能源甜菜BvGST基因在大肠杆菌体内对镉逆境的应答特性分析

为了进一步研究BvGST基因(LOC104898671)在能源甜菜耐受重金属镉逆境胁迫过程中的功能,明确其在细胞解毒镉离子中的作用和应答特性,本研究以780016B/12优为植物材料,通过RT-PCR的方法将BvGST基因克隆并构建于大肠杆菌原核表达载体pEASY-Blunt E1上,并转化到大肠杆菌BL21中。SDS-PAGE电泳表明,通过1 mmol/L IPTG诱导,在重组菌BL21总蛋白的25.9 kDa左右的位置上,获得了大肠杆菌his tag-BvGST的融合蛋白。当施加0.5 mmol/L CdCl2逆境胁迫后,表达BvGST蛋白的重组菌表现出优于对照菌的生长状态;与此同时,大量表达BvGST的大肠杆菌体内的GST酶活性也要远高于对照。这说明,能源甜菜BvGST基因通过在E. coli体内大量表达高活性的谷胱甘肽转移酶,来解毒细胞内由镉胁迫带来的氧化伤害,从而提高大肠杆菌的Cd耐受性。研究结果暗示了能源甜菜BvGST在镉逆境胁迫分子机制中发挥着重要作用。     

应用发酵罐高密度生产日本血吸虫重组蛋白Sj28GST工艺的研究

为了大量表达日本血吸虫重组蛋白Sj28GST,本文采用葡萄糖作为碳源,在不同的培养温度、种子液接种量、IPTG浓度、IPTG诱导时间等培养条件下进行高密度发酵表达日本血吸虫重组蛋白Sj28GST,分别用Bradford法和比浊法分析重组蛋白Sj28GST的浓度和重组大肠杆菌的密度.优化应用发酵罐高密度发酵表达日本血吸虫重组蛋白Sj28GST的培养温度、种子液接种量、IPTG浓度、IPTG诱导时机等培养条件.研究结果表明,在培养温度37℃、种子液接种量7%、溶解氧浓度保持20%以上、pH 7.2左右、用0.7mmol/L IPTG在重组大肠杆菌TB1开始培养后5 h进行诱导,细菌密度明显提高,获得的重组蛋白Sj28GST的表达产量最高,为74.32 mg/L发酵液,菌体密度达到13 OD左右.     

Post-harvest Regeneration of Lowland Black Spruce Forests in Northeastern Ontario

Success of natural regeneration has been a concern since the introduction of heavy machinery in harvesting. The objective was to compare the effect of three operational harvest methods careful logging around advanced growth (CLAAG), group seed tree (GST), and group seed tree followed by shearblading site preparation (SHE) on natural regeneration in the Clay Belt region of Ontario. A total of 30 stands, 562 cluster sample plots, were surveyed. Total density of black spruce regeneration did not differ, but height structure of black spruce regeneration did among harvest methods. The CLAAG method resulted in highest total regeneration density of other conifers. Decreasing density of other conifers from the CLAAG to GST to SHE sites indicated that the CLAAG method protected advance regeneration as expected and the SHE method removed advance regeneration in the path of the shearing blade. Both black spruce and other conifer regeneration densities increased with increasing time since harvest. Stocking of black spruce, all conifers, or all tree species did not differ significantly among harvest methods, nor did it change with time since harvest. Stocking was nonlinearly related to regeneration density. Models developed in this study predict that full stocking (i.e., 60%) can be reached based on regeneration density of 5000 stems per ha regardless of crop species choice preference. However, the existing stocking criterion for assessing black spruce regeneration may be problematic.     

微囊藻毒素对鲢鱼肝脏GST和CYP7A1基因表达的影响

为探究微囊藻毒素(MC)对鲢鱼肝脏谷胱甘肽S转移酶(GST)和胆固醇7α-羟化酶(CYP7A1)基因表达的影响,克隆了鲢鱼肝脏GST和CYP7A1基因的部分c DNA片段,并对饲养密度为1.3×108cell/L的有毒和无毒两种铜绿微囊藻水体中第2 d、第4 d和第6 d的鲢鱼肝脏取样,通过RT-PCR与Q-PCR技术分析肝脏GST和CYP7A1基因在MC影响下不同时间点的表达情况。结果显示,有毒铜绿微囊藻水体中鲢鱼肝脏GST相对表达量在第6 d显著下降,说明GST基因在第6 d时在MC解毒过程中开始发挥作用;鲢鱼CYP7A1相对表达量自第2 d开始显著下调并随时间延续维持在一个较低的水平,说明MC作用下CYP7A1表达受到抑制,可能对胆汁酸合成造成影响。为进一步研究鱼类微囊藻毒素去毒基因以及抑制对MC转运和吸收的关键基因提供依据。     

17-雌二醇对双齿围沙蚕幼虫SOD、CAT和GST活性的影响

将双齿围沙蚕（Perinereis aibuhitensis）幼虫暴露于不同浓度的17-雌二醇（0.1 gL-1、1 gL-1、10 gL-1、100 gL-1和1 000 gL-1）中,于第2 、第4 、第6 和第8 天取样,分别测定抗氧化酶[超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽硫转移酶（GST）]活性。结果表明,第2 天时10 gL-1浓度组显著诱导双齿围沙蚕CAT、SOD和GST活性,1 000 gL-1浓度组显著抑制其SOD和GST活性。第４天时各浓度组SOD和GST活性均被不同程度诱导。第6 天时低浓度组（0.1 gL-1和1 gL-1）显著抑制双齿围沙蚕CAT活性,高浓度组（100 gL-1和1 000 gL-1）则抑制其SOD活性。第8天时,除1 000 gL-1浓度组外,其余各组CAT活性均被不同程度诱导（P0.05）,而SOD活性则受到抑制,10 gL-1浓度组显著抑制其GST活性。试验结果显示,外源性17-雌二醇对双齿围沙蚕幼虫产生氧化胁迫,沙蚕幼虫SOD、CAT、GST对17-雌二醇的响应与其暴露浓度及时间有关。     

