The Construction Of Human TRAb Single Chain Fragments Variable Combinatorial Library

Objectives:The Graves Disease (GD) is a kind of organ specific autoimmune disease which is induced by immune response to self component from organism immune system under the interaction of both environmental and hereditary factors. TSH receptor antibody (TRAb) is a heterogeneous antibody which can result in cell proliferation and hyperfunction through different intra-cellular signal transduction pathway and is the most significant factor which causes GD. However, hitherto people still don't understand the effect on the mechanism and pathology alteration caused by the difference in the structure of TRAb family.In order to further investigate on the function of different antibody molecule in autoimmune disease on genetic level, my assignment is to construct a TRAb phage antibody library from GD patients and the application of TRAb-scFv combinatorial library may lay a solid foundation of the screening of thyroid stimulating antibody (TSAb) and TSH-binding antibody (TBAb) fragment. Methods:We extracted mRNA from the peripheral blood lymphocyte of GD patients whose TRAb and TSI were both positive ( detected by TRAb, TSI Assay Kit) . And cDNA synthesis was done by the Reverse Transcription System. Diverse libraries of immunoglobulin variable light chain (V_l) gene and variable heavy chain (V_H) gene were amplified by PCR technique using primes we designed. Because it was difficult to acquire V_h gene, consequently, we applied pUCm-T vector to construct the V_H library. Firstly, we cloned the amplified V_λ fragments into phagemid p3SCMH and electrotransformed into competent E.coli XL 1-Blue. Then, we constructed the V_λ, library. Secondly, we cloned the amplified V_H fragments into recombinant phagemid p3SCMH/V_λ and electrotransformed intocompetent E.coli XL 1-Blue. Thus, we connected Vx and VH with linker (Gly4Ser) 3 and then we constructed the TRAb-scFv combinatorial library from GD patients. Results:Using ultraviolet spectrophotometer we determined that OD26o/OD28o=1.9, and it testified that the mRNA we extracted was pure. PCR products were electrophoresis on the 1.5% agarose and Vxand Vh genes were prepared on the 38Obp and 420bp respectively in accordance with what we expected, and thus our primers were identified to be effective. Firstly, the Vx gene was inserted into phagemid p3SCMH to construct Vx library. The capacity of Vx library was 5.6 X 106, and the recombination rate was 90%. Secondly, the Vh gene was inserted into plasmid of Vx library to construct the single chain phage antibody library. The capacity of this library was 4.2 X 106, and the recombination rate was 62.5%. There were two difficulties we met in this experiment. On one hand, during constructing a phage antibody repertoire, I tried to optimize several cross-linking factors (such as voltage, electric resistance and capacitance etc.) within a single experiment system. Finally, the results of this study met the requirements of constructing a phage antibody repertoire after optimizing electrotransformation factors. On the other hand, another difficult point is the acquirement of Vh gene. Consequently, we applied pUCm-T vector to construct the Vh library successfully and the recombination rate was 80%. Conclusions:1. In this experiment, we extracted mRNA from peripheral blood lymphocyte of GD patients whose TRAb and TSI were both positive (detected by TRAb,TSI Assay Kit) . And cDNA synthesis was done by the Reverse Transcription System.2. Diverse libraries of immunoglobulin variable light chain ( Vl ) gene and variable heavy chain (Vh) gene were amplified by PCR technique using primes we...