APOBEC3A-HBc融合蛋白的表达及活性鉴定

Expression and activity identification of APOBEC3A-HBc fusion protein

目的构建载脂蛋白B mRNA编辑酶催化多肽3A(APOBEC3A)与不同长度和序列的乙型肝炎病毒核心蛋白(HBc)的融合蛋白真核表达载体,研究其表达能力及融合蛋白的细胞内定位及胞嘧啶脱氨基活性。方法采用In-Fusion重组克隆方法,将含有APOBEC3A的PCR产物分别和含有全长HBc、四种C端截短体HBc的PCR扩增片段连接克隆入真核表达载体pc DNA3.0。将重组载体分别转染HEK293 T细胞,用免疫荧光细胞化学染色法检测融合蛋白在HEK293 T细胞中的定位,Western blot法检测融合蛋白的表达水平;尿素变性PAGE法分析融合蛋白的胞嘧啶脱氨基活性。结果构建的五种融合蛋白表达载体,可在HEK293T细胞中表达五种融合蛋白,4种含HBc截短体编码的融合蛋白表达载体比含全长HBc编码的融合蛋白载体的表达能力强;含全长HBc的融合蛋白APOBEC3A-HBc主要定位于细胞质,而含HBc截短体的融合蛋白APOBEC3AHBc144 S、APOBEC3 A-HBc144 E、APOBEC3 A-HBc144 AAA和APOBEC3 A-HBc144 A均主要定位于细胞核中;HBc截短体融合蛋白的胞嘧啶脱氨基活性高于全长HBc融合蛋白。结论成功构建APOBEC3A与不同长度和序列HBc的融合蛋白真核表达载体,APOBEC3A与全长HBc及截短体HBc的融合蛋白在表达能力、细胞内定位以及胞嘧啶脱氨基活性具有明显差异。

Objective To construct the expression vector of the fusion protein of apolipoprotain B mRNA editing enzyme catalytic polypeptide-like 3 A（ APOBEC3 A） and hepatitis B virus core proteins（ HBc） with different sequences,and identify its expression,intracellular localization and cytosine deamination activity. Methods The APOBEC3 A gene and the coding sequence of HBc and four kinds of truncated HBc containing C-termimal domain were amplified by PCR. The APOBEC3 A with full-length HBc antigen or four kinds of truncated HBc were cloned into a eukaryotic expression vector pc DNA3. 0 by In-Fusion method,and confirmed by DNA sequencing. The recombinants were then transfected into HEK293 T cells. The expression and localization of the fusion proteins were detected by Western blotting and immunofluorescence cytochemistry.The cytosine deamination activity was analyzed by electrophoresis on a urea denaturing polyacrylamide gel. Results Five kinds of fusion protein were expressed in HEK293 T cells successfully,and the expression of the vectors containing the truncated HBc were higher than that of the APOBEC3 A-HBc vector containing the full-length HBc. The fusion protein of APOBEC3 A-HBc was mainly expressed in the cytoplasm of HEK293 T cells,and the fusion proteins of APOBEC3 A-HBc144 S,APOBEC3 A-HBc144 E,APOBEC3 A-HBc144 AAA,APOBEC3 A-HBc144 A were expressed mainly in the nuclei of HEK293 T cells. The cytosine deamination activity of the fusion proteins containing the truncated HBc was higher than that of the fusion protein APOBEC3 A-HBc. Conclusion The fusion protein expression vectors of APOBEC3 A and HBc with different lengths and sequences have been constructed successfully. The expressing ability,intracellular localization and cytosine deamination activity are obviously different between the fusion protein APOBEC3 A-HBc and the fusion proteins containing the truncated HBc.