Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( 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 anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris

BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING： A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital （Beijing, China） from January to July 2006. MATERIALS： Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS： Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES： Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS： Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION： The expression vector pPIC9K-scFvAβ was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris.

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

In order to enhance the glutathione peroxidase（GPX） catalytic activity of the selenium-containing single-chain variable fragments（Se-scFv）, a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional（3D） model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b（＋）, then the scFv protein was expressed in soluble form in Escherichia coli BL21（DE3） and purified by Ni2＋-immobilized metal affinity chromatography（IMAC）. The serine residue of scFv in the active site was converted into selenocysteine（Sec） with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.

Correctness and accuracy of template-based modeled single chain fragment variable (scFv) protein anti-breast cancer cell line (MCF-7)

Multiple sequence alignments can be used in the template-based modelling of protein structures to build fragment-based assembly models. Therefore, useful functional information on the 3D structure of the anti-MCF-7 scFv protein can be obtained using available bioinformatics tools. This paper utilises several commonly-used bioinformatics tools and databases, including BLAST (Basic Local Alignment Search Tool), GenBank, PDB (Protein Data Bank), KABAT numbering and SWISS-MODEL, to gain specific functional insights into the anti-MCF-7 scFv protein and the assembly of single-chain fragment variable (scFv) antibodies, which consist of a variable heavy chain (VH) and a variable light chain (VL) connected by the linker (Gly4-Ser)3. The linker has been built as a loop structure using the Insight II software. The accuracy of the loop structure has been evaluated using Root Mean Square Deviation (RMSD). The accuracies of the VL and VH template-based structures are enhanced by using the evaluation methods Verify3D, ERRAT and Ramchandran plotting, which measure the error in the residues. In the results, 100% of the light-chain residues scored above 0.2, whereas 88.5% of the heavy-chain residues’ scored above 0.15 in the Verify3D evaluation method. Meanwhile, using ERRAT, the alignments of both chains scored more than 70% in space. Additionally, the Ramchandran plot evaluation method showed large numbers of residues in the favoured areas in both chains;these findings demonstrated that all of the chosen templates were the best candidates.

Single Chain Fragment Variables Antibody binding to EGF Receptor in the Surface of MCF7 Breast Cancer Cell Line: Application and Production Review

In this review, single-chain fragment variable construction using phage-display technology as a promising anticancer immunotherapy technology is described. Cloning and the specific bio-panning selection with phage display technology, as well as the use of the epidermal growth factor receptor (EGFR) at the surface of MCF-7 cells as the antigen for the straightforward specific selection of single chain Fvs, are discussed. Moreover, phage display technologies and their application are important for vaccine production and immunotherapy against viruses and cancers. Furthermore, expression of the gene will cause the production and expression of the protein in prokaryotic and eukaryotic cells, which can be used to detect anti-cancer single chain fragment variables (scFvs). Finally, homology modelling is described to show the three-dimensional scFv structure that verifies the Complementary-Determining-Regions (CDRs) on the surface of the model.

Antigenic analysis of grass carp reovirus using single-chain variable fragment antibody against IgM from Ctenopharyngodon idella

Grass carp （Ctenopharyngodon idella） is an important species of freshwater aquaculture fish in China. However, grass carp reovirus （GCRV） can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic ca- pacity of the virus＇s structural proteins for preparing an effective vaccine has not been available. In this study, some sin- gle-chain variable fragment antibodies （scFv）, which could specifically recognize grass carp IgM, were selected from a con- structed mouse naive antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from vi- rus-infected grass carp was more than two times higher than that of the control group at 5-7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.

Context Information and Fragments Based Cross-Domain Word Segmentation

A new joint decoding strategy that combines the character-based and word-based conditional random field model is proposed.In this segmentation framework,fragments are used to generate candidate Out-of-Vocabularies(OOVs).After the initial segmentation,the segmentation fragments are divided into two classes as "combination"(combining several fragments as an unknown word) and "segregation"(segregating to some words).So,more OOVs can be recalled.Moreover,for the characteristics of the cross-domain segmentation,context information is reasonably used to guide Chinese Word Segmentation(CWS).This method is proved to be effective through several experiments on the test data from Sighan Bakeoffs 2007 and Bakeoffs 2010.The rates of OOV recall obtain better performance and the overall segmentation performances achieve a good effect.

Screening and identification of human Zn T8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library

Zinc transporter 8 （ZnT8） is a major autoantigen and a predictive marker in type 1 diabetes （T1D）. To investigate ZnT8-specific antibodies, a phage display library from TID was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment （scFv） phage display library consists of approximately 1 ~ l0s clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Topl0F＇ and then purified by affin- ity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

Energy Dependence of String Fragmentation Function and Ф Meson Production

The meson productions in Au+Au and/or Pb+Pb collisions at AGS, SPS, RHIC, and LHC energies have been studied systematically with a hadron and string cascade model LUCIAE. After considering the energy dependence of the model parameter α in string fragmentation function and adjusting it to the experimental data of charged multiplicity to a certain extent, the model predictions for meson yield, rapidity, and transverse mass distributions are compatible with the experimental data at AGS, SPS and RHIC energies. A calculation for Pb+Pb collisions at LHC energy is given as well. The obtained fractional variable in string fragmentation function shows a saturation in energy dependence. It is discussed that the saturation of fractional variable in string fragmentation function might be a qualitative representation of the energy dependence of nuclear transparency.

