针对干扰素-γ的新型核酸适配体的筛选及鉴定

 目的: 研发能特异性结合干扰素-γ（IFN-γ）的DNA适配体,为发展IFN-γ的新型检测技术提供基础。方法: 体外合成全长为59个碱基并含有21个随机寡核苷酸的单链DNA文库,以IFN-γ蛋白为靶标,利用指数富集的配体系统进化技术（SELEX）,从单链DNA文库中筛选能够选择性结合IFN-γ蛋白的核酸适配体;流式细胞术检测富集进度、适配体与IFN-γ蛋白结合特性;MFold软件预测二级结构。结果: 经多轮筛选获得了能够识别IFN-γ蛋白的DNA适配体B3-8,其能够选择性地结合IFN-γ蛋白,而与血清白蛋白的结合较弱。经检测其Kd值为185 nmol/L。结论: DNA适配体B3-8能选择性地识别IFN-γ蛋白,在研发针对IFN-γ的新型检测技术方面具有应用潜能。     

新型HER2适配体的筛选及鉴定

目的研发能特异性结合人表皮生长因子受体2(HER2)的DNA适配体,为开发针对HER2的新型肿瘤靶向诊疗技术提供依据。方法体外合成全长86个碱基并含有40个随机寡核苷酸的单链DNA文库,以HER2表位肽为靶标,利用指数富集的配体系统进化技术(SELEX),从单链DNA文库中筛选能够选择性结合HER2多肽的核酸适配体;流式细胞术检测富集进度、适配体与HER2蛋白及HER2阳性细胞的结合特性;MFold软件预测二级结构。结果经多轮筛选获得了能够识别HER2多肽的DNA适配体HA5,其能够选择性地结合HER2蛋白及HER2阳性的乳腺癌细胞,而不结合胰蛋白酶和HER2阴性细胞。结论 DNA适配体HA5能选择性地识别HER2阳性的乳腺癌细胞,在研发针对HER2的新型肿瘤靶向诊疗技术方面具有应用潜能。     

HIV-p24核酸适配体的筛选

 摘 要：目的 利用SELEX技术筛选HIV-p24的核酸适配体,为艾滋病的诊断和治疗奠定基础。方法 以重组p24为筛选靶,用SELEX技术从随机寡核苷酸库中筛选与HIV-p24结合的寡核苷酸,利用凝胶阻滞实验鉴定第12轮筛选到的寡核苷酸与HIV-p24的结合,再用Dot-blot法筛选出与HIV-p24结合的核酸适配体,并检测核酸适配体识别HIV-p24的特异性。结果 Dot-blot筛选到5条与HIV-p24有较强结合能力的核酸适配体,且均为不同的序列。特异性检测显示,18和26号配体只与HIV-p24特异性结合,与人血清白蛋白、牛血清白蛋白和脱脂奶粉均无明显结合。结论 成功筛选到2条特异结合HIV-p24的核酸适配体,为其应用于艾滋病诊断和治疗提供了实验基础。     

丙型肝炎病毒核心蛋白寡核苷酸适配子的筛选与鉴定

目的：筛选、鉴定抗HCV核心蛋白（C蛋白）的寡核苷酸适配子（aptamers）。方法：利用systematic evolution of ligands by exponential enrichment(SELEX)技术，以HCV C蛋白为靶分子，从体外合成的81bp随机单链DNA文库中筛选与HCV C蛋白特异结合的寡核苷酸适配子，并进行了解离常数（Kd）测定和适配子序列测定。再分别利用Clustal W软件包和DNA Folding Sever分析适配子的一级结构和二级结构。结果：经过9轮循环筛选，随机ssDNA库与HCV C蛋白的结合率从0.5%上升到32.5%。所有的一级结构没有共同的同源序列，但可分5个家族，每个家族具有共同的保守序列。二级结构分析表明，适配子形成的茎环、凸环结构可能是与HCV C蛋白结合的结构基础。其中寡核苷酸适配子C4与HCV C蛋白特异结合的亲和力最高，Kd值为68nmol/L。结论：利用随机寡核苷酸文库成功获得抗HCV C蛋白的寡核苷酸适配子。     

SELEX与适配体在蛋白质研究中的应用

指数富集的配基系统进化(SELEX)是一种从大容量寡核苷酸文库中经反复分离扩增步骤得到针对靶分子的高亲和力高特异性核酸配基—适配体的体外筛选技术。SELEX技术自1990年发展至今,已涌现出多种筛选模式和分离方法。筛选特异结合蛋白质的SELEX技术发展直接影响和指导了适配体在蛋白质功能调控方面的应用。文章综述了多种SELEX技术在筛选蛋白质方面的发展近况,适配体在蛋白质功能研究中的应用,筛选过程中关键性因素的确定及适配体的前后期修饰。     

