Cloning and expression of human anti-Rh(D) single-chain Fv fragment / 医学研究生学报
Journal of Medical Postgraduates
;
(12)2003.
Article
in Chinese
| WPRIM
| ID: wpr-586375
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Journal of Medical Postgraduates
Year:
2003
Type:
Article
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