pET15b-pep-1-p27mt的构建、表达及意义

构建细胞穿膜肽(cell-penetrating peptides,CPPs)PEP-1和细胞周期抑制蛋白p27mt的重组体,并在大肠杆菌中表达。将已构建的表达细胞周期抑制蛋白p27mt的基因,克隆入pET15b-pep-1原核表达载体,在大肠杆菌BL21(DE3)LysS内,以IPTG诱导融合蛋白表达。表达产物用SDS-PAGE及Western blot分析鉴定。结果:成功地构建PEP-1-p27mt融合蛋白原核表达载体,并在IPTG诱导下获得特异性的表达。PEP-1-p27mt融合蛋白表达载体的构建,为体内应用细胞周期抑制蛋白诱导肿瘤细胞凋亡提供了理论基础。     

柯萨奇病毒A组5型VP1蛋白原核表达及多克隆抗体的制备

目的 原核表达柯萨奇病毒A组5型(coxsackievirus A5,CV-A5)VP1蛋白,并制备抗VP1多克隆抗体(多抗),为CV-A5相关定性定量研究制备试剂。方法 逆转录PCR扩增N端截短的CV-A5VP1-ΔN56后,克隆至原核表达载体pGEX-6P-1,获得pGEX-6P-1-VP1-ΔN56。将其转化大肠埃希菌BL21(DE3),表达重组蛋白并纯化。用纯化的谷胱甘肽巯基转移酶-VP1-ΔN56融合蛋白经背部皮下免疫日本大耳白兔,制备多抗。结果 重组表达载体构建成功,融合蛋白以不可溶包涵体存在。ELISA、蛋白质印迹法检测表明,获得的兔多抗效价为107,可特异性识别重组和天然CV-A5 VP1蛋白。结论 成功制备重组CV-A5 VP1蛋白及特异性多抗。为中和抗原表征研究及VP1定性定量分析奠定了基础。     

人源性防御素HD-5的原核表达及抗真菌作用的初步研究

【摘要】目的 构建pQE-30Xa/HD-5原核表达载体,纯化重组蛋白并进行抗真菌活性的初步鉴定。方法 以pcDNA3.1(+)/HD-5为模板,聚合酶链反应（PCR）扩增编码HD-5成熟肽的基因。构建pQE-30Xa/HD-5重组表达载体,并对重组质粒进行酶切、基因序列分析。将鉴定正确的质粒转化入大肠杆菌M15后进行异丙基-D-硫代半乳糖苷(IPTG)诱导表达,对表达产物进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定。通过镍柱亲和层析纯化蛋白并进行蛋白复性,蛋白免疫印迹（Western blot）鉴定纯化产物。以KB纸片法初步验证纯化获得的重组蛋白对白色假丝酵母菌的抑菌活性。结果 成功克隆了HD-5基因并构建了重组质粒pQE-30Xa/HD-5。重组质粒在大肠杆菌M15中诱导表达出HD-5融合蛋白;Western blot分析结果显示纯化后的融合蛋白与目的蛋白相符;KB纸片法证实纯化后的融合蛋白对白色假丝酵母菌具有一定的抑菌活性。结论 成功构建HD-5原核表达载体,经诱导表达后纯化获得具有良好抑菌活性的HD-5融合蛋白。     

大鼠谷氨酰胺转运蛋白SNAT1-EGFP融合蛋白的构建与鉴定

目的利用分子生物学方法构建大鼠谷氨酰胺转运蛋白1重组质粒p EGFP-N1-SNAT1并进行鉴定。方法对载体p EGFP-N1和质粒p BK-CMV(Δ[1098-1300])-SNAT1双酶切,纯化后连接,构建重组质粒p EGFP-N1-SNAT1。用Western blot检测融合蛋白的表达,采用免疫荧光检测SNAT1在细胞膜上的表达和定位。结果成功构建重组质粒p EGFP-N1-SNAT1并正常表达、定位于细胞膜上。结论重组质粒p EGFP-N1-SNAT1的成功构建为研究SNAT1的结构和功能提供了有效工具。     

人纤溶酶原K5基因的结构改造及其在大肠杆菌中的表达、纯化与鉴定

目的 改造人纤溶酶原Kringle 5(K5)基因,构建RGDRGD-liteK5融合表达质粒,并表达纯化所得RGDRGD-liteK5蛋白.方法 以入纤溶酶原K5 cDNA为模板,通过PCR得到RGDRGD-liteK5基因片段,并克隆到质粒pGEXl-λT中,构建重组原核融合表达载体pGEX-RGDRGD-liteK5;在IPTG诱导下,观察融合蛋白在大肠杆菌Rosetta中的表达情况;利用亲合层析柱纯化表达产物,用Western blot分析鉴定表达产物.结果 PCR扩增得到274 bp的片段,并成功插入pGEX1-λT质粒;含重组质粒的大肠杆菌在IPTG诱导下表达了特异性的融合蛋白,其分子量为36 kD;获得的融合蛋白经凝血酶酶切后,得到了分子量约为10 kD的RGDRGD-liteK5蛋白;Western blot证实了表达产物的正确性.结论 成功地改造了人纤溶酶原K5基因,构建了重组融合表达质粒pGEX-RGDRGD-liteK5,并纯化了其表达产物.     

