利用噬菌体肽库序贯筛选组织因子靶向肽

目的: 建立一种高效的筛选跨膜受体靶向肽的噬菌体展示新方法,据此筛选出组织因子( TF) 高亲和力靶向肽。方法: 分别以纯化TF蛋白和具有TF高特异性表达的结直肠癌细胞株HT-29为靶标,用噬菌体七肽库进行序贯筛选(受体-细胞序贯筛选法,STRCA法),ELISA初步鉴定噬菌体克隆对HT-29亲和力。对噬菌体克隆进行测序,利用竞争抑制 ELISA比较各合成肽的TF结合力。重复实验检测筛选结果的可靠性。结果: 经过5轮筛选,与TF结合的噬菌体得到了富集,回收率由(2.25×10^-4)%增加到(1.32×10^-2)%;ELISA结果显示30个克隆中,阳性率为76.7%。噬菌体测序结果中,4种合成肽(分别命名为A、B、C、D肽)中A肽序列重复率为23.3%(7/30),B肽为23.3%(7/30),C肽为26.7%(8/30),D肽为10.0%(3/30);E肽为A、B肽合成的复合靶向肽。经竞争抑制 ELISA比较5种肽的TF亲和力,其IC₅₀分别为3.25 nmol/L、6.72 mol/L、3.24×10³ mol/L、2.08×10² mol/L和45.77 mol/L。重复实验所获得的阳性噬菌体克隆序列与第1次实验相比,重复率为33.3%。结论: STRCA法是一种理想的筛选跨膜受体靶向肽方法,采用该法获得的TF靶向肽对TF具有高度的亲和力。     

肿瘤血管靶向噬菌体展示肽的研究进展

肿瘤血管表达有正常血管内皮细胞没有或微量表达的蛋白,由于它们具有特征性的结构,故用噬菌体展示技术可以筛选出针对这些蛋白的特异性识别多肽。用此类肿瘤特异性识别多肽如RGD、NGR等作为抗肿瘤药物治疗的靶向分子,将其与药物、纳米颗粒或脂质体等结合起来,由此制备的纳米药物有望明显的抑制肿瘤的生长,并极大程度地减少化疗药物的毒副作用。除此之外,此类多肽还可用于肿瘤血管影像检测等领域的应用。     

单纯疱疹病毒囊膜糖蛋白模拟短肽的筛选

目的 获得可替代单纯疱疹病毒（HSV）囊膜糖蛋白行为该病毒亚单位疫苗免疫原的小片段短肽。方法 将1株具有强动物保护活性的抗HSV糖蛋白C型共同性单克隆抗体（mAb)1A12生物素化后，淘筛噬菌体随机12肽库，经4轮筛选后进行ELISA检测。随机挑取14个克隆DNA测序，得到保守序列（Motif)为－SG（L）RHII－,并进行了ELISA阻断实验。结果 携有此短肽的噬菌体可与mAb1A12特异结合，并可部分阻断抗体与HSV囊膜抗原的反应。结论所筛选到的保守序列可模拟mAb1A12针对的抗原表位，可能是HSV的替代抗原。这一研究方法有望使短肽替代庞大的糖蛋白长氨基酸序列，为研制更有效及更广谱的基因工程疫苗提供依据，也使研制基于抗原表位水平的特异诊断试剂成为可能。     

利用体内噬菌体展示技术筛选及鉴定肺癌特异性结合肽

目的利用体内噬菌体展示技术筛选并鉴定与肺癌特异性结合的多肽。方法用肺癌细胞A549接种裸鼠复制荷瘤动物模型,将随机肽库尾静脉注入裸鼠体内,循环15 min后取肿瘤组织中结合的噬菌体,如此进行3轮体内筛选后挑取噬菌体克隆,ELISA初步鉴定噬菌体克隆对肺癌细胞的亲和力、特异性。将阳性噬菌体克隆扩增、测序获得外源多肽氨基酸序列,化学方法合成多肽,鉴定多肽对肺癌细胞和组织的亲和力、特异性。结果ELISA结果显示,随机挑选的20个噬菌体单克隆中,1个对A549具有很强亲和力。测序获得多肽,命名为zp2。化学合成多肽zp2。竞争抑制、细胞免疫荧光和组织免疫荧光实验结果表明多肽zp2与肺癌细胞A549及肺癌组织特异性结合。结论利用体内噬菌体展示技术筛选出与肺癌细胞A549特异性结合的多肽,为肺癌诊断及治疗药物的研制奠定基础。     

