抗人死亡受体5单链抗体的构建表达纯化及活性分析

目的构建、表达、纯化抗人死亡受体5(DR5)单链抗体,并检测其诱导肿瘤细胞凋亡的活性。方法采用RT-PCR获取鼠源性抗人DR5单克隆抗体重链和轻链可变区基因序列,以一柔性连接肽连接二者,转入表达载体,以大肠杆菌表达融合蛋白,亲和色谱纯化后用MTT实验和凋亡检测试剂盒检测其凋亡诱导活性。结果获得的序列经比对为抗体重链和轻链可变区基因,表达纯化后的重组蛋白具有接近完整抗体的肿瘤细胞凋亡诱导活性。结论抗人DR5 scFv可作为诱导肿瘤细胞凋亡的候选药物,为肿瘤免疫学研究提供材料。     

抗人B-淋巴细胞刺激因子噬菌体单链抗体库的构建

目的构建抗人B-淋巴细胞刺激因子噬菌体单链抗体库。方法从B-淋巴细胞刺激因子免疫小鼠的脾细胞中抽提总RNA,经逆转录聚合酶反应合成cDNA,分别扩增小鼠重链可变区基因和轻链可变区基因。通过重叠延伸拼接法将重链可变区基因和轻链可变区基因组装成单链抗体基因,重组于载体pFUSE5,电转化E.coliXL 1-Blue。在辅助噬菌体援救下,构建成噬菌体单链抗体库。采用聚合酶链反应、核苷酸序列测定和酶联免疫分析鉴定单链抗体库的特征。结果本抗体库的噬菌体库容量约1×106,单链抗体基因插入效率为93%,存在序列差异性并含有抗B-淋巴细胞刺激因子单链抗体。结论成功地构建了抗人B-淋巴细胞刺激因子单链噬菌体抗体库,为筛选抗B-淋巴细胞刺激因子单链抗体药物奠定了良好的基础。     

原核细胞表达抗CD_3工程抗体

用RT—PCR方法 ,从分泌抗人CD3抗体的杂交瘤细胞中扩增抗体的重链和轻链可变区基因 ,并运用重叠延伸拼接法 (SOE)将其连接成单链抗体 (ScFv)基因 ,并克隆于带有Tac启动子的原核表达载体中 ,用斑点杂交方法筛选出阳性克隆。重组子在E .coilB1 2 1 (DE3)中获得高效表达且具有活性的单链抗体。     

应用核糖体展示技术高效筛选人源抗IgE单链抗体

本研究从哮喘患者外周血分离淋巴细胞、提取总mRNA,采用RT-PCR技术分别构建重链可变区VH(variable region of heavy chain)和轻链可变区VL(variable region of light chain)cDNA库,然后通过连接肽(Gly4Ser)3和特定引物将VH和VL cDNA基因组装成人源单链抗体(scFv)核糖体展示模板基因库。应用兔网织红细胞核糖体展示技术,单引物原位反转录技术,经3轮展示富集并回收针对人源IgE蛋白(免疫球蛋白E)的目的单链抗体基因;回收的抗体基因经双酶切后与pET22b(+)载体连接,转化至E.coli Rosseta(DE3)宿主细胞,应用菌落PCR结合Dot blotting技术快速鉴定阳性克隆,抗原ELISA法进一步鉴定阳性克隆。VH和VL基因库得到正确的构建,长度分别为400和710 bp,库容为1013。核糖体展示模板构建正确,长度为1 100 bp。该基因库经过3轮针对IgE蛋白的核糖体展示,目的基因得到了有效富集和回收。经过高效克隆、表达和鉴定,确定1株针对人IgE蛋白显示最强亲和力的阳性克隆菌pET-IgE-6。经测序和序列分析证实该单链抗体为人源抗体,序列未见国内外报道。结果表明,采用患者外周血构建人源抗体基因库,结合核糖体展示技术,可以成功获得高亲和力的人源单链抗体分子。该技术路线为快速获得具有药用价值的人源抗体提供了参考。     

