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Published on: December 23, 2016
[Construction and expression of anti-human AFP ScFv gene in BL-21 (DE3) E. coli]
Ping Zhang1, Mei-ying Cai, Zong-rong Zhao
1Department of Immunology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Objective:
To construct anti-human AFP single chain fragment variable (ScFv) gene, transform it into BL-21 (DE3) E. coli for expression, and identify its bioactivity.
Methods:
VH and VL genes of anti-human AFP monoclonal antibody were cloned by RT-PCR from hybridoma. The ScFv gene was spliced by sequence overlap extending (SOE) PCR, and then it was ligated into pGEM-T vector to be identified by endonuclease digestion, PCR and sequencing. ScFv gene was cloned into pET32 (a+) vector and transformed into BL-21 (DE3) E. coli. The positive clones were screened out by IPTG induction, and the ScFv antibody was purified to be identified by SDS-PAGE and competitive inhibition ELISA test.
Results:
The VH DNA consisted of 339 bases, coming from the mouse IgG gamma chain. The VL DNA consisted of 312 bases, coming from the mouse IgG kappa chain. The VH and VL genes were spliced by 45 bases coding a (G4S)3 flexible linker. The ScFv gene consisted of 696 bases. The ScFv antibody expressed by BL-21 (DE3) fused with TrxA tag protein and formed inclusions. The relative molecular mass of TrxA-ScFv fusion protein is about 40 x 10(3) and that of ScFv is about 24 x 10(3). The ScFv antibody has excellent activity tested by competitive inhibition ELISA, the TrxA-ScFv could inhibit about 41% of the McAb to bind antigen and ScFv could inhibit about 53%.
Conclusion:
We have successfully constructed an anti-human AFP ScFv gene with 696 bases; it can express in BL-21 with high activity.
