A novel technique for efficient construction of large scFv libraries.
The development of patient-tailored anti-tumor therapies requires large-scale production of antibodies for the purpose of screening specific tumor antibodies. Thus, the generation of a single chain fragment of variable region (scFv) library with a large repertoire is required for the selection of specific antibodies with high affinity. Presently, the generation of large scFv libraries is impeded by the low efficiency of cloning PCR fragments into phage display vectors, which is due to the low efficiency of the restriction digestion step required in the process. The aim of this study was to increase the efficiency of this critical step. We found methods that are formally believed to facilitate the digestion efficiency, such as adding longer tails to the primers or prolonging the incubation time, were inefficient. We then investigated the feasibility of using T/A cloning to improve the efficiency of cloning and found that when PCR fragments were first cloned into T-vector, and then subsequently subcloned into a phagemid, the cloning efficiency was dramatically increased. Our findings show that by utilizing this method the construction of a large scFv library can be easily accomplished.
Qin
Weijun
W
Department of Urology, Xijing Hospital, The Fourth Military Medical University, Xi'an, China.
Zhao
Aizhi
A
Han
Yueheng
Y
Wen
Weihong
W
Li
Yu
Y
Chen
Gang
G
Zhang
Zhuoli
Z
Wang
He
H
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004526:Efficiency; D005240:Feasibility Studies; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008969:Molecular Sequence Data; D019151:Peptide Library
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D004262:DNA Restriction Enzymes