Comparative Study On The Expression Of Two Nanobodies In Different Expression Systems

Nanobody is a new small antibody fragment.It is cloned from the VHH(variable domain of heavy chain of heavy chain antibodies).These nature-made nanobodies are well suited for various applications due to their favorable characteristics such as small size,ease of genetic manipulation,complete antigen binding site,good tissue penetration,high specificity,high affinity and solubility,overall stability,resistance to harsh conditions(e.g.,low p H,high temperature),and low immunogenicity.Engineered monoclonal antibody fragments have gained market attention due to their versatility and tailor-made potential and are now considered to be an important part of future immunobiotherapeutics,and it has vast develop prospects.The expression method and expression quality of the same nanobody in different expression systems have not been systematically studied in depth at present.In this thesis,7D12 and 9G8 Nanobodies were selected and expressed in Escherichia coli and Pichia pastoris respectively.This comparison laid the foundation for the large-scale industrialization of nanobodies.The specific content of the study is as follows:This study,genetic engineering methods were used to construct PET28a-7D12/PET28a-9G8 recombinant plasmid suitable for E.coli system expression and construct p GAPZa A-7D12/p GAPZa A-9G8 recombinant plasmid suitable for Pichia pastoris system expression.It provides the basis for subsequent expression of proteins.In this thesis,we first expressed the Nanobody 7D12 and used different methods to express in Escherichia coli BL21 and Pichia pastoris X33 respectively.On the basis of successful small-scale experiments,the 7D12 fermentation production,separation and purification were carried out on the pilot scale in cooperation with other researchers of the research group,and 7D12 samples with different purity were obtained for comparative analysis.The amount of the sample expressed in Pichia pastoris was significantly higher than that in E.coli.Furthermore,the Nanobody 9G8 was expressed in Escherichia coli BL21 and Pichia pastoris X33,respectively.Similarly,the amount of the sample expressed in Pichia pastoris was higher than that in E.coli.To sum up,Both Nanobodies have high expression levels in Pichia X33 and the experimental procedure is more simple and suitable for mass production.The replacement of genetic engineering drug expression systems is imperative,and the development of related technologies for the new generation of Pichia expression systems will greatly promote the development of the entire biomedical industry.The Pichia expression system has many advantages and it is a widely used eukaryotic expression system.With the further understanding and in-depth study of Pichia pastoris by the researchers,this expression system is bound to play an increasingly important role in the industrialization and commercialization of genetic engineering products.Based on the laboratory hardware and experimental basis,the conditions for nanobody expression in Pichia pastoris expression system were optimized to further increase the expression of nanobody.This will lay the foundation for connecting drug-related diseases afterwards.