噬菌体随机环7肽库筛选恶性疟原虫EBA-175的结合肽

目的从噬菌体随机环7肽库中筛选恶性疟原虫EBA175抗原的结合肽。方法以EBA175重组蛋白为靶筛选噬菌体随机环7肽库,通过ELISA、竞争抑制试验、Westernblot等方法鉴定获得的噬菌体短肽与EBA175之间的结合特性。对阳性克隆进行DNA序列测定,推导其氨基酸序列并与GPA氨基酸全序列进行了同源性比较。结果获得9株可与EBA175结合的阳性噬菌体克隆,序列分析显示为3种氨基酸序列,P1(MLLITIR)、P2(TRKLPRT)、P3(KRLMPLK)。其中出现频率最高的P1序列中LLI与EBA175的受体GPA的108110位氨基酸同源。竞争性ELISA显示展示序列P1的噬菌体能竞争抑制EBA175与其单抗的结合。结论获得了可与EBA175特异结合的阳性噬菌体短肽,·LLI··几位氨基酸可能对EBA175与GPA的结合起重要作用。     

细胞粘附因子-1与恶性疟原虫感染红细胞结合位点的鉴定

目的　探索恶性疟原虫感染红细胞(PRBCs)与细胞粘附因子1(ICAM1)之间的结合位点,研制治疗脑型疟的抗粘附药物。　方法　以抗ICAM1(I区)的单抗15.2为靶,采用亲和筛选法对噬菌体随机十二肽库进行3轮筛选,通过ELISA、竞争抑制试验、dotELISA及Westernblotting鉴定获得的噬菌体短肽与单抗15.2之间的结合特性。对阳性克隆进行DNA序列测定,推导其十二肽的氨基酸序列并与ICAM1氨基酸全序列进行同源性比较。　结果　经3轮亲和筛选后,结合噬菌体得到良好富集。从第3轮洗脱液铺制的琼脂板中随机挑取30个噬菌体单克隆,ELISA检测有26个为阳性,阳性率达86.7%。竞争性ELISA显示多数阳性噬菌体能竞争抑制ICAM1与15.2单抗结合。DNA及氨基酸序列分析表明半数以上的噬菌体克隆表达十二肽KLYLIAEGSVAA,该短肽中K(XX)L(XXX)GSV与ICAM1的64～73位aa有50%的同源性。　结论　阳性噬菌体表达的短肽是15.2单抗所识别的模拟表位,K··L···GSV几个氨基酸可能对ICAM1与PRBCs的结合起重要作用     

用噬菌体展示随机7肽库筛选血型A抗原模拟肽

目的:通过血型A单克隆抗体从噬菌体展示随机7肽库中筛选血型A抗原的模拟多肽。方法:通过对噬菌体随机7肽库进行3轮亲和筛选,从第三轮筛选后获得的洗脱物中挑选多个噬菌体克隆,采用ELISA方法鉴定阳性克隆,测序并推导噬菌体展示短肽的氨基酸序列,化学合成短肽、红细胞凝集抑制试验鉴定短肽模拟A抗原的能力。结果:三轮筛选后特异性噬菌体被富集了200多倍,对ELISA鉴定信号较强的16个噬菌体克隆DNA测序并推导氨基酸序列的结果显示两个克隆展示相同的序列FSYLPSH。化学合成肽能抑制A型红细胞与抗A的凝集作用。结论:肽FSYLPSH具有模拟血型A抗原表位的作用,具有代替天然血型A抗原在临床中应用的潜能。     

脂质A高亲和噬菌体融合肽的筛选及鉴定

以脂质A为靶标分子,对噬菌体展示环七肽库进行4轮亲合筛选,ELISA结合实验鉴定阳性克隆,对获得的阳性克隆进行DNA序列测定,推导短肽的氨基酸残基序列。结果4轮筛选后,随机挑取的20个噬菌体克隆中14个可与脂质A结合。测序发现这14个阳性克隆融合多肽中的8个序列一致,均为QTPLSST。认为QT-PLSST是一个可与脂质A高亲力结合的噬菌体多肽。该肽序列能否抑制脂多糖的活性有待进一步研究。     

应用噬菌体展示技术筛选脂多糖结合肽

以脂多糖(LPS)为靶标分子,对噬菌体展示环七肽库进行4轮亲合筛选,EL ISA结合试验鉴定阳性克隆,对获得的阳性克隆进行DNA序列测定,推导短肽的氨基酸残基序列。结果4轮筛选后,随机挑取的20个噬菌体克隆中11个可与LPS结合。测序发现这11个阳性克隆融合多肽中的7个序列一致,均为RW PLSST。提示本研究筛选到一个可与LPS高亲力结合的噬菌体多肽,该肽序列能否阻断LPS的活性有待进一步阐明。     

