从人源免疫抗体库筛选抗乙型肝炎病毒PreS1单链抗体

目的 获得高亲和力的全人源化抗乙型肝炎病毒PreS1的单链抗体。 方法 以健康供血者的外周血淋巴细胞为来源,以PreS1肽体外致敏后,借助噬菌体展示技术构建人源免疫单链抗体库,库容量7×10 8。 结果 以PreS1肽进行3轮固相筛选。获得了亲和力10 7～10 8mol/L的抗PreS1单链抗体。结论 通过体外致敏的方法构建人源免疫抗体库,可以快速获得高亲和力基因工程抗体。     

全人源肝癌噬菌体单链抗体基因的克隆及活性分析

从噬菌体单链抗体库中筛选克隆全人源肝癌抗体基因并进行活性鉴定.PCR鉴定阳性重组菌中人肝癌ScFv的插入率,以肝癌细胞SMMC-7721为抗原对所建抗体库进行4轮"吸附-洗脱-扩增"的亲和筛选.将筛选后的ScFv采用ELISA法鉴定其与人肝癌细胞的结合活性.ScFv基因插入率为70%.在亲和筛选过程中,肝癌噬菌体单链抗体得到富集,收获率逐轮提高,第4轮为第一轮的381倍.利用噬菌体抗体库技术筛选出了肝癌噬菌体单链抗体,且筛选后的抗体片段与人肝癌细胞有特异性的结合活性.     

抗体库优化策略对特异性抗肝癌单链抗体的人源化

目的:对特异性抗肝癌单链抗体(single chain variable fragment,scFv)DM同步进行人源化和优化,以获得亲和力高和免疫原性低的人源化抗肝癌单链抗体.方法:通过分子结构和序列分析,确定对抗原结合位点的构象有重要影响的骨架区(FR)残基,把难以判断回复突变残基的人、鼠源副本同时编入人源化抗体序列中;通过重叠延伸PCR技术合成人源化抗体全基因,利用噬茵体展示技术构建人源化组合抗体库;通过肝癌细胞筛选阳性克隆,ELISA方法测定各个克隆抗原结合活性;用硫氰酸盐洗脱法测定并比较亲本抗体和人源化scFv的相对亲和力.结果:成功构建人源化组合抗体库,实际库容4.77×10 5,包含由2 5不同重链和2 2不同轻链组成的128种人源化DM;经过筛淘及ELISA鉴定,获得HDM1、HDM2和HDM3 3个阳性克隆,相对亲和力指数分别为HDM1 1.6 mol/L、HDM2 1.2 mol/L和HDM3 2.2 mol/L.结论:抗体库优化法是一个高效、简便的人源化方法;获得的3株阳性克隆HDM1、HDM2、HDM3与亲本抗体DM同样具有良好的抗原结合特异性和较高的亲和力.     

抗弓形虫主要表面抗原1单链抗体的筛选及鉴定

目的采用重组抗原从人源化单链抗体库(Human single fold scFv library)中筛选和鉴定抗弓形虫主要表面抗原1(rSAG1)的单链抗体,对其特异性的弓形虫靶向作用进行分析,为弓形虫靶向生物治疗提供靶向分子。方法以重组SAG1融合蛋白作为包被抗原对噬菌体抗体库进行3轮固相筛选,获得抗rSAG1融合蛋白的阳性克隆;用纯化的原核表达载体pET-32C的亲和标签(Trx-His-Stag)、鼠可溶性抗原和大肠杆菌抗原对上述阳性克隆进行差异筛选,获得能特异性结合rSAG1的噬菌体克隆;差异筛选获得的特异性阳性克隆转染大肠杆菌HB2151进行诱导表达和聚丙烯酰胺凝胶电泳(SDS-PAGE)检测,获得能分泌抗rSAG1单链抗体(anti-rSAG1scFv)的阳性噬菌体克隆;DNA测序分析抗rSAG1的单链抗体基因序列。用Ni2 螯合柱亲和纯化rSAG1单链抗体,并用免疫印迹技术(Westernblot)对纯化的抗rSAG1单链抗体进行免疫反应性检测;免疫组化检测rSAG1单链抗体对弓形虫速殖子的靶向作用。结果经过rSAG1抗原的固相筛选后获得24株阳性克隆,差异筛选后获得5株能特异性结合rSAG1的噬菌体克隆,其中有3株分泌性表达抗rSAG1的单链抗体,DNA测序和Genbank比对表明其为3株不同的抗弓形虫主要表面抗原1的单链抗体;West-ernblot表明亲和纯化的单链抗体能较好结合rSAG1;免疫组化显示抗rSAG1单链抗体可与弓形虫速殖子结合。结论利用噬菌体抗体库技术获得的特异性人源rSAG1单链抗体与弓形虫速殖子有较强的结合能力,这对弓形虫的靶向生物治疗具有潜在应用价值。     

