利用噬菌体展示技术制备人血管生成素2单链抗体

目的 用纯化的人血管生成素2(Ang2)蛋白,通过噬菌体展示技术从非免疫小鼠噬菌体展示抗体库中淘选、鉴定Ang2单链抗体.方法 以纯化的人Ang2蛋白为抗原,对非免疫小鼠噬菌体展示抗体库进行富集和筛选,获得Ang2单链抗体,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot法检测目的蛋白的表达并测序.结果 经过3轮富集和筛选,获得了1个能特异性识别Ang2蛋白的阳性噬菌体克隆,DNA测序表明其含有完整的单链抗体基因片段,大小约750 bp,表达蛋白相对分子质量约33.7×103；通过SDS-PAGE进一步实现Ang2单链抗体(phage-Ang2-scFv)的鉴定.结论 成功分离并鉴定人Ang2-scFv,为进一步的功能学研究奠定了基础.     

前列腺癌噬菌体抗体库的构建及抗特异性膜抗原抗体的筛选

目的建立国人前列腺癌噬菌体Fab抗体片段库，筛选、鉴定抗前列腺特异性膜抗原（PSMA）的人源Fab抗体可变区的编码基因，为前列腺癌的诊断与基因治疗研究开辟新途径。方法20例前列腺癌患者外周血分离淋巴细胞，提取其总RNA，经RTPCR及半套式扩增得到免疫球蛋白全部轻链和重链Fd段基因，克隆进载体pComb3，并电转化大肠杆菌XLl-Blue，构建前列腺癌噬菌体Fab抗体片段库。采用噬菌体表面展示技术，以PSMA原核表达重组子pET30a（＋）-PSMA诱导表达并纯化后的PSMA为固相抗原，从噬菌体Fab抗体库中经过“吸附洗脱扩增”筛选过程，获得抗原结合活性和特异性较强的PSMA人源Fab可变区抗体基因片段的阳性克隆，并进行免疫检测及序列测定。结果成功构建前列腺癌噬菌体抗体Fab片段库，其轻链及重链Fd段与载体DNA的重组率分别为87％、79％，库容为2．32×10^7，滴度为8．7×10^13pfu／ml。筛选得到Fab抗体克隆的Fd段基因核苷酸序列为696bp，编码由232个氨基酸残基组成的多肽，与人免疫球蛋白γ链恒定区的同源性为98％；轻链κ基因核苷酸序列为630bp，编码由210个氨基酸残基组成的多肽，与κ链恒定区同源性为93％。结论成功构建了前列腺癌噬菌体抗体库，并获得了PSMA人Fab抗体可变区编码基因克隆，该克隆抗体基因具有典型的免疫球蛋白轻链和重链可变区的结构特点，基因编码产物具有与PSMA反应的免疫学活性和特异性。     

抗内毒素类脂A单链抗体基因的克隆、表达和初步鉴定

为探索 G 杆菌脓毒症的治疗问题,应用基因工程技术从本所制备的分泌抗内毒素单克隆抗体的杂交瘤细胞中提取 mRNA,然后反转录成 cDNA 第一链,用特异性轻、重链引物扩增反应产物,获得重链(340bp)和轻链(320bp)基因片段,经纯化后将轻链和重链基因用一多肽连接物连接,构成单链抗体基因片段。此单链抗体基因经再次 PCR 扩增后克隆入 PCANTAB_5噬菌粒中再转化于感受态的 TG_1大肠杆菌中,通过噬菌体抗体库技术筛选重组噬菌体,结果在 TG_1细菌中表达了9G_6单链抗体基因。ELISA 阻断试验初步证明了该单链抗体能阻断其亲本单克隆抗体对内毒素的结合。提示:抗内毒素单链抗体可为脓毒症的治疗提供一条途径。     

前列腺特异性膜抗原人源Fab抗体可变区基因的筛选与鉴定

目的:筛选、鉴定抗中国人前列腺特异性膜抗原(PSMA)的人源Fab抗体可变区的编码基因,为PSMA蛋白质生物学功能的研究及前列腺癌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PSMA原核表达重组子pET30 a(+)-PSMA诱导表达并纯化后的PSMA为固相抗原,从噬菌体Fab可变区抗体库中经过5轮“吸附-洗脱-扩增”筛选过程,获得抗原结合活性和特异性较强的PSMA人源Fab可变区抗体基因片段的阳性克隆,并对其进行免疫检测及序列测定。结果:筛选得到Fab抗体克隆的Fd段基因核苷酸序列为696 bp,编码由232个氨基酸残基组成的多肽,与人免疫球蛋白γ链恒定区的同源性为98%;轻链κ基因核苷酸序列为630 bp,编码由210个氨基酸残基组成的多肽,与κ链恒定区同源性为93%。结论:利用噬菌体抗体库技术,成功获得了PSMA人Fab抗体可变区编码基因克隆,该克隆抗体基因具有典型的免疫球蛋白轻链和重链可变区的结构特点,基因编码产物具有与PSMA反应的免疫学活性和特异性。     

