Amplification Of vh and vl Gene Fragments from RNA Source Of Single Human Plasma Cell
Subject Areas :Laya Esmati 1 , Jalil Fallah Mehrabadi 2 , Hamideh Rouhaninejad 3 , Masomeh Bazaz 4
1 - پژوهشکدة علوم و فناوری زیستی، دانشگاه صنعتی مالک اشتر، تهران
2 - پژوهشکدة علوم و فناوری زیستی، دانشگاه صنعتی مالک اشتر، تهران
3 - پژوهشکدة علوم و فناوری زیستی، دانشگاه صنعتی مالک اشتر، تهران
4 - پژوهشکدة علوم و فناوری زیستی، دانشگاه صنعتی مالک اشتر، تهران
Keywords: Human monoclonal antibody, Plasma cell, ScFv,
Abstract :
Inroduction and ObjectiveThe first generation of therapeutic monoclonal antibodies was isolated from mouse. One of the disadvantages of them was stimulating immune response in human. Fully human monoclonal antibodies are significantly considered due to their high efficiency and low immunogenic potential. Nowadays different kind of techniques such as phage display and single B cell technology are used to produce fully human mAb. The aim of this project was amplification of VH and VL genes from RNA source of Human plasma cell.Materials and Methods:With the aim of isolation and amplification of VH and VL regions, single cell RT-PCR reaction was performed. Single plasma cells were lysed by using cell lysis buffer. By using synthesized cDNA from plasma cells and antibody specific primers, antibody genes were amplified. Six pair of primers utilized to amplify the variable region of heavy chain (VH) and light chain (VL). Restriction sites and the linker sequences were placed on primer sequences due to respectively cloned in target plasmid and to link VH and VL. Results: Electrophoresis represented VH and VL fragments with 400 bp length were amplified by PCR. The VH and VL gene sequences were BLAST separately and showed 97% similarity among other antibodies gene sequence.Conclusion: Primer sets were selected and designed which contain linker sequence for ScFv construction, NcoI and NotI restriction sites in order to clone directly into an expression vector.
