Dissertation > Excellent graduate degree dissertation topics show

Construction of ScFv Phage Antibody Library Against Milbemycin Oxime

Author: ZhangXiao
Tutor: ChengLuoGen;LiuXianJin
School: Nanjing Normal University
Course: Genetics
Keywords: Milbemycin oxime enzyme linked immunosorbent assay (ELISA) Phage display antibody library Single-chain variable fragments (ScFv)
CLC: S859.84
Type: Master's thesis
Year: 2007
Downloads: 111
Quote: 0
Read: Download Dissertation

Abstract


Phage display antibody library technology, which has been widely used in the diagnosis and treatment of disease, and functional protein expression, was a high-performance, time saving, and effective technique for producing specific antibodies. Several high affinity antibodies against micro-molecular substances, such as pesticides or veterinarian, have been reported to be successfully obtained by phage display antibody technology, which gave it a bright prospect in the development of immunoassay for pesticide. In this study, a phage display antibody library against milbemycin oxime was successfully constructed, the objective was to develop a specific phage display library for screening antibodies against compounds with 16-membered macrocyclic backbone.1. Immunogen synthesizing and animal immunizing. The hapten 5-o-Succinoylmilbemycin-oxime were bound to BSA by the carbodiimide activation. Four Balb/c mice were immunized with MILO-BSA, after the titers of antiserum reached 1:25600. A MILO-OVA -based CI-ELISA was conducted with the antiserum, the inhibition curve show range for detection milbemycin oxime was between 20% and 70%.2. The obtained of immunoglobulin variable genes and splicing. The total RNA was extracted from spleen cells of mouse with the highest antiserum titers. Using the RT-PCR, immunoglobulin variable heavy chain region genes (V_h) and variable light chain region genes (V-l), which were about 360bp and 340bp respectively, are first amplified. The approximately mol V_H and V_L genes which have been purified were jointed together through splicing by overlap extension (SOE), using the fragments contain parts of the linker. The ScFv genes were about 750bp generated by PCR, using the primers that contain the slice sites of restriction endonuclease. 3. The construction of ScFv phage display antibody library. After digesting with SfiI and NotI, the ScFv gene fragments have been ligated into the phage vector pCANTAB5E that have been digested using the same enzymes and were transformed into competent E.coli TG1 cells. Plasmid electrophoresis showed that there were inserting fragments and the aimed fragments were amplified using PCR, with primers R1 and R2. The transformed bacterium were infected with helper phage M13K07, sedimented the cell and collected the supernant. Then a phage display antibody library containing 2.4×10 p.f.u/mL bacteriophage plaque has been successfully constructed .The reseach are help for establishing new screen methods of antibody library and antibody panning.

Related Dissertations

  1. The Dynamical Evaluation of Three Methods to Diagnose Mycoplasma Pneumoniae Infection in Children,R725.6
  2. Preparation of Monoclonal Antibodies against Cadium Ion and Study on the Immunological Characteristics,X830
  3. Establishment of an Indirect ELISA for Detection of Antibodies Against Haemophilus Parasuis and Screening of in Vivo Induced Antigen,S858.28
  4. The Quantitative Analysis and Clinical Significance of VEGF, bFGF, IGFBP-2 in Plasma and CSF of Patient with Glioma,R739.4
  5. Serum Brain-derived Neurotrophic Factor Level in Depressive Patients and Relative Analysis,R749.4
  6. Construction of Phage Display Single-chain Fv Antibody Library Against UV-attenuated Schistosoma Japonicum Cercariae and the Screening of the Sj SCA66-68kDa ScFv,R392
  7. Delivery Strategies in Mice for MAPs with a B-Cell Epitope of M3-M4 Loop in the Primary Subunit of NMDA Receptor,R392.1
  8. Construction of Human Immunoglobulin Combinatorial Library and Screening of Phage Antibodies to Platelet,R392
  9. Three major potato virus ELISA and RT-PCR detection technology,S435.32
  10. Construction of a Fully Synthetic Human Antibody Library and Preliminary Screening of Single Chain Fv Against BHL-1,R392
  11. Construction of Phage Display Library of Fab Antibodies Repertoies and Screening of Anti-Mouse Male Specific Antigen,Q78
  12. The Relationship between the HBV C Gene Mutation and the Celluar Immunity of the Host,R392
  13. Changes of IL-1β,TNF-α and IL-6 in Serum and Brain Following Whole-brain Irradiation in Rats,R818
  14. P185 ~ (erbB-2) proteins that target the single chain antibody screening,R73-3
  15. Study on Technology of Detecting of Two Main Virus of Potato with ELISA and RT-PCR,S435.32
  16. The Studies of the Anti-angiogenesis Drug Directed Against Human MMP-2 and Integrin αvβ3,R96
  17. Construction of Phage Display Antibody Library and Screening and Expression of the Single-chain Antibodies Against Breast Cancer Cells,R737.9
  18. Diagnostic Values and Clinical Significance of Detecting Soluble Fas and Soluble FasL of Pleural Effusions in Differentiating Tuberculous and Malignant Pleural Effusions,R446.6
  19. The pyrethroid pesticide immune and chromatographic analysis method,TQ450.2
  20. The Study on Microcystin-LR’s Development of Enzyme-linked Immunosorbent Assay,R115

CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Veterinary Pharmacology > Veterinary Toxicology > Chemical residues in animal foods
© 2012 www.DissertationTopic.Net  Mobile