2种Heymann肾炎受体相关蛋白融合蛋白的制备及其意义

目的:构建Heymann肾炎致病原受体相关蛋白(RAP)全长和羧基端多肽的原核融合表达载体,表达并纯化相应的融合蛋白.方法:通过RT-PCR法获得RAP全长的cDNA,将其与pGEM-T克隆载体连接并转化,对阳性克隆进行测序分析,设计针对RAP全长和羧基端带有EcoR Ⅰ和Xho Ⅰ双酶切位点的特异引物,通过PCR合成RAP全长359个氨基酸和羧基端108个氨基酸的DNA.纯化后与含有GST的pGEX-4T-1载体体外重组连接,亚克隆到感受态细胞BL21中,经IPTG诱导,表达的融合蛋白通过GST-Sepharose 4B亲和层析法纯化,并经SDS-PAGE和Western Blot检测.结果:酶切分析、PCR及DNA序列测定结果表明成功构建了重组原核表达载体pGEX-4T-1-RAP1083和pGEX-4T-1-RAP324,含有重组质粒的大肠杆菌BL21经诱导后表达出RAP全长70 ku和羧基端40 ku的融合蛋白.Western Blot结果显示分别在分子质量70ku、40ku处出现特异性条带,与理论相符.结论:成功构建了RAP1083、RAP324与pGEX-4T-1融合基因表达载体,融合蛋白在大肠杆菌中高效表达,为后续研究Heymann肾炎的分子发病机制奠定了基础.     

抗癫痫肽/GST融合表达载体的构建及蛋白表达

目的构建抗癫痫肽(anti-epilepsy peptide,AEP)/GST融合表达载体,原核表达GST-AEP融合蛋白。方法从克隆载体质粒pGEM-T/AEP中双酶切,得到AEP片段;通过中间载体puc18,转换酶位点;最后将AEP基因克隆入表达载体pGEX-4T-1质粒中,构建融合表达载体pGEX-4T-1/AEP;转化感受态大肠杆菌DH5α,经异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,获得GST-AEP融合蛋白;SDS-PAGE分析表达产物,确定蛋白质高表达条件;超声裂解法鉴定蛋白质可容性;凝胶薄层扫描确定蛋白质相对表达量。结果经酶切鉴定、测序证明,AEP基因已正确插入到pGEX-4T-1中,经IPTG诱导后,表达出相对分子质量约为36 000的融合蛋白,该融合蛋白以包涵体形式存在,约占总蛋白质的70%左右。结论pGEX-4T-1/AEP载体构建成功,融合蛋白在大肠杆菌中获得高效表达。     

人TIMP-2基因的克隆及其原核表达

目的 构建人TIMP-2重组表达载体,并在大肠杆菌BL21 (DE3)中表达.方法 提取人肝癌细胞(Hep G2)总RNA后,经RT-PCR扩增获得目的片段,插入克隆载体pMD18-T;酶切鉴定和测序后将TIMP-2导入原核表达载体pGEX-4T-1中,构建重组质粒pGEX-TIMP-2.将重组质粒转化至大肠杆菌菌株BL21 (DE3)中,IPTG诱导融合蛋白表达后用SDS-PAGE电泳检测并用western blot验证(蛋白质印迹法).结果 成功构建原核重组表达载体pGEX-TIMP-2,并在原核宿主BL21中大量表达融合蛋白GST-TIMP-2.结论 克隆人TIMP-2基因并在原核生物中大量表达.     

重组人白蛋白干扰素α-2b融合蛋白的抗HBV机制

IFN-α是目前临床上常用的抗HBV药物,但普通IFN-α半衰期只有4～6 h,患者不得不接受频繁的皮下注射,疗程至少半年.重组人白蛋白融合干扰素α-2b(rHSA-IFNα-2b)是通过生物工程技术,构建编码人血清白蛋白(HSA)和IFNα-2b的融合基因并在载体中获得高效表达而成的一种长效干扰素~([1]).本研究以HepG2 2.2.15细胞为模型,对rHSA-IFNα-2b的体外抗HBV作用进行了研究,为rHSA-IFNα-2b用于临床治疗慢性乙型肝炎提供实验依据.     

人胰升血糖素样肽1突变体基因的克隆及在原核细胞的表达

目的 构建人胰升血糖素样肽1(hGLP-1)突变体基因的原核表达载体,并在大肠杆菌BL21中进行表达.方法 用生物合成方法获得hGLP-1突变体基因8Val-hGLP-1,将其克隆于载体pGEX-4T-1中,构建pGEX-4T-1/8Val-hGLP-1融合表达载体,转化大肠杆菌BL21,酶切电泳以及测序鉴定阳性克隆,用SDS-PAGE和蛋白免疫印迹法检测其重组蛋白诱导表达情况.结果 重组质粒pGEX-4T-1/8Val-hGLP-1经双酶切电泳鉴定与测序分析证实构建成功,SDS-PAGE与蛋白免疫印迹分析证实其成功诱导表达重组融合蛋白.结论 成功地克隆了hGLP-1突变体基因8Val-hGLP-1,并在大肠杆菌中进行表达,为下一步获得含突变体基因的重组hGLP-1蛋白及其生物学功能研究奠定良好基础.     

