Construction And Expression Of Fusion Gene (RGD)₃ - DAB₃₉₁ Prokaryotic Expression Vector And Study Of Its Tumor Depression Effect In Vitro

Objective: This study investigated the expression of constructed recombinant fusion gene of the RGD peptide linking the absence of the diphtheria toxin receptor area before the 391 amino acids (DAB391) and its effect validation compared with the cyclic RGD peptide. To investigate those influence on proliferation and apoptosis of breast cancer cell lines : MCF-7 and MDA-MB-231 in vitro and discuss the mechanism in tumour targeted therapy.Methods: To construct specific targeting molecules (RGD-RGD-RGD) - DAB391 (The following abbreviated as (RGD)3- DAB391) prokaryotic expression vector and the non-RGD sequence of the prokaryotic expression vector DAB391 by recombinant method. To induce the expression of those proteins Selectively and purify the expressed proteins by Ni2 +-NTA metal affinity chromatography, then test those cytology functions after Purified protein refolding in vitro. Cell proliferation was evaluated by MTT assay. Cell apoptosis was detected by FACS.Results: constructed the recombinant prokaryotic expression vector (RGD)3- DAB391/pET-28a successfully. After induced by IPTG, an extra protein band located near the 45KD, approximately 25% of the total bacterial protein. After denaturation and purification, the putity of fusion protein (RGD)3- DAB391 may nearly reach 90%. By reduction / oxidation system refolding method, we finally gain a certain amount of the active protein: (RGD)3- DAB391. Some DAB391 active protein was also been obtained by the same way. Both (RGD)3- DAB391 fusion protein and RGD peptide could decrease the proliferation and increase apoptosis of breast cancer cell lines. Moreover, the fusion protein expressed more illustriously inhibiting tumour cells effect(P < 0.01). Yet, Refolded DAB391 protein had no effect on the two tumor cell(P>0.05).Conclusion: The fusion protein had been recombined and refolded successfully. It showed stronger anti-tumor effect of RGD peptide. This work may provide some new skills for the research of tumour molecular targeted therapy. Objective: To investigate the expression of Prox1 in human breast carcinoma and its relationship with clinical pathologic characters.Methods: The expression of Prox1 was detected by SP immunohistochemistry in 11 specimens of hyperplasia of mammary glands,80 specimens of breast cancer (30 with lymphatic metastasis and 50 without metastasis).Results: The expression of Prox1 in 11 specimens of hyperplasia of mammary glands was completely negative,but were 62.5%(50/80)of the carcinoma nests cells and 65.0%(52/80)of lymphatic endothelial cells in breast cancer specimens. The percentage of cells positive for Prox1 in breast cancer patients with metastasis (76.7%, 23/30) was higher than those without (54.0%, 27/50), as well as in lymphatic endothelial cells with metastasis (83.3%, 25/30) than those without (54.0%, 27/50). The differences of two groups have statistical significance (P<0.05). The expression of Prox1 in the lymphatic endothelial cells of lymphatic metastasis specimens was correlated with the numbers of invaded lymph nodes(P<0.05). However, no significant correlation was found between Prox1 expression level and the following clinical parameters: age, ER status, PR status, HER2 status(P>0.05). Positive correlation(r=0.452, P<0.001)of the Prox1 expression was found between carcinoma cells and lymphatic endothelial cells, and Prox1 expression in carcinoma cells and lymphatic endothelial cells were both positively correlated with TNM stage(r=0.589, P<0.001).Conclusion: The high expression of Prox1 may be correlated with the process of breast cancer progression and metastasis.