人噬菌体抗体库的构建和甲肝抗体的筛选

目的　构建人噬菌体抗体组合文库,筛选人单克隆抗体。方法　用RTPCR扩增人全套抗体基因片段,克隆于pComb3载体,电转化E.coli形成噬菌体抗体库;以固相化抗原淘筛抗体库,ELISA鉴定噬菌体抗体。结果　17对免疫球蛋白引物全部能够扩增出目的片段;经数次电转化构建了库容为693×107的抗体库,滴度为8×1014PFU/ml,Fab基因重组率为40%。以单抗捕获的甲肝抗原淘洗3轮,出现特异性富集;阳性克隆经直接ELISA和竞争抑制性ELISA实验证实具有良好的抗甲肝抗原特异性,无交叉反应性。结论　成功构建了抗体组合文库,从中获得抗甲肝抗原的特异性人抗体。     

全套抗体库

基因工程抗体被称为第三代抗体，其中全套抗体库（亦称为噬菌体抗体库）又较另两种某因工程抗体（嵌合抗体、改形抗体）优越，可实现单抗的人源化、小型化和多功能化，而且理论上若构建一个足够大容量的全套抗体库，便可从中筛选抗任何抗原的单克隆抗体。从而克服了杂交瘤单抗用于体内的局限性，将来可能代替杂交瘤单抗技术并可用于体内诊断和治疗人类疾病，本义就抗体库构建流程及其进展作一综述。     

噬菌体抗体库技术

噬菌体抗体库(surface display phage antibody library)技术用PCR扩增出抗体的全套可变区基因,通过噬菌体表面表达技术(surface display),把Fab段或单链抗体(single chain Fv,ScFv)表达在噬菌体表面,经过“吸附-洗脱-扩增”过程筛选并富集特异性抗体。这一技术将表型和基因型联系在一起,使识别抗原的能力和进行再扩增的能力结合在一起,是一极为高效的表达、筛 选体系,使单抗的制备根本改观,在生物技术领域极具潜能。     

噬菌体抗体库技术的构建及应用研究进展

噬菌体抗体库（phage antibody library）技术是由噬菌体展示技术发展而来的一项新型抗体制备技术。噬菌体抗体库技术的基础,噬菌体展示技术,发展自上世纪80年代中期。1985年,美国密苏里大学的Smith[1]首次证实丝状噬菌体fd基因组能通过基因工程的手段加以改造。     

黑素瘤患者噬菌体抗体库的构建与筛选

目的 :构建噬菌体抗体库并筛选抗黑素瘤细胞的人抗体。方法 :从 1例长期存活的黑素瘤患者的外周血中分离单个核细胞 (PBMC) ,提取总RNA ,并逆转录为cDNA。用PCR扩增多样性κ链基因和重链Fd段基因 ,分别构建κ链库和重链库 ,组合为Fab段噬菌体抗体库 ,用两种黑素瘤细胞系对抗体库进行筛选和鉴定。结果 :所构建的噬菌体抗体库的库容量达 1.2× 10 8,用两种黑素瘤细胞系进行亲和吸附筛选 ,获得针对其中 1种黑素瘤细胞系的特异性抗体。结论 :采用噬菌体抗体库技术 ,成功地从 1例长期存活的黑素瘤患者体内克隆到特异性抗黑素瘤细胞的抗体 ,为进一步的研究和应用奠定了基础     

噬菌体抗体库技术研究进展

应用ＰＣＲ技术从生物个体内扩增出整套抗体轻、重链Ｖ区基因，克隆λ噬菌体或噬粒表达载体系统，构建抗体表达文库，通过抗原亲和吸附→洗脱→繁殖（Ｐａｎｎｉｎｇ）从中筛选富集多种目的克隆，制备相应单克隆抗体。本文就此项技术研究新进展作一简要介绍。     

