溶藻弧菌HY9901血红素结合蛋白HutB的原核表达及纯化

为探究溶藻弧菌(Vibrio alginolyticus)HY9901血红素结合蛋白HutB作为疫苗候选抗原的可能性,采用PCR方法扩增V.alginolyticus HY9901 hutB基因全长序列,克隆到pMD18-T载体,经双酶切、连接后,定向插入到pET-28a(+)中,构建原核表达载体pET-HutB,转入大肠杆菌BL21(DE3)中进行IPTG诱导表达。SDS-PAGE验证后,优化诱导表达条件和纯化条件,并进行Western-blot分析。结果表明,HutB蛋白在E.coli中诱导表达的最优条件:0.4 mmol/L IPTG,37℃诱导4h,表达的蛋白分子量与预期大小相符,主要以包涵体的形式存在,300 mmol/L咪唑洗脱时效果最佳,能与鼠抗His-tag单抗特异性结合。     

人工湿地系统对泉河水质净化处理的实践应用

泉河是山东省济宁市汶上县一条重要的河流,工业点源、农业面源污染使得泉河污染,水质超标。为净化水质,建设了泉河河道走廊人工湿地工程,总长度23km,规模319.7hm2,总投资5170万元。主要采用河道滞留塘+表面流人工湿地组合工艺,处理汶上县污水处理厂外排水、沿河周边农村生活污水及农田退水,污水处理规模2.0万m3/d。工程2010年10月开工,2011年6月建成并正常运行。通过1a多的运行结果表明,该人工湿地工程具有较高的污染负荷去除率,实现了河道的生态修复和沿河生态环境治理,泉河生态环境得到了明显改善。     

Purification and characterization of a new thermostable κ-carrageenase from the marine bacterium Pseudoalteromonas sp. QY203

A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30℃ to 50℃ with the highest activity at 45℃ and pH7.2. Sodium chloride increased its activity markedly, and KCl increased its activity slightly. The divalent and trivalent metal ions including Cu2+ , Ni2+ , Zn2+ , Mn2+ , Al3+ and Fe3+ significantly inhibited its activity, while Mg2+ did not. CgkP remained 70% of original activity after being incubated at 40℃ for 48 h, and remained 80% of the activity after being incubated at 45℃ for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.     

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca²⁺, suggesting that the clotting reaction is catalyzed by a Ca²⁺-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.     

杂色鲍血蓝蛋白多克隆抗体的制备与血蓝蛋白35kDa片段鉴定

以杂色鲍(Haliotis diversicolor Reeve)为研究对象,通过密度梯度离心方法纯化得到高纯度血蓝蛋白,以其作为抗原皮下注射免疫新西兰大白兔,从而获得高效价的兔源多克隆抗血清。进一步通过Protein A抗原亲和纯化的方法对该抗血清纯化,最终获得效价更高、检测特异性更好的血蓝蛋白多克隆抗体。应用该抗体进行Western检测发现,鲍血淋巴中存在着多样的血蓝蛋白衍生产物;进一步结合质谱技术对其中35 kDa条带进行鉴定发现,其来源于血蓝蛋白I型亚基的H结构域。     

红笛鲷重组激活基因rag1原核表达条件的优化及纯化

克隆编码红笛鲷(Lutjanus sanguineus)RAG1蛋白(recombination activating protein1)活性核心区的基因序列,并与pET-28a(+)载体连接,构建原核表达载体pET-28a-RAG1,将其转入大肠杆菌BL21(DE3)菌株,利用IPTG进行诱导表达。为提高融合蛋白的表达效率,运用传统的实验方法对诱导条件进行优化。SDS-PAGE分析表明,在37℃条件下,利用0.1 mmol/L IPTG诱导8 h后,RAG1重组融合蛋白的表达量最大,相对分子质量与预测值相符,该蛋白主要以包涵体形式高效表达,利用His Trap HP亲和柱使其得到进一步纯化;Western blot分析显示,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,说明表达蛋白为目的蛋白。     

