Construction And Seletion Of Phage-displayed Antibody Library Against HRP-II Of Plasmodium Falciparum And The Expression,purification Of The Single Chain Antibody

Construction and selection of phage-displayed antibodylibrary against ILRP-II of Plasmodiumfalciparum and theexpression, purification of the single chain antibodyPostgraduateHou YunxiaAdvisorDong WenqiABSTRACTMalaria is estimated to kill nearly 2 million people each year. Rapid, accurate and affordable methods are needed for the diagnosis and treatment of malaria. The new-generation , antigen-capture tests are capable of detecting fewer parasites and of producing a result more rapidly, in which antibodies are the key reagents.Recombinant phage-displayed antibody technique is currently the most popular method for the selection of recombinant antibody fragments in vitro. This approach relies on a phage-display system in which fragments of antibodies are expressed as fusion proteins displayed on the phage surface. Using the polymerase chain reaction (PCR), immunoglobulin variable(V) region genes are first amplified from spleen cells. The VH (variable heavy) and VL(variable light) genes are then cloned into a phage vector and expressed as a fusion with a phage protein. Each recombinant phage genome contains the V genes that specify the antibody displayed on its surface. Since the displayed antibodies retain their antigen-binding capability, it is thus possible to enrich for recombinant phage expressing specific antibodies by affinity selection. With this approach, antibodies of defined specificity and affinity can be selected from a population. In this study, a phage-displayed antibody library against HRP-II of Plasmodium falciparum was constructed using this new and leading biotechnique. Eight clones expressing antibodiesagainst histidine-rich protein-II (HRP-II) were panned and screened from thelibrary.Using ANA Extraction kit and the series kits of the Recombinant PhageAnibody System (Phannacia), the genes of V. and V, were gained from thespleen cells of BALB/c mice immunized with HRP-II protein, Which wereabout 340bp and 300bp respectively, and then were jointed together with alinker DNA (Gly.Ser), by splicing overlap extension PCR and a 750bpdesired fragment(single chain Fv, ScFv) was generated. After digested withSfiI and NotI, the ScFv genes were ligated into the phagemid vectorpCANTAB 5E and transformed into comPetent E coli TG1 cells. Therecombinants were grown on the amp' plate. Plasmid electrophoresis showedthat there were insert fragments and the aimed fragments (~750bp) wereamplified using PCR. The results showed that we have successfullyconstrUcted a phage-displayed anibody library against Has-II ofPlasmodium faIciParum, with the middle content about l .2 X l06 which couldmeet the needs of diversity.Recombinan HRP-lI of P falciParum prepared by our laboratory, whichhas been proved to be a valuab1e target anigen fOr the diagnosis of Pf wasused for parming and screening ScFv anibody from the antibody libraryEight clones with good reactivity with HRP-II in ELISA were selected. Twoof them were chosen for further research. Soluble ScFv antibodies wereproduced and the expression conditions were optimized. Furthermore, thecharacteristics of anti-Har-II ScFvs were determined by Dot-ELISA andWestern blot. The products were purified by saturated an1monium sulfateand ion -exchange chIomatograPhy(Hi Trap Q l x5ml).The results demonstrated that phage display technology can be used as apowerful tool in making ScFv antibodies, and such antibodies have potentialas immunological reagents for malaria diagnosis.