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目的建立在肝组织内含双链环状乙型肝炎病毒(HBV)基因组DNA的小鼠模型。方法用PCR扩增HBV单拷贝线状基因组,将PCR产物转染Huh7、Hep G2以及293T细胞,以ELISA检测培养细胞上清中的HBsAg和HBeAg;用尾静脉高压水动力法将PCR产物注射C57BL/6J小鼠,以免疫组化检测小鼠肝组织中的HBc Ag,ELISA检测小鼠血清中的HBsAg、HBeAg。细胞转染以及小鼠体内实验均用质粒p AAV-HBV1.2作为阳性对照。结果 HBV基因组PCR产物转染Huh7、Hep G2以及293T细胞均可分泌表达HBsAg和HBeAg,其中HBeAg的检测值分别为质粒p AAV-HBV1.2转染细胞的88.7%、79.9%和53.7%;PCR产物注射小鼠后,肝组织检测到HBc Ag,血清中检测到HBsAg和HBeAg,HBsAg和HBeAg最长持续时间分别为59 d和38 d,血清中HBsAg和HBeAg水平以及持续时间与质粒p AAV-HBV1.2注射的小鼠总体相似。结论建立了一种新的HBV转基因小鼠模型,该模型制备方法简单,并且肝组织内存在与HBV ccc DNA类似的HBV基因组。
Objective To establish a mouse model of hepatitis B virus (HBV) genomic DNA in liver. Methods The HBV single copy linear genome was amplified by PCR. The PCR products were transfected into Huh7, Hep G2 and 293T cells. The HBsAg and HBeAg in the supernatant of the cultured cells were detected by ELISA. The PCR products were injected into C57BL / 6J mice to detect HBc Ag in mouse liver by immunohistochemistry, and ELISA to detect serum HBsAg and HBeAg in mice. Plasmid pAAV-HBV1.2 was used as a positive control for cell transfection and in vivo experiments in mice. Results HBsAg and HBeAg were secreted by Huh7, Hep G2 and 293T cells. The detected values of HBeAg were 88.7%, 79.9% and 53.7% of those transfected with plasmid pAAV-HBV1.2 respectively. PCR The results showed that the longest duration of HBsAg, HBeAg, HBsAg and HBeAg detected in serum was 59 d and 38 d respectively after the mice were injected with the mice. The level and duration of HBsAg and HBeAg in the serum were similar to those of plasmid p AAV- The mice injected with HBV 1.2 were generally similar. Conclusion A novel model of HBV transgenic mice was established. The method for preparing this model is simple, and HBV genome similar to HBV ccc DNA exists in liver tissue.