SLE患者自身抗体单链噬菌体抗体库构建及抗体活性检测

本文用ＳＬＥ病人外周血淋巴细胞抽提ＲＮＡ,采用ＲＴ ＰＣＲ反应以人的特异性ＨｕＶＨＢＡＣＫ、ＨｕＪＨＦＯＲ和ＨｕＶＬＢＡＣＫ、ＨｕＶＬＦＯＲ引物扩增人抗体的ＶＨ和ＶＬ基因片段,并将其与ｌｉｎｋｅｒ拼接成ＳｃＦｖ片段克隆于大肠杆菌,使其与噬菌体基因Ⅲ的产物以融合蛋白的形式在噬菌体表面表达,从而构建了ＳＬＥ病人的单链噬菌体抗体库,ＥＬＩＳＡ检测证明ＳｃＦｖ基因产物具有抗体活性。ＳＬＥ噬菌体抗体库的构建为ＳＬＥ发病机理、诊断和治疗研究打下了良好的基础。     

噬菌体抗体库技术研究进展

应用ＰＣＲ技术从生物个体内扩增出整套抗体轻、重链Ｖ区基因，克隆λ噬菌体或噬粒表达载体系统，构建抗体表达文库，通过抗原亲和吸附→洗脱→繁殖（Ｐａｎｎｉｎｇ）从中筛选富集多种目的克隆，制备相应单克隆抗体。本文就此项技术研究新进展作一简要介绍。     

大容量噬菌体抗体库的构建及鉴定

目的　构建大容量噬菌体单链抗体库 ,从中筛选人源单链抗体 (ScFv)。方法　从正常成人外周血和新生儿脐血分离淋巴细胞 ,用RT PCR扩增轻链可变区基因 (VL)和重链可变区基因(VH) ,通过重叠PCR法将VH 和VL 拼接形成ScFv基因 ,并克隆入噬菌体表达载体PDF ,得到ScFv初级噬菌体抗体库。以高MOI超感染cre 菌株BS136 5 ,通过loxp cre定位重组系统 ,介导轻重链的组合配对 ,得到大容量抗体库 ,用多种抗原对抗体库进行生物淘筛 ,鉴定抗体库的性能。结果　获得了 6×10 10 的大容量单链噬菌体抗体库。分别用卵清蛋白、胃蛋白酶、铁蛋白、人角蛋白、人TNF α、地高辛等6种抗原进行筛选 ,均得到多样性的特异性噬菌体抗体。结论　经loxp cre定位重组系统在单细胞内重组成功地构建了大容量单链噬菌体抗体库 ,初步尝试对 6种抗原进行筛选均获成功 ,提示该抗体库可用于制备具有应用前景的人源抗体     

噬菌体抗体库技术的发展及应用

近年来，治疗性抗体的临床应用对抗体的研究和改造提出了更高的要求。随着分子生物学的不断发展，抗体也由多克隆抗体、单克隆抗体发展到基因工程抗体阶段。噬菌体抗体库技术的出现，为抗体的发展提供了广阔的前景。     

TM-TNF-α模拟肽P18和S-TNF模拟肽P12的体外生物学活性的鉴定

目的：获得具有生物学活性的跨膜型TNF-α（TM-TNF-α）模拟肽。方法：以噬菌体扩增及PEG／NaCI沉淀法，大量制备所需的噬菌体展示肽。采用细胞毒实验、RT-PCR、Western blot印迹、凋亡检测实验分别检测TM-TNF-α模拟肽的杀瘤效应、对SODDmRNA表达水平、NF-κB核转位及致死亡方式的影响。结果：噬菌体展示肽P12和P18分别能模拟sTNF-α和TM-TNF-α的生物学活性。结论：获得能模拟TM-TNF-α的生物学活性的展示肽，为TM-TNF-α用于临床抗瘤治疗提供新线索。     

HIV-1 gp41 C-螺旋模拟位的筛选和鉴定

目的：筛选可作为小分子先导化合物的HIV-1 gp41 C螺旋模拟位，为开发抗HIV-1早期感染的小分子药物奠定基础。方法：以源于HIV-1 gp41端的肽N36为靶，对噬菌体环七肽库进行亲和筛选。利用ELISA鉴定噬菌体阳性克隆并对其进行DNA序列分析。结果：3轮筛选后随机挑取26个噬菌体克隆，ELISA鉴定表明有16个克隆可与N36结合，将其中10个克隆DNA测序并推导氨基酸序列，每一克隆至少含有2个疏水氨基酸，其中9个克隆均有WW，7个克隆有WWH保守序列。挑选阳性噬菌体克隆No.8进一步鉴定，游离N36肽阻断No.8克隆与固相化N36结合，C34肽竞争抑制N36肽与No.8克隆结合(IC50为12.5μg/ml)。结论：表达WW保守序列的肽模拟HIV-1 gp41C螺旋与N螺旋结合，该结果为设计针对HIV介导融膜的抑制剂提供技术支持。     

