抗人CD3单链抗体四聚体基因的构建及表达

为构建和表达抗人CD3单链抗体 (scFv) 人p5 3四聚功能域融合基因 ,选用人IgG3上游铰链区作为抗人CD3scFv和人p5 3四聚功能域之间连接的linker .利用递归PCR法扩增人IgG3上游铰链区与人p5 3四聚功能域融合基因 ,克隆入pUC18载体中构建pUC18 IgG3 p5 3克隆载体 .将抗人CD3scFv克隆入pUC18 IgG3 p5 3载体中 ,构建抗人CD3scFv 人p5 3四聚功能域融合基因 .经酶切鉴定及序列测定证实后 ,将融合基因克隆入真核表达载体pSecTag2 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亲和活性测定 .获得了抗人CD3scFv 人p5 3四聚功能域融合基因 ,基因全长 882bp ,可编码 2 94个氨基酸 ,与已发表的抗人CD3scFv、人IgG3上游铰链区和人p5 3四聚功能域基因cDNA序列一致 .表达产物经SDS PAGE和Western印迹实验证实为约 35kD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人外周血单个核细胞 (PBMC)细胞 ,亲和力高于scFv ,为进一步临床应用奠定基础     

基于狂犬病病毒G蛋白scFv介导的靶向shRNA制备与鉴定

【目的】探讨以狂犬病病毒G糖蛋白单链抗体介导的载体表达shRNA靶向制剂,靶向抑制狂犬病毒复制的可行性。【方法】应用PCR技术获得狂犬病毒G糖蛋白单链抗体scFv(G)和绿脓杆菌跨膜区-酵母DNA结合结构域ETA-GAL4基因,通过搭桥PCR法获得scFv(G)-ETA-GAL4(SEG)嵌合基因;克隆至原核表达载体pET28a(+),构建重组表达质粒pET28a(+)-scFv(G)-ETA-GAL4(pET28a-SEG);在大肠杆菌BL21(DE3)中经IPTG诱导表达,利用镍柱亲和层析法纯化包涵体,经复性、鉴定制得SEG蛋白;ELISA法检测表达蛋白与狂犬病毒特异结合活性;将SEG蛋白与含shRNA的质粒(pRNATU6.3-shRNA)连接制成靶向shRNA,接入100 TCID50狂犬病毒感染BHK-21细胞,35 h观察细胞中绿色荧光蛋白(GFP)表达情况;48 h用直接免疫荧光抗体试验测定复合物抑制病毒效果。【结果】克隆得到1557 bp的SEG蛋白编码基因,大肠杆菌中成功表达57 KDa的SEG蛋白,能与抗His的单克隆抗体发生特异性反应,SEG蛋白经镍柱纯化、复性后得率为2.8 mg/mL。ELISA试验证明SEG蛋白在一定浓度范围内与RV结合呈正相关。细胞试验表明GFP在细胞内得到表达;直接免疫荧光试验测定该复合物能抑制76%病毒复制。【结论】SEG蛋白能与携带shRNA的质粒结合,可运送该质粒至RV感染BHK-21细胞中,抑制狂犬病毒的复制。     

Rapid Purification of a New Humanized Single-chain Fv Antibody／Human Interleukin-2 Fusion Protein Reactive against HER2 Receptor

Overexpression of the proto-oncogene c-erbB-2, neuor HER2 has been shown in many human tumor cells,especially in breast cancer cells [1–5], which is thought tobe important in human carcinogenesis. c-erbB-2 geneencodes a 185 kD transmembrane glycoprotein …     

抗人整合素αvβ3-组织因子融合蛋白基因的构建和表达

目的:利用基因工程技术构建和表达抗人整合素αvβ3单链抗体(scFv)与人可溶性组织因子(sTF)的融合蛋白scFv-sTF。方法:用重叠延伸PCR技术扩增scFv基因,同时用组装PCR方法人工合成sTF基因,然后以酶切连接方式融合2个基因,并克隆至原核表达载体pQE80L中,构建表达质粒pQE80L-scFv-sTF,以重组子转化大肠杆菌M15诱导目的基因表达。结果:SDS-PAGE分析显示工程菌可以表达相对分子质量约55×103的融合蛋白scFv-sTF,Western印迹分析证实表达的目的蛋白具有6×His标签;经低剂量IPTG诱导和较低温度培养,scFv-sTF获得了可溶性表达;纯化回收目的蛋白,ELISA试验证实,该重组抗体分子具有良好的抗原结合活性。结论:构建并表达了抗人整合素αvβ3的单链抗体融合蛋白scFv-sTF,并验证了其抗原结合活性,初步证明它可以与抗原特异性结合,为进一步研究其抗肿瘤作用打下了基础。     

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.     

