Construction of a human pancreatic cancer cell line PANC1 stably over-expressing hB7-H3 gene

Abstract

Objective To construct the stably over-expressing hB7-H3 gene human pancreatic cancer cell line PANC1, and provide tools for further investigating the function of hB7-H3. Methods hB7-H3 gene fragment was inserted into lentiviral plasmid GV287 carrying GFP to construct recombinant hB7-H3-GV287 plasmid vector. 293T cells were transfected, and the GFP expression was evaluated under fluorescence microscopy. Western Blot wasused to detect the expression of hB7-H3 protein. Lentiviral vectors were packaged and the titer was determined. The recombinant hB7-H3 expressed lentivirus was used to infect PANC1 cell. Flow cytometry was applied to detecte the percentage of GFP and hB7-H3 positive cells. Real-time PCR and Western Blot was used to verify the mRNA and protein expression of hB7-H3. Self-cyclizing GV287 plasmid served as negative control (NC). Results PCR amplified fragment of recombinant plasmid was around 1 368 bp, and no amplified production of NC plasmid was observed. The DNA sequencing of recombinant plasmid was completely consistent with the designed fragment, indicating that hB7 H3-GV287 plasmid was successfully constructed. 293T cells transfected with recombinant plasmid expressed hB7-H3 protein, while those cells transfected with NC plasmid did not express hB7-H3 protein. The virus titer of lentiviral packaged recombinant hB7-H3 plasmid was 2×108 TU/ml. The percentage of hB7-H3 positive cells, hB7-H3 mRNA and protein expression in PANC1 cells infected with cells infected with hB7-H3 lentivirus was 94.3%, 5.09±0.24 and 2.85±0.27, respectively, which was obviously higher than 18.5%, 1.28±0.53 and 0.44±0.69 in cells infected with NC lentivirus, and the differences were statistically significant (P value <0.01). Conclusions A human pancreatic cancer cell line PANC1 stably over-expressing hB7-H3 was successfully constructed. Key words: Pancreatic neoplasms; Lentiviral infections; Gene expression; Cell line, tumor; hB7-H3