Isolation of human scFv antibody fragments against ABO blood group antigens from a phage display library

BACKGROUND AND OBJECTIVES: Phage display has proven very useful for the isolation of antibodies against a number of antigens. We used this technology to isolate scFv antibody fragments against A and B red blood cell antigens. MATERIALS AND METHODS: Phages from a phage display library were selected using unmodified red blood cells as a source of antigen. Bound phages were absorbed onto cells lacking the antigen of interest and used to infect Escherichia coli cells. Phages were rescued and assayed for specificity by enzyme-linked immunosorbent assay (ELISA). RESULTS: After several rounds of panning and subtraction on red blood cells, one anti-A and one anti-B human scFv antibody fragments were isolated. CONCLUSION: Isolation of anti-A and anti-B scFv antibody fragments on whole cells is an alternative method of obtaining antibodies against native cell-surface antigens.     

结肠癌患者淋巴结构建抗结肠癌噬菌体Fab抗体库

目的　构建 2个结肠癌患者自然致敏淋巴结抗体Fab段噬菌体呈现库。方法　取 2个结肠癌患者转移淋巴结 ,提取淋巴结总RNA ,逆转录PCR扩增重链Fd和κ轻链cDNA。依次将PCR产物插入载体 pComb3的相应部位 ,噬菌体VCSM13辅助感染。以点印迹检测噬菌体表面Fab的表达。 结果　所选 2种Ig亚类的重链Fd片段、2种κ轻链cDNA得到扩增。Fd片段和κ轻链均插入 pComb3的重组率为 40 %,Fab噬菌体表达库容量达 1.48× 10 6。噬菌体悬液的点印迹免疫染色显示有Fab表达。结论　构建了 2个结肠癌患者自然致敏淋巴结抗体Fab段噬菌体呈现库 ,为筛选结肠癌相关抗体奠定了基础。     

乙型肝炎病毒表面抗原人源单链可变区抗体基因的克隆与鉴定

目的 筛选,鉴定抗乙型肝炎病毒（ＨＢＶ）表面抗原（ＨＢｓＡｇ）蛋白的人源单链可变区抗体（ＳｃＦｖ）的编码基因,为细胞内表达小分子单链抗体的研究及抗ＨＢＶ的基因治疗研究奠定基础。方法 采用噬菌体表面展示技术,以氯化铯超速离心法纯化的ＨＢｓＡｇ蛋白为固相抗原,从噬菌体单链可变区抗体半合成库中经过５轮“吸附－洗脱－扩增”淘洗过程,获得抗原结合活性较强的ＨＢｓＡｇ人源单链可变区抗体阳性克隆,并对其进行免疫     

Study of a humanized inhibitory anti-platelet glycoprotein VI phage antibody from a phage antibody library

Objective: The aims of the study were to study the effect of anti-platelet glycoprotein (GP) VI auto-antibodies on platelet aggregation and use phage surface display technology to produce anti-platelet GPVI phage antibody fragment, which may be developed to inhibit platelet aggregation in the treatment of cardiovascular disease.

Methods: Plasma samples from patients with immune thrombocytopenia (ITP) were screened by monoclonal antibody immobilization of the platelet antigen assay and the platelet aggregation test for anti-platelet GPVI auto-antibody with an inhibitory effect. The humanized anti-platelet GPVI phage antibody was produced by phage surface display technology. The function of the phage antibody fragment against platelet aggregation was examined by the platelet aggregation test.

Results: Of 726 ITP patients, 2 (0.27%) patients’ plasma significantly inhibited platelet aggregation induced by collagen-1. After five rounds of selection, enrichment, and purification, a soluble phage antibody fragment was produced, which can inhibit platelet aggregation induced by collagen-1. The results demonstrate that only a few of the screened anti-platelet GPVI auto-antibodies showed an inhibitory effect on platelet aggregation.

Discussion: A completely humanized anti-GPVI soluble phage antibody can be produced by phage surface display technology. The antibody was able to specifically block collagen-induced platelet aggregation without influencing the aggregation responses to other agonists.

