1. Shahid Bahonar University of Kerman
Construction a synthetic phage display nanobody library from variable domain of human heavy chain immunoglobulins
Alibeiki M1 ,Golchin M1.
1Department of Phatobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran
Shahid Bahonar University of Kerman
Background
Single-domain antibodies (or called Nanobodies) are antibody fragments consisting of single monomeric variable antibody domains. Like a whole antibody, they are able to bind selectively to a specific antigen. These small molecules (12–15 kDa) have high target specificity and affinity for their targets and low inherent toxicity. The purpose of current study was construct a synthetic phage display nanobody library from variable domain of human heavy chain immunoglobulins with high diversity.
In conventional cloning method, pIT2 and pR2 vectors were digested by Nco I and Not I restriction enzymes. Next, antibody fragment genes from DAB library and pIT2 vector isolated from agarose gel and purified. Finally these fragments linked to each other using T4 ligase enzyme and incubation for overnight at 16 oC.
Cloning products were then transformed into TG1 E. coli cells.
Results
Methods
The results indicated variable domain of human heavy chains were amplified successfully and cloned in pIT2 vector.
To construct the library of human heavy chain nanobodies, human VH (V3-23/DP-47, 4H4b) genes were amplified by PCR from human synthetic DAB library. These fragments were cloned into pIT2 phagmid vector with Circular Polymerase Extension Cloning (CPEC) technique and conventional method using T4 ligase enzyme.
For CPEC technique, antibody fragment genes were amplified by PCR method using following primers and pfu enzyme. These primers had overlapping regions with cloning sites of pIT2 vector. Then amplified antibody fragments were purified from agarose gel and joined to vector by PCR. The library gene were then transformed into E. coli TG1 cells by electroporation.
Conclusion
It is possible to isolate specific nanobodies against different antigens from the constructed library for diagnostic and therapeutic proposes.
Figure 1: PR2
A: Lader
B: Cutting Plasmid
Figure 2: PIT2
A: Lader
B: Cutting Plasmid
Figure 3: PCR1
A: Lader
B: Product PCR1