RACE策略扩增EDAG-1基因3′端及序列分析

Strategy of Amplification of 3' End of EDAG-1 Gene by RACE and its Sequence Analysis

目的通过RACE(rapid amplification of cDNA Ends)技术分析K-562细胞株中高表达的EDAG-1基因3′端。方法采用Marathon-ReadyTMcDNA方法扩增HLCM cDNA(人白血病细胞株K-562,Marathon-ReadyTMcDNA)的EDAG-1基因3′端,将扩增的特异带回收纯化,构建到原核表达载体中,提取质粒并且测序分析。结果第1次扩增、第1次巢式PCR扩增未见明显特异带,2次巢式PCR结果可见200bp特异带,回收后连接到pMD18-T中,转化到大肠杆菌中并筛选阳性菌落,提取质粒测序,结果未发现该段序列存在点突变、插入或缺失。结论EDAG-1基因在白血病细胞株K-562中高表达的机制可能与3′端无关。

Objective To analyse the 3＇ end of EDAG-1 gene in K-562 ceil line by 3＇RACE. Methods We amplified the 3＇ end of EDAG-1 gene in HLCM cDNA （human leukemia ceil line K-562, chronic myelogenous Marathon-Ready^TM cDNA） by Marathon RACE. The amplified fragment was purified to construct the corresponding recombinant plasmid, subsequently the plasmid was sequenced to analyse the coding region. Results Although the specific fragments were not quite clear after the first PCR amplification and the first nested PCR reaction. 200 bp fragment was got in the second nested PCR amplification, and inserted into pMD 18-T vector. The recombinant pIasmid was transformed into JM109 ceils,and then sequenced. We didn＇t find point mutation,insertion or deletion in the 3＇ end of EDAG-1 gene in HLCM cDNA. Conclusion 3＇ end may not play a role in the mechanism of high expression of EDAG-1 in K-562 ceil line.