Production Of Monoclonal Antibody To NCOA5 And The Significance Of Its Expression In Breast Cancer

Objective:To produce monoclonal antibody specifically recognizing NCOA5 protein by hybridoma technique; To analyze the influence of NCOA5 on clinical and pathological parameters by detecting its expression in breast cancer specimens; To observe the effect on proliferation and migration of breast cancer cell lines after stably transfected with NCOA5 shRNA. Methods:NCOA5 C-terminal segement was amplified by PCR and inserted into the prokaryotic expression vector pET-41 a. Recombinant protein was expressed and the soluble fragment was purified by Ni-NTA column. The purity of the protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) followed by Coomassie Blue staining. Using hybridoma technique, the monoclonal antibody recognizing NCOA5 protein was produced, and the specificity of the antibody was verified by ELISA, Western blot, immunofluorescence and immunohistochemistry. The short segments of NCOA5 C-terminal fragment were amplified by PCR and inserted into pMAL-c2 X prokaryotic expression vector. The recombinant protein was expressed and applied to Western Blot to identify the range containing epitope recognized by the monoclonal antibody. NCOA5 expression level in breast cancer specimens was detected by immunohistochemistry using monoclonal antibody and the relationship between NCOA5 and clinicopathological parameters was analyzed. The breast cancer cell line MDA-MB-231 was transfected with NCOA5 shRNA, CCK-8 assay was applied to detect its effects on cell proliferation while wound healing and Transwell assay were required to mesure the change of cell migration. Results:The recombinant plasmid of NCOA5 C-terminal and pET-41 a vector was constructed and the fusion protein was expressed in bacteria with 1 mM IPTG induction and purified by Ni-NTA af?nity chromatography. SDS-PAGE and Coomassie blue staining showed a strong band at a molecular weight of about 69 kDa which means the fusion protein in the soluble fraction was successfully expressed and puri?ed. To screen MAb against NCOA5, hybridomas were detected for secretion by indirect ELISA and one stable clone which strongly reacted with NCOA5 was identified and designated as 8B11. The monoclonal antibody was produced in large scale and purified, the titer of 8B11 against NCOA5 was detected by indirect ELISA to be 1:32,000. The specificity of the 8B11 was verified by Western blot, immunofluorescence and immunohistochemistry. The recombinant plasmid of pMAL-c2 X vector and NCOA5-C-ter segments were constructed and the fusion protein was expressed in bacteria with 0.3 mM IPTG induction. Western blot showed 8B11 could recognize recombinant protein pMAL-c2X/NCOA5-C-ter-N’ which represented the amino acid between 291-434. Immunohistochemistry of breast cancer specimens showed the positive expression rate of NCOA5 correlates with larger tumor size, lymph node metastasis, late TNM stage, negative expression of ER or PR, with statistically signi?cant difference, but has no relationship with age, histologic grade and HER2 status. The breast cancer cell line MDA-MB-231 transfected with NCOA5 shRNA showed ef?ciently reduced level of endogenous NCOA5 protein and signi?cantly decreased cell proliferation and migaration. Conclusion:(1) The pET-41a/NCOA5-C-ter fusion protein was successfully expressed and puri?ed.(2) The monoclonal antibody 8B11 specifically recognizing endogenous NCOA5 protein was produced and characterized by Western blot, immunofluorescence and immunohistochemistry, the epitope recognized by 8B11 locates between 384-434 amino acid of NCOA5 protein.(3)NCOA5 is expressed in different cancer cell lines. The positive expression rate of NCOA5 correlates with larger tumor size, lymph node metastasis, late TNM stage, negative expression of ER or PR.(4) The breast cancer cell line MDA-MB-231 transfected with NCOA5 shRNA showed ef?ciently reduced level of endogenous NCOA5 protein and signi?cantly decreased cell proliferation and migaration.