SELEX技术筛选入rhTGF—βsRⅡ亲和核酸库方法的建立

目的建立SELEX技术筛选rhTGF-βsRⅡ单链DNA亲和核酸库的方法，为后续分离rhTGF-βsRⅡ的单一核酸适体奠定基础。方法体外构建了一个长度为108nt、含60个随机核苷酸序列的ssDNA随机库。将靶蛋白rhTGF-βsRⅡ预先吸附到固相栽体Phenyl-Agarose上，再与ssDNA随机库温育，洗脱未结合核酸片段，回收与rhTGF-βsRⅡ结合的ssDNA。行PCR扩增时，采用5’端生物素化的下游引物，使PCR产物偶联上生物素。变性聚丙烯酰胺凝胶电泳纯化PCR产物。利用Immobilized Streptavidjrr-Agarose捕获dsDNA，0．15N的NaOH使双链变性，释放非生物素标记的ssDNA，回收后用于下一轮筛选。随着筛选的进行，严谨度不断增加。结果经过8轮筛选后，富集库对靶蛋白的亲和力从0.76％上升到17.18％。结论成功建立了SELEX技术筛选rhTGF-βsRⅡ亲和核酸库的方法。     

泡沫细胞寡核苷酸适配子PM2的特异性研究

目的研究寡核苷酸适配子对动脉粥样硬化病变组织结合的特异性。方法将前期研究获得的特异性适配子命名为PM2,FITC标记后,荧光显微镜分别观察适配子与血管平滑肌细胞、巨噬细胞、内皮细胞、巨噬细胞源性泡沫细胞结合情况。采用高脂方法建立兔的动脉粥样硬化模型。利用PCR方法和荧光显微镜观察适配子与动脉粥样硬化病变结合情况。结果 PM2适配子与巨噬细胞源性泡沫细胞高度特异性结合,但不与血管平滑肌细胞、巨噬细胞、内皮细胞结合;PM2适配子能特异性结合动脉粥样硬化斑块组织,而不结合正常动脉组织结合。结论筛选出的PM2适配子特异性结合巨噬细胞源性泡沫细胞和动脉粥样硬化病变。     

利用肺癌特异性结合多肽ZS-6筛选肺癌相关标志物

目的利用本实验室已获得的与肺癌特异性结合的十二肽ZS-6从人类肺癌cDNA文库中筛选出肺癌相关标志物,为肺癌的临床早期诊断和靶向治疗奠定基础。方法以生物素标记的十二肽ZS-6为探针,筛选人类肺癌cDNA噬菌体展示文库,经过四轮减性筛选后,挑取与ZS-6特异性结合的噬菌体单克隆、扩增、纯化,再通过PCR技术扩增、产物纯化后,进行DNA测序,测序结果经过生物信息学分析。结果筛选出18个与ZS-6特异性结合的噬菌体克隆,测序结果显示其中1个为未知功能的新基因,其余的均已确定为肿瘤相关基因。结论从人类肺癌cDNA噬菌体展示文库中筛选出潜在的肺癌肿瘤标志物,为肺癌的早期诊治奠定基础。     

脂联素对巨噬细胞源性泡沫细胞ABCA1及胆固醇含量的影响及机制

目的探讨脂联素对RAW264.7巨噬细胞源性泡沫细胞ABCA1及胆固醇含量的影响及其可能的机制。方法体外培养RAW264.7细胞,加入20 mg/L氧化低密度脂蛋白(ox-LDL)共同孵育48 h,将其诱导成泡沫细胞,加入不同浓度(0、1、5、10μg/mL)的脂联素干预24 h,RT-PCR测定ABCA1 mRNA的表达,高效液相色谱测定细胞内胆固醇含量。观察脂联素对泡沫细胞中ABCA1表达的影响。结果脂联素显著增加RAW264.7巨噬细胞源性泡沫细胞ABCA1 mRNA的表达(P<0.05),并增加细胞内胆固醇含量,且呈浓度依赖性(P<0.05)。结论脂联素可以增加巨噬源性泡沫细胞ABCA1转录水平,促进胆固醇流出,延缓AS的发生发展。     

