Bioluminescence imaging of hepatitis B virus enhancer and promoter activities in mice

By bioluminescence imaging and hydrodynamic gene transfer technology, the activities of hepatitis B virus (HBV) promoters and the effects of HBV enhancers on these promoters in mice under true physiological conditions have been assessed. Our studies reveal that either of the two HBV enhancers can stimulate HBV major promoter activity in hepa 1-6 cells (in vitro) and in mouse liver (in vivo), and the enhancer effects on the three promoters (S1, S2 and X promoter) are markedly greater in vivo than in vitro. The two HBV enhancers have no cooperative action on HBV promoters in vitro or in vivo.     

Development of antibody-based assays for omega-conotoxin MVIIA

Omega-conotoxin MVIIA (CTX MVIIA) is a specific peptide blocker of the N-type voltage-sensitive calcium channel in neurons. The synthetic version of CTX MVIIA, Ziconotide, has been recently approved by FDA for management of severe and chronic pains. Currently, the chemical synthetic CTX MVIIA has been analyzed by RP-HPLC, and there are no chemical or immunological assays available for determination of the peptide. In this article, we report a novel method for preparation of polyclonal antibody against CTX MVIIA, and the antibody-based assays for the analysis of CTX MVIIA. The DNA sequences encoding the conotoxin were chemically synthesized and then cloned into the expression vector pGEX-2T. The GST fusion protein of CTX MVIIA was expressed in E. coli BL21 (DE3) with induction of IPTG. The purified fusion protein was used to immunize the male rabbits with standard protocols. The produced antiserum was purified through anion-exchange chromatography. Another thioredoxin (Trx) fusion protein of CTX MVIIA was employed to cross-examine the antibody against the conotoxin. Our Western blot and ELISA results show that the polyclonal antibody was capable of binding the conotoxin parts of both GST and Trx fusion proteins, and the antibody titer is 1:8192. Thus, the assays based on this antibody are useful for the conotoxin analysis.     

人IFN-g体外释放检测法的建立及其在结核病诊断中的应用

本研究旨在建立人IFN-g体外释放检测法, 用于人结核病的特异性诊断。克隆表达了人IFN-g基因, 利用纯化的重组IFN-g免疫小鼠, 获得两株高效价的单克隆抗体。用所获得的单克隆抗体及兔抗IFN-g多克隆抗体建立了检测人IFN-g的夹心ELISA, 检测灵敏度达到31.25 pg/mL。采集111位结核病阳性病人与292位临床健康对照者肝素抗凝全血,利用结核菌特异性抗原ESAT-6/CFP-10融合蛋白体外刺激外周血淋巴细胞释放IFN-g, 用所建立的夹心ELISA及商品化试剂盒平行检测所有样本, 结果表明两种方法的检测结果相符。结核患者的检测灵敏度为95.5%, 健康对照的阳性检出率为16.7%, 患者与健康对照的阳性检出率差异极显著(P<0.01), 证实所建立的方法灵敏度与特异性均很高, 具有良好的应用前景。     

Microbial metabolism of 1-aminoanthracene by Beauveria bassiana

The carcinogen and mutagen, 1-aminoanthracene, was efficiently metabolized by the fungal strain Beauveria bassiana ATCC 7159 to yield three new metabolites identified as 1-acetamido-5-[(4′-O-methyl-β-d-glucopyranosyl)oxy]anthracene, 1-acetamido-8-[(4′-O-methyl-β-d-glucopyranosyl)oxy]anthraquinone, and 1-acetamido-6-[(4′-O-methyl-β-d-glucopyranosyl)oxy]anthraquinone, together with 1-acetamidoanthracene and 1-acetamidoanthraquinone. Formation of these metabolites suggests that the metabolic pathways of 1-aminoanthracene in B. bassiana ATCC 7159 involve acetylation, oxidation, hydroxylation, and O-methylglucosylation.     