抗内毒素类脂A单链抗体基因的克隆、表达和初步鉴定

为探索Ｇ－杆菌脓毒症的治疗问题，应用基因工程技术从本所制备的分泌抗内毒素单克隆抗体的杂交瘤细胞中提取ｍＲＮＡ，然后反转录成ｃＤＮＡ第一链，用特异性轻、重链引物扩增反应产物，获得重链（３４０ｂｐ）和轻链（３２０ｂｐ）基因片段，经纯化后将轻链和重链基因用一多肽连接物连接，构成单链抗体基因片段。此单链抗体基因经再次ＰＣＲ扩增后克隆入ＰＣＡＮＴＡＢ５噬菌粒中再转化于感受态的ＴＧ１大肠杆菌中，通过噬菌体抗体库技术筛选重组噬菌体，结果在ＴＧ１细菌中表达了９Ｇ６单链抗体基因。ＥＬＩＳＡ阻断试验初步证明了该单链抗体能阻断其亲本单克隆抗体对内毒素的结合。提示：抗内毒素单链抗体可为脓毒症的治疗提供一条途径。     

利用噬菌体肽库筛选高转移潜能人肝癌细胞结合肽

目的筛选高转移潜能人肝癌细胞特异性结合肽。方法以高转移潜能人肝癌细胞系HCCLM3作为靶细胞，低转移潜能人肝癌细胞系MHCC97L为吸附细胞对噬菌体随机7肽库进行差减筛选，采用噬菌体结合实验和抗噬菌体免疫细胞化学染色对阳性克隆的特异性进行鉴定。结果经3轮筛选，随机挑选48个噬菌体克隆进行DNA序列分析，展示AWYPLPP肽的噬菌体被高度富集77％（37／48）；噬菌体结合实验显示，与低转移潜能人肝癌细胞系MHCC97L、PLC／PRE／5和无转移潜能入肝癌细胞系Hep3B以及正常入肝细胞系CCLl3相比，AWYPLPP噬菌体特异性与高转移潜能人肝癌细胞系HCCLM3、HCCLM6、MHCC97H和MHCC97结合（P〈0．01）；抗噬菌体免疫细胞化学染色证实AWYPLPP噬菌体特异性靶向高转移潜能人肝癌细胞系HCCLM3、HCCLM6、MHCC97H和MHCC97。结论从噬菌体肽库中获得与高转移潜能人肝癌细胞特异性结合肽，可作为肝癌转移复发靶向治疗研究的载体。     

抗TAG-72抗原单链抗体的原核表达及对人肝癌的检测

目的探讨抗肿瘤相关糖蛋白-72（TAG-72）单链抗体的原核表达及与4种肝癌细胞系和肝癌组织的特异性亲和力。方法将抗TAG-72单链抗体的cDNA片段插入噬菌粒载体pCANTAB5E,获得噬菌粒载体TAG-72-scFv-pCANTAB5E。将后者转化入E.coliHB2151,经β,D异丙基硫代半乳糖苷（IPTG）诱导表达获得抗TAG-72单链抗体蛋白。采用细胞培养及免疫细胞/组织化学方法 ,检测4种肝癌细胞系及石蜡包埋的肝癌组织中TAG-72抗原的表达。结果 SDS-PAGE及Western blot法证实,抗TAG-72单链抗体蛋白分子得以正确表达。抗TAG-72单链抗体可与肝癌细胞系SMMC7721、HepG2和HHCC结合,表明这3种细胞表达了特异性肿瘤抗原;抗TAG-72单链抗体不能结合BEL7402细胞。40例肝癌组织中TAG-72抗原表达阳性率Ⅰ期、Ⅱ～Ⅲ期分别为23.08%（3/13）和62.96%（17/27）,在正常肝组织标本中无TAG-72抗原表达,TAG-72抗原在正常肝组织及肝癌组织中表达的差异有统计学意义（P〈0.05）。结论 TAG-72抗原在肝癌细胞或肝癌组织中有特异性表达,有可能成为判断肝癌的标志物。     

