转录抑制因子Bmi-1基因正、反义真核表达载体的构建及鉴定

Amplification and construction of its sense and antisense eukaryotic expression vector of transcriptional repressor Bmi-1 gene

关键词:  转录抑制因子  基因克隆  表达载体

Key words:  transcriptional repressor Bmi-1  genetic cloning  recombination

摘要:

［目的］克隆转录抑制因子Bmi-1基因并构建其正、反义真核表达载体,为研究其功能及其作用机制奠定基础。［方法］根据Bmi-1基因已知序列，设计合成一对引物，上下游引物分别加入XhoI和ClaI的酶切位点，用反转录PCR（RT-PCR）方法从人红白血病细胞株（K562）总RNA中扩增编码Bmi-1的基因片段（1017 bp），插入克隆载体pMD18-T,构建pMD18-Bmi-1载体，经限制性核酸内切酶谱证实后，再克隆至逆转录病毒载体pLNCX2中，构建pLNCX2- Bmi-1真核表达载体，并经测序证实。然后以pLNCX2- Bmi-1为模板，物扩增Bmi-1基因起始密码子及编码锌指结构区域上下171 bp长度的核苷酸片段，并反向连接于表达载体（pLNCX2）上。［结果］DNA测序表明编码序列与读框完全正确，RT-PCR扩增出和目的片段相同的片段,正义DNA片断长度为1029 bp,反义片断长度为171 bp, Bmi-1基因被成功扩增并被克隆至真核表达载体pMD18-T和pLNCX2中。［结论］Bmi-1正义及反义片断被成功扩增，并成功构建了Bmi-1正、反义重组真核表达载体，为进一步研究该基因打下了基础。

Abstract:

［Objective］ To construct a recombinant eukaryotic expression vector of transcriptional repressor Bmi-1 gene. ［Methods］Using the total RNA extracted from a human erythroleukemia cell line (K562) as the template, the Bmi-1gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers containing the restriction sites of XhoI and ClaI. The amplified fragment of Bmi-1 gene digested with XhoI and ClaI was subcloned into cloning vector pMD18-T and then into expression vector pLNCX2. The recombinant plasmid was identified by restriction endonuclease analysis and PCR. ［Results］ The amplified sense and antisense DNA fragment was about 1029 bp and 171 bp in length, and enzyme digestion and PCR analysis showed that Bmi-1 gene had been successfully cloned into the eukaryotic expression vector pMD18-T and pLNCX2. ［Conclusion］ The sense and antisense fragment of Bmi-1 gene has been successfully amplified and the reconstruction eukaryotic expression vector has been successfully constructed.