应用噬菌体肽文库筛选McAb F3特异性结合肽

目的：应用噬菌体展示肽文库筛选可与抗汉坦病毒囊膜蛋白单抗F3特异性结合的配体肽。方法：采用protein-A亲和层析纯化的F3单抗为筛选配基，对噬菌体展示的随机12肽文库进行生物亲和淘洗，夹心ELISA、竞争ELISA鉴定筛选克隆的结合特性，并进一步对阳性克隆进行序列测定和分析。结果：通过3轮生物淘洗，能被抗体捕获的噬菌体克隆为91．7％（11／12）；ELISA测定显示筛选到的噬菌体短肽能与F3单抗特异性结合；序列分析表明7个阳性克隆氨基酸序列相同，均为－MHGPTKNQMWHT，同源性分析显示该序列与HTNV／SEOV M蛋白G2区第750－759位氨基酸有较高的同源性，结论：获得了具有良好结合活性的模拟表位肽，为基于表位水平的HFRSV（肾病综合征出血热病毒）特异性分子多肽疫苗的设计提供了重要依据。     

利用体内噬菌体展示技术筛选膀胱癌特异性结合肽

目的:利用体内噬菌体展示技术筛选与人膀胱移行细胞癌特异性结合的小分子多肽。方法:将人膀胱移行细胞癌细胞BIU87接种于裸鼠体内,制备膀胱癌荷瘤小鼠模型,尾静脉注射噬菌体展示环七肽库,然后筛选与膀胱移行细胞癌特异性结合的含外源多肽的噬菌体,经过3轮体内筛选后,免疫组织化学法及ELISA法鉴定单克隆噬菌体对BIU87的亲和力。提取阳性单克隆噬菌体单链DNA进行测序,并推导出外源多肽氨基酸序列,化学合成多肽、制备分子探针后采用激光扫描共聚焦显微镜术及流式细胞术鉴定多肽对膀胱癌细胞和组织的特异性。结果:3轮体内筛选后,噬菌体富集率达到4.334×102倍。免疫组化结果显示,肿瘤组织中噬菌体肽的含量随着每一轮筛选呈增长趋势,且结合逐渐增强,同时由于噬菌体经肝、肾代谢,可见肝脏结合大量非特异性的噬菌体。ELISA结果显示,随机挑选的30个单克隆噬菌体斑中,有24个阳性噬菌体,其中10个噬菌体对BIU87有较强的亲和力,对其测序并推导出3种多肽序列,重复率最高的序列CSSPIGRHC(8/10)命名为NYZL1。化学合成FITC-C6-NYZL1,通过激光扫描共聚焦显微镜术、流式细胞术均证明多肽NYZL1可以特异性结合膀胱癌细胞。结论:利用体内噬菌体展示技术筛选出了与人膀胱移行细胞癌特异性结合的小分子多肽NYZL1,为膀胱癌早期诊断和靶向治疗提供一定的理论依据。     

突变肽库的构建及其应用--噬菌体展示短肽的改造

1990年, 由Parmley和Smith[1]提出并建立的噬菌体展示技术(phagedisplaytechnique, PDT)是以噬菌体或噬菌粒为载体, 表达蛋白、单链抗体、酶及短肽。由此衍生的多种噬菌体展示库, 可用于研究蛋白质之间或蛋白与非蛋白之间相互作用, 如抗原与抗体的相互作用。然而, 在对噬菌     

VEGF受体结合抑制肽的筛选及其特性鉴定

目的　筛选能抑制VEGF与血管内皮细胞表面受体结合的VEGF模拟短肽 ,探讨小分子短肽作为血管内皮细胞生长抑制剂、肿瘤疫苗的可行性。方法　以抗 VEGF单克隆抗体分别对噬菌体随机 7肽、12肽库进行筛选。通过硝酸纤维膜斑点印迹法进行阳性克隆鉴定。用噬菌体克隆免疫小鼠检验这种短肽可否模拟VEGF的抗原决定簇诱发机体产生能与VEGF结合的抗体 ,研究小鼠抗血清、噬菌体表达的 7肽和 12肽以及人工合成 7肽对人脐静脉血管内皮细胞 (HUVEC)生长的抑制作用。结果　对肽库进行 3轮筛选后 ,噬菌体克隆具有良好的富集效果。硝酸纤维膜斑点印迹法证实阳性率达到 10 0 % ,测序结果显示噬菌体上的短肽有共同序列 :GWYYDAL、VASAVFYSALVE。这两种噬菌体短肽免疫小鼠能激发产生高效价的抗 VEGF抗体。噬菌体表达的 7肽和 12肽对HUVEC的生长有明显的抑制作用。由短肽GWYYDAL阳性克隆免疫的抗血清对VEGF诱导的内皮细胞生长起抑制作用 ,人工合成的 7肽 (GWYYDAL)呈剂量依赖性阻拮VEGF促HUVEC增殖作用。结论　从噬菌体肽库中筛选到的噬菌体短肽GWYYDAL、VASAVFYSALVE模拟了VEGF的抗原表位 ,为研究VEGF受体结合抑制肽和设计肿瘤疫苗奠定了实验基础。     

