多拷贝表达转鲑鱼降钙素基因酵母的急性、亚急性毒性

口服转鲑鱼降钙素基因酵母为鲑鱼降钙素的应用开辟了一条新的途径.为评价转鲑鱼降钙素基因酵母的安全性,将转基因酵母分别灌胃昆明种小鼠和Wistar大鼠进行急性毒性实验(7 d)、亚急性毒性实验(8周).急性毒性实验结果表明灌喂转基因酵母对小鼠无致死效应(LD50>10 000 mg/kg);亚急性毒性实验中高(2.0 g/kg)、低(0.5 g/kg)剂量组动物的一般状况、体重、主要脏器系数、血液学指标及血液生化指标与各对照组比较,均未见明显差异(P>0.05),组织病理切片检查未发现病理性变化.实验结果说明转鲑鱼降钙素基因酵母不会对实验动物造成不良影响,是安全无毒的.图3表3参16     

北京官厅水库水体甲状腺激素干扰效应检测及特征

应用重组人甲状腺激素受体(hTR)基因酵母快速测试方法,检测北京官厅水库水体类/抗甲状腺激素干扰效应,并分析其地理分布、季节分布特征以及水体甲状腺激素干扰化合物(TDCs)组成特征.结果表明:官厅水库水体检出了显著的抗甲状腺激素干扰效应,与其它研究区域相比,效应处于较低到中等水平;入库河流对库区水质产生显著影响,且入库河流水质受季节影响显著,因此,库区水体不同季节甲状腺激素干扰水平存在差异;C18固相萃取柱对抗甲状腺激素干扰效应的去除率在61.9%~92.3%,表明官厅水库水体中的甲状腺激素抑制活性物质多集中在有机组分,加入30mg/L的EDTA后TR抑制活性显著降低,表明官厅水库水体中TDCs除有机化合物外可能还包含重金属类无机化合物.     

In vitro thyroid disrupting e ects of organic extracts from WWTPs in Beijing

It is generally known that there are many endocrine disrupting compounds (EDCs) in the e uents from wastewater treatment plants (WWTPs). Most research has focused on the occurrence of estrogenic or androgenic activities, while ignoring that there are environmental chemicals disrupting thyroid system, which is essential for growth and development in both humans and animals. In the present work, a two-hybrid yeast assay was conducted to evaluate the removal e ciencies of agonistic or antagonistic thyroid receptor (TR) mediated e ects in di erent treatment processes of three WWTPs located in Beijing. We found no TR agonistic, but TR antagonistic activities in all processes from the WWTPs. The TR antagonistic activities in organic extracts of water samples were then calibrated regarding to a known TR-inhibitor, amiodarone hydrochloride (AH). The observed concentration of TR disrupting substances ranged from 2.35 10??8 to 6.19 10??7 mol/L AH in Gaobeidian WWTP, 3.76 10??8 to 8.75 10??8 mol/L AH in Lugouqiao WWTP, and 4.80 10??9 to 2.55 10??8 mol/L AH in Beixiaohe WWTP. Of the three WWTPs, the removal rates were 92.7%, 42.2%, and 23.1% respectively. Industrial sewage may contain more TR disrupting substances compared with domestic sewage. The recipient waters were found to contain considerable concentrations of TR disrupting substances that may cause adverse e ects on the exposed organisms.     

