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.     

猪流行性腹泻病毒(PEDV)M蛋白原核表达载体的构建

为消除M蛋白抑茵现象使其得到稳定表达,本试验采用两种策略:一是将M基因截短得到编码M蛋白膜外区的基因序列(M'),构建重组表达载体pGEX-6p-M';二是将M基因改造,在其N端和C端同时加上GST基因,即构建pGEX-6p-M-GST重组表达栽体.     

水稻VDAC3蛋白的可溶性外源表达及纯化

[目的]获得外源表达的可溶性水稻VDAC3蛋白.[方法]将osvdac3基因克隆到含有GST标签的pGEX-4T-1原核表达载体中,转化入BL21表达菌株,经IPTG诱导,并使用SDS-PAGE和Western Blot检测分析表达产物,通过Glutathione Resin亲和层析系统纯化获得较纯的目的蛋白.[结果]试验成功构建了pGEX-4T-1-osvdac3原核表达载体并转化大肠杆菌.通过SDS-PAGE和Western Blot试验证实56 kD处的蛋白条带是具有可溶性表达的VDAC3蛋白.[结论]经Glutathione Resin亲和层析系统纯化得到可溶性带GST标签的VDAC3蛋白.该研究结果为水稻VDAC蛋白的功能研究进一步奠定了基础.     

Arsenic-induced responses in freshwater teleosts

The environment is currently polluted by thousands of chemicals or xenobiotics introduced into the environment by man to meet the demands of the modern era. Every day we encounter this negative side of human civilization, but have done little to lessen the rate of pollution. Although the entire biosphere is polluted it is water resources that are the most polluted because water is the ultimate sink for many contaminants. Thus, fish are the most vulnerable of all animal species. They are helpless because they cannot avoid the polluted habitat and face this contamination by default. Nevertheless, fish are found to survive under extreme conditions when their natural habitat has been compromised to a great extent. However, fish are highly sensitive to small environmental changes and their populations gradually dwindle if pollution continues unabated. However, we know that there are instances when water is cleaned and the rate of repopulation by different fish species has gained momentum, restoring the ecological balance. Thus, fish are considered reliable bioindicators of water pollution and fish ecotoxicology has received much attention in recent years, and fish toxicology has been able to defend a significant position in the arena of xenobiotics research over the years. This review deals with some of the major intoxication and detoxication signals manifested by fish exposed to arsenic (As), which is presently one of the most worrying metalloids in water pollution.     

大豆GST基因家族全基因组筛选、分类和表达

利用生物信息学手段,结合公共大豆基因组数据库和大豆发育表达芯片数据获得大豆GST家族基因序列、蛋白序列和染色体位置等信息并进一步对基因的组织表达等进行分析。结果显示大豆中含94个GST家族基因,根据系统发育分析将这些GST基因分成5个亚家族;定位分析表明,94个GST基因分布于大豆的16条染色体上。表达分析结果表明,14个不同发育阶段,大多成员至少在一个组织中表达,11差异表达的基因中有7在根中表达,另外4在其它部位优势表达,基因表达具有一定特异性。本研究为进一步研究GST家族的功能及其抗逆利用提供基础。     

Resistance in the highly DDT-resistant 91-R strain of Drosophila melanogaster involves decreased penetration,increased metabolism,and direct excretion

Resistance to 4,4′-dichlorodiphenyltrichloroethane (DDT) in the 91-R strain of Drosophila melanogaster is extremely high compared to the susceptible Canton-S strain (>1500 times). In addition to enhanced oxidative detoxification, the 91-R strain also has a reduced rate of DDT penetration, increased levels of reductive and conjugative metabolism, and substantially more excretion than the Canton-S strain. Contact penetration of DDT was ∼30% less with 91-R flies, which also had significantly more cuticular hydrocarbons and a thicker, more laminated cuticle compared to Canton-S flies, possibly resulting in penetration differences. DDT was metabolized ∼1.6-fold more extensively by 91-R than Canton-S flies, resulting in dichlorodiphenyldichloroethane (DDD), two unidentified metabolites and polar conjugates being formed in significantly greater amounts. 91-R flies also excreted ∼4-fold more DDT and metabolites than Canton-S flies. Verapamil pretreatment reduced the LD₅₀ value for 91-R flies topically dosed with DDT by a factor of 10-fold, indicating that the increased excretion may involve, in part, ATP-binding cassette (ABC) transporters. In summary, DDT resistance in 91-R is polyfactorial and includes reduced penetration, increased detoxification and direct excretion.     