Conclusions: Results of the present study suggest that very few anti-platelet GPVI auto-antibodies inhibit the aggregation function of platelet. The humanized anti-platelet GPVI produced by phage surface display technology is promising to be used to inhibit platelet aggregation in the treatment of cardiovascular disease.     

人源性大肠癌Fab段噬菌体抗体库的免疫组化鉴定

目的：鉴定基因工程抗体库技术构建的人源性大肠癌自然致敏噬菌体Fab段抗体库与人大肠癌组织及体外培养的大肠癌细胞的特异性结合活性。方法：通过免疫组化的方法检测抗体库对人大肠癌标本及体外培养的大肠癌lovo细胞的结合活性，并取绒毛状腺瘤、胃癌、食管癌及正常肠粘膜组织和体外培养的胃癌、肝癌细胞进行对照研究。结果：30例大肠癌有22例发生了阳性反应；肠息肉、胃癌、食管癌、正常肠粘膜各取20例，发生阳性反应的分别为13例、5例、4例和1例，体外培养的胃癌、肝癌细胞未发生阳性反应。结论：大肠癌噬菌体抗体库能较特异性地与人大肠癌组织及体外培养的大肠癌细胞结合。     

人源性抗结核分枝杆菌噬菌体展示单链抗体文库的构建

目的 构建人源抗结核分枝杆菌噬菌体展示单链抗体文库,为结核特异性单链抗体的筛选奠定基础. 方法 从抗结核分枝杆菌抗体阳性患者淋巴细胞中提取总RNA,逆转录成cDNA,PCR扩增获得抗体的重、轻链可变区基因,然后拼接装配成单链抗体基因,重组于噬菌粒载体pCANTAB5S,转化大肠埃希菌E.coli TG1,以辅助噬菌体拯救后,构建人源性抗结核分枝杆菌噬菌体展示单链抗体库. 结果 成功构建人源性抗结核分枝杆菌噬菌体展示单链抗体文库,库容量达107. 结论 以结核患者淋巴细胞免疫球蛋白可变区基因片段和噬菌粒展示载体pCANTAB5S为基础,成功构建人源性抗结核分枝杆菌噬菌体展示单链抗体库,并达到建库标准,可进一步从中筛选获得结核特异性单链抗体.     

丙型肝炎病毒非结构蛋白3人源单链抗体的筛选与鉴定

目的 筛选、鉴定抗丙型肝炎病毒（ＨＣＶ）非结构蛋白（ＮＳ）３的人单链抗体（ＳｃＦｖ）,以解决鼠单抗蝗免疫原性问题,为ＨＣＶ的基因治疗研究开辟新途径。方法 采用噬菌体表面展示技术,以重组的ＨＣＶＮＳ３为包被抗原,从噬菌体单链楞变区抗体库中经过５轮“吸附－洗脱－扩增”筛选过程,获得抗原结合活性较强的ＨＣＶＮＳ３人单链抗体（ＳｃＦｖ片段）阳性克隆,并对其进行免疫体及序列测定。结果 筛选出来的ＳｃＦｖ片段     

应用噬菌体展示技术筛选原发性肝癌血清结合蛋白

目的:通过筛选原发性肝癌血清蛋白结合蛋白,探究肝癌的分子发病机制.方法:应用噬菌体展示技术,以肝癌患者的血清作为固相筛选分子,对T7 Select噬菌体人肝细胞cDNA文库进行5轮"吸附-洗脱-扩增"的筛选过程,经噬斑的聚合酶链式反应(PCR)扩增后,对阳性PCR产物直接进行序列测定和同源性分析.结果:噬菌体经富集后,随机挑取48个噬斑进行PCR扩增,得到5种大小不同的PCR片断,序列测定后经序列同源性分析,结果发现与肝癌患者血清蛋白结合的蛋白有以下5种:人核仁螺旋体磷酸化蛋白NOLC1,人高度迁移基核小体结合域1(HMGN1),人依赖ATP的DNA连接酶1(LIG1),人小EDRK-丰富因子2(SERF2)和A-kinase anchor protein 9 isoform.结论:用噬菌体人肝cDNA文库筛选得到了肝癌血清蛋白结合蛋白,为进一步阐明肝癌的发生及发病机制奠定了基础.     