猪丹毒丝菌C43065株表面保护性抗原AN端保护区在大肠杆菌中的表达

目的：在大肠杆菌中表达猪丹毒丝菌C43065株表面保护性抗原A（SpaA）N端保护区（SpaA-N）,并检测其抗原性。方法：利用PCR方法从猪丹毒丝菌C43065株基因组中扩增出spaA基因片段,构建pMD18-spaA重组质粒并对插入片段进行测序;以pMD18-spaA重组质粒为模板,PCR扩增得到spaA-N基因片段,构建重组表达质粒pGEX-spaA-N,经序列测定证实正确后转化大肠杆菌BL21（DE3）,再经IPTG诱导表达GST-SpaA-N融合蛋白并纯化。结果：扩增得到的spaA基因长1881bp,编码由626个氨基酸残基构成的多肽;SDS-PAGE和Western印迹检测结果表明,诱导表达获得相对分子质量约64000的GST-SpaA-N融合蛋白,该融合蛋白能与相应抗体发生特异性反应。结论：获得了在大肠杆菌中可溶性表达的GST-SpaA-N融合蛋白,为进一步研究猪丹毒丝菌免疫保护性抗原奠定了基础。     

A recombinant anti-erbB2, scFv-Fc-IL-2 fusion protein retains antigen specificity and cytokine function

Elevated erbB2 expression is detected in many in situ and invasive human ductal carcinomas. Anti-erbB2 antibody directed at the extracellular domain of erbB2 can result in an antitumor response in some patients with tumors overexpressing erbB2 oncoprotein. By combining interleukin 2 (IL-2) activities with a tumor specific antibody, immunotherapy of tumors might be more effective in the future. In this study, a fusion protein consisting of erbB2 single chain antibody (scFv), Fc fragment of human IgG1 and IL-2 was constructed. The molecular weight of fusion proteins is 66 kDa, only one third of whole antibody-IL-2 fusion protein or 44% whole Ig molecule. The fusion proteins retained the activities of both antigen binding and IL-2. The scFv-Fc-IL-2 fusion protein may have advantages over whole antibody-IL-2 fusion proteins, such as smaller molecule, better activity of penetration, more favorable pharmacokinetic properties.     

Baculoviral display of functional scFv and synthetic IgG-binding domains

Viral vectors displaying specific ligand binding moities such as scFv fragments or intact antibodies hold promise for the development of targeted gene therapy vectors. In this report we describe baculoviral vectors displaying either functional scFv fragments or the synthetic Z/ZZ IgG binding domain derived from protein A. Display on the baculovirus surface was achieved via fusion of the scFv fragment or Z/ZZ domain to the N-terminus of gp64, the major envelope protein of the Autographa californica nuclear polyhedrosis virus, AcNPV. As examples of scFv fragments we have used a murine scFv specific for the hapten 2-phenyloxazolone and a human scFv specific for carcinoembryonic antigen. In principle, the Z/ZZ IgG binding domain displaying baculoviruses could be targeted to specific cell types via the binding of an appropriate antibody. We envisage applications for scFv and Z/ZZ domain displaying baculoviral vectors in the gene therapy field.     

Improved expression characteristics of single-chain Fv fragments when fused downstream of the Escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain

The expression of single-chain Fv fragments (scFv) targeted to the periplasm of Escherichia coli often results in very low yields of soluble protein frequently accompanied by host cell growth arrest and sometimes lysis. Single-chain antibody fragments (scAb) are scFv with a human kappa light chain constant (HuCkappa) domain attached C-terminally and share similar problems of expression. By fusing the E. coli maltose-binding protein (mbp) gene either 3' or 5' to a scAb specific for the herbicide atrazine, a reduction in growth arrest was observed that was dependent on the order of gene fusion. The scAb-mbp fusion delayed the onset of growth arrest following induction while the mbp-scAb fusion appeared to ablate growth arrest completely. Cell fractionation revealed barely detectable levels of scAb-mbp in the periplasm while mbp-scAb was detected at equivalent levels as scAb in the periplasmic compartment, indicating that periplasmic scAb solubility is unrelated to propensity to cause growth arrest. IMAC purification of scAb and mbp-scAb proteins followed by liquid competition ELISA revealed the IC(50) for atrazine to be approximately 1 nM for both proteins demonstrating that 5'-mbp fusion does not alter antigen binding. The equivalent scFv and mbp-scFv vectors expressed far less material in both periplasmic and insoluble fractions indicating that the HuCkappa domain can have a positive effect on scFv expression when expressed either alone or as a mbp fusion. The ablation of growth arrest by a 5'-mbp fusion and enhancement of expression by a 3'-HuCkappa domain fusion were extended to a second scFv specific for the herbicide diuron. Therefore, by expressing scFv as tripartite fusions (mbp-scFv-HuCkappa) enhanced levels of soluble periplasmic expression can be achieved without causing growth arrest of the host cell, realizing the potential for constitutive expression of hapten-binding scFv in the E. coli periplasm.     

Anti-CD3 scFv-B7.1 fusion protein expressed on the surface of HeLa cells provokes potent T-lymphocyte activation and cytotoxicity.

The targeting of tumor cells by cytotoxic T lymphocytes is a promising strategy for biotherapy, but T cells require 2 signals via the T-cell receptor - CD3 complex and CD28 molecules for activation. To bridge the gap between cytotoxic T lymphocytes and tumor cells, our objective in this study was to describe the construction and the cell surface-anchored expression of a fusion protein, anti-CD3 scFv-B7.1, derived from inserting a fusion gene encoding anti-CD3 scFv and the extra-cellular domain of B7.1 fused by the splicing by overlap extension method into a mammalian expression vector, pDisplay. Transfection of the recombinant vector by electroporation into HeLa cells resulted in the production of protein migrating at approximately 57 kDa under reducing conditions. The expressed fusion protein could bind to T lymphocytes and induce strong T-cell activation. Meanwhile, a potent cytotoxicity was induced in the mixed culture of T-cell-modified tumor cells in a 96 h methyl-thiazolyl-diphenyl tetrazolium bromide assay. Our results indicate that this bifunctional protein, through activating T lymphocytes to lyse homologous human carcinomas, may be of potential value for T-cell-based immunotherapeutical treatment protocols in vivo.     

A yeast platform for the production of single-chain antibody-green fluorescent protein fusions

Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 microg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)3 linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.     

A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions

Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 μg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)₃ linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.     

Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater.

Comamonas acidovorans YM1609 secreted a polyhydroxybutyrate (PHB) depolymerase into the culture supernatant when it was cultivated on poly(3-hydroxybutyrate) [P(3HB)] or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] as the sole carbon source. The PHB depolymerase was purified from culture supernatant of C. acidovorans by two chromatographic methods, and its molecular mass was determined as 45,000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures below 37 degrees C and at pH values of 6 to 10, and its activity was inhibited by diisopropyl fluorophosphonate. The liquid chromatography analysis of water-soluble products revealed that the primary product of enzymatic hydrolysis of P(3HB) was a dimer of 3-hydroxybutyric acid. Kinetics of enzymatic hydrolysis of P(3HB) film were studied. In addition, a gene encoding the PHB depolymerase was cloned from the C. acidovorans genomic library. The nucleotide sequence of this gene was found to encode a protein of 494 amino acids (M(r), 51,018 Da). Furthermore, by analysis of the N-terminal amino acid sequence of the purified enzyme, the molecular mass of the mature enzyme was calculated to be 48,628 Da. Analysis of the deduced amino acid sequence suggested a domain structure of the protein containing a catalytic domain, fibronectin type III module as linker, and a putative substrate-binding domain. Electron microscopic visualization of the mixture of P(3HB) single crystals and a fusion protein of putative substrate-binding domain with glutathione S-transferase demonstrated that the fusion protein adsorbed strongly and homogeneously to the surfaces of P(3HB) single crystals.     

Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures

A synthetic gene encoding an anti-phytochrome single-chain Fv (scFv) antibody bearing an N-terminal signal peptide has been used to transform tobacco plants. Immunoblot analysis showed that transformed plants accumulate high levels of scFv protein, accounting for up to 0.5% of the total soluble protein fraction, which could be extracted by simple infiltration and centrifugation of leaf tissue. A substantial proportion of the scFv protein extracted in this way was found to possess antigen-binding activity. Callus cell suspension cultures derived from transformed plants secrete functional scFv protein into the surrounding medium. Compared with the levels of scFv protein observed in plants expressing the native scFv gene, the incorporation of an N-terminal signal peptide, to target the scFv to the apoplast, results in elevated accumulation of the protein.     

两种人源化单链抗体-尿激酶融合基因的构建与表达

Ｓｃｕ ＰＡ 32Ｋ是单链尿激酶 (Ｓｃｕ ＰＡ)分子的Ｎ端肽段被水解后的产物 ,其分子量小但具有与Ｓｃｕ ＰＡ相同的体内外生物活性[1] 。在过去的工作中 ,本实验室利用噬菌体表面呈现技术筛选到 1株对人纤维蛋白特异的鼠源单链抗体[2 ] ,并构建了鼠抗人交联纤维蛋白单链抗体—Ｓｃｕ ＰＡ 32Ｋ融合基因。为解决该融合基因在大肠杆菌中的高表达问题 ,通过在大肠杆菌中表达构建的一系列融合基因的缺失突变体 ,初步认定Ｓｃｕ ＰＡ 32Ｋ基因中两个连排的大肠杆菌稀有密码子ＡＧＧ(精氨酸 )是影响该融合基因表达的主要因素。用ＰＣＲ定位诱变法…     

具有谷胱甘肽过氧化物酶活性的含硒单链抗体酶制备

 利用RT PCR从分泌有谷胱甘肽结合部位的单克隆抗体杂交瘤细胞株 2F3中 ,扩增出单抗重链可变区和轻链可变区基因 .经DNA测序后 ,用Linker(Gly4 Ser1) 3 构建成单链抗体 (scFv)表达载体pTMF scFv ,将重组质粒pTMF scFv转化到大肠杆菌BL2 1(DE3) ,实现了单链抗体的高效表达 .表达的单链抗体占菌体总蛋白 2 5%～ 30 % .该重组蛋白以包涵体形式存在 ,分子量为 30kD .经过金属螯合亲和层析纯化、复性和凝胶过滤纯化 ,得到电泳均一的单链抗体 .再经化学诱变 ,得到含硒单链抗体酶 ,其谷胱甘肽过氧化物酶活性为 330 0U μmol.采用荧光滴定法测定了单链抗体对谷胱甘肽的结合常数     

Biological effects of anti-ErbB2 single chain antibodies selected for internalizing function

Two internalizing monovalent single chain antibody fragments (scFv), C6.5 and F5, that recognize distinct ErbB2 extracellular domain (ECD) epitopes, and their bivalent forms dbC6.5 and F5(scFv')(2), were compared to the growth-inhibiting anti-ErbB2 antibody Herceptin/trastuzumab, in either its bivalent (Her) or monovalent (4D5Fab') form, for their abilities to induce biological responses in the ErbB2-overexpressing breast cancer cells, SkBr-3. Assays compared internalization by receptor-mediated endocytosis, effects on cell cycling and culture growth, and interference with intracellular MAPK and PI3K signaling pathways. We found no correlation between ErbB2 epitope affinity or valency on degree of antibody-induced endocytosis, since all the scFv were able to internalize better than Her. Unlike Her, neither the monovalent or bivalent forms of the internalizing scFv had any sustained effect on cell growth. Basal levels of MAPK and PI3K signaling in SkBr-3 cells were not inhibited by up to 8 h scFv treatment, while decreased MAPK and PI3K signals were noted within 8 h of Her treatment. In summary, antibody-induced ErbB2-mediated endocytosis is not a surrogate marker for resultant biological response, as it shows no correlation with cell cycle, culture proliferation, or intracellular kinase signal induction by internalizing antibodies. Thus, the enhanced endocytotic property of scFv like C6.6 and F5 in conjunction with their absence of any growth or signaling impact on ErbB2-overexpressing cells favors their choice as ErbB2 targeting moieties for intracellular delivery of novel cancer therapeutics.