Constructing CXCR4 lentiviral vector and transfecting breast cancer cells / 实用医学杂志

Objective To construct lentivirus containing CXCR4 gene and transfect MCF-7 cells , and obtain CXCR4 high-expressing MCF-7 cells. Methods CXCR4 gene was amplified by RT-PCR to construct CXCR4/pSin-EF2, which was transfected into HEK293T cells with psPAX2 and pMD2G vector for lentivirus packing. Packaged lentivirus was used to transfect human breast cancer cells MCF-7, with empty lentivirus as control. CXCR4 mRNA and protein expression levels were detected by RT-PCR and Western blot before and after transfection. And flow cytometry was used to detecte cell surface CXCR4 expression. Results The recombinant plasmid CXCR4/pSin-EF2 was constructed successfully,identified by double digestion and sequencing, and transfected into HEK293T cells to obtain high-titer lentivirus. RT-PCR and Western blot confirmed that the expression of CXCR4 in MCF-7 cells increased significantly after CXCR4 lentivirus transfection. Flow cytometry results showed that the CXCR4 positive rate increased from 26.78% to 99.29%, while there is no significant difference in CXCR4 expression between vector-transfected MCF-7 cells and non-transfected MCF-7 cells. Conclusion CXCR4 lentivirus and the breast cancer cell line with high and stable expression of CXCR4 (MCF-7CXCR4) were successfully constructed.

Soluble expression and activity evaluation of SDF-1/54R, a specific antagonist of CXCR7 / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a soluble prokaryotic expression vector of the CXCR7-specific antagonist SDF-1/54R and evaluate its activity.</p><p><b>METHODS</b>SDF-1/54r gene amplified by PCR was inserted into the soluble expression vector pET-41a+ engineered with GST fusion tag, and the recombinant vector was transformed into E. coli strain BL21 (DE3). After IPTG induction of E. coli, the expressed recombinant protein was purified with GST affinity chromatography purification system and confirmed by SDS-PAGE and Western blotting assay. The target protein SDF-1/54R was obtained after digestion of the purified product with enterokinase. Breast cancer MCF-7 cells with high expression of CXCR7 was treated with SDF-1/54R and the cell proliferation and metastasis was evaluated with MTT and chemotaxis assays.</p><p><b>RESULTS</b>The target protein SDF-1/54R obtained showed an obvious inhibitory effect on the proliferation and metastasis of MCF-7 cells as confirmed by MTT and chemotaxis assays.</p><p><b>CONCLUSION</b>SDF-1/54R is a good antagonist of CXCR7 and shows a potential value as an effective anti-cancer agent.</p>