应用噬菌体展示随机12肽库筛选诺如病毒抗原模拟表位

目的:利用Ph.D.-12噬菌体展示肽库筛选诺如病毒(NoV)的抗原模拟表位。方法:包被与GⅡ.4、GⅡ.6、GⅡ.17型NoV具有高特异性及中和能力较强的单链可变片段抗体(scFv),用Ph.D.-12噬菌体展示肽库进行3轮生物淘选。ELISA鉴定淘选所得噬菌体与scFv的结合活性及其与NoV P蛋白的竞争作用;阳性克隆测序后进行生物信息学分析,合成多肽鉴定其抗原性。结果:发现1段与GⅡ.6 VP1区同源性较高的氨基酸序列“MG-D-W”,综合分析提示其可能为GⅡ.6 NoV的抗原模拟表位,且合成的包含“MG-D-W”的多肽可竞争抑制P蛋白与人类组织血型抗原(HBGAs)受体的结合。结论:“MG-DW”是与NoV单链抗体高亲和力的肽段,可能模拟了GⅡ.6 NoV与scFv结合的抗原表位。

森林破碎化的社会经济驱动力分析——以美国阿拉巴马州为例

以基于植被变化追踪遥感模型所生成的1999年森林干扰产品为基础,采用森林破碎化分析模型在5×5窗口分析尺度下将美国阿拉巴马州森林归并为内部、孔洞、边界、斑块、过渡及未确定6种破碎化成分。同时,以行政县为单位采集森林像元的平均高程、森林像元的平均坡度、居民受教育程度、人口密度及人均收入等与破碎化成分建立统计关联。结果表明:除平均坡度外,其余4因子均与森林破碎化成分显著相关,人口密度、人均收入及受教育程度是阿拉巴马州森林破碎化重要的社会经济驱动力;所识别的驱动因子对不同的破碎化成分空间变动的解释能力不一,其中对于孔洞森林变异的解释能力最强,达65.8%。统计检验表明所建立的破碎化驱动力模型对于归纳阿拉巴马州森林破碎化成因是有效和可靠的。

伏马菌素B_1特异单链抗体的同源建模及分子对接模拟研究

目的分析伏马菌素B1(Fumonisin B1,FB1)与其特异单链抗体的分子互作模式。方法通过同源建模构建和优化抗FB1单链抗体的三维结构,结合Procheck和Verify 3D等方法评价得到稳定的抗体模型,利用分子对接研究单链抗体与其抗原FB1的结合特性、疏水性和表面静电力作用。结果单链抗体的互补决定区参与同其抗原FB1的结合,抗体与抗原之间不仅形成稳定的氢键,疏水性和静电力的匹配也很好。结论氢键结合力、分子间疏水相互作用和静电力的共同作用,使得单链抗体形成一个能够与FB1高度互补的相互作用区域,并在抗体与抗原的特异性识别及结合稳定性等方面起着关键作用。

表达于大肠杆菌中的人源性抗CTLA4单链抗体复性方法探索

探索表达于大肠杆菌中的人源性抗CTLA4单链抗体(Anti-CTLA4-scFv)的体外复性方法.采用稀释和透析两种复性方式,并分析影响复性得率的各种因素如复性时间、温度、适宜的氧化-还原体系对其的影响.通过非还原电泳确定复性效果,并测定蛋白含量.结果显示:含0.15 mol·L-1NaCl、1 μmol·L-1氧化型谷胱甘肽和3μmol·L-1还原型谷胱甘肽的50 mmol·L-1Tris-HCl(pH 8.0)缓冲液作为Anti-CTLA4-scFv的复性液,4℃下透析复性48～54 h,可获得具有天然构象的蛋白质.该方法复性蛋白得率高,从3.9 g菌体中可复性获得28 mg蛋白.

具有GPX活性单链抗体的制备及其抗氧化效应

为实现单链抗体的可溶性表达,制备具有谷胱甘肽过氧化物酶(GPX)活力的单链抗体,在单链抗体表达载体pTMF 2F3上去除原2F3基因N端非必需的18个氨基酸,采用新的表达载体pRose质粒,在2F3的N端引入13个氨基酸的前导肽,转入BL21(lysS)中,使其分泌到大肠杆菌的周质腔中得以表达,获得可溶性表达产物.产物经过分离纯化、WesternBlot印迹和硒化测活确定具有GPX活性的鼠单链抗体,其GPX活力为2530U/μmol.以脂质过氧化、细胞存活率和细胞膜完整性为指标的抗氧化实验研究表明,具有GPX活性的单链抗体对大鼠乳鼠表皮细胞有抗紫外线损伤的作用,是膜脂质过氧化的有效抑制剂.