人源性大肠癌Fab段噬菌体抗体库的筛选与鉴定

目的　通过大肠癌组织及体外培养的大肠癌细胞抗原对人源性大肠癌Fab段噬菌体抗体原始库的反复筛选 ,鉴定抗体库的特性并检测其与人大肠癌相关抗原的结合活性。方法　PCR鉴定阳性重组菌xL1 B1ue中人大肠癌相关抗体Fab段的插入率 ,构建人大肠癌Fab段原始库 ,提取 3例用于构建原始抗体库的致敏大肠癌组织抗原 ,13例非致敏大肠癌组织抗原及 3种体外培养的大肠癌细胞株Lovo、HT 2 9、LS 174T的抗原各自等体积混合 ,利用 3组混合抗原分别对原始抗体库进行 3轮筛选 ,将筛选后的三个三级库等体积混和后通过ELISA法鉴定其与人大肠癌组织及体外培养的大肠癌细胞的结合活性 ,取胃癌、食管癌及正常肠粘膜组织和体外培养的胃癌、肝癌细胞进行对照研究。结果　N片段及κ链的插入率分别为 40 %和 70 % ,Fd片段及κ链均插入质粒Pcomb3的重组率为 2 8% ,Fab段基因库库容为 2 .1× 10 6,3组大肠癌混合抗原分别对原始抗体库进行 3轮筛选后 ,抗体库均得到了不同程度的富集 ,ELISA显示富集后的Fab段抗体库对大肠癌组织及体外培养的大肠癌细胞均有较特异性的结合活性。结论　利用噬菌体抗体库技术筛选出了相关抗大肠癌Fab段抗体 ,且筛选后的抗体片段与人大肠癌相关抗原有较特异性的结合活性     

人源性抗-HBc单链噬菌体抗体库的构建

目的 构建人源性单链噬菌体抗体库，为筛选人源性抗—HBc单链抗体奠定基础。方法 利用逆转录—聚合酶链反应(RT—PCR)和噬菌体表面展示技术，直接从乙肝病毒核心抗体(抗—HBc)阳性患者淋巴细胞中提取总RNA，逆转录成cDNA；合成全套人抗体可变区引物扩增抗体可变区基因，并将重、轻链可变区基因进行拼接装配成单链抗体(ScFv)基因，重组于噬菌粒载体叶pHEN1，转化抑制型大肠埃希菌E.coliTG1，以辅助噬菌体援救后，构建成人源性单链噬菌体库。结果 成功地构建了人源性抗—HBc单链噬菌体库，库容量达10^6。结论 利用RT—PCR和噬菌体表面展示技术可以成功构建人源性单链抗体库，并达到建库标准，可进一步从中筛选人源性单链抗体。     

Screening for PreS specific binding ligands with a phage displayed peptides library

ABM: To construct a random peptide phage display library and search for peptides that specifically bind to the PreS region of hepatitis B virus (HBV). METHODS: A phage display vector, pFuse8, based on the gene 8 product (pⅧ) of M13 phage was made and used to construct a random peptide library. Ecoli derived thioredoxin-PreS was purified with Thio-bond beads, and exploited as the bait protein for library screening. Five rounds of bio-panning were performed. The PreS-binding specificities of enriched phages were characterized with phage ELISA assay. RESULTS: A phage display vector was successfully constructed as demonstrated to present a pⅧ fused HBV PreSl epitope on the phage surface with a high efficiency. A cysteine confined random peptide library was constructed containing independent clones exceeding 5±108 clone forming unit (CFU). A pool of phages showing a PreS-binding specificity was obtained after the screening against thio-PreS with an enrichment of approximately 400 times. Five phages with high PreS-binding specificities were selected and characterized. Sequences of the peptides displayed on these phages were determined. CONCLUSION: A phage library has been constructed, with random peptides displaying as pVIII-fusion proteins. Specific PreS-binding peptides have been obtained, which may be useful for developing antivirals against HBV infection.     