人工进化丙型肝炎病毒C和E1区噬菌体展示文库的构建及初步筛选

目的构建人工进化丙型肝炎病毒(HCV)C和E1区基因噬菌体展示文库,并进行初步筛选。方法应用DNA改组技术进行不同基因型的HCV C和E1区基因的人工进化,克隆于噬菌体载体,以辅助噬菌体M13K07援救后,构建噬菌体展示文库,应用HCV C和E1区单克隆抗体进行初步筛选。随机选取筛选后的20个噬菌体克隆,用高滴度HCV阳性血清通过双抗体夹心酶联免疫吸附法进行抗原抗体结合反应,以正常人血清作为对照。结果人工进化HCV C和E1区噬菌体展示文库库容达1．64×106,重组率为0．86。以C和E1区单克隆抗体淘筛4轮,噬菌体展示文库得到特异性富集。筛选获得12个阳性克隆。结论所构建的噬菌体展示文库的库容和多样性符合筛选的要求。筛选获得的阳性克隆具有较好的抗原抗体结合反应活性,表现出较好的亲和力。     

抗SSA噬菌体抗体库的构建及鉴定

目的　用噬菌体展示技术构建抗SSA单链抗体库。方法　分离抗SSA抗体阳性病人外周血单个核细胞 ,提取RNA并反转录cDNA。扩增免疫球蛋白重链可变区 (VH)和κ链可变区(Vκ)。通过重叠聚合酶链反应 (PCR)用连接片段将VH和Vκ体外连接成单链抗体 (single chainFv ,scFv)基因。而后用SfiⅠ和NotⅠ酶切并与噬菌粒 pHEN2 连接 ;将 pHEN2 scFv电转至大肠杆菌TG1,建立抗SSAscFvcDNA库。随机挑选克隆用PCR扩增插入片段了解插入效率。用辅助噬菌体VCS M13感染含 pHEN2 scFv的TG1,产生scFv噬菌体抗体。用酶联免疫吸附 (ELISA)检测构建的scFv噬菌体抗体库中有无抗SSA抗体存在。结果　与VH和Vκ支架结构互补的引物可扩增出 30 0～ 4 0 0bp大小VH和Vκ片断 ;重叠PCR体外连接成约 80 0bp大小scFv基因 (VH linker Vκ)。将scFv基因克隆至 pHEN2 ,电转TG1后计数克隆数为 3 0× 10 7cfu。随机挑选 12个克隆 ,其中 11个克隆PCR扩增出插入片段 ,插入效率约为 91%。用辅助噬菌体VCS M 13感染含 pHEN2 scFv的TG1后 ,产生 1 2× 10 14 pfu/ml噬菌体抗体颗粒。抗SSAELISA试剂盒检测其抗SSA活性 ,scFv噬菌体抗体的A值比相同稀释度的VCS M13的A值高 2 0～ 2 2倍。结论　建立的抗SSAscFv噬菌体抗体库含有的独立克隆数为 3 0× 10     

抗日本血吸虫噬菌体抗体库的筛选和阳性克隆的鉴定

目的　研制抗日本血吸虫循环抗原SjMAg的单链抗体。　方法　用日本血吸虫成虫代谢抗原SjMAg包板,对已构建的抗日本血吸虫噬菌体抗体库进行3轮富集,然后用ELISA法从富集的次级抗体库中筛选阳性克隆,最后借助ELISA、SDSPAGE和Western印迹等方法,根据阳性克隆表达产物与其它4种吸虫抗原反应的情况,进行特异性鉴定。　结果　从随机挑取的72个克隆中筛选到阳性克隆6个,经交叉筛选和鉴定最终获得特异性抗日本血吸虫阳性克隆2个。　结论　用固相富集和ELISA筛选法可以从抗体库中有效地筛选到抗日本血吸虫循环抗原的阳性克隆,获得的抗体克隆可用于抗日本血吸虫循环抗原单链抗体的制备。     

