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HBc Ag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p GBKT7-HBc Ag。p GBKT7-HBc Ag分别与p GADT7-3G和p GADT7-3F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HBc Ag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。
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/A3 G and HBcAg proteins.METHODS：The cDNAs of human A3 F and A3 G 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/-A3 G and pGBKT7-HBcAg were constructed.To ascertain the direct interactions between HBcAg and A3 F or A3 G,PGBKT7-HBcAg was co-transformed into yeast AH109 with pGADT7-A3 F or pGADT7-A3 G,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 A3 F or A3 G 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 A3 F or A3 G.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 A3 F and A3 G had no evident direct interaction with HBcAg.The results of Co-IP and western blot indicated A3 F and A3 G had interactions with HBcAg.CONCLUSION：We demonstrated the interactions between A3F/A3 G and HBcAg,but the interactions might not be realized by direct bind of the proteins.
出处
《癌变．畸变．突变》
CAS
CSCD
2015年第3期182-186,共5页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
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)