抗内毒素类脂A单链抗体基因的克隆,表达和初步鉴定

为探索Ｇ杆菌脓毒症的治疗问题，应用基因工程技术从本所制备的分泌抗内毒素单克隆抗体的杂交瘤细胞中提到ｍＲＮＡ，然后反转录成ｃＤＮＡ第一链，用特异性轻、重锭引物扩增反应产物，获得重链（３４０ｂｐ）和轻链３２０ｂｐ基因片段，经纯化后将轻链和重链基因用一多肽连接物连接，构成单链抗体基因片段。此单链抗体基因经再次ＰＣＲ扩增后克隆入ＰＣＡＮＴＡＢ５噬菌粒中再转化于感受态的ＴＧ１大肠杆菌中，通过噬菌体抗体库技术     

抗PSA/抗CD3两种双特异性单链抗体的活性研究

目的:研究并比较已成功构建的抗PSA/抗CD3两种双特异性单链抗体的生物学活性。方法:利用流式细胞仪和51Cr释放试验评价抗PSA/抗CD3双特异性单链抗体的抗原亲和活性和体外介导杀伤靶细胞的效果。建立前列腺癌裸鼠模型,分为非治疗组、对照组、双特异性单链抗体(BsAb)组和多价双特异性单链抗体(mBsAb)组,分析两种双特异性单链抗体在体内介导细胞毒T细胞对肿瘤细胞杀伤的能力。结果:流式细胞仪结果显示:BsAb与LNCaP细胞和Jurkat细胞的阳性结合率分别为56.3%和55.4%;mBsAb与LNCaP细胞和Jurkat细胞的阳性结合率分别为74.0%和83.0%。在体外,有细胞毒T细胞存在时两种抗体均可引起前列腺癌细胞的裂解,裂解效率分别与抗体浓度和T细胞与靶细胞比例呈正相关。与对照组比较,接种前列腺癌细胞的裸鼠在体内注射激活的细胞毒T细胞的同时分别接受两种抗体的治疗后,肿瘤生长均明显受到抑制(P<0.05)。在亲和力、体外杀伤和体内抑制肿瘤生长等方面,mBsAb的活性明显优于BsAb。结论:抗PSA/抗人CD3 BsAb及其四聚体均具有良好的生物学活性,单链抗体四聚体的形成可以明显改善抗体的生物学活性。     

抗肝癌单链免疫毒素在肝癌细胞系SMMC-7721中的表达

目的 构建分泌型抗肝癌单链免疫毒素真核表达载体并在人肝癌细胞系SMMC-7721中表达。方法 采用PCR方法在抗肝癌sFv的5′端引入引导序列使其能够在真核细胞中表达并分泌，在其下游连接人TNF-α基因，构建分泌型抗肝癌单链免疫毒素基因，并将该基因克隆人带有GFP报告基因的真核表达载体，用磷酸钙共沉淀法转染人肝癌细胞系SMMC-7721进行瞬时表达。结果 DNA序列分析证实在sFv的5′端引入正确的60bp引导肽序列，酶切鉴定证明成功构建了分泌型抗肝癌单链免疫毒素融合GFP真核表达载体，并在SMMC-7721细胞中成功地表达了融合荧光蛋白。结论 成功构建并表达了分泌型抗肝癌单链免疫毒素融合GFP基因，为肝癌免疫基因治疗的进一步研究奠定了基础。     

Construction and preliminary screening of a human phage single-chain antibody library associated with gastric cancer.

BACKGROUND: The aim of this study was to construct a phage library of human single-chain antibodies associated with gastric cancer and screen such a library for CEA binding scFv. MATERIALS AND METHODS: The cDNA library of antibody variable regions was constructed using mRNA from metastatic lymph nodes or spleen of patients with stomach cancer by RT-PCR. These cDNA were assembled into a single-chain format and cloned into phagemid pCANTAB-5 and then transformed into Escherichia coli TG1. The scFv gene library was rescued by M13KO7 helper phage. CEA and the viable CEA-positive gastric cancer cell line MKN-28 were used to screen the phage antibody library. Indirect and tumor cell ELISA was used to determine the specificity of phage antibody. Fixed cell immunofluorescence and live cell FACS analysis were used to further characterize the binding of phage scFv. RESULTS: After transformation into E. coli TG1, 2.5 x 10(7) cfu/microg ampicillin-resistant clones grew. Sequences of those positive insert clones showed that the V(H) genes were derived from the V(H) III subgroup, while the V(L) genes belonged to the V(kappa) III subgroup. After four rounds of panning, the titer of eluted binding phage increased 135- to 158-fold and ELISA results showed that 20/95 clones can bind CEA and 47/95 clones can bind fixed tumor cells. Immunofluorescence and FACS analysis results showed that these phage scFv fragments could bind CEA-positive cells. CONCLUSIONS: We successfully constructed a human phage antibody library from lymph nodes of stomach cancer patients. Such kinds of library prove useful for generating tumor-antigen-specific human antibody fragments.     

