Development Of GFP-Based Small Molecular Antibody Against TLH Of Vibrio Parahaemolyticus

Vibrio pararahaemolyticus(V.P)is a human pathogen that is widely distributed in the marine environments.This organism is frequentlyisolated from a variety of raw seafoods,particularly shellfish.The thermolabile hemolysin gene(tlh)is widely existed in all most vibrio parahaemolyticus strains,and it was used as a potential target for detecting the V.P.Green fluorescent protein(GFP)is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range,and contains a typical beta barrel structure,consisting of eleven ?-sheets linked by eleven loops with a chromophore in the center.Besides,GFP could be used as a scaffold for fluorobody development,and the stable beta-barrel structure and the ability to give multiple color fluorescence prompted the interest in developing a new class of antibody using GFP frame as template.In this study,the different regions of an anti-TLH scFv were inserted into the loop 5 of GFP,and the binding activities of these antibodies were analyzed by ELISA.The main content was composed of three sections as fellows:The different antibody fragments(LCDR3,HCDR3,VL,VH and scFv)genes were inserted into the loop 5 of GFP scaffold,and the expressed and purified proteins were used to identify the interaction to TLH antigen.ELISA results showed that the G5-LCDR3 has the highest binding activity to TLH,with the binding activity being higher than all the others.Although the G5-HCDR3 derived from HCDR3 insertion reacted with TLH,the binding activity is lower than G5-LCDR3.In fact,these proteins expressed from VL,VH and scFv insertions showed a very low binding activity to TLH.The above result demonstrated that loop 5 is feasible for insertion of CDR3 to produce the functional GFP-Abs,while the other insertions are not ideal.To understand the interaction between G9-HCDR3 and TLH antigen,all the amino acids of HCDR3 were mutated to alanine by site-directed mutagenesis for scanning the key amino acids that responsible for binding.Compared to the wild-type G9-HCDR3,the binding activity of the seven mutated proteins(L/A,D1/A,Y1/A,W/A,Y2/A,F/A and D2/A)decreased greatly,showing very low binding activity to TLH in all the HCDR3 mutants.When all the seven amino acids were mutated to lysine,only the mutated binding activity of mutanted protein(L/K)has increased greatly,showing the highest binding activity to TLH in all the HCDR3 mutants.To achieve dual loop insertion,the loop of LCDR3 and HCDR3 from anti-TLH scFv and the loop of CDR3 containing R2/K mutant were inserted into positions of loop 5 and loop 9 in different formats,respectively,and the binding activities of expressed proteins were analyzed by ELISA.While the dual insertion mutants 9L-5L and 9L-5L(R2/K)retain higher binding activities than the single loop insertion,and the dual insertion mutant 9L-5L(R2/K)showed the highest binding activity to TLH.However,the other dual insertions showed low binding activity to TLH,and the fluorescence intensity of those were diminished substantially.In conclusion,the created antibodies based on GFP scaffold could recognize TLH specifically.This study provided a new method to enhance the affinity of the antibody,and also solved the engineering antibody's insoluble problem.