摘要
目的:采用长度为245 bp的PML-RARα融合基因片段构建一种新的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从NB4细胞的RNA中扩增出包含PML-RARα基因融合点的长度为245 bp的基因片段,通过RT-PCR从Jurkat细胞中扩增hIL-2基因,并将两基因片段分别克隆到pIRES质粒中,通过酶切和测序验证重组质粒的正确性。将重组质粒转染A549细胞,通过RT-PCR检测该质粒在真核细胞中的转录情况,利用点杂交和ELISA技术检测目的蛋白的表达。结果:Nhe I/Mlu I和Sal I/Not I双酶切证明重组质粒中含有相应大小的PML-RARα融合基因片段和hIL-2基因,测序结果证明重组质粒中插入片段的碱基序列均完全正确。将构建的pIRES-PML-RARα245-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并翻译出相应蛋白。结论:成功构建了含有245 bp的PML-RARα基因与hIL-2基因的真核双表达载体,该载体能够在真核细胞中正常转录并翻译出PML-RARα和hIL-2蛋白,为利用DNA疫苗治疗急性早幼粒细胞白血病提供了更丰富的资料。
Objective：PML-RARα gene segment with the length of 245 bp was used to construct a eukaryotic coexpression plasmid containing PML-RARα gene and human interleukin-2（hIL-2）gene.Methods：PML-RARα fusion gene segment with the length of 245 bp 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 pIRES plasmid respectively to construct a recombinant plasmid pIRES-PML-RARα245-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 pIRES 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 PMLRARα and hIL-2 genes was successfully constructed.And this recombinant plasmid has the function of transcription and translation in eukaryotic cells in vitro,which will provide more rich materials for the research of DNA vaccine used in the treatment of APL.
出处
《沈阳医学院学报》
2012年第2期69-72,共4页
Journal of Shenyang Medical College
基金
广东省科技计划项目(No.2005B50301016)
广州市科技计划项目(No.2005Z1-E4011
2009Z1-E161)
