pComb3X vector

Prostate cancer is the most common type of cancer in men.Antibody-mediated cancer therapy depends on the identification of selected molecular targets.Prostate-specific membrane antigen(PSMA)is a potential molecular target in prostate cancer and is abundantly expressed in this type of cancer.The variable fragment of heavy-chain antibodies(VHH)is the smallest(approximately 120 amino acids)antibody fragment capable of binding to an antigen.In addition to their small size,VHHs are generally more stable(especially under heating conditions)and more soluble than traditional antibodies.VHHs can bind to their targets with high affinity and can be produced in high yields by simple methods.In this study,researchers used phage display technology to produce high-affinity VHHs targeting PSMA.Camels were immunized with recombinant PSMA and then a VHH phage library was generated.After panning and screening,the VHHs with the highest affinity for PSMA were selected and expressed in their soluble form.Selected VHHs were then characterized and their affinity for PSMA was assessed.

In this study,the VHH gene and pComb3x vector(purchased from Creative Biogene)were digested with sfil restriction enzyme and purified.The digested VHH gene was ligated to phagemid pComb3x by T4 DNA ligase.The ligation product was transferred into competent E.coli XL1 blue by electroporation.Library size was determined by plating 10μL of transformants on 100μg/mL ampicillin LB agar plates.Colonies were counted to calculate library size and PCR was performed to confirm VHH clones.The VHH fragment cloned into the pComb3x phagemid vector showed a library size of 3×106.PCR analysis revealed the presence of the VHH gene in 95%of the clones.Phage obtained from each round of panning were used in an ELISA reaction to detect rPSMA(Figure 1A).The light absorption at 450 nm reached 1.4 in the fourth round of biopanning.The colonies number 3,10,and 18 showed the highest affinity towards rPSMA(Figure 1B).

Figure 1.(A)Biopanning process(enrichment)by polyclonal phage ELISA.(B)Single colonies selected from the fourth stage of biopanning.(Zare H,et al.,2014)