CAJ | 학술논문

急性早幼粒细胞白血病(APL)具有特征性染色体易位,产生的PML-RARα融合基因在其发生发展中有重要作用.PML-RARα融合蛋白在细胞内被中性粒细胞弹性蛋白酶(neutrophil elastase,NE)切割为PML突变蛋白(核定位信号NLS缺失)和RARa突变体(NLS-RARα,包含有PML的核定位信号),这两段蛋白质在APL的发生中可能具有重要作用.为进一步研究NLS-RARα的生物学功能,运用酵母双杂交技术在白血病cDNA文库中筛选与其作用的蛋白质.首先PCR技术扩增NLS-RARα编码序列.克隆至诱饵载体pGBKT7,测序鉴定后将其转化酵母AH109.免疫印迹检测到诱饵蛋白表达后,将含有诱饵载体的AH109与含有白血病cDNA文库的酵母Y187交配,在含有X-α-gal的营养缺陷性培养基上选择和筛选二倍体酵母.经回转实验和测序分析验证得到8个与NLS-RARα相互作用的蛋白质.为进一步验证这些相互作用,克隆其中的JTV-1蛋白,利用间接免疫荧光,GST pull-down和免疫共沉淀技术成功验证了它与NLS-SARα的相互作用.为进一步探讨APL的发生机制提供了新的线索.
급성조유립세포백혈병(APL)구유특정성염색체역위,산생적PML-RARα융합기인재기발생발전중유중요작용.PML-RARα융합단백재세포내피중성립세포탄성단백매(neutrophil elastase,NE)절할위PML돌변단백(핵정위신호NLS결실)화RARa돌변체(NLS-RARα,포함유PML적핵정위신호),저량단단백질재APL적발생중가능구유중요작용.위진일보연구NLS-RARα적생물학공능,운용효모쌍잡교기술재백혈병cDNA문고중사선여기작용적단백질.수선PCR기술확증NLS-RARα편마서렬.극륭지유이재체pGBKT7,측서감정후장기전화효모AH109.면역인적검측도유이단백표체후,장함유유이재체적AH109여함유백혈병cDNA문고적효모Y187교배,재함유X-α-gal적영양결함성배양기상선택화사선이배체효모.경회전실험화측서분석험증득도8개여NLS-RARα상호작용적단백질.위진일보험증저사상호작용,극륭기중적JTV-1단백,이용간접면역형광,GST pull-down화면역공침정기술성공험증료타여NLS-SARα적상호작용.위진일보탐토APL적발생궤제제공료신적선색.
Acute promyelocytic leukemia (APL) is characterized by the generation of the prototypic promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα), an oncogenic fusion protein due to chromosomal translocation. In a human myeloid cell line, PML-RARα is cleaved by neutrophil elastase (NE) to produce the mutational PML [nuclear localization signal (NLS) deletion and RARα (NLS-RARα, containing NLS of PML), both of which may play an important role in APL pathogenesis. The yeast two-hybrid technique was used to screen the intracellular proteins interacting with NLS-RARα, which may be involved in NLS-RARα signaling. The NLS-RARα coding sequence was amplified by polymerase chain reaction method and was cloned into the bait plasmid pGBKT7 vector, which, after the confirmation by sequencing, was transformed into yeast AH109 and the subsequent expression of bait plasmid was proved by Western-blot. The transformed yeast AH109 was mated with yeast Y187 (containing leukemia cDNA library plasmids pACT2) in medium. Diploid yeast was plated on synthetic dropout nutrient medium containing X-α-gal for screening. After being reintroduced into yeast AH109 and sequenced to verify the expression of ORF, eight positive colonies were obtained, among which one containing JTV-1 was cloned. The interaction between NLS-RARα and JTV-1 was further supported by indirect immunofluorescence, GST pull-down and co-immunoprecipitation, respectively. These findings brought some new clues for the further exploration of NLS-RARα signaling to APL.