Screening,Preparation And Identification Of Adenoviral Vector Specific Single Domain Antibodies

Adenovirus is a DNA virus that usually causes minor infections of the upper respiratory tract,lower respiratory tract,gastrointestinal tract or ocular epithelium.With the development and integration of various disciplines such as immunology,molecular biology and molecular virology,the application of adenovirus at the molecular level has become more and more widespread,especially adenovirus as a vaccine vector,which not only has a high safety profile,but also stimulates the body to produce cellular and humoral immunity.The aim of this study was to prepare adenovirus vector-specific single-domain antibodies for adenovirus identification and purification.We first immunized adult healthy double humps with adenovirus vectors rAd26 and ChAd63 as antigens,collected their lymphocytes by jugular vein blood collection,extracted total lymphocyte RNA,reverse transcribed into cDNA,amplified the VHH gene fragment using PCR,ligated to the cloning vector of pMECS,and then transformed into E.coli TG1 receptor cells,and successfully constructed A 1.8×10~9 single domain antibody library of adenoviral vector specific phage was successfully constructed.Through two rounds of enrichment and screening,22 positive clones were screened by Phage ELISA,from which two clones with the highest affinity were selected and named sdAb 5 and sdAb 31,respectively,for further experimental studies.The sdAb 5 and sdAb 31 were ligated to the prokaryotic expression vector pET-25b-SBP,induced and purified to obtain anti-adenovirus vector-specific single-domain antibodies.It specifically binds adenovirus rAd26,ChAd63 and HAd5 by ELISA and cellular immunofluorescence assay.Finally,we established an effective adenovirus immunoaffinity purification method by immobilizing the single domain antibody onto Sepharose microbeads,which can be used to selectively capture adenovirus from cell culture media.The preparation of adenovirus-specific single-domain antibodies provides the basis for a one-step immunoaffinity purification and identification of adenovirus.