The Research On The Expression Systems Of Nanobodies VHH10 And VHH16 And The Analysis Of Thermostability Of Nanobodies

Heavy chain antibodies?HcAb?without light chains are naturally produced by camelids.The single domain antigen-binding fragment of HcAb is referred to as VHH or nanobody?Nb?,which is the smallest antigen-binding entity at present.Several characteristics such as low molecular weight,high stability,and low immunogenicity make nanobodies have great value in the basic research,diagnosis and treatment.Despite the fact that nanobodies have better solubility compared to conventional antibodies,the same kind of nanobody has different yields in different expression systems and different nanobodies have different yields in the same expression system.So it's necessary to optimize nanobodies'expression systems to increasing nanobodies yields.In order to obtain the optimal expression system of VHH10 and VHH16,the E.coli expression system was selected on the basis of previous studies(pET-28a vector fused with DsbA-DsbA^mut).The yields of the nanobodies in E.coli were 642 mg/L and 348mg/L,respectively.The fusion protein DsbA-DsbA^mut-VHH10 and DsbA-DsbA^mut-VHH16 were digested with enterokinase to obtain natural monomeric antibodies.The final monomer yields were 14.7 mg/L and 8.9 mg/L,respectively.Then,VHH10 and VHH16 were investigated to have good antigen binding activity and high thermostability by sandwich Elisa.Finally,Bacillus subtilis and Pichia pastoris were used to express VHH10 and VHH16,and nanobodies were purified by ion exchange chromatography and affinity chromatography.The yields of VHH10 and VHH16 were2.3 mg/L and 1.8 mg/L in Bacillus subtilis,respectively.The final yields of VHH10 and VHH16 were 3.8 mg/L and 2.5 mg/L in Pichia pastoris,respectively.The fermentation cycle of E.coli is shorter and the final yield is higher than the other two expression systems,although it is necessary for E.coli expression system to carry out enzyme digestion and secondary purification.At the same time,DsbA-DsbA^mut dosen't affect the antigen-binding activity and the thermostability of nanobodies.This strategy can provide reference for the expression of other nanobodies.