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目的:筛选参与APOBEC3B羧基端脱氨酶及抗乙肝病毒(hepatitis B virus,HBV)活性的重要区域和关键位点。方法:定点突变构建羧基端活性中心附近的3个螺旋区段(α2、α3、α4)的缺失突变体和α4螺旋区内(D316、K320、E321、R327、D328)位点的突变体;Southern blot筛选APOBEC3B抑制HBV复制的重要区段和关键位点;不同DNA变性PCR(differential DNA denaturation PCR,3D-PCR)和克隆测序进行验证。结果:APOBEC3B羧基端脱氨酶活性中心周围的α3和α4螺旋区缺失后,其抗病毒活性消失;D316位点突变后APOBEC3B的脱氨酶活性和抗病毒活性消失。结论:APOBEC3B羧基端的D316位点是APOBEC3B脱氨酶和抗病毒的关键位点。
Objective: To screen important regions and key sites involved in APOBEC3B carboxy-terminal deaminase and hepatitis B virus (HBV) activity. METHODS: Three helical segments (α2, α3, α4) deletion mutants and α4 helix region (D316, K320, E321, R327, D328) mutants were constructed by site-directed mutagenesis. blot screening APOBEC3B inhibition of HBV replication of the important sections and key sites; different DNA denaturation PCR (differential-PCR denaturing PCR, cloning and sequencing to verify. Results: The deletion of α3 and α4 helices around APOBEC3B carboxyl terminal deaminase disappeared, and the deamination activity and antiviral activity of APOBEC3B disappeared after D316 site mutation. Conclusion: The D316 site of APOBEC3B carboxyl terminal is the key point of APOBEC3B deaminase and antiviral.