Site-directed mutagenesis and protein expression of ABCA3 gene in A549 cells / 中国当代儿科杂志

ABSTRACT

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>