核酸适配体在肿瘤学中的应用研究进展

核酸适配体是一类通过指数级富集的配体进化技术(SELEX)获得的能以较高的亲和力与各类生物靶标特异性相结合的单链DNA或RNA。利用细胞-SELEX(Cell-SELEX)技术筛选出来的核酸适配体越来越多的作为靶向分子应用在肿瘤细胞检测、靶向药物载体和siRNA的研究中。体内外研究证明,核酸适配体作为靶向分子能用于肿瘤细胞的体外检测和肿瘤组织的体内成像,并能显著提高药物或siRNA对肿瘤细胞的杀伤作用。     

肝脏去唾液酸糖蛋白受体特异性RNA适配子的SELEX筛选与鉴定

目的 获得能够特异性高亲和力结合肝脏特异性去唾液酸糖蛋白受体(asialoglycoprotein receptor, ASGPR)的RNA适配子,为开发诊断和治疗肝脏疾病的靶向性试剂和药物奠定基础.方法 合成一个长度为115 nt含有25个随机序列的单链DNA随机文库,通过体外转录构建出单链RNA适配子随机文库,以肝脏ASGPR大亚基为靶蛋白,采用SELEX(systematic evolution of ligands by exponential enrichment)技术筛选具有高亲和力的ASGPR特异性RNA适配子;通过膜结合测定实验、凝胶阻滞实验鉴定筛选适配子对靶蛋白的特异性和亲和力.结果 经过12轮筛选获得了具有高亲和力的肝脏ASGPR特异性RNA适配子.结论 成功地筛选出了具有高亲和力的肝脏ASGPR特异性RNA适配子库.     

Progress in aptamers for cancer research

核酸适配体是一类通过指数级富集的配体进化技术(SELEX)获得的能以较高的亲和力与各类生物靶标特异性相结合的单链DNA或RNA.利用细胞-SELEX (Cell-SELEX)技术筛选出来的核酸适配体越来越多的作为靶向分子应用在肿瘤细胞检测、靶向药物载体和siRNA的研究中.体内外研究证明,核酸适配体作为靶向分子能用于肿瘤细胞的体外检测和肿瘤组织的体内成像,并能显著提高药物或siRNA对肿瘤细胞的杀伤作用.     

人慢性粒细胞白血病融合蛋白BCR-ABL适配子筛选及结构分析

目的:筛选、鉴定人慢性粒细胞白血病融合蛋白BCR-ABL适配子。方法:利用SELEX(Systematic evolution of ligands by exponential)技术,以高纯度融合蛋白BCR-ABL为靶分子,从体外化学合成的长度为90 bp的随机单链DNA文库中来筛选与融合蛋白BCR-ABL特异性结合的寡核苷酸适配子,并进行解离常数(Kd)值测定和适配子序列测定,再分别用Clustal W软件包和DNA Folding Sever分析适配子一级结构及二级结构,以酶联仪测定OD450值,根据OD值高低判定适配子亲和力大小。结果:经过13轮筛选,随机ss DNA文库与融合蛋白的亲和率从0.3%上升到47.1%,所有的一级结构没有共同的同源序列,二级结构分析结果显示,茎和环等二级结构可能是适配子和融合蛋白BCR-ABL结合的基础,其中,寡核苷酸适配子A2与BCR-ABL亲和力最高,kd值达72 nmol/L。结论:利用随机寡核苷酸文库筛选技术成功获得抗融合蛋白适配子,为临床上治疗和预防慢性粒细胞白血病提供一定的参考。     

SELEX技术筛选结核分枝杆菌MPT64蛋白DNA适配子方法学研究

目的 利用指数富集配体系统进化(SELEX)技术筛选能与结核分枝杆菌MPT64蛋白特异结合的寡核苷酸适配子.方法 体外合成长度为78个核苷酸的随机ssDNA文库,利用SEL-EX技术筛选,以MPT64蛋白为靶物质进行12轮筛选,利用生物素-亲和素显色系统检测寡核苷酸与蛋白的结合性.结果 SELEX技术筛选MPT64蛋白适配子的技术体系:PCR扩增的最佳退火温度为65℃;文库优化时,最佳Mg2+的浓度为1.5 mmol/L;不对称PCR法制备ssDNA时,Mg2+的浓度为0.75 mmol/L;以酶联板为介质筛选时,在10轮后筛选达到饱和,且随着筛选轮数的增加,PCR扩增产物的电泳条带逐渐单一、致密,亲和性检测显示第10轮获得的适配子库,比初始的文库亲和性吸光度(A)值增加了9.18倍.将适配子库克隆,随机挑取10个单克隆子,利用混合夹心法检测其与MPT64的亲和性,亲和性范围分布在0.572～1.606之间.结论 已初步筛选到与MPT64蛋白高亲和性结合的DNA适配子.     