抗人肺癌噬菌体展示可溶性ScFv的筛选与鉴定及其序列测定

目的制备抗人肺癌单链抗体（single chain Fv ferment antibody,ScFv）,并对抗体生物学特性进行初步研究。方法以人肺腺癌细胞系A：为抗原,对5F一11杂交瘤细胞噬菌体抗体库进行富集和筛选。以人肺腺癌细胞系A2和正常人淋巴细胞为抗原,进行酶联免疫吸附试验,从富集后的噬菌体抗体库中筛选出只与A2细胞结合的阳性克隆。筛选的噬菌体克隆转染大肠埃希菌HB2151,得到可溶性单链抗体分泌克隆。可溶性抗体分泌克隆测序。应用ELISA、竞争性ELISA、SDS．PAGE及蛋白质印迹法对其中的2A7．1克隆进行初步鉴定。结果以肺腺癌细胞A2为抗原进行了4轮富集。进一步筛选得到18个仅识别A：细胞而与人淋巴细胞无反应的融合抗体分泌克隆。转染大肠埃希菌HB2151后筛选到能与Az细胞特异结合的可溶性抗体分泌克隆2A7．1。竞争性ELISA结果显示,5F-11能强烈抑制2A7-1与A2细胞的结合。SDS-PAGE蛋白质印迹法显示得到大小约为30X10。的单链抗体。结论通过噬菌体抗体技术成功分离到了鼠单抗5F-11的可溶性ScFv分泌克隆,为进一步的抗体应用研究奠定了基础。     

丙型肝炎病毒核心蛋白人源单链可变区抗体的筛选与鉴定

目的 筛选、鉴定抗丙型肝炎病毒（HCV）核心蛋白的人源单链可变区抗体（ScFv)。方法 采用噬菌体表面展示技术，以重组的HCV核心蛋白为包被抗原，从噬菌体单链可变区抗体库中经过3轮“吸附－洗脱－扩增”筛选过程，获得抗原结合活性较强的HCV核心蛋白特异性人源单链可变区抗体片段阳性克隆，并对其进行免疫学及核苷酸序列测定。结果 筛选得到的ScFv片段具有抗HCV核心蛋白的特异性，基因序列分析结果表明符合人源单链可变区抗体基因序列的结构特征。结论 利用噬菌体抗体库技术，成功获得HCV核心蛋白的特异性人源单可变区抗体的编码基因。     

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。     

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library

AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.     

Preparation of single chain variable fragment of MG(7) mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.     

肝细胞癌人源抗c-Met抗体Fab的筛选及鉴定

目的 从大容量人源Fab抗体库中筛选全人源抗c-Met抗体,并对抗体与肝癌细胞的结合活性进行初步鉴定.方法 利用Met-Fc融合蛋白对大容量人源Fab抗体库进行固相筛选,经过5轮固相筛选,随机挑选30个克隆经酶联免疫吸附法差减鉴定,阳性克隆进行可溶性表达,用c-Met表达阳性的人肝癌细胞株鉴定抗c-Met抗体Fab的结合活性.结果 Western blot、免疫荧光结果显示,c-Met分子表达于SMMC721、BEL7402人肝癌细胞膜上;从大容量人源Fab抗体库中筛选出1株抗c-Met抗体Fab(AM2-26),经免疫共沉淀、荧光激活细胞分类术、免疫荧光分析,结果显示AM2-26与人肝癌细胞表面c-Met分子有较好的结合活性.结论 从人源Fab抗体库中筛选的AM2-26能够与肝癌细胞表面c-Met分子特异性结合,为研制用于肝癌生物治疗的靶向药物,提供了候选分子.     