丙型肝炎病毒非结构蛋白NS3抗原模拟表位的筛选和鉴定

目的 筛选丙型肝炎病毒(HCV)非结构蛋白NS3(HCV NS3)特异性噬菌体模拟表位，为抗—HCV的疫苗研究探索新途径。方法 以抗—HCV NS3的单克隆抗体作为固相筛选分子，对人工合成的噬菌体随机七肽库进行3轮“吸附—洗脱—扩增”的筛选过程，随机挑取60个克隆，经噬菌体酶联免疫吸附法(ELISA)鉴定并进行交叉反应实验以及竞争抑制性结合实验，最后对所选克隆进行DNA序列分析，以确定HCV NS3抗原的模拟表位。结果 经噬菌体富集后，从随机筛选的60个克隆中得到13个阳性克隆，确定氨基酸序列SRNTxKL为HCV NS3的模拟表位。结论 用噬菌体七肽库成功筛选得到HCV NS3的模拟表位。     

囊虫抗原模拟表位的筛选和序列分析

目的探讨能否从与兔抗蚯蚓血清免疫球蛋白(EIg)结合后的噬菌体12肽库中筛选囊虫抗原模拟表位。方法噬菌体12肽库经过EIg筛选1轮后,再以囊虫病患者血清免疫球蛋白(CIg)作为靶分子进行3轮吸附-洗脱-扩增,随机挑取24个蓝色噬菌斑扩增;分析其抗原性及诊断囊虫病的价值;并测定核苷酸序列分析同源性。结果24个克隆中有17个克隆可与囊虫病患者混合血清发生免疫反应,其中6个A491值较高的克隆ELISA和囊虫抗原DotELISA的抗体阳性率无显著性差异(P>0.05);与其它寄生虫病的交叉反应率明显低于囊虫抗原(P<0.05),说明所获克隆具有诊断囊虫病的潜在价值。挑选其中2个克隆(C3、C5)测定核苷酸序列,结果显示这两个抗原模拟表位的氨基酸序列具有结构同源性。结论将EIg结合后的肽库作为源肽库经过3轮筛选,得到对囊虫病具有较高潜在诊断价值的抗原模拟表位。     

人类白细胞抗原-B*2704/B*2705重链拮抗肽的筛选和初步鉴定

目的 从噬菌体展示随机12肽库筛选人类白细胞抗原(HLA)-B·2704及B·2705重链拮抗肽并做初步鉴定.方法 用HLA-B*2704/B*2705重链胞外区蛋白分别筛选噬菌体展爪随机肽库,酶联免疫吸附试验(ELISA)鉴定阳性克隆,DNA测定确定氨基酸序列,免疫荧光和流式细胞术鉴 ,定噬菌体克隆分别与HLA-B*2704及B*2705细胞株结合的特异性.结果 经3轮筛选,获得12个HLA-B·2704拈抗肽,共有5种序列,分别为HTSFCSTHLCLI(×4),QHCSPTLCQIHR(×5),ARCTITL-CYLSN(×1),YGLCTDWYCHIT(×1),YPLCDAILCRLP(×1);10个B*2705拮抗肽共有4种序列,分别为:①SHCSPHWCALPF(×6);②HLCSNSLCLLPW(×2);③EPMCSWFWCTLP(×1);④WTCSPLLCTWGA(×1).比对分析表明,B*2704与B*2705拮抗肽的序列是基本一致的,均含有CS(T)TXXL(W)CXL表位.展示有B*2704拮抗肽的噬菌体克隆可与HLA-B*2704细胞株结合,阳性率为43.55%;而B*2705拈抗肽噬菌体克隆与HLA-B·2705细胞株的阳性结合率为45.69%.结论 筛选获得的HLA-B27拈抗肽的噬菌体克隆具有一定的亲和力,可与表达于细胞株表面的B*2704和B**2705分子特异性结合,而与正常B细胞不结合,因而表现出一定的结合特异性.     

丙型肝炎病毒非结构蛋白NS5抗原模拟表位的筛选和鉴定

目的 筛选丙型肝炎病毒非结构蛋白NS5（HCV NS5）特异性噬菌体模拟表位，为抗HCV的疫苗研究探索新途径。方法 以抗－HCV NS5的单克隆抗体作为固相筛选分子，对人工合成的噬菌体随机七肽库进行5轮“吸附－洗脱－扩增”的筛选过程，随机挑取30个克隆，经噬菌体酶联免疫吸附法（ELISA）鉴定并进行交叉反应实验以及竞争抑制性结合实验，最后对所选克隆进行DNA序列分析，以确定HCV NS5抗原的模拟表位。结果 经噬菌体富集后，从随机筛选的30个克隆中得12上阳性克隆，确定氨基酸序列QIRPTRQ为HCV NS5的模拟表位。结论 用噬菌体七肽库成功筛选得到HCV NS5的模拟表位，为用HCV模拟表位探索HCV感染的研究创造了条件。     

