Sreening And Application Of High Affinity Anti-EGFP-Nanobody

Green fluorescent protein(GFP)is the most commonly used tool protein in the current life science research.Because of its non-toxic characteristics,it has unique advantages in molecular labeling and tracer imaging technology.Nano antibody is known to have the smallest binding activity.It has the characteristics of easy expression,high specificity and strong stability.It is widely used in detection,diagnosis,treatment and other fields.In this study,phage display technology was used to construct a phage display library successfully.The high affinity positive clones were obtained by the improved bio-panning method.The VHH and EGFP gene was expressed in E.coli.The nanobodies were coupled with HRP enzyme and magnetic beads respectively for IP experiment and Western blot verification.The main experimental results are as follows:1.Alpaca was immunized with EGFP antigen,the titer of serum was monitored,leukocyte were isolated from the peripheral blood of Alpaca after four times of immunization,extracted total RNA.The phage display library was obtained by nested PCR and electrical transformation.A phage library with titer of 4 × 1013 CFU/mL was obtained after helper phage rescue2.The 87 positive clones were screened by the improved bio-panning method.The 10 unique kinds of nanobody sequences were obtained after sequencing and comparing;the 4-28 VHH gene which has highest binding activity was expressed in E.coli.Fortebio octet was used to study the interaction,the 4-28 nanobody affinity up to 9.67 × 10-11 M.3.The detection range is 1:10000-1:25000(concentration 100-40 ng/mL),and has higher sensitivity;4.Pet25b-b13-EGFP recombinant vector was constructed and expressed successfully,the working concentration is 3.125 ?g/mL.And the nanobody was coupled with magnetic beads and applied to immunoprecipitation successfully.The results showed that the magnetic beads could reduce the reaction steps and shorten the reaction time in immunoprecipitation.