脂联素单链抗体基因的克隆及表达

目的 克隆人脂联素单链抗体基因并进行表达。方法提取分泌抗脂联素单克隆抗体杂交瘤细胞株的总RNA,通过RT-PCR技术,扩增VH和VL,与质粒pUC19载体连接,形成克隆,并对其进行鉴定分析。结果抗体重、轻链可变区扩增拼接后的重、轻链各约为340bp、350bp,基因全长约710bp,将拼接产物插入pUC19质粒中转化大肠杆菌JM109,得到数个克隆,经酶切分析约含710bp片段,与拼接结果基本相同,经测定特异性较好,与其他杂蛋白及载脂蛋白没有交叉反应。结论抗人脂联素单链抗体基因的克隆和表达较为成功。     

抗CD5单链抗体的基因构建、蛋白纯化及活性鉴定

目的构建高表达的抗CD5单链抗体(scFv anti-CD5)工程菌,为开展以抗CD5单链抗体为构件的肿瘤免疫治疗研究奠定基础。方法从Genbank中获得抗CD5单克隆抗体H65的重链可变区(VH)和轻链可变区(VL)的基因序列,用连接肽的编码序列连接,用重叠PCR技术获得目的基因,酶切后插入pET22b(+)构成重组质粒。测序正确的重组质粒转入E.coliBL21(DE3)诱导表达,产物经Ni-NTA柱纯化后复性,流式细胞术和Western blot-ting检测活性。结果获得序列正确的重组质粒,工程菌表达量高,产物复性后具有识别CD5抗原的活性。结论成功构建了高表达scFv anti-CD5的工程菌,为scFv anti-CD5的应用奠定了基础。     

电鳐乙酰胆碱酯酶单链抗体基因的克隆及表达(英文)

目的　欲用计算机模拟及分子对接技术揭示抗乙酰胆碱酯酶单克隆抗体 3F3抑酶的分子基础 ,但 3F3分子远大于乙酰胆碱酯酶 (AChE) ,难以操作 ,故拟用其单链抗体为工具进行研究。本研究旨在设计、克隆并表达抗AChE单链抗体Sc3F3基因 ,推演重组单链抗体的氨基酸顺序 ,并证明纯化的重组单链抗体有抑制AChE的作用。方法　使用分泌抗电鳐AChE单克隆抗体 3F3(3F3Ab)的杂交瘤细胞提取总RNA。用RT PCR技术扩增重链可变区 (VH)cDNA及轻链可变区 (VL)cDNA基因 ,通过连接肽(Gly4 Ser) 3 基因将VH 基因和VL 基因连接 ,制成单链抗体基因 (ScFv)。将ScFv基因插入载体pCANTAB 5E用于表达。在大肠杆菌中表达的 3F3单链抗体(Sc3F3)用SephadexG75凝胶过滤和QAE SephadexA 5 0离子交换层析纯化。AChE与抗体的免疫反应性用ELISA法测定。AChE活性用微量羟胺比色法测定。结果　测序结果证明所克隆的ScFv基因是功能重排基因 ,长度为 72 3bp。重、轻链基因长度分别为 35 7bp和 32 1bp。ScFv基因 (含连接肽基因 )所编码的纯化的重组Sc3F3的分子量为 31.6ku。Sc3F3与AChE反应良好 ,但对AChE活性的抑制明显弱于 3F3Ab。Sc3F3的抑制效力约为 3F3Ab的 1/10。结论　在计算机模拟及分子对接研究中使用本研究设计的单链抗体Sc3F3应是可靠的     

鼠抗人CD3噬菌体基因工程单链抗体的初步制备

本研究应用PCR方法，从小鼠杂交瘤细胞中扩增鼠抗人CD3抗体重、轻链可变区基因，经DNA接头连接后与噬菌粒pCANTAB5重组；转化大肠杆菌形成抗体可变区基因文库。通过文库的“抢救”、亲和筛选、再转染、克隆化及鉴定，得到高亲和力鼠抗人CD3噬菌体基因工程单链抗体。     