Antigen-Induced Proliferative Response to Murine Thymus Cells in Vitro

Thymus cells from 5- to 6-week-old normal (unimmunized) BALB/c mice showed an increased incorporation of [(3)H]thymidine in the presence of 2,4-dinitrophenyl-bovine serum albumin, fluorescein-bovine serum albumin, and bovine serum albumin (BSA) in tissue culture. The concentrations of antigen (BSA and haptenated proteins) required for stimulation were approximately 25- to 50-fold higher than those of the nonspecific mitogen, concanavalin A. In contrast to the stimulation by concanavalin A, which was maximal at 24 to 72 h, the stimulation by antigen was most marked earlier in the culture period (6 to 24 h). The BSA response was diminished to a statistically significant degree (especially at low BSA concentrations) in thymocytes from animals injected 72 h previously with BSA, indicating that the stimulation is immunologically specific.     

Electrochemically assisted co-precipitation of protein with calcium phosphate coatings on titanium alloy

A bovine serum albumin protein-containing calcium phosphate coating (BSA/brushite) was prepared by electrochemically assisted co-precipitation onto a hydroxyapatite (HA) coated Ti-6Al-4V surface. Electrochemically assisted co-precipitation of BSA/brushite coatings onto HA resulted in a 70-fold increase in BSA inclusion compared to simple adsorption, and was subsequently released by a slower mechanism (15% loss over 70 h). Thus, this electrochemically assisted co-precipitation technique provides an efficient method of protein incorporation at physiological temperature, with a potential for sustained release of therapeutic agents as may be required for metallic implant fixation.     

Hollow and degradable polyelectrolyte nanocapsules for protein drug delivery

Biodegradable hollow capsules encapsulating protein drugs were prepared via layer-by-layer assembly of water-soluble chitosan and dextran sulfate on protein-entrapping amino-functionalized silica particles and the subsequent removal of the silica. In order to enhance the encapsulated efficiency and decrease its burst release, we designed this new system to fulfill these two goals. Bovine serum albumin (BSA), which was used as model protein, was entrapped in the nanocapsules. This system demonstrated a good capacity for the encapsulation and loading of BSA. The burst release was decreased to less than 10% in phosphate-buffered saline within 2 h. No significant conformation change was noted from the released BSA in comparison with native BSA by using circular dichroism spectroscopy. Cell viability study suggested that the nanocapsules had good biocompatibility. The drug release kinetics mechanism is Fickian diffusion. These kinds of novel composite nanocapsules may offer a promising delivery system for water-soluble proteins and peptides.     

An enzyme-linked immunosorbent assay for the detection of autoantibodies to albumin

The development of an enzyme-linked immunosorbent assay (ELISA) for anti-albumin autoantibodies (AAA), using immobilized monomeric or glutaraldehyde-polymerized human, bovine or egg albumin, is described. Major problems in detection by the ELISA of AA against human albumin (HSA) were due to high 'non-specific' binding with the commercial anti-human immunoglobulin antisera used and to interference by IgM/HBs circulating complexes. However, it was found that AAA are not species-specific and that these problems may be overcome using immobilized bovine (BSA) or egg (EggA) albumin. AAA were found to have a similar affinity for BSA as for HSA but slightly lower for EggA, while AAA affinities for the monomeric forms were lower than for the corresponding polymeric albumins. All sera from the 28 normal subjects tested were found to contain both IgM- and IgG-AAA. Patients with acute hepatitis B (n = 23) had significantly lower titres of IgM-AAA than normal subjects, as did chronic HBV carriers with (n = 33) or without (= 17) underlying liver disease, while IgG-AAA titres were reduced only in the acute hepatitis patients. These findings support the concept that AAA have a normal physiological function (probably for removal of effete albumin molecules) and that, in HBV infection, there is a decrement in titres that may be related to the clearance of the virus.     