抗人膀胱癌单链抗体的构建及表达的研究

目的 为膀胱癌的导向诊断和治疗提供免疫原性更低的单链抗体。方法 从1株分泌鼠抗人膀胱癌单克隆抗体的杂交瘤细胞株BDI-1中分离出总RNA。     

抗人AFP单链抗体的制备、鉴定及其免疫学活性的测定

目的 构建具有免疫学活性的抗人甲胎蛋白单链抗体ScFv (single chain fragment of variety region,ScFv),为进一步的研究奠定实验基础.方法 采用RT-PCR 技术,从分泌抗人AFP 单克隆抗体的杂交瘤细胞中扩增mAb 的VH、VL 基因,并进一步将其组装成VH-Linker-...     

抗前列腺特异抗原/抗人CD3双特异性单链抗体基因在HeLa细胞中的表达及亲和活性测定

目的　在HeLa细胞中表达抗前列腺特异抗原 (PSA ) /抗人CD3双特异性单链抗体(scFv)融合基因 ,并进行亲和活性测定。方法　在已经构建抗PSA/CD3双特异性scFv融合基因基础上 ,将融合基因克隆入真核表达载体pSecTag2 B中 ,并转染HeLa细胞进行表达。表达产物纯化后 ,用流式细胞仪进行亲和活性测定。结果　经SDS PAGE和Western印迹实验证实 ,表达产物的Mr约为 65 0 0 0。纯化后经流式细胞仪检测 ,可特异性地结合PC 3细胞和人外周血单个核细胞(PBMC)。结论　获得了可与PC 3细胞和PBMC特异结合的抗PSA/CD3双特异性scFv ,为进一步临床应用奠定了基础。     

Antitumor effect of an HER2-specific antibody-toxin fusion protein on human prostate cancer cells

BACKGROUND: HER2/neu has been implicated in the oncogenesis of human prostate cancer. Clinical studies have suggested that overexpression of HER2 may be one of the indicators of poor prognosis in prostate cancer patients. METHODS: We used Western blot analysis to examine the expression of HER2 in a panel of established human prostate cancer cell lines and used an MTT assay to evaluate the cytotoxicity on these cells of a recombinant fusion protein consisting of an HER2-specific single-chain antibody and the Pseudomonas exotoxin A, scFv(FRP5)-ETA. RESULTS: LNCaP cells express high levels of HER2 protein. Exposure of LNCaP cells to scFv(FRP5)-ETA caused remarkable cell death. In contrast, PC3M cells, which express an undetectable level of HER2 protein, were resistant to scFv(FRP5)-ETA-induced cytotoxicity. MDA PCa 2a, MDA PCa 2b, and DU145 cells express low-to-medium levels of HER2 protein and showed an HER2 level-dependent response to scFv(FRP5)-ETA-induced cytotoxicity. The scFv(FRP5)-ETA-induced cytotoxicity of LNCaP cells could be inhibited by an anti-HER2 monoclonal antibody (mAb), which downregulated the levels of HER2 protein, indicating the specificity of scFv(FRP5)-ETA in inducing cytotoxicity in LNCaP cells. Using an apoptosis ELISA, we demonstrated that scFv(FRP5)-ETA induced apoptosis in LNCaP cells. The apoptosis was inhibited by the presence of dihydrotestosterone (DHT) in culture medium. Exposure of LNCaP cells to scFv(FRP5)-ETA caused reduction in the level of the prostate-specific antigen (PSA). CONCLUSIONS: Our data suggest that scFv(FRP5)-ETA might be a useful agent for the treatment of human prostate cancer cells with high levels of HER2 expression.     