梭曼单链抗体的基因克隆和表达

成功地扩增了有机磷毒剂梭曼类似物１－羟基－１－对胺基苯基甲膦酸单频哪基醇酯与血蓝蛋白结合物免疫小鼠脾细胞中抗体重链和轻链基因片段，构建了单链抗体可变区基因（ＶＨ－ｌｉｎｋｅｒ－ＶＬＤＮＡ），并将该基因克隆到噬菌体表达载体ｐＣＡＮＴＡＢ５Ｅ的外壳蛋白ｇ３ｐ基因中，使单链抗体以融合蛋白的形式在Ｅ．ｃｏｌｉＴＧ１中表达在噬菌体表面，构成噬菌体抗体库．通过免疫亲和淘洗和竞争抑制酶联免疫吸附测定法筛选，得到１２株克隆，其分泌的单链抗体与梭曼有结合活性．     

梭曼单链抗体酶EP_6一级结构的确定和三维结构的模建

以梭曼水解反应过渡态类似物 O1-甲基 - O2 -(1 ,2 ,2 -三甲基丙基 ) - 2 -羟基 - 5-硝基苯基甲基膦酸为半抗原得到了梭曼单链抗体酶 EP6.对这一株抗体进行了核苷酸序列的测定 ,测序结果表明这一单链抗体基因全长 71 4bp,其中重链长 351 bp,编码1 1 7个氨基酸 ,轻链长 31 8bp,编码 1 0 6个氨基酸 ,二者由编码 (Gly4 Ser) 3的 45bp的连接肽基因相连 .重 ,轻链均为开放读框 ,有明确的四个框架区和三个互补决定区 (CDR)以及抗体特征性的两个半胱氨酸残基 ,表明重 ,轻链的氨基酸序列符合鼠抗体可变区特征 .在 SGI工作站上对 EP6的三维结构进行了同源模建 .计算机模型显示 EP6表面包含一个由重轻链 CDRs围成的 ,可能用于半抗原结合和催化活性的长裂缝 ,其中轻链的 CDR3(L- CDR3)和重链三个 CDRs(H- CDRs)都可能参与抗原或半抗原的接触 ,L- CDR3和 H- CDR2的贡献较大 ,对抗体酶功能可能起重要作用     

APPβ分泌酶切割位点特异性单链抗体的制备和鉴定

目的制备针对人淀粉样蛋白前体(APP)β分泌酶切割位点的特异性单链抗体(ScFv),并进行鉴定。方法设计扩增单克隆抗体细胞株重链和轻链可变区基因片段VH和VL的引物,利用RT-PCR技术,从本室制备的抗人APPβ分泌酶切割位点单克隆抗体细胞株中扩增出VH和VL基因片段,然后通过重叠引物延伸法(SOE)将VH和VL基因拼接成ScFv基因片段,再将其亚克隆入原核表达载体pET-28a中,转化E.coli BL21(DE3)诱导表达,目的蛋白经镍柱纯化和复性后,用SDS-PAGE、ELISA和Western blot等方法对其检测分析。结果成功从一株抗人APPβ位点单克隆抗体细胞株2H10中扩增出VH和VL并拼接成ScFv片段,片段长744 bp,编码248个氨基酸。PCR、酶切和测序表明表达载体构建成功,转化E.coli BL21(DE3),经IPTG诱导可以表达出约29 ku的目的蛋白,主要为包涵体形式,经镍柱纯化和复性后,获得纯度达90%以上的ScFv蛋白,ELISA和Western blot检测表明可溶性ScFv可以与人APPβ分泌酶切割位点序列短肽和全长APP结合。结论成功构建并表达人APPβ分泌酶切割位点的特异性单链抗体,为进一步研究其生物学活性奠定基础。     

PCR定点诱变改造低效价抗胶质瘤单克隆抗体的初步报告

目的：抗胶质瘤单克隆抗体建株初期效价为1：10^5,经长期传代培养后效价下降至低于1：10^4,为探索其分子机制并将其改造为有活性的单链抗体,为构建靶向基因转移载体奠定基础。方法：通过RT－PCR克隆该单抗可变区基因,发现轻链可变区CDR1区出现一终止密码子,为此利用PCR定点诱变技术将其突变为该亚组相应的氨基酸,进一步构建单链抗体表达载体,并在大肠杆菌表达。结果：改造后的单链抗体能高效表达,Western blot和免疫荧光证实能与相应的膜抗原特异结合。结论：成功地改造并构建单链抗体,可用于胶质瘤靶向基因治疗。     

