乳糖诱导重组别藻蓝蛋白(rAPC)在大肠杆菌(Escherichia coli)中的表达

采用廉价的乳糖替代IPTG诱导重组别藻蓝蛋白(rAPC)在大肠杆菌(Escherichiacoli)JM109中表达,对诱导产物表达的培养基、培养条件、诱导剂的浓度和诱导时机进行了优化,将优化条件用于5L自控发酵罐进行高密度培养。结果表明,对rAPC表达的条件进行优化后,乳糖诱导rAPC的表达率可达26.8%,与IPTG的诱导结果接近;在高密度培养中,OD600最大达21.8。研究结果可为乳糖替代IPTG大规模生产rAPC奠定基础。     

乳糖诱导重组别藻蓝蛋白基因在大肠杆菌中的表达

以重组别藻蓝蛋白表达工程菌JM109（DE3）／pET28-APC作为研究对象，对于JM109（DE3）菌株在乳糖诱导下表达重组蛋白的规律进行了研究。比较分析了不同生长阶段进行诱导，最佳乳糖浓度、诱导持续时间和诱导温度等参数对重组蛋白表达的影响。实验结果表明，采用JM109（DE3）为宿主菌，经过条件优化，乳糖诱导重组蛋白的表达量可以达到IPTG诱导的水平；较低的诱导培养温度能有效提高可溶性重组蛋白的表达。最后在摇瓶发酵结果的基础上，实现了乳糖诱导工程菌的5L全自动发酵罐培养。研究结果为在大肠杆菌大规模发酵中，运用廉价、无毒的乳糖作为诱导剂提供了一定的借鉴。     

Faecal indicator bacteria in New Zealand freshwater fish: a pilot study

Recent studies have suggested that poikilothermic animals, such as fish, may represent a previously overlooked source of the faecal indicator bacteria (FIB) used for the assessment of water quality. However, quantitative studies of FIB in poikilotherms are scarce. We investigated the presence of FIB in the faeces of five freshwater fish species. E. coli and enterococci were detected in 71 and 76% of faecal samples, respectively. Concentrations were highly variable, with maximum concentrations (2.1?×?10⁴ E. coli and 1.3?×?10⁶ enterococci per gram of faecal material) up to four orders of magnitude higher than present in the water column. FIB were not detected in faecal samples from marine fish. Our findings suggest that FIB ingested from the environment may be capable of replication within the fish gut, making fish a potential source and transport mechanism for FIB in New Zealand freshwaters. This may have implications for water quality monitoring.     

cDNA cloning and expression of a cytochrome P450 1A (CYP1A) gene from the hermaphroditic fish Rivulus marmoratus

We previously described the genomic structure of the cytochrome P450 1A (CYP1A) gene from the hermaphroditic fish Rivulus marmoratus [Kim, I.-C., Kim, Y.J., Yoon, Y.-D, Kawamura, S., Lee, Y.-S., Lee, J.-S., 2004a. Cloning of cytochrome P450 1A (CYP1A) genes from the hermaphroditic fish Rivulus marmoratus and the Japanese medaka Oryzias latipes. Mar. Environ. Res. 58, 125–129]. To further characterize R. marmoratus CYP1A, we cloned the cDNA sequence of a CYP1A gene from this species and also expressed its recombinant protein in an E. coli system. We exposed R. marmoratus to 4-nonylphenol, and found a small induction of CYP1A mRNA in the treated animals. In this paper, we discuss the characteristics of R. marmoratus CYP1A gene as well as its potential use in a biomonitoring assay.     

A Sensitive and Versatile Multiplex PCR System for the Rapid Detection of Enterotoxigenic (ETEC), Enterohaemorrhagic (EHEC) and Enteropathogenic (EPEC) Strains of Escherichia coli

Although Escherichia coli is widely distributed in the marine environment, only a small percentage are pathogenic to humans. Nonetheless, the widespread occurrence of waterborne infections of E. coli origin in humans has become one of the major health problems worldwide. To date, several types of enterovirulent E. coli have been recognized as the aetiologic agents of various gastrointestinal infections in humans. The most commonly encountered are those belonging to the enterotoxigenic (ETEC), enteroinvasive (EIEC), enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) subtypes. In order to better determine the health risks that are associated with exposure to some of these specific subtypes, we have developed a very sensitive multiplex PCR system for the rapid detection and typing of ETEC, EHEC and possibly EPEC strains of E. coli in the aquatic environment. The target genes chosen for this investigation included: the PHO-A housekeeping gene (present in all E. coli); the LT1, LT2 and ST1 genes of ETEC; the VT1 and VT2 verotoxin, and EAE virulence genes of EHEC and EPEC, respectively. Six pairs of oligonucleotide primers were designed to simultaneously amplify internal fragments of these genes by multiplex PCR to generate PCR products that could be analysed and confirmed with relative ease by gel electrophoresis and HincII enzyme digestion. The results showed that the six sets of PCR primers were highly specific for their target genes and produced specific amplimers of the expected size from several control strains of E. coli – ATCC 35401 (LT1+/ST1+); SA53 (LT2+/VT2+); and O157 (VT1+/VT2+/EAE+). The detection sensitivity of the multiplex PCR system for the six target genes in an E. coli cell mixture was optimized and enhanced by preincubating serially diluted cells in Luria-Bertani broth for 6 h prior to PCR analysis. The results obtained indicated a detection sensitivity of 10° CFU (of each strain) per 100 μl reaction. Multiplex PCR analysis of seawater samples collected from four sewage-polluted sites in Hong Kong indicated the presence in all four samples of E. coli bacteria that were positive for LT1, ST1, VT1 and EAE virulence genes. Overall, the data indicated that the multiplex PCR system described in this study is a potentially very useful and powerful method for routine monitoring and risk assessment of water quality.     

