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Mol Med Rep. 2015 Jul;12(1):1213-8. doi: 10.3892/mmr.2015.3561. Epub 2015 Mar 27.

Design, expression and characterization of single chain Fv, Mms13 and the single chain Fv‑mms13 fusion protein.

Author information

1
Department of Basic Medicine, Weifang Medical College, Weifang, Shandong 261053, P.R. China.
2
Department of Pharmacy and Bioscience, Weifang Medical College, Weifang, Shandong 261053, P.R. China.

Abstract

Single chain Fv (scFv) antibodies are attractive as tumor-targeting vehicles due to their smaller size compared with intact antibody molecules. Mms13 is a putative membrane anchor protein of magnetosome. The present study fused the scFV gene of type Ⅳ collagenase to mms13 using the splicing by overlap extension polymerase chain reaction technique. The genes of scFv, mms13 and the scFv-mms13 fusion gene were cloned into a pET30a(+) vector to construct pET30a(+)-scFv, pET30a(+)-mms13 and pET30a(+)-scFv-mms13 expression vectors. The three protein compositions were confirmed by DNA sequencing and western blot analysis, and their cellular locations were determined using SDS-PAGE. The results of enzyme-linked immunosorbent assays and immunofluorescence demonstrated that the ScFv and ScFv-mms13 fusion proteins bound to the type Ⅳ collagenase and the antigen-associated cancer cells SMMC-7721, MCF-7 and HepG2 cells, in a dose-dependent and saturable manner. Although the immunoreactivities of ScFv-mms13 to the type Ⅳ collagenase and associated tumor cells were marginally lower than the corresponding scFv (3G11), considerable binding ability to the antigen by ScFv-mms13 remained.

PMID:
25824464
DOI:
10.3892/mmr.2015.3561
[Indexed for MEDLINE]

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