蛋白激酶AMPK高效制备及活性检测方法的建立

目的 建立高效的腺苷酸依赖的蛋白激酶（AMPK）的制备方法,并建立1种基于酶联免疫吸附（ELISA）法的AMPK的激酶活性检测方法以用于药物筛选。方法 利用大肠杆菌表达系统,将AMPK的α、β和γ 3个亚基与钙调素依赖性蛋白激酶β(CAMKK β)共表达,重组AMPK被CAMKK β磷酸化修饰并激活,进而纯化得到具有激酶活性的重组AMPK蛋白复合体。根据AMPK底物乙酰辅酶A羧化酶（ACC）的磷酸化修饰位点（Ser79）设计合成生物素标记的多肽（SAMS）作为AMPK激酶的底物。在ATP存在的情况下AMPK对生物素标记的SAMS多肽进行磷酸化修饰,反应产物转移到链霉亲和素包被的酶标板中,SAMS多肽通过链霉亲和素结合到酶标板。以能够识别SAMS磷酸化修饰的抗体作为一抗进行ELISA检测,SAMS的磷酸化修饰水平反应AMPK的激酶活性。结果 利用大肠杆菌表达系统成功得到有激酶活性的AMPK蛋白复合体,建立了ELISA检测AMPK的激酶活性方法。通过对AMPK激活剂A-769662检测,证明该方法可以用于AMPK激活剂/抑制剂的筛选。结论 通过构建大肠杆菌共表达系统,成功获得了AMPK激酶...     

一种可用于天然产物粗提物的HIV-1逆转录酶抑制剂筛选方法

人类免疫缺陷病毒（HIV）逆转录酶是成功的获得性免疫缺陷综合症（AIDS）治疗药靶，HIV逆转录酶抑制剂是AIDS治疗中必不可少的药物。现有药物的副作用和耐药性的快速出现促使加快寻找新HIV逆转录酶抑制剂的研究工作。天然产物是新药的重要来源，如能够对天然产物粗提物进行HIV逆转录酶抑制活性筛选将极大地提高发现新HIV逆转录酶抑制剂的效率，但目前还未见可用于天然产物粗提物的HIV逆转录酶抑制活性的筛选方法。本文依据HIV逆转录酶以RNA为模板催化DNA合成的原理，利用biotin-dUTP和digoxigenin-dUTP在逆转录反应过程中随机参入DNA，形成digoxigenin和biotin双标记的DNA，借助digoxigenin-anti-digoxigenin系统捕获digoxigenin标记DNA，再通过生物素．链霉亲和素系统将铕螯合物标记在DNA上，以时间分辨模式测定结合的铕螫合物的荧光强度，得到HIV-1逆转录酶的活性，该方法可直接用于天然产物粗提物的HIV逆转录酶抑制活性的筛选，而不受内源性生物素的干扰。     

人源抗BLyS单链抗体的筛选及其构建与表达

目的:从人源单链抗体库中筛选抗B细胞刺激因子(BLy S)的单链抗体基因、构建表达载体并实现单链抗体的表达。方法:以哺乳动物细胞展示型人源Sc Fv抗体库为起始文库,通过流式细胞术进行分选,获得荧光强度最强、比例为0.1%的阳性细胞。从候选细胞中提取质粒并转化至DH5α中进行扩增,得到质粒转染293T细胞作为下一轮筛选所需的抗体库。经过依次降低抗原浓度进行了3轮分选得到2个候选的Sc Fv序列。经序列分析,选择其中一种单链抗体基因,利用基因工程技术构建分泌型表达质粒,转染293E细胞并通过镍亲和层析纯化获得B-10 Sc Fv抗体蛋白并通过Fortie Bio Octet QK进行亲和力分析。结果:经过3轮筛选获得了全新的抗BLy S Sc Fv抗体序列,成功构建并表达了anti-Bly S Sc Fv抗体蛋白,该单链抗体与的BLy S亲和常数为3.08 nmol·L-1。结论:从哺乳动物细胞展示型人源Sc Fv抗体库中成功获得了可结合BLy S的全新单链抗体基因序列,该单链抗体与BLy S具有较高的亲和力,这为后续的活性研究以及产品开发奠定了基础。     

基于特异性结合多肽ZS-1的肺癌新肿瘤标志物的筛选

目的：利用本实验室已获得的与肺癌特异性结合的十二肽ZS-1（EHMALTYPFRPP）从人类肺癌cDNA文库中筛选出肺癌相关标志物,并进行初步确认,为肺癌的临床早期诊断和靶向治疗奠定基础。方法：本实验以ZS-1为探针,通过将偶联生物素的多肽ZS-1固化在亲和素处理的酶标板,筛选人类肺癌cDNA噬菌体展示文库,经过三轮减性筛选后,挑取与ZS-1特异性结合的噬菌体单克隆、扩增、纯化,再通过PCR技术扩增、产物纯化后,进行DNA测序,测序结果经过生物信息学分析,随后采用组织芯片技术对相关生物大分子进行了检测。结果：通过生物信息学分析首次发现与ZS-1结合的肺癌细胞膜受体蛋白为KIAA1199,随后以KIAA199抗体检测人肺癌组织芯片证实,KIAA1199的阳性检出率为32.8%。结论：从人类肺癌cDNA噬菌体展示文库中筛选出潜在的肺癌肿瘤标志物KIAA1199,为肺癌的早期诊治奠定基础。     

