pIRES-PML-RARα-hIL-2重组质粒的构建和表达研究  

Study on construction and expression of the eukaryotic coexpression plasmid containing PML-RARαgene and human IL-2 gene

作　　者：胡刚[1] 李扬秋[1] 周羽竝[2] 陈少华[1] 杨力建[1] 岑东芝[1] 

机构地区：[1]暨南大学医学院血液病研究所 [2]暨南大学医学院生化教研室,广州510632

出　　处：《癌症进展》2008年第4期357-361,366,共6页Oncology Progress

基　　金：中德生物技术合作项目(CHN02/319);广东省科技计划项目(2005B50301016);广州市科技攻关计划(2003J1-I0011;2005Z1-E4011);国务院侨办重点学科建设基金资助

摘　　要：目的构建PML-RARα融合基因与人白介素-2(hIL-2)基因真核双表达质粒。方法利用RT-PCR技术从NB4细胞的RNA中扩增出PML-RARα融合点附近的部分基因片段,通过RT-PCR扩增Jurkat细胞中的hIL-2基因,并分别将两基因片段连接到pIRES质粒的多克隆位点(MCS)A和B中,构建真核双表达质粒。利用酶切和序列分析方法验证所构建质粒的正确性。将构建成功的重组质粒转染A549细胞,通过RT-PCR检测重组质粒在真核细胞中的转录情况,利用点杂交、ELISA检测目的蛋白的表达情况。结果NheⅠ/MluⅠ和SalⅠ/NotⅠ双酶切证明重组质粒中含有相应大小的PML-RARα基因片段及hIL-2基因,序列分析证明重组质粒中插入片段的碱基序列均完全正确。将所构建的pIRES-PML- RARα-hIL-2质粒转染A549细胞后经RT-PCR鉴定表明,插入到重组质粒中的PML- RARα和hIL-2基因能够在真核细胞中进行正常转录。经点杂交和ELISA检测显示重组质粒在真核细胞中可表达hIL-2蛋白。结论成功构建了pIRES-PML-RARα-hIL-2质粒,该重组质粒能够在真核细胞中正常转录并表达PML—RARα和hIL-2蛋白。To construct a eukaryotic coexpression aplsmid containing PML-RARα gene and human IL-2 （hIL-2） gene. Methods PML-RARα fusion gene segment was amplified from NB4 cell line by RT-PCR, and the whole hIL-2 gene was amplified from Jurkat cell line by RT -PCR. Both PCR products were cloned into plRES plasmid respectively to construct a recombinant plasmid pIRES-PML-RARα- hIL- 2. The recombinant plasmid was identified by double enzyme cutting and sequence analyzing. Results Restriction analysis （ Nhe I/Mlu I, Sal I/Not I ） and sequence analysis confirmed that the length and the sequence of the fragments inserted in multi-clone site （MCS） A and MCS B of plRES plasmid were absolutely correct. Both mRNA and protein expressed by recombinant plasmids in A549 cell lines could be correctlly detected. Conclusions A recombinant eukaryotic coexpression plasmid containing PML-RARα and hIL-2 genes was successfully constructed. This recombinant plasmid has the function of transcription and translation in eukaryotic cells in vitro.

关 键 词：PML-RARΑ HIL-2 急性早幼粒细胞白血病 DNA疫苗 

分 类 号：R733.71[医药卫生—肿瘤]