四聚功能域提高前列腺特异抗体亲和力的作用

目的探讨人p53四聚功能域在提高抗前列腺特异抗原(PSA)／抗人CD3双特异性单链抗体(scFv)亲和力方面的作用。方法利用递归聚合酶链反应(PCR)法扩增人IgG3上游铰链区与人p53四聚功能域融合基因,克隆入pUC19载体中构建pUC19／IgG3／p53克隆载体。将抗 PSA／CD3双特异性scFv克隆入pUC19／IgG3／p53载体中,构建多价抗PSA/CD3双特异性scFv融合基因。将融合基因克隆入真核表达载体pSeeTag2-B中,转染HeLa细胞进行表达,表达产物纯化后利用流式细胞仪进行活性测定。结果获得了多价抗PSA/CD3双特异性scFv融合基因,基因全长1638 bp,可编码546个氨基酸,与已发表的抗PSA／CD3双特异性scFv和人p53四聚功能域基因cDNA序列一致。表达产物经SDS-PAGE和Western印迹实验证实为约67×103的特异蛋白条带,纯化后经流式细胞仪检测可以特异性地结合PC-3细胞和人外周血单个核细胞(PBMC),亲和力高于双特异性scFv。结论人IgG3上游铰链区／p53四聚功能域基因与抗PSA／CD3双特异性 scFv基因融合后表达产物的功能性亲和力大大提高,为提高抗体的功能性亲和力开辟了新的思路。     

Single-chain antibody fragment-based adsorbent for the extracorporeal removal of beta2-microglobulin

BACKGROUND: Dialysis-related amyloidosis (DRA) is a frequent complication of end-stage renal disease (ESRD) that has been associated with the accumulation of beta2-microglobulin (beta2-m). Removal of beta2-m results in the loss of important proteins due to the nonspecific nature of current therapies. Although whole antibodies can potentially be used to confer specificity to beta2-m removal from blood, single-chain variable region (scFv) antibody fragments could potentially offer several advantages as immunoadsorption ligands due to their size, genetic definition, ability to be expressed by microbes, and amenability for in vitro evolution. METHODS: An antihuman beta2-m scFv was constructed from the BBM.1 hybridoma and expressed by a yeast display vector. The binding affinity of the wild-type scFv fragment was quantified by flow cytometry analysis. Soluble scFv was expressed by a yeast secretion vector, purified, and immobilized onto agarose beads. The binding capacity of the immunoadsorbent was measured by equilibrating samples with saturating quantities of fluorescent beta2-m in serum. RESULTS: The displayed scFv possessed a nanomolar affinity (KD= 0.008 +/- 0.004 mg-beta2-m/L). The immunoadsorbent exhibited an adsorption site density of 0.41 +/- 0.01 mg beta2-m/mL settled gel. Under saturating conditions, the mass ratio of adsorbed beta2-m to immobilized antibody is 70% greater than any previous literature report for whole antibodies. Preliminary specificity experiments suggest that the scFv-based immunoadsorbent is specific toward human beta2-m. CONCLUSION: Recombinant DNA technology was successfully used to engineer an scFv-based immunoadsorbent. Use of immobilized scFvs during hemodialysis may minimize loss of valuable proteins and facilitate the removal of macromolecules that are significantly larger than the molecular weight cut-off of the membrane.     

抗人膀胱癌抗独特型抗体的制备和鉴定

目的：制备抗人膀胱癌抗独特型抗体并进行体外实验鉴定。方法：提取膀胱癌抗原（Ag)。应用膀胱癌抗原通过杂交瘤技术制备抗人膀胱癌单克隆抗体（Ab1),Ab1经胃蛋白酶水解制备Ab1的F（ab)2片段，以F（ab)2片段免疫新西兰白兔，其血清经凝胶纯化后获抗人膀胱癌抗独特型抗体（Ab2)。再应用ELISA等方法进行鉴别。结果：Ab2与Ab1呈阳性反应，与BALB/C小鼠r球蛋白呈阴性反应；Ab2与Ag竞争结合Ab1；Ab2抑制Ab1与Ag的结合。结论：Ab1是针对膀胱移行细胞癌的单克隆抗体，Ab2是具有膀胱癌抗原内影像的抗独特型抗体。     

原发性肝细胞癌消减杂交文库构建及差异表达基因筛选

目的构建人原发性肝细胞癌（HCC）消减杂交文库，筛选差异表达基因。方法以癌组织为检测者（tester）、癌旁组织为参照者（driver），应用抑制性消减杂交（SSH）方法构建消减杂交cDNA文库，随机挑选56个阳性克隆进行鉴定、测序及同源性分析。结果文库获得130个阳性克隆，鉴定96％有200～1500bp插入片断。获得的35个基因中包括已知基因26个，功能未知基因5个，新基日4个。已知基因中包含酶、细胞信号转导、基因调控因子、转录调节因子、癌基因、抗凋亡因子及细胞的黏附与生长调节因子等相关基因。结论应用SSH方法成功构建HCC差异表达基因cDNA文库，筛选出低丰度和新基因在内的HCC差异表达基因。