P-糖蛋白结合肽的筛选及合成肽鉴定

目的 筛选人膀胱癌P-糖蛋白特异性结合肽并合成鉴定.方法 利用表达获得的P-糖蛋白胞外段融合蛋白为靶蛋白,采用酶联板法筛选噬菌体随机12肽库,化学合成该特异性肽序列,免疫细胞化学方法进行鉴定.结果 从噬菌体随机肽库中筛选获得了与P-糖蛋白特异性结合的噬菌体阳性克隆,测序获得特异性结合肽序列.免疫细胞化学结果显示特异性合成肽可与耐药细胞BIU-87/ADM结合,而与敏感细胞BIU-87不结合.结论 筛选获得的结合肽具有一定的亲和力,可与特定的肿瘤细胞结合,表现出一定的肿瘤特异性;P-糖蛋白结合肽的筛选,为人膀胱癌多药耐药的靶向治疗结论提供实验依据.     

应用噬菌体肽文库筛选mAb F3特异性结合肽

目的 应用噬菌体展示肽文库筛选可与汉坦病毒囊膜蛋白中和性单抗（mAb） F3特异性结合的配体肽。方法 以F3mAb为筛选配基，对噬菌体展示和随机12肽文加进行3轮生物亲和淘选；用夹心ELISA和竞争ELISA鉴定筛选克隆的结合特性，并进一步对阳性克隆进行序列测定和分析。结果 通过3轮生物淘选，能被抗体捕获的噬菌体克隆为21／22，ELISA测定显示，筛选到的噬菌体短肽能与F3mAb特异性结合。序列分析表明，7个阳性克隆氨基酸序列相同，均为－MHGPTKNQMWHT；同源性分析显示，该序列与HTNV／SEOV M蛋白G2区第750－759位氨基酸有较高的同源性。结论 本研究为基于表位水平的HFRSV特异性分子多肽疫苗的设计提供了重要的依据。     

A study of possible heterogeneity in Duchenne muscular dystrophy

One possible explanation for the apparently high birth incidence of Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, is genetic heterogeneity. As a first step in possibly demonstrating genetic heterogeneity, affected boys were sub-divided into those with and without severe mental handicap. In those with severe mental handicap, ages at onset and of becoming confined to a wheelchair were later, the fall in SCK level with age was less marked, and the urinary excretion of certain aminoacids was greater than in affected boys with normal intelligence. Though the number of subjects investigated was relatively small (15 in each group) and further studies are therefore needed, the results suggest that DMD may not be a single disease entity.     

噬菌体表达短肽模拟脂多糖类脂A表位的研究

目的：得到拟类脂Ａ的六肽。方法：用抗类脂Ａ的单克隆抗体筛选噬菌体随机六肽库,经四轮筛选后进行ＥＬＩＳＡ检测,用得到的１５个阳性克隆分别免疫小鼠,并将使小鼠产生抗ＬＰＳ抗体的１０个克隆进行测序,将保守序列进行肽合成。结果：得到保守序列为Ｌｙｓ－Ｐｈｅ－Ｓｅｒ－Ｓｅｒ－Ａｒｇ,即ＫＦＳＳＲＸ。固相合成此小肽可与其１Ｂ６抗体结合,且此结合可被ＬＰＳ抑制。结论：此六肽可模拟脂多糖或类脂Ａ表位。     

Screening of deletions and RFLP analysis in Turkish DMD/BMD families by PCR

Gökgöz N, Kuseyri F, Topalolu H, Yüksel-Apak M, Kirdar B. Screening of deletions and RFLP analysis in Turkish DMD/BMD families by PCR.
Clin Genet 1993: 43: 261–266. © Munksgaard, 1993
We have screened 76 DMD and 5 BMD patients for deletions, using two separate Multiplex gene amplification systems. The use of both systems together revealed deletions in 52% of the cases in the Turkish population. The majority of these deletions (33/37) were found to be localized within the central region of the dystrophin gene. The remaining deletions were mapped to the proximal hotspot. Deletion end-points were identified by PCR and/or by Southern blot analysis with cDNA probes, and exceptions to the Open Reading Frame (ORF) hypothesis are discussed. PCR-based techniques to screen the pER.T87.15/XmnI, pERT87.15/BamHI, and pERT87.8/TaqI polymorphisms were used for linkage analysis in the Turkish DMD/BMD families, and approximately 70% of the mothers at risk were found to be informative for at least one of these polymorphisms studied.     

