PML-RARα/pIRES和PML-RARα/pSecTag重组载体的构建和鉴定

Construction and identification of PML-RARα/pIRES and PML-RARα/pSecTag recombinant vectors

目的构建PML-RARα基因重组表达质粒,为发展PML-RARα基因疫苗提供实验依据。方法利用RT-PCR方法从急性早幼粒细胞白血病细胞株NB4细胞中扩增出465bp的位于PML-RARα融合基因的框内结构的片段,克隆至真核表达载体pIRES和分泌载体pSecTag后,经酶切和序列分析鉴定重组质粒的构建情况。结果经酶切鉴定和序列测定表明,PML-RARα目的基因已正确插入真核表达载体中,构成了目的重组载体。结论成功构建了PML-RARα/pIRES和PML-RARα/pSecTag重组真核表达质粒,可进一步用于PML-RARα基因疫苗的实验研究。

Objective To construct the PML-RARα gene recombinant plasmid, which is expected to be used as a modified DNA vaccine for acute promyelocytic leukemia. Methods A segment of 465 bp around PML-RARα gene in frame was amplified from human acute promyelocytic leukemia cell line NB4 cells by RT-PCR, and the PCR products were further cloned into eukaryon expression vector plRES and secretion vector pSecTag by cloning technique. The recombinat vector then was confirmed by double enzyme cutting and sequence analyzing. Results Restriction enzyme cutting and sequence analysis confirmed thai the length and the sequence of the PML-RARα gene fragment was inserted into PIRES or pSecTag plasmid. Conclusion The recombinant eukaryon expression plastaid PML-RARα/pIRES and PML-RARα/pSeeTag are successfully constructed, which can be used in the research of PML-RARα gene vaccine.