The Construction And Characterization Of RP215-VH Single Domain Antibody

ObjectiveThe aim of this study was to develop the novel recombinantsdAb-RP215antibody based on the variable regions of the RP215monoclonal antibody (RP215-mAb) against CA215,a pan cancer markerexpressed in various human tumor tissues, and to examine their biologicalactivity in breast cancer cell lines.Methods1. The reconstructed RP215-VH fragments were digested with theappropriate restriction enzymes and subcloned into the pET32a(+) vectordigested with the same enzymes to create new expression vector,pET32a(+)-RP215-VH. The identity of the inserted fragment was confirmedby appropriate restriction enzymes analysis followed by automated DNAsequencing. The pET32a(+)-RP215-VH were transformed into E. coliBL21(DE3), and express in large scale. The bacterial pellet were collectedand resuspension, and then sonicated on ice. The supernatant was purifiedusing Ni-NTA His-Bind IMAC column. The products of RP215-VHantibodies were confirmed by SDS-PAGE and Western-blot.2．The specific binding reactivity of RP215-VH antibodies against target antigen-containing breast cancer cells (MB231, MCF7, BT549,MB468, and SK-BR-3) were examed by Immunofluorescence staining,Western-blot, ELISA and Competitive ELISA.3. To determine RP215-VH function in breast cancer in vitro, CCK8assay, cell cycle analysis,cell apoptosis assay were used.Results1.The recombinant pET32a(+)-RP215-VH was successfullyconstructed and comfirmed as31KDa by SDS-PAGE and Western-blot.2. Immunofluorescence analysis showed that red color was detected inbreast cancer cells (MB231, MCF7, BT549, SK-BR-3, MB468), but nofluorescence was detected in controls (HBL100).3. The results of western blot showed25-75KDa immunoreactive bandsin the human breast cancer cells (MB231、MCF7、BT549), but no band wasdetected in controls.4. In ELISA, RP215-VH showed best binding activity to antigenCA215during dilution ranging from1/25to1/200in four breast cancer celllines (BT549, MB231, MCF7and Sk-BR-3).5. In Competitive ELISA,competitive inhibition rate is53.9%、55.6%、31.4%、10.6%、11.3%、4.6%respectively during dilution ranging from1/25to1/200。6. To assess the effect of the antibodies on cell cycle progression andapoptosis, MB231cells were treated with RP215-VH, stained with propidium iodide (PI) and analyzed by flow cytometry, which showed thatthe percentage of cells in the G0/G1phase increased significantly from50.21%to63%（p<0.05）and increased apoptotic cells in antibody-treatedMB231cells（p＞0.05）.ConclusionThe recombinant pET32a(+)-RP215-VH was successfully constructedand could bind to antigen CA215of breast cancer cells, showingimmunoreactivity. Flow cytometric analysis of cell cycle demonstrate thatRP215-VH obviously increased the number of MCF7cells in the G0-G1phase from48.6%to63%（p<0.05）.Our results showed that sdAb-RP215have excellent immunoreactivity andlocalize accurately to breast cancercells in membrane-bound form, suggesting their potential as tumortargetingantibodies for breast cancer therapy.