A survey of manifesting carriers of Duchenne and Becker muscular dystrophy in Wales

Manifesting carriers of Duchenne and Becker muscular dystrophy are uncommon but well described. Such patients are of particular importance with regard to the differential diagnosis from autosomal recessive limb-girdle muscular dystrophy. All mothers of affected males known to the Genetic Register of Muscular Dystrophy Families in Wales were contacted, and 167 out of a possible 190 were examined. It was estimated from pedigree and creatine kinase analysis that 119 out of the 167 were carriers of the Duchenne/Becker gene. Three manifesting carriers were identified, giving the proportion affected as 3/119 = 2.5%. We estimate the prevalence of manifesting carriers to be 1 in 100,000 of the female population, a figure comparable to the prevalence of autosomal recessive limb-girdle muscular dystrophy. During the period of the survey, several other women with similar clinical findings but without an appropriate family history were seen. We strongly suspect that some of these are also manifesting carriers of the Duchenne/Becker gene.     

Early Marker of Myocardial Deformation in Children with Duchenne Muscular Dystrophy Assessed Using Echocardiographic Myocardial Strain Analysis

PurposeAs cardiomyopathy is more prevalent and currently the leading cause of death in Duchenne muscular dystrophy (DMD), early detection of myocardial involvement is important. The purpose of this study was to analyze myocardial strain in DMD children, for the possibility of early detection of myocardial dysfunction.ResultsEpicardial longitudinal strain was lower in DMD (DMD: -9.3±3.8%; control: -12.3±4.3%; p=0.012). Radial strain (DMD: 24.1±11.1%; control: 37.3±25.9%; p=0.027) and strain rate (SR) (DMD: 1.68±0.91; control: 2.42±0.84; p=0.006) on parasternal short axis view were lower in DMD. Circumferential strains in the endocardium (DMD: -17.5±4.7%; control: -24.2±5.3%; p<0.001), myocardium (DMD: -12.7±3.8%; control: -18.0±4.0%; p<0.001), and epicardium (DMD: -8.4±4.0%; control: -12.2±5.0%; p=0.006) were significantly decreased in DMD. Circumferential SRs were lower in the endocardial (DMD: -1.46±0.38; control: -1.78±0.27; p=0.002) and myocardial layers (DMD: 1.02±0.27; control: -1.28±0.22; p=0.001).ConclusionIn DMD patients, deteriorations in myocardial circumferential strain might be an indicator for predicting cardiomyopathy.     

Results of Duchenne muscular dystrophy family screening in practice: leaks rather than cascades?

McGowan R, Challoner BR, Ross S, Holloway S, Joss S, Wilcox D, Holden ST, Tolmie J, Longman C. Results of Duchenne muscular dystrophy family screening in practice: leaks rather than cascades? Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations in the gene that encodes the protein dystrophin. Approximately 2 of 3 affected boys inherit their mutation from their carrier mother whereupon other female relatives are at risk of carrying the mutation. Female carriers are also at risk of developing cardiomyopathy and regular cardiac screening is recommended. Clinical genetics services offer genetic counselling and carrier tests for consenting relatives of DMD patients known as ‘cascade screening’. We retrospectively analysed data from two genetics centres, West of Scotland and South East Thames where the latter centre operated a computer‐held DMD register. Over the period, 1971–2008, a total of 843 potential carriers, in 195 West of Scotland families, were tested: 16% of 1st degree relatives and 48% of 2nd degree and more distant relatives were not tested. In South East Thames, a total of 1223 potential carriers in 349 families were tested: 49% of 1st degree and 65% of 2nd degree and more distant relatives were not tested. These data are similar to Becker muscular dystrophy/DMD carrier screening results recently reported from the Netherlands. Retrospective results from three countries indicate that despite efforts to offer extended cascade screening, significant numbers of potential carriers of DMD remain unaware of their reproductive and health risks.