Cloning and sequence analysis of death associated protein kinase gene ORF and DAPK1 inducing Raji cell apoptosis

AIM: Open reading frame(ORF) of death associated protein kinase1(DAPK1) gene was cloned for studying on tumor forming and metastasis.METHODS: Based on nucleotide sequence of DAPK1 gene from GenBank, a pair of primers was designed. DAPK1 gene ORF was transfected into Raji cells in expression vector pcDNA3.1(+) with lipofectamine reagent. Morphologic assessment of apoptosis was performed with fluorescence microscope cytotoxicity and cell viability was assayed by MTT. RESULTS: DAPK1 gene ORF was amprified from K562 cells by RT-PCR. It was cloned into plasmid pMD18-T and sequenced. There were seven mutation in 4 300 bp nucleotide sequence relatively to DAPK1 nucleotide sequence from GenBank, but six was synonymous mutation and one was single nucleotide polymorphism. 4 300 bp nucleotide of DAPK1 gene ORF was transfected into Raji cells. DAPK1 gene expression was detected in 48 h after it was transfected into Raji cells. Then Raji cells showed apoptosis. CONCLUSION: Large fragment gene was cloned by RT-PCR and transfected into Raji cells successfully. Over-expression of DAPK1 gene induced Raji cells apoptosis.