用随机肽库筛选胃癌相关抗原MGb2—Ag模拟肽表位

的　运用肽库筛选技术寻找胃癌单克隆抗体ＭＧｂ２ 所识别的胃癌相关抗原的短肽模拟表位。方法　以胃癌单克隆抗体ＭＧｂ２ 作为筛选配基，对呈现于噬菌体衣壳蛋白ｐⅧ上的两个九肽库进行了亲和富集和免疫筛选；阳性克隆用荧光标记和硝酸纤维素膜斑点印迹证实结合活性；随机抽取部分阳性克隆进行了ＤＮＡ 测序分析。结果　经ＤＮＡ 测序分析推导出相应的氨基酸序列并加以比较分析，得到具有保守性的表位信息。结论　具有相对保守性的ＶＣＸＸＤＧＶ 可能为胃癌单克隆抗体ＭＧｂ２所识别的表位基序。     

微小隐孢子虫T7噬菌体展示文库的构建

目的为筛选微小隐孢子虫保护性抗原基因,构建了微小隐孢子虫T7噬菌体展示文库。方法用Trizol试剂提取微小隐孢子虫总RNA,分离纯化mRNA,经反转录合成双链cDNA。在双链cDNA末端加上定向EcoRⅠ/HindⅢ接头,用EcoRⅠ和HindⅢ消化接头,使形成两端分别带有EcoRⅠ和HindⅢ粘性末端的双链cDNA。经Mini Column纯化,收集400bp以上的双链cDNA片段,将其连接于带有EcoRⅠ和HindⅢ末端的T7Select 10-3b载体,经体外包装后,以BLT5403为受体菌构建T7噬菌体展示文库。结果经测定,库容量为1.2×107pfu/mL,重组率为96.7%,扩增后文库滴度为2.4×1010pfu/mL。对随机挑取的100个噬菌斑进行PCR鉴定,92%的插入片段大于400bp。结论成功构建了微小隐孢子虫T7噬菌体展示文库。     

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。     

噬菌体抗体库的构建及人源抗内毒素类脂A抗体的筛选

目的寻找一种治疗内毒素血症及其并发症较为有效的途径.方法从感染革兰阴性杆菌J7患者体内含有内毒素类脂A抗体的淋巴细胞中提取mRNA,经反转录再从IgG重链Fd两端及轻链通用引物分别扩增Fab基因片段,依次插入到噬菌体载体pCOMR3中,电穿孔转入大肠杆菌XL-blue,经辅助病毒VCSM13感染,重组噬菌体溶源裂解.结果Fab抗体表达于噬菌体CPⅢ的N端,噬菌体抗体库的容量为4.8×106,筛选出抗内毒素类脂A抗体,经LPS蛋白对噬菌体抗体库进行3轮淘选,使抗内毒素类脂A的特异性抗体得到100倍的富集.结论通过直接和竞争ELISA实验筛选出3株结合活性好的特异性抗体,为下一步应用研究奠定了基础.     

人源性大肠癌自然致敏噬菌体抗体Fab呈现库的构建与筛选

目的构建大肠癌病人自然致敏淋巴结抗体 Fab 段噬菌体呈现库,初步筛选相关抗大肠癌抗体。方法取大肠癌病人转移淋巴结,提取淋巴结总 RNA,逆转录 PCR 扩增重链 Fd 和 k 轻链 cDNA。依次将 PCR 产物插入载体 pComb3的相应部位,构建人源性大肠癌噬菌体 Fab 基因库,并应用噬菌体表面呈现技术对该抗体库进行淘选及鉴定。结果所选2种 Ig 亚类的重链 Fd 片段、2种 k 轻链 cDNA 得到扩增。Fd 片段和 k 轻链均插入 pComb3的重组率为40%,Fab 噬菌体表达库容达1.48×10~6。经3轮淘选,抗体库得到约120倍的富集。3轮抗体库的点印记免疫染色均显示有 Fab 表达:酶联免疫吸附实验显示其与大肠癌抗原有结合活性。结论成功构建了大肠癌病人自然致敏淋巴结抗体 Fab 段噬菌体呈现库,利用噬菌体抗体库技术,可筛选相关抗大肠癌抗体。     

