ABCA3基因定点突变及其在A549细胞株中的表达

Site-directed mutagenesis and protein expression of ABCA3 gene in A549 cells

摘要:

目的 探讨构建与新生儿呼吸窘迫综合征相关的ABCA3 基因突变体c.875A>T(p.E292V)和c.2169G>A(p.M723I)及其绿色荧光表达载体的方法,并观察其在人肺腺癌(A549)细胞株中的表达。方法 应用重叠延伸PCR 法构建ABCA3 基因的两个突变体E292V 和M723I,运用限制性酶切连接等技术构建两个突变体的绿色荧光表达载体,使用脂质体Lipofectamine 2000 将构建的载体瞬时转染到A549 细胞株,使其在体外表达,在荧光显微镜下观察转染效率,利用激光共聚焦显微镜观察重组子的表达情况。结果 构建的两个突变体E292V 和M723I 经测序分别证实ABCA3 基因cDNA 第875 位碱基A 变为T 和第2169 位碱基G 变为A,重组子转染A549 细胞后,野生型和突变型基因的蛋白质都在细胞内成功表达。结论 成功构建了ABCA3 基因野生型及突变型的绿色荧光表达载体,并使其在A549 细胞表达,为后续实验提供了条件。

Abstract:

Objective To study the protocol of construction of the mutation E292V and M723I of hABCA3 gene associated with neonatal respiratory distress syndrome, as well as their eukaryotic green fluorescent protein expression rectors, and to examine the expression of mutation proteins in human lung carcinoma epithelial cells (A549). Methods Site-directed mutagenesis method based on overlap extension PCR was used to introduce mutations in the two sites which were E292V and M723I in the ABCA3. The PCR fragments were subcloned to PEGFP-C2 vectors to construct the eukaryotic green fluorescent protein expression rectors. A549 cells were transiently transfected with the recombinants using Lipofectamine 2000 and the transfection efficiency was confirmed through GFP signal. The expression and location of recombinants were detected by FV1000 laser scanning microscope. Results Direct sequence analysis confirmed an A to T transition at position 875 in E292V and a G to A transition at position 2169 in M723I. Recombinants were transfected to A549 cells and both wild type and mutant ABCA3 proteins were expressed in the cytoplasm. Conclusions The eukaryotic green fluorescent protein expression rectors of wild type and mutant ABCA3 gene were constructed and they were successfully expressed in A549 cells. This experiment provides a basis for subsequent research.