Receptor-mediated abeta amyloid antibody targeting to Alzheimer's disease mouse brain

The goal of this work is the reduction in the Abeta amyloid peptide burden in brain of Alzheimer's disease (AD) transgenic mice without the concomitant elevation in plasma Abeta amyloid peptide. An anti-Abeta amyloid antibody (AAA) was re-engineered as a fusion protein with a blood-brain barrier (BBB) molecular Trojan horse. The AAA was engineered as a single chain Fv (ScFv) antibody, and the ScFv was fused to the heavy chain of a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this fusion protein was designated cTfRMAb-ScFv. The cTfRMAb-ScFv protein penetrates mouse brain from blood via transport on the BBB TfR, and the brain uptake is 3.5% of injected dose/gram brain following an intravenous administration. Double transgenic APPswe,PSEN1dE9 mice were studied at 12 months of age. The mice were shown to have extensive Abeta amyloid plaques in cerebral cortex based on immunocytochemistry. The mice were treated every 3-4 days by intravenous injections of either saline or the cTfRMAb-ScFv fusion protein at an injection dose of 1 mg/kg for 12 consecutive weeks. The brain Aβ1??2 concentration was reduced 40% in the fusion protein treated mice, without any elevation in plasma Aβ1??2 concentration. No cerebral microhemorrhage was observed in the treated mice. These results show that brain-penetrating antibody pharmaceutics can be developed for brain disorders such as AD following the re-engineering of the antibody as a fusion protein that is transported across the BBB via receptor-mediated transport.     

Expression and characterization of human rheumatoid factor single-chain Fv

The variable region of heavy chain [V(H)] of human rheumatoid factor (hRF) IgM was connected with the variable region of light chain [V(L)] with the peptide-linker (GGGSGGGSGGGS) by genetic engineering method and the single-chain Fv (scFv) was expressed in E. coli. On design, scFv and scFv (tag) were planned; the latter had a detection marker at the carboxyl-terminal. These scFvs were expressed as inclusion bodies in E. coli, purified in the presence of 8 M urea by gel filtration and renatured to the active form in vitro. As a control, the Fv, non-covalently associated V(H) and V(L) fragments, was also constructed. The 3 derivatives showed almost the same binding activity to rabbit-IgG to which hRF is cross-reactive. ScFv (tag) was the most stable against urea among the 3 derivatives.     

梭曼单链抗体酶EP6一级结构的确定和三维结构的模建

以梭曼水解反应过渡态类似物O¹-甲基-O²-(1，2，2-三甲基丙基)-2-羟基-5-硝基苯基甲基膦酸为半抗原得到了梭曼单链抗体酶EP₆. 对这一株抗体进行了核苷酸序列的测定，测序结果表明这一单链抗体基因全长714 bp, 其中重链长351 bp，编码117个氨基酸，轻链长318 bp，编码106个氨基酸，二者由编码(Gly₄Ser)₃的45 bp的连接肽基因相连. 重, 轻链均为开放读框，有明确的四个框架区和三个互补决定区（CDR）以及抗体特征性的两个半胱氨酸残基，表明重，轻链的氨基酸序列符合鼠抗体可变区特征. 在SGI工作站上对EP₆的三维结构进行了同源模建. 计算机模型显示EP₆表面包含一个由重轻链CDRs围成的, 可能用于半抗原结合和催化活性的长裂缝，其中轻链的CDR3（L-CDR3）和重链三个CDRs(H-CDRs)都可能参与抗原或半抗原的接触，L-CDR3和H- CDR2的贡献较大，对抗体酶功能可能起重要作用.     

