中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
3期
228-233
,共6页
陈婉南%陈金烟%黄清玲%林建银%林旭
陳婉南%陳金煙%黃清玲%林建銀%林旭
진완남%진금연%황청령%림건은%림욱
乙型肝炎病毒%RNA剪接%酵母双杂交
乙型肝炎病毒%RNA剪接%酵母雙雜交
을형간염병독%RNA전접%효모쌍잡교
Hepatitis B virus%RNA splicing%Yeast two-hybrid
目的 筛选与单剪接型2.2 kb乙型肝炎病毒(HBV)剪接特异性新蛋白相互作用的肝细胞蛋白.方法 PCR扩增单剪接型2.2 kb HBV剪接特异性新基因TPss并克隆于诱饵载体pGBKT7,在证实TPss蛋白不具有自激活作用的前提下,以酵母双杂交系统筛查与TPss蛋白相互作用的肝细胞蛋白,进而通过哺乳动物细胞双杂交实验验证候选肝细胞蛋白与TPss蛋白在Huh7和HepG2肝细胞中的相互作用.结果 构建酵母双杂交诱饵载体pGBKT7-TPss,Western blot显示其在酵母中表达TPss蛋白.酵母双杂交筛选及哺乳动物细胞双杂交证实TPss蛋白可与4种肝细胞蛋白相互作用,即组织蛋白酶B、微粒体环氧化物水解酶、组织蛋白酶D与纤维蛋白原γ链.结论 TPss可与多种肝细胞蛋白相互作用.
目的 篩選與單剪接型2.2 kb乙型肝炎病毒(HBV)剪接特異性新蛋白相互作用的肝細胞蛋白.方法 PCR擴增單剪接型2.2 kb HBV剪接特異性新基因TPss併剋隆于誘餌載體pGBKT7,在證實TPss蛋白不具有自激活作用的前提下,以酵母雙雜交繫統篩查與TPss蛋白相互作用的肝細胞蛋白,進而通過哺乳動物細胞雙雜交實驗驗證候選肝細胞蛋白與TPss蛋白在Huh7和HepG2肝細胞中的相互作用.結果 構建酵母雙雜交誘餌載體pGBKT7-TPss,Western blot顯示其在酵母中錶達TPss蛋白.酵母雙雜交篩選及哺乳動物細胞雙雜交證實TPss蛋白可與4種肝細胞蛋白相互作用,即組織蛋白酶B、微粒體環氧化物水解酶、組織蛋白酶D與纖維蛋白原γ鏈.結論 TPss可與多種肝細胞蛋白相互作用.
목적 사선여단전접형2.2 kb을형간염병독(HBV)전접특이성신단백상호작용적간세포단백.방법 PCR확증단전접형2.2 kb HBV전접특이성신기인TPss병극륭우유이재체pGBKT7,재증실TPss단백불구유자격활작용적전제하,이효모쌍잡교계통사사여TPss단백상호작용적간세포단백,진이통과포유동물세포쌍잡교실험험증후선간세포단백여TPss단백재Huh7화HepG2간세포중적상호작용.결과 구건효모쌍잡교유이재체pGBKT7-TPss,Western blot현시기재효모중표체TPss단백.효모쌍잡교사선급포유동물세포쌍잡교증실TPss단백가여4충간세포단백상호작용,즉조직단백매B、미립체배양화물수해매、조직단백매D여섬유단백원γ련.결론 TPss가여다충간세포단백상호작용.
Objective To screen the liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome. Methods The splicing-specific gene TPss generated by the 2.2 kb singly spliced variant of HBV genome was amplified by PCR and cloned into the bait vector pGBKT7. After exclusion of self-activatian capacities of TPss protein, a two-hybrid library screening was performed using a pre-transformed human liver cDNA library to screen the liver cell proteins interacting with TPss. Mammalian two-hybrid assay was also done to further confirm the interactions between the bait and prey proteins in Huh7 and HepG2 hepatacytes. Results TPss gene with the size of 336 bp was successfully amplified and cloned, and the TPss protein expressed well in AHI09 yeast cells. Four liver cell proteins interacting with TPss, i. e. , cathepsin B, epoxide hydrolase 1, cathepsin D and fibrinogen gamma chain, were screened by yeast two-hybrid assay and further confirmed by mammalian two-hybrid assay. Conclusion TPss could interact with liver cell proteins.
