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Construction and Expression of Fusion Gene (RGD)3 - DAB391 Prokaryotic Expression Vector and Study of Its Tumor Depression Effect in Vitro

Author: ZhangYiQun
Tutor: MaDing
School: Huazhong University of Science and Technology
Course: Obstetrics and Gynaecology
Keywords: (RGD) 3 - DAB391 fusion protein RGD peptide MCF-7 cells MDA-MB-231 cells Breast Cancer Prox1 Tumor metastasis Lymphatic endothelial cells
CLC: R737.9
Type: Master's thesis
Year: 2010
Downloads: 17
Quote: 0
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Abstract


[Objective] This study investigates the functional comparison between the diphtheria toxin before the RGD peptide receptor deletion build 391 amino acid (DAB391) of recombinant fusion gene expression and effect of verification and cyclic RGD peptide. Breast cancer cell line MCF-7 and MDA-MB-231 proliferation and apoptosis process, to investigate the mechanism of action targeting tumor therapy. 【Methods】 specific targeting molecules (RGD-RGD-RGD) - DAB391 (hereinafter abbreviated as (RGD) 3 - DAB391) prokaryotic expression vector containing RGD sequence DAB391 prokaryotic expression vector, constructed by recombinant methods select Induction of protein expression with Ni2-NTA affinity chromatography The method of the expressed protein was purified after the renaturation of the purified protein was detected by in vitro cytological its features. MTT assay comparing different molecules on the tumor cell proliferation inhibitory effect on tumor cells apoptosis induced differences detected by flow cytometry technology. [Results] successfully constructed recombinant prokaryotic expression vector (RGD) the 3 - DAB391/pET-28a, the target protein can be expressed after IPTG induction relative molecular weight of about 45,000, and its production accounts for about 25% of the E. coli total protein. After denaturation, can be obtained after purification and approximately 90% purity (RGD) 3 - DAB391 fusion protein expression product. The reduction / oxidation system refolding methods, eventually obtained a certain amount of active protein: (RGD) 3 - DAB391. It also obtained active DAB391 protein. The reorganization (RGD) 3 - DAB391 fusion protein and RGD peptide on tumor cell proliferation inhibition, a stronger role and the fusion protein (P lt; 0.01); these two molecules may increase tumor cell apoptosis , the effect is more apparent and the fusion protein (P lt; 0.01). No significant role (P gt DAB391 protein refolding after the two tumor cell; 0.05) Conclusion】 successfully build and get active (RGD) 3 - DAB391 fusion protein, shows stronger than RGD peptide tumor-killing effect, further study of small molecules targeting peptide provides a new approach to the application of targeted therapy of malignant tumors. 【Objective】 To investigate the expression of Prox1 in breast cancer tissue and its relationship with clinicopathological factors. 【Methods】 Immunohistochemical SP method detected 11 cases of benign breast hyperplasia and 80 cases of invasive ductal carcinoma tissue (including 30 patients with lymph node metastases, 50 patients without lymph node metastases) in Prox1 expression of. [Results] Prox1 in benign breast hyperplasia were negative expression in breast cancer cells and lymphatic endothelial cells express positive rates were 62.5% (50/80) and 65.0% (52/80), which positive rate of lymph node metastasis were 76.7% (23/30) and 83.3% (25/30) was significantly higher than that without lymph node metastasis-positive rate of 54.0% (27/50) and 54.0% (27/50), two differences between the groups was statistically significant (P lt; 0.05); and have metastasis, lymphatic the Prox1 expression of endothelial cells and the number of lymph node metastasis related (P lt; 0.05), but the expression of ER, PR, HER- 2 regardless of age. Cancer cells and lymphatic endothelial cells Prox1 expression in breast cancer tissue were positively correlated (r = 0.452, P lt; 0.001), and both the expression and TNM stage were positively correlated (r = 0.589, P lt; 0.001) . [Conclusion] Prox1 high expression in breast cancer may be associated with lymphatic metastasis of breast cancer are closely related.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Breast tumor
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