In vitro thyroid disrupting effects of organic extracts from WWTPs in Beijing

It is generally known that there are many endocrine disrupting compounds (EDCs) in the effluents from wastewater treatment plants (WWTPs). Most research has focused on the occurrence of estrogenic or androgenic activities, while ignoring that there are environmental chemicals disrupting thyroid system, which is essential for growth and development in both humans and animals. In the present work, a two-hybrid yeast assay was conducted to evaluate the removal efficiencies of agonistic or antagonistic thyroid receptor (TR) mediated effects in different treatment processes of three WWTPs located in Beijing. We found no TR agonistic, but TR antagonistic activities in all processes from the WWTPs. The TR antagonistic activities in organic extracts of water samples were then calibrated regarding to a known TR-inhibitor, amiodarone hydrochloride (AH). The observed concentration of TR disrupting substances ranged from 2.35 × 10-8 to 6.19 × 10-7 mol/L AH in Gaobeidian WWTP, 3.76 × 10-s to 8.75 x 10-s mol/L AH in Lugouqiao WWTP,and 4.80 × 10-9 to 2.55 × 10-s mol/L AH in Beixiaohe WWTP. Of the three WWTPs, the removal rates were 92.7%, 42.2%, and 23.1% respectively. Industrial sewage may contain more TR disrupting substances compared with domestic sewage. The recipient waters were found to contain considerable concentrations of TR disrupting substances that may cause adverse effects on the exposed organisms.     

利用唐鱼雌激素受体构建环境雌激素重组酵母测评系统的研究

利用唐鱼雌激素受体基因片段构建重组酵母,用以筛选环境雌激素类化学物质.实验先将唐鱼雌激素受体terα基因片段插入载体pGADT7中构建表达质粒p GADT7/TERα,同时将雌激素效应元件(ere)片段插入pMP206载体中构建报告质粒pMP206/ERE-Lac Z;然后把表达质粒和报告质粒共转化到酵母AH109中,经筛选成功构建了由唐鱼雌激素受体调控的表达lac Z基因的重组酵母AHpTERα/ERE.结果表明,所构建的重组酵母AHpTERα/ERE在不同浓度17β-雌二醇(E2)的诱导下,β-半乳糖苷酶的活性呈现出明显的剂量-效应关系,其EC50为(0.521±0.700)nmol·L-1.与DMSO对照组相比,重组酵母在17β-雌二醇诱导下有明显的β-半乳糖苷酶活性增强的现象.在不同浓度17α-乙炔基雌二醇(EE2)、壬基酚(NP)及其混合物、双酚A(BPA)、17β-雌二醇(E2)(阳性对照)的诱导下,重组酵母均呈现出剂量-效应关系,且灵敏度大小为E2EE2NPBPA.结果表明,本研究成功构建了重组基因酵母测评系统,初步判定该重组酵母可应用于环境雌激素的筛选.     

镉、汞、锌3种重金属的雌激素样作用（Estrogenic Activity of Cadmium, Mercury, and Zinc）

为探讨镉、汞、锌3种重金属的雌激素样作用,采用双杂交酵母法测定了醋酸镉、氯化汞、醋酸锌单独作用时对重组基因酵母β-半乳糖苷酶诱导活性的EC50值.实验结果表明,醋酸镉浓度为1.0×10-5mol·L-(1实验浓度范围10-7~10-2mol·L-1),氯化汞浓度为5.0×10-7mol·L-(1实验浓度范围10-9~10-5mol·L-1),醋酸锌浓度为1.0×10-4mol·L-(1实验浓度范围10-9~10-2mol·L-1)时对酵母β-半乳糖苷酶诱导活性最大,分别达到1.3、0.95和1.1U.不能求出镉、汞、锌单独作用时对β-半乳糖苷酶诱导活性的EC50值.一味计算EC50值不仅难以评价重金属的雌激素活性,而且限制重组受体基因酵母法的普遍运用.一些重金属通过MCF-7细胞法(E-screen)呈阳性,通过重组hERα酵母法YES检测呈阴性,有可能是重金属通过与ERβ作用而产生效应.若构建出含ERβ基因的双杂交酵母菌株,与现有的方法互补将能完善环境雌激素的体外测评系统.     

