pComb3XSS vector

Tetanus is a leading cause of death in the developing world and is caused by the tetanus neurotoxin.Recombinant antibodies against tetanus neurotoxin can be used in tetanus treatment.Phage display of antibody fragments from immune human antibody libraries using single-chain constructs that bind variable fragments(scFv)has been one of the most important techniques in antibody engineering.The purpose of this study was to generate a library of variable region single-chain fragments(scFv)and select specific antibodies with high affinity for tetanus toxin.A phagemid library of immunized human single-chain fragment variable(HuscFv)antibodies was displayed on pill of filamentous phage.After three rounds of panning,scFv clones were selected against the tetanus toxin antigen.Selected scFv clones were analyzed for their inhibitory effect on tetanus toxin binding to ganglioside GT1b.The researchers found that six HuscFv clones inhibited toxin binding to ganglioside GT1b.These selected antibodies may be used in the treatment of tetanus.

In this study,the variable regions of the heavy(VH)and light(VL)antibody genes were amplified by PCR.The amplified VH and VL products were electrophoresed and the expected fragment size of 400 bp was observed(Figure 1A).These variable fragments were used in overlap extension PCR reactions to generate scFv fragments.The scFv product(750 bp)was confirmed by agarose gel electrophoresis(Figure 1A).The overlap extension PCR product was cloned into the pComb3Xss vector(purchased from Creative Biogene),and the library was transformed into TG1 E.coli.The total scFv library contained 1×105 clones.

Figure 1.Electrophoresis gel showing the Ig light chain(V_L)and heavy chain(V_H)domains.(Khalili E,et al.,2015)