APOBEC-3F和APOBEC-3G与乙肝核心抗原的相互作用研究

doi:10.3969/j.issn.1004-616x.2015.03.005

癌变·畸变·突变 ›› 2015, Vol. 27 ›› Issue (3): 182-186.doi: 10.3969/j.issn.1004-616x.2015.03.005

APOBEC-3F和APOBEC-3G与乙肝核心抗原的相互作用研究

王革非, 曾祥兴, 李蕊, 苏景华, 刘俊, 周艳琳, 李康生

汕头大学医学院微生物学与免疫学教研室, 广东汕头 515041

收稿日期:2014-01-06 修回日期:2014-12-23 出版日期:2015-05-30 发布日期:2015-05-30
通讯作者: 李康生,E-mail:ksli@stu.edu.cn E-mail:ksli@stu.edu.cn
作者简介:王革非,E-mail:gfwangcn@gmail.com
基金资助:
国家自然科学基金(31170852,81001322,81172795);广东省优秀青年教师培养计划(Yq2013077)

Interactions between human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide -3F/-3G and hepatitis B core antigen

WANG Gefei, ZENG Xiangxing, LI Rui, SU Jinghua, LIU Jun, ZHOU Yanlin, LI Kangsheng

Department of Microbiology and Immunology, The Key Immunopathology Laboratory of Guangdong Province, Shantou University Medical College, Shantou 515041, Guangdong, China

Received:2014-01-06 Revised:2014-12-23 Online:2015-05-30 Published:2015-05-30

摘要/Abstract

摘要：

目的:人载脂蛋白B mRNA编辑酶催化多肽APOBEC-3F(A3F)和APOBEC-3G(A3G)可抑制HBV复制,并参与HBV基因组的超突变,其中HBcAg被认为是潜在作用位点,为此本文研究A3F和A3G与HBcAg可能存在的蛋白相互作用方式。方法:RT-PCR克隆人A3F和A3G的cDNA,并构建相应酵母双杂交表达载体pGADT7-A3F/-A3G;PCR克隆HBV ayw亚型HBcAg基因,构建其酵母双杂交表达载体pGBKT7-HBcAg。pGBKT7-HBcAg分别与pGADT7-3G和pGADT7-3F共转化AH109酵母菌,利用营养二缺平板及四缺平板培养及α-半乳糖苷酶活性测试,检测HBcAg与A3F和A3G的直接作用情况。构建含有HBcAg、人A3F和A3G基因的多种标签融合真核表达质粒,脂质体法转染HeLa细胞后进行免疫共沉淀实验(Co-IP),并通过Western blot检测免疫共沉淀产物确定HBcAg与A3F和A3G之间是否存在间接相互作用。结果:酶切及DNA测序分析表明成功克隆HBcAg、人A3F和A3G,并成功构建酵母双杂交、真核表达及亚细胞定位相关的表达质粒。通过酵母双杂交实验对α-半乳糖苷酶定性和定量检测显示,A3F和A3G与HBcAg无显著的直接相互作用。Western blot及Co-IP实验结果表明A3F和A3G与HBcAg均存在相互作用。结论:A3F和A3G蛋白与HBcAg蛋白之间确实存在相互作用,推测这种作用不是通过两个蛋白直接结合实现的。

关键词: APOBEC-3F, APOBEC-3G, 乙肝核心抗原, 酵母双杂交, 免疫共沉淀

Abstract:

