The Construction And Qualification Of Ribosome Display Library Of FMDV ScFv

Single-chain variable fragment which constants of the heavy variable chain (VH) and the light variable chain (VL) is a small molecular antibody. And the VH and the VL are connected by a flexible linker. Because of small molecular, low immunogenicity and so on, the single-chain variable fragment becomes a new style genetic engineering antibody on molecule diagnosis and molecule therapeutics. The ribosome display technology is cell-free completely, which is used to scan the fusion protein in vitro. Using ribosome display technology and in experiments with lymphocyte of rabbit immunized by FMDV Asiaâ… antigen, the ribosome display library of FMDV ScFv was constructed by PCR. And the library was sequenced and analyzed by transcription and expandability analysis. The capacity of library was determined to test the rationality of ribosome display library.The RNA was extracted from the spleen of rabbit which was immunized by the FMDV Asiaâ… . The VH and VL were amplified. The ScFv was constructed by over-lapping PCR. The overlapping gene belonged to the linker gene-(Gly4Ser)3. The sequence was analyzed by BLAST in the Genbank database. The identities of VH and VL were 81% and 93% respectively. And the linker was found in the sequence. The properties of ScFv amino acid were also analyzed. The homology modeling of 3D structure was done by SWISS-Model of Swiss-pdb viewer. The Ramachandram Plot suggested that the structure of ScFv modeled was reasonable. Then, the constant region of rabbit antibodies light chain (Ck) was amplified. By BLAST analysis, the identity of Ck was over 82% so far as to 100%.The result showed that Ck belonged to the constant region of light chain of rabbit antibodies. The Ck was used as a spacer region. The ScFv and Ck were connected by over-lapping PCR. The myc-tag was the overlapping region. Meanwhile, the ribosome display library was constructed by PCR, added a series of elements. And the capacity of ribosome library was 1013 theoretically. The ribosome display library was tested by sequence analysis and the result showed that there were the elements of ribosome display library which contained the T7 promoter, SD sequence, 5'stem loop structure, 3'stem loop structure and two restriction enzyme sites(Notâ… and Ncoâ… ). And the ScFv and Ck in library sequence were aligned with the sequencing ScFv and Ck. The result of alignment confirmed that the VH and VL gene of antibody were variable and the Ck gene was constant. And the transcription in vitro analysis confirmed that using LiCl to purify mRNA was feasible and the Ribo m7G Cap Analog could enhance the transcription. In conclusion, the ribosome display library of FMDV ScFv constructed was reasonable.