乙型肝炎病毒e抗原在肝细胞内作用蛋白AK026018亚细胞定位的研究

Subcellular localization of a novel HBeAg interacting protein AK026018 in hepatocytes

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 investigate the subcellular localization of a novel hepatitis B virus e antigen (HBeAg) interacting protein AK026018 in hepatocytes. Methods: The AK026018 coding DNA fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) technique from HepG2 cell. The expressive vector of pEGFP-AK was constructed by routine molecular biological methods. HepG2 and 293T cells were transfected with pEGFP-N1 and pEGFP-AK, respectively by (using) lipofectamine. The expression was detected by fluorescence microscope and confocal fluorescence microscope. Results:The expressive vector was constructed and confirmed by DNA sequencing analysis and restriction enzyme digestion.The green fluorescence could be seen in cytoplasm in transfected pEGFP-AK cells and was distributed uniformly in whole cell in transfected pEGFP-N1 cells respectively. Conclusion:The expression vector was constructed successfully. Protein AK026018 was distributed mainly in cytoplasm.