Immunotoxins in the treatment of hematologic malignancies

Immunotoxins, composed of protein toxins connected to cell binding ligands including monoclonal antibodies and growth factors, have been developed for several decades to target hematologic malignancies. Protein toxins from either plants or bacteria are extremely potent based on their enzymatic inhibition of protein synthesis and induction of apoptosis. Plant toxins, particularly ricin, are useful for chemically conjugating to monoclonal antibodies, and have shown clinical activity in several types of lymphoma and leukemia. Their dose is generally limited by vascular leak syndrome. Bacterial toxins have been used to produce single chain fusions with either growth factors or recombinant antibody fragments. These agents are smaller in size (55-65 kDa) and exit the bloodstream much more rapidly than the chemical conjugates, and generally do not cause severe vascular leak syndrome. The only approved drug containing a protein toxin is denileukin diftitox, a fusion of human interleukin 2 with truncated diphtheria toxin. Denileukin diftitox has shown efficacy in cutaneous T-cell lymphoma, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Recombinant immunotoxin BL22 is an anti-CD22 Fv fragment fused to truncated Pseudomonas exotoxin; it induces complete remissions in a high percentage of patients with chemoresistant hairy cell leukemia. The anti-CD25 recombinant immunotoxin LMB-2 is active in several CD25+ hematologic malignancies. Several other recombinant immunotoxins are undergoing preclinical development for other target antigens expressed on hematologic malignancies.     

Technology of compact MAb and its application for medicinal plant breeding named as missile type molecular breeding

Single chain fragment-variable (scFv) enhanced solasodine glycoside accumulation in Solanum khasianum hairy root cultures transformed by the ScFv solamargine (As)-scFv gene. The scFv protein was expressed at a high level in inclusion bodies of E. coli. After being renatured, the scFv protein was purified in a one-step manner by metal chelate affinity chromatography. The yield of refolded and purified scFv was 12.5 mg per 100 ml of cell culture. The characteristics of the As-scFv expressed in E. coli and transgenic hairy roots were similar to those of the parent monoclonal antibody (MAb). The expression of scFv protein provides a low cost and a high yield of functional scFv antibody against solamargine. The full linear range of the ELISA assay using scFv was extended from 1.5-10 μg/ml. The expressed anti-solamargine scFv protein could be useful for determination of total solasodine glycoside content in plant samples by ELISA. Solasodine glycoside levels in the transgenic hairy root were 2.3-fold higher than that in the wild-type hairy root based on the soluble protein level and binding activities. The As-scFv expressed in S. khasianum hairy roots enhanced solasodine glycosides accumulation and provide a novel medicinal plant breeding methodology that can produce a high yield of secondary metabolites.     

人源抗HBsAg单链抗体与α干扰素融合蛋白酵母表达载体的构建

目的构建以人HBsAg单链抗体(HBScFv)为导向作用、α干扰素为治疗作用的融合蛋白酵母表达载体pPICZ-αB/ScFv-IFN-α。方法通过重叠PCR构建目的蛋白质基因,再亚克隆到酵母表达载体pPICZαB中,DNA测序后,转化巴氏毕赤酵母宿主菌GS115 ,菌落PCR、高浓度Zeocin抗性筛选鉴定重组酵母,重组酵母经甲醇诱导后,通过SDS PAGE分析鉴定表达产物。结果DNA测序结果显示构建的目的基因片段(HBScFv IFN-α)的序列与原设计序列相符合;菌落PCR结果显示目的基因已整合到酵母菌中;诱导表达后,SDS PAGE结果显示在相对分子质量约4 4 0 0 0处可见目的蛋白质表达带。结论成功构建了抗HBsAg单链抗体与α干扰素融合蛋白酵母表达载体     

抗HBsAg单链抗体与白细胞介素-2融合蛋白的构建与序列测定

目的:构建以人抗乙肝表面抗原（HBsAg）单链抗体（ScFv）为导向作用,白细胞介素-2（IL-2）为治疗作用的融合蛋白原核表达载体pQE 40/ScFv-IL-2。方法:应用PCR技术将抗HBsAg单链抗体与白细胞介素-2在分子水平上进行基因重组,构建中间载体pGEM7Zf（+）/ScFv-IL-2,经酶切鉴定及序列测定正确后,将目的基因片段克隆到pQE 40表达载体中,转化大肠杆菌,经IPTG诱导表达。结果:SDS-PAGE结果显示在相对分子质量约4.3×104Da处,可见蛋白表达带。以鼠抗人IL-2单克隆抗体经免疫印迹验证,该表达蛋白为所设计的融合蛋白。结论:融合蛋白ScFv-IL-2的成功构建及表达,为融合蛋白的纯化和研究开发新型的乙肝导向治疗的基因工程药物打下良好的基础。