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Isolation of scfv antibodies of rP27^(Kip1) from phage display libraries constructed from immunized and non-immunized repertoires

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Science China(Life Sciences) 1999年第4期42卷 376-382页

作者：曹跃琼乔守怡袁有忠黄建生赵寿元 1. State Key Laboratory of Genetic Engineering Institute of Genetics Fudan University 200433 Shanghai China 2. Department of Hematology Second Military Medical University 200433 Shanghai China

Through mRNA extract, RT and a series of PCR, phage antibody libraries were constructed from rP27kip1-immunized and non-immunized mice. After only one round of selection with rP27kip1, clones from each library were chosen randomly and digested by Tag I and Hinf I. 11 of 64 clones from the immunized animal had consistent restriction pattern, while none of the 64 clones from the non-immunized animal had, except that one had the same fragments pattern as that of the 11 clones. The 12 fragments were expressed in E. coli BL2l(DE3)/pET-28b( + ) system. ELISA showed that some of the fragments could bind to rP27kip1 specifically. All these results implied that specific antibody can be obtained by genetic engineering without hybridoma or many rounds of growth and panning selection.

关键词： rP27^(Ktp1) phage antibody libraries scfv expression in E. coli.

Antitumor effects of an engineered and energized fusion protein consisting of an anti-CD20 scfv fragment and lidamycin

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Science China(Life Sciences) 2011年第3期54卷 255-262页

作者： FANG Hong MIAO QingFang ZHANG ShengHua CHENG Xin XIONG DongSheng ZHEN YongSu Institute of Medicinal Biotechnology Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China Institute of Hematology & Hospital of Blood Diseases Chinese Academy of Medical Sciences and Peking Union Medical College Tianjin 300020 China Cancer Institute & Hospital Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100021 China

Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other ***20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma,is an attractive target for the therapy of B-lymphoid *** (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical *** this study,we prepared an engineered fusion protein,scfv-LDP,consisting of an anti-CD20 scfv fragment and the apoprotein LDP of LDM using DNA *** purification and refolding,scfv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining *** energized fusion protein scfv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and *** assay revealed potent cytotoxicity of scfv-LDP-AE to CD20-positive Raji and Daudi cells,with IC 50 values of 1.21×10-11 and 6.24×10-11 mol L-1,*** in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also *** were given intravenously on day 14 and 21 after tumor *** terms of maximal tolerated doses,scfv-LDP-AE at 0.3 mg kg-1 suppressed tumor growth by 79.3%,and LDM at 0.05 mg kg-1 by 68.6% (Pscfv-LDP-AE could be a potential candidate for tumor-targeting therapy.

关键词： lymphoma CD20 lidamycin scfv energized fusion protein

抗人P185erbB2的scfv-Fc融合蛋白的表达及免疫功能分析

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Acta Biochimica et Biophysica Sinica 2003年第4期35卷 371-374页

作者：解志刚郭宁施明冯建男于鸣孙瑛勋沈倍奋军事医学科学院基础医学研究所军事医学科学院基础医学研究所北京100850

为提高鼠源单抗用于体内治疗的效应功能及降低人抗鼠抗体反应 ,将编码抗人P185 erbB2 单抗轻、重链可变区 (VL、VH)融合构建的单链抗体 (scfv)基因片段与人IgG1的Fc区基因片段融合构建了scfv Fc融合基因 ,并将其克隆到哺乳动物细胞表达... 详细信息

为提高鼠源单抗用于体内治疗的效应功能及降低人抗鼠抗体反应 ,将编码抗人P185 erbB2 单抗轻、重链可变区 (VL、VH)融合构建的单链抗体 (scfv)基因片段与人IgG1的Fc区基因片段融合构建了scfv Fc融合基因 ,并将其克隆到哺乳动物细胞表达载体pCIDN中。用重组载体转染CHO细胞 ,通过G4 18筛选获得稳定高表达克隆。更换无血清培养基培养 ,经重组蛋白质A亲和层析柱纯化scfv Fc融合蛋白。融合蛋白质在还原SDS PAGE中表现为5 2kD的条带 ;与SK BR 3细胞裂解液共培育可特异性地沉淀出P185 erbB2 蛋白 ;FACS分析表明融合蛋白质识别P185 erbB2 蛋白的胞外段 ;ELISA测定融合蛋白质对细胞表面抗原P185 erbB2 的亲和常数为 7.5× 10 -10 (mol/L) -1。构建scfv Fc融合基因 ,将其克隆到表达载体 ,进一步稳定表达抗P185 erbB2 的scfv Fc融合蛋白 ,为进一步的体外、体内研究鼠源单抗治疗效果创造了条件。