Physico-chemical,bacteriological and health hazard effect analysis of the water in Taladanda Canal,Paradip area,Odisha, India

The physico-chemical and bacteriological parameters of the water in Taladanda canal and associated water-borne diseases,from which the dwellers have suffered,were studied,by using statistical method. Overuse and the addition of the wastes with sources from urbane industrial sectors,as well as the decrease in water level have caused the canal water quality declined drastically and subsequently led to extensive eutrophication and bacterial contamination. According to the water sample analytical results,the water is lightly acidic with the pH value of 4.5~6.7. The measured indexes,such as total dissolved solid (TDS),electrical conductivity (EC),total suspended solid (TSS),Mn,Zn,Al,Fe,Cu,Cr,and Hg etc,mostly have very high concentrations which are higher than permissible limit,indicating that the canal water is completely unsuitable for human consumption. Furthermore,the biological analysis shows that the total coliform (TC) is in the range of 45.9~30.2 in per 100 mL water in April,30.5~25.3/100 mL in July and 52.9~35.4/100 mL in December,respectively. Similarly,fecal coliform (FC) ranges from 12.8 to 10.1,10.5~7.5 and 13.1~6.4 per 100 mL water in the months of April,July and December respectively. As a result,people who use the water have suffered from different water-borne diseases. On the basis of disease data derived from hospital observations in a period of three years,there had been 4 284 people affected by different water-borne diseases from 2016 to 2018.     

不同种类纳米零价铁的毒性比较研究

纳米零价铁是一种高效的环境修复材料,可以处理多种污染物;然而,纳米粒子的尺寸效应可能导致其在自然界中存在潜在毒性风险。选择几种常用包覆型、负载型和裸露的纳米铁,通过大肠杆菌的耐受性实验,比较3种纳米铁的毒性。研究表明,负载型纳米铁的分散性最好,而裸露纳米铁最差。3种纳米铁虽然对大肠杆菌都表现出毒性,但是负载型纳米铁的毒性最小。通过毒性减缓的机理分析,说明纳米铁改性后阻止了纳米颗粒与细菌的直接接触,这是空间位阻效应的作用。研究结果进一步证实了在使用纳米材料前应充分评估潜在毒性和环境效应的重要性。     

正常菌与可吸入矿物细颗粒作用致突变性研究

采用Ames试验分别对蒙脱石、石英、方解石、钠长石4种可吸入矿物细颗粒及其与大肠杆菌、表皮葡萄球菌相互作用48 h后的混悬液作为受试物进行检测,分析IMG是否具有致突变性,及其与人体正常细菌群作用后,IMG/正常菌复合体是否有致突变性毒力改变。结果显示石英以及石英/大肠杆菌复合体对TA98、TA100菌株Ames试验呈阳性反应;石英/表皮葡萄球菌复合体对TA98菌株Ames试验呈阳性反应;而蒙脱石、方解石、钠长石纯矿及其与正常菌作用后的复合体Ames试验结果均为阴性。结果表明石英具有直接致突变作用;石英与正常菌菌作用后复合体依然具有致突变性,但石英表现出的碱基置换突变作用在与表皮葡萄球菌作用后消失,其机制有待进一步研究。     

溶藻弧菌asp基因在大肠杆菌中表达活性研究及条件优化

为进一步研究溶藻弧菌碱性丝氨酸蛋白酶(ASP)蛋白生物学活性和功能,将构建的pET23d-asp在大肠杆菌BL21(DE3)中表达,对表达产物进行纯化分析,并优化表达条件。结果表明:在咪唑洗脱缓冲液浓度为100 mmol/L时,表达的可溶性ASP被大量洗脱,纯化的ASP相对分子质量约为42 000,具有蛋白活性;在诱导温度28℃、异丙基-β-D硫代半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)浓度1 mmol/L及诱导时间16 h时,表达的活性ASP蛋白最多。     

Water quality in New Zealand rivers: current state and trends

River water quality, particularly in lowland catchments, is a matter of concern to the New Zealand public. We assessed river water quality and biological state and trends using data from more than 900 monitoring sites. Parallel state and trend analyses were carried out using all sites and a subset of lowland river sites. Median water-quality state in urban and pastoral land-cover classes was poorer than in exotic forest and natural land-cover classes, and lowland sites in the urban and pastoral classes had the poorest water quality. Nutrient and Escherichia coli concentrations increased and visual clarity and Macroinvertebrate Community Index scores decreased as proportions of catchments in high-intensity agricultural and urban land cover increased. Ten-year trends (2004–2013) indicated recent improvements in ammoniacal nitrogen, dissolved reactive phosphorus and total phosphorus in the pastoral and urban classes, possibly reflecting improved land management. In contrast, trends in nitrate-nitrogen in the exotic forest and cool-dry/pastoral classes indicated worsening conditions.