CAJ | 학술논문

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。
목적：탐토구건여신생인호흡군박종합정상관적ABCA3기인돌변체c.875A>T（p.E292V）화c.2169G>A（p.M723I）급기록색형광표체재체적방법，병관찰기재인폐선암（A549）세포주중적표체。방법응용중첩연신PCR법구건ABCA3기인적량개돌변체E292V화M723I，운용한제성매절련접등기술구건량개돌변체적록색형광표체재체，사용지질체Lipofectamine 2000장구건적재체순시전염도A549세포주，사기재체외표체，재형광현미경하관찰전염효솔，이용격광공취초현미경관찰중조자적표체정황。결과구건적량개돌변체E292V화M723I경측서분별증실ABCA3기인cDNA제875위감기A변위T화제2169위감기G변위A，중조자전염A549세포후，야생형화돌변형기인적단백질도재세포내성공표체。결론성공구건료ABCA3기인야생형급돌변형적록색형광표체재체，병사기재A549세포표체，위후속실험제공료조건。
ObjectiveTo 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 lfuorescent 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 lfuorescent protein expression rectors. A549 cells were transiently transfected with the recombinants using Lipofectamine 2000 and the transfection efifciency was conifrmed through GFP signal. The expression and location of recombinants were detected by FV1000 laser scanning microscope.ResultsDirect sequence analysis conifrmed 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 lfuorescent 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.