Development of a novel C1q immunoadsorbent for removal of circulating immunecomplexes: quantitative isolation of hepatitis B virus surface antigen and immunecomplexes

A technique for large scale production of human C1q from plasma by affinity chromatography on an anti-C1q column is described. Affinity purified C1q was covalently coupled to a newly developed agarose polyacrolein microsphere beads immunoadsorbent. This immunoadsorbent was utilized for quantitative removal of artificially formed bovine serum albumin (BSA)-anti-BSA immune complexes (IC). The C1q affinity column was then used for the isolation of immunecomplexes containing hepatitis B virus (HBV) surface antigen (HBsAg) from serum of an HBsAg carrier. Identical columns may be utilized for quantitative removal of a variety of IC from blood of patients with infectious and autoimmune diseases, as well as neoplastic diseases. Furthermore, dissociated immunecomplexes will provide an additional source for purification of specific antigens.     

Effect of vitamin A on the systemic and local antibody responses to intragastrically administered bovine serum albumin.

The effects of vitamin A on the immune response to bovine serum albumin (BSA) were studied in adult mice. Treatment with vitamin A by the intragastric or parenteral routes markedly increased the local as well as the systemic antibody response to different concentrations of the antigen (BSA). In contrast, in animals given BSA alone, antigen concentrations above a certain dose resulted in a decreased or even absent anti-BSA response. These studies suggest that vitamin A may be an appropriate adjuvant in oral immunization.     

Interactions between calcium, phosphate, and albumin on the surface of titanium.

The deposition of calcium phosphate on chemically polished commercially pure titanium immersed in Hank's balanced salt solution (HBSS) with bovine serum albumin (BSA) (concentrations 0 and 4 mg/mL) has been investigated. Electrochemical techniques, 125I labeling of albumin, scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy were used. A tricalcium phosphate layer with a thickness of ca. 1 microm was formed for periods of immersion in HBSS ranging between 1 and 2 weeks. A concentration of 4 mg/mL of BSA prevented its formation, even for periods as long as 1 month. In the absence of BSA, the electrochemical behavior of titanium specimens was significantly affected by the length of immersion time, reflecting the changes that slowly occur on their surface. In the presence of BSA, the surfaces maintained most of their original electrochemical activity. Surface studies have shown that calcium and phosphate become incorporated in the surface at very early stages of immersion. Albumin, which was rapidly adsorbed on titanium, was slowly desorbed when titanium was placed in HBSS. Protein and phosphate may coexist on the same surface, but initially adsorbed albumin molecules prevent the precipitation of a thick layer of tricalcium phosphate.     

Bovine serum albumin antibodies as a disease marker for hepatitis E virus infection

This report evaluates the significance of antibody/bovine serum albumin (BSA) interactions as a risk factor for the diagnosis of acute hepatitis E. Serum samples from 40 patients with acute hepatitis E and from 40 age/sex matched healthy adult subjects were tested for IgA, IgG, and IgM by ELISA and by turbidimetric assay. BSA was used as a target to characterize changes in levels of interacting immunoglobulins. Initial results obtained before removal of antibodies that interacted with BSA suggested that HEV patients had increased levels of IgM in their sera. It was found that normal individuals had mean IgA, IgG, and IgM levels of 2.55 mg/mL, 9.80 mg/mL, and 1.73 mg/mL, respectively while HEV patients had mean levels of 2.66 mg/mL, 10.04 mg/mL, and 2.01 mg/mL ($P < .26$ , $P < .32$, and $P < .0004$). However, the mean level of IgM in HEV-infected sera after purification from antibodies that interacted with BSA was determined to be 1.72 mg/mL indicating that there was no significant difference in IgM level in HEV patients compared to normal individuals ($P < .6$). The presence of antibodies that interact with BSA might serve as a diagnostic tool for detection of high-risk patients.     

Investigation of slow dynamics of the sulfhydryl in the solution and gel states of bovine serum albumin: A vector electron paramagnetic resonance study

Serum albumin has one reactive sulfhydryl (Cys-34) that is one of the important binding sites. Cys-34 is located in the crevice on the surface of the albumin molecule and is therefore restricted in its motion. Bovine serum albumin (BSA) Fraction V forms a transparent gel at pD 4.0 (F-form) in D2O at protein concentrations above 7% (BSA*-gel). We studied the molecular motion of Cys-34 on BSA in the solution and gel states by the vector electron paramagnetic resonance (EPR) method using a maleimide spin label. The rotational correlation times of the spin label bound to Cys-34 in the BSA solution and BSA*-gel were in the order of 10(-6) and 10(-5) s, respectively. A longer rotational correlation time of the Cys-34 spin label in the BSA*-gel suggested that the gel network formed in BSA may drastically slow the motion of Cys-34. The integrated value obtained from the vector EPR spectra also showed an extremely dramatic slowing of the Cys-34 spin label during the gel formation. On the other hand, the values for order parameter and the inclination of the principal axis (z) of the Cys-34 spin label to the rotational axis (mu) were the same in the BSA solution and BSA*-gel.