人乳腺癌细胞系MCF-7/W和MCF-7/ADM细胞中阿霉素结合蛋白的分析

目的通过比较野生型和耐药型人乳腺癌细胞系MCF-7中阿霉素(ADM)结合蛋白的差异,探讨耐药型MCF-7的多药耐药机制。方法酯化脱水法制备生物素化ADM,HPLC纯化,MS分析确证。以ADM耐药指数≥200的MCF-7细胞(MCF-7/ADM)和野生型MCF-7细胞(MCF-7/W)为研究材料,采用链亲和素-亲和甄别磁珠法从两种细胞全蛋白中分离出与ADM结合的蛋白。经SDS-PAGE,肽质谱指纹图谱(MALDI_TOF_MS)分析ADM结合蛋白的种类。细胞迁移实验比较MCF-7/W和MCF-7/ADM细胞的运动能力。结果生物素化ADM、ADM对MCF-7/W的IC50分别为0.796μmol.L-1和0.547μmol.L-1。分别从MCF-7/W和MCF-7/ADM中采用亲和甄别磁珠法获得ADM结合蛋白20种及17种,SDS-PAGE分析两者有一条差异条带,序列分析共同蛋白为myosin,beta-actin,alpha-actin,keratin。MCF-7/W和MCF-7/ADM细胞在培养72h后,前者的迁移速度较快。结论除了因方法学灵敏度限制未能解析的结合蛋白外,MCF-7细胞内的主要ADM结合蛋白是细胞骨架蛋白和参与细胞运动的蛋白。ADM与MCF-7细胞中的骨架蛋白结合后,对细胞的迁移运动有一定的影响作用。     

特异性结合白色念珠菌（1，3）-β-D-葡聚糖单链 DNA 适配子的筛选及其应用

目的：通过指数富集的配基系统进化技术（systematic evolution of ligands by exponential enrichment ,SELEX）筛选能与高亲和力（1,3）‐β‐D‐葡聚糖特异性结合的适配子,并用该适配子建立双适配子夹心酶联寡聚核苷酸吸附试验（enzyme‐linked oligonucleotide assay ,ELONA）来对深部真菌感染血浆进行辅助诊断。方法提取白色念珠菌 ATCC10231株细胞壁（1,3）‐β‐D‐葡聚糖,通过 SELEX 筛选获得能与（1,3）‐β‐D‐葡聚糖特异性结合的高亲和力单链 DNA 适配子,并用该适配子建立双适配子夹心ELONA 来检测深部真菌感染血浆中的（1,3）‐β‐D‐葡聚糖。结果从白色念珠菌细胞壁中成功提取了高聚合度（1,3）‐β‐D‐葡聚糖,并通过体外酶解获得可溶性低聚合度（1,3）‐β‐D‐葡聚糖作为筛选靶标。用SELEX 进行12轮筛选后,从初始单链 DNA 文库中获得2个高亲和力适配子 AU1和 AD1,检测发现它们并非结合于（1,3）‐β‐D‐葡聚糖的同一表位。双适配子夹心 ELONA 检测深部真菌感染血浆中（1,3）‐β‐D‐葡聚糖的特异性和敏感度分别为91．94％和92．31％。结论从初始单链 DNA 文库中成功获得可与（1,3）‐β‐D‐葡聚糖特异结合的高亲和力适配子,为深部真菌感染新型诊断试剂的研发奠定了基础。     

Identification of ssDNA aptamers specific for anti-neuroexcitation peptide III and molecular modeling studies: Insights into structural interactions

Twelve ssDNA aptamers specific for a novel recombinant anti-neuroexcitation peptide (ANEPIII) were identified using the SELEX method from a 79-nucleotide ssDNA pool to purify ANEPIII in a more efficient way. To further understand the binding modes between ssDNA and ANEPIII, fully flexible dinucleotides were docked onto the homology-modeled ANEPIII structure. AutoDocking identified favorable binding sites on ANEPIII for nucleotides, which was valuable for designing more potent ligands.     