温度两阶段控制策略发酵生产重组角质酶

为进一步提高重组Bacillus subtilis WSHB06-07发酵生产角质酶的产量和生产强度,在pH两阶段控制策略的基础上,考察了温度(27～40℃)对菌体生长和产酶的影响.研究发现,37℃适于菌体生长而30℃适于菌体产酶.通过分析发酵过程中菌体比生长速率及产物比合成速率的变化,确定了温度两阶段控制策略,即0～4 h时控制温度37℃,4 h后将温度调至30℃.通过采用这一优化策略,角质酶酶活和生产强度分别可达312.5 U/mL和13.02 kU L-1 h-1,相比恒定温度37℃控制模式下分别提高了83.4%和10.9%.图6表2参13     

长江(南京段)环境雌激素的污染特征

利用固相萃取技术和重组基因酵母检测了长江(南京段)四个典型断面环境雌激素污染状况.结果表明,除秦淮新河断面外,江心洲断面、三汊河断面和大桥断面均有环境雌激素的检出,雌二醇当量分别为0.38、0.24和0.45 ng·L~(-1).与国内外地表水环境雌激素检测结果相比,长江(南京段)典型断面雌激素活性水平为较低至中等.对江心洲断面和大桥断面,甲醇洗脱组分的雌激素活性最大,其次为丙酮洗脱组分,二氯甲烷洗脱组分的雌激素活性最小;对三汊河断面,丙酮洗脱组分的雌激素活性最大,其次为二氯甲烷洗脱组分,甲醇洗脱组分的雌激素活性最小.这可能是污水来源不同所致,生活污水导致大量极性雌激素污染物的输入,而工业污水则主要引入了中等至弱极性雌激素污染物.三个雌激素活性阳性检出断面主要活性洗脱组分的雌激素活性均大于混合样的雌激素活性,这可能是由于样品混合后各种结构上不同的污染物之间的交互作用掩盖了实际的雌激素活性.     

含vgb基因的重组毕赤酵母菌对重金属离子的吸附解吸特征

酵母因为其比表面积大、密度高被广泛用于水体中重金属的吸附.本文研究比较野生毕赤酵母KM71及含有vgb基因的重组毕赤酵母KM71对重金属Ni²⁺、Cd²⁺和Pb²⁺的吸附解吸特性,并研究混合金属离子对酵母菌吸附性能的影响.不同金属离子均在2 h达到吸附解吸平衡,酵母菌对不同种类金属离子的吸附具有选择性,温度和pH变化对毕赤酵母吸附重金属影响较小.重组毕赤酵母菌对重金属的抗性更强.通过对环境因素及复合剂的优化,得到对重金属镉、镍、铅具有高效吸附性能的重组酵母菌,重组酵母菌对单元素溶液中Ni²⁺、Cd²⁺和Pb²⁺的吸附量分别为10.56、17.68和20.14 mg·g^-1,对三元金属混合溶液中的Ni²⁺、Cd²⁺和Pb²⁺的吸附量分别为8.28、9.89和19.99 mg·g^-1.通过条件优化重组毕赤酵母对Ni²⁺、Cd²⁺和 Pb²⁺的吸附率分别提高了15.14%、21.04%、18.47%,吸附量分别提高了11.26、13.43、22.90 mg·g^-1.本试验为促进酵母菌在处理重金属污染废水中的广泛应用提供了科学依据.     

降解-示踪质粒的构建及性能评价研究

在生物强化中采用报告基因技术对降解质粒进行标记,可实现对基因工程菌和降解基因的原位监测。通过基因克隆技术,将绿色荧光蛋白基因(gfp)和阿特拉津脱氯水解酶基因(atzA)重组,构建降解-示踪功能质粒,实现对阿特拉津降解质粒的示踪标记,并对转化获得的基因工程菌绿色荧光蛋白表达及阿特拉津降解能力进行评价。结果表明将gfp和atzA基因克隆至质粒载体pUC18所获得的重组质粒,经检测含有atzA基因,具有氨苄青霉素抗性,并可表达绿色荧光蛋白。携带重组质粒的基因工程菌具有阿特拉津降解活性;且在活性污泥中产生绿色荧光现象,可通过原位观察进行监测。