乙型肝炎病毒preS2蛋白在转基因小鼠肝脏中的表达

Expression of hepatitis B virus preS 2 protein in transgenic mice liver

目的 :分析乙型肝炎病毒 pre S2蛋白在 3′末端缺失的 pre S/ S基因转基因小鼠肝脏中的分布及其病理学作用。方法 :采用原核显微注射法将质粒 pc DNA3.1- pre S/ St注射入小鼠受精卵雄原核 ,制备转基因小鼠。PCR法在基因组水平筛选 3′末端缺失的 pre S/ S基因转基因小鼠首建者 (founder)及后代 ;免疫组织化学法在蛋白水平检测 pre S2蛋白在这些小鼠中的表达 ;H- E染色分析转基因小鼠肝组织的病理学变化。结果 :经原核显微注射法将目的片段注射入受精卵雄原核后 ,共出生 15只新生小鼠 ,其中存活 7只 ,经 PCR检测后获得 2只 founder转基因小鼠 ,命名为 C5 7- Tg N (pre S/ St) SMMU。免疫组织化学检测发现转基因小鼠肝细胞质中有 pre S 2蛋白表达 ,H- E染色发现转基因小鼠肝组织中央静脉周围有淋巴细胞聚集。将这 2只转基因小鼠与正常同系异性小鼠交配 ,进行传代培育 ,PCR法筛选阳性转基因小鼠 ,目前已传至 F2 代。 结论 :本研究成功建立了稳定遗传 3′末端缺失的 pre S/ S基因并表达 pre S2蛋白的转基因小鼠 C5 7- Tg N((pre S/ St) SMMU,它将是体内研究 3′末端缺失的 pre S/

Objective: To analyze the distribution of preS 2 protein and the pathological changes in liver tissue of hepatitis B virus 3′ truncated preS/S gene transgenic mice. Methods: After restriction enzyme digestion, the pcDNA3.1 preS/S t plasmids, including 3′ truncated preS/S gene and CMV promoter were microinjected into male pronuclei of mice zygotes. The pups and offsprings were evaluated by polymerase chain reaction (PCR) at genomic DNA level. Expression of preS/S t gene in transgenic mice were confirmed by immunohistochemistry. H E staining was used to analyze the pathological changes. Results: Following microinjection of coding sequence of pcDNA3.1 preS/S t, the embyros were transferred to oviducts of pseudopregnant females. Fifteen pups were born and 7 of them survived, two of them were verified to integrate the preS/S t gene in their genomic DNA by PCR assay, named C57 TgN( preS/S t)SMMU. With immunohistochemistry, preS 2 protein was detected in hepatocytes cytoplasm of C57 TgN( preS/S t)SMMU transgenic mice. Lymphocyte effusion was found near the central vein of liver tissue and NP of hepatocytes increased in transgenic mice by H E staining. Then the transgenic mice were mated with normal mice to establish transgenic mice strains. Now one of them had F 2 offsprings. Conclusion:We have established preS/S t gene transgenic mice C57 TgN( preS/S t)SMMU, which is a valuable animal system to study the roles of preS/S t gene in development of hepatocellular carcinoma in vivo .