Market and Its Interpretation

In order for China to play a bigger, more positive role in the world, it is important for China to have a healthy capital market. This perception motivates us to examine the health of China''s capital market, especially the severity of the overall loss of the listed companies in China and the effects of accounting irregularities on the losses. We show the overall loss of the listed companies was very severe, in particular, crisis-like behavior emerged in the fourth quarter of 2002, 2004, 2005, and 2008. We further observe that loss in the fourth quarter was much greater than the average loss of the first three quarters in the same year. The most straightforward interpretation of this loss pattern is that companies underreported losses in the first three quarters, to boost their stock values in most time of the year. However, in the fourth quarter, accounting balance of the whole year dictated that the reported loss in the fourth quarter had to be much greater than the actual loss. Fortunately, such irregularity has been greatly reduced, thanks to the accounting reforms in China in 2007.     

含甲肝病毒基因的重组痘苗病毒在动物体内产生针对甲肝病毒的中和抗体

用DNA重组技术得到的含甲肝病毒基因的重组痘苗病毒,可在家兔体内产生ELISA竞争抑制与中和抗体。基础免疫后,动物体内竞争抑制抗体滴度为10,加强免疫后达到80。由重组病毒产生的抗体中和指数比甲肝病毒产生者略低。     

Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis

A novel δ-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The δ-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF486523","term_id":"1121787749","term_text":"EF486523"}}EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 α-helices (domain I); 213 residues forming three antiparallel β-sheets (domain II); and 134 residues forming a β-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC₅₀s) were 8.98 μg/ml for the expressed Cry7Ca1, 0.87 μg/ml for the activated toxin 1, and 4.43 μg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.     

海洋芽孢杆菌（Bacillus marinus）B-9987菌株抑制病原真菌机理的研究

摘要：【目的】：探讨海洋芽孢杆菌（Bacillus marinus）B-9987菌株的代谢产物BMME-1,对植物病原真菌茄链格孢菌的抑菌作用机理。【方法】分别使用分光光法、气相色谱-质谱GC-MS联用技术、红外光谱法等,检测了BMME-1处理病原真菌后,菌体渗透性、细胞壁及细胞膜成份的变化。【结果】BMME-1对茄链格孢菌的抑菌中浓度（MIC50）为6.2 mg/L,最小杀菌浓度（MFC）为50 mg/L,在MIC50浓度或高于此浓度处理靶标菌,将导致菌体蛋白质、核酸等大分子物质的外流;处理菌株葡聚糖结     

乏情和发情初产母猪下丘脑–垂体–卵巢轴中lincRNAs表达谱比较分析

初产母猪断奶后能否正常发情对养猪生产影响重大,也是初产母猪被淘汰的主要原因。本研究以乏情和发情初产母猪为研究对象,首次利用RNA-seq技术对其下丘脑-垂体-卵巢轴中的基因间长链非编码RNAs(long intergenic noncoding RNAs,lincRNAs)进行筛选比较,得到lincRNAs的表达图谱,并对其特征和功能进行了初步分析。结果显示,在乏情和发情初产母猪下丘脑–垂体–卵巢轴中鉴定得到3519个lincRNAs,以发情组为对照共有17个lincRNAs存在差异表达,其中12个表达上调,5个表达下调(FC≥2,P<0.05)。选择4个差异表达的lincRNAs经qRT-PCR验证,其表达水平与测序结果基本一致。对这17个差异表达的lincRNAs进行GO分析、KEGG通路分析及lincRNA-mRNA共表达网络分析,发现这些lincRNAs主要与猪卵母细胞减数分裂成熟、卵巢细胞分化及颗粒细胞凋亡等生殖活动相关。本研究结果丰富了猪lincRNAs数据资源,为进一步深入研究初产母猪的生殖机能提供了理论依据。     

Functions of poly-gamma-glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance

Poly-γ-glutamic acid (γ-PGA) is an important biopolymer with greatly potential in industrial and medical applications. In the present study, we constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens LL3 strain with considerable γ-PGA production, which was carried out by single, double, and triple markerless deletions of three degradation genes pgdS, ggt, and cwlO. The highest γ-PGA production (7.12 g/L) was obtained from the pgdS and cwlO double-deletion strain NK-pc, which was 93 % higher than that of wild-type LL3 strain (3.69 g/L). The triple-gene-deletion strain NK-pgc showed a 28 % decrease in γ-PGA production, leading to a yield of 2.69 g/L. Furthermore, the cell morphologies of the mutant strains were also characterized. The cell length of cwlO deletion strains NK-c and NK-pc was shorter than that of the wild-type strain, while the ggt deletion strains NK-g, NK-pg, NK-gc, and NK-pgc showed longer cell lengths. This is the first report concerning the markerless deletion of γ-PGA degradation genes to improve γ-PGA production in a glutamate-independent strain and the first observation that γ-glutamyltranspeptidase (encoded by ggt) could be involved in the inhibition of cell elongation.