Identification of targeting peptides for ischemic myocardium by in vivo phage display

Therapies selectively targeting ischemic myocardium could be applied by intravenous injection. Here, we report an approach for ischemic tissue-selective targeting based on in vivo screening of random peptide sequences using phage display. We performed in vivo biopanning using a phage library in a rat model of ischemia-reperfusion and identified three peptide motifs, CSTSMLKAC, CKPGTSSYC, and CPDRSVNNC, that exhibited preferential binding to ischemic heart tissue compared to normal heart as well as other control organs. The CSTSMLKAC sequence was capable of mediating selective homing of phage to ischemic heart tissue. The CSTSMLKAC peptide was then made as a fusion protein with Sumo-mCherry and injected intravenously in a mouse model of myocardial ischemia-reperfusion injury; subsequently, bio-distribution of Sumo-mCherry-CSTSMLKAC was measured with quantitative ELISA. The targeting peptide led to a significant increase in homing to ischemic left ventricle compared to tissues from non-ischemic left ventricle, the right ventricle, lung, liver, spleen, skeletal muscle, and brain (all p < 0.001). These results indicate that the peptide sequence CSTSMLKAC represents a novel molecular tool that may be useful in targeting ischemic tissue and delivering bioengineered proteins into the injured myocardium by systemic intravenous administration.     

东方田鼠肺脏T7噬菌体展示cDNA文库的构建

目的构建东方田鼠肺脏T7噬菌体展示cDNA文库，为筛选东方田鼠抗血吸虫感染抗性相关基因奠定基础。方法  用TRIzol试剂提取东方田鼠肺脏总RNA，分离纯化mRNA，经反转录合成双链cDNA。在双链cDNA末端加上定向EcoR I／HindⅢ接头并用EcoR I和HindⅢ消化，使其两端分别带EcoR I和HindⅢ黏性末端。用Mini Column纯化，收集300 bp以上的双链cDNA片段，再连接于带有EcoR I和Hind Ⅲ末端的T7 Select 10-3b载体，经体外包装后，以BLT5403为受体菌构建T7噬菌体展示cDNA文库。结果  经测定，库容量为1.5×106 pfu，扩增后文库滴度为1.1×1012pfu/ml。对从原始文库中随机挑取的100个噬菌斑进行PCR签定，重组率为91%，阳性克隆片段长度分布在200-1 500 bp，其中有90%的插入片段>300 bp。结论成功构建了东方田鼠肺脏T7噬菌体展示cDNA文库。     

Cross-clade HIV-1 neutralization by an antibody fragment from a lupus phage display library

A single-chain fragment containing antibody V domains (scFv) isolated from a lupus antibody library displayed the ability to bind gp120 and the conserved gp120 determinant composed of residues 421-436. The scFv neutralized R5 and X4-dependent HIV-1 strains from clades B, C, and D. The lupus repertoire may be useful as a source of neutralizing antibodies to HIV.     

Peptides selected from a phage display library with an HIV-neutralizing antibody elicit antibodies to HIV gp120 in rabbits, but not to the same epitope

Monoclonal antibodies specific for the conserved CD4 binding site region of the HIV envelope protein gp120 were used to select phage from two different random peptide display libraries. Synthetic peptides were made with sequences corresponding to those displayed on the selected phage, and peptide-protein fusions were expressed that contained the selected phage-displayed peptide sequence and either the N-terminal domain of the phage pIII protein or the small heat shock protein of Methanococcus jannaschii or both. For monoclonal antibody 5145A, these constructs containing the selected peptide sequences were all capable of specifically inhibiting the binding of 5145A to HIV-1 gp120. Rabbits immunized with peptide-protein fusions produced antisera that bound to recombinant HIV-1 gp120, but did not bind to HIV-infected cells nor neutralize HIV. The antisera also did not compete with CD4 or antibodies to the CD4 binding site for binding to gp120.