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【目的】构建LYRM1基因绿色荧光蛋白融合载体,进而观察LYRM1基因编码蛋白在细胞内的定位情况。【方法】运用RT-PCR技术从人网膜脂肪组织中分离LYRM1基因的完整编码框,将其亚克隆到绿色荧光蛋白表达载体pEGFP-N2,脂质体转染3T3-L1前体脂肪细胞,激光共聚焦显微镜下观察融合蛋白的表达情况。【结果】PCR、酶切鉴定及测序结果表明重组质粒构建正确。激光共聚焦显微镜下观察到pEGFP-N2转染的细胞中绿色荧光均匀分布于整个细胞,而pEGFP-LYRM1转染的细胞中绿色荧光主要集中在细胞核区域。【结论】成功构建了LYRM1绿色荧光蛋白融合载体,LYRM1编码蛋白在细胞中定位于胞核中。
【Objective】 The green fluorescent protein fusion vector of LYRM1 gene was constructed and the localization of LYRM1 gene in cells was observed. 【Method】 The complete coding sequence of LYRM1 gene was isolated from human omental adipose tissue by RT-PCR, subcloned into the green fluorescent protein expression vector pEGFP-N2, liposome transfected 3T3-L1 precursor adipocytes, Confocal laser scanning microscope was used to observe the expression of the fusion protein. 【Result】 PCR, restriction enzyme digestion and sequencing results showed that the recombinant plasmid was constructed correctly. Under the confocal laser scanning microscope, the green fluorescence of pEGFP-N2 transfected cells was evenly distributed throughout the cells, while the green fluorescent of pEGFP-LYRM1 transfected cells was mainly concentrated in the nucleus. 【Conclusion】 The LYRM1 green fluorescent protein fusion vector was successfully constructed, and the LYRM1-encoding protein was localized in the nucleus in the cells.