摘要
目的 :探讨死亡相关蛋白激酶 (DAPK1)在肿瘤的形成、转移中所起作用 ,克隆DAPK1基因的开放读码序列 (ORF)。方法 :根据GenBank提供的核苷酸序列 ,自行设计引物 ,用RT -PCR从K562细胞中扩增DAPK1基因的ORF ;用质脂体将pcDNA3 1(+ ) -DAPK1转染到Raji细胞 ,Hoechst 3 3 2 58染色观察细胞凋亡 ,MTT法观察DAPK1基因对细胞生存的影响。结果 :DAPK1基因的ORF克隆到质粒pMD18-T ,测序鉴定 ;分析结果显示 ,从K562细胞成功扩增 43 0 0bp的DAPK1基因ORF有 7处突变 ,其中 6处是同义突变 ,1处是基因的单链核苷酸多态性 ;DAPK1基因的ORF克隆到真核表达载体pcDNA3 1(+ ) ,转化到Raji细胞 ;转染 48h后 ,可以检测到DAPK1的表达 ,并可以观察到细胞发生凋亡。结论 :大片段的基因可以采用RT -PCR法直接克隆 ,采用常规的转染技术可以将大片段基因转染到宿主细胞 ;成功克隆具有活性功能DAPK1基因ORF 。
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.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2004年第1期88-93,共6页
Chinese Journal of Pathophysiology
基金
国家教委留学回国人员启动基金资助项目[2 0 0 0 ] 4 79
