title: Design, expression and characterization of single chain Fv, Mms13 and the single chain Fv‑mms13 fusion protein.
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.
authors.affiliation: Department of Basic Medicine, Weifang Medical College, Weifang, Shandong 261053, P.R. China.
authors.forename: Deng
authors.initials: D
authors.lastname: Kong
authors.affiliation: Department of Basic Medicine, Weifang Medical College, Weifang, Shandong 261053, P.R. China.
authors.forename: Xiaoke
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Pharmacy and Bioscience, Weifang Medical College, Weifang, Shandong 261053, P.R. China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Pharmacy and Bioscience, Weifang Medical College, Weifang, Shandong 261053, P.R. China.
authors.forename: Xueyun
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Basic Medicine, Weifang Medical College, Weifang, Shandong 261053, P.R. China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Basic Medicine, Weifang Medical College, Weifang, Shandong 261053, P.R. China.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Ji
authors.affiliation: Department of Basic Medicine, Weifang Medical College, Weifang, Shandong 261053, P.R. China.
authors.forename: Xinhua
authors.initials: X
authors.lastname: Fu
authors.affiliation: Department of Basic Medicine, Weifang Medical College, Weifang, Shandong 261053, P.R. China.
authors.forename: Shouxun
authors.initials: S
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018093:Gelatinases / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D061986:MCF-7 Cells; D055944:Magnetosomes / Q000737:chemistry / Q000378:metabolism; D042201:Magnetospirillum / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D058990:Molecular Targeted Therapy / Q000379:methods; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D018093:Gelatinases