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建立萤火虫荧光素酶(Fluc)为报告基因标记的乙型肝炎病毒(HBV)小鼠模型。通过水动力转染10μg质粒pGL3-CP-Fluc-HBV1.2分别导入C57BL/6和BALB/c小鼠肝细胞内,定期取血检测HBsAg和HBV-DNA水平,活体成像检测Fluc在小鼠的表达。在BALB/c小鼠中,血清HBsAg表达水平在转染后第1w内迅速升高,随后快速下降。而在C57BL/6小鼠中,HBsAg表达水平下降较缓慢。血清病毒滴度在转染后第1d平均达到3.89×104copy/ml,在第4d和第7d达到最高,分别为1.61×105和1.31×105copy/ml,随后开始下降,在转染的第70d仍维持在103 copy/ml以上。免疫组化检测C57BL/6小鼠肝脏HBcAg的表达,结果显示在转染后第3dHBcAg高水平表达,第135dHBcAg仍有表达,但表达水平降低。活体荧光成像实时检测小鼠肝脏Fluc,结果表明两组小鼠均能持续高水平表达Fluc,转染后第7周Fluc的表达量仍然超过106 photon/s。我们建立的报告基因标记的HBV小鼠模型能够稳定表达HBV标志物和Fluc,为进一步进行HBV的疫苗评价提供了基础。
A firefly luciferase (Fluc) reporter gene-tagged hepatitis B virus (HBV) mouse model was established. The C57BL / 6 and BALB / c mouse hepatocytes were transfected with 10μg plasmid pGL3-CP-Fluc-HBV1.2 by hydrodynamic method, and the serum levels of HBsAg and HBV-DNA were detected periodically. Fluc in vivo expression. In BALB / c mice, the level of HBsAg in serum increased rapidly within the first week after transfection, then decreased rapidly. In C57BL / 6 mice, HBsAg expression decreased more slowly. Serum viral titers reached 3.89 × 104 copies / ml on the 1st day after transfection and reached the highest on the 4th and 7th days respectively, which were 1.61 × 105 and 1.31 × 105 copies / ml, respectively, and then began to decrease. At the 70th day after transfection Maintain at 103 copy / ml above. The expression of HBcAg in the liver of C57BL / 6 mice was detected by immunohistochemistry. The results showed that HBcAg was highly expressed on the 3rd day after transfection, and was still expressed on the 135th day, but the expression level was decreased. Live Fluorescence Real-time detection of mouse liver Fluc showed that both groups of mice continued to express Fluc at high levels, and Fluc expression still exceeded 106 photon / s at the 7th week after transfection. We established a reporter gene-tagged HBV mouse model that stably expresses HBV markers and Fluc, providing the basis for further vaccine evaluation of HBV.