N末端Strep蛋白标签表达载体的构建和应用

目的构建携带N末端Strep(NS)蛋白标签的表达载体;在表达载体中构建衣原体RNA聚合酶亚基重组蛋白并表达。方法采用PCR的方法,通过引物引入NS蛋白标签和新的多克隆位点替代p ET21c-DH质粒中原有的T7蛋白标签和多克隆位点,选择新引入的Not I酶切位点进行PCR产物的环化自连,转化DH-5α细菌后筛选阳性菌株,PCR法和基因测序法鉴定新构建的表达载体;在新构建表达载体的Bam H I和Sal I位点之间分别插入衣原体RNA聚合酶核心酶的α、β、β'亚基,获得表达NS-α、NS-β、NS-β'融合蛋白的表达载体,转化表达菌株Arctic Express,筛选阳性表达菌株,并用考马斯亮蓝染色、Western blot等法鉴定融合蛋白的表达情况。结果成功构建了携带NS蛋白标签的p ET21c-NSMCS载体,并成功将其应用于衣原体RNA聚合酶核心酶亚基融合蛋白的构建及表达。结论获得稳定表达NS-α、NS-β、NS-β'融合蛋白的表达载体,为研究衣原体基因转录调控奠定了良好的基础。     

原癌基因Pokemon的克隆原核表达及纯化

目的克隆小鼠Pokemon(POKeryhroidmyeloidontogenicfactor)基因并进行原核表达及重组蛋白的纯化。方法应用反转录聚合酶链反应(RT-PCR)扩增目的基因片段,克隆人pGEM-T-easy载体,经鉴定后,以限制性核酸内切酶分别消化重组质粒及原核表达载体pET-30a(+),体外定向连接,进行PCR、内切酶酶切及DNA序列分析,阳性重组表达质粒进一步转化到大肠杆菌E.coliBL21(DE3)中诱导表达,经Ni-NTA亲和层析纯化融合蛋白,Westernblotting检测其特异性。结果限制性核酸酶切及序列分析表明重组表达质粒包含Pokemon基因编码区,阅读框架无移位;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示有预期相对分子质量为36000的重组蛋白表达,经亲和层析纯化后,融合蛋白的纯度达到90%以上;Westernblotting印迹表明纯化的融合蛋白具有特异的免疫反应特性。结论采用基因克隆技术成功构建了Pokemon原核表达载体,并获得了高纯度的重组蛋白,为后期抗体的制备及进一步研究该基因与肿瘤发生的关系奠定了基础。     

Chinese 1基因型弓形虫棒状体蛋白 ROP16的基因克隆表达及生物信息学分析

目的：构建弓形虫Chinese 1基因型TgCtWh3（以后均简称Wh3）株棒状体蛋白（ rhoptry protein ,ROPs）16的原核和真核重组表达质粒及其3D结构。方法参照ROP16序列分别设计引物,采用PCR从弓形虫Chinese 1基因型Wh3株基因组DNA中扩增出编码ROP16的基因片段,克隆至pMD18-T载体;经PCR及测序分析鉴定;阳性克隆的质粒分别亚克隆至原核表达载体pET-28a和真核表达载体pEGFP-C2,分别转化大肠杆菌BL21和DH5α,PCR和酶切鉴定转化菌落插入的序列;将构建的原核表达菌株经IPTG诱导,SDS-PAGE和免疫印迹分析融合蛋白的表达;将构建的真核重组质粒经脂质体转染293T细胞,观察其在细胞中表达;采用生物信息学方法分析构建了蛋白的3 D结构。结果各组均PCR扩增出约2．1 kb ROP16基因的特异片段,序列检测结果均正确;分别亚克隆到原核表达载体pET-28a和真核表达载体pEGFP-C2中,成功构建了Chinese 1基因型弓形虫棒状体蛋白ROP16的原核表达质粒和真核表达质粒;原核表达质粒在大肠杆菌中表达了ROP16的融合蛋白;真核表达质粒在293T细胞中成功表达,成功构建出ROP16基因的3D结构图。结论以pET-28a和pEGFP-C2为载体,分别成功构建并表达了ROP16的原核和真核重组质粒,并构建出其3D结构图。     

