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目的对乙型肝炎病毒(HBV)DNA聚合酶反式激活蛋白1(HBVDNAPTP1)的生物活性进行检测。方法构建pcDNA3.1(-)/myc-His A-HBVDNAPTP1载体,并使用该载体对单核细胞性白血病细胞系THP-1进行转染。通过Western blot蛋白印迹法对HBVDNAPTP1表达进行检测。使用抑制消减杂交技术(SSH)在pGEM-T Easy系统中构建THP-1细胞HBVDNAPTP1反式激活cDNA基因文库。在对cDNA序列进行测序后,将测序结果与GenBank中的序列进行BLAST对比检测分析。结果某些序列(如CIP4)可能参与细胞凋亡过程。THP-1细胞内的CIP4mRNA和蛋白表达分别通过实时定量反转录聚合酶链式反应(RT-PCR)和Western blot蛋白印迹法进行分析。HBVDNAPTP1能够在转录水平和翻译水平使CIP4的表达下调。结论 HBVDNAPTP1可能参与单核细胞凋亡起始的正向调节。本实验研究结果揭示了HBVDNAPTP1的生物功能,并为HBVDNAPTP1的调节机制的进一步探索提供了新证据。
Objective To detect the biological activity of hepatitis B virus DNA polymerase transactivator 1 (HBVDNAPTP1). Methods The pcDNA3.1 (-) / myc-His A-HBVDNAPTP1 vector was constructed and the monocytic leukemia cell line THP-1 was transfected with this vector. HBVDNAPTP1 expression was detected by Western blot. Construction of THP-1 cell transactivation cDNA gene library of HBVDNAPTP1 in pGEM-T Easy system using Suppression Subtractive Hybridization (SSH). After sequencing the cDNA sequence, the sequencing results were compared with the sequences in GenBank for BLAST analysis. The results of some sequences (such as CIP4) may be involved in the process of apoptosis. CIP4 mRNA and protein expression in THP-1 cells were analyzed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot Western blotting. HBVDNAPTP1 is able to down-regulate the expression of CIP4 at the transcriptional and translational levels. Conclusion HBVDNAPTP1 may play a positive role in the initiation of monocyte apoptosis. The results of this experiment revealed the biological function of HBVDNAPTP1 and provided new evidence for the further exploration of the regulatory mechanism of HBVDNAPTP1.