应用噬菌体随机肽库研究细粒棘球蚴抗原模拟表位

用抗细粒棘球蚴B抗原(EgB)多克隆抗体筛选噬菌体随机7肽库。经过5轮筛选后,噬菌体回收率从第1轮的4.15×10-5增加到第5轮的4.30×10-2,说明阳性克隆得到富集。随机挑取60个蓝色噬菌斑进行扩增,对其核苷酸序列进行测定分析并与EgB进行同源性比较。采用ELISA法对阳性噬菌体与抗EgB多克隆抗体结合特性进行检测,共检测出45个与抗EgB多克隆抗体结合的克隆株(阳性率为75%),其递呈的七肽序列与EgB的同源性低。采用ELISA法检测所选多肽对棘球蚴病所致过敏性休克患者抗血清的反应性,结果显示,细粒棘球蚴病患者抗血清与45个阳性噬菌体克隆反应的吸光度(A410值)比其与7肽库反应的A410值大2倍以上,该45个阳性噬菌体克隆与EgB多克隆抗体的结合是特异的。提示成功分析细粒棘球蚴囊液B抗原肽表位以及模拟表位。     

人髓系细胞触发受体-1模拟多肽的筛选和鉴定

目的寻找能特异性与人髓系细胞触发受体-1（TREM-1）蛋白结合并抑制其传导通路的多肽。方法克隆、表达和纯化TREM-1功能区蛋白，并以之为诱饵蛋白，筛选随机噬菌体展示肽库。经过4轮生物淘洗，通过ELISA方法和单核细胞ELISA方法分析噬菌体克隆与TREM-1蛋白的亲和力。化学合成模拟多肽，检测其对盲肠结扎穿刺（CLP）小鼠的治疗效果。结果成功找到5种噬菌体克隆，ELISA法和细胞ELISA均阳性。应用化学方法合成的模拟多肽HYGMTHPNTMsH能降低CLP小鼠的死亡率。结论模拟多肽HYGMTHPNTMSH对脓毒症小鼠有保护作用。     

Identification of Hitherto Undefined B-Cell Epitopes by Antibodies in the Sera of Vitiligo Patients Using Phage-Display Peptide Library

A random 12 mers phage library was used to screen a pool of immunoglobulin fractions obtained from vitiligo patients. Subsequent to panning experiments, a panel of affinity selected phage from vitiligo patients were obtained. This panel was tested using an ELISA for their reactivity with pooled sera from patients and normal controls. Among the 16 randomly selected clones, two of clones showed distinct positive reactivity with the patient's sera compared with controls. The peptides displayed by these phages expressed the following amino acid sequences: SHMPLANQYQWA and NHVQAWEQFWDS. Thus, screening with phagedisplayed random peptide library of vitiligo sera can reveal peptide sequences that mimic vitiligo-related self-antigen.     

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGb1 to screen a phage-displayed random peptide library fused with coat protein pⅢ in order to get some information on mimotopes.METHODS: Through affinity enrichment and ELISA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times ofenrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGb1-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGb1-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0±1.6)%to (39.0±2.7)%.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.     

Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGbl to screen a phage-displayed random peptide library fused with coat protein plII in order to get some information on mimotopes.lV~37BODS: Through affinity enrichment and EUSA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGbl-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGbl-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0&#177;1.6) %to (39.0&#177;2.7) %.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.     

日本血吸虫模拟短肽诱导小鼠的免疫保护性研究

目的　研究日本血吸虫模拟短肽对小鼠的免疫保护效果,预测其在抗血吸虫感染中的作用。　方法　用粗提纯大鼠血清IgG为配基对噬菌体肽库进行3轮免疫学筛选。随机挑取噬菌体克隆,检测其特异性并进行序列分析;用阳性克隆免疫小鼠,以40条日本血吸虫尾蚴攻击感染,感染后42d剖杀取虫,计算减虫率和减卵率;用ELISA测定免疫鼠抗体反应。　结果　经过3轮筛选,特异性噬菌体得到有效富集;自动测序仪测序获得2个模拟肽分子;将其用于免疫小鼠后,各免疫组与对照组相比,2个模拟肽混合免疫组的减虫率为34.9%P<0.05,肝内减卵率为67.6%(P<0.001)。 2个模拟肽分子分别免疫小鼠 ,各组的减虫率为31.0%(P<0.05)和14.5%(P>0.05)及肝内减卵率为61.2%(P<0.001)和35.7%(P<0.05)。ELISA检测其免疫组鼠血清特异性IgG抗体滴度均大于1∶6 400以上。　结论　利用噬菌体表面呈现技术获得的2个模拟肽分子均可诱导部分抗日本血吸虫感染的免疫保护力