ABSTRACT
Human arrest defective 1(hARD1) is an acetyltransferase catalyzing the N-terminal acetylation of proteins after translation. The high expression of hARD1 could be an indicator of the breast cancer. In current study, we produced an anti-hARD lp monoclonal antibody that could specifically recognize ARD1 in breast cancer tissues by using the immunohistochemical assay. The full-length His-tag hARD1 protein (1-235 aa) was over-expressed in Escherichia coli, and purified recombinant protein was injected into Balb/c mice to perform immunization procedure. Eight stable positive monoclonal cell lines were isolated. ELISA results demonstrated that all light chains of antibodies were kappa, and the heavy chains displayed three subtypes IgG1, IgG2a and IgG2b, respectively. A monoclonal antibody, which could specifically recognize hARD1 protein in breast cancer tissues, was identified by screening different cancer tissues using antibody-specificity method. Further, the specificity of the antibody was confirmed by Western blotting analysis. Our study would facilitate breast cancer diagnosis by using this ARD1 monoclonal antibody in clinic. Also, this antibody could be used as an important tool for further investigating the role of ARD1 in tumorigenesis.
Subject(s)
Animals , Female , Humans , Mice , Acetyltransferases , Genetics , Allergy and Immunology , Antibodies, Monoclonal , Genetics , Allergy and Immunology , Biomarkers, Tumor , Allergy and Immunology , Breast Neoplasms , Allergy and Immunology , Metabolism , Pathology , Escherichia coli , Genetics , Metabolism , Immunization , Mice, Inbred BALB C , N-Terminal Acetyltransferase A , N-Terminal Acetyltransferase E , Recombinant Proteins , Genetics , Allergy and ImmunologyABSTRACT
Human arrest defective 1 (hARD1) is an acetyltransferase; its physiological significance remains unclear. To explore the relationship between ARD1 protein and tumors, we detected the hARD1 protein in tumor tissues in vivo. We cloned hARD1 gene from Hela cell and construct recombinant plasmid pET28b-hARD1. The recombinant plasmid was transformed into E. coli BL21 (DE3)plysS. hARD1 protein was expressed by inducing with IPTG(1 mmol/L) and purified up to 95% through Ni2+ chelation affinity chromatography. We used the purified hARD1 protein as antigen immunized the Balb/c mice and obtained the hARD1 specific polyclonal antiserum. Through immunohistochemical analysis of different tumor tissues in vivo, we found that hARD1 expressed at high frequency in breast cancer, prostate cancer and lung cancer, especially, hARD1 expression frequency in breast cancer was up to 70%, which is higher than in the other tumors. These results indicate that the high expression level of hARD1 could be an indicator of the breast cancer. This new finding would be a foundation to further explore the relationship between breast tumor and hARD1.