谷胱甘肽-S转移酶-中期因子融合蛋白原核表达及多克隆抗体的制备

目的 构建谷胱甘肽-S-转移酶(GST)和中期因子(MK)融合蛋白的原核表达质粒,并表达和纯化蛋白,制备多克隆抗体.方法 通过RT-PCR技术从人胃癌组织中扩增入MK编码序列,克隆入表达载体pGEX-1λT中,获得表达质粒pGEX-MK,并在大肠杆菌BL21 (DE3)中经IPTG诱导表达,通过亲和层析纯化表达的GST-MK融合蛋白,并以重组蛋白免疫兔子.结果 成功构建了GST-MK融合蛋白的原核表达载体,经诱导表达纯化得到GST-MK融合蛋白.免疫兔子后取多抗血清以间接ELISA检测效价达1∶64 000,Western blotting分析显示多克隆抗血清对MK蛋白特异结合.结论 MK在大肠杆菌中成功表达及其多克隆抗体的获得,为研究MK生物功能奠定了基础.     

重组人PD-L1在大肠杆菌BL-21中的表达、纯化和鉴定

目的 分离纯化人程序死亡蛋白-1(PD-LI)基因,制备重组人PD-L1蛋白,为进一步研究PD-L1在肿瘤免疫逃逸中的作用和机制建立基础.方法 通过RT-PCR分离纯化人PD-L1基因,并将其克隆到原核表达质粒pGEX-4T一1中,经酶切,测序鉴定分析后,转化大肠杆菌BL21(DE3),经IPTG诱导表达.产物经GST蛋白纯化系统进行纯化后,用10%SDS-PAGE及Western Blot分析鉴定.结果 经RT-PCR分离纯化的PD-L1 cDNA分子由645 bp构成,编码相对分子量约25 KD的蛋白,并与GST形成融合蛋白.GST-PD-L1融合蛋白相对分子量约46 KD.Western Blot结果显示,该蛋白能被兔抗GST和抗PD-L1抗体识别.结论 成功构建了PD-L1原核表达载体,并在大肠杆菌BL21中获得高效表达.     

Nachweis einiger Heilmittel in der Kniegelenksynovialflüssigkeit

Zusammenfassung Mittels Gaschromatographie und Dünschichtchromatographie wiesen die Autoren 11 Substanzen nach, welche durch Injektion oder nach Verabreichung per os in die Kniegelenksynovialflüssigkeit eindrangen. In ihrer Aufstellung konnten sie eine direkte Beziehung zwischen Struktur sowie chemischphysikalischen Eigenschaften der Substanz und ihrer Fähigkeit, aus dem Blut in die Kniegelenksynovialflüssigkeit einzudringen, nicht nachweisen, außer der Tatsache, daß Substanzen mit starker Affinität zu Eiweißstoffen erst in höheren Dosen nachweisbar waren.     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Emerging drugs for epilepsy

Epilepsy affects ≤ 1% of the world's population. Antiepileptic drugs (AEDs) are the mainstay of treatment, although more than a third of patients are not rendered seizure free with existing medications. Uncontrolled epilepsy is associated with increased mortality and physical injuries, and a range of psychosocial morbidities, posing a substantial economic burden on individuals and society. Limitations of the present AEDs include suboptimal efficacy and their association with a host of adverse reactions. Continued efforts are being made in drug development to overcome these shortcomings employing a range of strategies, including modification of the structure of existing drugs, targeting novel molecular substrates and non-mechanism-based drug screening of compounds in traditional and newer animal models. This article reviews the need for new treatments and discusses some of the emerging compounds that have entered clinical development. The ultimate goal is to develop novel agents that can prevent the occurrence of seizures and the progression of epilepsy in at risk individuals.     

盐酸达泊西汀中甲磺酸甲酯、甲磺酸乙酯及甲磺酸异丙酯的顶空毛细管GC-ECD法测定

建立了衍生化顶空毛细管气相色谱-电子捕获检测器(ECD)法测定盐酸达泊西汀中的甲磺酸甲酯(MMS)、甲磺酸乙酯(EMS)和甲磺酸异丙酯(IMS).应用碘化钠衍生技术,使用PW-5毛细管柱,载气为氮气,ECD检测,程序升温.MMS、EMS和IMS分别在0.03～0.30、0.05～0.50和0.05～0.50 μg/ml浓度范围内线性关系良好,平均回收率分别为63.5％、100.3％和96.2％,最低检测限分别为0.30、0.50和0.50 ng/ml.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.