| Phage display antibody library technology has been widely used in the diagnosis and treatment of disease, and functional protein expression, which gave it a bright prospect in the development of immunoassay.Somatostatin(SS)consisting of 14 amino acid resides is one of the most important neuropeptides secreted from hypothalamus nucleus, which inhibits the secretion of growth hormone (GH) and results in depression of the growth and lactation. SS has been considered a key endocrine hormone in physiological regulation of animals produced for meat and milk purpose. Therefore, it is important to establish the immunoassay methods and techniques for study on the growth regulation mechanism, and for development of new techniques to promote growth and lactation. There have been few reports about micromolecule single chain recombination monoclonal antibodies so far. Furthermore, there have been no reports about selection of ScFvs against somatostatin by phage display, too.SS was coupled with bovine serum albium (BSA) to obtain an artificial SS antigen which was used to immunized mice. Spleens were obtained from both non-immunized and artificially immunized mice and used to extract and purify RNA respectively. Two sources of RNA were used to amplify antibody genes for the natural and immune library respectively. Two ScFv libraries were constructed via phage display technique with two sources of RNA. The main results are shown as follows:1. Immunogen synthesis and animal immunization.The hapten somatostatin was coupled with BSA to make a complete antigen SS-BSA by the carbodiimide activation. The Balb/c mice were immunized with SS-BSA several times until the titer of antibodies reached to 1/2560.2. The obtainning and splicing of immunoglobulin variable genes. The complete RNA was extracted from spleen cells of non-immunized mice and the immunized mice with the highest antibody 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, were first amplified. The approximately same mole of V_H and V_L genes purified were jointed together through splicing by overlap extension (SOE), using the fragments containing parts of the linker in immune single chain antibody library construction. The single strand DNA was amplified via asymmetry PCR for natural single chain antibody library construction.The single chain fragment variable genes were about 750bp generated by PCR, using the primers that contain the slice sites of restriction endonuclease. The result indicated that the asymmetry PCR is better than the traditionary ligated method.3. The construction of ScFv phage display antibody library. After digesting with SfiI and NotI, the ScFv gene fragments was ligated with the phage vector pCANTAB5E and transformed into competent Ecoli.TG1 cells via electroporation. Plasmid electrophoresis showed that the inserting fragments and the aimed fragments were amplified using PCR.The transformed bacterium were infected with helper phage M13K07. After sedimenting the cell, collected thesupernatant .Then the phage display antibody library had been successfully constructed. The natural single chain antibody library contained 9.8×10~7 independent clones after forty times of electroporation, the immune single chain antibody library contained 9.3×10~7 independent clones after five times of electroporation. It is clear that the latter method is more efficiency than the former, the job to constructe the immune antibody is less than the natural one.In a word, we take some method in researching single chain antibody by construcing ScFv phage library, which is able to be used in set up of radioactive immunoassay methods, enzyme-immunoassay methods, immunohistochemistry assays and immunoachip against somatostatin in the future. |
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