恶性疟原虫感染的红细胞膜表面蛋白1模拟肽的筛选及鉴定

目的 筛选恶性疟原虫感染的红细胞膜表面蛋白1（PfEMP?鄄1）的噬菌体表位模拟肽。 方法 细胞间粘附分子ICAM-1模拟12肽（KLYLIAEGSVAA）能模拟ICAM-1分子与疟原虫感染红细胞结合的功能，以展示该短肽的噬菌体为靶，采用差减筛选法（subtraction method）对噬菌体环7肽库进行3轮筛选，通过ELISA、竞争抑制试验鉴定获得的噬菌体短肽与ICAM-1之间的结合特性。对阳性克隆进行DNA及氨基酸序列分析并与PfEMP-1氨基酸序列进行同源性比较。 结果 ELISA筛选22个克隆有3个为阳性克隆，氨基酸序列分析显示2个克隆的展示的短肽序列为C-ITAVPVR-C，另1为C-DIMGGYN-C。同源性分析未发现2短肽序列与野生型MC株恶性疟原虫PfEMP-1的氨基酸序列有同源性。但竞争抑制试验显示3个阳性克隆均可与15.2单抗间互相竞争抑制与ICAM-1分子的结合。 结论 获得2种PfEMP-1噬菌体构象表位模拟肽，两短肽能与ICAM-1分子特异性结合。     

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。     

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。     

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。     

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     

一种新型转铁蛋白受体结合肽的筛选与鉴定

目的以转铁蛋白受体(TfR)为靶标,从噬菌体随机七肽库淘选TfR结合肽并进行鉴定。方法经过三轮筛选,从平板上挑取分隔良好的特异性TfR结合肽单克隆,采用ELISA初步鉴定后行扩增测序及多肽合成,采用流式细胞仪检测人肝癌HepG2细胞表面TfR表达、采用免疫荧光实验及激光共聚焦实验行噬菌体与细胞共定位鉴定、采用短肽—噬菌体竞争抑制实验检测短肽对噬菌体的竞争抑制率,以正常肝脏L-O2细胞为对照。结果特异性TfR结合肽克隆最终富集度达80倍;初步检测到20个阳性克隆,其中9号克隆对TfR亲和力最高,测序表明其展示短肽序列为AHLHNRS,以碱性氨基酸为主,经HPLC检测纯度为99.91%;HepG2细胞表面TfR表达显著高于L-O2细胞;免疫荧光实验及激光共聚焦实验均显示9号噬菌体(P9)出现在HepG2细胞表面,而对照L-O2细胞无此现象;短肽与噬菌体P9对TfR的结合存在竞争关系,并且呈浓度依赖性,而野生型噬菌体M13与L-O2不存在此现象。结论本研究获得一条与TfR具有特异性结合能力的TfR结合肽;此为进一步构建肿瘤和脑源性疾病靶向治疗药物奠定了基础。     

噬菌体展示肽库在乳腺癌血清肿瘤标志物筛选中的应用

目的用噬菌体随机12肽库对乳腺癌患者的血清进行差异性筛选,以获得乳腺癌特异性的新的肿瘤标志物。方法对噬菌体随机12肽库进行三轮差异性筛选,用ELISA法测定噬菌体克隆对乳腺癌患者血清结合的特异性。结果经过三轮筛选,噬菌体富集率逐轮递增,经47例乳腺癌患者和正常人血清的ELISA法检测验证,获得一株与乳腺癌患者血清特异较好的噬菌体,经DNA测序和推导,其短肽序列为短肽(QVSAEHKVQGFW)。结论成功筛选到能与乳腺癌患者血清高亲和力特异结合的12肽序列,为今后研究乳腺癌肿瘤标志物奠定了基础。     

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.     

噬菌体表达的短肽模拟蚯蚓与日本血吸虫共同表位研究

目的采用噬菌体呈现技术,获得蚯蚓与血吸虫的共同表位。方法依次以日本血吸虫病患者血清(SjIg)、蚯蚓免疫兔血清(LtIg)和LtIg、SjIg作为靶分子,以噬菌体12肽库的第三轮扩增肽库为源肽库,进行2轮吸附-洗脱-扩增免疫筛选。每轮随机挑取蓝色噬菌斑各21个,用ELISA方法检测其抗原性,并对其反应性较好的阳性克隆进行测序。结果获得4个阳性克隆可与SjIg、LtIg较好的结合。得到3个阳性克隆的氨基酸序列,它们有蚯蚓与血吸虫共同的线性表位。结论结果说明,从12肽库筛选蚯蚓与寄生蠕虫抗原共同表位是可行的,同时也为获得寄生蠕虫共同抗原提供了一条新的途径。     

