仙台病毒HN基因核酸疫苗的构建及初步鉴定

目的：构建用于防治仙台病毒感染的核酸疫苗并初步鉴定。方法：使用逆转录一聚合酶链反应(RT-PCR)，从新分离的仙台病毒天津株基因组中扩增出HN全部编码序列，将其插入pcDNA3的Pcmv下游，构建仙台病毒HN基因核酸疫苗。PCR、酶切和测序进行鉴定。结果：鉴定证实了核酸疫苗的构建。结论：构建好的核酸疫苗为下一步的体外表达和动物免疫实验奠定基础。     

单纯疱疹病毒Ⅱ型gD糖蛋白核酸疫苗的构建及免疫观察

目的：构建用于防治单纯疱疹病毒Ⅱ型（HSV-Ⅱ）引起的生殖器疱疹的DNA疫苗。并探讨其免疫保护作用。方法：利用PCR法从HSV-Ⅱ Say株中扩增出gD糖蛋白基因的全部编码序列，将其插入pcDNA3中，构建HSV-Ⅱ gD核酸疫苗，用PCR法、酶切法和测序法进行鉴定；将重组质粒转染COS-7细胞，用原位ELISA检测表达产物；对BALB／c小鼠进行肌肉免疫，用微量细胞中和试验、ELISA、脾T淋巴细胞增殖反应和病毒攻击试验检测免疫效果。结果：构建的HSV-Ⅱ gD核酸疫苗具有良好的免疫原性，对小鼠具有保护作用。结论：本实验成功构建出了具有免疫防御效应的HSV-Ⅱ gD核酸疫苗。     

结核分枝杆菌Ag85B真核表达质粒的构建及其表达

目的：构建结核分枝杆菌Ag85B真核表达质粒并表达。方法：从结核分枝杆菌（H37Rv）基因组中扩增出Ag85B编码基因，经限制性内切酶消化后，定向克隆入pcDNA3.1( )中。采用脂质体法将pcDNA/Ag85B转染COS－7细胞，采用RT－PCR、ELISA和斑点印迹法检测其表达。结果：扩增出Ag85B基因，经双向DNA序列测定，与Genbank注泵的序列一致；重组质粒转染COS－7细胞后经检测证实，该基因能在真核细胞中表达。结论：成功地构建了pcDNA/Ag85B质粒，其在真核细胞中表达的蛋白具有良好的抗原性。     

野生型犬尿氨酸酶表达载体的构建及酶活性检测

目的构建野生型犬尿氨酸酶(KYNU)的真核表达载体,并分析其在HEK293细胞中的表达及其酶活性。方法抽提人肝脏细胞的总RNA,通过反转录聚合酶链反应(RT-PCR)法获得KYNU基因全长cDNA,将野生型KYNU基因克隆到pcDNA载体质粒中,获得野生型pcDNA-KYNU重组表达质粒,经酶切鉴定和测序验证后转染HEK293细胞,用蛋白质印迹法技术检测野生型KYNU在细胞中的表达,用高效液相色谱法(HPLC)检测HEK293细胞表达的野生型KYNU酶蛋白的活性。结果通过测序及酶切鉴定野生型pcDNAKYNU重组表达质粒扩增后的PCR产物电泳结果显示构建成功,蛋白质印迹法结果显示转染有重组野生型cDNA-KYNU质粒的HEK293细胞能表达KYNU蛋白,HPLC结果显示野生型KYNU酶存在活性。结论成功构建野生型pcDNA-KYNU的真核表达载体,野生型重组质粒在HEK293细胞中能够表达KYNU并具有一定的酶活性。     

HPV_(16)E7-HSP_(70)融合表达质粒的构建及鉴定

采用分子克隆技术 ,首先将 HSP70 基因片段克隆到真核表达载体 pc DNA3 .1ˉ上 ,获得 pc DNA-HSP重组质粒 ,然后采用 PCR扩增技术 ,定点突变编码 HPV1 6 E7蛋白 C末端锌指结构的基因序列 ,将该突变的 E7基因插入 pc DNA-HSP重组质粒 ,通过粘端连接的方式构建 pcd-HPV1 6 E7-HSP70 融合表达质粒 ,最后通过限制性内切酶酶切、PCR和 DNA测序等技术鉴定。结果显示 ,重组融合表达质粒经酶切、PCR和 DNA测序证明其突变位点、碱基及阅读框架均准确无误。     

携带人PPARα基因高效真核表达载体的构建及其意义

目的构建可以高效表达人过氧化物酶体增殖物激活受体(αhPPARα)的真核细胞表达载体,为筛选作用于PPARα受体的药物提供分子平台。方法将HepG2细胞总RNA经逆转录-聚合酶链式反应(RT-PCR)克隆hPPARα全长基因,用BamHⅠ、SalⅠ双酶切后,与相同双酶切的pIRES2-EGFP载体连接,构建phPPARα-IRES2-EGFP重组质粒,用酶切及基因测序鉴定重组质粒中hPPARα基因的完整性和可靠性;重组质粒转染293细胞,荧光显微镜观察EGFP报告基因表达强度,并对转染细胞的hPPARα表达进行荧光定量PCR及免疫细胞化学检测。结果经酶切和测序证实重组质粒构建正确,并在转染的293细胞中获得hPPARα的高效表达。结论成功构建重组质粒phPPARα-IRES2-EG-FP,为基于hPPARα受体靶点的药物筛选平台的建立提供了高效表达hPPARα的重组载体。     