关键词： Her2/neu scfv 融合蛋白质

Human scfv antibody fragments specific for hepatocellular carcinoma selected from a phage display library

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World Journal of Gastroenterology 2005年第26期11卷 3985-3989页

作者： BingYu MingNi Wen-HanLi PingLei WeiXing Dai-WenXiao YuHuang Zhen-JieTang Hui-FenZhu Guan-XinShen DepartmentofImmunology TongjiMedicalCollegeHuazhongUniversityofScienceandTechnologyWuhan430030HubeiProvinceChina DepartmentofInfectiousDiseases TongjiHospitalTongjiMedicalCollegeHuazhongUniversityofScienceandTechnologyWuhan430030HubeiProvinceChina

AIM:To identify the scfv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scfv phage display library. METHODS: A large human naive scfv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scfv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scfv gene sequence. The specific scfv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scfv antibody fragments in *** HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scfv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scfv antibody fragments were successfully expressed in *** HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scfv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

关键词： scfv Biopanning HCC IMAC Phage display

抗CD3 scfv基因的真核表达及其生物学活性鉴定

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细胞与分子免疫学杂志 2004年第5期20卷 552-555页

作者：杨章民胡劲松来宝长王一理司履生西安交通大学生命科学与技术学院癌症研究所陕西西安710061

目的 :真核表达抗CD3单链抗体 (scfv) ,并研究其生物学活性。方法 :将编码抗CD3scfv的DNA片段插入真核表达载体pDisplay中。对所构建重组表达载体进行测序确认后 ,以电穿孔法将重组质粒导入Hela细胞 ,以原位杂交法检测抗CD3scfv的表达 ,... 详细信息

目的 :真核表达抗CD3单链抗体 (scfv) ,并研究其生物学活性。方法 :将编码抗CD3scfv的DNA片段插入真核表达载体pDisplay中。对所构建重组表达载体进行测序确认后 ,以电穿孔法将重组质粒导入Hela细胞 ,以原位杂交法检测抗CD3scfv的表达 ,以3 HTdR掺入法检测其在体外对T细胞的活化作用 ,以MTT比色法观察转染的Hela细胞与T细胞混合培养后 ,诱发的细胞毒性T细胞的杀伤作用。结果 :成功地构建了抗CD3scfv的真核表达载体 ,并在Hela细胞中获得表达。所分泌的抗CD3scfv在抗CD2 8mAb存在的条件下 ,能够刺激T细胞活化。将转染的Hela细胞与T细胞混合培养能够诱发CTL的杀伤作用。结论 :真核表达的抗CD3scfv具有刺激T细胞活化的活性。

关键词：抗CD3 scfv 真核表达生物学活性

高效构建卵清白蛋白scfv噬菌体文库及其筛选

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中国生物工程杂志 2018年第11期38卷 25-31页

作者：郎巧利余琳何麒麟葛良鹏杨希重庆市畜牧科学院农业部养猪科学重点实验室养猪科学重庆市市级重点实验室重庆市医用动物资源的开发与利用工程技术研究中心

目的:建立一种高效噬菌体文库构建方法,获得抗鸡卵清蛋白(ovalbumin,OVA)的单链抗体(scfv)噬菌体展示文库,筛选鉴定获得OVA单链抗体。方法:用OVA蛋白免疫Balb/C小鼠,选取血清抗体效价高的小鼠提取脾脏RNA,利用RT-PCR方法扩增获得小鼠重... 详细信息

目的:建立一种高效噬菌体文库构建方法,获得抗鸡卵清蛋白(ovalbumin,OVA)的单链抗体(scfv)噬菌体展示文库,筛选鉴定获得OVA单链抗体。方法:用OVA蛋白免疫Balb/C小鼠,选取血清抗体效价高的小鼠提取脾脏RNA,利用RT-PCR方法扩增获得小鼠重链和小鼠轻链基因。通过无缝连接酶一步将小鼠重链基因、轻链基因和linker DNA连接起来,插入噬菌体表达载体中,构建OVA scfv噬菌体展示文库。测定文库容量,对文库进行富集筛选,ELISA鉴定阳性克隆,测序后构建真核表达载体,转入Expi-CHO悬浮细胞进行真核表达,利用Western blot进行鉴定。结果:成功获得库容量为1. 2×10^7cfu的OVA scfv噬菌体展示文库,并从中筛选出8个阳性克隆,选取效价最高的2号克隆,在Expi-CHO悬浮细胞中表达获得可溶性抗体。结论:建立了一种高效构建scfv噬菌体文库的方法,筛选获得高结合活性的OVA单链抗体,并成功进行了真核表达,为OVA ELISA检测试剂盒的研制奠定了基础。