从噬菌体肽库中筛选弓形虫抗原模拟表位初探

以弓形虫感染兔血清免疫球蛋白（Ig）作为靶分子，免疫筛选噬菌体随机7肽库和12肽库。经3轮筛选，随机挑取70个噬菌体克隆用ELISA检测其特异性，共获得43个阳性克隆。对23个ELISA反应较强的克隆进行测序，根据序列分析选择有代表性的克隆A7作为模拟表位抗原进行ELISA检测。对47份弓形虫感染兔血清进行ELISA检测，结果32份为阳性，敏感性为68.1%；155份健康人血清中弓形虫抗体呈阳性者10份，特异性为93.5%。从噬菌体随机肽库中能筛选到弓形虫抗原模拟表位有诊断弓形虫病的潜在价值。     

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±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.     

Mapping regions containing binding residues within functional domains of Plasmodium vivax and Plasmodium knowlesi erythrocyte-binding proteins

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Whereas Plasmodium vivax and Plasmodium knowlesi use the Duffy blood group antigen, Plasmodium falciparum uses sialic acid residues of glycophorin A as receptors to invade human erythrocytes. P. knowlesi uses the Duffy antigen as well as other receptors to invade rhesus erythrocytes by multiple pathways. Parasite ligands that bind these receptors belong to a family of erythrocyte-binding proteins (EBP). The EBP family includes the P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi beta and gamma proteins, which bind alternate receptors on rhesus erythrocytes, and P. falciparum erythrocyte-binding antigen (EBA-175), which binds sialic acid residues of human glycophorin A. Binding domains of each EBP lie in a conserved N-terminal cysteine-rich region, region II, which contains around 330 amino acids with 12 to 14 conserved cysteines. Regions containing binding residues have now been mapped within P. vivax and P. knowlesi beta region II. Chimeric domains containing P. vivax region II sequences fused to P. knowlesi beta region II sequences were expressed on the surface of COS cells and tested for binding to erythrocytes. Binding residues of P. vivax region II lie in a 170-aa stretch between cysteines 4 and 7, and binding residues of P. knowlesi beta region II lie in a 53-aa stretch between cysteines 4 and 5. Mapping regions responsible for receptor recognition is an important step toward understanding the structural basis for the interaction of these parasite ligands with host receptors.     

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.     

恶性疟原FCC-1 HN株EBA-175和HRP-II基因片段在HeLa细胞中的表达及鉴定

目的　在 Ｈｅ Ｌａ 细胞中表达恶性疟原虫红细胞结合蛋白 Ｅ Ｂ Ａ－ １７５ 和富组氨酸蛋白 Ｈ Ｒ Ｐ－ Ｉ Ｉ, 进一步研究其生物学功能和免疫学特性。方法　将 Ｅ Ｂ Ａ－ １７５ 和 Ｈ Ｒ Ｐ－ Ｉ Ｉ 部分基因串接插入 Ｐ Ｃ Ｄ Ｎ Ａ３ 真核表达载体, 获得重组表达质粒 Ｐ Ｃ Ｄ Ｎ Ａ３ Ｅ Ｂ Ａ１７５ － Ｈ Ｒ Ｐ Ｉ Ｉ。采用磷酸钙－ Ｄ Ｎ Ａ 共沉淀法将重组质粒转染 Ｈｅ Ｌａ 细胞进行体外表达, 对表达产物进行 Ｓ Ｄ Ｓ－ Ｐ Ａ Ｇ Ｅ、 Ｄｏｔ－ Ｅ Ｌ Ｉ Ｓ Ａ 和 Ｗｅｓｔｅｒｎ － ｂｌｏｔ 鉴定。结果　表达产物占表达蛋白总量的１６４ ％ , 相对分子量与预设计的４８ｋ Ｄａ 基本相符。表达产物与鼠抗恶性疟原虫血清及构建的重组质粒免疫鼠血清产生特异免疫反应。结论　表达载体 Ｐ Ｃ Ｄ Ｎ Ａ３ 在 Ｈｅ Ｌａ 细胞内可表达出具有一定免疫学活性的恶性疟原虫重组抗原蛋白。     

