Construction And Preliminary Identification Of Phage Antibody Library For Autoimmune Antibodies

Autoimmune disease means body's immune response against autoantigen which led to self-tissue injury, it is common but difficult to treat. Its salient features are multiple autoantibodies, and immune pathological damage against self-tissue by ways such as immune complexes, resulting in injury almost all over the body. The pathogenesis of autoimmune diseases are complex, involving many factors such as genetic, infection, sex hormones, immune dysfunction. So far, there are many questions about autoimmunity don't have answers, for example, how to start autoimmunity, the genetic background , the damage mechanism and immunologic recognition of autoimmunity, and the pathopoiesis of autoantibodies. Generally speaking, autoimmune disease is caused by the number abnormality and dysfunction of immune cell, which led to immune functional disorder, followed by the appearance of multiple autoantibodies, but the specific mechanism of autoimmune disease is still unknown. The research on autoan tibodies is always the key point of AID.Now, the diagnosis of autoimmune disease is limited, the main diagnostic code is the clinical symptom together with one or more high titer autoantibodies. The acknowledged specific antibodies of AID are seldom, while some adjunct antibodies are not specific for AID. Further more, the detection level of AID in China is varied, and there isn't a specific index for early diagnosis, as well as specific drug, all of these have influence on diagnosis and treatment of AID. Therefore, purified antibodies with high affinity and high specificity have great value in our research on autoimmune disease.The preparation and purification of antibody have become the focus of immunology for many years, and the development of antibody have been under way for more than 100 years, which were divided three periods , namely, the period of polyclonal antibody, the period of monoclonal antibody and the period of humanized antibody. So far, the phage antibody library technology is the most sophisticated, the most widely used technology for antibody humanized. With the help of this technology, the gene of foreign protein or polypeptide was fused with the gene of coat protein of phage, so that the foreign protein or polypeptide expressed on the surface of the phage, achieving the unity of genotype and phenotype. Until now, several phage antibody library against HBV, tumor cells and so on have been successfully constructed, and antibodies with high affinity and high specificity were obtained. These antibodies were of great value.In this study, we used peripheral blood mononuclear cell of 55 autoimmune disease patients to construct a phage antibody library against AID, and obtained antibody against double-stranded DNA by panning and solid-phase antigen assay. Our study hope to lay the foundation for further panning and function research.Methods:1. The peripheral blood mononuclear cell of 55 autoimmune disease patients were isolated, then total RNA was extracted and reverse transcription to cDNA .The quality of cDNA mixture was verified by reference gene GADPH;2. By using the template cDNA verified above, human immunoglobulinκ/λlight chains and heavy chains Fd genes were amplified by polymerase chain reaction (PCR);3. The single colone of XL1-Blue was picked to prepare the competent cell by using Hepes solution;4. The light chain genes were first cloned into pComb3Hss vector to construct a human recombinant light chain library;5. The heavy chain genes were subsequently inserted into the corresponding sites of recombinant light chain library to generate a combinatorial H+L+pComb3Hss plasmid;6. The combinatorial plasmid transformed into E.coli.XL1-Blue, then E.coli.XL1-Blue was infected by helper phage M13KO7. A random combinatorial library was expressed on the surface of filamentous phage;7. The phage antibody library were panninged for four rounds by using anti-dsDNA ELISA kit, determinated the titer after each round;8. Identified the phage antibodies after four rounds panning by indirect ELISA. Plasmid DNA isolated from positive clone was cut off gIII genes. After ligated itself, the recombinant plasmid transformed E.coli.XL1-Blue, then XL1-Blue was induced by IPTG to product soluble Fab.9. Finally, the antigenic specificity of soluble antibody Fab was identified by anti-dsDNA ELISA kit and anti-U1RNP ELISA kit.Results:1. The total RNA extracted was 7-10 ug, and the quality of RNA was good. The cDNA was verified well, too;2. Human immunoglobulin light chainsκ,λand heavy chain Fd genes (MW 660bp) were obtained by PCR successfully. The constructed light chains library size is 4×105 and the recombination frequency is 80 percent;3. The constructed heavy chains library size is 6×106 and the recombination frequency is 70 percent, PCR showed that both light chain and heavy chain are inserted in the recombinatorial library and the library was successfully constructed.4. The titer of eluted phage after the first bound was 1.1×105 , and the titer after both the second and the third bound raised remarkably, the eluted phages were enriched 218 fold after the forth rounds panning.5. Two unique clones were isolated from the human Fab fragment library. It was proved the recombinant plasmid had been cut off gIII genes and ligated by itself through electrophoresis after digested by XhoI.6. The results of indirect ELISA showed that all of six clones of Fab were dsDNA positive while U1RNP negative, indicating the soluble antibody Fab had specific to bind with dsDNA.Conclution:We gained cDNA mixture successfully by reverse transcription technology from total RNA of peripheral blood mononuclear cells of fifty-five autoimmune disease patients with high titer autoantibodies and antinuclear antibodies in the serum. A human antibody Fab fragment library against AID was constructed based on it. Subsequently, human Fab fragment phage antibody library against double-stranded DNA was constructed successfully by phage display technology. Through four rounds panning of the antibody library, soluble Fab fragment was obtained and identified antibody activity against double-stranded DNA. The construction of phage antibody library against AID lay the foundation for the further panning of more autoantibodies with high affinity as well as their function research.