目的:利用噬菌体7肽库进行体外快速差减筛选(biopanning and rapid analysis of selective interactive ligands,BRASIL),从而获得卵巢癌细胞株HO-8910细胞表面特异性结合肽。方法:以中国仓鼠卵巢细胞株CHO为吸附细胞,卵巢癌细胞株HO-8...目的:利用噬菌体7肽库进行体外快速差减筛选(biopanning and rapid analysis of selective interactive ligands,BRASIL),从而获得卵巢癌细胞株HO-8910细胞表面特异性结合肽。方法:以中国仓鼠卵巢细胞株CHO为吸附细胞,卵巢癌细胞株HO-8910为靶细胞,利用噬菌体展示技术联合差减筛选策略,经过连续5轮生物淘洗,随机挑选13个噬菌体单克隆进行DNA测序,利用噬菌体竞争结合实验、ELISA实验、细胞免疫染色、细胞免疫荧光、合成多肽的竞争抑制实验鉴定阳性噬菌体的亲和性及特异性。结果:筛选出的噬菌体NPMIRRQ与卵巢癌细胞株HO-8910有较高的亲和性及特异性。结论:阳性噬菌体表面展示的多肽NPMIRRQ可能为卵巢癌的早期诊断和转移复发监测提供新思路。展开更多
利用肽配体库技术(CPLL)研究了蛋清中的低丰度蛋白质组。考察了盐浓度和洗脱顺序差异对CPLL富集效率的影响。结果表明:盐浓度越高,蛋清蛋白质溶液的离子相互作用越强。利用洗脱溶液解离差异,采用先HOS(有机水溶液)后尿素CHAPS(3-[3-(胆...利用肽配体库技术(CPLL)研究了蛋清中的低丰度蛋白质组。考察了盐浓度和洗脱顺序差异对CPLL富集效率的影响。结果表明:盐浓度越高,蛋清蛋白质溶液的离子相互作用越强。利用洗脱溶液解离差异,采用先HOS(有机水溶液)后尿素CHAPS(3-[3-(胆酰胺丙基)二甲氨基]-1-丙磺酸内盐)的洗脱顺序,溶菌酶能够得到高效分离。保持蛋清的原p H 8.8,25 mmol/L KH2PO4,150 mmol/L Na Cl,尿素CHAPS洗脱,是CPLL提供高效富集的前提。通过分析CPLL富集前后的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)条带差异,采用线性离子阱(LTQ)质谱在蛋清中检出45种低丰度蛋白质,其中两种未见报道。对蛋清中低丰度蛋白质亚细胞的定位分析发现,蛋清蛋白质的分泌蛋白居多。研究结果表明,CPLL技术能够实现蛋清中低丰度蛋白质的高效富集,分析深度达到亚细胞水平。展开更多
A new method for the rapid and efficient screening of affinity ligands to biological targets is reported. The fusion peptide of influenza virus A was used as the model target and immobilized on the PGMA beads. Antisen...A new method for the rapid and efficient screening of affinity ligands to biological targets is reported. The fusion peptide of influenza virus A was used as the model target and immobilized on the PGMA beads. Antisense peptide YRSKQA of fusion peptide was chosen as the lead compound. The special positional scanning peptide libraries were designed based on YRSKQA and synthesized by utilizing solid phase peptide synthesis manually. The libraries were YRSKQX, YRSKXA, YRSXQA, YRXKQA, YXSKQA and XRSKQA, where X represented 18 L-amino acids(except for Cys and Trp). Each library was screened by affinity chromatography. The eluates from the fusion peptide affinity column were collected and analyzed by RP-HPLC and MS, respectively, in order to determine the kind of X at each position. After the preferred residues of six positions were decided, the two preferred peptide sequences, GRGKHK and TRGKHK, were obtained. The dissociation constants of GRGKHK, TRGKHK and YRSKQA, were 3.35×10 -6, 5.24×10 -6 and 1.15×10 -5 mol·L -1, respectively. The preferred peptides showed the higher affinity binding to immobilized fusion peptide than the lead peptide.展开更多
