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目的:建立可表达乙型肝炎病毒X蛋白的HBx基因(adr亚型)转基因小鼠模型,以研究x基因的功能。方法:采用基因重组技术构建含有CMV启动子和HBx基因(adr亚型)开放阅读框(ORF)的真核表达载体pcDNA3-HBx。该载体经Sal I限制性内切酶线性化后,琼脂糖电泳回收目的片段,用原核显微注射法将其注射入雄原核,制备转基因小鼠。复合PCR法在基因组水平筛选HBx基因转基因小鼠founder;免疫组织化学法在蛋白水平检测X蛋白在这些小鼠中的表达情况。结果:成功构建了HBx基因真核表达载体pcDNA3-HBx。经原核显微注射法将目的片段注射入受精卵雄原核后,出生并存活了11只新生鼠,经PCR检测后获得5只founder转基因小鼠,命名为C57-TgN(HBx)SMMU。免疫组织化学检测发现5只PCR阳性小鼠的肝细胞质中均有X蛋白的表达。将这5只转基因小鼠与正常同系异性小鼠交配,进行传代培育,复合PCR法筛选阳性转基因小鼠,目前已传至F4代。结论:本研究成功地产生了稳定遗传HBx基因并表达X蛋白的转基因小鼠C57-TgN(HBx)SMMU,它将有利于体内研究HBx基因在肝细胞癌发生中的作用。
OBJECTIVE: To establish a transgenic mouse model of HBx gene (adr subtype) expressing hepatitis B virus X protein to study the function of x gene. Methods: The eukaryotic expression vector pcDNA3-HBx containing CMV promoter and HBx gene (adr subtype) open reading frame (ORF) was constructed by gene recombination technology. After the vector was linearized with Sal I restriction endonuclease, the target fragment was recovered by agarose electrophoresis and injected into the prokaryotic nucleus by pronuclear microinjection to prepare transgenic mice. The multiplex PCR method was used to screen the HBx gene transgenic mice at the genomic level. Immunohistochemistry was used to detect the expression of X protein in these mice at the protein level. Results: The HBx gene eukaryotic expression vector pcDNA3-HBx was successfully constructed. After injecting the target fragment into the pronucleus of the fertilized ovum by pronuclear microinjection, 11 newborn mice were born and survived. Five founder transgenic mice were obtained after PCR detection, and named as C57-TgN (HBx) SMMU. Immunohistochemistry showed that the expression of X protein was found in the liver cytoplasm of 5 PCR-positive mice. The five transgenic mice were crossed with normal male mice and subcultured, and the positive transgenic mice were screened by multiplex PCR. At present, they have been transmitted to F4 generation. CONCLUSIONS: This study successfully generated a transgenic mouse C57-TgN (HBx) SMMU that stably inherited the HBx gene and expressed the X protein, which will be useful for studying the role of the HBx gene in hepatocellular carcinoma in vivo.