Construction and Identification of Transfection of Human Chk1/2Gene in Gastric Cancer MGC803 Cells

Objective To construct the eukaryotic expression vector and human gastric cancer MGC803 cells of human Chk1/2 gene for investigating the function of Chk1/2.Methods A pair of primer smatching each end of Chk1 and Chk2 cDNA and supplementing with appropriate endonuclease sites were designed according to the sequence published in NCBIGenBank.The Chk1 and Chk2 mRNA were extracted from human gastric cancer MGC803 cells,and used as the template to synthesize the first strand of cDNA.The Chk1 and Chk2 gene with complete expression sequence were amplified,which were cloned into pcDNA 3.1(+) vector after being double digested by endonucleases.The recombinants were identified with specific PCR and sequencing.The recombinant plasmid were transfected into MGC803 cells via lipofectamine,and after G418 selection,the expression of Chk1/2 were detected by RT-PCR and Western blot.Results The results of specific PCR showed that a 1.4 kb and 1.6kb fragment had been cloned into pcDNA3.1(+) vector,which was further identified by sequencing and NCB BLAST analysis.The stable clones transfected with 2 vectors were respectively constructed.As identified by RT-PCR and Western blot,the expression of Chk1/2 in MGC803 cells were significantly increased as compared with that in control groups.Conclusion Eukaryotic expression plasmid pcDNA3.1(+)/Chk1/2 and MGC803 cells of overexpression Chk1/2 have been constructed successfully,which may facilitate further research of Chk1 and Chk2 in G2/M arrest induced by diallyl disulfide.