抗镰刀菌单链抗体在大肠杆菌中可溶性表达条件的研究

通过优化诱导表达条件,实现抗镰刀菌单链抗体在大肠杆菌周质中高效可溶性表达。将抗镰刀菌单链抗体FvSG7转化到大肠杆菌XL1-Blue中,确定诱导表达培养基,在不同的诱导温度、诱导剂IPTG的浓度和诱导时间条件下培养重组大肠杆菌,通过Western杂交和ELISA检测分析单链抗体的可溶性表达情况以及抗体的活性。重组大肠杆菌培养至OD600nm为0.5时加入终浓度为0.1 mmol/L IPTG,25℃诱导表达2 h可获得最大量的可溶性单链抗体。通过对诱导温度、IPTG浓度和诱导时间等表达条件的优化,可以显著提高Fv SG7抗体在大肠杆菌XL1-Blue周质中的表达量。

大肠杆菌表达的人源性抗CTLA4单链抗体三种复性方法比较

比较表达于大肠杆菌中的人源性抗细胞毒性T淋巴细胞抗原4单链抗体(A n ti-CTLA 4 scFv)的三种体外复性方法的复性效率。稀释、透析和亲和柱上三种复性方式复性A n ti-CTLA 4 scFv,采用B rad ford法分析蛋白复性得率,采用间接细胞EL ISA法检测所得蛋白的活性。透析复性蛋白得率最高,稀释复性蛋白得率其次,亲和柱上复性蛋白得率最低;透析复性所得蛋白结合活性是稀释复性的1.95倍,是亲和柱上复性所得蛋白活性的4.13倍(无谷胱甘肽氧化还原对G SH/G SSH)及3.63倍(有G SH/G SSH)。结论:采用含0.15 m o l/L N aC、l1 mm o l/L G SSH和3 mm o l/L G SH的50 mm o l/L T ris-HC l(pH 8.0)缓冲液作为A n ti-CTLA 4 scFv的复性液,4℃下透析复性48 h,可获得较高复性得率和结合活性。

人源化抗Cry1B毒素蛋白单链抗体的原核表达及生物学活性测定

利用含有重组噬菌粒的噬菌体直接侵染大肠杆菌(Escherichia coli)HB2151,原核分泌表达了抗苏云金芽孢杆菌(Ba-cillus thuringiensis,Bt)Cry1B毒素蛋白的单链抗体(single chain-variable fragment,scFv),经纯化、鉴定抗原结合活性后,建立了Cry1B毒素蛋白的ELISA检测方法。方法以展示scFv抗体的重组噬菌体感染E.coli HB2151,经PCR和基因测序检测克隆scFv基因片段的完整性,用SDS-PAGE方法检测scFv在E.coli HB2151宿主菌中的表达水平,用ELISA测定法检测scFv的抗原结合活性,通过时间梯度优化获得可溶性表达蛋白的最佳培养时间。结果表明:经PCR、DNA电泳及基因测序等,均证实重组噬菌体对E.coli HB2151宿主菌侵染成功,SDS-PAGE电泳结果表明scFv抗体表达成功,纯化后的蛋白质量浓度为132μg.mL-1。以纯化的scFv蛋白为基础建立了对Cry1B毒素蛋白的间接竞争ELISA法,方法的抑制中质量浓度(IC50)为1.398μg.mL-1,最低检测限(IC10)为0.025 7μg.mL-1,线性检测范围为0.5～5.0μg.mL-1,scFv对Cry1C的交叉反应率为7.51%;与Cry1Ab、Cry1Ac的交叉反应率均小于0.1%;在培养温度30℃,1 mmol.L-1IPTG诱导条件下,scFv在E.coliHB2151宿主中的最佳诱导表达时间为12 h。本研究成功地将抗Cry1B毒素蛋白的scFv在E.coli HB2151中进行了可溶性表达,获得了具有抗原结合活性的scFv融合型抗体,为实际生产应用与试剂盒研发提供了基础。

抗癌胚抗原单链抗体的原核表达及对人胃癌的检测

目的:探讨T84．66单链抗体的原核表达及对6 种胃癌细胞系及胃癌组织的特异性亲和力．方法:将抗CEA单链抗体T84．66的cDNA插入噬菌粒载体pcANTAB5E,获得噬菌粒载体T84．66-scFv-pCANTAB5E．将后者转化入 E．coli HB2151,经β,D异丙基硫代半乳糖苷 (IPTG)诱导表达．采用细胞培养及免疫细胞／组织化学方法,检测胃癌细胞中及石蜡包埋的胃癌组织中的癌胚抗原表达．结果:SDS-PAGE及Western blot证实,T84．66 单链抗体蛋白分子正确表达．T84．66单链抗体可结合KATOIII,HGC-27和MKN45,表明这3种细胞表达了特异性肿瘤抗原;单链抗体不能结合SGC7901,GC803,BGC823．42例胃癌组织癌胚抗原阳性率早期和进展期分别为55％(6／11)和61％(19／31),在正常胃黏膜组织标本中无表达,二者之间存在显著性差异 (P<0．05)．结论:KATOIII等胃癌细胞系可表达癌胚抗原．后者在胃癌组织表达水平较高,而正常胃组织不表达．