关键词：鸡卵清蛋白 scfv 噬菌体展示真核表达

Preparation and Identification of scfv and bsFv against Transferrin Receptor

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2008年第6期28卷 621-625页

作者：刘静肖代雯周小鸥文雪代红王志华沈昕代维杨道锋沈关心 Department of Immunology Tongji Medical CollegeHuazhong University of Science and Technology Clinical Laboratory Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital Department of Microbiology Jiujiang Medical College Department of Infectious Diseases Tongji HospitalTongji Medical CollegeHuazhong University of Science and Technology

To obtain single chain variable fragment （scfv） and bivalent single chain variable fragment （bsFv） against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy （VH） and FR4 of variable light （VL）, respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scfv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scfv protein once induced by IPTG in the host bacteria. To express scfv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scfv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scfv and bsFv respectively. Flow cytometry showed that scfv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scfv.

关键词： gene sequence G4S linker scfv bsFv transferrin receptor

天然抗角蛋白单克隆抗体3B4的噬菌体呈现的scfv的构建及表达

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细胞与分子免疫学杂志 2005年第4期21卷 470-472页

作者：管海宏付萌李巍夏汝山王刚刘玉峰第四军医大学西京医院皮肤科陕西西安710032

目的:构建并表达噬菌体呈现的天然抗角蛋白单克隆抗体(mAb)3B4的单链抗体(scfv),并测定其活性。方法:分别以pMD18TmAb3B4VH和pMD18TmAb3B4VL为模板进行PCR,扩增mAb3B4的VH和VL基因,然后将其依次插入噬菌体表达载体pscMH中。经DNA序列测... 详细信息

目的:构建并表达噬菌体呈现的天然抗角蛋白单克隆抗体(mAb)3B4的单链抗体(scfv),并测定其活性。方法:分别以pMD18TmAb3B4VH和pMD18TmAb3B4VL为模板进行PCR,扩增mAb3B4的VH和VL基因,然后将其依次插入噬菌体表达载体pscMH中。经DNA序列测定正确后,转化大肠杆菌,用辅助噬菌体VCSM13超感染诱导表达scfv;用ELISA检测其与亲本mAb3B4的抗原结合活性的改变。结果:对扩增的mAb3B4的VH和VL基因进行测序,结果表明VH基因与原序列完全一致,VL基因在骨架区FR1有1个碱基发生突变。用VCSM13超感染后能检测到scfv的表达,其与亲本一样具有多反应性,但抗原结合模式不完全相同。结论:成功地构建并表达了天然抗角蛋白mAb3B4噬菌体呈现的单链抗体,表达的scfv与mAb3B4的抗原结合活性有一定的差异,为探讨天然自身抗体的抗原结合模式奠定了基础。

关键词：天然抗角蛋白自身抗体 scfv 多反应性

Generation of a Canine-origin Neutralizing scfv Against Canine Parvovirus

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Journal of Northeast Agricultural University(English Edition) 2020年第3期27卷 45-52页

作者： Pi Xue-lei Wang Yu-yang Zou Yi-meng Guo Xiao-chen Kang Kai Wang Meng-xia Li De-shan Ren Gui-ping Biopharmaceutical Lab College of Life SciencesNortheast Agricultural UniversityHarbin 150030China Key Laboratory of Agricultural Biological Functional Gene Northeast Agricultural UniversityHarbin 150030China

Canine parvovirus(CPV)is a high morbidity and lethality virus which causes severe enteric disease in *** an attempt to gain canine-origin neutralizing antibodies against CPV,single chain variable fragment(scfv)bacteria display libraries against the protective antigen VP2 were constructed and ***2 specific scfvs were selected following three rounds of screening *** scfvs were characterized by FCM and *** scfvs showed high affinity and specific binding to ***,the neutralizing activity of the antibody was preliminarily identified by CPV(100 TCID_(50))in vitro and scfv-2 showed the neutralizing ability with a titer of *** study generated the first canine-origin neutralizing scfv against *** neutralizing scfv would be constructed into full-length antibody in the *** study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.

关键词： canine parvovirus VP2 protein canine-origion antibody library scfv