噬菌体展示TM-TNF-α模拟肽体内杀瘤效应的研究

目的　研究噬菌体展示TM -TNF- α模拟肽的体内杀瘤效应及机制。方法　大量制备所需的噬菌体展示肽,比较不同展示肽对小鼠的肝癌细胞H2 2的体外杀瘤效应,挑取杀瘤效应最好的展示肽并选择其合适的滴度进行体内实验。小鼠接种H2 2细胞3d后,于肿瘤接种部位皮下注射噬菌体展示肽,观察肿瘤的生长情况。结果　噬菌体展示TM- TNF -α模拟肽在体内能明显抑制肿瘤的生长(P <0 .0 1)。且抑瘤率与展示肽之间呈剂量依赖关系。HE染色发现展示肽治疗组肿瘤组织中有大量淋巴细胞浸润,而sTNF- α治疗组除淋巴细胞浸润还可见中性粒细胞、浆细胞浸润。结论　直接注射噬菌体展示TM -TNF -α模拟肽可在体内有效杀瘤,其杀瘤机制可能不同于分泌型TNF -α。     

The skewed TCR-BV repertoire displayed at the maternal-fetal interface of women with unexplained pregnancy loss

PROBLEM: The study was undertaken to investigate T-cell receptor (TCR) variable beta (BV)-chain usage at maternal-fetal interface and explore the relationship between the skewed TCR-BV usage and unexplained pregnancy losses. METHOD OF STUDY: A total of 57 patients with unexplained pregnancy loss including 39 cases with unexplained spontaneous abortion and 18 cases with unexplained recurrent spontaneous abortion (RSA) were chosen in Renji Hospital, Shanghai Second Medical University matched with 41 women with normal pregnancies in first trimester between September 2002 and November 2003. A high-resolution spectratyping analysis of complementarity-determining region 3 (CDR3) was used to detect and compare the degree and pattern of TCR-BV repertoire usage at the maternal-fetal interface between patients with pregnancy loss and normal controls. RESULTS: There were two comparisons of TCR usage performed between patients and controls, which included the degree (mean value of every TCR-BVn expression) and the pattern (skewed TCR-BVn frequency). The skewed TCR-BVn is defined as an absolute BVn usage of > 5% calculated from the formula or a double increased BVn usage compared with the mean value of normal BVn. According to the degree of TCR-BV usage, BV2 (P = 0.046), BV10 (P = 0.016), and BV11 (P = 0.030) in spontaneous abortion group and BV19 (P = 0.038) in RSA group showed higher usage, while BV5.2 (P = 0.006 and P = 0.046) in both abortion groups showed significantly lower usage when compared with normal controls. About the pattern of skewed TCR-BV distribution, we found that TCR-BV2, -3, -6, and -7 were the four most common BV families in deciduas of patients with both types of abortion and normal controls. Women with spontaneous abortion demonstrated higher frequency of BV10 (P = 0.035) and lower frequencies of BV4 (P = 0.002) and BV5.2 (P = 0.003) in comparison with controls. In RSA, higher frequencies of BV15 (P = 0.018), BV19 (P = 0.049), and BV20 (P = 0.018), in the mean time, lower frequencies of BV4 (P = 0.026) and BV7 (P = 0.018) distributions were verified. CONCLUSIONS: Our results suggested that a significant skewed TCR-BV repertoire occurred at the maternal-fetal interface with patients undergoing abortion. The specific skewed usages of TCR-BV might be associated with the susceptibility to unexplained pregnancy loss.     

