摘要
目的真核细胞内,DNA甲基化调控在转座子沉默、基因表达调控、甚至病毒感染过程中起关键作用,其中HIV-1启动子的甲基化修饰在病毒潜伏感染中扮演重要角色。为明确载脂蛋白B mRNA编辑酶催化亚基3A(APOBEC3A,A3A)对HIV-1启动子5'LTR上CpG岛甲基化程度的调节作用,本研究构建并筛选出A3A异构体A3A Isotype a(A3Aa)和A3A Isotype b(A3A-b)高表达真核载体,分析其去甲基化功能。方法从THP-1细胞mRNA中扩增得到A3A-a和A3A-b的c DNA,分别克隆至真核表达载体pASIB-HA和pCAGGS-HA。转染HEK-293T细胞,蛋白印迹法检测A3A-a和A3A-b的蛋白表达,并筛选出A3A-a和A3A-b的高表达载体。接着将A3A-a和A3A-b高表达载体与实验室构建并修饰的HIV-1启动子甲基化载体p-me LTR-EGFP共转染HEK-293T,检测指示蛋白EGFP在细胞水平中的表达,评价A3A-a和A3A-b的去甲基化调节作用。结果 PCR结果、酶切鉴定和测序结果均表明A3A真核表达载体pASIB-HA-A3A1-a、pCAGGS-HA-A3A2-a、pASIB-HA-A3A1-b、pCAGGS-HA-A3A2-b构建成功。经过筛选,pCAGGS-HA-A3A2-a和pCAGGS-HA-A3A2-b中A3A蛋白表达量最高,并用于后续实验。甲基化载体共转染实验显示,相较于对照A3G转染组,A3A-a和A3A-b组中EGFP的表达量明显上调。结论本研究在细胞水平上证实A3A分子Isotype a和Isotype b均可识别DNA中5-甲基化胞嘧啶,催化HIV-1启动子CpG岛去甲基化,为后续研究A3A对HIV潜伏感染调控奠定了基础。
Objective In eukaryotes, DNA methylation is involved in transposon silencing, gene regulation and viral infection, within which methylation of HIV-1 promoter plays an important role in HIV-1 latent infection. In order to determine the demethylation effect of APOBEC3 A(A3 A) on CpG island of HIV-1 5'LTR promoter, the eukaryotic vectors expressing A3 A isotype a and A3 A isotype b were constructed and selected, and A3 A mediated DNA-demethylation was further characterized. Methods The c DNA of A3 A-a and A3 A-b were amplified from THP-1 mRNA and cloned into eukaryotic expression vectors pASIB-HA and pCAGGS-HA. The HEK-293 T cells were transfected, and the protein expression of A3 A-a and A3 A-b was detected by Western blot. The A3 A-a and A3 A-b highly expressive vectors were screened and were then,the A3 A-a and A3 A-b co-transfected with the HIV-1 methylated promoter p-me LTR-EGFP, which was constructed and modified by our laboratory, into the HEK-293 T, and the expression of EGFP was detected.Results The results of PCR, enzymatic identification and sequencing showed that the A3 A eukaryotic expression vector pASIB-HA-A3 A1-a, pCAGGS-HA-A3 A2-a, pASIB-HA-A3 A1-b, pCAGGS-HA-A3 A2-b were successfully constructed. After screening, A3 A proteins were highly expressed in pCAGGS-HA-A3 A2-a and pCAGGS-HA-A3 A2-b, which could be used in following experiments. The co-transfection experiment showed that in comparison with A3 G transfected control group, the fluorescence intensity and expression level of EGFP in A3 A-a and A3 A-b groups were significantly increased. Conclusion This study indicated that both A3 A isotype a and isotype b could recognize the 5-methylcytosine and catalyze the demethylation of CpG island in a HIV-1 promoter, providing a function for further study on the role of A3 A in HIV latency infection.
引文
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