摘要
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)中央人民政府。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)中央人民政府,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 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)中央人民政府 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)中央人民政府, 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.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2002年第11期1184-1187,共4页
Academic Journal of Second Military Medical University
基金
国家"九五"攻关项目(TJ99-LA01)
国家自然科学 基金资助项目(39670811)
上海市科学技术发展基金项目 (994919033).
