Cloning and sequence analysis of death associated protein kinase gene ORF and DAPK1 inducing Raji cell apoptosis
- VernacularTitle:DAPK1基因开放读码序列的克隆、序列分析及诱导Raji细胞凋亡
- Author:
Haitao ZHANG
;
Zhenyu ZHU
;
Qiongmei JI
;
Minyou LI
;
Jianquan MA
;
Nianc LIANG
- Publication Type:Journal Article
- Keywords:
Death associated protein kinase;
Apoptosis;
M ethylation;
Raji cells;
K562 cells
- From:
Chinese Journal of Pathophysiology
1986;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: Open reading frame(ORF) of death associa ted protein kinase1(DAPK1) gene was cloned for studying on tumor forming and met astasis.METHODS: Based on nucleotide sequence of DAPK1 gene f rom GenBank, a pair of primers was designed. DAPK1 gene ORF was transfected into Raji cells in expression vector pcDNA3.1(+) with lipofectamine reagent. Morphol ogic assessment of apoptosis was performed with fluorescence microscope cytotoxi city 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 rel ativel y to DAPK1 nucleotide sequence from GenBank, but six was synonymous mutation and one was single nucleotide polymorphism. 4 300 bp nucleotide of DAPK1 gene O RF was transfected into Raji cells. DAPK1 gene expression was detected in 48 h a fter it was transfected into Raji cells. Then Raji cells showed apoptosis.CONCLUS ION: Large fragment gene was cloned by RT-PCR and transfected into Raji cells successfully. Over-expression of DAPK1 gene induced Raji cells apoptosis.
