Construction And Identification Of Human Single Chain Antibody Against Fusion Protein Of Human Respiratory Syncytial Virus

Objective:The worldwide spread human respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract illness in infants and children. The mechanism of host immune protection against RSV infection remains unknown. So far no safe and effective vaccines are available. Among the encoded 11 viral proteins by RSV genome, the fusion glycoprotein (F) and attachment glycoprotein (G) are the only two neutralization antigens. F protein owns highly conserved amino-acid identity between different group's viruses, which is the most important target antigen in vaccine project, elicits cross-protective immunity. In this research, the human single-chain Fv fragment (scFv) antibody library was constructed by phage display technology and the specific scFvs against RSV F protein were obtained by screening the human scFv library.Methods: Peripheral blood lymphocytes (PBL) were separated from ten healthy donors, and the total RNA was extracted from the PBL. The variable heavy (VH) and variable light (VL) genes were amplified by RT-PCR and then the scFv genes, obtained through SOE-PCR, were cloned into the vector pCANTAB5E, and electroporated into competent E.coli TG1 cells to prepare a scFv phage display library. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for RSV F were enriched after five rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. The gene of the positive clone was identified by nucleic acid sequence analysis. After infecting E.coli HB2151 with the positive phage clone, soluble scFv was prepared, and then confirmed by Western and Dot blots.Result: The VH (370 bp) and VL (320 bp) genes were amplified by RT-PCR and then the scFv (750 bp) genes were obtained through SOE-PCR. After the scFv genes were cloned into the vector pCANTAB5E and electroporated into competent E.coli TG1 cells, a scFv phage display library containing 1.8×107clones was gained. Recombinant phages specific for RSV F were enriched to 108 times after five rounds of biopanning and 18 antigen-positive clones were selected from the enriched clones by phage ELISA. DNA sequence analysis of the clone E4 with the highest OD indicated that the variable region gene of the clone was highly homologous with human immunoglobulin gene family. The expression of the scFv E4 was confirmed by Western and Dot blots.Conclusion: From the human phage display library, the specific scFv for RSV F protein was selected and confirmed. This study laid a firm foundation for further investigation on the biological activity of the scFv.