结核分枝杆菌H37Rv结合肽的筛选研究

目的 应用噬菌体展示随机7肽文库,筛选MTB标准株H37Rv的结合肽,并初步鉴定其与MTB的结合能力.方法 以H37Rv灭活菌体为靶分子,应用噬菌体展示随机7肽文库进行筛选,并于第2～4轮的筛选中加入耻垢分枝杆菌灭活菌体进行反筛,经4轮生物淘选后,随机选取单噬菌体进行测序,间接酶联免疫吸附测定(ELISA)法鉴定阳性克隆.将亲和性最强的阳性单噬菌体所展示的短肽进行体外合成及荧光标记,观察其与16种分枝杆菌标准株及3种非分枝杆菌(铜绿假单胞菌、金黄色葡萄球菌和白色念珠菌)的结合活性.结果 通过4轮生物淘选,能与靶分子特异性结合的噬菌体得到明显富集.单噬菌体测序分析共获得5种共同序列.间接ELISA检测出5个单噬菌体均为阳性克隆,其中单噬菌体H8与H37Rv及耻垢分枝杆菌的亲和性均较强.荧光显微镜下观察到,荧光标记H8可与H37Rv结合,与包括耻垢分枝杆菌在内的其他15种分枝杆菌有一定亲和力,而与3种非分枝杆菌均不结合.结论 利用噬菌体展示随机肽库技术淘选可获得MTB标准株H37 Rv 的结合肽,与MTB的结合活性及特异度较高,这为以标记肽为基础探索新的MTB体外检测方法提供了思路.

Abstract：

Objective To screen the Mycobacterium tuberculosis H37Rv binding peptide using phagedisplayed random peptide libraries, and to analyze the binding capacity of the peptide with Mycobacterium tuberculosis. Methods lnactive Mycobacterium tuberculosis H37 Rv was used for screening of the binding peptide from the Ph. D. -7 peptide library, and Mycobacterium smegmatis was used for reverse screening during the 2nd to 4th rounds of screening. After 4 rounds of screening, single phages were randomly selected for DNA sequencing. The selected clones were tested by indirect enzyme linked immunosorbent assay (ELISA). The peptide of positive clone, which showed the highest affinity, was synthesized in vitro with fluorescent markers. The specific combination of the peptide with 16 mycobacterium standard strains and 3 other microbes ( Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans) were observed by fluorescence microscopy. Results After 4 rounds of biopanning, remarkable enrichment of the phages that specifically bind with target molecules were observed. Single phages were randomly selected for sequencing analysis and 5 sequences were obtained. Five phages with different sequences were detected using indirect ELISA and all of them were found to be positive clones. Phage 8 showed the highest affinity with target molecule. The peptide of phage H8 was synthesized in vitro with fluorescent markers, and it was confirmed that the peptide could bind with H37Rv and other 15 mycobacterium including Mycobacterium smegmatis, but not with 3 other microbes. Conclusions By using phage-displayed random peptide libraries, we obtained the binding peptide of H37 Rv. It was shown that the peptide could bind with Mycobacterium tuberculosis specifically, which provided a new way for the detection of Mycobacterium tuberculosis in vitro.     

Evidence for erythrocyte-binding antigen 175 as a component of a ligand-blocking blood-stage malaria vaccine

The ligands that pathogens use to invade their target cells have often proven to be good targets for vaccine development. However, Plasmodium falciparum has redundant ligands that mediate invasion of erythrocytes. The first requirement for the development of a successful ligand-blocking malaria vaccine is the demonstration that antibodies induced to each ligand can block the erythrocyte invasion of parasites with polymorphic sequences. Because of P. falciparum's redundancy in erythrocyte invasion, each ligand needs to be studied under artificial conditions in which parasite invasion is restricted in its use of alternative pathways. Here we investigate the role of erythrocyte-binding antigen 175 (EBA-175), a parasite ligand that binds to sialic acid on glycophorin A, in the invasion of erythrocytes by 10 P. falciparum clones under conditions in which invasion is partially limited to the EBA-175-glycophorin A pathway, using chymotrypsin-treated erythrocytes. We show that the ability to invade erythrocytes for both sialic acid-independent and sialic acid-dependent pathways requires the EBA-175-glycophorin A pathway for erythrocyte invasion. Importantly, antibodies against region II of EBA-175 from the 3D7 clone blocked invasion of chymotrypsin-treated erythrocytes by >50% by all parasite clones studied, including those with multiple different mutations described in the literature. The one exception was FCR3, which had a similar sequence to 3D7 but only 30% inhibition of invasion of chymotrypsin-treated erythrocytes, indicating alternative pathways for invasion of chymotrypsin-treated erythrocytes. Our findings suggest that antibodies to region II of EBA-175, as one component of a ligand-blocking malaria vaccine, are largely unaffected by polymorphism in EBA-175.