Construction And Primary Screening Of A Large Nonimmune Human Fab Fragment Phage Antibody Library

Antibody, which is employed extensively in diagnosis and treatment of illness aswell as laboratory research, plays an important role in the field of modem medicalscience. In the seventies of the twentieth century, Kohler and Milstein prepared themonoclonal antibody (McAb) of hybridoma by means of cell fusion and applied it intoprevention, diagnosis and treatment of human diseases. However, the property ofprovoking strong Human anti-mouse Antibody (HAMA) immune responses restrictedtheir application clinically. In the nineties, the technology of Phage antibody libraryresolves the problems of generating human McAb by hybridoma approach.Recombinant phage-display library technique synthesizes virtually the entirerepertoire of a person's antibody genes of variable regions by PCR technique and forexpressing the encoded antibodies on the surface of a phage vector. It is a valid approachto get human antibody through constructing phage display library and aim to haverecombinant antibodies by panning the library in vitro. PCR is used to amplify fromimmunoglobulin all the variable fragments of heavy chain and light chain which then isrecombined into appropriate phagemid vector, and finally is displayed on the surface ofphage by fusion with phage protein, so it is very convenient to select specific antibodieswith different antigens. Furthermore, those phage antibody libraries with large capacityand high diversity have the advantage of obtaining antibodies with high affinity. Phagedisplay technology makes people can simulate the whole process of antibodiesproduction in vitro, it supplies a simple and highly effective operation system, thus it hasimportant theoretic signification and application foreground.In this project, by using the leading biotechnology of phage display antibody library,we constructed a large naive human Fab phage antibody library.Firstly, amplifying the fragments of heavy (Fd) and light chain genes of antibodiesby RT-PCR: total RNA was extracted from the peripheral blood lymphocytes of thenonimmunized healthy donors and various disease patients. RNA integrality was keptwell shown in agarose gel electrophoresis, and cDNA was synthesized by ReverseTranscriptase (RT) from total RNA with the oligo (dT)₁₈. Heavy and light chain appropriate 5' and 3' primers were used for PCR amplification of human heavy chain Fdfragment and light chain gene, An aliquot of the PCR products was run on 1ï¼…agarosegel, which was about 700bp.Secondly, cloning light chain genes into pComb3XSS: the light chain product ofPCR (isolated by agarose gel electrophoresis) was cut with an excess of the restrictionenzymes Sacâ… /Xabâ… and was ligated with Sacâ… /Xabâ… -linearized pComb3XSS vector,then was electrotransformed into E.Coli XL1-Blue. After transformation, XL1-Bluesamples were withdrew for plating to determine the rate of transformation. The insertionof target light fragments were detected by digesting with restriction endonucleases fromten random XL1-Blue monoclones. The plasmids with light insertion were namedpComb3XSS+L.Finally, cloning heavy chain Fd fragment into pComb3XSS+L to construct Fabantibodies libraries and primary panning: the phagemids abstracted from amplified E.coliwere cut with endonucleases Speâ… /Xhoâ… , as well as digested. Fd fragment gene. Therecombinant phagemids were then electroporated into competent E. coli XL1-Blue cells.Under the super-infection of helper phage VCSM13, theFab phage library with highdiversity was successfully constructed by cloning of Fd fragments into the phagemidcollection containing the light chain repertoires. Ten random XL1-Blue monoclones wereselected to identify recombinant ratio by digesting with restriction endonucleases. Theinsertion of target Fd fragments were detected by digestion with Speâ… /Xhoâ… , the Fabfragments' insertion were detected by digestion with Sacâ… /Speâ… . The plasmids with Fdtogether with light insertion were named pComb3XSS+(H+L). Panning consists of tworounds of binding phage antibody against HAV antigen immobilized to the well of anELISA plate. Analysis of antibody fragment-displaying phage pools by kit ofenzyme-linked immunoassay for detecting serum HAV IgG. The recombinant plasmids(Fd+L) were extracted randomly from one clone. Their sequences of Heavy chain Fd andL chain DNA fragments were assayed and their homology with human Ig were analyzed.The Fab phage antibody library was successfully constructed, capacity of librarywas1.26×10¹¹ pfu/ml and recombinant ratio was 90ï¼…. The results showed that the sequence of Fd fragment had the homology of 92ï¼…withγconstant region and 95ï¼…forlight chain withλ, constant region of human immunoglobulin; the variable regionsequences of the Fd and L chain belonged to human VH1 gene family and a subgroup of1gG. Phage pool obtained in second round of panning was tested in a competitiveinhibition ELISA, the rate of inhibition was 38.61ï¼….This research is an exploration and sample of high molecular biology techniqueresearch. The completion of this research provides a new mind and technical flat for theselection of all kinds of antibodies. This new phage antibody library is set to become avaluable source of antibodies to many different antigens and to plays a vital role in theresearch of our lab.