CAJ | 학술논문

目的：以血清中乙型肝炎病毒（HBV）DNA为模板，克隆构建HBV X蛋白质的真核表达载体pcDNA-HBx。方法：设计扩增HBV X基因的In-Fusion PCR引物，采用高保真DNA聚合酶分两段扩增HBV X基因序列；采用In-Fusion克隆技术，将两段X基因扩增片段一步克隆入经Hind III和Xba I双酶切的pcDNA3.0真核表达载体，以构建重组真核表达载体pcDNA-HBX；采用Hind III和Xba I双酶切和DNA序列测序筛选鉴定重组载体；pcDNA-HBx转染HepG2细胞，Western blot检测pcDNA-HBx转染细胞的HBx蛋白质表达。结果：In-Fusion PCR引物分两段扩增获得全长HBx基因序列；In-Fusion获得克隆的pcDNA-HBx经双酶切筛选得到阳性重组质粒，测序分析证实插入序列正确；转染pcDNA-HBX的HepG2细胞， Western blot证实表达HBx蛋白质。结论：以血清HBV DNA为模板克隆HBV X基因，构建了表达HBV HBx蛋白质的真核表达载体，为HBx蛋白质的生物学功能研究提供支持。
목적：이혈청중을형간염병독（HBV）DNA위모판，극륭구건HBV X단백질적진핵표체재체pcDNA-HBx。방법：설계확증HBV X기인적In-Fusion PCR인물，채용고보진DNA취합매분량단확증HBV X기인서렬；채용In-Fusion극륭기술，장량단X기인확증편단일보극륭입경Hind III화Xba I쌍매절적pcDNA3.0진핵표체재체，이구건중조진핵표체재체pcDNA-HBX；채용Hind III화Xba I쌍매절화DNA서렬측서사선감정중조재체；pcDNA-HBx전염HepG2세포，Western blot검측pcDNA-HBx전염세포적HBx단백질표체。결과：In-Fusion PCR인물분량단확증획득전장HBx기인서렬；In-Fusion획득극륭적pcDNA-HBx경쌍매절사선득도양성중조질립，측서분석증실삽입서렬정학；전염pcDNA-HBX적HepG2세포， Western blot증실표체HBx단백질。결론：이혈청HBV DNA위모판극륭HBV X기인，구건료표체HBV HBx단백질적진핵표체재체，위HBx단백질적생물학공능연구제공지지。
Objective: to produce recombinant HBx protein in HepG2 cells by eukaryotic plasmid expression vector system with serum HBV DNA template. Method: HBX1 and HBX2, two HBV X gene sections, were amplified from human serum HBV DNA samples by the high fidelity PCR polymerase with In-Fusion primers, and cloned into the eukaryotic expression plasmid, pcDNA3.0, restriction digested by Hind III and Xba I. The recombinant plasmid with DNA sequencing has identiifed by double restriction enzyme analysis with Hind III and Xba I. Expressed the HBx protein in HepG2 transfected pcDNA-HBx, and detected with Western blot. Result: Two fragment HBX1 and HBX2 of HBV X gene were cloned exactly into pcDNA3.0 as pcDNA-HBx used In-Fusion clone technology and expressed HBx protein in hepG2 cell. Conclusion:the HBV X gene, from serum HBV DNA template, were recombinant into the eukaryotic expression plasmid pcDNA-HBx, which can express HBx protein in hepG2 cell. This study provided an experiment condition for researching the biological function of HBx protein.