论文部分内容阅读
目的:确定肝细胞内乙型肝炎病毒e抗原(HBeAg)作用蛋白AK026018的亚细胞定位,初步研究该蛋白的生物学功能。方法:应用逆转录聚合酶链反应(RTPCR)技术,从HepG2细胞中扩增编码AK026018蛋白的全基因,构建真核表达载体pEGFPAK,转染HepG2、293T细胞系,在激光共聚焦荧光显微镜下与转染空载体pEGFPN1的细胞比较观察。结果:经限制性内切酶分析和DNA序列测定鉴定构建的重组表达载体正确。通过激光共聚焦荧光显微镜观察,转染了重组载体pEGFPAK的细胞绿色荧光信号较集中分布于胞浆中,而空载体pEGFPN1转染的细胞绿色荧光信号在整个细胞中均匀分布。结论:成功分离了AK026018全基因序列并构建了真核表达载体,该基因表达产物亚细胞定位于细胞浆中。
Objective: To determine the subcellular localization of hepatitis B virus e antigen (HBeAg) protein AK026018 in hepatocytes and to study the biological function of this protein. Methods: The full-length gene encoding AK026018 protein was amplified from HepG2 cells by reverse transcription-polymerase chain reaction (RTPCR). The eukaryotic expression vector pEGFPAK was constructed and transfected into HepG2 and 293T cell lines. Cells transfected with empty vector pEGFPN1 were observed. Results: The constructed recombinant expression vector was identified by restriction endonuclease analysis and DNA sequencing. By confocal laser scanning microscopy, the green fluorescence signal of the recombinant vector pEGFPAK was more concentrated in the cytoplasm, while the green fluorescence signal of the transfected vector pEGFPN1 was uniformly distributed throughout the cells. CONCLUSION: The complete AK026018 gene sequence was successfully isolated and an eukaryotic expression vector was constructed. The subcellular localization of this gene in the cytoplasm was determined.