小叶章湿地系统对污水中磷的净化模拟研究

2008年5～9月,在三江平原小叶章(Calamagrostis angustifolia)湿地中,通过在模拟桶中人工加入3种浓度的含磷污水进行湿地系统对污水中磷的净化模拟研究.结果表明,小叶章湿地系统对污水中正磷酸盐的净化效率较高,平均净化率为85.26%;小叶章主要生长过程中的各生长阶段对湿地系统净化污水中正磷酸盐效率的影响显著(n=60, p<0.05);在小叶章慢速生长阶段至衰老阶段(7月8日～9月30日),湿地系统对污水中正磷酸盐的净化率逐渐降低;小叶章湿地系统对污水中有机磷的净化效率较差,平均净化率为11.64%;生长阶段对小叶章湿地系统净化污水中有机磷效率的影响显著(n=60, p<0.01);小叶章慢速生长阶段至衰老阶段,湿地系统对污水中有机磷的净化率逐渐降低.磷输入浓度由5 mg/L增加到10 mg/L时,磷输入浓度增加对小叶章湿地系统净化污水中正磷酸盐效率的影响不明显,对净化污水中有机磷效率的影响显著(n=60, p<0.01),且在小叶章不同生长阶段,其影响也不同.在小叶章快速生长阶段(6月10日～7月7日);磷输入浓度由10 mg/L增加到20 mg/L时,小叶章湿地系统净化污水中正磷酸盐的效率显著降低(n=24, p<0.01),净化污水中有机磷的效率显著升高(n=24, p<0.05).     

紫菜中类菌孢素氨基酸纯化工艺的优化及其抗紫外辐射作用研究

为了筛选纯化紫菜类菌孢素氨基酸(mycosporine-like amino acid,MAAs)的离子交换树脂,优化纯化工艺,并研究纯化后MAAs体外抗紫外辐射活性,本研究通过静态动力学吸附及解析实验,筛选纯化树脂;结合动态动力学吸附及解析单因素实验建立响应面法对SA-2型阳离子树脂纯化MAAs的二次多项式模型,对MAAs的纯化工艺进行优化;通过建立大肠杆菌、金黄色葡萄球菌的复合紫外线(UVA+UVB)损伤模型,分析纯化后MAAs样品的体外抗辐射活性。结果表明:静态动力学吸附及解析实验筛选出SA-2型阳离子树脂为纯化MAAs的最佳树脂,吸附率和解析率分别为86.01%和56.63%;吸附的最佳工艺条件为:上样液质量浓度3 g/L、上样流速1 m L/min、上样液pH为6、洗脱液体积分数为3%、洗脱流速为1.5 m L/min、洗脱液pH为6;在此条件下吸附率为79.21%,洗脱率为64.02%;纯化后的MAAs样品能有效抑制紫外辐射而造成的损伤,将菌种失活速度分别降低了39.10%和40.58%,表现出显著的抗紫外辐射活性。     

A Challenge of Water Purification in Dokai Bay, Japan

A history of water pollution and countermeasures for water purification in Dokai Bay, Japan are reviewed. Now, Dokai Bay suffers from the occurrences of red tides and oxygen-deficiency in summer. In order to prevent the occurrences of red tides and oxygen-deficiency, an ecological numerical model has been developed. The model experiments forecast that when the phosphorus or nitrogen load from land will be cut by 90% or 95%, respectively, red tides and oxygen-deficiency will not occur in Dokai Bay. If the industrial and sewage facilities are not sufficient to cut the nutrients load to the necessary degree, we have to consider other countermeasures such as cultivating bivalves and algae in order to decrease the nutrients concentration in the bay. International co-operation related to coastal zone management is also discussed.     

Insect communities of Phragmites habitats used for sewage purification: Effects of age and area of habitats on species richness and herbivore-parasitoid interactions

In 1994, 28 sewage purification plants based on common reed Phragmites australis were analyzed, to study fragmentation effects on diversity and on food web structure of insect communities. Purification plants differed in age (2 to 11 years) and area (10 to 2,500 m²). Both age and area were significantly correlated with species richness of herbivores. Populations of the two most abundant herbivores, the gall makers Lipara pullitarsis (Diptera, Chloropidae) and Giraudiella inclusa (Diptera, Cecidomyiidae), increased significantly with age and size of the Phragmites habitats, i.e. the purification plants. Incidence curves showed that only habitats older than 6 years and larger than 100 m² have a 50% probability of finding these two most abundant gall makers, and can correspondingly be considered to contribute to insect species conservation. Natural enemies were even more susceptible to habitat fragmentation than their phytophagous hosts or prey, thereby hampering possible biocontrol. Percent parasitism of both the Lipara and Giraudiella gall makers significantly increased with habitat age.