In silico designing and expression of novel recombinant construct containing the variable part of CD44 extracellular domain for prediagnostic breast cancer.

Abstract

CD44, as a tumor-associated marker, can be used to detect stem cells in breast cancer. While CD44 is expressed in normal epithelial cells, carcinoma cells overexpress CD44. In the current study, we designed a recombinant protein that included the variable component of the CD44 (CD44v) extracellular domain to apply in clinical diagnosis of breast cancer. A total of 100 CD44v amino-acid residues were determined, and the structure was examined using bioinformatics tools. The construct was inserted into the PET28a vector and transformed in E. coli BL21(DE3). A nearly 12 kDa fusion protein was obtained by Ni-NTA affinity metal chromatography. Recombinant CD44v was examined by Western blotting, ELISA, and immunohistochemistry (IHC) assays. The findings revealed that the structure of rCD44v was stable, and its antigenic domain was exposed. The recombinant CD44v was confirmed by western blotting, and the presence of antibodies against recombinant CD44v protein in the patient's serum was detected by the ELISA. Our data demonstrated a link between CD44v serum levels and the prevalence of breast cancer. Assessments of antiCD44v antibodies with rCD44v could be a useful tool for identifying breast cancer in its early stages, which can lead to better outcomes.