Construction of a human pancreatic cancer cell line PANC1 stably over-expressing hB7-H3 gene
10.3760/cma.j.issn.1674-1935.2017.04.007
- VernacularTitle:稳定过表达hB7-H3基因的人胰腺癌PANC1细胞株的构建
- Author:
Dongbao LI
;
Dechun LI
;
Hua ZHAO
;
Xin ZHAO
- Keywords:
Pancreatic neoplasms;
Lentiviral infections;
Gene expression;
Cell line,tumor;
hB7-H3
- From:
Chinese Journal of Pancreatology
2017;17(4):243-247
- CountryChina
- Language:Chinese
-
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.