OBJECTIVE: APOBEC-3F (A3F) and -3G (A3G) inhibit viral replication of HBV and participate in hypermutation of HBV genome. HBcAg is considered as a potential interaction site. Therefore, we studied the interactions between A3F/A3G and HBcAg proteins. METHODS:The cDNAs of human A3F and A3G were cloned by RT-PCR from PHA-stimulated PBMC, and DNA of HBcAg was cloned by PCR from ayw subtype HBV. Then the yeast two hybrid expression plasmids pGADT7-A3F/-A3G and pGBKT7-HBcAg were constructed. To ascertain the direct interactions between HBcAg and A3F or A3G, pGBKT7-HBcAg was co-transformed into yeast AH109 with pGADT7-A3F or pGADT7-A3G, then the transformed yeast cells were cultured in synthetic double drop-out medium (DDO) and quadruple drop-out medium (QDO) containing X-α-gal, and Alpha galactose glucoside enzyme activity tests were carried out. The eukaryotic expression plasmids, containing HBcAg or A3F or A3G respectively, were constructed and transfected into HeLa cells, then co-immunoprecipiation (Co-IP) and western blot were performed to ensure the indirect interactions between HBcAg and A3F or A3G. RESULTS:The results of restriction endonuclease analysis and DNA sequencing showed that the plasmids involved were constructed successfully. Yeast two hybrid experiments with alpha galactosidase qualitative and quantitative assays displayed that A3F and A3G had no evident direct interaction with HBcAg. The results of Co-IP and western blot indicated A3F and A3G had interactions with HBcAg. CONCLUSION:We demonstrated the interactions between A3F/A3G and HBcAg, but the interactions might not be realized by direct bind of the proteins.

Key words: APOBEC-3F, APOBEC-3G, HBcAg, yeast two hybrid, co-immunoprecipitation

中图分类号:

Q344

王革非, 曾祥兴, 李蕊, 苏景华, 刘俊, 周艳琳, 李康生. APOBEC-3F和APOBEC-3G与乙肝核心抗原的相互作用研究[J]. 癌变·畸变·突变, 2015, 27(3): 182-186.

WANG Gefei, ZENG Xiangxing, LI Rui, SU Jinghua, LIU Jun, ZHOU Yanlin, LI Kangsheng. Interactions between human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide -3F/-3G and hepatitis B core antigen[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2015, 27(3): 182-186.

参考文献

[1] Turelli P, Mangeat B, Jost S, et al. Inhibition of hepatitis B virus replication by APOBEC3G[J]. Science, 2004, 303 (5665):1829.
[2] Zhao DJ, Wang XF, Lou GH, et al. APOBEC3G directly binds hepatitis B virus core protein in cell and cell free systems[J]. Virus Res, 2010, 151(2):213-219.
[3] Baumert TF, Rosler C, Malim MH, et al. Hepatitis B virus DNA is subject to extensive editing by the human deaminase APOBEC3C[J]. Hepatology, 2007, 46(3):682-689.
[4] Guarnieri M, Kim KH, Bang G, et al. Point mutations upstream of hepatitis B virus core gene affect DNA replication at the step of core protein expression[J]. J Virol, 2006, 80(2):587-595.
[5] Rosler C, Kock J, Kann M, et al. APOBEC-mediated interference with hepadnavirus production[J]. Hepatology, 2005, 42(2):301-309.
[6] Harris RS, Bishop KN, Sheehy AM, et al. DNA deamination mediates innate immunity to retroviral infection[J]. Cell, 2003, 113(6):803-809.
[7] Navaratnam N, Sarwar R. An overview of cytidine deaminases [J]. Int J Hematol, 2006, 83(3):195-200.
[8] Cullen BR. Role and mechanism of action of the APOBEC3 family of ntiretroviral resistance factors[J]. J Virol, 2006, 80(3):1067-1076.
[9] Suspene R, Guetard D, Henry M, et al. Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo[J]. Proc Natl Acad Sci USA, 2005, 102(23):8321-8326.
[10] Nguyen DH, Gummuluru S, Hu J, et al. Deamination-independent inhibition of hepatitis B virus reverse transcription by APOBEC3G[J]. J Virol, 2007, 81(9):4465-4472.
[11] Nguyen DH, Hu J. Reverse transcriptase- and RNA packaging signal-dependent incorporation of APOBEC3G into hepatitis B virus nucleocapsids[J]. J Virol, 2008, 82(14):6852-6861.
[12] Kim J, Park HC, Gedi V, et al. Yeast-hybrid based high-throughput assay for identification of anthraxlethal factor inhibitors[J]. Biochem Biophys Res Commun, 2011, 404(1):517-522.
[13] Golemis EA, Brent R. Fused protein domains inhibit DNA binding by LexA[J]. Mol Cell Biol, 1992, 12(7):3006-3014.

APOBEC-3F和APOBEC-3G与乙肝核心抗原的相互作用研究

Interactions between human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide -3F/-3G and hepatitis B core antigen

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics

本文评价