乙型肝炎病毒x基因转基因小鼠模型的建立及培育

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。

Establishment of transgenic mouse model harboring hepatitis B virus x gene

Objective: To establish a mouse model harboring hepatitis B virus x gene (adr subtype) for studying the function of hepatitis X protein. Methods: Expression vector pcDNA3-HBx, containing CMV promoter and hepatitis B x gene open reading frame (ORF) , was constructed by recombination DNA technique. After restriction enzyme digestion, the coding elements were microinjected into male pronuclei of mouse zygotes. The new born pups were evaluated by multiplex poly-merase chain reaction (PCR). Expression of x gene in transgenic mouse founders were confirmed by immunohistochemistry. Results: Expression vector pcDNA3-HBx was constructed. It was identified by restriction endonucleases digestion and confirmed by DNA sequencing. The sequence of HBx gene was coincident with the reported. Following microinjection the emby-ros were transferred to oviducts of pseudopregnant females. Eleven pups were born and survived, 5 of them were verified to integrate the HBx gene in their genomic DNA. With immunohistochemistry, X protein was detected mainly in hepatocytes cytoplasm of C57-TgN (HBx)SMMU transgenic mice. Then the transgenic mice were mated with normal mice to establish transgenic mice strains. Now one of them had F4 offsprings. Conclusion: HBx (adr subtype) transgenic mice C57-TgN (HBx)SMMU, harboring hepatitis B virus x gene are generated and X protein is expressed. It is a valuable animal system to study the roles of HBx gene in the course of HBV infection and in development of hepatocellular carcinoma in vivo.