单抗MGb1的噬菌体呈现型ScFv片段的制备

目的 制备胃癌单抗ＭＧｂ１的噬菌体呈现型单链可变区片段（ＳｃＦｖ）,为获得用于胃癌体内诊疗研究的靶向载体分子奠定基础。方法 从杂交瘤细胞分离ｍＲＮＡ,扩增抗体重,轻链可变区（ＶＨ和ＶＬ）ｃＤＮＡ,经ｌｉｎｋｅｒＤＮＡ连接形成ＳｃＦｖ ＤＮＡ。将ＳｃＦｖＤＮＡ与ｐＣＡＮＴＡＢ５Ｅ的连接产物转化于大肠杆菌ＴＧ１,经Ｍ１３ＫＯ７感染后,获得重组噬菌体抗体ＳｃＦｖ。经亲和筛选和ＥＬＩＳＡ检测获得呈现ＭＧｂ     

Isolation of anti-CD22 Fv with high affinity by Fv display on human cells

In vitro antibody affinity maturation has generally been achieved by display of mouse or human antibodies on the surface of microorganisms (phage, bacteria, and yeast). However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. An ideal system would select and improve antibodies in a mammalian cell environment where they are naturally made. Here we show that human embryonic kidney 293T cells that are widely used for transient protein expression can be used for cell surface display of single-chain Fv antibodies for affinity maturation. In a proof-of-concept experiment, cells expressing a rare mutant antibody with higher affinity were enriched 240-fold by a single-pass cell sorting from a large excess of cells expressing WT antibody with a slightly lower affinity. Furthermore, we successfully obtained a highly enriched mutant with increased binding affinity for CD22 after a single selection of a combinatory library randomizing an intrinsic antibody hotspot. Important features are that one display selection cycle requires only 1 week, and transfection of cells in a single 100-mm dish produces 10(7) individual clones so that a repertoire of 10(9) is feasible under current experimental conditions.     

Construction of non-covalent single-chain Fv dimers for hepatocellular carcinoma and their biological functions

AIM: To create new diabodies with improved binding activity to antigen of the variable light - variable heavy (VH-VL) oriented single-chain Fv dimers genes (scFv).METHODS: The linker between VH and VL genes was shortened to 3-5 amino acid residues and cloned into the vector pCANTAB5E. The recombinant plasmids were transformed into TG1 cells and sequenced. The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibodies. Expressed products were identified by SDS-PAGE, Western blotting, size exclusion gel chromatography (SEC), ELISA and immunohistochemistry.RESULTS: Three scFv (scFv-3, scFv-4, scFv-5) were constructed successfully with binding ability to hepatocellular carcinoma 3.5-6 fold greater than their parental scFv. The single-chain Fv dimer (scFv-5, termed BDM3) with the best binding ability was successfully expressed in Yeast pichlia, as shown by. SDS-PAGE and Western blotting. SEC results suggested the molecular weight of the expressed products was about 61 kDa. Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv, still having 50% binding activity even after 16 h incubation as 37°C. The purified dimers were bound specifically to the tumor antigen of HCC.CONCLUSION: we have generated scFv dimers by shortening a series of linkers to 3-5 amino acid residues in VH-linker-VL orientation, resulting in highly stable and affinity-improved dimeric molecules. These will become an attractive targeting moiety in immunotherapeutic and diagnostic applications for HCC.     

Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

AIM:To construct the natural immune Fab antibody phagedisplay libraries of colorectal cancer and to select antibodiesrelated with colorectal cancer.METHODS:Extract total RNA from tissue of local cancermetastasis lymph nodes of petients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and lightchain k and the amplification products were insertedsuccessively into the vector pComb3 to construct the humanlibraries of Fab antibodies.They were then panned by phagedisplay technology.By means of Dot immunoblotting andEUSA,the libradse were identified and the Fab phageantibodies binding with antigens of colorectal cancer wereselected.RWSULTS:The amplified fragments of Fd and k gained byRT-PCR were about 650bp.Fd and k PCR products weresubsequently inserted into the vector pComb3,resulting in arecombination rate of 40% and the volume of Fab phagedisplay library reached 1.48×10~6.The libraries wereenriched about 120-fold by 3 cycles of adsorption-elution-multiplication(penning).Dot immunoblotting showed Fabexpressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activitieswith antigens of colomctal cancer.CONCLUSION:The nstuml immune Fab antibody phagedisplay libraries of colorectal cancer were constructed.Theycould he used to select the relstive antibodies of colorectalcancer.     

A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.     

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.