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Construction of Dengue Virus-specific Full-length Fully Human Antibody Libraries by Mammalian Display Technology

Author: WenYangMing
Tutor: CaoHong; ZhouChen
School: Southern Medical University,
Course: Pathogen Biology
Keywords: dengue virus mammalian cell surface display full-length fullyhuman antibody library secondary antibody library
CLC: R373.33
Type: Master's thesis
Year: 2013
Downloads: 2
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Abstract


Antibody is an important part of the human immune system that can provide the body with effective immune protection through clearing foreigner such as microorganisms,parasites.With the advance of modern biotechnology,numerous monoclonal antibodies have been developed and the side effects have been improved significantly.Monoclonal antibodies were first developed in the1970s by hybridoma technology and have been used more and more widely in various fields,including diagnostics and clinical treatment of human diseases, environmental monitoring etc.Because these antibodies were come from mouse,human anti-mouse antibody (HAMA) was induced when applied in human.Over the past20years,researchers have used the techniques of molecular biology to clone full repertoires of human antibody genes.By inserting such genes into an expression vector,a variety of antibody libraries have been constructed and numerous antigen-specific antibodies identified from the libraries.Currently,phage display is the most developed and widely used technology for screening and selecting specific antibodies from library through the so-called panning procedure.Humanization of mouse monoclonal antibodies(mAbs) and direct selection of fully human antibodies have dramatically advanced the development of antibody therapeutics.Phage display has some obvious disadvantages.For example,it cannot display a full-length antibody,but only a fragment (scFv or Fab).During the past10years,many scientists have exercised considerable effort in the attempt to develop mammalian cell surface display technology.Coupled with fluorescence activated cell sorting (FACS),some antigen-specific antibodies were successfully screened and identified from mammalian antibody libraries displayed on the cell surfaces. However,the diversity of mammalian display library developed with these techniques is limited to the range of104-106,which does not meet the requirements for the screening of high-affinity antibodies with biological function.It is therefore desirable to construct antibody libraries more effectively with a diversity of more than109to display full-length antibodies,especially human antibodies for the screen of antibody therapeutics.Dengue virus can cause dengue fever (DF),dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).DHF/DSS is the serious development of the disease with high mortality rates.Because the infection mechanism is so complicated and has not been illustrated,there are no specialized effective clinical treatments and effective dengue vaccine currently.It will be of significance to construct dengue virus-specific antibody libraries and screen specific antibodies with high specificity from the libraries for the development of both vaccine and antibody therapeutics.Using total RNA isolated from peripheral blood lymphocytes of a health donor recovered from infection of Dengue virus as starting material,whole set of antibody genes were amplified by RT-PCR and then the antibody genes were inserted into the mammalian cell expression vector pDGB-HC-TM.Full-length fully human primary antibody gene libraries have been constructed with a combinatory diversity of 1.46×109.The secondary antibody display library was constructed with a size of2.4x105and a background of1.2%.The library antibody was detected on transfected CHO cells surface.Part I:Construct of Dengue virus-specific full-length fully human primary antibody display librariesObjective:To construct the Dengue virus-specific full-length fully human primary antibody display libraries.Methods:Human peripheral blood mononuclear cells(PBMCs) were isolated from a health donor recovered from infection of Dengue virus by gradient centrifugation.The total RNA was isolated from the purified PBMC using RNA Easy kit.The heavy chain variable domain and full length kappa chain were amplified by two-step RT-PCR using specific forward and reverse primers. The vector pDGB-HC-TM and RT-PCR amplified DNA fragments were digested by proper restriction enzymes;target fragments were purified by gel purification kit and ligated together to construct the libraries.The ligation was performed at16℃for24hours.The libraries were transformed into DH-5a competent cells.The transformation efficiency and library sizes were calculated.The transfection of libraries into CHO cells was carried out in12-well plate.The antibody expression on cell surface was detected by FACS and the data were analyzed using FCS Express V3software.Results and discussion:Six PCR reactions were carried out to amplify antibody genes,3for heavy chain variable domain (VH),and3for full length kappa chain.The PCR products were separated and purified by electrophoresis.The sizes of these fragments are about0.45kb for VH and0.75kb for kappa chain.Total4μg of target PCR fragments were collected.After digestion by BsmBI,the VH fragments and vector pDGB-HC-TM were ligated and the primary heavy chain library was constructed with a size of1.8×105.The light chain fragments and the vector pDGB-HC-TM were digested by SfiLAfter libation and transformation,the primary light chain library was constructed with a library size of1.45×104.10heavy chain clones and10light chain clones were randomly picked up for sequence and expression analysis.Results show that7heavy chain clones and8light chain clones contain right coding regions for unique amino acid sequences.The heavy chain and light chain gene libraries were co-transfected into the CHO cells.The expression of full length antibodies on CHO cells surface were analyzed by FACS.The results show that37%of the cells expressed detectable antibodies.Conclusion:Two full-length fully-human mammalian display antibody libraries were constructed with a combinatory diversity of1.46x109[(1.45×104×80%)x(1.8×105x70%)].PartⅡ:Construct of the secondary Dengue virus-specific full-length fully human antibody library and analysis of antibody display on mammalian cells surfaceObjective:To construct the secondary Dengue virus-specific full-length fully-human antibody libraryMethod: The primary VH library was digested by BsmBI and the primary light chain library was digested by SfiI.The vector pDGB4was digested by both BsmBI and SfiI.The0.45kb VH fragments,0.70kb light chain fragments,5kb and3kb vector fragments were purified respectively.These4fragments were ligated by4-way ligation.After transformation of libraries into competent bacteria cell,the transformation efficiency and library size were calculated.The antibody library was transiently transfectedinto CHO cells and the antibody expression was analyzed by FACS.Result and discussion:The secondary antibody display library was constructed with a size of2.4×105and a background of1.2%.The library antibody was detected on transfected CHO cells surface.Conclusion:A secondary Dengue virus-specific antibody display libraries was constructed successfully.

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