Cloning and expression of human anti-Rh(D) single-chain Fv fragment / 医学研究生学报

ABSTRACT

Objective: To construct a phage display library of human single-chain Fv antibodies against blood group Rh(D) substance. Methods: Combining phage display library techniques, isolated total RNA from B lymphoblastoid cell lines secreting anti-Rh(D) antibodies was used for the synthesis of the first strand of cDNA, V_ H and V_ L genes were amplified by 2nd PCR and linked together by splicing overlap extension (SOE) with the use of a (Gly_ 4Ser)_ 3 linker. The resulted scFv genes were then cloned into pCANTAB5E vectors and displayed on the phage. Phage clones were selected using intact red cells as a source of antigen. After 4 rounds of "binding-elution-enrichment", each clone was assayed for specificity by Dot ELISA. Results: A phage antibody library, with the sink size being 1.2?107, was obtained. The percentage of full-length scFv gene inserted into phage DNA was 0.80. Rescued by helper phage, a phage scFv library with titer of 3?108 pfu/ml was established. Specific phages with scFv were acquired after 4 rounds of panning, one clone exhibiting specific binding to Rh+ cell was identified by Dot ELISA. Conclusion: A strategy for construction phage antibody library by means of phage display technique was practicable, which would be useful in screening engineered antibodies against human Rh (D) blood group substances.