HBX基因逆转录病毒载体的构建及在LO_2细胞内的稳定表达  

Construction of a retroviral vector carrying HBX gene and its expression in LO_2 human hepatocytes

作　　者：张婷[1,2] 赵迎泽[1,2] 罗进勇[1] 卢晓娟[1,2] 文巍[1,2] 冯涛[1,2] 

机构地区：[1]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016 [2]重庆医科大学分子医学与肿瘤研究中心,重庆400016

出　　处：《南方医科大学学报》2010年第7期1526-1529,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30771925)

摘　　要：目的构建乙型肝炎病毒X基因(HBX)逆转录病毒载体,并使其在人正常肝细胞LO2内的进行稳定表达。方法 PCR扩增HBX基因,克隆到逆转录病毒载体质粒pSEB-Flag中,构建表达HBX的重组逆转录病毒载体质粒pSEB-Flag-HBX,通过通过PCR、酶切、测序验证HBX基因后;体外将重组质粒pSEB-Flag-HBX与包装质粒pAmpho共同转染人胚胎肾上皮细胞系293T细胞,包装获得携带HBX基因的重组逆转录病毒,并感染靶细胞人正常肝细胞LO2,稻瘟菌素(Blasticidin)筛选,获得成功转入HBX的LO2细胞。通过RT-PCR和Westernblot进一步验证HBX基因及HBx蛋白在LO2细胞内的表达。结果 (1)成功获得携带HBX基因的阳性重组质粒pSEB-Flag-HBX;(2)HBX基因被逆转录病毒成功地导入靶细胞LO2细胞,并实现稳定表达,RT-PCR检测到HBXmRNA在靶细胞中的表达,Westernblot检测到HBx蛋白在靶细胞LO2-HBx细胞中的表达。结论成功构建了携带HBX基因的重组逆转录病毒载体,感染人正常肝细胞LO2后能够稳定表达HBx蛋白,为进一步探讨HBx在肝癌发生发展中的作用提供了较为理想的实验模型。Objective To construct a retroviral vector carrying HBX gene and investigate its expression in LO2 human hepatocytes. Methods HBX gene was amplified by PCR and subcloned into the retroviral vector pSEB-Flag to construct a retroviral plasmid （pSEB-Flag-HBX） expressing HBX. The HBX gene insert was confirmed by restriction enzyme digestion, PCR and DNA sequencing. The recombinant retroviruses carrying HBX gene were generated in 293T cells co-transfected with pSEB-Flag-HBX and the packaging plasmids pAmpho, and used to infect LO2 human hepatocyte. After selection with blasticidin, the mRNA and protein expressions of HBx were determined by the reverse transcription-PCR and Western blotting, respectively. Results The retroviral plasmid （pSEB-Flag-HBX） carrying HBX was constructed successfully. The recombinant retrovirus efficiently delivered HBX gene into LO2 human hepatocyte, resulting in stable expression of HBX mRNA and HBx protein as shown by RT-PCR and Western blotting, respectively. Conclusion The recombinant retrovirus pSEB-Flag-HBX has been successfully constructed, which is capable of delivering the target gene HBX into LO2 human hepatocytes and results in stable expression of HBx to serve as an ideal model to study the effect of HBx on the development of hepatocelular carcinoma.

关 键 词：HBX LO2 逆转录病毒 肝癌 

分 类 号：R373[医药卫生—病原生物学]