Construction And Screening Of Human Specific Fab Phage Display Antibody Library Against Human Adenovirus Type 3 And 7

[Objective] To construct a human specific Fab phage antibody library for screening neutralizing antibody against human adenovirus type 3 and 7.[Methods] The lymphocytes were isolated from a volunteer,whose anti-Adv3 and anti-Adv7 neutralizing antibody were titrated equally as 1:1151.The total RNA was extracted from the isolated lymphocytes and the c DNA was synthesized.The genes of variable regions of light and heavy chains were amplified by a set of human IgG variable regions antibody primers.And then the genes of constant regions of light and heavy chains were amplifide from phagmids carrying the templates respectively.Light chain and Fd heavy chain were obtained by the first SOE-PCR,and then the full-length Fab gene were obtained by the second SOE-PCR.Ligate the Fab gene to phagmid p Comb3 X and the ligation products were electroporated into competent E.coli XL1-Blue cells.The final transformed cells were infected with M13K07 helper phage to yield recombinant phage antibody library of Fab.After panning four rounds by purified Adv3 and Adv7 viruses paticles respectively,the specific Fab antibody phages were enriched.The fourth round clones were tested by CLEIA to select the positive clones.Four positive clones binding to adenovirus type 7 were obtained,and named as 5C,5C,9F and 9H.Positive clones were sequenced and their homology with human Ig G were analyzed.The variable regions of light and heavy chains of positive clones were amplified and then cloned into expressing vectors of Igκ and Ig H respectively.The full human antibody was expressed in 293 T cells and tested by CLEIA,SDS-PAGE.[Results] We construct two antibody libraries according to the different light chains,The capacity of Fab phage antibody gene library was 3.0×109(κlight chain)and the other was 1.8×109(λlight chain).Insertion rate of Fab gene inserts were near to 100%.The specific antibodies were enriched after four rounds screening.Four clones,5C,5D,9F,9H specifically recognizing Adv7 were obtained and identified by CLEIA.And four clones also recognized Adv3 、 Adv14 、 Adv55 identified by CLEIA.Sequencing analysis showed that four clones were different.Referring to IMGT/V-QUEST database,sequence analysis indicates that two clones with κ light chain,two clones with λ light chain.The variable heavy domains(VH)and variable light domains(VL)were highly homologous with human embryonic Ig heavy chain variable region sequences and light chain variable region sequences respectively.The genes of variable regions of κLight of 5C and 9H were successfully cloned into expressing vectors of Igκ,and heavy chains of 5C,9F and 9H were successfully cloned into expressing vectors of Ig H.After matched by light chains and heavy chains,six full human Ig G antibodies were expressed in 293 T cells and tested by CLEIA and SDS-PAGE.CLEIA experiments showed that these antibodies were expressed.Among these antibodies,two antibodies expressed with higher production specifically recognized Adv7,and they also recognized Adv3、Adv14、Adv55 tested by CLEIA.[Conclusion] We successfully constructed human Fab phage display antibody library specifically against human adenovirus type 3 and 7,and four specific antibodies against adenovirus type 7 are obtained,which provide new insight to the development of novel diagnosis and treatment reagents for adenovirus type 7 infection in children.The neutralizing activity need to be identitied further for reason of low expression production.The methods of antibody library construction and antibody screening provide a foundation for the research of neutralizing antibodies against Adv3 and Adv7 in the future.