Exploring the Utility of ssDNA Aptamers Directed against Snake Venom Toxins as New Therapeutics for Snakebite Envenoming

Snakebite is a neglected tropical disease that causes considerable death and disability in the tropical world. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapy for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. In this study, we sought to explore the potential of ssDNA aptamers as toxin-specific inhibitory alternatives to antibodies. As a proof of principle model, we selected snake venom serine protease toxins, which are responsible for contributing to venom-induced coagulopathy following snakebite envenoming, as our target. Using SELEX technology, we selected ssDNA aptamers against recombinantly expressed versions of the fibrinogenolytic SVSPs ancrod from the venom of C. rhodostoma and batroxobin from B. atrox. From the resulting pool of specific ssDNA aptamers directed against each target, we identified candidates that exhibited low nanomolar binding affinities to their targets. Downstream aptamer-linked immobilised sorbent assay, fibrinogenolysis, and coagulation profiling experiments demonstrated that the candidate aptamers were able to recognise native and recombinant SVSP toxins and inhibit the toxin- and venom-induced prolongation of plasma clotting times and the consumption of fibrinogen, with inhibitory potencies highly comparable to commercial polyvalent antivenoms. Our findings demonstrate that rationally selected toxin-specific aptamers can exhibit broad in vitro cross-reactivity against toxin isoforms found in different snake venoms and are capable of inhibiting toxins in pathologically relevant in vitro and ex vivo models of venom activity. These data highlight the potential utility of ssDNA aptamers as novel toxin-inhibiting therapeutics of value for tackling snakebite envenoming.     

Cell-based aptamer selection for diagnosing cancer and predicting cancer progression

Antibodies are excellent molecular recognition agents for a wide range of applications therefore they have been used heavily in clinical assays such as disease diagnosis. More recently, aptamers have emerged as alternative capturing agents in a variety of applications including medical diagnosis, environmental toxicity detection, targeted drug delivery and viral therapeutics. Aptamers are ssDNA or RNA that form three dimensional structures and bind to the target molecules such as peptide, protein or small molecules. Aptamers are generated by in vitro process called “SELEX (Systematic Evolution of Ligands by Exponential Enrichment)”. Conventionally, SELEX is performed with immobilized target molecules such as proteins in column, filter or beads. However, for some targets like membrane proteins, it is very difficult or almost impossible to immobilize the target proteins in their active conformation. However, cell-based aptamer selection technology explain how it can be better than standard immobilization methods in brief. Here, we described the cell-specific aptamers selecting technology, called cell-based SELEX, for diagnosing disease and predicting disease progression, especially in the case of complex disease, like cancer.     

核酸适配体在抗肿瘤药物主动靶向传递中的应用

核酸适配体是通过指数级富集的配体系统进化技术(systematic evolution of ligands by exponential enrichment,SELEX)从合成的大容量单链随机寡核苷酸文库中筛选并富集的对某些靶点具有高特异性和高结合率的小分子DNA或RNA片段.核酸适配体具有直接抑制肿瘤细胞增殖的作用,通过结构修饰可增强在体内的稳定性.此外,它可与载药纳米结构键合,用于肿瘤诊断和治疗.本文综述了核酸适配体的肿瘤靶点、筛选方法及其在肿瘤诊断和治疗中的应用.     

Low efficient generation of ssDNA aptamer using chemical modified spacer primer

Aptamers are oligonucleotides (ssDNA or RNA) with an appropriate size of 100 bps that bind with high affinity and specificity to a wide range of target molecules, including virtually any class of protein, drugs or small organic/inorganic molecules. The in vitro selection process referred to as SELEX provides a powerful tool to identify specific aptamers with high affinity and even discriminate between closely related targets. Aptamers have various applications such as analytical tools, disease diagnosis and prediction, pharmaceutical research, drug development, therapy and even for environmental monitoring. Nowadays, with the development of SELEX methods, generation of aptamer becomes more efficient, less time consuming and even automatically. The whole SELEX process includes binding, separation, and nucleic acid amplification. As amplification of nucleotides is an important process in successive SELEX, we will compare several methods for generation of aptamer in this report.     

U937泡沫细胞中细胞间粘附分子—1的表达及欧芹素乙的抑制作用

目的：研究在人类单核细胞系U937泡沫细胞中,细胞间粘附分子－1（ICAM－1）的表达水平,观测欧芹素乙（欧前胡内酯,imperatorin,IMP)对ICAM－1表达的抑制作用。方法：将U937细胞与80mg/L氧化低密度脂蛋白孵育48h,建立U937泡沫细胞模型,在培养基中预加入不同浓度的IMP（0,25,50,100μmol/L）,采用Western blotting检测ICAM－1的蛋白表达;采用Northern blotting检测ICAM－1的mRNA水平。结果：泡沫细胞中ICAM－1的表达显著高于正常U937细胞。ICAM－1的蛋白和mRNA水平分别是正常U937的15和10倍。经IMP50和100μmol/L预处理后,泡沫细胞中ICAM－1的高表达被显著抑制。当IMP浓度达到100μmol/L时,ICAM－1的蛋白水平降低了79％,mRNA水平降低了74％。结论：经氧化低密度脂蛋白孵育后,U937泡沫细胞中ICAM－1呈现高表达,IMP能显著抑制这种表达。