The aminopeptidase ERAAP shapes the peptide repertoire displayed by major histocompatibility complex class I molecules

Major histocompatibility complex (MHC) class I molecules present thousands of peptides to allow CD8(+) T cells to detect abnormal intracellular proteins. The antigen-processing pathway for generating peptides begins in the cytoplasm, and the MHC molecules are loaded in the endoplasmic reticulum. However, the nature of peptide pool in the endoplasmic reticulum and the proteolytic events that occur in this compartment are unclear. We addressed these issues by generating mice lacking the endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP). We found that loss of ERAAP disrupted the generation of naturally processed peptides in the endoplasmic reticulum, decreased the stability of peptide-MHC class I complexes and diminished CD8(+) T cell responses. Thus, trimming of antigenic peptides by ERAAP in the endoplasmic reticulum is essential for the generation of the normal repertoire of processed peptides.     

噬菌体突变库技术在蛋白质改造中的应用

噬菌体展示突变蛋白库是以噬菌粒为载体,对蛋白质序列中的位点(特异或随机)在DNA水平上进行突变而构建的次级文库。突变蛋白库构建策略包括:寡核苷酸介导的直接位点突变、盒式突变、DNA shuffling以及错倾PCR。蛋白突变库主要应用于提高蛋白亲和力、延长半衰期、确定蛋白关键残基。     

抗恶性疟SERA噬菌体抗体库的构建及单链抗体的筛选与鉴定

目的 研制用于疟疾简便,快速诊断试剂盒的试剂。方法 利用噬菌体抗体库技术,构建抗恶性疟原虫丝氨酸重复抗原（SERA）的噬菌体抗体库。经3轮“吸附－洗脱－扩增”的富集反应后,从中筛选抗SERA的阳性克隆株,并进行可溶性诱导表达,最后用ELISA和Western blot等进行鉴定。结果 成功地构建了中等库容的噬菌体抗体库,并从中筛选到9株抗SERA的阳性克隆株,表达的单链抗体的相对分子质量大小约为31kDa,能与SE47‘抗原起特异性结合反应。结论 成功地构建了抗SE47‘的噬菌体抗体库,从中筛选制备的单链抗体为研制疟疾快速诊断试剂盒奠定了基础。     

肾综合征出血热患者血清汉坦病毒基因的PCR-ELISA方法建立

目的：建立检测肾综合征出血热（HFRS）患者血清标本中HV基因的PCR－ELISA方法。方法：设计并合成互补于HV S基因片段的标记引物。对108例HFRS患者血清进行巢式RT－PCR扩增，并用酶免方法分析扩增产物。结果：108例患者不同病日采集的血清标本的巢式RT－PCR平均检出率仅为62．0％，而PCR－ELSA平均检出率为81．5％，结论：PCR－ELISA用于早期HFRS患者的HV基因检测是一种理想的检测方法。     

重组人G－CSF免疫小鼠全套单链抗体噬菌体表面展示文库的构建

利用全套噬菌体抗体表面展示技术,绕过杂交瘤技术,从重组人G－CSF免疫的小鼠脾淋巴细胞中提取总RNA,反转录成cDNA后,用抗体可变区PCR混合引物进行全套抗体重、轻链可变区（VH和VL）基因的扩增。经重叠延伸反应,在体外随机装配成单链抗体（ScFv）。将其克隆至噬菌粒载体pCANTAB5E中,电转化含SupE的     

Scavenger receptor block as strategy for the identification of bone marrow homing phages by panning in vivo random peptide phage displayed libraries

Surface molecules exclusively expressed by cells of the bone marrow (BM) are candidate targets for delivering therapeutic agents to this tissue. To identify ligands specific for the BM, we performed a series of pannings in vivo with random peptide phage displayed libraries (RPPDL). We could show that phages bind to bone marrow endothelium (BME) independently of the peptide insert, suggesting that the BM, similarly to spleen and liver, is part of the reticulo-endothelial system (RES). Furthermore, this strong "natural" affinity to the BME was abrogated by polyanions, indicating that phage trapping by this endothelium is mediated by scavenger receptors (SR). To circumvent interference by SR, polyinosinic acid was administered before phage panning in vivo. This led to the identification of a consensus motif that confers binding specificity for a subpopulation of hemopoietic marrow cells. Thus, SR inhibition, by avoiding phage trapping by the endothelium, seems to allow phage particles to extravasate and reach parenchymal cells. Accordingly, this panning strategy in vivo may be useful for the identification of targeting motifs specific for cells located in the extravascular space of various tissues.