毒死蜱致2种作物中可溶性蛋白及相关酶的活性变化

为探讨农药"毒死蜱"与韭菜中解毒代谢酶的关系,本试验以毒死蜱喷施韭菜,研究了不同作用时间韭菜中可溶性蛋白质含量,谷胱甘肽-S-转移酶(GST)、谷胱甘肽过氧化物酶(GSH-PX)、酸性磷酸酯酶(ACP)、碱性磷酸酯酶(AKP)活性的变化动态并以小白菜与之对照。结果表明:韭菜和小白菜可溶性蛋白在受到毒死蜱作用时,其蛋白含量变化动态型式基本一致,呈立即升高随后降低又升高的趋势,并分别在14和5d恢复到正常水平;GST,GSH-PX的活性在韭菜中的变化不明显,在小白菜中程上升趋势;正常情况下2种蔬菜中ACP的活性是AKP的100倍左右,受胁迫时韭菜两磷酸酯酶变化不大,小白菜的两磷酸酯酶活性在作用后期迅速下降。毒死蜱在小白菜中的作用时间比韭菜短;小白菜的解毒代谢酶活性较韭菜高;小白菜能够更好的保护细胞膜结构并维护自身功能的完整。     

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.     

氰氟草酯、杀虫安亚致死剂量对金鱼Carrasius auratus和麦穗鱼Pseudorasbora parva肝脏酯酶及谷胱甘肽-S-转移酶活性的影响

研究了 10 %千金乳油 (有效成分 :氰氟草酯 )和 78%杀虫安可溶性粉剂对金鱼和麦穗鱼肝脏酯酶及谷胱甘肽-S-转移酶 (GST)活性的亚致死剂量效应。发现杀虫安 (0 .2 34mg· L-1)对两种鱼的 GST活性均具诱导作用 ,而氰氟草酯 (1,2 mg· L-1)仅诱导了麦穗鱼的 GST活性 ;当杀虫安与氰氟草酯混合处理时 ,对麦穗鱼 GST活性的诱导效应最为明显。在一定的浓度范围内 ,氰氟草酯 (1,2 mg· L-1)和杀虫安 (0 .117,0 .2 34mg· L-1)均可诱导金鱼肝脏酯酶活性 ;对麦穗鱼肝脏酯酶而言 ,杀虫安为诱导作用 ,氰氟草酯则抑制其活性。研究结果表明 :两种酶的活性直接或间接地受供试药剂的影响 ,两种试鱼对氰氟草酯和杀虫安的生物反应存在差异。     

GST pull-down验证新城疫病毒基质蛋白与鸡importinβ1蛋白的相互作用

旨在利用GST pull-down技术验证新城疫病毒(Newcastle disease virus,NDV)基质(matrix,M)蛋白与鸡importinβ1蛋白在体外的相互作用。根据NDV ZJ1株M基因和鸡importinβ1基因序列设计引物,通过PCR扩增获得NDV M基因和鸡importinβ1基因,将其分别定向插入到原核表达载体pGEX-6p-1和pET-32a(+)构建重组原核表达载体pGEX-6p-M、pET-32a-importinβ1,然后转化至大肠杆菌BL21(DE3)后进行IPTG诱导表达,SDSPAGE检测重组蛋白的表达情况,并对包涵体重组蛋白进行变性和复性处理。然后以GST-M重组蛋白为诱饵蛋白,His-importinβ1重组蛋白为猎物蛋白,利用GST pull-down技术验证M蛋白与importinβ1蛋白的相互作用。结果表明,成功构建了重组原核表达载体pGEX-6p-M和pET-32a-importinβ1,将其转化至大肠杆菌BL21(DE3)获得了正确表达。SDS-PAGE电泳检测显示GST-M重组蛋白主要以包涵体形式存在,而His-importinβ1重组蛋白以可溶性和包涵体两种形式存在。利用蛋白重折叠试剂盒获得了有活性的GST-M重组蛋白,将其作为诱饵蛋白,可以捕获并检测到His-importinβ1重组蛋白,但是GST标签蛋白不能结合His-importinβ1重组蛋白。利用GST pull-down技术证实了NDV M蛋白与鸡importinβ1蛋白在体外具有直接的相互作用,这为深入研究importinβ1蛋白在NDV M蛋白细胞核定位的分子机制以及在NDV复制和致病中的作用奠定了基础。