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目的研究B、C基因型乙型肝炎病毒X蛋白(HBx)功能差异。方法B、C基因型乙型肝炎病毒X基因真核表达载体(分别为pcDNA3.1-XB及pcDNA3.1-XC)及空白载体pcDNA3.1/Hygro(-)以脂质体2000分别转染Chang细胞,转染细胞2d后以流式细胞仪检测凋亡率;转染细胞以潮霉素筛选,14d后形成的抗性细胞集落以冷甲醇固定、Gi msa染色并计数;各载体与指示载体pCMVβ共转染细胞,转染2d后裂解细胞并检测胞内β-半乳糖苷酶的活性。所有数据均以配对t检验进行分析。结果各载体转染Chang细胞后细胞的凋亡率为pcDNA3.1-XC>pcDNA3.1-XB>pcDNA3.1/Hygro(-),形成的抗潮霉素细胞集落数为pcDNA3.1/Hygro(-)>pcDNA3.1-XB>pcDNA3.1-XC,细胞内β-半乳糖苷酶活性为pcDNA3.1-XB+pCMVβ>pcDNA3.1-XC+pCMVβ>pcDNA3.1/Hygro(-)+pCMVβ。结论B基因型HBx反式激活能力高于C基因型HBx,而抗细胞增殖及致细胞凋亡能力都低于C基因型HBx,HBx这些功能差异可能与B、C基因型乙型肝炎病毒致病性差异有关。
Objective To study the functional differences of hepatitis B virus X protein (HBx) between B and C genotypes. Methods Eukaryotic expression vectors of hepatitis B virus X gene (pcDNA3.1-XB and pcDNA3.1-XC respectively) and blank vector pcDNA3.1 / Hygro (-) were transfected by lipofectamine 2000 Chang cells. The apoptosis rate was detected by flow cytometry after transfected cells for 2 days. The transfected cells were screened by hygromycin. The colonies of resistant cells formed after 14 days were fixed with cold methanol and stained with Gi msa. The vector pCMVβ was cotransfected into cells, and after 2 days of transfection, the cells were lysed and the intracellular β-galactosidase activity was detected. All data were analyzed by paired t-test. Results The apoptosis rate of cells transfected with Chang cells was pcDNA3.1-XC> pcDNA3.1-XB> pcDNA3.1 / Hygro (-), and the number of hygromycin resistant cells formed was pcDNA3.1 / Hygro (-)> pcDNA3.1-XB> pcDNA3.1-XC and the intracellular β-galactosidase activity was pcDNA3.1-XB + pCMVβ> pcDNA3.1-XC + pCMVβ> + pCMVβ. Conclusion The HBx transactivation ability of B genotype is higher than that of C genotype HBx, but the ability of antiproliferation and apoptosis is lower than that of C genotype HBx. The functional differences between HBx and B genotype may be related to hepatitis B virus Disease related to differences.