日本血吸虫多价核酸疫苗的构建及免疫保护试验

利用PCR技术获得日本血吸虫GST抗原基因,通过分子克隆技术将GST、FABP基因克隆至pVAX1载体,并将其克隆至舍有Sj23基因表达核的plRESneo栽体,构建重组质拉pVAX-GST、pVAX-GST-FABP、pIRESneo-Sj23和pIRESneo-GST-FABP-Sj23.将50只昆明小鼠随机分成5组,每组10只,分别以肌肉注射质粒pIRESneo-GST-FABP-Sjz3、plRESneo-Sj23、pVAX-GST、pVAX-GST-FABP和空白质粒plRESneo,50μg/只,免疫2次,每次间隔21 d.测定小鼠免疫前后血清抗体水平,30 d经腹部皮肤感染血吸虫尾蚴,45 d后剖杀小鼠,计数各组小鼠虫卵数及成虫数.结果表明.重组质粒pVAX-GST、plRESneo-Sj23、pVAX-GST-FABP和pIRESneo-GST-FABP-Sj23均能诱导小鼠产生特异性的IgG抗体,plRESnecrGST-FABP-Sj23免疫组所诱导的IgG水平最高;减虫率分别为26.1%、30.8%、33.2%和47.5%,减卵率分别为25.4%、45.0%、48.4%和69.8%.pIRESneo-GST-FABP-Sj23的减卵率和减虫率明显高于其他DNA疫苗.结果表明,日本血吸虫多价核酸疫苗能产生较强的免疫反应,并能获得较好的免疫保护效果.     

拟南芥IAA7的原核表达及稳定性分析

以p GEX–KG为基本骨架,构建了拟南芥IAA7蛋白的原核表达载体,用IPTG诱导IAA7在3种大肠杆菌表达菌株Rosetta、BL21和Tuner中表达,利用GST SefiroseTM resin亲和树脂分离纯化GST–IAA7融合蛋白,并分析重组蛋白在体外保存时的稳定性。结果表明:GST–IAA7融合蛋白在Rosetta菌株中于25℃和0.4 mmol/L IPTG诱导下表达较好;蛋白酶抑制剂苯甲基磺酰氟(PMSF)可显著延长GST–IAA7在体外保存的时间,最后利用凝血酶切除GST标签后获得了纯化的IAA7蛋白质。     

氧化损伤与遗传毒性的检测研究概况

概述了氧化损伤与遗传毒性的分子毒理学评价手段。简单介绍和讨论了活性氧自由基(ROS)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、谷胱甘肽解毒酶(GST)、丙二醛含量(MDA)、DNA损伤等生物学指标在毒理学领域的研究现状,重点探讨了彗星实验技术(SCGE)在检测遗传毒性方面的应用情况。     

Comparison of in vitro activities of biotransformation enzymes in pig, cattle, goat and sheep

In vitro activities of cytochromes P450 (7-alkyl/aryloxyresorufin dealkyl(aryl)ases, testosterone hydroxylase/oxidase, 6-chlorzoxazone hydroxylase, 7-methoxy-4-trifluoromethyl-coumarin demethylase, and lauric acid hydroxylases), reductases of carbonyl group (toward metyrapone, daunorubicin, glyceraldehyde, and 4-pyridine-carboxaldehyde) and conjugation enzymes (p-nitrophenol-UDP-glucuronosyl transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-tranferase) in young adults, males, non-castrated (N=6) farm animals were studied and compared. Presence of proteins cross-reacting with anti-human CYP3A4, CYP2C9, and CYP2E1 IgG was detected in all farm species. Bovine microsomes differed from other microsomes of farm species in very high 7-ethoxyresorufin-O-deethylase activity (CYP1A1/2). Significantly higher 7-methoxy-4-trifluoromethyl-coumarin demethylase (2-3 times) and 12-lauric acid hydroxylases (4-10 times) activities (probably corresponding to CYP2C and CYP4A, respectively) were found in ovine microsomes. The highest 6beta-testosterone hydroxylase activity, which is usually considered to be a CYP3A activity marker, was found in pig. Reductases of all farm animals display considerable ability to reduce carbonyl group of xenobiotics. Significant differences in level and activity of many biotransformation enzymes tested suggest that extrapolation of pharmacokinetic data obtained in one species to another (even related) could be misleading.     

桔小实蝇BdorGSTO1基因的克隆及发育表达分析

为研究桔小实蝇(Bactrocera dorsalis Hendel.) Omega家族GST蛋白在发育过程的功能。采用RT-PCR和RACE技术克隆获得了桔小实蝇Omega家族GST基因的cDNA全长序列,命名为BdorGSTO1。该基因开放阅读框全长750 bp,编码249个氨基酸,分子量约为28.52 KD,等电点为6.46。氨基酸序列比对表明BdorGSTO1与刺舌蝇(Glossina morsitans morsitans)ADD18952的序列一致性最高,一致性值为70%,与豌豆蚜(Acyrthosiphon pisum)GST蛋白NP_001155757的一致性最低,一致性值为38.9%。荧光定量PCR分析表明BdorGSTO1在不同发育时期都有表达,且在1天蛹时表达量达到最高峰,随着蛹的发育其表达量逐渐降低,推测BdorGSTO1在桔小实蝇蛹的发育过程特别是在化蛹过程中发挥重要作用。