携带小鼠 KLF4 基因重组慢病毒的构建及对 RAW264.7 细胞增殖的影响

目的 构建小鼠 Krüppel 样因子 4（KLF4）慢病毒表达载体,并建立 KLF4 过表达小鼠腹腔巨噬细胞 RAW264.7 细胞株。方法 采用聚合酶链式反应（PCR）技术扩增目的基因 KLF4 后,构建重组质粒 pLVX-KLF4,并 通过 PCR、双酶切和测序方法对其进行鉴定。重组质粒与 pSPAX2、pMD2.G 辅助质粒通过 Lipofectamine® 3000 共转 染 293T 细胞,包装病毒并测定病毒滴度。将获得的慢病毒感染 RAW264.7 细胞,实时定量 PCR 法检测 KLF4 mRNA 的表达。分选流式细胞仪分选 GFP 阳性 RAW264.7 细胞。流式细胞术检测 KLF4 对 RAW264.7 细胞周期的影响。 EdU 法检测 KLF4 对 RAW264.7 细胞增殖的影响。结果 经 PCR、双酶切鉴定和测序证实,成功构建了包含小鼠 KLF4 基因的慢病毒穿梭质粒,RT-PCR 证实 Lenti-KLF4 感染的 RAW264.7 细胞中 KLF4 mRNA 表达高于未感染的 对照组 RAW264.7 细胞（P＜0.05）。初次浓缩后测定小鼠 KLF4 基因重组慢病毒的滴度为 2.05×108 TU/mL。分选出 KLF4 过表达的 RAW264.7 细胞。KLF4 过表达的 RAW264.7 细胞周期变化显示为 S 期延长,G0/G1 期缩短。EdU 检 测显示 KLF4 过表达的 RAW264.7 细胞增殖活性增高。结论 成功构建了 KLF4 的慢病毒表达载体,并建立 KLF4 过表达的 RAW264.7 细胞株,KLF4 过表达促进了 RAW264.7 细胞的增殖。     

CTX-M-38型β-内酰胺酶的克隆与表达

目的构建新型β-内酰胺酶CTX-M-38的表达载体。方法应用PCR扩增CTX-M-38基因全长编码序列,经NdeI、XhoI酶切后连接至pET-26b(+)表达载体,重组质粒经酶切及DNA测序确证后,转入大肠埃希菌BL21(DE3),IPTG诱导表达。超声破碎法提取表达蛋白产物,检测其活性,等电聚焦电泳检测蛋白的等电点(pI)。结果PCR扩增获得894bp的产物,重组表达载体经NdeI、XhoI酶切及DNA测序后表明,目的基因已成功接入表达载体,重组菌的粗提物经头孢硝噻吩检测显示具有β-内酰胺酶活性,显示载体[pET-26b(+)/CTX-M-38]构建成功。蛋白pI为8.4。结论β-内酰胺酶CTX-M-38在原核表达细胞中实验了基因重组表达,为进一步分析酶的特性提供条件。     

一种对趋化因子受体4具有表型剔除效应的重组腺毒表达载体的构建及其体外检测

目的构建含有人趋化因子突变体SDF-1α/54及内质网定位肽段KDEL基因细胞内趋化因子突变体的重组腺病毒表达载体,并在体外探讨对CXCR4的作用。方法以质粒pcDNA3.1/SDF-1为模板,通过PCR扩增获得SDF-1α/54/KDEL基因全长序列。PCR产物回收后经酶切,定向插入腺病毒穿梭质粒,获得重组质粒pAdTrack-CMV-SDF-1α/54/KDEL。通过酶切、PCR及插入片段测序鉴定,将正确重组体pAdTrack-CMV-SDF-1α/54/KDEL转化E.coliBJ5183并进行细菌内同源重组,然后筛选阳性克隆,提取质粒,将此重组腺病毒质粒分别进行酶切、线性化、纯化,用脂质体Lipofectamine2000介导转染293细胞。制备AdSDF-1α/54/KDEL病毒上清并测定其滴度,将病毒上清转染293细胞,应用RT-PCR分析SDF-1α/54/KDEL的表达。重组病毒感染MCF-7后,MTT法检测其细胞毒性;流式细胞仪检测重组腺病毒对肿瘤细胞表面CXCR4表达的影响。结果通过细菌内同源重组法构建了含SDF-1α/54/KDEL目的基因的重组腺病毒载体AdSDF-1α/54/KDEL,RT-PCR鉴定表明经重组腺病毒转染的293细胞可有效转录SDF-1α/54/KDEL,证实其在293细胞中高效表达并产生6×109PFU/mL的重组腺病毒;AdSDF-1α/54/KDEL处理的乳腺癌细胞,MOI在200以内无细胞毒性对细胞生长生长无影响,但能显著降低肿瘤细胞表面CXCR4的表达量。结论本次构建的细胞内趋化因子突变体重组腺毒表达载体AdSDF-1α/54/KDEL的构建成功,并具有对乳腺癌细胞系MCF-7具有表型剔除作用。     

pmRNA IRES-hKDR(Ig1-3)重组质粒的构建、表达及鉴定

目的:利用基因工程技术构建pmRNA IRES-hKDR(Ig1-3)重组质粒,为其应用于肿瘤的基因治疗奠定基础.方法:以本实验室构建保存的pcDNA3.1 hKDR为模板,PCR扩增hKDR(Ig1-3)cDNA片段后用Nco Ⅰ和Xho Ⅰ酶切后插入pmRNA IRES多克隆位点的相应位点中,经PCR、酶切和测序鉴定其序列正确性,然后用试剂盒体外转录出相应的mRNA,用脂质体转染COS-7细胞,经G418筛选后通过免疫组化和Western blot检测该融合蛋白.结果:PCR、酶切和测序证实pmRNA IRES-hKDR(Ig1-3)构建成功,体外转录出相应的mRNA,免疫组化和Western blot检测出其蛋白表达.结论:成功构建含pmRNA IRES-hKDR(Ig1-3)的真核表达重组质粒,有助于进一步研究其抗肿瘤作用.     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.