文摘目的用蛋白质组学方法寻找先兆子痫患者血清蛋白的差异表达。方法 5例重度先兆子痫患者血清和5例正常妊娠对照血清分别混合。每个混合样本血清用亲水亲脂柱(hydrophilic-lipophilic balance,HLB)和多肽配体库磁珠(peptide ligandlibrary bead,PLLB)方法去除表达丰富蛋白,使低含量蛋白表达,洗脱液用1D胶联合液相色谱质谱串联技术(liquidchromatography tandem mass spectrometry,LC-MS/MS)对蛋白表达进行定量分析;结果 HLB方法共有28种蛋白发生差异表达;PLLB方法共有51种蛋白发生差异表达,包括白介素17F(IL-17F)和铜蓝蛋白(ceruloplasimin,CP)等。结论 PLLB联合1D胶-LC-MS/MS是鉴定血清中差异表达蛋白的一种有效方法;炎性反应因子可能与先兆子痫的发病机制有关。
文摘目的:利用噬菌体7肽库进行体外快速差减筛选(biopanning and rapid analysis of selective interactive ligands,BRASIL),从而获得卵巢癌细胞株HO-8910细胞表面特异性结合肽。方法:以中国仓鼠卵巢细胞株CHO为吸附细胞,卵巢癌细胞株HO-8910为靶细胞,利用噬菌体展示技术联合差减筛选策略,经过连续5轮生物淘洗,随机挑选13个噬菌体单克隆进行DNA测序,利用噬菌体竞争结合实验、ELISA实验、细胞免疫染色、细胞免疫荧光、合成多肽的竞争抑制实验鉴定阳性噬菌体的亲和性及特异性。结果:筛选出的噬菌体NPMIRRQ与卵巢癌细胞株HO-8910有较高的亲和性及特异性。结论:阳性噬菌体表面展示的多肽NPMIRRQ可能为卵巢癌的早期诊断和转移复发监测提供新思路。
文摘利用肽配体库技术(CPLL)研究了蛋清中的低丰度蛋白质组。考察了盐浓度和洗脱顺序差异对CPLL富集效率的影响。结果表明:盐浓度越高,蛋清蛋白质溶液的离子相互作用越强。利用洗脱溶液解离差异,采用先HOS(有机水溶液)后尿素CHAPS(3-[3-(胆酰胺丙基)二甲氨基]-1-丙磺酸内盐)的洗脱顺序,溶菌酶能够得到高效分离。保持蛋清的原p H 8.8,25 mmol/L KH2PO4,150 mmol/L Na Cl,尿素CHAPS洗脱,是CPLL提供高效富集的前提。通过分析CPLL富集前后的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)条带差异,采用线性离子阱(LTQ)质谱在蛋清中检出45种低丰度蛋白质,其中两种未见报道。对蛋清中低丰度蛋白质亚细胞的定位分析发现,蛋清蛋白质的分泌蛋白居多。研究结果表明,CPLL技术能够实现蛋清中低丰度蛋白质的高效富集,分析深度达到亚细胞水平。
文摘A new method for the rapid and efficient screening of affinity ligands to biological targets is reported. The fusion peptide of influenza virus A was used as the model target and immobilized on the PGMA beads. Antisense peptide YRSKQA of fusion peptide was chosen as the lead compound. The special positional scanning peptide libraries were designed based on YRSKQA and synthesized by utilizing solid phase peptide synthesis manually. The libraries were YRSKQX, YRSKXA, YRSXQA, YRXKQA, YXSKQA and XRSKQA, where X represented 18 L-amino acids(except for Cys and Trp). Each library was screened by affinity chromatography. The eluates from the fusion peptide affinity column were collected and analyzed by RP-HPLC and MS, respectively, in order to determine the kind of X at each position. After the preferred residues of six positions were decided, the two preferred peptide sequences, GRGKHK and TRGKHK, were obtained. The dissociation constants of GRGKHK, TRGKHK and YRSKQA, were 3.35×10 -6, 5.24×10 -6 and 1.15×10 -5 mol·L -1, respectively. The preferred peptides showed the higher affinity binding to immobilized fusion peptide than the lead peptide.
