Limk1不同突变体HA融合表达载体构建及在细胞内定位

A Study on the Construction,Expression and Intracellular Localization of Different Mutants of Limk1 Fusion Expression Vector

目的:构建LIM激酶(Limk)1不同突变体的HA融合表达载体,并在真核细胞中表达,观察这些突变体在细胞内定位。方法:利用聚合酶链式反应(PCR)扩增Limk1全长序列,构建HA-Limk1真核表达质粒;利用定点突变策略对HA-Limk1进行核定位信号(NLS)缺失突变,命名为Limk1-NLS(del);用合成两条互补序列退火获得目标片段插入载体的策略构建Limk1-NLS;将2表达质粒转染HepG2细胞,细胞免疫荧光方法以及核质分离后用western-blot检测其在细胞中定位。结果:经测序鉴定Limk1及其不同突变体序列正确,在HepG2细胞中高表达;其中HA-Limk1在细胞质、细胞核均有表达,Limk1-NLS(del)主要定位于细胞质,Limk1-NLS主要定位于细胞核。结论:成功构建了Limk1全长及不同突变体的HA融合表达载体,初步明确突变体在细胞内的定位。

Objective： To construct eukaryotic cell expression vectors of different Limkl mutants fused with HA and to study the intracellular localizations of the mutant. Methods： Using polymerase chain reaction （PCR） for amplification of Limkl full sequence, constructing HA-Limkl Eukaryotic Expression Vector; using fixed-mutation to perform NLS deletion mutation, and named it as Limkl- NLS （del） ; using annealed complementary sequence to gain biotinylated target, to insert it in measure- ment building of Limkl-NLS; Mutants of Limkl gene transfected into HepG2 cells,. The intracellular localizations of these mutants were detected with immunofluorescence and western-blot followed to cyto- plasm-nucleoplasm separation. Results： HA-fused Limkl mutants highly expressed in HepG2 ceils. HA-Limkl expressed in both cytoplasm and nucleus. The Limkl-NLS （del） mainly localized in cyto- plasm. The Limkl-NLS mainly localized in nucleus. Conclusions： Limkl full length and different HA fusion expression vectors are successfully constructed, preliminarily define localization of mutants in nucleus.