bcr/abl基因b3-a2型接合区cDNA的分离和序列分析

ISOLATION AND SEQUENCING ANALYSES OF THE b3a2-TYPE JUNCTION REGION cDNA IN bcr/abl GENE

利用分子生物学方法,我们将K562细胞林bcr/abl mRNA 0.3kb的接合区cDNA片段用聚合酶链反应扩增并插入到质粒pGEM-4-Z载体中。经限制酶切分析和荧光标记M13引物法DNA序列测定,证实重组质粒pB3A2中有0.3kb的插入片段与K562 mRNA接合区序列完全一致。我们分离到的接合区DNA可用于bcr/abl基因的检测及其反义核酸抑制的研究。

Chronic myeloid leukemia (CML) is a hematological malignancy characterized by a reciprocal translocation t (9;22), that results in the fused bcr/abl gene and the chimeric 8.5kb transcript. The transformative activity of the bcr/abl gene has been confirmed by introducing bcr/abl constructs in mice. Furthermore, leukemic colony formation is selectively suppressed when CML blast cells are exposed to synthetic oligodeoxynucleotides complementary to bcr/abl gene junction region. We amplified the bcr/abl gene junction region from K562 cell RNA using a pair of synthetic primers by RT-PCR technique and inserted it into pGEM-4Z plasmid. The PCR product was a 0.3 kb DNA fragment. Restriction analyses of the recombinant plasmids suggested that we had got the clones of the target sequence. Results from sequencing analyses supported that the plasmid, pB3A2, contained a 0.3kb fragment homologous to the b3a2-type junction region cDNA of bcr/abl gene. The plasmid pB3A2 may provide a good source of probes and antisense plasmids in further studies on selective killing of CML cells.