Construction of a lentiviral expression vector carrying EphB4 and its expression in colon cancer cells / 医学研究生学报

ABSTRACT

Objective Studies show that the abnormal expression of EphB4 plays an important role in the development and progression of colon cancer .The present study aims to provide some experimental evidence for the gene therapy of colon cancer by con -structing a lentiviral expression vector carrying the homo EphB4 gene and further establishing colon cancer cell lines with stable overex-pression of EphB4. Methods A series of oligonucleotides (oligo) encoding the homo EphB4 gene were ligated together by PCR and then cloned into a lentiviral expression vector pLenti 6.3-MCS-IRES2-EGFP.After confirmed by sequencing , the vector pLenti6.3-EphB4-IRES2-EGFP and its helper vectors were mixed and co-transfected into 293 T cells to obtain recombinant virus containing the EphB4 gene.The lentiviral titer was detected and the resulting recombinant lentiviruses carrying EphB4 or control viruses only carrying green fluorescence protein (GFP) were used to infect the human colon cancer cell lines .The expression of GFP was determined under the inverted fluorescence microscope and the level of EphB 4 mRNA in the infected cells detected by qPCR . Results The lentiviral expression vector pLenti6.3-EphB4-IRES2-EGFP carrying correct homo EphB4 gene sequence was successfully constructed .The titer of the recombinant EphB4 lentiviral supernatant Lenti6.3-EphB4 was 1 ×108 TU/mL.The expression of GFP was observed in the trans-duced cells under the fluorescence microscope , and that of EphB4 mRNA in the transfected SW480 and Coca-2 cells was significantly up-regulated as compared with the control and blank groups . Conclusion The homo EphB4 gene was successfully amplified and cloned.A lentiviral expression vector was successfully constructed , and so were colon cancer cell lines stably overexpressing EphB 4, which may shed light on the lentivirus-mediated genetic therapy for colon cancer .