黑色素瘤抗原-3原核重组质粒的构建及表达

目的 构建MAGE-3目的 基因原核重组表达质粒pGEX-4T-1-MAGE-3并检测其在大肠杆茵(E.coli)B121(DE3)中的表达.方法 RT-PCR法制备MAGE-3目的 基因,连接入原核表达载体pGEX-4T-1.构建MAGE-3目的 基因的原核重组表达质粒pGEX-4T-1-MAGE-3并测序,将该质粒转化E.coli BL21(DE3)菌株,经IPTG诱导,12%SDS-PAGE和Western Blot表达鉴定.结果 扩增出349bp的MAGE-3 目的 基因并构建了原核重组表达栽体pGEX-4T-1-MAGE-3;测序结果与GenBank收录序列相一致;在E.coli BL21(DE3)中检测到含该重组表达载体的转化菌表达出分子量约35kD的融合蛋白并证实其为目的 蛋白.结论 成功构建的原核重组表达栽体pGEX-4T-1-MAGE-3及其所表达的融合蛋白,为以MAGE-3为基础的肽疫苗及特异诊断试剂的研制提供抗原打下了基础,为后续实验提供了依据.     

人骨唾液酸蛋白酵母工程细胞的构建与表达

目的构建表达重组人骨唾液酸蛋白 (rhBSP)的巴斯德毕赤酵母工程细胞 ,实现rhBSP的酵母胞外分泌性高表达。方法通过PCR扩增获得人BSP基因后 ,构建重组表达载体pPICZaA hbsp ,经电转化导入到毕赤酵母GS115菌中 ,获得GS115 pPICZaA hbsp工程菌 ,通过Zeocin高抗性筛选和摇瓶发酵筛选出高表达rhBSP的菌株 ,并进行高密度发酵提高表达量。结果rhBSP分子量接近 6 6 0 0 0 ,产物可达上清液总蛋白的 80 % ,浓度约 0 .2 4 8g L。结论基因工程菌GS115 pPICZaA hbsp能高效地表达rhBSP并分泌到胞外     

重组鼠干扰素α4在毕赤酵母中的表达与鉴定

目的 本研究旨在获得稳定、高表达的重组鼠干扰素α4.方法 通过密码子优化对天然的鼠干扰素α4进行优化,采用全基因合成方法合成了rmIFNα4,构建了pPIC9k-rmIFNα4质粒,DNA测序无误后转至毕赤酵母GS115中,经蛋白表达筛选获得了稳定、高表达的基因工程菌.经离子交换柱层析及疏水凝胶层析纯化可获得rmIFN...     

基因变构人白细胞介素-2克隆构建和原核表达

目的构建基因变构IL-2穴88N→R雪的重组克隆熏并将其在原核系统中进行高效表达,为研究基因变构IL-2穴88N→R雪的生物学活性奠定基础。方法分离人体T细胞经PHA刺激活化后,提取细胞的RNA为模板,逆转录构建cDNA文库,经套式PCR扩增出编码IL-2的基因,插入T-A载体后获得编码天然IL-2的克隆,核苷酸测序证实成功后,自行设计含有突变位点的引物,利用重组PCR技术,进行定点诱变构建基因变构IL-2穴88N→R雪的重组克隆熏DNA测序确认变构成功。将改构后的IL-2基因重组于原核pGEX-4T-2质粒上,转化宿主菌E.coli.DH5α中,经IPTG诱导后表达IL-2变构体,表达产物经SDS-PAGE电泳分析。结果获得了人类天然IL-2的编码基因克隆和基因变构IL-2的编码基因克隆,并将变构IL-2穴88N→R雪在大肠杆菌中获得了高效表达。结论IL-2基因的成功定点变构及其在原核生物中获得稳定高效的表达,为进一步在真核细胞中表达以及研究制备低毒、高效的新型基因变构IL-2药物奠定基础。     

单纯疱疹病毒1型糖蛋白B胞外区基因片段的克隆、表达及初步鉴定

构建单纯疱疹病毒1型糖蛋白B(HSV1gB)胞外区基因片段的重组原核表达质粒,并获得HSV1gB胞外区基因的表达.选用真核和原核细胞均偏爱的密码子,化学合成含信号肽序列的HSV1gB蛋白胞外区基因序列,用PCR方法扩增编码HSV1gB胞外区1～696 aa的基因,利用DNA重组技术将其定向插入到原核表达载体pGEX4T-2上,转化大肠杆菌TG1菌株,经IPTG诱导表达及SDS-PAGE鉴定分析.SDS-PAGE检测显示,表达的HSV1gB/GST 融合蛋白分子质量为96 ku.利用亲和层析方法,纯化获得了表达的目的蛋白.ELISA结果表明表达产物具有较好的抗原性和特异性.可溶性重组HSV1gB蛋白的表达成功为单纯疱疹病毒亚单位疫苗的研究奠定了基础.     

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.