A novel anti‐prion protein monoclonal antibody and its single‐chain fragment variable derivative with ability to inhibit abnormal prion protein accumulation in cultured cells

mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121–231. Both mAbs were cross‐reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137–143 of MoPrP and buried in PrP^C expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP^Sc in cultured scrapie‐infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132–217 and this epitope was exposed on the cell surface PrP^C. mAb T2 showed an excellent inhibitory effect on PrP^Sc accumulation in vitro at a 50% inhibitory concentration of 0.02 μg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2‐producing N2a58 cells induced a clear inhibitory effect on PrP^Sc accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP^Sc accumulation.     

抗四环素单链抗体基因的构建与序列测定

目的：构建抗盐酸四环素的单链抗体（scFv）基因。方法：以抗盐酸四环素单克隆抗体杂交瘤细胞株的总RNA为模板,用RT-PCR法扩增全套抗体轻、重链基因;经重叠延伸反应,以编码柔性多肽（GIy4Ser）3的基因为接头,将轻、重链基因组装为完整的seFv基因,并克隆到pGEMT-Easy载体中进行测序分析。结果：所克隆的四环素scFV基因全长为735 bp,为VH-Linker-VL结构,VH基因为354bp,Linker为（Gly4Ser）3多肽的核酸序列,VL基因为336 bp。结论：构建了抗四环素的单链抗体基因,为进一步用于四环素的残留枪测奠审某础．     

Mammalian expression of single chain variable region fragments dimerized by Fc regions

To promote application of a single chain variable region fragment (sFv) in immunoglobulins, a sFv gene was connected to an IgG1 Fc gene, designated as a sFvc gene, and used for transfection of Sp2/0. As a result, the sFvc protein was found to be secreted in a dimeric form. It is thus felt that the sFvc protein, which mimicks the shape of a naturally occurring antibody, can be simple and useful to reproduce divalency and Fc-associated effecter functions as seen in a natural antibody.Abbreviations Abbreviations sFv single chain variable region fragment - Fc constant region of immunoglobulin - sFvc single chain variable region fragment with an Fc region     

单链抗体融合蛋白的构建及应用

通过重组DNA技术将单链抗体(scFv)基因与其他效应蛋白基因融合在一起,经表达后可以得到具有scFv特性和所融合的效应蛋白活性的scFv融合蛋白。这种融合蛋白已应用于许多领域的研究中,并已显示出较高的价值。本就scFv融合蛋白的构建和应用做一综述。     

Degradation of proteins upon storage at near-neutral pH: Indications of a proteolytic/gelatinolytic activity associated with aggregates

Background

The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation.

Methods

Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions.

Results

Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of ‘gelatinolytic’ activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity.

Conclusions

Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic.

General significance

The study suggests that a bi-directional cause–effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.     

噬菌体单链抗体文库的构建及筛选

噬菌体抗体是继多克隆抗体、单克隆抗体之后兴起的第3代基因工程抗体.噬菌体抗体库技术是抗体基因文库技术和噬菌体表面展示技术相结合形成的一项新技术与方法,在生物科学领域极具潜力.现主要就近年来该技术在抗体基因扩增、抗体库的构建、筛选方法等方面的进展进行综述.     

Assessment of the effect of triton X‐114 on the physicochemical properties of an antibody fragment

The effect of Triton X‐114 on the physicochemical properties of a single‐chain antibody fragment (scFv) has been studied. According to the far UV circular dichroism spectroscopy, the secondary structure of the recombinant antibody was not significantly affected by the presence of Triton. From the antibody tertiary structure analysis, it was found that the surfactant could be located around the tryptophan molecules accessible to the solvent, diminishing the polarity of its environment but maintaining most of the protein structure integrity. However, in certain conditions of high temperature and high concentration of denaturant molecules, the presence of TX could compromise the antibody fragment stability. These results represent a previous step in designing scFv purification protocols and should be considered prior to developing scFv liquid–liquid extraction procedures. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:554–561, 2014     

抗TNF-α单链抗体噬菌体展示文库的建立和鉴定

应用噬菌体展示技术构建抗肿瘤坏死因子α(tumornecrosis factor α,TNF-α)单链抗体(single chain Fv,scFv)文库,从中筛选抗TNF-αscFv并进行鉴定.利用重组人TNF-α(rhTNF-α)免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸反应将VH和VL基因拼接成scFv基因,以SfiⅠ/NotⅠ位点定向插入pCANTAB 5E噬菌粒载体,转化E.coli TG1,构建了库容为4.6×108的抗TNF-α单链抗体库.对抗体库进行3轮富集筛选后,ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析.结果表明,抗TNF-αscFv基因序列长774bp,编码258个氨基酸.将此阳性克隆转化E.coliHB2151,IPTG诱导可溶性scFv的表达,经SDS-PAGE和Western印迹分析,scFv的分子量约为28kD.经亲和纯化后的scFv可与rhTNF-α结合,并可中和由rhTNF-α引起的L929细胞毒性.本文利用噬菌体抗体库筛选到了高亲和力的抗TNF-αscFv,为研制临床免疫治疗的新型抗体奠定了实验基础.     

抗CD5单链抗体基因克隆和表达

以分泌抗CD5单克隆抗体的杂交瘤细胞poly(A) mRNA为模板,通过RTPCR扩增出抗CD5单克隆抗体的重链可变区(VH)和轻链可变区(VL) cDNA片段组装出抗CD5单链抗体(ScFv) cDNA片段。该ScFv片段被克隆到pCANTAB 5E载体上,以E.coli TG1为宿主,进行噬菌体表面呈现。通过Molt4细胞表面分子CD抗原,对噬菌体表面呈现的ScFv进行免疫亲和富集筛选。经细胞ELISA鉴定,得到4株高亲和力克隆。DNA序列分析得知,单链抗体全长732碱基,其中VH为339碱基,VL为300碱基。抗CD5 ScFv在E.coli HB2151中以可溶形式分泌表达,产物主要分布于周质之中,占周质中总蛋白的20%。     

利用植物质体转基因技术高效表达抗人源白介素6单链抗体

白介素6 (interleukin-6, IL-6)是与包括癌症在内的多种疾病相关的一种多功能细胞因子,免疫疗法利用抗人源IL-6抗体来治疗相关疾病取得了很好的治疗效果。植物作为生物反应器可以有效降低药用蛋白的生产成本,同时积累功能蛋白。通过基因枪轰击和再生筛选,得到了2个在烟草质体中表达了鼠源抗人源IL-6单链抗体(single chain variable fragment, scFv)的独立株系,并用Southern blotting鉴定了质体转化烟草的同质化状态。抗人源IL-6 scFv基因在质体转基因烟草中成功转录和翻译,功能性抗人源IL-6 scFv在质体转基因烟草叶片中的含量占到总可溶性蛋白的1%,达到41 mg/kg鲜重。另外,质体转基因烟草的表型与野生型烟草相比并没有显著差异,它们具有相似的生长速率、成熟植株的株高以及果荚数目。抗人源IL-6 scFv的高表达量也表明了利用质体转基因植物低成本生产scFv的潜力。     

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.     

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1–14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1–14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1–14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1–14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.