Variations of enzyme activities in the haemocytes of scallop Chlamys farreri after infection with the acute virus necrobiotic virus (AVNV)

Acute virus necrobiotic virus (AVNV) is one of the main pathogens for large scale mortality of Chinese scallop Chlamys farreri. In this paper, C. farreri were infected by different dilutions of AVNV supernatant (5(0), 5(-1), 5(-2), 5(-3), 5(-4), 5(-5), 5(-6), 5(-7), respectively), and dead individuals were counted every day for 15 days. Samples from groups of 5(-3) and 5(-5) were taken every day till 15 days and the activities of acid phosphatase (ACP), alkaline phosphatase (ALP), superoxide dismutase (SOD), myeloperoxidase (MPO), phenoloxidase (PO), peroxidase (POD) and catalase (CAT) in haemocytes were measured. The results of virus challenge showed that survival rates of scallops in groups of 5(0) and 5(-1) decreased sharply after the first day and died out completely on the 5th and 4th day, respectively. In other groups (5(-2), 5(-3), 5(-4), 5(-5), 5(-6) and 5(-7)), survival rates decreased gradually till 6 or 7 days, then kept steady till 15 days, and they were dose-dependent, increasing from 12% to 80% as the dose decreased from 5(-2) to 5(-7) viral supernatant. In the control group, survival rate was 88%. Enzyme activities for groups of 5(-3) and 5(-5) illustrated that activities of ACP, SOD, MPO, PO in groups of 5(-3) and 5(-5) were significantly higher than the control group in the first 9 or 10 days, and went back to the control group levels gradually after 10 days. Moreover, their activities in group of 5(-3) varied more than that in the group of 5(-5), especially activities of MPO, PO. Differently, the activities of POD and CAT were reduced or induced by virus infection and showed no regular trends in the experiments. The activity of ALP was not detected.     

城市污水处理系统的菌群分析和除氮基因工程出发菌的选择

目的:对脱氮污水处理工艺的活性污泥的菌群组成进行分析,以期获得适合于脱氮基因工程改良的出发菌.方法:首先采用平板稀释法对活性污泥进行菌落计数,并对分离到的菌落进行详细的生化鉴定,对其中的优势菌-假单胞菌进行脱氮能力测定,并分析其对常作为筛选标志的抗生素的药物敏感性.结果:发现在采用该工艺的活性污泥中,优势菌为假单胞菌、肠杆菌、莫拉菌和不动杆菌,分别占总菌数的23%、16%、16%和12%.根据菌群分析的结果,从中选择了两株耐药性弱、脱氮能力强的菌作为基因改良的出发菌.结论:本研究阐明了活性污泥的菌群构成,获得了两株适合基因工程改良的菌株,为日后脱氮基因工程菌的构建奠定基础.     

Comparison of Gene Expression Profiles of Fenneropenaeus chinensis Challenged with WSSV and Vibrio

Microarray technique was used to analyze the gene expression profiles of shrimp when they were challenged by WSSV and heat-inactivated Vibrio anguillarum, respectively. At 6 h post challenge (HPC), a total of 806 clones showed differential expression profile in WSSV-challenged samples, but not in Vibrio-challenged samples. The genes coding energy metabolism enzyme and structure protein were the most downregulated elements in 6 h post WSSV-challenged (HPC-WSSV) tissues. However, a total of 155 clones showed differential expression in the Vibrio-challenged samples, but not in WSSV-challenged samples. Serine-type endopeptidase and lysosome-related genes were the most upregulated elements in tissues 6 h post Vibrio challenge (HPC-Vibrio). Totally, 188 clones showed differential expression in both 6 and 12 HPC-WSSV and HPC-Vibrio samples. Most of the differentially expressed genes (185/188) were downregulated in the samples of 12 HPC-WSSV, whereas upregulated in the samples at 6 and 12 HPC-Vibrio and 6 HPC-WSSV. The expression profiles of three differentially expressed genes identified in microarray hybridization were analyzed in hemocytes, lymphoid organ, and hepatopancreas of shrimp challenged by WSSV or Vibrio through real-time PCR. The results further confirmed the microarray hybridization results. The data will provide great help for us in understanding the immune mechanism of shrimp responding to WSSV or Vibrio. Wang and Li contributed equally to this work.